Book of Abstracts List of Committees

Organising Committee Steering Committee

Chairs Ian Adcock, UK John Hamilton, Australia Ian Ahnfelt-Ronne, Denmark Eric Morand, Australia Gareth Bowen, UK Michel Chignard, France Gary Anderson, Australia John Hamilton, Australia Gareth Bowen, UK Gordon Letts, USA Andrew Cook, Australia Lisa Marshall, USA Michael Hickey, Australia Tineke Meijers, Canada Gordon Letts, USA Tatsutoshi Nakahata, Japan Alan Lewis, USA Wim van den Berg, The Netherlands Lisa Marshall, USA Kouji Matsushima, Japan Amy Roshak, USA Glen Scholz, Australia Ross Vlahos, Australia

Young Investigator Award Committee Program Committee

Chair: Chair: Glen Scholz, Australia Michael Hickey, Australia

Laurent Audoly, Canada Andrew Cook, Australia Susan Brain, UK John Hamilton, Australia John Schrader, Canada Lisa Marshall, USA Vincent Lagente, France Eric Morand, Australia Kouji Matsushima, Japan Glen Scholz, Australia Ross Vlahos, Australia

For enquiries after the Congress please contact the Congress Secretariat: ICMS Pty Ltd Attention: 7th World Congress on Infl ammation 2005 84 Queensbridge Street Southbank Vic 3006 Australia P: +61 3 9682 0244 F: +61 3 9682 0288 E: infl [email protected] W: www.infl ammation2005.com Contents

Sunday 21 August 2005 Abstract No. Page Title

Morning 1001 — Plenary 1: Peter Doherty 1002 -1004 — Symposium 1: Chronic Obstructive Pulmonary Disease 1005 - 1008 S 85 Symposium 2: Arthritis Afternoon 1010 - 1013 S 85 Focus Group 1: Understanding Infl ammation through Genetics, Genomics and Proteomics 1014 - 1018 S 86 Focus Group 2: Asthma 1019 - 1022 S 88 Focus Group 3: The Immunoregulatory Response 1023 -1027 S 89 Focus Group 4; Structure-Based Drug Design 1028 - 1032 S 90 Focus Group 5: Chronic Obstructive Pulmonary Disease 1033 - 1037 S 90 Focus Group 6: Rheumatic Diseases

Monday 22 August 2005 Abstract No. Page Title

Morning 2001 S 92 Plenary 2: Marc Feldmann 2002 — Research Highlights: Alberto Mantovani 2003 - 2006 — Symposium 3: Breaking Steroid Resistance 2007 -2010 S 92 Symposium 4: Infl ammation and Regeneration 2011 - 2015 S 92 Focus Group 19: Biomarkers and Imaging Afternoon 2016 – 2021 S 94 Focus Group 7: Cytokines 2022 – 2026 S 95 Focus Group 8; Leukocyte Traffi cking 2027 – 2032 S 96 Focus Group 9: Infl ammation and Cancer 2033 – 2038 S 97 Focus Group 10: Receptors/Signalling 2039 – 2043 S 98 Focus Group 11: Coagulation 2044 – 2049 S 99 Focus Group 12: Young Investigators

Tuesday 23 August 2005 Abstract No. Page Title Morning 3001 S 102 Plenary 3: Luke O’Neill 3002 — Research Highlights: Michael Karin 3003 – 3006 S 102 Symposium 5: Macrophages and Infl ammation – The Key to Much Pathology 3007 – 3011 S 103 Symposium 6: Matrix Metalloproteinases in Tissue Remodelling Afternoon 3012 –3016 S 104 Focus Group 13: Signalling in Infl ammation 3017 – 3021 S 105 Focus Group 14 Obesity/Diabetes and Infl ammation 3022 – 3025 S 106 Focus Group 15; Infl ammation in the Central Nervous System 3026 – 3030 S 106 Focus Group 16: Receptors and Innate Immunity 3031 – 3035 S 108 Focus Group 17: Atherosclerosis 3036 – 3039 S 109 Focus Group 18: Renal and Transplantation Immunology

Wednesday 24 August 2005 Abstract No. Page Title Morning 4001 –4004 S 110 Plenary 4: Diverse Mechanisms and Impact of Infl ammatory Responses 4005 – 4007 S 110 Plenary 5: Late Breaking Clinical Data on New Anti-Infl ammatory Drugs

Please note: No abstracts have been received for some sessions and the session titles are therefore not included in this supplement. Abstract numbers are identical to poster board numbers.

Posters will be on board from Sunday 21 August until the conclusion of the Congress on Wednesday 24 August. Abstracts for General Poster Topics

Sunday 21 August 2005 Presenters by Boards Abstract No. Page Topic 10:30 – 11:00 12:30 – 13:30 15:30 – 16:00 5001 -5041 S 115 Rheumatic Diseases

5042 – 5062 S 127 The Immunoregulatory Response

5063 - 5067 S 134 Structure-Based Drug Design

5068 - 5087 S 135 Asthma

5088 - 5096 S 141 Understanding Infl ammation Through Genetics and Protemics 5097 - 5106 S 143 Chronic Obstructive Pulmonary Disease

Monday 22 August 2005 6001 – 6031, 6033, 6037 S 147 Cytokines

6034, 6036, 6038-6052, 6084 S 156 Receptors/Signalling

6053-6054 S 161 Coagulation

6055-6070 S 162 Infl ammation and Cancer

6032, 6035, 6071-6083, 6085 S 155 Leukocyte Traffi cking

6086-6089 S 171 Diversity of Infl ammation

Tuesday 23 August 2005 7001-7044 S 173 Signalling in Infl ammation

7045-7067 S 185 Receptors and Innate Immunity

7068-7076 S 192 Obesity/Diabetes and Infl ammation

7077-7100 S 194 Infl ammation in the Central Nervous System

7101-7120 S 201 Atherosclerosis

7121-7125 S 207 Renal and Transplantation Immunology Young Investigator Award Finalists

S 212 Introduction

Mini Paper 1 2044 S 213 IL-23 Drives The Expansion Of Pathogenic Th IL-17 Cells Required For Autoimmune Infl ammation

Mini Paper 2 2048 S 215 Directed Gp130-Mediated Signalling Dissociates Infl ammation From Fibrosis In Bleomycin-Induced Lung Injury

Mini Paper 3 2045 S 217 A Novel Role For Annexin 1 In Macrophage Phagocytosis

Mini Paper 4 2046 S 219 Signalling Pathway Of TNF-α-Induced AQP3 Expression In Human Gingival Epithelial Cells: Implications In The Pathogenesis Of Periodontitis

Mini Paper 5 2047 S 221 Joint Damage And DC Function During Rheumatoid Arthritis Is Strictly Controlled By Fc Gamma Receptor Iib

Mini Paper 6 2049 S 223 Immune Complexes Alter Cerebral Microvessel Permeability: Roles Of Complement And Leukocyte Adhesion

Author Index S 225 Plenary Talks, Symposia and Focus Groups Infl amm. res. Supplement 2 (2005) Sunday 21 August 2005 S 85

1006 1012

Signalling Platforms that Mediate the Infl ammatory Response RNAi library screening based approaches to target identifi cation for Infl ammatory Diseases Christopher A McCulloch, University of Toronto, Canada Moitreyee Chatterjee-Kishore, Wyeth Research, United States IL-1-induced signals may serve in host defense or, paradoxically, contrib- ute to infl ammatory tissue injury in arthritis, periodontitis and lung fi brosis. Whole genome transcriptional and proteomic profi ling efforts that help IL-1 stimulates Ca2+ release and expression of the early response genes identify novel signaling pathway components and targets are powerful c-fos and c-jun as well as multiple cytokines and infl ammatory factors tools for drug discovery. However, much of the data obtained from expres- that drive extracellular matrix degradation via mitogen activated protein sion profi ling experiments provides information about genes and proteins kinase pathways. IL-1-induced signaling in fi broblasts and synoviocytes whose expression correlates with a functional phenotype. RNAi technol- requires the formation of protein complexes in spatially restricted cellu- ogy has emerged as a revolutionary new method of targeted gene silencing lar domains termed focal adhesions. Peptides based on the structure of and can greatly accelerate the process of gene functionalization and help the anti-adhesive domains of tenascin, osteonectin and thrombospondin validate the role of genes in causation of the phenotype under study. In the cause dispersion of focal adhesions. These peptides also block IL-1-in- last two years, libraries of chemically synthesized (siRNA) or expressed duced calcium fl uxes and ERK (but not JNK or p38. We have found that (shRNA) gene knockdown reagents have been developed and used to di- the protein tyrosine phosphatases PTP-alpha and SHP-2 are enriched in rectly identify genes which when knocked down directly affect phenotypes focal adhesions and there regulate IL-1-induced calcium signaling. This of interest. signaling pathway is dependent on the association of SHP-2 with PLC- We are using the power of RNAi library based gene knockdown to iden- gamma 1. We also found that the endoplasmic reticulum proteins calnexin tify genes that are of direct relevance to various infl ammatory phenotypes. and calreticulin physically associate with focal adhesions. Focal adhesion- Initial hits from these screens are then prioritized using expression and endoplasmic reticulum interactions are required for calcium release from functional profi ling and classifi ed using literature-mining tools such as In- the endoplasmic reticulum and subsequently, for activation of ERK. As genuityTM. Details of the RNAi library screening workfl ow and examples Ras signaling to ERK involves sequestration of Ras to endoplasmic re- of analysis of siRNA library screen data using various molecular profi ling ticulum membranes, the co-localization of focal adhesions with the endo- techniques will be presented. plasmic reticulum provides an attractive mechanism for optimizing signal transduction. We suggest that the spatial approximation of these organelles creates molecular staging sites for the effi cient conversion of upstream 1013 IL-1 signals into the activation of the protein kinase pathways that lead ultimately to ERK activation. Two Novel, Highly Upregulated Nuclear Factors in Infl ammation

Natasha C Foster, Centre for Immunology, St Vincents Hospital, Sydney, 1010 Australia Kristina Warton, Centre for Immunology, St Vincents Hospital, Sydney, ENU mutagenesis based screens Australia Wendy A Gold, Centre for Immunology, St Vincents Hospital, Sydney, Matthew Cook1,2, Carola Vinuesa1, Keats Nelms2, Chris Goodnow1,2 Australia Australian National University1 and Phenomix Australia2, Canberra ACT. Keith K Stanley, Centre for Immunology, St Vincents Hospital, Sydney, Australia Treatment for autoimmune diseases such as systemic lupus erythemato- sus and rheumatoid arthritis (RA) with global immunosuppression is often The aim of this study was to characterise two novel genes induced by pro- unsuccessful and frequently associated with side effects. Improved treat- infl ammatory cytokines in human endothelial cells. Both genes are up- ment will depend on targeting more precisely the key pathogenic path- regulated over 30 fold following treatment with IL-1?. They correspond ways. Strategies to identify the genetic basis of these pathways include to separate genes located less than 100kb apart on the same chromosome, investigation of mice with rare spontaneous mutations that give rise to and they share 78% homology at the amino acid level. We have named autoimmune phenotypes. Mice with engineered null mutations have also them NLFs, as they are Nuclear Localised Factors. The induction of NLF1 been informative although this approach is limited by a priori assumptions and NLF2 by infl ammatory stimuli was ablated by the proteasomal in- and only reveals the consequences of genetic loss-of-function. ENU muta- hibitor lactacystin, suggesting a requirement for NF-kB in infl ammation genesis is a method for screening the entire genome for novel pathogenic induced transcription. Futhermore, two sequences with homology to the pathways. After administration to male mice, ENU induces approximately NF-?B consensus site in the promoter of NLF1 were found to be required one point mutation every 1-2 Mb of genome in spermatogonial stem cells. for IL-1? upregulation. NLF1 and NLF2 fusion proteins localised to the Both gain- and loss-of-function (including hypomorphic) mutations re- nuclear compartment but were excluded from areas of dense heterochro- sult in dominant and recessive traits in G1 and G3 offspring, respectively. matin. Further, examination of the sub-nuclear localisation of NLF1 and About 1 in 1000 sperm contain a point mutation at a given locus so allelic NLF2 showed they accumulated in the nucleolus, suggesting a role in gene series can be identifi ed. We have used ENU mutagenesis to investigate transcription or RNA processing. Overexpression of NLF1 in HMVEC autoimmune disease by screening G3 mice for clinically obvious pheno- led to cell detachment from substratum, and real time RT-PCR analysis types, such as diarrhoea, and relevant serological abnormalities such as an- showed that Rnd1 and GEM-GTPase, two Rho kinases upregulated by IL- tinuclear autoantibodies. In addition, a modifi er screen is in place in which 1?, were also upregulated in NLF transfected cells. We propose that NLF1 a mouse model of RA is mutated to identify alleles that protect against and NLF2 are members of a new gene family of nuclear factors with a role disease and should suggest therapeutic strategies. Causative mutations in regulating genes that control cell architecture. have been mapped by conventional F2 outcrosses to reveal novel disease- associated alleles, which provide signifi cant clues to disease mechanisms. For example, a mutation induced in Roquin, a gene whose function was previously unknown, has elucidated a pathway for censoring high-affi n- ity autoantibodies in the periphery, which when defective leads to a lupus phenotype. S 86 Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

strong predictor of atopic asthma. This is the type of asthma that is likely 1016 to persist into adult life and preventing this type of asthma is likely to have an impact on disease prevalence. Immunoprophylaxis While these three wheezing phenotypes are easily distinguishable in longi- tudinal birth cohorts, they are not so easy to distinguish when faced with an Peter Sly, TVW Telethon Institute for Child Health Research, Australia individual wheezy child. However, the successful development of asthma preventative strategies is predicated on being able to determine, with a A major goal of asthma research is disease modifi cation or true prophy- reasonable degree of certainty, which children are likely to have persistent laxis. None of the currently available asthma therapies can be considered asthma. Longitudinal cohort studies have shown that the overlap between to be disease modifying but rather achieve symptom relief and disease sup- asthma and atopy is incomplete in children entering school. In Perth, WA, pression at best. Major improvements have occurred in asthma manage- approximately 40% of 6-year-old children are atopic, defi ned by at least ment over recent decades through the understanding that persistent airway one positive allergen skin prick test, and 40% reporting current wheeze. infl ammation underlies persistent asthma and through the concentration on However, only half of those with wheeze are atopic and only half of the anti-infl ammatory preventative therapies. While this management strategy atopic children wheeze. has produced genuine reductions in asthma severity and mortality and im- In attempt to clarify these relationships further we have examined data provements in quality of life for patients, it is not likely to achieve major from two longitudinal birth cohorts. The fi rst cohort is a community-based reduction in asthma prevalence and will not remove the disease burden birth cohort that was not selected on any asthma or atopic criteria. Almost from our community. True reduction in the prevalence of asthma requires 2000 children were assessed at approximately 6 years of age, determining different strategies, with a change of emphasis from disease control to the prevalence of wheeze in the past 12 months, current asthma (doctor di- disease prevention. Approaches to disease prevention including second- agnosed asthma plus current wheeze plus current asthma medication) and ary prevention, ie strategies designed to "switch off" or reverse a disease atopic status (skin prick tests to house dust mite, cat, grasses and moulds). process once it has become established and primary prevention, in which Atopy was considered as a quantitative variable by deriving a severity the disease is prevented from occurring in the fi rst place. A number of score based on wheal sizes for the individual allergens and the number of asthma prevention strategies have been proposed and trialed with varying positive tests. Both the risk of current asthma within the atopic population success. However, before considering asthma prevention strategies it is and the severity of asthma increased with increasing atopy severity over- necessary to review current theories on how asthma develops and becomes all. This relationship was driven by sensitization to house dust mite and persistent. cat, but not by sensitization to grasses or moulds. The second birth cohort The vast majority of asthma begins in childhood, however, not all children consists of 250 children at high risk of allergic sensitization and asthma diagnosed with asthma will have persistent asthma, with many losing their by virtue of having at least one parent with allergies and asthma. When symptoms during their school years. Clearly prevention strategies need to these children were assessed at 5 years of age, the prevalence of atopy, be directed against the types of asthma that are likely to become persistent. current asthma and of both were similar to those seen in the community Thus identifying these types of asthma is a key factor to developing suc- cohort. In an attempt to better defi ne atopic asthma a sub-group underwent cessful preventative strategies. inhalation challenge with either house dust mite or grass allergen. Twenty Epidemiological studies in children, particularly those employing longitu- six atopic children were challenged together with 11 non-atopic controls. dinal birth cohorts, recognize at least three distinct wheezing phenotypes Twelve atopics (46.2%), but no controls, had a positive challenge; all hav- in children: transient infantile wheeze; viral-associated wheeze and atopic ing both early and late-phase responses. The major factors predicting a asthma. Wheezing is common in the fi rst year of life, with at least one positive response amongst the atopics were larger wheal size [odds ratio episode of wheeze reported from approximately 30% to 60% of infants, 2.5 (95% CI 1.2-5.3)] and persistent atopy (ie positive at both 2y and 5y) depending of the characteristics of the study population. Many of these [18.0 (1.8-18.9)]. Receiver-operator characteristics analysis suggested a infants will only have a single episode of wheeze. Transient infantile wheal size on 4.5 mm as giving the best balance between sensitivity (0.75) wheeze is characterized by wheeze in the fi rst year of life with resolution and specifi city (0.86), with a positive predictive value of 82% and a nega- of wheeze before the third birthday. The major risk factors identifi ed for tive predictive value of 80% for a positive inhalation challenge. Adding this type of wheeze are maternal smoking during pregnancy and young clinical features such as current wheeze and other allergic features (atopic maternal age (defi ned as < 20y in western populations). The presumed dermatitis and allergic rhinoconjunctivitis) to wheal size increased the mechanism underlying maternal smoking is an interference with airway positive and negative predictive values to close to 100%. growth by the concentration of nicotine in the fetal circulation in infants The data from these two cohorts are complementary and suggest that the of smoking mothers. Infants born with smaller airways are more likely best predictors of atopic asthma in young children are the degree and se- to wheeze when confronted with any respiratory insult. The loss of the verity of allergic sensitization, the presence of early persistent sensitization wheezing tendency with growth is explained by increasing airway size and evidence of clinical expression of atopy. Armed with this information with growth. it should be possible to select children early in life who are likely to have The second epidemiologically distinct wheezing phenotype in children is persistent asthma, ie the children most likely to benefi t from asthma pre- viral-associate wheeze. This phenotype is arguably the most common seen ventative strategies. in children, especially in those in the preschool and early school years. The development of atopic asthma can be considered to occur in two Typically these children wheeze with viral infections but may not have per- phases; an initiation phase in early childhood and a disease consolida- sistent symptoms between episodes. Parents frequently report one episode tion phase that occurs later in life. During the initiation phase susceptible following closely upon the previous, especially during the winter months. children program Th-2 polarised immunological memory against inhal- Current paediatric asthma treatment guidelines recommend preventative ant allergens, as opposed to a modifi ed form of Th1-lioke memory that is (anti-infl ammatory) treatments for children who have episodes occurring characteristic of non-atopics. However, allergic sensitization per se is not at least once a month, meaning that the majority of these children are treat- suffi cient to drive the development of asthma. Additional infl ammatory ed with these therapies. Cohort studies show that this type of asthma is not insults to the lungs seem to be required. These insults may come from re- associated with an increased prevalence of atopy and that these children peated viral lower respiratory infections and/or exposure to environmental are likely to lose their symptoms during their school years. This phenotype irritants such as environmental tobacco smoke, air pollution or air tox- is likely to be responsible for the majority of children thought to "grow out ics. Infl ammatory insults to the lungs occurring during the process of lung of" their asthma. growth and development may cause respiratory symptoms in their own The third distinguishable phenotype is atopic asthma. These children are right, but when combined with allergic sensitization and repeated allergen likely to have a family history of allergic sensitization and of clinical atop- exposure markedly increase the risk of developing persistent asthma. Dur- ic disease. They may present with wheezing during their preschool years, ing the initiation phase allergen-driven Th-2 mediated infl ammation drives although those with the "worse" childhood asthma may present within the early development of asthma. This is followed by airway remodel- the fi rst year of life. These children are also likely to have other allergic ing and heightened bronchial responsiveness to a range of "non-specifi c" features and, in some series, sensitization to egg protein early in life is a environmental stimuli, characteristic of the disease consolidation phase Infl amm. res. Supplement 2 (2005) Sunday 21 August 2005 S 87 of persistent asthma. Removal of the allergen-specifi c or Th-2 mediated infl ammation during this stage does not appear to result in true disease 1017 modifi cation and it is likely that other infl ammatory cascades involving airway smooth muscle, myofi broblasts and fi brotic mechanisms are driv- Development and use of mouse models of viral exacerbations of ing this process. Following on from this logic, asthma preventative strate- asthma and chronic obstructive pulmonary disease (COPD) gies will need to be enacted during the disease initiation phase of asthma development to be effective. Rosa C Gualano, CRC for Chronic Infl ammatory Diseases, The Universi- There have been a number of asthma preventative strategies proposed. The ty of Melbourne, Australia primary preventative strategies trialed to date have primarily been based Ruth A Park, CRC for Chronic Infl ammatory Diseases, The University of on allergen avoidance to prevent allergic sensitization. These strategies Melbourne, Australia have been disappointing in that they have failed to reduce allergen expo- Jessica E Jones, CRC for Chronic Infl ammatory Diseases, The University sure, failed to prevent the development of asthma, or more recently actual- of Melbourne, Australia ly increased the prevalence of allergic sensitization. Dietary interventions Michelle J Hansen, CRC for Chronic Infl ammatory Diseases, The Uni- have been proposed and some studies carried out. One strategy that shows versity of Melbourne, Australia promise based on small, underpowered studies is supplementation of ma- Ross Vlahos, CRC for Chronic Infl ammatory Diseases, The University of ternal and or infant diet with Omega-3 polyunsaturated fatty acids. Modi- Melbourne, Australia fi cation of infant immune responses to allergen have been demonstrated Steve Bozinovski, CRC for Chronic Infl ammatory Diseases, The Universi- but large-scale clinical trials are required to know whether this strategy ty of Melbourne, Australia will ultimately prove to be of benefi t. Immunologically-based secondary Gary P Anderson, CRC for Chronic Infl ammatory Diseases, The Univer- prevention strategies have included attempts to suppress the expression of sity of Melbourne, Australia atopic disease in the airways or the switching off of allergic sensitization via immune deviation. There are insuffi cient data to know whether these Asthma and COPD are chronic lung conditions where cross talk between strategies are likely to be of any benefi t. many cell types leads to persistent, harmful infl ammation. Genetic and We have proposed an alternative approach to primary asthma prevention environmental factors contribute to both disorders and most COPD pa- by inducing "high-zone" tolerance to the common aeroallergens that ap- tients are long term smokers. In both conditions, exacerbations are a major pear to be important drivers of asthma in children. This approach, that cause of time off work, use of health resources and death. Most COPD we have named Oral Mucosal ImmunoProphylaxis (OMIP) is based on and asthma exacerbations are associated with a respiratory virus infection decades of animal research showing that exposure of the oropharyngeal and current treatments are of little benefi t. The molecular mechanisms mucosa to high doses of allergen results in an initial stimulation of the behind viral exacerbations of asthma and COPD are poorly understood immune system with the generation of Th-2 responses and the production and a diverse range of respiratory pathogens are implicated. However, of low levels of allergen-specifi c IgE antibodies, followed rapidly by the exacerbations are potentially treatable. development of immunological tolerance. Recent data in humans, show- Using an ovalbumin sensitization protocol to induce an asthma-like state, ing biphasic dose response curves to allergen exposure, supports this con- and a short term cigarette smoke exposure protocol, we have developed cept. Recent epidemiological data shows that high exposure to animals in mouse models with many features of asthma and COPD. These mice are early life protects against the development of allergic sensitization and/or then infected with an intermediate virulence strain of live infl uenza virus. asthma. This effect does not appear to be explained by exposure to bacte- We have studied many aspects of lung infl ammation and in general, the rial products that may be associated with animals, however, a role for such “exacerbation” mice are worse off than control mice given virus alone or exposures cannot be excluded. In addition, the increase in allergic sensiti- subjected to the sensitization or smoke protocols without virus infection. zation to house dust mite in the recent study from the UK where exposure Infl ammatory parameters of interest include cellularity of the bronchoal- to house dust mite was reduced to very low levels, is supportive. Further veolar lavage (BAL, a washout of the lungs), lung virus titres, CD4+ and evidence in support of this concept comes from the natural history of the CD8+ lymphocyte profi les, lung proteases and induction of mRNA and immunological responses to food allergens. There is evidence of almost protein for pro-infl ammatory cytokines. The mouse exacerbation models universal recognition of and response to food allergens within the fi rst year have great potential for identifi cation of therapeutic targets and improving of life. This results in the production of allergen-specifi c IgE antibodies our knowledge of these debilitating disorders. that, in most children, remain in the subclinical range and are not associ- ated with clinical symptoms. These responses are 'switched off", presum- ably by continued exposure to the allergens and are no long present in most 1018 children after the second or third year of life. We have evidence from our cohort studies of similar processes occurring with aeroallergens. In our Aberrant Activation of the Src-family Kinase Hck Results in high-risk cohort 70 children had positive skin prick tests to aeroallergens, Spontaneous Pulmonary Infl ammation and an Enhanced Innate primarily house dust mite, at two years of age. Half of these children had Immune Response in a T-cell Independent Manner lost these responses by the time they were fi ve years old. Based on these principles, we are in the process of commencing a proof-of- Matthias Ernst, Ludwig Institute for Cancer Research, PO Royal Mel- concept study in which children at high risk of developing allergic sensiti- bourne Hospital, VIC3050, Australia zation to aeroallergens and atopic asthma, but who are not yet sensitized, Melissa Inglese, Ludwig Institute for Cancer Research, PO Royal Mel- will receive OMIP with a combination of aeroallergens or placebo daily for bourne Hospital, VIC3050, Australia 12 months. These children will then be followed for a further three years to Jessica Jones, Dept of Pharmacology, University of Melbourne, determine whether true tolerance has been induced and whether the devel- VIC3050, Australia opment of asthma has been prevented. Should this proof-of-concept study Dianne Grail, Ludwig Institute for Cancer Research, PO Royal Melbour- prove successful a series of further questions will need to be investigated. ne Hospital, VIC3050, Australia These include: what is the appropriate dose and how long should treat- Steven Bozinovski, University of Melbourne, Australia ment be continued; is "consolidation" therapy, as used in immunotherapy, Ross Vlahos, Dept of Pharmacology, University of Melbourne, VIC3050, required; and does OMIP prevent sensitization to bystander allergens? Australia Acknowledgements Gary P Anderson, Dept of Pharmacology, University of Melbourne, A large number of people have made major contributions to the data I have VIC3050, Australia presented. These include, but are not limited to: Patrick Holt, Richard Loh, Bengt Bjorksten, Ulrich Wahn, Hugh Sampson, Nicholas deKlerk, Merci Aberrant activation of Src tyrosine kinases family members is generally Kusel, Tonia Douglas, Jacqui Jospeph-Bowen and Felicity Flack. considered to confer oncogenic activity. To identify the consequence of constitutive activation of the myelomonocyte-specifi c family member Hck, we generated HckF “knock-in” with a Y499F substitution of the C- S 88 Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) terminal, negative regulatory tyrosine residue (Ernst et al., JEM 2002). Homozygous HckF mice spontaneously show an asthma-like lung pathol- 1021 ogy characterized by extensive eosinophilic and mononuclear infi ltrates within the parenchyma, alveolar airspaces and around blood vessels, as Myxomaviral Serpin Reactive Center Loop Sequence Determines well as epithelial mucus metaplasia in conducting airways. Lungs in ag- Outcome of Vascular Infl ammatory Responses ing HckF mice showed mild emphysema and fi brosis resulting in altered lung function and respiratory distress, associated with body wasting and Erbin Dai, Robarts Research Institute, Canada occasional pulmonary adenocarcinomas. Transnasal challenge of HckF Kasinath Viswanathan, Robarts Research Institute, Canada mice with LPS induced an exaggerated pulmonary innate immune re- Yuming Sun, University of Western Ontario, Canada sponse, characterized by excessive release of MMP2/9 and TNFa. HckF Lying Liu, Robarts Research Institute, Canada mice were also highly sensitive to LPS mediated endotoxemia, and mu- Peter C Turner, University of Florida, United States tant macrophages and neutrophils exhibited enhanced effector functions Richard W Moyer, Inversity of Florida, United States in vitro (e.g. NO and TNFa production, chemotaxis and degranulation). Grant McFadden, University of Western Ontario, Canada Genetic ablation of TNFa partially prevented development of the lung dis- Alexandra R Lucas, Robarts Research Institute, Canada ease, while bone marrow reconstitution of diseased HckF mice with wild- type cells ameliorated the lung pathology. However, established disease Background: The thrombolytic serine protease cascade is intricately in- in HckF mice was insensitive to treatment with steroids or neutralizing volved in activation of infl ammatory responses. The urokinase-type plas- antibodies against CD4 or IL5. minogen activator (uPA) and its receptor, uPAR are up regulated at sites Based on the functional association of Hck with leukocyte integrins, we of vascular injury. Serp-1, a myxomaviral serpin, targets uPA/uPAR, dis- propose that constitutive Hck activation mimic adhesion-dependent prim- playing exceptional anti-infl ammatory and anti-atherogenic activity in a ing of leukocytes and causes spontaneous infl ammatory lung disease in an wide range of animal models. The reactive center loop (RCL) amino acid IL5 and T-cell-independent way. Thus, uncontrolled activation of Hck sequence, the signature of serpins defi nes the precise activity of each ser- may skew innate immunity from a reversible physiological host defense pin. We have examined the role of the Serp-1 RCL sequence and muta- response to one causing irreversible tissue damage and Hck may therefore tions that mimic known mammalian and viral serpins in animal models serve as a potential novel pharmacological target. of infl ammation, thrombosis and vascular remodeling. Methods: Throm- bosis, infl ammation, remodeling and plaque growth were assessed after infusion of Serp-1, Serp-1 chimeras, anti-apoptotic viral serpins (Serp-2 1020 and CrmA) and mammalian serpin, PAI-1 in wild type C57BL/6, PAI- 1-/- and uPAR-/- defi cient mouse aortic transplants. Results: Altering the Concurrent NKT Cell-Dependent Leukemia Eradication and P1-P11, scissile bond, Arg-Asn sequence compromised Serp-1 anti-in- Immune Regulation Following Stem Cell Mobilization with Potent fl ammatory activity, P11 Thr increased thrombosis and P1 Met increased G-CSF Analogues remodeling. Substitution of the P2-P7 arm with alanine (X6), converted the anti-infl ammatory activity of Serp-1 to a diametrically opposed, exces- Kelli P MacDonald, The Queensland Institute of Medical Research, sive pro-infl ammatory response with mononuclear cell activation, throm- Australia bosis and aneurysm formation (P<0.03). PAI-1 produced marked throm- Edward S Morris, The Queensland Institute of Medical Research, Aus- botic responses (P<0.0001), while CrmA increased plaque and aneurysm tralia formation. uPAR defi ciency either blocked anti-infl ammatory activity or Geoff R Hill, The Queensland Institute of Medical Research, Australia exacerbated infl ammation. Conclusions: (1) the Serp-1 RCL sequence is unique, with profound anti-infl ammatory and anti-atherogenic actions (P Stem cell mobilization with G-CSF has immunomodulatory actions and <0.01). (2) Alteration of the P1-P11 (Arg-Asn) sites blocks anti-infl amma- we have studied new more potent anologues in relation to regulatory T tory activity and altering the P2-P7 sequence induces powerful pro-infl am- cells, antigen presenting cells (APC) and NKT cells. Stem cell mobiliza- matory responses, indicating exquisite Serp-1 RCL sequence specifi city tion with the novel G-CSF and Flt-3L chimeric cytokine, Progenipoietin-1 for anti-infl ammatory actions. (ProGP-1), signifi cantly reduces CD4 dependent graft-versus-host disease (GVHD) due to the generation of regulatory APC and T cell populations that are characterized as monocyte precursors and CD4+CD25+ T cells re- 1022 spectively. These cell populations differentially require IL-10 for the gen- eration of T cell dependent regulation. We have now studied the graft-ver- An essential role for hPGD 2 synthase derived cyclopentenone sus-leukaemia (GVL) effects after transplantation of stem cell mobilized prostaglandins in the resolution of T cell driven immune responses by ProGP-1, which are essential for translation to clinical practice. NKT cells have pivotal roles in immune regulation and tumor immunosurveil- Seema G Trivedi, Experimental Pathology Group, William Harvey Rese- lance, and we now show that donor stem cell mobilization with ProGP-1 ach Institute, United Kingdom markedly expands and activates donor NKT cells which have enhanced Ravindra Rajakariar, William Harvey Research Institute, United King- functional responses to α-galactosylceramide. In a murine model of alloge- dom neic stem cell transplantation (SCT), donor NKT cells promoted host DC Justine Newson, The Rayne Institute, University College London, United activation and enhanced perforin-restricted CD8 T cell cytotoxicity against Kingdom host-type antigens. Following leukaemic challenge, donor treatment with Paul R Colville-Nash, SW Thames Institute for Renal Research, United ProGP-1 signifi cantly improved overall survival when compared to G- Kingdom CSF or control, attributable to reduced GVHD mortality and paradoxical Derek W Gilroy, The Rayne Institute, University College London augmentation of GVL effects. Enhanced cellular cytotoxicity was depen- dent on donor double-negative NKT cells, and leukaemia clearance was The resolution of infl ammation is a highly co-ordinated and active event profoundly impaired in recipients of NKT cell-defi cient grafts. Enhanced whose dysregulation may result in chronic infl ammation. Type IV delayed cytotoxicity and GVL effects were not associated with Flt-3L signalling type hypersensitivity (DTH) is an example of acute resolving infl ammation or effects on dendritic cells, but were reproduced by prolonged G-CSF re- driven by T-lymphocytes. While a great deal is known about the factors ceptor engagement with pegylated G-CSF. Thus, modifi ed G-CSF signal- that control its initiation, little is known about the endogenous signals that ling during stem cell mobilization prevents CD4 dependent GVHD, whilst bring about the resolution of such DTH responses. Arguably, understand- paradoxically augmenting NKT cell dependent CD8 cytotoxicity and GVL ing these mechanisms will provide insight into chronic debilitation diseas- effects. GVHD and GVL are thus effectively separated, greatly expanding es like rheumatoid arthritis, where T-lymphocytes play a pathogenic role. the potential applicability of allogeneic SCT for the therapy of malignant As prostaglandin D2 and its metabolite the cyclopentenone prostaglandin, disease and the induction of tolerance prior to solid organ transplantation. 15d-PGJ2, are essential for the resolution of acute pleuritis, we questioned Infl amm. res. Supplement 2 (2005) Sunday 21 August 2005 S 89

whether such pro-resolution properties of PGD2 and its metabolites ex- JAK activity is also associated with a number of immune diseases includ- tended to T cell mediated responses. We examined the course and severity ing rheumatoid arthritis, psoriasis and asthma. Accordingly, the develop- of a methylated BSA (metBSA)- induced DTH reaction in mice defi cient ment of JAK2 specifi c inhibitors has tremendous clinical relevance. Criti- in- and over expressing haematopoietic PGD2 synthase(hPGD2S), the en- cal to the function of JAK2 is its protein tyrosine kinase (PTK) domain. zyme that synthesises PGD2. Paw swelling was greater in knockouts than We report the crystal structure of the JAK2 PTK domain in complex with in controls, with infl ammation failing to resolve. Throughout the course of a high affi nity, pan-JAK inhibitor that appears to bind via an induced fi t -/- infl ammation lymph node cells from hPGD2S mice exhibited signifi cant- mechanism. This inhibitor was buried deep within a constricted ATP-bind- ly heightened antigen and mitogen specifi c proliferative responses after 72 ing site, in which extensive interactions, including residues that are unique -/- hours of culture compared to WT cells. Moreover, hPGD2S lymphocytes to JAK2 and the JAK family, are made with the inhibitor. We present a generated more IL-2 in response to metBSA and comparatively less IL-4 structural basis of high affi nity JAK-specifi c inhibition that will undoubt- than did controls. In contrast, paw swelling in transgenics was barely de- edly provide an invaluable tool for the further design of novel, potent and tectable, with no difference in lymph node cell function when compared to specifi c therapeutics against the JAK family.

WT mice. These data implicate hPGD2S- derived eicosanoids as negative regulators of the adaptive immune system, possibly controlling the bal- ance of Th1 versus Th2-type lymphocytes. Ex vivo studies revealed that 1026 the protective effects of hPGD2S came from 15d-PGJ2 and not parental

PGD2. These protective effects were not mediated through either PPARγ Novel Insights into Functions of Immediate-Early Genes In Vascular or PPARα, but through the inhibition of NF-κB. Permeability and Infl ammation using Catalytic DNA

Levon M Khachigian, University of NSW, Australia 1023 Alla Waldman, University of NSW, Australia Roger Fahmy, University of NSW, Australia Virtual screening approaches to disrupting receptor signalling / Michael Perry, University of NSW, Australia Victorian Synchrotron Carolyn Geczy, University of NSW, Australia Guishui Zhang, University of NSW, Australia Michael W Parker, St. Vincent’s Institute of Medical Research, Australia Increased vascular permeability and leukocyte infi ltration are key infl am- Signalling events across cellular membranes are quite often initiated by matory processes underlying the pathogenesis of a diverse range of dis- protein-protein interactions. We have been studying a number of protein- orders including atherosclerosis, asthma, psoriasis, rheumatoid arthritis protein receptor interactions with the aim of understanding how signals are and sepsis. Our understanding of the transcription factors regulating these transmitted across membranes and how these signals might be modulated processes is limited. DNAzymes are RNA-cleaving phosphodiester-linked as a basis for the discovery of new drugs. DNA-based enzymes that seek out and cleave their target mRNA in a Historically, it has been the dogma that small molecule inhibitors of pro- gene-specifi c fashion. We demonstrate that DNAzymes (Dz13) targeting tein/protein interactions are unlikely, given the usually large interfaces in- the G1311-U junction in the mRNA of the immediate-early gene c-Jun volved. In recent years, however, a number of protein/protein interactions localize to blood vessels in the ears of Balb/c mice and block the increase have been inhibited with small, drug-like molecules. Although protein- in vascular permeability following passive cutaneous anaphylactic chal- protein interfaces are often large, most of the binding energy can be attrib- lenge and administration of vascular endothelial growth factor (VEGF165) uted to small hotspots and there sometimes exist crevices to which small in the dorsal skin of athymic nude mice. Dz13 suppressed retinal neo- molecules can bind with reasonable affi nity. Examples of recent successes vascularization in a C57BL/6 mouse model of retinopathy of prematurity. in the area include small molecule inhibitors of the interaction between the Further, it abrogated monocyte-endothelial adhesion in a co-culture model, tumour suppressor p53 and its cognate ubiquitin ligase, mdm2, the IL-2/ and abolished leukocyte rolling, adhesion and extravasation through en- IL-2Ralpha interaction, the Bcl-XL interaction with BAK, and the HIF-1 dothelium in the mesenteric vasculature of Sprague-Dawley rats super- alpha interaction with p300. fused with interleukin-1beta (IL-1beta). Dz13 blocked IL-1beta-induc- We have developed a procedure to screen virtual libraries of compounds ible endothelial ICAM-1 and VCAM-1 expression but had no effect on for inhibitors of protein/protein interactions. We have assembled a library PECAM-1. Dz13 also suppressed neutrophil infi ltration in the lungs of of over 4 million commercially available compounds. Inclusion of drug- Balb/c mice challenged with lipopolysaccharide (LPS). These fi ndings, like fi lters reduces the size of the library to about 1 million compounds. which follow our recent report of Dz13 inhibition of angiogenesis and tu- Potential inhibitors are bought commercially and their activity examined mour angiogenesis (1), provide the fi rst demonstration of c-Jun regulating using standard assays. In this talk I will describe our recent results in the leukocyte-endothelial cell adhesion and infi ltration and suggest that c-Jun discovery of compounds to inhibit protein ligand-receptor interactions DNAzymes may be useful as novel gene-specifi c therapeutic tools in acute with a particular focus on infl ammation targets. infl ammatory diseases. (1) Zhang et al. 2004. J. Natl. Cancer Inst. 96:683. 1024 1027 The structural basis of Janus Kinase 2 inhibition by a potent and specifi c pan-Janus kinase inhibitor Discovery of p38-alpha MAP Kinase Inhibitor SCIO-469

Isabelle S Lucet, Monash University, Australia Sundeep Dugar, Scios Inc., United States Emmanuelle Fantino, Cytopia Pty Ltd, Australia Michelle Styles, Cytopia Pty Ltd, Australia In recent years there have been several reports of efforts to fi nd small Andrew W Wilks, Cytopia Pty Ltd, Australia molecule inhibitors of p38 MAP kinase (MAPK) as potential therapy for Jamie Rossjohn, Monash University, Australia several disease areas. p38a MAPK is an intracellular soluble serine threo- nine kinase which is activated in response to stress, growth factors and JAK2, a member of the Janus kinase (JAK) family of Protein Tyrosine cytokines, such as IL-1b and TNF-a. It is considered to have an important Kinases (PTKs), is an important intracellular mediator of cytokine signal- patho-physiological role in diseases, such as rheumatoid arthritis, where ling. Mutations of the JAK2 gene are associated with hyperproliferative chronic infl ammation is said to play a causal role. More recently, a role has syndromes such as Polycythemia Vera, whilst rearrangements of the gene been described for the utility of p38 inhibition in a number of oncologic are seen in hematological cancers such as atypical Chronic Myelogenous settings in which the modulation of the immune response to facilitate anti- Leukemia and Acute Lymphoblastic Leukemia. Furthermore, aberrant S 90 Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) tumor activity, either singly or in combination with chemotherapy has been the infl ammatory changes in balb/c, sv129, A/J and c57/bl6 mice after reported to provide clinically benefi cial effects. an acute exposure to cigarette smoke (CS). Mice were exposed to 1R3F, This presentation will describe the structure-activity studies of a series of UKY Research Cigarettes for 3 days (40 mls of CS per minute). For dose- compounds that led to the identifi cation of SCIO-469. The presentation response studies mice were exposed to 2-5 cigarettes and for time course will include data on the profi ling studies of these indole based piperazine studies mice were exposed to 4 cigarettes daily and then culled 6, 24, 48 amide inhibitors of p38 kinase that led to the selection of SCIO-469 as or 72h after the third exposure. In all 4 strains, CS dose-dependently in- a clinical candidate. SCIO-469 is presently in phase II clinical trials, for creased BAL fl uid (BALF) neutrophils numbers and neutrophil chemokine evaluation as a potential treatment for rheumatoid arthritis. levels (KC and MIP-2) in BALF or tissue. The cytokine changes were greatest at 6h while the neutrophil increases peaked 24h after the third exposure. There was an increase in BALF macrophages in c57/bl6 mice 1031 at 3 and 6h post-exposure (p < 0.05), while macrophages were only sig- nifi cantly increased 72h after the last exposure in A/J and balb/c mice (p Advanced murine models of COPD < 0.05). BALF lymphocytes were signifi cantly increased in c57/bl6 and balb/c mice 24-72h post-exposure (p < 0.01) and 48-72h in A/J’s ( p < Ross Vlahos, CRC for Chronic Infl ammatory Diseases, The University of 0.01). The infl ammatory changes were greatest in the balb/c strain, so they Melbourne, Australia were chosen to directly compared the effects of budesonide (Bud; 0.3-3 Steven Bozinovski, University of Melbourne, Australia mg/kg), N-acetylcysteine (NAC; 100-1000 mg/kg) and rofl umilast (Rof; Michelle J Hansen, CRC for Chronic Infl ammatory Diseases, The Uni- 1-10 mg/kg) on CS-induced infl ammation. Balb/c’s were dosed orally, versity of Melbourne, Australia 1h before each exposure to 5 cigarettes for 3 days (exposure period ~ 45 Rosa C Gualano, CRC for Chronic Infl ammatory Diseases & The De- minute/day) and culled 24h after the last exposure. Bud and NAC had partment of Pharmacology, The University of Melbourne, Australia no effect on neutrophil infi ltration, although there appeared to be a trend Gary P Anderson, CRC for Chronic Infl ammatory Diseases, The Univer- towards inhibition in the NAC-treated animals. Rof dose-dependently in- sity of Melbourne, Australia hibited CS-induced infl ammation with signifi cant inhibition achieved at both 3 and 10 mg/kg (p < 0.05). These data suggest that this modelling COPD is characterised by persistent airfl ow limitation and an abnormal system may be relevant for investigating the early mechanisms involved in lung infl ammatory response to noxious particles. This abnormal response the CS-mediated infl ammation associated with COPD. consists of neutrophilia, macrophage accumulation, increased protease expression and induction of various cytokines and chemokines. There is fi brosis, obstruction of small airways and emphysema with airspace en- 1036 largement and destruction of lung parenchyma. COPD patients also have signifi cant weight loss due to peripheral muscle wasting. Cigarette (cig) Effi cacy of a novel, orally active MIF antagonist in a model of smoking is the major cause of COPD and there is no satisfactory therapy. rheumatoid arthritis A better understanding of the cellular and molecular responses induced by cig smoke may yield new molecular targets for the development of Eric Morand, Monash University, Australia therapeutics. Our group has developed unique mouse models of COPD to Peter Tapley, Cortical Pty Ltd, Australia investigate the cellular and molecular mechanisms underlying COPD. By Pamela Hall, Monash University, Australia comparing outcomes from short- and long-term cig smoke exposure pro- Lisa di Nezza, Cortical Pty Ltd, Australia tocols we found that short-term protocols may be a useful predictor of the Tom Gilbert, Monash University, Australia development of emphysema and such models may be useful in identifying Yuanhang Yang, Monash University, Australia therapeutic targets. We have exposed mice to cig smoke for 4 days and Basil Danylec, Monash University, Australia found an increase in infl ammatory cells, proteases, cytokines and chemo- Magdy Iskander, Monash University, Australia kines. Mice exposed to cig smoke for 4 and 12 weeks had similar BALF cellularity to those observed in the 4-day protocol, with the added feature The cytokine macrophage migration inhibitory factor (MIF) has a well-es- of emphysema. In addition, mice exposed to both short- and long-term tablished role in the pathogenesis of RA. Computer modeling of MIF pro- cig smoke lost signifi cant body weight that was associated with muscle tein Xray crystallographic data led to the design and synthesis of a novel wasting. In our unique in vivo models of COPD exacerbations, smoke- MIF antagonist small molecule compound, COR100140, which we tested exposed mice infected with live infl uenza had signifi cantly more macro- in a model of RA. phages, neutrophils and CD8+ T-lymphocytes in BALF than mice infected Arthritis was induced by intra-articular injection of mBSA in pre-immu- with infl uenza only. Proteomic and microarray analysis of lungs obtained nized mice and assessed by clinical and histological scoring. Mice re- from our models have identifi ed numerous cig smoke-induced proteins and ceived COR100140 by IP injection or orally. T cell activation was assessed genes. In conclusion, the cellular and molecular mechanisms affected by by examination of footpad DTH, and antigen-specifi c T cell proliferation cigarette smoke in mice may have relevance to COPD and these models ex vivo. Cultured human RA synovial and dermal fi broblasts were treated may be used to identify candidate therapeutic targets for COPD. with COR100140 in vitro. Relative to vehicle-treated controls, IP administration of COR100140 (15 mg/kg) resulted in signifi cantly reduced joint swelling (P<0.05) and his- 1032 tological evidence of synovitis, exudates, cartilage degradation and bone damage (P<0.05). Furthermore, COR100140 treatment was associated An acute, steroid-insensitive model of cigarette smoke-induced with reduced footpad DTH (P<0.05) and ex vivo T cell activation (P<0.05). infl ammation in mice Oral COR100140 (25 mg/kg) was similarly effective, with signifi cant in- hibition of joint swelling (P<0.05) and histological evidence of infl am- Gillian Kinnear, Novartis Institute of Biomedical Research, United mation and damage (P<0.05). The magnitude of therapeutic responses to Kingdom COR100140 were similar to those observed in MIF -/- mice. In contrast, Daniel Wyss, Novartis Institute of Biomedical Research, United Kingdom etanercept (4 or 12 mg/kg) did not inhibit disease in this model. In vitro, Andy Nicholls, Novartis Institute of Biomedical Research, United King- COR100140 signifi cantly inhibited IL-1-induced human RA synovial fi - dom broblast proliferation (P<0.05), and IL-1-induced fi broblast expression of Christopher S Stevenson, Novartis Institute of Biomedical Research, cyclooxygenase-2 (P<0.05). United Kingdom Small molecule MIF antagonist compounds represent a potential new ther- apeutic class, the fi rst oral anti-cytokine drugs. COR100140 is effective COPD is a smoking-related disorder associated with an abnormal infl am- in an animal model of RA. These data strongly support the development matory response of the lung that is steroid-insensitive. Here, we describe of MIF antagonist compounds as therapeutics in RA. Moreover, activity Infl amm. res. Supplement 2 (2005) Sunday 21 August 2005 S 91 of COR100140 in a model in which etanercept is ineffective is consistent with potential application in anti-TNF resistant RA.

1037

Therapeutic application of NFkB decoy deoxyoligonucleotides on joint destruction of CIA monkeys

Tetsuya Tomita, Department of Orthopaedics, Osaka University Graduate School of Medicine, Japan Hideo Hashimoto, Department of Orthopaedics, Osaka University Gra- duate School of Medicine, Japan Yasuo Kunugiza, Department of Orthopaedics, Osaka University Gradua- te School of Medicine, Japan Naruya Tomita, Division of Nephrology, Department of Internal Medici- ne, Kawasaki Medical College, Japan Ryuichi Morishita, Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Japan Hideki Yoshikawa, Department of Orthopaedics, Osaka University Gra- duate School of Medicine, Japan

Purpose: Recently transfection of cis element double-stranded oligode- oxynucleotides (ODN) (= decoy) as a new powerful tool in a new class of anti-gene strategy for gene therapy has been reported. The purpose of this study is to evaluate the preclinical therapeutic effi ciency and safety of NFkB decoy ODN using CIA in cynomolgus monkey. Methods: Male cynomolgus monkeys were immunized with bovine type II collagen in complete Freunds’ adjuvant. One week later, monkeys were re- ceived intradermal booster injection. Five monkeys were given intraarticu- lar injection of NFkB decoy ODN into bilateral wrist (total 10 joints) every 2 weeks from day 7 up to 11 weeks. For the vehicle group (n=5, 10 joints), the same volume of scrambled decoy (SD) ODN was administrated with same method. Blood samples for biochemical parameters and x-rays of wrist joints were collected every 2 weeks, starting from just before the fi rst immunization. Biochemical parameters were also examined in this study. At 14 weeks, the monkeys were sacrifi ced for histologic evaluation for joints and other major organs. To study the effect of NFkB decoy ODN the joint synovium was harvested at 35 days. The level of TNFa and IL-1b was determined using ELISA kit. Results: Radiographic examination showed marked suppression of joint destruction with NFkB decoy ODN treated (NFkB D) group compared with untreated or SD group. The histologic examination showed hyperplasia of synovitis with severe destruction of articular cartilage and bone in untreated and SD group. However, slight sy- novitis was confi rmed in the joints, the articular cartilage and subchondral bone was almost intact in 70% in the joints of NFkB D group. There were signifi cant differences in the average expression level of TNFa and Il-1b in the synovium among these groups (p<0.01). Conclusion: We demonstrated that consecutive intraarticular injection of NFkB decoy ODN suppressed the severity of joint destruction. S 92 Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

and subsequent process including the migration, survival and functional 2001 maturation of differentiated cells. It has been reported that after spinal cord injury (SCI), the endogenous NSCs do not differentiate into neurons but TNFα blockade : validation of importance of cytokines in medicine into astrocytes, resulting in the formation of glial scars. Based on reports that the expression of IL-6 and the IL-6 receptor is sharply increased in the Marc Feldmann, Kennedy Institute of Rheumatology Division, Faculty of acute stages after spinal cord injury, and that IL-6 may serve as a factor Medicine, Imperial College London, United Kingdom strongly inducing the differentiation of NSCs into astrocytes, we examined the effects of an antibody to the IL-6 receptor in cases of SCI, and found TNFα, discovered in cancer and cachexia studies, is the most rapidly re- that the antibody indeed suppressed secondary injury (caused by infl am- leased cytokine after any stress. It is thus the body's 'fi re alarm' and initi- matory reactions) and glial scar formation, facilitating functional recovery. ates the protective recruitment of leucocytes to the site of injury or infec- In this paper, we present the data from this investigation and discuss the tion. This process, however, can also lead to disease. Excess or persistent relationship between IL-6 signals and SCI. leucocytes in local sites can cause tissue damage, and is an important step in a variety of chronic infl ammatory disease. This concept has been vali- dated by the success of TNF blocking therapy in close to a million patients 2011 worldwide, in several approved indications, including rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, psoriasis and psoriatic arthritis. Proteomics and hematopoiesis The mechanisms of TNF blockade and how it causes benefi t will be re- viewed, as are potential and real problems. Approaches to resolve these Anthony D Whetton, Christie Hospital, University of Manchester., United issues will be discussed. Kingdom Richard Unwin, Christie Hospital, University of Manchester., United Kingdom 2007 John R Griffi ths, Christie Hospital, University of Manchester., United Kingdom Infl ammation-associated regeneration: Fibrotic and granulomatous Andrew Pierce, Christie Hospital, University of Manchester., United diseases Kingdom Duncan Smith, Christie Hospital, University of Manchester., United Kouji Matsushima, Graduate School of Medicine, The University of Kingdom Tokyo, Japan Proteomics is a set of techniques that can be employed to advance many Fibrosis and granuloma formation are typical features of chronic infl am- areas of biology. Advances in mass spectrometry (MS) plus sequencing mation which are extreme ways of host defense to shut in danger signals. of complete genomes have enabled rapid progress in this area. MS meth- Fibrocytes expressing CXCR4 and CCR7 in the circulation are considered ods now permit the sensitive determination of protein identity via peptide to be mobilized by chemokines to infl ammation sites at which fi brocytes sequencing. Relative quantifi cation of proteins from several samples can differentiate into fi broblats. Angiotensin-angiotensin receptor system also be achieved using isotope-coding strategies. We have employed pro- seems to be critically involved to up-regulate CCR2 expression on fi bro- teomics approaches to gain new insights into the regulation of stem cell blasts, and MCP-1 which is a ligand of CCR2 not only recruits macro- commitment events and leukaemic transformation. Specifi c pathways in- phages to infl ammation sites but also acts on fi broblasts to produce high volved in BCR/ABL mediated transformation have been elucidated using 2 amount of collagen type I/III and make tight ECM. On the other hand, dimensional electrophoresis and mass spectrometry. Furthermore gel-free granuloma has been considered to be an endpoint of chronic infl ammation, relative quantifi cation procedures (isobaric tags for relative quantifi cation, and typically consist of macrophages and epithelioid cells surrounded by ITRAQ), using novel peptide labelling methods plus liquid chromatogra- CD4+T lymphocytes. We have recently revealed the existence of dendritic phy/tandem MS hold great promise for proteomic analyses. This has been cells at the center of granuloma experimentally induced by Propionibacte- demonstrated in a study of hematopoietic stem cell transformation by the rium acnes in mice and active participation of dendritic cells in granuloma TEL/PDGFR oncogenic tyrosine kinase, where novel insights into the formation. In this symposium, I will overview current understanding of transformation process are acquired.The value and sensitivity of an MS the mechanisms of fi rosis and granuloma formation, and then introduce based relative quantifi cation approach in investigating cell development is our recent analyses of the involvement of chemokine-chemokine receptor shown in a study of changes in the nuclear proteome during the process systems in murine fi brosis and granuloma models. of embryonal stem cell development to haemangioblasts via mesoderm commitment. This work demonstrates that deep proteomic penetration can be achieved with populations enriched using fl ow cytometry. Mass spec- 2010 trometry is also able to identify post-translational modifi cation events. The sensitivity required is an issue and an approach to mapping phosphoryla- Infl ammation and regeneration of injured spinal cord: effects of the tion sites in proteins using a novel MS methods, employing a hybrid triple stem cell transplantation and blockade of IL-6 signaling quadrupole/linear ion trap mass spectrometer has many advantages. This technique can approach phosphorylation site mapping with a sensitivity in Hideyuki Okano, Department of Physiology, Keio University School of the femtomole region allow gel bands to be used as starting material, mak- Medicine & CREST, JST, Japan ing phosphoprotein analysis a more accessible technique. Seiji Okada, Department of Physiology, Keio University School of Medi- cine & CREST, JST, Japan Masaya Nakamura, Department of Orthopedic Surgery, Keio University 2012 School of Medicine & CREST, JST, Japan Yoshiaki Toyama, Department of Orthopedic Surgery, Keio University The application of advanced emerging technologies to novel School of Medicine, Japan biomarker discovery, target validation and earlier clinical Proof of Principle in Pharma R & D Recent progress in the stem cell biology has led much insight into new therapeutic interventions aiming for the regeneration of the damaged cen- Harsukh Parmar, Director, Discovery Medicine, Respiratory & Infl am- tral nervous system (CNS). The major strategies can be classifi ed into two mation, Astrazeneca R & D., United Kingdom subgroups; 1) activation of endogenous NSCs, and 2) cell transplantation therapies. In either of these strategies, it is crucial to understand the under- The post-genomic technologies required to identify and validate molecular lying mechanisms of maintenance, activation and differentiation of NSCs targets from the human genome include: Infl amm. res. Supplement 2 (2005) S 93

1. Biomarkers, Surrogates and Diagnostics linked to the disease process identifi ed using Genomics, Proteomics, “Cellomics”, Metabonomics, 2014 Physiomics, Pharmacogenomics and Toxicogenomics 2. Biopathway analysis giving an understanding of metabolic, signalling, Cytokine secretion and phagosome formation are spatially linked in gene regulatory, protein-protein and disease process interactions and macrophages 3. Bioinformatics linking multiple biological databases from gene expres- sion to mRNA and protein expression to disease processes and clinical Rachael Z Murray, Institute of Molecular Biosciences, University of outcomes Queensland, Australia These technologies need to be applied in a holistic manner to address is- Jennifer L Stow, Institute of Molecular Biosciences, University of sues around human target validation and proof of principle in man and Queensland, Australia provide insights and dissection of disease processes and patient segmenta- tion. The defi nition of critical interventional points to maximally modify Temporal secretion of the proinfl ammatory cytokine TNF is a key func- the disease process and clinical outcomes is a key strategy in dealing with tion of LPS activated macrophages; however, the intracellular machinery a large number of potential targets now available to the pharma industry and pathway required to secrete TNF is largely undefi ned. SNARE pro- for various diseases. Examples will be given how development of these teins are required for a vesicle to dock with the target membrane. Cog- novel biomarker technologies have been applied to accelerate R & D and nate SNAREs, on the target membrane (Q-SNAREs) and on the donor improve disease understanding, identify segments of patients most suited membrane (R-SNAREs) form a trans-SNARE complex bringing the two to certain therapies and creating various potential approaches to person- bilayers in close proximity for subsequent fusion. Microarray and Western alised medicine. analysis data show specifi c SNAREs are temporally regulated by LPS. By Disease Modifi cation remains a key aim in certain diseases such as Asth- overexpression and mutation we demonstrate novel, rate-limiting roles for ma, COPD, Osteoarthritis and we need to devise various methodologies, SNAREs from different parts of the traffi cking pathway. The Q-SNAREs technologies and therapeutic targets that can address this unmet medical Vti1b and Stx6 form a complex, are found on similar sized Golgi-derived need. Technology, Software and Informatic Tools need to be developed in carriers to that of TNF and are individually rate limiting for delivery to parallel to address the scientifi c and regulatory needs for disease modifi ca- the plasma membrane. We have previously shown a Q-SNARE complex tion and investments often need to be sustained for long periods of time to containing Stx4 and SNAP23 at the plasma membrane to regulate cytokine deliver the fi nal outcomes. An illustration of this process will be given for secretion and have now identifi ed VAMP3, found on recycling endosomes, selected diseases in infl ammation. as the R-SNARE for both this and the Golgi derived carrier Q-SNARE complex containing Vti1b and Stx6. VAMP3 is able to regulate exocytic traffi cking and secretion of TNF. Newly synthesised TNF is transport from 2013 the Golgi via VAMP3 positive recycling endosomes to the cell surface. VAMP3 is known to be involved in the delivery of recycling endosomes Nanodetection of Myeloperoxidase in the Dextran Sulfate Model of to pseudopods during phagocytosis. We now show TNF is being delivered Colitis these sites with the VAMP3 positive membrane during nascent phagosome formation presumably to aid rapid secretion of TNF. Overall we show the Sreekant Murthy, Drexel University, United States differential regulation and function of SNAREs at strategic steps uniquely Elisabeth Papazoglou, Drexel University, United States adapts the secretory pathway to ensure maximal and rapid secretion of Som Tyagi, Drexel University, United States TNF via VAMP3 positive recycling endosomes. During phagocytosis the Kambiz Pourrezaei, Drexel University, United States focal exocytosis of TNF occurs at nascent phagosomes along with the Amolkumar Karwa, Drexel University, United States VAMP3 positive recycling endosomes showing a novel link between these two arms of the innate immune response. The quantifi cation of Myeloperoxidase (MPO), a lysosomal enzyme which is an excellent biomarker of infl ammation in autoimmune and infl amma- tory diseases requires tedious extraction techniques and often yields incon- 2015 sistent results. The objective of this project is to image and quantitatively measure the concentration of MPO conjugated with Quantum Dots (QDs) Imaging of activated leukocytes using integrin targeting phage in the lamina propria of mice colon with dextran sulfate (DSS) induced display peptide derivatives colitis. The DSS model of colitis presents clinical disease and histological appearance reminiscent of human ulcerative colitis and colitis associate- Tanja-Maria Ranta, Department of Biological and Environmental Sci- colon cancer. QDs are novel nanometer size fl uorescent markers, with dis- ences, University of Helsinki, Finland tinct advantages of high quantum yield and excellent photostability mak- Heli Valtanen, Cancer Targeting Technologies Ltd., Finland ing them great tools for dynamic monitoring of disease progression. MPO Sami Tw(SatT*06Maria 0(nj/F6 1 Tf0 t of Biological and Eeguand Eegir)37.1(onmental Sci-)TJ0 -1.2 TD(ences, 4a)TJ0 -1.2 TDg eNond)UniT*TDg eNonSci- is an important indicator of neutrophils and reactive oxygen species pres- ent in acute infl ammation. Antibodies specifi c to MPO were conjugated to Quantum Dots and tested in vivo in acute infl ammation and in the Dextran Sulfate (DSS) model for colitis. The resulting fl uorescence intensity was then used as a measure of concentration of MPO and in turn infl amma- tion. Experiments were performed to test the effi ciency and specifi city of these antibody-bound QDs. Infl ammation was induced in the colon of Swiss Webster mice. The colon was exposed to QD conjugates, washed, excised, and frozen. Histological slides (10 uM thickness) were prepared and imaged with a Leica confocal microscope. The images showed the presence of QDs complexing with MPO in the areas of infl ammation. QDs were absent in control colon tissue devoid of infl ammation. Our results show that QD bioconjugates specifi c for MPO can be designed and used to characterize infl ammation. At present we are quantitating and mapping out the progression of infl ammation and tissue damage during the course of the disease. We are also extending our methodology to simultaneously image additional infl ammation and cancer biomarkers for the early detec- tion of cancer in this model. S 94 Monday 22 August 2005 Infl amm. res. Supplement 2 (2005) sion of monocytic cells to intercellular adhesion molecule-1 (ICAM-1), the major ligand of beta-2 integrins on infl amed endothelium, thereby restrict- 2018 ing an important step in leukocyte extravasation and tissue infi ltration. Biodistribution of the phage display peptide LLG and different conjugates Acute dextran sulphate sodium (DSS) -induced colitis is exacerbated with this targeting moiety were studied in varied animal models by external in granulocyte macrophage-colony stimulating factor (GM-CSF) imaging and tissue sampling. Experiments in mice and rabbits show that defi cient mice the LLG peptide does not have any specifi c accumulation sites in normal tissues and is rapidly cleared from the blood through the kidneys. In neu- YingHua Xu, Deparment of Pathology, University of Sydney, Australia trophil infi ltration model, where peritonitis is induced with thioglycolate, Nicholas H Hunt, Deparment of Pathology, University of Sydney, Aus- a concomitant injection of LLG in the blood stream greatly reduced the tralia number of neutrophils infi ltrating into the peritoneal cavity, demonstrating Shisan Bao, Deparment of Pathology, University of Sydney, Australia the beta-2 integrin blocking effect of the peptide. Recently we have used conjugated LLG-peptide in several animal infl am- Infl ammatory bowel disease (IBD) is a chronic uncontrolled infl amma- mation and infection models to image the accumulation of activated leuko- tory condition of the intestinal mucosa. Current leading hypotheses for cytes to sites of injury. Excellent targeting signals have been observed. the aetiology of IBD emphasise the involvement of complex interactions between genetic predisposition, immunological and environmental factors, especially a dysregulated immune response to normal intestinal microfl o- 2017 ra. Accumulating evidence indicates that impairment of mucosal cytokine secretion plays a crucial role in the pathogenesis of IBD. GM-CSF is best The Critical Role of Endogenous Granulocyte Colony-Stimulating viewed as a major regulator governing the functions of granulocytes and Factor (G-CSF) and Neutrophils during Collagen-Induced Arthritis macrophages. GM-CSF-/- mice provide a unique opportunity to investi- gate the role of GM-CSF in the development of IBD. GM-CSF-/- mice Jo L Eyles, The Walter and Eliza Hall Institute of Medical Research, and wild-type (Wt) mice were given 2.5% (w/v) DSS in drinking water Parkville, Australia for 7 days. Clinical symptoms, pathological changes, and colonic cyto- Kate E Lawlor, The Walter and Eliza Hall Institute of Medical Research, kine productions were examined. After 7 days DSS challenge, GM-CSF- Parkville, Australia /- mice developed more severe acute colitis than Wt mice, refl ected by a Ian K Campbell, The Walter and Eliza Hall Institute of Medical Research, greater bodyweight loss, more faecal bleeding, aggravated epithelial injury Parkville, Australia and infl ammatory cell infi ltration. Signifi cantly lower constitutive colonic Ian P Wicks, The Walter and Eliza Hall Institute of Medical Research, levels of IL-4, IL-6, IL-17 and TNF-α were found in GM-CSF-/- mice Parkville, Australia compared to their Wt counterparts. However, colonic IL-17 and TNF-α levels were signifi cantly increased in DSS-treated GM-CSF-/- mice but Background: Granulocyte colony-stimulating factor (G-CSF) is a key not in DSS-treated Wt mice. The level of IL-6 was increased by 2.5-fold regulator of neutrophil production. We have previously shown that G-CSF and 5-fold in the colon of DSS-treated GM-CSF-/- and DSS-treated Wt defi cient (G-CSF -/-) mice have reduced collagen-induced arthritis (CIA), mice, respectively. No signifi cant changes in colonic IL-4 and IFN-γ were suggesting an important in vivo role for G-CSF during infl ammatory ar- detected in either strain of mouse following DSS treatment. These results thritis. These studies investigate the role of endogenous G-CSF and neu- suggest that GM-CSF contributes to colonic cellular immune response and trophils during infl ammatory arthritis by using gene knock-out mice, bone cytokine regulation in acute DSS-induced colitis, and that a loss of fi ne- marrow chimeras, and neutralising antibodies in CIA. tuning of local cytokine production results in more severe infl ammation Methods: G-CSF -/- or G-CSF receptor defi cient (G-CSFR -/-) mice were in the gut of GM-CSF-/- mice. This information may be useful for both compared to wild-type (WT) mice. For CIA, mice were immunised with clinical treatment and /or investigation of basic chick type II collagen in CFA and evaluated clinically (1-3, mild-severe; maximum score per mouse, 12) and histologically. To determine which cells produce/respond to G-CSF during CIA, bone marrow chimeras were 2019 generated which had either a haemopoietic or joint compartment that was WT or knockout with respect to G-CSF or G-CSFR. To investigate the Distinct intracellular transport pathways for the secretion of TNF role of neutrophils during disease progression in CIA, DBA/1 mice (the and IL-6 in macrophages most responsive strain) received a neutrophil-depleting mAb (RB6.8C5) or isotype control mAb (rat IgG2b) every 3 days for 12-14 days, starting Anthony P Manderson, Institute of Molecular Biosciences, University of from the day of disease onset. Queensland, Australia Results: Similar to G-CSF -/- mice, G-CSFR -/- mice showed few clini- Jennifer L Stow, Institute of Molecular Biosciences, University of cal signs of arthritis compared to wild-type (WT) mice (day 60 incidence, Queensland, Australia 7.1% vs. 61.1%; average clinical score, 0.1 ± 0.1 vs. 3.6 ± 1.1 respectively, n > 14 mice per genotype). Bone marrow chimeras with either a G-CSFR- LPS triggers macrophages to synthesise and secrete proinfl ammatory cy- /- haemopoietic compartment or a G-CSF-/- non-haemopoietic compart- tokines such as TNF and IL-6. To accommodate the secretion of TNF a ment were protected against CIA. Neutrophil depletion in DBA/1 mice number of SNAREs, proteins that regulate vesicle docking and fusion, after disease onset dramatically halted disease progression (clinically and are upregulated in macrophages and function in the transport of TNF be- histologically). tween the Golgi complex and cell surface. In contrast, little is known about Conclusion: During CIA, non-haemopoietic cells are the major producers the machinery responsible for delivery of IL-6 to the cell surface. IL-6 of G-CSF, and haemopoietic cells are the primary responders to G-CSF. is temporally secreted with a partially overlapping profi le to that of TNF Neutrophils are critical for maintaining joint infl ammation during CIA. in IFN-primed LPS-stimulated macrophages. Intracellular staining of ac- Therefore, G-CSF defi ciency may confer protection in CIA via reduced tivated macrophages demonstrated that IL-6 colocalises with TNF in the neutrophil production, function and/or survival. These fi ndings demon- perinuclear Golgi complex but not in post-Golgi compartments. TNF and strate that endogenous G-CSF is an important mediator of joint infl am- IL-6 were also found in different populations of Golgi-derived vesicles mation and strengthen the rational for therapeutic targeting of G-CSF in budded in vitro. Overexpression and mutation of two SNAREs proteins infl ammatory arthritis. required for the traffi cking and secretion of TNF demonstrated that these same SNAREs do not function in IL-6 delivery to the cell surface. Our results show that although TNF and IL-6 are both synthesised in the bio- synthetic pathway the subsequent traffi cking routes and SNARE-mediated delivery to the cell surface are distinct. Thus there is potential for separate Infl amm. res. Supplement 2 (2005) Monday 22 August 2005 S 95 transport and regulation of these two proinfl ammatory cytokines in macro- Furthermore, the expression of Fn14 on other mesenchymal progenitor phage mediated immunity. cells suggests a broad involvement of this pathway in other tissue injury and disease settings. 2020 2023

Characterization of IL-17 secreting T-helper cells (ThIL-17) Novel Mechanisms of Leukocyte Recruitment Joanna J Listopad, CRBA Dermatology, Schering AG, Berlin, Germany Khusru Asadullah, CRBA Dermatology, Schering AG, Berlin, Germany Michael J. Hickey, Centre for Infl ammatory Diseases, Department of Wolf-Dietrich Doecke, CRBA Dermatology, Schering AG, Berlin, Ger- Medicine Monash University many Leukocyte recruitment is of key importance to the infl ammatory response. Recent data indicate that IL-23, a new member of IL-12 cytokine family, Evidence from our experiments suggest that macrophage migration in- is of major importance for T cell-dependent infl ammation. IL-23 selec- hibitory factor (MIF) and anti-neutrophil cytoplasmic antibodies (ANCA) tively triggers the secretion of IL-17, a T cell cytokine with main effects are mediators with previously-unrecognised function in directly inducing on infl ammatory system, and induces the differentiation of IL-17-secreting leukocyte recruitment in vivo. MIF is a proinfl ammatory cytokine with

T-helper cells (ThIL-17). The purpose of our study was the characterization established roles in several models of infl ammatory disease. We have pre- of this new Th cell population. viously shown that MIF promotes leukocyte recruitment in the context of Human Th cells were differentiated from naive CD4+ cells by anti-CD3/ infl ammatory stimulation. The mechanism for this has remained unclear, CD28 stimulation in the presence of IL-23, IL-12 or other cytokines and therefore we examined the possibility that MIF may promote leukocyte hormones. mRNA and cytokine receptor expression were determined by recruitment directly. Exposure of the cremaster muscle to MIF for 4 hours TaqMan-PCR and FACS. Cytokine secretion was measured after PMA/ resulted in a signifi cant increase in leukocyte adhesion within postcapillary Ionomycin or PHA stimulation. venules, and specifi c recruitment of CD68+ monocytes to the perivascular i) In comparison to IL-12-induced Th1 cells, IL-23-triggered ThIL-17 cells tissue. These observations indicate that MIF may promote infl ammation show increased IL-17 secretion whereas the production of IL-2, IFNg, by directly inducing recruitment of monocytes. Secondly, in vitro evi- TNFa, and GM-CSF is diminished. Expression of T-bet and Foxp3 is low- dence suggests that ANCA can induce neutrophils to undergo adhesion. To er in ThIL-17 than in Th1 cells. No differences are found for IL-4, IL-10 and assess this possiblilty in vivo, we treated mice with anti-sera against one of GATA-3. A differential cytokine receptor expression is seen. the antigens recognized by ANCA, myeloperoxidase. These antisera in- ii) Presence of IFNg during Th differentiation inhibits and IL-10 and GM- duced rapid leukocyte adhesion within glomerular capillaries, as observed

CSF may increase the differentiation of ThIL-17 cells. Strong stimulatory by intravital microscopy. However this only occurred in mice in which a effects on ThIL-17 differentiation are seen for glucocorticoids and proges- systemic infl ammatory response had been induced. These fi ndings sug- terone. gest that ANCA may promote microvascular pathology in the glomerulus iii) Small, but signifi cant effects of cytokines on PHA-induced IL-17 se- by inducing adhesion of leukocytes. However this is most likely to occur cretion of PBMC are observed. Whereas IL-23 and IL-4 lead to a slight under conditions of systemic infl ammatory activation. increase, IL-12, IFNg and GM-CSF decrease IL-17 secretion. The effects of hormones depend on dosage. Low concentrations of glucocorticoids, progesterone and epinephrine tend to increase PHA-induced IL-17 secre- 2024 tion, high concentrations have a rather suppressive effect.

ThIL-17 cells represent a new Th subset with distinct cytokine profi le. Be- Natalizumab, a Humanized Antibody Against alpha-4 Integrin for side IL-23, other cytokines and hormones infl uence ThIL-17 differentiation the Treatment of Autoimmune disease and IL-17 secretion of T cells. The induction of IL-17-secreting T cells by stress hormones may be relevant for infl ammation in trauma and stress. Roy R Lobb, Albor Biologics Inc., United States

The integrins alpha-4 beta-1 (VLA-4) and alpha-4 beta-7 are expressed on 2021 leukocytes and allow their recruitment to infl ammatory sites. Pre-clinical data in models of multiple sclerosis, infl ammatory bowel disease, rheu- Regulation of Skeletal Muscle Precursor Cells by Proinfl ammatory matoid arthritis and other autoimmune diseases suggested that an anti- Tweak/Fn14 pathway body to the human alpha-4 Integrin chain might show effi cacy in human disease. Natalizumab, a humanized antibody against the alpha-4 integrin Timothy S Zheng, Biogen Idec, Inc., United States chain, blocks the adhesive function of both integrins. Clinical data on the effects of Natalizumab in Multiple Sclerosis and Crohn’s disease will be Infl ammation participates in tissue repair through multiple mechanisms in- described. cluding directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand Tweak, we observed that Tweak binds to all progenitor cells of the 2025 mesenchymal lineage and triggers NFkB activation, suggesting a possible role of this proinfl ammatory cytokine in modulating progenitor cell biol- Endogenous Corticosteroids On Neutrophil Mobilization: Effect On ogy. Using both the established C2C12 cell line and primary mouse muscle Hematopoesis And Cellular Interactions myoblasts, we demonstrated that Tweak promoted their proliferation and consequently inhibited their terminal differentiation. Importantly, primary Danielle Maia Cavalcanti, University of São Paulo, Brazil myoblasts isolated from mice defi cient in the Tweak receptor Fn14 ex- Primavera Borelli, University of São Paulo, Brazil hibited signifi cantly reduced proliferative capacity and aberrant myotube Celina Maria Lotuffo, University of São Paulo, Brazil formation. Following cardiotoxin injection, a known trigger for satellite Jorge Mario Ferreira, Butantan Institute, Brazil cell-driven skeletal muscle regeneration, Fn14 knockout mice exhibited Regina Pekelman Markus, University of São Paulo, Brazil defective expansion of muscle myoblasts, altered infl ammatory response Sandra Helena Poliselli Farsky, Cidade University, Brazil and delayed muscle fi ber regeneration compared to wild-type mice. These results indicate that the Tweak/Fn14 pathway is a novel regulator of skel- Introduction: The present work investigated the effects of EC on hema- etal muscle myoblast properties and illustrate an important mechanism by topoesis and on neutrophil-endothelial interactions. Methods: Adrenal- which infl ammatory cytokines infl uence tissue regeneration and repair. ectomized (ADX), sham-operated (SO) or normal (N) male Wistar rats S 96 Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

(180-220g) were employed to evaluate: 1) hematological parameters, by Louise M Judd, University of Melbourne, Australia determining the numbers of leukocytes in bone marrow and in circulation; 2) in vitro adherence of circulating neutrophils to cultured endothelium; Tissue infl ammation accompanies or is a necessary precursor for induction 3) expression of adhesion molecules on circulating leukocytes by fl ow ci- of cancers of the gastrointestinal tract, in particular the stomach, colon tometry and on postcapillary endothelium from cremaster muscle by im- and pancreas. This presentation will focus on gastric cancer, in particular munohistochemistry. Results: Defi ciency of EC did not alter the produc- the role of dysregulated infl ammatory cytokine signaling in the initiation tion of blood cells, but accelerated the neutrophil maturation and promoted and progression of intestinal-type gastric neoplasia. Both mouse models of neutrophilia (70%vs controls). Neutrophils from ADX rats adhered lesser gastric tumourigenesis and data from human pre-cancerous stomach biop- to the normal endothelium (90% vs controls), and endothelium from ADX sies infected with the bacterium Helicobacter pylori will be used to demon- animals presented more ability of adhering normal neutrophils (59%vs strate the importance of infl ammatory events in cancer pathogenesis, and controls). L-selectin expression on cells at the last phase of maturation emphasise possible new targets for clinical intervention. in bone marrow was impaired (30% vs controls), but it was increased on circulating neutrophils (100% vs controls). β2 integrin expression was unaffected. Expressions of ICAM-1 and PECAM-1 on endothelium 2030 were higher (about 25% vs controls) in ADX animals. Conclusions: EC modulate neutrophil-endothelial interactions by interfering with neutrophil Impact of infl ammation on drug clearance pathways in cancer: maturation process and with expressions of adhesion molecules. However, Tumour-associated infl ammation represses hepatic CYP3A function different control on adhesion molecules expression on neutrophil and en- and expression of drug transporter genes dothelium can be addressed depending on each phase of recruitment. Fi- nancial support: FAPESP (grants 03/09410-5), CNPQ. Graham R Robertson, Cancer Pharmacology Unit, ANZAC Research Institute, Australia Kellie Slaviero, Sydney Cancer Centre, Royal Prince Alfred Hospital, 2026 Australia Rohini Sharma, Molecular Pharmacology, Westmead Millennium Institu- A Role for Platelet-Derived P-Selectin in Glomerular Leukocyte te, Australia Recruitment Chris Liddle, Molecular Pharmacology, Westmead Millennium Institute, Australia Michael P Kuligowski, Monash Institute For Medical Research, Monash Stephen Clarke, Cancer Pharmacology Unit, ANZAC Research Institute, University, Australia Australia Richard Kitching, Monash Institute For Medical Research, Monash University, Australia A major challenge in pharmacogenomics is the narrow therapeutic index Michael J Hickey, Centre for Infl ammatory Diseases, Monash University, of anti-cancer treatments, as toxicity due to variable drug clearance is Australia a common cause of treatment failure. CYP3A4 is the major pathway of human drug metabolism, responsible for clearance of over half of all anti- Many renal diseases result from leukocyte-mediated infl ammation in the cancer agents. Recently, we observed that reduced CYP3A4 activity in kidney. However the mechanisms of leukocyte recruitment to glomeruli cancer patients results in enhanced toxicity of anti-cancer drugs, and was are poorly characterised. Therefore the aim of this study was to character- also correlated to the degree of systemic infl ammation. Unlike other in- ize the mechanisms of glomerular leukocyte recruitment, using intravital fl ammatory states, the mechanism of this interaction in cancer has yet to be microscopy. Mouse kidneys were rendered hydronephrotic by unilateral elucidated. To investigate hepatic expression of the human CYP3A4 gene ureteric ligation. Twelve weeks later, mice were anaesthetized, leukocytes in the presence of cancer, we used a transgenic mouse model incorporat- stained with rhodamine 6G, and the glomerular vasculature examined via ing the regulatory region of the CYP3A4 gene attached to lacZ. The EHS intravital microscopy. Leukocyte recruitment to glomeruli was induced by sarcoma was injected i.m. into the quadricep of transgenic CYP3A4/lacZ i.v. administration of sheep anti-mouse glomerular basement membrane mice. Transgene-derived b-galactosidase activity was visualised by histo- antibodies (anti-GBM). Immunohistology of hydronephrotic and non- chemical staining of liver slices. Markers of infl ammation included cyto- hydronephrotic kidney showed that anti-GBM was deposited to a similar kine levels and hepatic expression of Serum Amyloid protein P (SAP), the degree in both kidneys. Also expression of inducible infl ammatory mol- major mouse acute phase protein. Assessment of CYP3A function by the ecules, such as complement (C3), and P-selectin did not differ. Anti-GBM midazolam sleep test showed that tumour-bearing mice had longer mid- induced signifi cant leukocyte recruitment to glomeruli within 1 hour. Leu- azolam-induced anaesthesia compared to control mice. Hepatic CYP3A4 kocytes were observed to adhere in glomerular capillaries without under- and mouse CYP3A11 expression was decreased by 60% and 80% in tu- going prior rolling interactions with the endothelium. Leukocyte recruit- mour-bearing animals, while SAP expression was increased 8 fold. The ment was eliminated by function-blocking antibodies against CD18 and expression of a number of drug transporter genes including mrp2, mrp3, ICAM-1. Similar reductions in adhesion were observed using mAb direct- Oatp2 and Oatp-c were also down-regulated. In addition, serum IL-6 con- ed against P-selectin, PSGL-1, and in P-selectin-/- mice. Depletion of circu- centrations were elevated in tumour-bearing mice. CONCLUSION This lating platelets also eliminated glomerular leukocyte adhesion. However, study has demonstrated for the fi rst time that cancer-associated infl amma- administration of wild type platelets restored anti-GBM-induced leukocyte tion transcriptionally represses drug clearance pathways. The transgenic adhesion in P-selectin-/- mice. These data indicate that anti-GBM-induced mouse model of human CYP3A4 regulation, coupled with explant tumour leukocyte recruitment to glomeruli occurs in the absence of detectable roll- models, permits pre-clinical testing of anti-infl ammatory interventions ing, via a mechanism involving specifi c roles for the CD18-ICAM-1 path- aimed at reducing toxicity by normalising drug metabolism and transport. way, platelets and P-selectin. These data also show that platelet-derived P-selectin is critical for leukocyte adhesion within glomeruli. 2031

2028 Targetting tumor-infi ltrating macrophages for tumor regression

Cancer in the gastrointestinal tract Bernadette Scott, Monash Institute of Medical Research, Monash Univer- sity, Australia Andrew S Giraud, University of Melbourne, Australia Xiang Liu, Monash Institute of Medical Research, Monash University, Meegan Howlett, University of Melbourne, Australia Australia Brendan Jenkins, Ludwig Institute, Australia Bruce WS Robinson, Department of Medicine, University of Western Matthias Ernst, Ludwig Institute, Australia Australia, Australia Infl amm. res. Supplement 2 (2005) Monday 22 August 2005 S 97

Nico van Rooijen, Vrije Universiteit, VUMC Department of Molecular Cell Biology, Faculty of Medicine, The Netherlands 2036 Paul Hertzog, Monash Institute of Medical Research, Monash University, Australia Disintegrins: integrin selective ligands able to activate integrin- coupled signaling and to modulate leukocyte functions Macrophages, which are often a predominate infi ltrate in both experimen- tal and human tumors, may be perfect targets for anti-tumor therapies as, Christina Barja-Fidalgo, Universidade do Estado do Rio de Janeiro, not only are they an interface between the innate and the adaptive immune Brazil system, but, when activated, they can have tumoricidal activity. Marta S De Freitas, Universidade do Estado do Rio de Janeiro, Brazil We have well-established murine models of malignant mesothelioma that Ana Lucia J Coelho, Universidade do Estado do Rio de Janeiro, Brazil mimic the human disease in terms of aetiology and histopathological fea- Andrea Mariano-Oliveita, Universidade do Estado do Rio de Janeiro, tures. Macrophages are the predominant infi ltrate, forming almost 50 per- Brazil cent of established tumor tissue. To determine whether these macrophages Edward Helal, Universidade do Estado do Rio de Janeiro, Brazil contribute to tumorigenicity in vivo, we undertook a kinetic analysis of Cezary Marcinkiewicz, Temple University- Philadelphia, United States the macrophages during tumor growth. We found that tumor-infi ltrating Russolina Zingali, Universidade Federal do Rio de Janeiro, Brazil macrophages (TIMS) changed phenotypically with tumor progression by down-regulating MHC class II expression. We have also shown that these Disintegrins (Dis) are a family of cystein-rich low-molecular weight pep- macrophages produced IL-10, a prototypic cytokine associated with pro- tides that often contain an RGD sequence, a cell attachment site of a large tumorigenic activities of TIMs. Indeed, neutralisation of IL-10 in vivo number of adhesive extracellular matrix (ECM) and cell surface proteins, reduced tumor growth and prevented down modulation of MHC class II recognized by the integrins. They are selective and competitive antagonists expression. Using the well-established technique of liposome encapsu- of integrins, being potent inhibitors of platelet aggregation and cell-cell/ lated clodronate for the in vivo depletion of macrophages, we found that cell-ECM interactions. In neutrophils (PMN) integrins are involved in ad- the physical deletion of these immune modulatory macrophages did indeed hesion, activation migration phagocytosis and oxidants production. T-cell prevent tumor growth. This process however did not alter the immune interaction with ECM is critical for differentiation, migration and activa- response to the tumor. Together these experiments support the idea that by tion. We have reported that monomeric RGD-Dis [Jarastatin (JT): aMb2, determining the nature of the relationship between tumor cells and TIMs, a5b1,avb3; kistrin (KR: avb3); fl avoridin (FL:a5b1, avb3); and echistatin appropriate strategies can be undertaken to disrupt this interaction and pro- (Ech: a5b1, avb3)] and the heterodimeric MLD/VGD-Dis, EC3 and VLO5 mote tumor eradication. (a4b1, a9b1) selectively interact with integrin on human PMN and lym- phocytes, interfering with cell functions, through the activation of integ- rin-coupled intracellular signaling pathways. All RGD-Dis inhibited PMN 2032 migration induced by fMLP, in vitro. However, only JT and the MLD- Dis were also chemotactic for PMN, an effect blocked by anti-amb2 and CSF-1 Regulation of Macrophage Adhesion, Motility and Promotion anti-a9b1 antibodies, respectively. Chemotactic peptides increased actin of Tumour Invasion and Metastasis polymerization and activated FAK, PI3K and MAPK pathways in PMN. VLO5 and JT, but not EC3, induced IL-8 production and protected PMN Fiona J Pixley, Albert Einstein College of Medicine, United States from spontaneous, caspase-dependent, apoptosis, via Erk-2 activation. E Richard Stanley, Albert Einstein College of Medicine, United States The RGD-Dis, FL, KR and Ech, binding to a5b1 and avb3 integrins on lymphocytes, triggered T-cell activation. They increased CD69 expression Colony Stimulating Factor-1 (CSF-1), the primary factor regulating mac- and actin polymerization, activating FAK and NFkB nuclear translocation, rophage survival, proliferation and differentiation, also regulates morphol- via PI3K. However, only FL increased proliferation of T-cells in response ogy, adhesion and motility and promotes the progression of mammary to ConA. The data suggest that specifi c interactions of Dis with integrins tumours. Tumour-associated macrophages have been shown to participate present on cell surface might differentially interfere with important cel- in a paracrine loop with mammary carcinoma cells to induce co-migratory lular functions as mobility, cell cycle differentiation and gene expression behaviour of tumour cells and macrophages, leading to tumour invasion (FAPERJ, CNPq, SR2/UERJ) and metastasis. The effects of CSF-1 are mediated by the CSF-1 receptor tyrosine kinase (CSF-1R), which contains eight tyrosine residues demon- strated to be phosphorylated upon receptor activation. Each phosphotyro- 2037 syl residue recruits signalling molecules to the receptor to propagate the pleiotrophic downstream signal. Residues important for macrophage adhe- A Novel role for SOCS3 in Cartilage destruction via induction of sion and motility have not yet been identifi ed although Y559, Y721 and chondrocyte desensitization towards IGF-1 Y974, which bind Src family kinases, PI3K and Cbl respectively, are likely to be involved. The overall aim of this study is to delineate the mecha- Ruben L Smeets, Radboud University, The Netherlands nisms by which CSF-1 exerts regulatory control of macrophage adhesion, Sharon Veenbergen, Rheumatology research and Advanced Therapeutics, motility and invasive capacity and thereby enhances tumour invasion and Department of Rheumatology, Radboud University Nijmegen Medical metastasis. To examine the role of CSF-1R signalling in macrophage adhe- Centre, The Netherlands sion and motility, we have retrovirally expressed specifi c tyrosine mutant Onno J Arntz, Rheumatology Research and Advanced Therapeutics, CSF-1R molecules in CSF-1R-null macrophages derived from the CSF- Department of Rheumatology, Radboud University Nijmegen Medical 1R-/- mouse. Macrophages expressing individual mutant receptors are Centre, The Netherlands currently being examined by a wide range of microscopic techniques for Miranda B Bennink, Rheumatology Research and Advanced Thera- alterations in adhesion and motility. In addition, the ability of these mutant peutics, Department of Rheumatology, Radboud University Nijmegen receptors to promote tumour progression via the paracrine interaction of Medical Centre, The Netherlands the mutant receptor-bearing macrophages with mammary carcinoma cells Leo AB Joosten, Rheumatology research and Advanced Therapeutics, will be examined using in vitro and in vivo invasion assays that we have Department of Rheumatology, Radboud University Nijmegen Medical developed. Preliminary fi ndings will be discussed. Centre, The Netherlands Wim B van den Berg, Rheumatology Research and Advanced Thera- peutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, The Netherlands Fons AJ van de Loo, Rheumatology Research and Advanced Thera- peutics, Department of Rheumatology, Radboud University Nijmegen Medical Centre, The Netherlands S 98 Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Chondrocyte desensitization towards Insulin-like growth factor (IGF)-1 In conclusion, altogether, our results suggest that Lnk modulates EC in- is an important contributing mechanism for cartilage destruction during fl ammatory responses via a mechanism involving the molecule HO-1 and experimental arthritis. Previous studies revealed that Interleukin(IL)-1 ex- the PI3-kinase/Akt pathway. posure of patellar cartilage explants results in decreased IGF-1 induced proteoglycan (PG) synthesis. In this study we explored the role of Sup- pressor of Cytokine signaling (SOCS)3 in the IL-1 induced IGF-1 desen- 2039 sitization of chondrocytes.IGF-1 induced stimulation of chondrocyte PG synthesis was either determined by 35S-sulphate incorporation into PGs or Tissue Factor Biology via mRNA expression analysis of aggrecan using Q-PCR. In vivo IGF-1 nonresponsiveness of patellar chondrocytes could be elicited by zymo- Nigel Mackman, The Scripps Research Institute, United States zan-induced arthritis (ZIA). This phenomenon could be mimicked in IL-1 primed articular cartilage, revealing an 85% decrease in IGF-1 induced Tissue factor (TF) is the primary cellular initiator of blood coagulation. 35S-sulphate incorporation.In iNOS-/-mice the effect of IL-1 induced IGF-1 Binding of plasma FVII/VIIa to TF forms a complex that leads to the desensitization was still present, revealing the involvement of a secondary generation of a number of serine proteases, such as FXa and thrombin. IL-1 induced factor, other than NO. Articular chondrocytes derived from Thrombin plays a central role in blood coagulation by cleaving fi brinogen arthritic knee joints highly expressed SOCS3, as could be observed by and activating platelets by cleavage of various protease activated receptors immunohistochemistry. Incubation of a murine chondrocyte cell line (H4) (PARs). In endotoxemia and sepsis, TF expression within the vasculature with IL-1 revealed a signifi cant increase of SOCS3 mRNA and protein. In leads to intravascular coagulation. Importantly, inhibition of TF in animal contrast to IL-1, another proinfl ammatory cytokine IL-18 did not induce models of sepsis reduces coagulation and infl ammation. Our laboratory SOCS3 expression in H4 chondrocytes. Furthermore, incubation of patel- studies the cell types that express TF during endotoxemia and how the co- lar cartilage with IL-18 did not affect the IGF-1 response. Via adenoviral agulation cascade enhances infl ammation. Our data suggest that multiple overexpression of SOCS3 in H4 chondrocytes, IGF-1 induced aggrecan pathways mediate the crosstalk between coagulation and infl ammation. expression was signifi cantly reduced. Furthermore, SOCS3 overexpression inhibited IGF-1 signaling at the receptor level, which could be observed in decreased Insulin Receptor Substrate(IRS)-1 tyrosine phosphorylation. 2040 With this study we show that besides IL-1 induced NO production, IL-1 induced SOCS3 expression is a novel effector mechanism of IGF-1 desen- Activated Protein C: modulation of the endothelial-leukocyte sitization in chondrocytes, resulting in increased cartilage damage. interface

Brian W Grinnell, Lilly Research Laboratories, United States 2038 Protein C is a plasma protease that when activated plays a central role The adaptor molecule Lnk controls TNFa-induced endothelial cell in modulating the function of the vascular endothelium and its interface activation via a mechanism implicating Heme Oxygenase-1 and the with the innate immune system. A recombinant form of human activated PI3-K/Akt pathway protein C, drotrecogin alfa (activated), has shown effi cacy in a number of preclinical models of thrombosis and ischemia, and reduces mortal- Juliette Fitau, INSERM U643, France ity in patients with severe sepsis. Studies have begun to elucidate the Gaelle Legaud, INSERM U643, France mechanism for the multifunctional role of APC in modulating not only Flora Coulon, INSERM U643, France coagulation, but infl ammatory and apoptotic processes. From gene profi l- Gwenola Boulday, INSERM U643, France ing to pharmacology studies, drotrecogin alfa (activated) appears to di- Beatrice Charreau, INSERM U643, France rectly modulate endothelial dysfunction by blocking cytokine signaling, functional cell adhesion expression, vascular permeability, and preventing Lnk belongs to an adaptor molecule family, including APS and SH2-B, the induction of apoptosis. Moreover, APC, via EPCR/PAR-1 mediated that participates in intracellular signalling by protein-protein interactions. mechanisms, also appears to directly modulate leukocyte migration and Lnk has no enzymatic activity but is implicated in the differentiation path- adhesion. The ability of APC to suppress pro-infl ammatory pathways way of different cell types as well as in the activation pathway of T lym- and enhance cellular survival mechanisms suggests a role in the complex phocytes. Previously, we have shown that Lnk is expressed in endothelial adaptive response at the vessel wall that protects from vascular insult and cells (ECs) and regulated by the pro-infl ammatory cytokine, TNFa. prolongs endothelial, cellular, and organ survival. Coupled with analyses The aim of this study was to evaluate the role of the Lnk protein in the in sepsis models and proteomic studies of patient samples, the emerging TNFa-mediated signaling pathway of ECs. Lnk was overexpressed in hu- data further suggest that APC effectively modulates the complex changes man primary ECs derived from umbilical cords (HUVECs) using a recom- that occur during multi-system activation and dysfunction in sepsis. binant adenovirus. Quantitative RT-PCR, fl ow cytometry and western blot analysis were used to analyse the effect of Lnk on endothelial activation and to identify the signalling pathway and mechanisms involved. 2041 Our results indicate that Lnk overexpression did not induce the adhesion molecules VCAM-1 and E-selectin in ECs but, in contrast, reduced the Tissue factor signalling in innate and adaptive immunity TNF-mediated VCAM-1 expression by 50% (at both the mRNA and pro- tein levels) while ICAM-1 expression was unmodifi ed. This effect was in- Peter G Tipping, Monash University, Australia dependent of the NFkB pathway. However, Lnk induced a 7 fold-increase Laveena Sharma, Monash University, Australia in heme oxygenase-1 (HO-1) expression by ECs, suggesting that the anti- infl ammatory properties of HO-1 are responsible for the down-regulation Tissue factor (TF) is a single chain transmembrane glycoprotein with of VCAM-1. We further showed that HO-1 expression did not correlate structural homology to type II cytokine receptors. The extracellular do- with an activation of the classical regulatory pathways: ERK1 and 2, JNK main is essential for initiation of coagulation and has been show to play and p38 MAPK. Interestingly, Lnk induced Akt phosphorylation(at Ser important roles in embryogenesis and tumor metastasis. The function of 473) in ECs. The treatment of ECs with the specifi c PI3-Kinase inhibitor, the intracellular domain is less clearly defi ned. The involvement of the LY249002, decreased Akt activation as well as HO-1 expression in cells intracellular domain of TF in innate and adaptive immune responses has expressing Lnk. These results showed that the PI3-K/Akt pathway plays a been studied in vitro and in vivo. Using a monocytic cell line (U937 cells) key role in HO-1 regulation independently of the MAPKs pathway. stably transfected to express either full length or cytoplasmic domain de- fi cient TF, binding of factor VIIa to the extracellular domain induced in- tracellular Ca2+ fl uxes that were dependent on the presence of intracellular Infl amm. res. Supplement 2 (2005) Monday 22 August 2005 S 99 domain. Absence of the intracellular domain also signifi cantly reduced and S100A10 on the cell surface as shown by fl ow cytometry and fl uores- PMA stimulated IL-1 and TNF production by U937 cells. In mice with cence microscopy. Binding of plasmin to annexin A2 and S100A10 on the genetic deletion of the cytoplasmic domain, thioglycolate induced recruit- monocyte membrane was verifi ed by biotin transfer from plasmin labeled ment and activation of peritoneal macrophages was also reduced compared with a trifunctional cross-linker to the receptor proteins. The signifi cance to macrophages from wild type mice. Following endotoxin challenge cy- of this binding was verifi ed in functional assays, where antibodies directed toplasmic domain defi cient mice showed enhanced survival and blunted against annexin A2 or S100A10 inhibited the chemotaxis elicited by plas- systemic infl ammatory responses, despite similar dysregulation of coagu- min, but not that induced by fMLP. Further, downregulation of annexin A2 lation. Activation of the pro-infl ammatory transcription factor NFκB was or S100A10 in monocytes by antisense oligodeoxynucleotides impaired attenuated. The involvement of the cytoplasmic domain of TF in adaptive the chemotactic response to plasmin, but not that to fMLP. Antisense oli- immune responses was also studied in mice. Following active immuniza- godeoxynucleotides similarly decreased the TNF-α release in plasmin-, but tion, cytoplasmic domain defi cient mice showed attenuated systemic an- not in LPS-stimulated monocytes. At the molecular level, stimulation with tibody production and impaired cutaneous delayed type hypersensitivity plasmin, but not with catalytically inactivated plasmin, induced cleavage in response to local antigen challenge. Leukocyte rolling, adhesion and of annexin A2. Substitution of lysine to alanine in position 27 abolished transmigration in post capillary venules following local antigen challenge the cleavage of recombinant annexin A2 in vitro, thus allowing identifi ca- in sensitized mice was impaired in the absence of the cytoplasmic domain tion of the plasmin cleavage site. of TF. These studies suggest a contribution of the cytoplasmic domain of Together, these data identify the annexin A2 heterotetramer as signaling TF to innate and adaptive immune responses. The mechanisms of TFsig- receptor on human peripheral monocytes activated by plasmin via proteo- nalling in leukocytes remains to be fully elucidated. lytic cleavage. Supported by the Deutsche Forschungsgemeinschaft SFB 451. 2042 2044 Fibrin Deposition and The Plasminogen Activators in Infl ammation IL-23 drives the expansion of pathogenic ThIL-17 cells required for Andrew D Cook, Arthritis and Infl ammation Research Centre, Depart- autoimmune infl ammation ment of Medicine, Royal Melbourne Hospital, and CRC for Chronic Infl ammatory, Australia Claire L Langrish, DNAX Research, United States Yi Chen, DNAX Research, United States Accumulation of fi brin in the synovium in rheumatoid arthritis represents Wendy M Blumenschein, DNAX Research, United States one of the most striking pathologic features of rheumatoid synovitis, pre- Jeanine Mattson, DNAX Research, United States sumably resulting from an altered balance between coagulation and fi bri- Jonathon D Sedgwick, DNAX Research, United States nolysis. Degradation of fi brin is primarily mediated by the serine protease Terrill McClanahan, DNAX Research, United States plasmin, which is formed upon cleavage of plasminogen by the plasmino- Robert A Kastelein, DNAX Research, United States gen activators (PAs), urokinase-PA (u-PA) and tissue type-PA (t-PA). In Daniel J Cua, DNAX Research, United States addition to fi brin removal, the PAs have also been implicated in tissue remodelling and cell migration occurring during both physiologic and Interleukin-23 (IL-23) is a heterodimeric cytokine composed of a unique pathologic processes. However, the precise roles of u-PA and t-PA in fi bri- p19 subunit, and a common p40 subunit shared with IL-12. We have previ- nolysis in during infl ammation and arthritis is unclear. We have shown that ously demonstrated that IL-23-defi cient mice are resistant to experimental in a monoarticular arthritis model involving direct intra-articular injection, autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA) both t-PA and u-PA are protective. In contrast, for the chronic systemic and infl ammatory bowel disease (IBD), highlighting an important role collagen-induced arthritis model, u-PA is deleterious, while t-PA is protec- for this cytokine in autoimmune pathogenesis. Although IL-12 and IL-23 tive. In both arthritis models, absence of t-PA results in increased severity share a common p40 subunit, they promote two distinct immunological of arthritis with increased fi brin deposition. In several peritonitis models, pathways which have separate but complementary functions. IL-12 is im- absence of u-PA had no effect on the resulting infl ammatory response portant for the development of Th1 cells that are essential for host defense whereas absence of t-PA led to the adherence of macrophages to fi brin and tumor suppression. In contrast, IL-23 is a critical factor required for deposits in/or the cavity wall resulting in fewer macrophages recoverable the development and expansion of an alternative activated effector CD4+ from the peritoneal cavity. The data highlight the differing roles for t-PA T cell population-characterized by the production of IL-17, IL-17F, TNF and u-PA in infl ammatory responses and arthritis. and IL-6. Gene expression analysis of these IL-23-driven T cells (termed ThIL-17) identifi ed a unique expression pattern of pro-infl ammatory cyto- kines and other novel factors, distinguishing them from the IFN-gamma/ 2043 Th1 pathway. Using murine EAE passive transfer studies, we confi rm that these novel IL-23-dependent ThIL-17 cells are highly pathogenic and Plasmin-Induced Monocyte Activation Requires the Annexin A2 essential for the establishment of organ-specifi c infl ammation associated Heterotetramer for Proinfl ammatory Signaling with CNS autoimmunity. In addition, treatment with monoclonal antibod- ies against IL-23p19 prevented acute and relapsing EAE by suppressing Tatiana Syrovets, University of Ulm, Germany invasion of these pathogenic ThIL-17 cells into the central nervous system, Yves Laumonnier, University of Ulm, Germany suggesting IL-23 as a potential therapeutic target for the treatment of in- Kai Pitterle, University of Ulm, Germany fl ammatory autoimmune diseases. Thomas Simmet, University of Ulm, Germany This work was performed at DNAX Research Inc, 901 California Avenue, Palo Alto, CA 94304, USA. We have previously identifi ed plasmin as a potent proinfl ammatory ac- CLL present address: Roche Palo Alto LLC, 3431 Hillview Avenue, Palo tivator of human peripheral monocytes inducing lipid mediator release, Alto, CA 94304, USA. expression of proinfl ammatory genes including TNF-α and monocyte che- JDS present address: Eli Lilly and Company, Lilly Corporate Center, In- motaxis. We have also shown that the plasmin-induced monocyte stim- dianapolis, IN 46285, USA. ulation is critically dependent on the proteolytic activity of the plasmin molecule. Although plasmin triggers activation of various intracellular sig- naling mechanisms, the identity of the plasmin receptor is still obscure. Here we present evidence that the annexin A2 heterotetramer, composed of two molecules of annexin A2 and S100A10 each, is essential for plasmin- induced signaling in human monocytes. Monocytes express annexin A2 S 100 Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

as epithelial cell lines (CA 9-22 and KB) following addition of recombi- 2045 nant human TNF-α (rhTNF-α) was determined by northern blot and SDS- PAGE, respectively. The activations of MAPKs pathway and phosphory- A novel role for Annexin 1 in macrophage phagocytosis lation of the CRE-binding protein (CREB) transcription factor on serine 133 by which rhTNF-α affected AQP3 were examined by western blotting. Simon Yona, William Harvey Research Institute, University of London, Intense staining of AQP3 spread from connective tissues with active in- United Kingdom fl ammatory cells infi ltration to entire epithelial layer in a severe periodon- Sigrid Heinsbroek, Sir William Dunn School of Pathology, Oxford Uni- titis lesion. AQP-3 density increases gradually and confi nes to the granular versity, United Kingdom layers of the epithelia in an early abscess formation. AQP3 mRNA and Siamon Gordon, Sir William Dunn School of Pathology, Oxford Universi- protein expression were increased at 4-and 6-hour following addition of ty, United Kingdom rhTNF-α. We show that cooperation of the 55-kDa TNF receptor (TNF-R Mauro Perretti, William Harvey Research Institute, University of London, 55) is required for a full blown response to rhTNF-α. FACs analysis reveal United Kingdom that Ca-9-22 AQP-3 knocked down by transfection of AQP-3 small inter- Roderick J Flower, William Harvey Research Institute, University of fering RNAs inhibited signifi cantly the TNF-α elicited intercellular adhe- London, United Kingdom sion molecule (ICAM)1 expression. The kinase inhibitor profi le showed a new AQP3 signaling pathway that mediates JNK, p38 MAPK, ERK1/2 Myeloid cells have abundant levels of the glucocorticoid-regulated protein and CREB phosphorylation. Our studies indicate that AQP3 activation by annexin 1 (ANX-A1), this is particularly true for macrophages (Mø). Us- TNF-α is a novel mechanism involved in periodontal disease. ing conventional gene targeting technology we have generated a mouse strain lacking the ANX-A1 gene (Hannon et al., 2003). Here we have used this tool to investigate the role of ANX-A1 during Mø phagocytosis. 2047 ANX-A1-/- and littermate wild type (WT) mice (6-8 weeks old) were killed and bone marrow extracted, washed and cultured for one week in The inhibitory Fc gamma receptor IIb tunes the rate of radiological RPMI 1640 with 10% FCS and 15% L929-cell conditioned medium to joint damage and modulates dendritic cell function in rheumatoid obtain bone marrow derived Mø (BMDMø). arthritis BMDMø were incubated for 2h at 37°C with FITC zymosan or Neisse- ria. Cells were washed prior to fi xation. Fluorescence was analyzed fl ow Timothy R Radstake, Radboud University Nijmegen Medical Center, The cytometry. In order to determine a better understanding of events during Netherlands phagocytosis BMDMø were also incubated for 1 h at 37°C with zymosan, Barbara Franke, Radboud University Nijmegen Medical Center, The this was recorded by real time confocal microscopy. In addition, BMDMø Netherlands were incubated with LPS (10 ng/ml), zymosan or Neisseria for 2, 4, 12, 24 Mark Wenink, Radboud University Nijmegen Medical Center, The Ne- h at 37°C, cytokine release was measured by ELISA. therlands ANX-A1-/- BMDMø exhibited a reduced particle association, with all par- Nabbe C Nabbe, Radboud University Nijmegen Medical Center, The ticles tested. For instance, cell association after 30 min with zymosan in Netherlands WT cells yielded 1944 ±54 MFI relative to 1450±48 MFI in the ANX-A1- Marieke J Coenen, Radboud University Nijmegen Medical Center, The /- cells, this phenomenon was confi rmed by real time microscopy. Netherlands Incubation with Zymosan or Neisseria for 6 h provoked a substantial Wim B van den Berg, Radboud University Nijmegen Medical Center, The increase from basal in TNF-? production in ANX-A1-/- cells. By 24 h Netherlands Zymosan, Neisseria or LPS provoked a substantial increase by in IL-6 Pilar Barrera, Radboud University Nijmegen Medical Center, The production. This data suggests the absence of ANX-A1 augments the in- Netherlands fl ammatory response. Piet L van Riel, Radboud University Nijmegen Medical Center, The In conclusion, this data demonstrates that the lack ANX-A1 is functionally Netherlands associated with a reduced phagocytic potential in Mø and supports the existence of a tonic inhibitory role for endogenous ANX-A1 in controlling Background. Fc gamma receptors (FcgRs) recognize immune complexes Mø activation. (ICs) and coordinate the immune response by modulation of dendritic cell Hannon R et al., (2003) Faseb J 17, 253-255 (DC) function. By studying the effect of the functional FcgRIIb variant on This work was supported by a PhD. studentship from Nuffi eld the disease severity and DC function we aimed to unravel the role of the inhibitory FcgR in rheumatoid arthritis. Methods. Genotyping was performed in all RA patients (n=246) participat- 2046 ing in an early RA inception and healthy blood donors (n=269) were re- cruited. Demographic data, disease severity and progression were assessed Signaling pathway of TNF-α-induced AQP3 expression in human at baseline and after 3 and six years of disease. Genotyping of the 695T>C gingival epithelial cells: Implications in the pathogenesis of polymorphism in FCGR2B was based on RFLP and direct sequencing. periodontitis Monocyte-derived dendritic cells were cultured using standardized proto- cols. Flowcytometry was performed to determine the expression of FcgRI Salunya Tancharoen, Kagoshima University Graduate School of Medical (CD64), IIa/b (CD32) and III (CD16) and the expression of fl uorescent and Dental Science, Japan ICs. The antigen uptake capacity was studied by measuring the uptake Kenji Matsushita, United States capacity of FITC-ICs. TNFa, IL-6, IL-10 and IL-12 production levels by Takashi Matsuyama, Japan DCs were measured during LPS mediated activation with or without the Ikuro Maruyama, Japan presence of ICs. Mitsuo Torii, Japan Results. Here we report that the FcgRIIb functional variant is associated with a signifi cantly higher rate of joint radiological damage after 3 ((mean Aquaporin3 (AQP3) is membrane channel proteins implicated in water ± SD) 95.0 vs 50.1; P < 0.0001) and 6 years (125.0 vs 76.0; P < 0.0001) absorption in human epithelial cell surfaces. TNF-α plays a central role of RA compared to patients carrying the wild type variant. In vitro data in gingival infl ammation with dramatic changes in fl uid homeostasis. We shows that DCs from patients carrying the functional variant of FcgRIIb hypothesized that TNF-α could alternate an expression of AQP3-related fail to display the inhibitory DC phenotype after FcgR-mediated trigger- human periodontitis. Gingival biopsies were collected from periodontitis ing as seen upon triggering of DCs from patients carrying the FcgRIIb patients, including periodontal abscess tissues, as well as from periodon- wild type. In accordance with this fi nding, DCs from patients carrying the tally healthy subjects for immunohistochemistry analysis. AQP3 mRNA FcgRIIb functional variant have a diminished FcgRII-mediated antigen and protein expression on human primary gingival epithelial cell, as well uptake compared to DCs carrying the wild type. All FcgRs were expressed Infl amm. res. Supplement 2 (2005) Monday 22 August 2005 S 101 at similar levels between DCs with the FcgRIIb functional variant and wild an infl ammatory response in the brain using the reverse passive Arthus type suggesting that the functional FcgRIIb variant has an altered func- (RPA) model to localize IC formation to the pial microvasculature of mice. tion. Alterations in microvascular permeability were determined by quantitating Discussion. Our data underscore the importance of the inhibitory FcgRIIb leakage of FITC-dextran across the pial microvasculature, and leukocyte and DCs in the disease progression of RA and shed new light on FcgRs as recruitment was assessed using intravital microscopy. Initiation of the RPA potential therapeutic targets to modulate DC function to combat autoim- response in the pial microvasculature induced IC formation and C3 deposi- mune conditions. tion in perivascular regions within minutes. Immune complex formation induced dose-dependent changes in cerebral microvascular permeability with higher doses also increasing leukocyte adhesion in pial post-capillary 2048 venules within 30 minutes. Depletion of C3 prevented the RPA-induced alterations in microvascular permeability and leukocyte adhesion. P-se- Directed gp130-Mediated Signaling Dissociates Infl ammation from lectin was required for RPA-induced leukocyte adhesion and microvascu- Fibrosis in Bleomycin-Induced Lung Injury lar permeability, as both responses were abrogated in P-selectin-defi cient mice. Similarly, blockade of the leukocyte b2-integrin (CD18) inhibited Robert JJ O'Donoghue, Asthma and Allergy Research Institute, Australia leukocyte adhesion and was associated with reduced microvascular per- Gary P Anderson, University of Melbourne, Australia meability. These fi ndings demonstrate that ICs are capable of rapidly in- Steven Bozinovski, University of Melbourne, Australia ducing infl ammatory responses in the cerebral microvasculature, with the Jessica Jones, Univeristy of Melbourne, Australia complement pathway and leukocyte recruitment playing critical roles in Matthias Ernst, Ludwig Insitute for Cancer Research, Australia microvascular injury. Darryl A Knight, Asthma and Allergy Research Institute, Australia Steven E Mutsaers, Asthma and Allergy Research Insitute, Australia

Recently our laboratory demonstrated differences in IL-6/gp130-medi- ated STAT and ERK signaling in lung fi broblasts obtained from normal subjects and patients with idiopathic pulmonary fi brosis (IPF). This study aimed to evaluate the contribution of gp130-mediated signaling to the development of pulmonary fi brosis in a murine model. Bleomycin or sa- line was administered intranasally to wild-type (wt) mice and mice with a mutant gp130 molecule that directs signaling through the ERK pathway (gp130ΔSTAT). The effect of bleomycin on lung architecture, infl ammation, collagen production, transforming growth factor beta (TGF-β) and matrix metalloproteinase (MMP) activity was examined in bronchoalveolar la- vage fl uid and lung tissue at 3, 30 and 60 days after treatment. The cellular composition of infl ammatory and fi brotic foci in gp130ΔSTAT and wt mice was characterised by immunohistochemistry. gp130ΔSTAT mice showed a marked infl ammatory response compared with wt 3 days after bleomycin treatment but no signs of fi brosis after 30 and 60 days as refl ected histo- logically and in changes in collagen accumulation. Despite an elevated infl ammatory response TGF-β activity was reduced compared with wt, however MMP activity was markedly increased suggesting active remod- elling in the lungs of gp130ΔSTAT mice. To determine if the infl ammatory response protected gp130ΔSTAT mice from fi brosis wt animals were depleted of bone marrow and reconstituted with gp130ΔSTAT bone marrow and sub- sequently challenged with bleomycin or saline. These chimeric mice were not protected from fi brosis suggesting that the activity of gp130ΔSTAT bone marrow-derived infl ammatory cells alone does not ameliorate the fi brotic effects of bleomycin. This study has demonstrated gp130-mediated ERK signaling prevents bleomycin-induced lung fi brosis, which occurs through mechanisms dissociated from the enhanced infl ammatory response.

2049

Immune complexes alter cerebral microvessel permeability: Roles of complement and leukocyte adhesion

Karyn J Lister, Centre for Infl ammatory Diseases, Monash University, Australia Michael J Hickey, Centre for Infl ammatory Disease, Monash University, Australia

Immune complexes (ICs) have been implicated in several autoimmune dis- eases, particularly systemic lupus erythematosus (SLE). Numerous studies of the mechanisms of IC-mediated infl ammation in peripheral microvas- culatures have demonstrated that ICs induce rapid changes in leukocyte traffi cking and microvascular permeability. However, very little is known regarding the capability of ICs to induce similar infl ammatory responses in the immuno-privileged environment of the brain. This is of relevance as the brain is among the most commonly affected organs in SLE. Therefore, the objective of this study was to investigate the ability of ICs to induce S 102 Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

therapeutic strategy to reduce synovial infl ammation and joints destruc- 3001 tion in RA.

Signal transduction comes of age 3004 Luke A O'Neill, Trinity College Dublin, Ireland CSF-1 and the regulation of Macrophage Function As in all complex biological processes, infl ammation is driven by signal transduction pathways triggered inside cells which culminate in an altered E Richard Stanley, Albert Einstein College of Medicine, United States phenotype in the infl amed organism. Cells activated during infl ammation Xu-Ming Dai, United States can therefore be considered as having an 'infl amed phenotype' whereby Sayan Nandi, United States banks of genes are activated leading to the production of infl ammatory Fiona J Pixley, Albert Einstein College of Medicine, United States mediators such as cytokines and chemokines, or cells such as mast cells Violeta Chitu, United States and neutrophils are activated to release infl ammatory products. Great Yee-Guide Yeung, United States progress has been made in the elucidation of signalling pathways involved in their production and action. To limit infl ammation, the infl ammatory Colony stimulating factor-1 (CSF-1) locally and humorally regulates the mediators themselves are being targetted. In addition the targeting of sig- survival, proliferation, differentiation and function of mononuclear phago- nalling pathways has been the focus of much attention. The success of cytic cells. It is expressed as a secreted glycoprotein, a secreted proteogly- targetting signalling can be seen in the effi cacy of glucocorticoids, which can and a membrane-spanning, cell-surface glycoprotein. The biological interefere with signalling processes in infl ammation. Notable discoveries activity of these CSF-1 isoforms has been studied using transgenic mice in include the description of NF-kappaB as the key infl ammatory transcrip- which synthesis of each is driven by the CSF-1 promoter and fi rst intron tion factor, and stress activated protein kinases such as p38 and JNK being on the CSF-1-defi cient, Csf1op/Csf1op background. Transgenic mice ex- identifed as important stabilisers of mRNAs encoding infl ammatory me- pressing each isoform in a normal tissue-specifi c and developmental pat- diators. The speed at which signalling pathways are being discovered for tern exhibit distinct phenotypic differences, indicating differential effects recently discovered receptors such as the Toll-like receptors (TLRs)which of each CSF-1 isoform. They are being used to study the role of CSF-1 in are important initiators of infl ammation during infection, and the defi n- development and in disease. ing of the multiple interacting partners of proteins in signalling cascades Targeted inactivation of the CSF-1 receptor (CSF-1R) revealed that all presents a real challenge, both in terms of synthesising the information of the effects of CSF-1 are mediated by the CSF-1R encoded by the c- being acquired, and the evaluation of this knew knowledge for the drug fms protooncogene. On the FVB background, osteoclast-defi cient Csf1r-/ discovery process. In addition, recent work in signalling increasingly re- Csf1r- mice die within 4 weeks, have frequent spontaneous bone fractures, veals complex negative and positive regulatory processes which control impaired cortical bone formation, delayed bone mineralization, delayed the response. Disease might therefore result in either an over-activation of cartilage resorption, disorganized bone matrix and disorganized and round- a given pathway, and/ or a breakdown in the control mechanisms. A key ed osteoblasts. This osteoblast phenotype is due to a cell non-autonomous question is how useful this deluge of information will be for the effort to effect of the CSF-1R in the osteoclast and is indicative of regulation of discover new drugs. osteoblast behavior by the osteoclast during bone development. Proteomic and genetic approaches are being used to elucidate the identity and function of signaling proteins downstream of the CSF-1R that regulate 3003 the functions of mature macrophages. Several of these proteins regulate macrophage adhesion and motility. The methods being used and the ac- Pathogenesis of rheumatoid arthritis tions of proteins identifi ed using these approaches will be discussed.

Paul P Tak, AMC/University of Amsterdam, The Netherlands 3005 Rheumatoid arthritis (RA) primarily targets the joints, although a variety of extra-articular manifestations may occur. B cells, T cells, and dendritic Macrophage lineage differentiation cells play a key role in driving the immunological process, ultimately re- sulting in infi ltration of effector cells like macrophages in synovial tissue. Pieter JM Leenen, Dept. Immunology, Erasmus MC, Rotterdam, The This process is mediated by the coordinated action of a variety of infl am- Netherlands matory mediators. Leukocytes adhere to the activated endothelium, lead- Tatjana Nikolic, Dept. Immunology, Erasmus MC, Rotterdam, The ing to migration across the endothelial barrier into the synovium. A com- Netherlands plex network of molecules, including EGF-TM7 receptors, coordinate cell Sacha B Geutskens, Dept. Immunology, Erasmus MC, Rotterdam, The migration and retention, by working in concert to induce an infl ammatory Netherlands response. EGF-TM7 receptors, such as EMR2 and CD97, are predomi- Douglas A Drevets, Dept. Medicine, Oklahoma University Health Sci- nantly leukocyte restricted cell-surface proteins, which possess extended ences Center and VAMC, Oklahoma City, OK, United States extracellular regions containing variable numbers of N-terminal epidermal growth factor (EGF)-like domains. CD97 is found on a broad range of Macrophages are involved in virtually all homeostatic processes, but they leucocytes, whereas expression of EMR2 is restricted to myeloid cells, are best known for their essential functions in host defense. Since the con- including monocytes, macrophages, dendritic cells (DCs), and granulo- stitution of the mononuclear phagocyte system, the prevailing view is that cytes. We have found a close association between CD97+ macrophages they originate from myeloid bone marrow precursors, which mature into and CD55+ fi broblast-like synoviocytes in the intimal lining layer. This blood monocytes and subsequently into tissue mononuclear phagocytes. observation suggests a possible role of the CD97-CD55 interaction in mac- In this presentation, I will give an overview of work from our lab aimed rophage retention and activation at this site. Interestingly, aberrant CD97 at the characterization of the various developmental stages. Furthermore, expression in the synovium is accompanied by detectable levels of soluble results from others and us suggest that the general developmental para- CD97 in the synovial fl uid. EMR2 and CD97 are abundantly expressed digm outlined above is probably not as universal as originally thought. on myeloid cells in RA synovial tissue where their cognate ligands der- In particular, recent insights into the differentiation of the closely related matan sulphate and CD55 are detected. Increased or altered expression of dendritic cells (DC) have indicated that precursor cells from myeloid or dermatan sulphate might be involved in the retention of activated EMR2 lymphoid origin may develop into DC with similar phenotypes and func- positive macrophages and DCs in the synovial sublining, promoting syno- tions. Moreover, some populations may be self-maintained in the steady vial infl ammation. Interference with these pathways might provide a novel state by local proliferation of cells that are probably seeded in embryonic life. In this light, we have observed that macrophage precursors inhabit the Infl amm. res. Supplement 2 (2005) Tuesday 23 August 2005 S 103 mouse embryonic pancreas before the development of genuine monocytes. In the adult mouse peripheral blood, a marked heterogeneity appears to 3008 exist among monocytes. This relates to their maturation stage: immature monocytes, which have been recruited recently from the bone marrow, re- MMP / TIMP imbalance in the development of fi brosis spond to acute infl ammatory triggers from the environment, while more mature monocytes that have resided longer in the circulation do not. In- Annie Pardo, Universidad Nacional Autónoma de México, Mexico stead, those have the phenotype of immature DC. Investigating the devel- opmental aberrancies in autoimmune-prone NOD mice, we have found Lung fi brotic remodeling is characterized by fi broblast/myofi broblast ac- that NOD precursors show a preferential maturation into pro-infl ammatory tivation, and excessive extracellular matrix (ECM) accumulation leading macrophages rather than DC. Furthermore, NOD mononuclear phagocytes to progressive organ dysfunction and usually terminal outcome. Studies in show essential defects in their adhesive and chemokine-mediated migra- humans and experimental models support a critical role for matrix metallo- tory properties, which probably contribute to their inability to maintain proteinases (MMPs) and their tissue inhibitors (TIMPs) in the development tolerance and prevent the development of autoimmunity. of pulmonary fi brosis. By oligonucleotide microarrays, an overexpression of several MMPs, primarily, MMP-7, MMP-1, MMP-2 and MMP-9 have been found in idiopathic pulmonary fi brosis (IPF). One of the most up- 3006 regulated genes is MMP-7 whose immunoreactive protein is mainly local- ized in reactive epithelial cells and bound to ECM. This enzyme is able Macrophages and the chronicity of Infl ammation to degrade several matrix and substrates, such as proteoglycans, laminin, fi brin/fi brinogen, and others. Furthermore, matrilysin knockout mice are John A Hamilton, University of Melbourne, Australia dramatically protected from pulmonary fi brosis in response to intratra- cheal bleomycin, suggesting that MMP-7 plays an important role in lung Acute infl ammatory reactions are usually benefi cial as part of host ho- fi brogenesis and that it can be a potential therapeutic target. MMP-1 is meostasis. The reasons for the maintenance of deleterious chronic infl am- found primarily in reactive alveolar epithelial cells as well as in bronchio- matory lesions, such as a rheumatoid joint, are unknown. Macrophages lar epithelial cells lining honeycomb cystic spaces. Moreover, MMP-1 is are a feature of such lesions and their numbers can correlate with disease virtually absent in fi broblasts of the interstitial fi brotic areas where col- severity. It is proposed that such increases in macrophage numbers can be lagens are being deposited. The role of epithelial MMP-1 is presently un- caused by shifting the balance from apoptosis to survival and possibly also known but it might be related with cell migration. Considering that one by local proliferation. Evidence will be presented for the signifi cance of of the main substrates of collagenases are fi brillar collagens, the lack of a dysregulated cytokine loop involving colony stimulating factors (CSFs) the expression of this enzyme in interstitial fi broblasts might explain in to help explain the increased numbers of macrophages in a wide range of a simplistic way the presence of scars that do not undergo resorption. By chronic infl ammatory lesions; evidence will also be given for a role for the contrast, TIMPs are usually found in the interstitial compartment. TIMP-1 persistent signalling provided by many different types of poorly degrad- is expressed by interstitial cells associated to fi brous tissue and TIMP-2 able stimuli, which cannot be readily cleared. is specifi cally expressed by myofi broblasts within fi broblast foci. TIMP-3 binds to the ECM and is primarily localized to the elastic lamina of vessels and in fi broblasts. Therefore, despite the high expression of some MMPs 3007 in IPF, the strong interstitial localization of the TIMPs as compared to interstitial collagenases, supports the notion that a non-degrading fi brillar The role of macrophage elastase (MMP-12) in the infl ammatory collagen microenvironment is present in this disease. response in the airways

Claude P Bertrand, AstraZeneca, United Kingdom 3010 Soazig Nenan, France Vincent Lagente, INSERM U620, Université de Rennes 1, France Effect of neutrophil depletion on MMPs/TIMP-1 imbalance in bleomycin-induced pulmonary fi brosis in mice Macrophage elastase (MMP-12) is a metalloproteinase able to degrade ex- tracellular matrix components such as elastin. As other MMPs, MMP-12 Boris Manoury, INSERM U620, Université de Rennes 1, Rennes, France, may be involved in acute and chronic lung injury. However, its precise France role in the infl ammatory cascade of the airways during obstructive diseases Soazig Nénan, INSERM U620, Université de Rennes 1, Rennes, France, is not clearly understood. We demonstrated that rhMMP-12 instilled in France mice airways is able to induce severe infl ammatory cell recruitment with Isabelle Guénon, INSERM U620, Université de Rennes 1, Rennes, Fran- an early accumulation of neutrophils associated with an increase in pro- ce, France infl ammatory cytokines and in gelatinases followed by a relatively stable Vincent Lagente, INSERM U620, Université de Rennes 1, Rennes, Fran- recruitment of macrophages in the lungs over a period of about ten days. ce, France Recent studies suggest that resident alveolar macrophages and recruited Elisabeth Boichot, INSERM U620, Université de Rennes 1, Rennes, neutrophils are not essential to induce the delayed macrophage recruit- France, France ment. However, epithelial cells could be the target for MMP-12 in our model. We also reported that a corticosteroid, dexamethasone, a PDE4 in- Pulmonary fi brosis is characterized by excessive deposition of extracellular hibitor, rolipram and a non selective MMP inhibitor, marimastat were able matrix resulting in respiratory failure. The turn over of extracellular matrix to reverse some of these infl ammatory events. These data indicate that our is partially regulated by proteases such as metalloproteinases (MMPs) and MMP-12 model could highlight some of the infl ammatory response seen their inhibitors (TIMPs). We investigated the impact of polymorphonucle- in COPD and could be used for the pharmacological evaluation of new ar neutrophils (PMN) depletion on the MMP/TIMP-1 imbalance and the anti-infl ammatory mechanism of action. Because of its ability to induce development of bleomycin-induced fi brosis in mice. C57BL/6 mice were an infl ammatory response and tissue remodeling, it may be possible to injected i.p. with 200μL of rabbit anti-mouse PMN antibody. Control mice consider MMP-12 as an essential component of the process leading to the received vehicle solution. One day and 14 days after bleomycin adminis- development of the disease. tration (0.3 mg/mouse), bronchoalveolar lavages (BAL) were performed and lungs were removed for analysis. Administration of anti-PMN anti- bodies blunted the neutrophil infl ux detected in BAL and in whole blood one day after bleomycin administration (89 % decreased; P < 0.05). At day 14, hydroxyproline content refl ected the collagen deposition in the lung tissue was increased both in anti-PMN treated and control mice, without S 104 Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) difference between groups. At day one, bleomycin elicited a raise in pro- MMP-9 level in BAL that was signifi cantly attenuated in anti-PMN de- 3014 pleted mice, whereas bleomycin increased TIMP-1 release in both groups in a similar manner. Pro-MMP-9 activity was signifi cantly correlated with The Lyn tyrosine kinase is a critical regulator of innate and adaptive the neutrophil count in the BAL fl uids (P = 0.0003). Anti-PMN adminis- immune responses tration elicits stronger IL-6 level in BAL of bleomycin-treated mice. Anti- PMN administration had no effect on MMP-2 that was induced in BAL by Margaret L Hibbs, Ludwig Institute for Cancer Research, Australia bleomycin, both at day 1 and day 14. Higher RNA levels were observed Sarah-Jane E Beavitt, Ludwig Institute for Cancer Research, Australia in PMN-treated mice at day one for MMP-9 and MMP-2 and at day 14 for Joanna M Kemp, Ludwig Institute for Cancer Research, Australia MMP-2 only. At day 14, bleomycin elicited a raise of TIMP-1 protein and David M Tarlinton, Walter and Eliza Hall Institute of Medical Research, RNA levels regardless to anti-PMN treatment, whereas MMP-9 returned Australia to basal level. PMN-depletion and the associated modifi cations in pro- Gary P Anderson, University of Melbourne, Australia MMP-9 / TIMP-1 imbalance in lung during the early infl ammatory phase Kenneth W Harder, Ludwig Institute for Cancer Research, Australia do not alter susceptibility to bleomycin-induced pulmonary fi brosis. Members of the Src-family of protein tyrosine kinases play critical roles in the regulation of immune system function, and cellular homeostasis. Most 3011 members of this family are involved in transmitting signals that result in cellular activation, and perturbation of these signals has been associated Induction Of ADAMTS-4 (Aggrecanase-1) Gene Expression By with malignant transformation. By contrast, the Lyn tyrosine kinase is Interleukin-1 In Articular Chondrocytes Is Mediated By Mitogen- duplicitous in nature, acting as a signal amplifi er and also as a signal in- Activated Protein Kinases hibitor through its ability to mobilize phosphatases to inhibitory receptors at the plasma membrane where they promote signal termination. Lyn’s Rasheed Ahmad, Department of Medicine and Research Centre of CHUM role in this phenomenon has been most comprehensively studied in the B Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada lymphocyte lineage, where Lyn has been found to be largely dispensable Mohammed El MABROUK, Department of Medicine and Research for positive regulation of signaling, but critically important for signal at- Centre of CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, tenuation. Canada Lyn has been implicated as both a facilitator and inhibitor of signaling Judith Sylvester, Department of Medicine and Research Centre of CHUM pathways that play a role in allergic infl ammation, although its role in Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada asthma is unclear as Lyn is not expressed in T cells. We have found that Ataf Chaudry, Department of Medicine and Research Centre of CHUM Lyn-defi cient mice develop a severe, persistent infl ammatory asthma-like Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada syndrome with lung eosinophilia, mast cell hyper-degranulation, hyper Francois Huynh, Department of Medicine and Research Centre of IgE, and Th2-polarizing dendritic cells. While Lyn-defi ciency gives rise CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada to a Th2-biased immune response, enhanced Lyn activity leads to height- Muhammad Zafarullah, Department of Medicine and Research Centre of ened sensitivity to endotoxin and the development of lung pathology that CHUM Notre-Dame Hospital, Montreal Quebec H2L 4M1, Canada resembles chronic obstructive pulmonary disease. Analysis of the role of dendritic cells in these phenotypes has shown that Lyn plays a critical role PURPOSE: Interleukin-1 (IL-1β) is the main proinfl ammatory stimulant in the maturation of this lineage, and regulates the production of IL-10 of cartilage catabolism in arthritis. Aggrecanases (ADAMTS are enzymes and IL-12. These results show that Lyn plays a central role in regulating implicated in tissue remodeling and cleavage of aggrecan, a major pro- the switch between Th1- and Th2-biased immune responses, and suggest tein of cartilage matrix responsible for its compressive stiffness. IL-1 also that Lyn, or components of the Lyn-dependent signaling machinery may inhibits matrix synthesis. We investigated the molecular mechanisms of prove to be valuable targets for therapeutic manipulation of dendritic cell- IL-1 signal transduction leading to ADAMTS-4 RNA induction in human induced immune responses. articular chondrocytes. METHODS: Confl uent normal human knee articular chondrocytes main- tained in serum-free medium were pretreated either with different pharma- 3015 cological inhibitors or transiently transfected with antisense oligonucle- otides or small interfering RNAs (siRNAs) and stimulated further for 24 PI3Kg - A Novel Target for Rheumatoid Arthritis. Closing the loop h with IL-1β (10 ng/ml). ADAMTS-4 and glyceraldehyde-3-phosphate - From genetic validation and rationale drug design to therapeutic dehydrogenase (GAPDH) RNA levels were analyzed by RT-PCR. proof of concept RESULTS: IL-1β induced ADAMTS-4 RNA in high-density human chondrocyte monolayer cultures. Pretreatment with specifi c inhibitor of Thomas Ruckle, Serono Pharmaceutical Research Institute, Serono ERK-MAPK, U0126 and transfection with antisense ERK or its siRNA International S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, partially inhibited IL-1-induced expression of ADAMTS-4 RNA. Protein Switzerland 38 and JNK inhibitors, SB203580 and SP600125 also down-regulated Montserrat Camps, Serono Pharmaceutical Research Institute, Serono such induction. A leukotreine and c-Fos (component of activating protein International S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, or AP-1 transcription factor) inhibitor, nordihydroguiaretic acid (NDGA) Switzerland suppressed the ADAMTS-4 RNA induction. Further, AP-1 and nuclear Hong Ji, Serono Pharmaceutical Research Institute, Serono International factor kappa B (NF-κB) transcription factors inhibitors, pyrrolidine dithio- S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland carbamate (PDTC) and curcumin partially or completely inhibited aggre- Felix Rintelen, Serono Pharmaceutical Research Institute, Serono canase-1 induction. The levels of internal control, GAPDH RNA remained International S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, constant. Switzerland CONCLUSIONS: These results suggest the involvement of ERK-, p38- Vittoria Ardissone, LCG-RBM, Serono International S.A., Via Ribes 1, and JNK-MAPKs as well as AP-1 and NF-κB transcription factors in the 10010 Colleretto Giacosa, Italy IL-1 induction of ADAMTS-4 in chondrocytes. Inhibition of these targets Rocco Cirillo, LCG-RBM, Serono International S.A., Via Ribes 1, 10010 by the specifi c pharmacological or genetic agents could be useful for re- Colleretto Giacosa, Italy ducing ADAMTS-4 driven cartilage resorption in arthritis. Matthias K Schwarz, Serono Pharmaceutical Research Institute, Serono International S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland Infl amm. res. Supplement 2 (2005) Tuesday 23 August 2005 S 105

Christian Rommel, Serono Pharmaceutical Research Institute, Serono International S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, 3019 Switzerland Infl ammation and insulin resistance Small molecule inhibitors targeting class I PI3K isoforms have vast po- tential use for the treatment of infl ammatory and autoimmune disorders as Joseph Proietto, University of Melbourne, Australia well as cancer and cardiovascular diseases. However, the lack of specifi c- ity, isoform selectivity and poor biopharmaceutical profi le of PI3K inhibi- There is a surprising emerging association between obesity, insulin resis- tors has so far hampered rigorous disease-oriented and pharmacologically tance, and features of a low-grade infl ammatory state. C-reactive protein, relevant target validation. PI3Kg plays a crucial role in mediating leuko- a marker of infl ammation used in clinical practice, is elevated in obese cyte chemotaxis as well as mast cell degranulation, mainly in response to subjects. The mechanism for the elevated circulating acute phase proteins G-protein coupled receptor activation and hence represents a high value and cytokines in obesity is unclear and requires further study. Adipocytes target for autoimmunity and infl ammation. and pre-adipocytes have features of cells involved in the infl ammatory re- In our laboratories we have studied PI3Kg defi cient mice in two murine sponse, such as production of cytokines and proteins of the complement models of rheumatoid arthritis (RA). We show that these mice are largely pathway and pre-adipocytes have the capacity to be effi ciently differen- protected in both these models, which feature the effector phase of RA, re- tiated into macrophages. It has recently been demonstrated that in obe- sulting from impaired neutrophil migration. These fi ndings further validate sity there is also a macrophage infi ltrate in the adipose tissue that may PI3Kg as a therapeutic target. be initiated by signals from fat-laden adipocytes including the release of Exploiting the interface of Molecular Medicine, Medicinal Chemistry and cytokines. The mechanisms responsible for initiating the infl ammatory Experimental Pharmacology, we have developed potent and selective small signals from adipocytes are not known but a clue is given by our recent molecule PI3Kg inhibitors, that upon oral treatment suppress the progres- fi nding that insulin stimulates IL-6 mRNA levels 8 fold in muscle tissues sion of joint infl ammation and cartilage damage in murine models of RA, from insulin resistant rats but not from normal control animals. We hypoth- reproducing the protective effects exhibited by PI3Kg defi cient mice. SAR esised that insulin stimulates IL-6 mRNA production by a mechanism that of two generations of inhibitors, supported by X-ray crystallography and remains normally insulin sensitive in muscle cells in which the metabolic structure based design, has led to the identifi cation of the lead compound actions of insulin are impaired. This is consistent with the fi nding that in 1 (Ki = 7.8 nM), active in animal models of cell recruitment, mast cell obese type 2 diabetics, the insulin resistance in muscle tissue affects the activation and infl ammation. PI-3 kinase pathway but not the MAP kinase pathway. The failure of insu- Our results identify selective PI3Kg inhibitors as potential therapeutics for lin to signifi cantly stimulate IL-6 production in muscle cells with normal the treatment of RA insulin sensitivity is probably due to the fact that normal activation of PI-3 kinase inhibits IL-6 production. It has been shown that inhibition of PI-3 kinase using LY294002 increased IL-6 protein secretion 3 fold in human 3016 monocytes, consistent with this hypothesis.

Topical nuclear factor kappa B decoy - An effective and safe therapeutic for atopic dermatitis 3020

Rolf O Ehrhardt, Corgentech, Inc., United States Control of obesity and glucose homeostasis by Th1/Th2 cytokines Maya Dajee, Corgentech, Inc., United States Michael S Rolph, Arthritis and Asthma Research Program, Garvan Insti- Atopic Dermatitis (AD) is the most common chronic skin infl ammatory tute of Medical Research, Australia disease. Long-term use of topical corticosteroids and topical calcineurin Trina So, Australia inhibitors raises increasing concerns about systemic immunosuppression Tim Young, Australia and malignancy. The nuclear factor kappa B (NF-kB) transcription factor Ross Laybutt, Australia plays a central role in the progression and maintenance of AD. This study Zoe A Commandeur, Garvan Institute of Medical Research, Australia explores the possibility of using topical NF-kB decoy (NFkBD) as a thera- Paul S Foster, The University of Newcastle & Hunter Medical Research peutic alternative for AD. The effi cacy and skin penetration of a high affi n- Institute, Australia ity, specifi c NFkBD was examined in several animal models. In a dustmite Gregory J Cooney, Australia antigen induced AD mouse model (NC/Nga), topical NFkBD (0.1-1.0%) Juan-Carlos Molero, Australia produced a dose-dependent reduction of ear swelling (up to 80%), simi- Charles R Mackay, Garvan Institute of Medical Research, Australia lar to topical betamethasone treatment. NFkBD also shows dose-depen- dent effi cacy in a TPA-induced dermatitis murine model. In both models, Obesity and associated metabolic complications affect over 50% of the NFkBD decreased expression of pro-infl ammatory cytokines IL-1b, IL-6, adult population in the USA, with Australia not far behind. Recently, obe- TNFa and TSLP by an average of 60-80%. Both topical NFkBD and beta- sity has been shown to have a substantial infl ammatory component. For methasone therapy dramatically reduced mononuclear cells infi ltration and example, TNF and IL-6 production is enhanced in obesity, and adipose epidermal proliferative indices. In contrast, comparative gene expression tissue of obese subjects has a marked macrophage infi ltrate. profi ling study between betamethasone and NFkBD treatment on infl amed We hypothesized that obese infl ammation is a polarized “type 1” cytokine ears illustrate diverse and very distinct regulation of target genes. Interest- environment. To test this, IL-4, IL-13 and IFNg mRNA expression was ingly, severe rebound of infl ammation was seen after cessation of beta- measured in adipose tissue of lean and obese mice. IFNg mRNA expres- methasone, however, the NFkBD therapeutic benefi t was maintained for sion was increased 10-fold in adipose tissue of mice with diet-induced at least 15 days post-withdrawal of drug treatment. Unlike betamethasone obesity, while IL-4 and IL-13 could not be detected in adipose tissue of that induces skin atrophy of 15%, prolonged NFkBD application fails to lean or obese mice. To test the role of IFNg in obesity, wild-type (WT) show any such side effect. In addition, betamethasone, but not NFkBD, and IFNg KO mice were fed normal chow or a high fat diet (HFD), fol- down-regulates major components of extracellular matrix of the skin, like lowed by metabolic assessment. WT mice fed a HFD became obese. The Collagen I and III (50%), tenacin C (40%) elastin (30%). Furthermore, IFNg KO mice were completely protected from diet-induced obesity and twenty-three weeks of topical NFkBD decoy application (Q.I.D.), on its metabolic complications such as impaired glucose tolerance. chemically induced skin cancer murine model does not increase skin pap- Although IL-4 and IL-13 were not detected in adipose tissue, we have illoma formation and prevents the formation of large tumors. In summary, uncovered an unexpected metabolic role for one or both of these cytokines. the present study illustrates the potential of topical NFkBD as a novel, IL-4Ra KO mice, which cannot signal to IL-4 or IL-13, developed a sub- effective and safer therapeutic agent for the treatment of AD and other stantial defect in systemic glucose homeostasis, despite maintaining nor- infl ammatory skin diseases. mal insulin sensitivity. The impaired glucose homeostasis is due to a defect S 106 Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) in insulin release, and pancreatic islets from IL-4Ra KO mice released rived neurotrophic factor (BDNF) and other neurotrophic factors in vitro. 5-fold less insulin in response to glucose. We are currently undertaking Furthermore, BDNF is expressed in different types of infl ammatory cells detailed metabolic profi ling of IL4Ra and IFNg KO mice to pinpoint the in brain lesions of patients with acute disseminated leukoencephalopathy source and mechanism of action of these cytokines. or multiple sclerosis. Intriguingly, the corresponding receptor TrkB is also Thus, type 1 and type 2 cytokines play opposing roles in metabolic homeo- expressed, especially in neurons in the immediate vicinity of MS plaques stasis, most likely by distinct molecular mechanisms. The fi ndings provide and in reactive astrocytes in lesions, but not in infl ammatory cells. This insights into the relationship between infl ammation and metabolism, and suggests that BDNF and its receptor are involved in immune-mediated suggest novel approaches for development of new therapies for metabolic neuroprotective interactions in MS, supporting the idea that immune cells disease. produce both damaging and protective factors in MS lesions. The concept of neuroprotective autoimmunity has obvious implications for the therapy of multiple sclerosis and other neuroimmunological diseases. 3021

Anti-infl ammatory Properties of Novel Antidiabetic Compound 3025 BLX-1002, Which Lowers Body Weight Gain in Obese Mice Interferon-γ and Sonic Hedgehog Signaling in Infl ammation, Deben Dey, Bexel Pharmaceuticals Inc.,, United States Developmental Deviation and Tumour Formation in the Brain Bindu Pandey, Bexel Pharmaceuticals Inc.,, United States Madhavi Anumula, United States Iain L Campbell, The University of Sydney, Australia Abhijeet Nag, United States Partha Neogi, United States Sonic hedgehog (Shh) is essential for the fate and proliferation of granule Bishwajit Nag, United States neuron precursors during development of the cerebellum. Dysregulated Shh signaling is linked to pathological consequences including medullo- BLX-1002 is an amino acid conjugated small molecule that has a distinct blastoma. IFN-α-stimulated STAT2-independent signaling in the neonatal mode of action as compared to any known antidiabetic compound. In db/ mouse brain results in profound granule neuron hyperplasia with medul- db mice, BLX-1002 (12.5 mg/kg) signifi cantly lowered blood glucose lev- loblastoma co-existent with a Th-1-like immune response that includes els (40%) without any change in body weight gain. It has reduced serum T-cell infi ltration and IFN-γ gene expression in the brain. Furthermore, triglycerides (43%), free fatty acid(20%)levels compared to vehicle treated Shh signaling in these brains showed signifi cant ectopic activation in gran- animals. BLX-1002 signifi cantly improved oral glucose tolerance in Diet ule neurons supporting an autocrine growth stimulatory circuit. A direct Induced Obesity model and reduced systolic blood pressure, triglyceride role for IFN-γ in causing this phenotype was identifi ed using bigenic mice levels in fructose fed rats. In streptozotocin induced type I diabetes model, with temporal control of IFN-γ gene expression in the brain driven by a blood glucose and triglycerides are reduced after BLX-1002 treatment. tetracycline controllable promoter. Inducible expression of IFN-γ in the When orally administered, it has strong inhibitory effect on lipopolysa- developing brain resulted in disturbance in the development of the cerebel- charide (LPS) induced serum TNF-alpha(72%)and IL-6(42%) produc- lum with extensive proliferation of the granule neurons, medulloblastoma tion compared to vehicle group. Inducible nitric oxide synthatase (iNOS), and ectopic activation of the Shh signaling pathway. The role of IFN-γ as another pro-infl ammatory protein was also inhibited by BLX-1002 in a regulator of Shh gene expression was examined in cultured granule neu- RAW264.7 cells upon LPS challenge. In carrageenan induced paw edema rons derived from embryonic mouse brain. While the type I IFN, IFN-α, model, BLX-1002 (50mg/kg) is able to reduce 30-40% of PAW infl amma- was without effect, treatment with IFN-γ led to signifi cant induction of the tion compare to controls and the effect was similar extent to dexametha- Shh gene. IFN-γ induced Shh mRNA expression was dependent on signal- sone dose (5mg/kg, oral).BLX-1002 has no affi nity to PPARα, γ and δ as ing via STAT1 but not STAT2 and is consistent with the Shh promoter con- shown by various transactivation and radioligand binding assays. It does taining an IFN-γ-activated GAS regulatory element. Thus, IFN-γ mediates not induce adipogenesis, aP2, adiponectin expression in cultured adipo- STAT1-dependent induction of Shh in early granule neurons driving the cytes. Aldose reductase, a key enzyme for polyol pathway and responsible perpetual growth and eventual neoplastic transformation of these cells to for diabetic retinopathy and neuropathy is also inhibited by BLX-1002 medulloblastoma. These studies highlight a potential role for the immune (IC50 4.07 μM) where quercitrin is kept as positive control(IC50 0.352 system in the pathogenesis of developmental disorders and tumorigenesis μM). Recently it is demonstrated that BLX-1002 reduces the cellular of the CNS and perhaps other organs involving dysregulated Shh signaling energy charge and activates AMPK in liver and skeletal muscles. These mediated by IFN-γ. Supported by NIH Grant MH62231 results suggest that BLX-1002 is a novel, orally active anti-diabetic com- pound which has strong anti-infl ammatory properties and can be useful for the treatment of diabetes and associated complications. Its potential as a 3026 candidate for the treatment of Metabolic Syndrome-X is currently being evaluated in Clinical studies. Expression of Toll-like Receptors in Rheumatoid Arthritis Synovial Tissue and Assessment of TLR Involvement in Mouse Collagen- Induced Arthritis 3023 Keith P Ray, GlaxoSmithKline R&D, RA Disease Biology Unit, Stevena- Protective immunity in CNS demyelination ge, United Kingdom Jacky Buckton, GlaxoSmithKline R&D, RA Disease Biology Unit, Ste- Reinhard Hohlfeld, Institute for Clinical Neuroimmunology, University of venage, United Kingdom Munich, Germany John Spaull, GlaxoSmithKline R&D, RA Disease Biology Unit, Stevena- ge, United Kingdom “Autoreactive” T cells are physiological components of the immune sys- Ginette R Squires, GlaxoSmithKline R&D, RA Disease Biology Unit, tem. It has been proposed that under certain circumstances, some of these Stevenage, United Kingdom autoreactive T cells may have a protective function in infl ammatory CNS diseases, especially multiple sclerosis (MS) (reviewed in Kerschensteiner The family of mammalian Toll-like receptors (TLRs) has evolved to re- et al., Neurotrophic cross-talk between the nervous and immune systems: spond to pathogen associated molecules and to activate protective pro- Implications for neurological diseases. Ann. Neurol. 53: 292-304, 2003). infl ammatory mechanisms required for host defence. The importance of Recent studies support this notion by demonstrating that a) myelin-specifi c TLRs in chronic diseases such as rheumatoid arthritis (RA) remains to be T cells show neuroprotective effects in vivo, and b) activated antigen-spe- established. However, evidence that TLRs can infl uence the production cifi c human T cells and other immune cells produce bioactive brain-de- of clinically relevant cytokines including TNFa and IL-1b and may con- Infl amm. res. Supplement 2 (2005) Tuesday 23 August 2005 S 107 tribute to mechanisms of autoimmunity suggests potential for roles in RA Fc receptors are one of the major receptor families involved in immunity as disease pathogenesis. To investigate disease associated expression, TLR a consequence of their role in the binding of antibody immune complexes. mRNA levels were assessed in synovial tissue using quantitative PCR and There is increasing evidence from human and mouse studies that they have protein distribution examined by immunohistochemistry (IHC). Normal a primary role in the development of tissue destruction in autoimmune synovium and RA disease tissue with mild, moderate or severe infl am- diseases such as rheumatoid arthritis. We have used several approaches matory involvement were selected. Involvement of TLR4 and TLR9 in to understand their role in autoimmunity and to develop potential new a model of collagen induced arthritis (CIA) was investigated using TLR therapeutic strategies. Xray crystallographic and drug design studies have defi cient mice. Arthritis was induced by immunisation with chick type II generated a number of potential new chemical entity therapeutics and also collagen in complete Freund’s adjuvant. revealed new information on receptor organization. In addition, we have Expression of TLRs (mRNA) was increased in RA synovial tissue, with engineered recombinant soluble forms of the receptor as well as generated highest levels in the most activated specimens associated with increased monoclonal antibodies as potential biologic therapeutics. Our animal data proinfl ammatory cytokines (mRNA). IHC showed increases in TLR clearly indicates that the major FcgammaRIIa of humans is likely to have a expression in both synovial lining layer and sub-intimal tissue. Results dominant and early role in the development of autoimmune pathology. suggest increased TLR expression may relate to the increased numbers of infi ltrating infl ammatory cells. In mouse CIA no differences in disease incidence, progression or pathol- 3029 ogy were seen between WT and TLR4 defi cient (C57Bl/10ScN) mice. TLR9 KO mice had a lower incidence of arthritis than their WT litter- Regulation of Toll-like receptor mediated infl ammatory responses by mates, however, there were no differences in severity judged visually or controlling protein kinase stability histologically. No signifi cant differences in joint cytokines, serum anti- collagen antibody levels or splenocyte responsiveness to collagen were Dominic De Nardo, The University of Melbourne, Australia measured between WT- and TLR- defi cient arthritic mice. Results demon- Paul Masendycz, The University of Melbourne, Australia strate that TLR9 but not TLR4 plays an important role in this model of CIA Lena Cross, The University of Melbourne, Australia by a mechanism that remains to be elucidated. John A Hamilton, The University of Melbourne, Australia Glen M Scholz, The University of Melbourne, Australia

3027 Toll-like receptors (TLRs) serve crucial roles in innate immunity by me- diating the activation of macrophages by microbial pathogens. Different Small Molecule TLR7 and 8 Agonists for Treatment of Viral TLRs are activated by specifi c components of pathogens. For example, Infection and Cancer TLR4 is activated by bacterial lipopolysaccharide (LPS), whereas CpG DNA triggers the activation of TLR9. The protein kinase IRAK-1 is a key Mark A Tomai, 3M Pharmaceuticals, United States component of TLR signaling pathways due to its TLR ligand-induced in- teraction with TRAF6, which subsequently leads to the activation of MAP Imidazoquinolines are a group of immune response modifi er compounds, kinases and various transcription factors. Interestingly, IRAK-1 is degrad- characterized ed following TLR activation and this has been proposed to contribute to as TLR7/TLR8 agonists. Structure-activity relationship studies have led the induction of a tolerant state in macrophages by limiting further TLR- to the mediated signaling. development of a diverse group of compounds characterized by the cell Using a mass spectrometric-based approach we have identifi ed a cohort of types they chaperones and co-chaperones, including Hsp90 and Cdc37, which bind to activate and the cytokine profi les they generate. These molecules activate IRAK-1 but not IRAK-4 in 293T cells. Pharmacologic inhibition of Hsp90 NFkB via led to a rapid decline in the expression level of IRAK-1, whereas overex- TLR7 and/or TLR8. TLR7-selective agonists activate B-cells with mini- pression of Cdc37 enhanced the activation and oligomerization of IRAK-1 mal effects on myeloid-derived cells. In contrast, TLR8-selective agonists in 293T cells. Signifi cantly, inhibition of Hsp90 in macrophages resulted preferentially activate myeloid DC and monocytes with minimal effects in the rapid degradation of IRAK-1 but not IRAK-4. Concomitant with the on pDC and B-cells. TLR7-selective agonists are more effective induc- loss of IRAK-1 expression was a reduction in the activation of p38 MAP ers of type I IFN-alpha and weaker inducers of TNF-alpha as compared kinase and Erk1/2 following stimulation with the TLR ligands LPS and to TLR8-selective agonists. Overall, activation of innate immune cells CpG DNA. Moreover, TLR ligand-induced expression of proinfl amma- through TLR7 or TLR8 results in the production of cytokines, co-stimula- tory cytokines was also profoundly reduced. We conclude that the level tory markers and activation of T-cells consistent with the hallmarks of an of on-going support provided to IRAK-1 by Hsp90 and Cdc37 directly immunomodulator. infl uences the magnitude of TLR-mediated macrophage activation. Thus, modulation of Hsp90 or Cdc37 expression and/or activity by infl ammatory cytokines or stress stimuli (e.g. fever) may represent a novel mechanism for governing the magnitude of the infl ammatory response of macrophages to microbial pathogens. 3028

Understanding Fc Receptor Structure and Function and the 3030 development of new therapeutic strategies for treatment of immune complex-induced tissue destruction A novel splice variant of interleukin-1 receptor associated kinase-1 plays a dominant negative role in Toll/IL-1R-induced infl ammatory Mark Hogarth, Austin Research Institute, Australia signaling Paul Ramsland, Austin Research Institute, Australia Geoffrey A Pietersz, Austin Research Institute, Australia Navin Rao, Johnson & Johnson Pharmaceutical Research & Develop- Maree S Powell, Austin Research Institute, Australia ment, United States Bruce D Wines, Austin Research Institute, Australia Steven Nguyen, Johnson & Johnson Pharmaceutical Research & Deve- Tessa Bradford, Austin Research Institute, Australia lopment, United States William Farrugia, Austin Research Institute, Australia Karen Ngo, Johnson & Johnson Pharmaceutical Research & Develop- PeckSzee Tan, Austin Research Institute, Australia ment, United States Wai-Ping Fung-Leung, Johnson & Johnson Pharmaceutical Research & Development, United States S 108 Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

The interleukin-1 (IL-1) receptor associated kinase 1 (IRAK1) is a member Richard Bucala, Department of Medicine and Pathology, Yale University, of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor School of Medicine, United States (TIR) family signaling cascade. We have identifi ed a novel splice variant, Lin Leng, Department of Medicine and Pathology, Yale University, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. School of Medicine, United States IRAK1c expression was confi rmed by both RNA and protein detection. Christian Weber, Department of Cardiovascular Molecular Biology, Although both IRAK1 and IRAK1c are expressed in most tissues tested, University Hospital RWTH Aachen, Germany IRAK1c is the predominant form of IRAK1 expressed in the brain. Un- like IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated Atherosclerosis is an infl ammatory response of the arterial wall to “in- by IRAK4. However, IRAK1c retains the ability to strongly interact with jury“, which is prominently driven by cytokines. The infl ammatory media- IRAK2, MyD88, Tollip and TRAF6. Overexpression of IRAK1c sup- tor macrophage migration inhibitory factor (MIF) is a unique cytokine that pressed NFκB activation and blocked IL-1β-induced IL-6 as well as LPS was recently associated with atherogenesis. Here, we have investigated and CpG-induced TNFα production in multiple cellular systems. Mecha- whether MIF has a role in spontaneous atherosclerosis by studying apo- nistically, we provide evidence that IRAK1c functions as a dominant nega- lipoprotein E-defi cient (ApoE-/-) mice treated with neutralizing anti-MIF tive by failing to be phosphorylated by IRAK4, thus remaining associated monoclonal antibody and comparison with isotype IgG-treated controls. with Tollip, and blocking NFkB activation. The presence of a regulated After 14 weeks, the aortas and heart valves were analyzed for infl amma- alternative splice variant of IRAK1 that functions as a kinase dead, domi- tory status, macrophage content and plaque areas. MIF expression in the nant negative protein adds further complexity to the variety of mechanisms aortic wall was elevated upon spontaneous atherogenesis, with foam cells that regulate TIR signaling and the subsequent infl ammatory response. representing a major source. Importantly, MIF blockade led to a marked reduction in intimal Mac-1-positive macrophages. Similarly, treatment with anti-MIF antibody led to a reduction of a variety of infl ammatory 3032 mediators typically associated with atherosclerosis including fi brinogen, MIF and IL-6. Importantly, the local aortic expression of ICAM-1, MMP- Cytokines and Atherosclerosis 2, TNF, IL-12, and CD40L was reduced by MIF blockade, as were the levels of the phospho-c-Jun and C-EBP transcription factors. The observed Alex Bobik, Baker Heart Research Institute, Australia strong reduction of infl ammatory parameters by anti-MIF treatment was associated with a small reduction in aortic plaque area. Thus, in this mouse Accumulating evidence indicates that atherosclerosis is a chronic infl am- model of spontaneous atherogenesis, MIF plays an important role in the matory disease state regulated by pro-infl ammatory and anti-infl ammatory development of intimal infl ammation by enhancing the expression of a cytokines. We recently identifi ed HMGB1 as a novel pro-infl ammatory variety of infl ammatory mediators and by leading to a strong increase in cytokine in human atherosclerotic lesions that appears to account for many intimal macrophages. To investigate the mechanism by which MIF may of the elevations in other pro-infl ammatory cytokines such as TNF-alpha, contribute to the increase in macrophage content, the effect of MIF on IL-1beta and IL-6 in atherosclerotic lesions and may also contribute to monocyte adhesion and migration was studied. In vitro fl ow assays with lipid accumulation by macrophages. HMGB1 is highly expressed by mac- HAoECs revealed that treatment with MIF led to a marked elevation in rophages in fatty streaks and fi brofatty lesions; however, expression is monocyte recruitment and Transwell migration assays showed that MIF greatest in fi brofatty lesions. Expression is regulated by other pro-infl am- potently enhanced monocyte migration. Of note, both processes were matory cytokines including interferon-gamma, tumor necrosis factor-al- found to be dependent on the chemokine receptor CXCRII. It will be im- pha, and TWEAK and also transforming growth factor-beta. HMGB1 can portant to decipher the molecular link between MIF-induced monocyte/ in turn stimulate their secretion by macrophages, thereby amplifying pro- macrophage activation and CXCRII-dependent processes. infl ammatory cytokine concentrations in lesions. Cytokines stimulating HMGB1 secretion initially increase its expression and redistribution from the nucleus to the cytoplasm, effects that can be attenuated by inhibitors 3035 of MEK1/MEK2, protein kinase C and PI-3kinase/Akt. In atherosclerotic lesions HMGB1 partially colocalises with RAGE suggestion that part of Intracellular viral serpin, Serp-2, inhibits apoptosis of T lymphocytes its actions in the lesions may be via this receptor. Secreted HMGB1 acti- - Linking infl ammation and apoptosis in atherogenesis vates and also stimulates vascular smooth muscle cell migration and down regulates the expression of components of the reverse cholesterol transport Kasinath Viswanathan, Robarts Research Institute, Canada system in macrophages-ABCA1 and Apo E. Our studies on anti-infl am- Lying Liu, Robarts Research Institute, Canada matory cytokines, in particular TGF-beta/Smad expression and associated Erbin Dai, Robarts Research Institute, Canada responses in human atherosclerotic lesions indicate that this signaling Peter C Turner, University of Florida, United States pathway is impaired in vascular smooth muscle cells of fi brofatty lesions, Richard W Moyer, University of Florida, United States suggesting that these cells are mostly under the infl uence of pro-infl am- Alexandra R Lucas, Robarts Research Institute, Canada matory cytokines such as HMGB1, promoting development of vulnerable lesions. Introduction: The role of apoptosis in arterial infl ammation is still not clearly understood. Apoptosis of endothelial, monocyte and smooth muscle cells has been found to cause increased plaque growth and even 3034 rupture. Apoptotic effector, Granzyme B secreted by CTLs has been iden- tifi ed in chronic rejections and alveolar infl ammation. The myxoma viral MIF is a potent mediator of spontaneous atherogenesis: Role for intracellular serpin, Serp-2, inhibits granzyme B and caspase-1 in vitro. MIF in infl ammatory cytokine and adhesion molecule expression and Our previous studies have shown this protein to attenuate infl ammation CXCRII-dependent monocyte adhesion and migration and apoptosis in an arterial angioplasty model, but the cellular target that links apoptosis and infl ammation has not been defi ned. In order to deci- Jürgen Bernhagen, Department of Biochemistry and Molecular Cell pher the exact target for Serp-2 inhibition of infl ammation, we have tested Biology, University Hospital RWTH Aachen, Germany the role of the anti-infl ammatory protein, Serp-2 in extrinsic and intrinsic Regina Krohn, Department of Cardiovascular Molecular Biology, Uni- apoptotic pathways and in infl ammatory responses. Methods: Apoptosis versity Hospital RWTH Aachen, Germany of THP-1 monocytes, Jurkat T cells and Endothelial cells were induced by Ivan Georgiev, Department of Biochemistry and Molecular Cell Biology, Fas L, granule-mediated apoptosis, staurosporine and Camptothecin and University Hospital RWTH Aachen, Germany the effects of Serp-2 and its two reactive center loop mutants 294A and Anke Burger-Kentischer, Fraunhofer IGB Stuttgart, Germany 294E was assessed by caspase 3 and granzyme B activity. Results: Serp-2 Heike Goebel, Department of Pathology, University of Freiburg, Germany blocked the apoptosis caused by camptothecin in T cells and monocytes and reduced the level of intracellular granzyme B in cytotoxic T lympho- Infl amm. res. Supplement 2 (2005) Tuesday 23 August 2005 S 109 cyte mediated apoptosis of T cells. Serp-2 showed no effect on FasL and merulonephritis it plays a pathogenetic role, probably by its potent mac- sturosporine induced apoptosis. Serp-2 showed association with T cells as rophage activating functions and by promoting class switching to “Th1” revealed by FACS analysis. Antibody to Granzyme B reversed the action IgG subclasses. However, in organ specifi c renal autoimmunity, while IL- of Serp-2 in granule-mediated but not in camptothecin induced apoptosis. 12p40 is pathogenetic, IFN-g is protective, probably due to its regulatory Also Serp-2 attenuated monocyte adhesion to endothelial cells. Conclu- effects on T cells. Future improved and more specifi c treatments for glo- sion: Serp-2 blocks apoptosis of monocytes and T cells, the immune cells merulonephritis are likely to require a more detailed understanding of the recruited in the early infl ammatory response, responsible for progression pathogenesis of different forms of the disease, mechanisms to switch off of plaque. Serp-2 mediates anti-apoptotic activity inpart through the gran- local infl ammation and treatments that selectively modify the underlying zymeB pathway. The present data suggests that apoptosis of immune cells abnormal nephritogenic immune response. may play a major role in infl ammation. 3039 3037 Mice Overexpressing Latent TGF-b1 Are Protected against Controlling rejection in xenotransplantation Crescentic Glomerulonephritis

Anthony J d'Apice, St Vincent's Health, Australia Xiao R Huang, Baylor College of Medicine, United States Allen G Li, Oregon Health & Science University, United States Xenotransplantation has made enormous progress in the last 15 years. Pre- Xiao J Wang, Oregon Health & Science University, United States viously, a transplant of a pig heart or kidney to a primate was rejected Hui Y Lan, Baylor College of Medicine, United States within minutes by a process called hyperacute rejection (HAR). This was overcome by genetic modifi cation of the pig donor by inserting human Mice Overexpressing Latent TGF-b1 Are Protected against Crescentic regulators of complement. The fi rst such transgenic pig expressing decay Glomerulonephritis. accelerating factor (CD55) was a remarkable success. Organs from CD55 Huang XR, Li AG, Wang XJ, Lan HY. Dept. of Medicine, Baylor College transgenics escaped hyperacute rejection in most (but not all) cases. This of Medicine, Houston, TX, and Dept. of Dermatology, Oregon Health & was subsequently improved by adding another complement regulator gene Science University, Portland, OR, USA. (CD46 or CD59), and HAR was overcome. Unfortunately, such organs TGF-b has been shown to play a critical role in suppression of immune re- were regularly rejected by a new and vigorous form of rejection called sponse. We reported here that mice overexpressing a human latent TGF-b1 acute vascular rejection (AVR). This was associated with anti-donor anti- on skin were protected against crescentic glomerulonephritis (GN) in an bodies and involved intragraft thrombosis at least at a microvascular level. accelerated model of anti-glomerular basement membrane (GBM) GN. At The next major development was the elimination of the Gal epitope. This is day 14 after i.v. injection of a sheep anti-mouse GBM Ab, wild-type (Wt) a blood group like terminal disaccharide (galactose- alpha-1,3 - galactose) mice developed crescentic GN, accounting for 25±1.4% of glomeruli with present on cell surface glycoproteins and glycolipids especially of endo- crescentic formation. In contrast, TGF-b transgenic (Tg) mice showed a thelial cells. The gene which encoded the pig galactosyltransferase which substantial inhibition of glomerular crescentic formation (9±4%, p<0.001 generates this epitope was knocked out by homologous recombination and vs Wt mice). Inhibition of crescentic GN in TGF-b Tg mice was associated cloning. Transplants of organs from Gal KO pigs were fi rst reported in with a signifi cant inhibition of macrophage and T cell accumulation in the Nature Medicine in January 2005. Like complement regulator transgenic diseased kidney (50-60 % reduced, p<0.01 vs Wt mice). Proteinuria was orgens, these avoided HAR but did not overcome AVR completely. It was signifi cantly reduced by more than 50% in TGF-b Tg mice compared to evident that microvascular thrombosis is still a signifi cant problem and Wt mice (p<0.001). Interestingly, while there was no difference in immune graft survival is insuffi cient to warrant clinical trials. Current efforts to deposition of sheep anti-mouse GBM Ab and mouse immunoglobulin in overcome the thrombosis related problems will be described including the glomeruli between Wt and Tg mice, deposition of C3 was signifi cantly generation of furtehr genetically modifi ed pigs. reduced in TGF-b Tg mice (50% reduced, p<0.001 vs Wt mice). Results from this study suggest that TGF-b may play a renoprotective role in a mouse model of crescentic GN. Inhibition of cellular immune response 3038 and complement-mediated renal injury may be the key mechanisms by which latent TGF-b1 prevents crescentic GN. Findings from this study Immune Modulation in Glomerulonephritis provide evidence that latent TGF-b1 may have therapeutic effect on cres- centic GN. Richard Kitching, Centre for Infl ammatory Diseases, Monash University, Australia

The glomerulonephritides are a common cause of end-stage renal failure in humans. They are a collection of heterogeneous diseases with variable manifestations and outcomes, that are characterised by infl ammatory in- jury of the glomerulus induced in most cases by abnormal or deregulated immune responses. The function of the glomerulus as a high fl ow, high pressure fi lter contributes to its vulnerability to different mechanisms of injury, including both cell mediated and humoral effectors. While it has long been recognized that humoral injury is important in a number of forms of glomerulonephritis, a signifi cant role for Th1 effector cells and macrophages in rapidly progressive forms of glomerulonephritis is emerg- ing. Using a planted antigen system, severe “crescentic” glomerular injury has been demonstrated to be a manifestation of a Th1 nephritogenic cell- mediated immune response directed by IL-12p40 with important effector contributions from GM-CSF, TNF, IL-1b and to some degree IL-18. This lesion can be negatively regulated by endogenous/exogenous IL-4 and IL- 10, but not by IL-13. Both immune cells and local renal cells contribute to injury. Costimulatory molecules are important in both the initiation of immune responses and the localisation of leucocytes within glomeruli. The role of IFN-g in glomerulonephritis is complex. In some forms of glo- S 110 Wednesday 24 August 2005 Infl amm. res. Supplement 2 (2005)

ACR 20=73.1 vs 39.7%; ACR 50=48.3 vs 18.2%; ACR 70=28.8 vs 6.1% 4001 (p<0.001 for all); 42.5 vs 9.9% of patients had low disease activity (Dis- ease Activity Score 28 [DAS28] ≤3.2) and 23.8 vs 1.9% were in remission Leukocyte traffi cking to infl ammatory sites- different mechanisms in (DAS28 <2.6), abatacept vs placebo. At 1 year, abatacept signifi cantly different organs inhibited the progression of erosions (p=0.029), joint space narrowing (p=0.009) and total score (p=0.012) vs placebo. Abatacept was generally Paul Kubes, University of Calgary Medical Center, Canada safe and well tolerated with a low incidence of infusion reactions and seri- ous infections vs placebo (3.9% vs 2.3%). The accepted paradigm for leukocyte recruitment includes a multi-step CONCLUSIONS: These data demonstrate the clinical benefi t of the selec- cascade including initial tethering of leukocytes to endothelium via selec- tive co-stimulation modulator abatacept in decreasing RA signs, symptoms tins followed by fi rm adhesion via integrins. However this work was pri- and disease progression in patients with an inadequate response to MTX. marily established in fl ow chamber assays in vitro and in post-capillary ve- nules of mesentery and cremaster muscle microvasculatures stimulated by various cytokines or chemokines. However, the microvasculatures of the 4006 lung, liver and brain differ signifi cantly from the cremaster muscle. In the case of the liver, shear forces are low and a signifi cant amount of recruit- Phase 1 clinical and biomarker experience with recombinant human ment occurs in the narrow sinusoids. To date a molecular mechanism for IL-18 (SB485232) neutrophil recruitment into these sinusoids has not been elucidated while novel adhesive mechanisms may be involved in lymphocyte recruitment Kevin Koch, GlaxoSmithKline, United States into sinusoids. Although endothelium is critical to leukocyte recruitment M Mohammed, United States into lung capillaries, the adhesion does not include selectins or integrins Linda M Thurmond, United States in situations like endotoxemia. Finally, in the brain microvasculature, the Paul Godilot, United States shear forces are very high and selectins appear to play a dominant role in Theresa M Conway, United States leukocyte recruitment. In addition, platelets also contribute to leukocyte Michael J Robertson, United States adhesion in the brain microvasculature in certain infl ammatory conditions. James W Mier, United States It is clear that the selectin-dependent and integrin-dependent leukocyte re- Henry Koon, United States cruitment is critical for leukocyte recruitment in some but not all organ John M Kirkwood, United States systems and evaluation of individual organs for leukocyte recruitment in infl ammation is absolutely necessary. IL-18 belongs to the Th1 family of cytokines. Recombinant murine IL- 18 has shown anti-tumor activity in mouse xenografts and recombinant human IL-18 is now in early phase clinical trials. Biomarker and clinical 4005 activity data from the fi rst two human trials of this immunotherapeutic agent will be described. Effi cacy and safety of the selective co-stimulation modulator In Study 1, 21 patients with renal cell carcinoma (RCC) and 4 with meta- abatacept with methotrexate for treating rheumatoid arthritis: static melanoma (MM) received a single cycle of 5 consecutive daily IV 1-year clinical and radiographic results from the Phase III AIM doses ranging from 3-1000 mcg/kg/2h. In Study 2, 6 patients with MM (Abatacept in Inadequate responders to Methotrexate) trial and 3 with RCC received repeated cycles of this regimen at 100, 500, and 1000 mcg/kg every 28 days. Tolerability, immunogenicity, pharmacoki- S Hall, Cabrini Medical Centre, Malvern, Victoria, Australia netics, cytokine and cell activation biomarkers, and preliminary anti-tumor P Hanrahan, Goatcher Clinical Research Unit, Royal Perth Hospital, activity were assessed to defi ne a biologically active dose range for Phase Perth, Australia II clinical testing. Plasma IL-18 and cytokine/protein biomarkers were TT Cheng, Chang Gung Memorial Hospital, Kaohsiung, Taiwan measured by ELISA or cytokine multiplex biochip on Day 1 (Study 1) and HY Lin, Veterans General Hospital, Taipei, Taiwan Days 1 and 5 of Cycles 1 and 2 (Study 2). Cell activation biomarkers were R Westhovens, Department of Rheumatology, Universitaire Ziekenhuizen measured by fl ow cytometry on Days 1-15 (Study 1) and 1-8 (Study 2). Leuven, Leuven, Belgium Plasma rhIL-18 was comparable between cycles, and daily dosing with L Moreland, The University of Alabama School of Medicine, Birming- a 36h half-life resulted in 2.5-fold accumulation. rhIL-18 clearance was ham, AL, United States time- and concentration-dependent due to induction of, and saturable P Emery, Department of Rheumatology and Rehabilitation, University of binding to IL-18 binding protein (BP). BP is a high-affi nity circulating Leeds, Leeds, United Kingdom modulator of rhIL-18 activity induced through IFN-gamma. Plasma con- A Russell, University of Alberta Hospital, Edmonton, Alberta, Canada centrations of INF-gamma, GMCSF, TNF-alpha, IP-10, MIG, and MCP-1 J Kremer, Center for Rheumatology, United States were modestly increased after dosing, but attenuated within and between cycles. Lymphopenia was apparent 6h after the fi rst dose in a cycle, co- PURPOSE: The AIM trial assessed the effi cacy and safety of the selec- incident with increased expression of FasL and CD69 on CD4, CD8, and tive co-stimulation modulator abatacept over 1 year in rheumatoid arthritis NK cells, and CD11b on NK cells and monocytes, which were not attenu- (RA) patients with an inadequate response to methotrexate (MTX). ated between cycles. Clinical activity was observed as tumor reduction in METHODS: AIM was a 1-year, randomized, double-blind, placebo-con- 3 patients, and suggested by prolonged stable disease (6-12 months) in 6 trolled, multicenter Phase III trial of a fi xed dose of abatacept approxi- patients. Biomarkers and clinical activity suggest bell-shaped dose- and mating 10 mg/kg plus MTX vs placebo plus MTX in patients with active concentration-response relationships. RA despite MTX treatment. Study medication was administered on Days rhIL-18 induces infl ammatory cytokines and cellular activation consistent 1, 15, 29 and every 28 days thereafter. Co-primary endpoints included with the limited Phase I clinical activity observed. This novel cytokine is American College of Rheumatology (ACR) 20 responses at 6 months and now undergoing Phase II evaluation for clinical effi cacy. structural damage progression (Genant-modifi ed Sharp score) at 1 year. Secondary endpoints included ACR 50 and 70 responses at 6 months and all ACR responses at 1 year. RESULTS: A total of 433 and 219 patients were randomized and treated with abatacept or placebo, respectively, with 385 (88.9%) of the abatacept group and 162 (74.0%) of the placebo group completing 1 year. Baseline characteristics were similar. At 6 months, ACR 20=67.9 vs 39.7%; ACR 50= 39.9 vs 16.8% and ACR 70=19.8 vs 6.5% for abatacept vs placebo (p<0.001 for all). At 1 year, ACR responses increased with abatacept: Infl amm. res. Supplement 2 (2005) Wednesday 24 August 2005 S 111

4007

BMS-561392 - A TNFalpha Convertase Inhibitor for Treating Rheumatoid Arthritis

James M Trzaskos, Bristol-Myers Squibb, United States

The clinical success of anti-TNFalpha biologics in treating rheumatoid arthritis and other infl ammatory diseases highlights the vital role of TN- Falpha in the pathology of these conditions. TNFalpha is initially synthe- sized as a 26-kDa membrane bound precursor form that is processed to a 17-kDa secreted, soluble cytokine. The enzyme responsible for this cleav- age is TNFalpha converting enzyme (TACE), a member of the ADAM (a disintegrin and metalloproteinase) family of protein processing enzymes (ADAM-17). We have identifi ed a series of TACE inhibitors that block TNFalpha processing and demonstrate anti-infl ammatory activity in pre- clinical models of arthritis. BMS-561392 is the most advanced member of this series and has entered clinical development for the treatment of rheumatoid arthritis. Phase I single ascending dose and multiple ascending dose studies have been completed in normal healthy volunteers. In addi- tion, a double blind placebo-controlled pilot study (n = 22) evaluating the safety and tolerability of BMS-561392 over 14 days in adult RA subjects has been conducted. The results of preclinical and clinical evaluation of BMS-561392 will be reviewed. General Poster Topics Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 115

conditions and cartilage damage. Triggering of TLRs leads to release of 5001 proinfl ammatory cytokines like IL-1b, a crucial cytokine in pathogenesis of RA driving cartilage and bone destruction. Therefore, TLRs may be NSAIDs and thiocyanate can each induce arthritis in rats (!) and involved in either initiation or chronicity of arthritis. In this study we in- prevent acquisition of tolerance vestigated the contribution of TLR2 and TLR4 in IL-1b-driven joint in- fl ammation, cartilage and bone erosion. C57BL/6 wild type (WT), BL/6 Michael W Whitehouse, Dept Medicine, University of Queensland, TLR2 knock-out (KO), C57BL/10 WT and BL/10 TLR4 KO mice were Brisbane, Australia intraarticularly injected with 3.106 PFU of mouse IL-1b adenoviral vector (AdmIL-1b) or control vector (Ad5del70-3). Infl ammatory cell infi ltration, Purpose: To evaluate effects of SCN- or NSAIDs on acquisition of toler- cartilage proteoglycan (PG) depletion and cartilage and bone destruction ance to 2 arthritigens in rats. were microscopically examined 7 days after injection. Prolonged expres- Background: Thiocyanate (SCN-) is the detoxifi cation product of HCN sion of mIL-1b in mice knee joint induced a severe arthritis in WT animals, present in tobacco smoke. NSAIDs may be continuously consumed for exhibiting pathophysiologic changes resembling those in human RA. High pain relief in arthritis. amounts of infl ammatory cells, predominantly polymorphonuclear cells, Methods: were present in the knee joint cavities of both WT and KO mice. Severe Stage 1: tolerance to a) mycobacterial arthritigens (MA) and b) cartilage PG-depletion, cartilage and bone destruction were observed in WT mice, collagen (C-II) was induced by replacing mineral oil in Freund’s adju- comparable with those in TLR2 KO mice. Interestingly, in TLR4 KO mice, vants with non-arthritigenic oils (NAO) e.g. phytol, isopropyl myristate, cartilage PG-depletion and destruction were signifi cantly reduced. TLR4 acetulan. Female Wistar rats were fi rst challenged with MA/NAO or C- KO mice showed also a marked reduction in bone erosion, although this II/NAO and any signs of arthritis recorded after three weeks. NSAIDs reduction was not signifi cant. These data strongly suggest that TLR4 is were administered for six days only beginning two days before inoculating involved in IL-1b- driven joint pathology. Both cartilage and bone destruc- tolerogenic formulations of MA or C-II. NaSCN- (1 mg/ml) was included tion were reduced in TLR4 KO mice, although joint infl ammation was in tap water for 14 days. comparable to WT mice. This indicates the involvement of potential TLR4 Stage 2: Non-arthritic animals were then rechallenged with either MA ligands, generated by IL-1b-induced cartilage degradation, in chronic RA. in squalane or C-II with Incomplete Freund’s adjuvant to probe whether Our data point out that TLR4 may be a novel therapeutic target in RA. animals had become tolerant/immuno-unresponsive. Results: Animals given NSAIDs (mg/kg) = aspirin (150 b.i.d.), ibuprofen (100 b.i.d.), celecoxib (20) or prednisolone (10) developed arthritis i.e. 5003 these drugs prevented acquisition of tolerance to both MA and C-II. By contrast untreated controls or animals given sodium salicylate (150 b.i.d), Transcriptional Activation of IGF-1 Production in Human Cartilage paracetamol (150 b.i.d.) or Lyprinol®(20) did not develop arthritis (stage Explants Concomitant with the Prevention of IL-1 induced 1) and remained tolerant (stage 2). Catabolism by a Natural Product Extract, IGFlex SCN- both a) induced arthritis at stage 1 and b) abolished acquired toler- ance when given only during stage 2 to untreated controls from stage 1 Mark JS Miller, Albany Medical College, United States i.e. no drugs. Salahuddin Ahmed, United States Conclusion: Paul J Bobrowski, United States 1. NSAIDs, steroids and smoking might each confound strategies to in- Tariq M Haqqi, United States duce selective tolerance to C-II, and perhaps other putative arthritigens, in patients with chronic arthritis e.g. RA. A combination of two natural products extract were tested individually 2. In future studies for inducing tolerance in patients, it may be best to re- and collectively, for their ability to directly promote the IGF-1 in human strict concurrent use of analgesics to those that don’t inhibit prostaglandin cartilage and limit IL-1 induced catabolism. Vincaria‚, is derived from synthesis within the immune system. cat’s claw (Uncaria guianensis) and is a well-described inhibitor of NF- kB. RNI 249, is derived from Lepidium meyenii, with ethnomedical ap- plications for fertility and vitality. Both are South American medicinal 5002 plants, and their combination is referred to as IGFlex. Human cartilage samples were procured from surgical specimens with consent, and were IL-1-driven cartilage and bone destruction are dependent on Toll-like evaluated as explants or as primary chondrocytes prepared after enzymatic receptor (TLR) 4, but not TLR2 digestion of cartilage matrix. Assessments: IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) Shahla Abdollahi, Rheumatology research laboratory and Advanced production, under basal conditions and in the presence of IL-1b. RNI 249 Therapeutics, University Medical Centre Nijmegen, The Netherlands enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, Leo AB Joosten, Rheumatology research laboratory and Advanced The- an effect that was further enhanced to 3.8 fold by vincaria co-administra- rapeutics, University Medical Centre Nijmegen, The Netherlands tion. Enhanced basal IGF-1 production by RNI 249 alone and together Mieke F Roelofs, Rheumatology research laboratory and Advanced The- with vincaria, was confi rmed in both explants and in primary chondro- rapeutics, University Medical Centre Nijmegen, The Netherlands cytes. IL-1b-induced cartilage matrix degradation was quantifi ed as GAG Monique M Helsen, Rheumatology research laboratory and Advanced release. RNI 249 or Vincaria almost completely prevented this catabolic Therapeutics, University Medical Centre Nijmegen, The Netherlands action. IL-1b exposure completely shutdown IGF-1 production in carti- Birgitte Oppers-Walgreen, Rheumatology research laboratory and lage, however both RNI 249 and Vincaria protected IGF-1 production in Advanced Therapeutics, University Medical Centre Nijmegen, The the presence of IL-1b, in an additive manner. Cartilage NO production was Netherlands dramatically enhanced by IL-1b, a response that both Vincaria and RNI Cora H Arndtz, Rheumatology research laboratory and Advanced Thera- 249 partially attenuated (25%). In summary, the identifi cation of agents peutics, University Medical Centre Nijmegen, The Netherlands that promote autocrine production of IGF-1 in cartilage, even in the face of Timothy RD Radstake, Rheumatology research laboratory and Advanced potent infl ammatory cytokine IL-1b exposure, has remarkable therapeutic Therapeutics, University Medical Centre Nijmegen, The Netherlands potential. When linked to the concomitant functional sequelae - prevention Wim B van den Berg, Rheumatology research laboratory and Advanced of the catabolic events - this approach has signifi cant potential in the treat- Therapeutics, University Medical Centre Nijmegen, The Netherlands ment of debilitating joint diseases.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by sys- temic, chronic joint infl ammation. Toll-like receptors (TLRs) have been reported to recognize several endogenous ligands produced under stress S 116 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

responses specifi ed as Th1-type. As interferon-gamma (IFNg)-secreting 5004 Th1 cells are suspected to promote autoimmune diseases, a shift of Th1 responses towards Th2 responses can be expected to ameliorate arthritis. Rat Models of Arthritis: Similarities, Differences, Advantages, and However, the results of therapy studies in human and experimental diseases Disadvantages in the Identifi cation of Novel Therapeutics remain confl icting. Therefore, we investigated the role of IFNg in murine antigen-induced arthritis (AIA), a model with homologies to human RA. Lisa R Schopf, Millennium Pharmaceuticals, United States Methylated bovine serum albumin(mBSA)-induced AIA was evaluated in Anneli Savinainen, Millennium Pharmaceuticals, United States IFNg-defi cient (IFNg-/-) or wild-type mice by clinical, histological, and Michelle DuPont, Millennium Pharmaceuticals, United States immunological parameters. Th cells isolated from immunized wild-type Julie Kujawa, Millennium Pharmaceuticals, United States mice were separated into IFNg+ and IFNg- Th cells (cell-surface expres- Matthew Silva, Millennium Pharmaceuticals, United States sion). These cells were then transferred into naive mice exposed in parallel Tim Ocain, Millennium Pharmaceuticals, United States to a single intraarticular injection of mBSA for arthritis induction. Further- Bruce Jaffee, Millennium Pharmaceuticals, United States more, the stimulatory capacity of IFNg+ and IFNg- Th cells was tested Karen Anderson, Millennium Pharmaceuticals, United States in vitro in cocultures with synovial fi broblasts. IFNg-/- mice developed a more severe arthritis than wild-type mice (signifi cantly higher knee joint Rat adjuvant (AA) and collagen-induced (CIA) polyarthritis models, and swelling, stronger infl ammation and joint destruction), with reduced IgG more recently the monoarticular streptococcal cell wall (SCW) model have levels, but increased cytokine production (IL-2, IL-4, IL-5) and enhanced been used extensively to evaluate novel therapeutic approaches to treat proliferation of lymph node cells upon antigen stimulation. Congruently, RA. All 3 models share mechanistic and pathologic features with each the transfer of IFNg- Th cells from immunized into naive mice led to a other as well as with human RA. Most notably, they are all T cell- and signifi cantly more severe arthritis than IFNg+ cells. In vitro, IFNg- Th macrophage-driven. All have been linked to pro-infl ammatory molecules cells had stronger stimulatory effects for MMPs and IL-6 in synovial fi bro- regulated by the NFkB pathway, such as TNFa, IL1b, IL6, COX2, iNOS blasts, whereas IFNg+ Th cells were more potent NO inducers. In conclu- and MMPs, and exhibit elevations in serum acute phase proteins. Differ- sion, the lack of IFNg in Th cells exerted higher disease-promoting effects ences in joint destruction, cytokine and infl ammatory mediator production in vivo as well as a higher stimulatory capacity to synovial fi broblasts in and paw edema between the animal models are mostly quantitative not vitro, suggesting a protective role of IFNg in the murine arthritis model. qualitative. As a general rule, the AA model is the most robust, followed by CIA and then SCW. However, the aggressive nature of all of these ani- mal models needs to be considered when comparing to human RA. The 5006 AA model has been the most extensively used, thereby having the largest database which aids in the prediction to human effi cacy, and its robust Extracellular reduction of T cells due to a mutation in Ncf1 increases nature allows for ease in compound ranking. CIA has an advantage due to arthritogenicity in rats the antibody component and the directed nature of the cartilage destruc- tion. The SCW model most closely resembles an arthritic fl are, has fewer Kyra A Gelderman, Dept of Medical Infl ammation Research, Lund Uni- systemic complications, and requires much less compound when given versity, Lund, Sweden therapeutically. We have characterized and compared these 3 animal mod- Malin Hultqvist, Dept of Medical Infl ammation Research, Lund Universi- els by examining paw swelling, NFkB-regulated gene expression in joints, ty, Lund, Sweden joint histopathological changes, serum acute phase proteins and biomark- Peter Olofsson, Dept of Medical Infl ammation Research, Lund Universi- ers of cartilage and bone integrity, and have used imaging technologies to ty, Lund, Sweden quantify bone destruction. Despite demonstrable differences between the Jens Holmberg, Dept of Medical Infl ammation Research, Lund Universi- models, we have found that the effi cacy of reference compounds such as ty, Lund, Sweden cyclosporine A and methotrexate is comparable in all three. Furthermore, Rikard Holmdahl, Dept of Medical Infl ammation Research, Lund Univer- therapeutic administration of our small molecule inhibitor of IKK2 gener- sity, Lund, Sweden ated similar ED50 values in all of these arthritic models. Through positional cloning of the PIA4 QTL in rats, we found that a poly- morphism in Ncf1 (part of the NADPH oxidase complex) increases ar- 5005 thritis severity. A mutation in mouse Ncf1 exerted similar effects. These mutations resulted in decreased production of reactive oxygen species Protective effects of interferon-gamma in experimental arthritis (ROS). Expression of Ncf1 and ROS production was found in APC’s but not in T cells. However, T cells from immunized mutated but not from Ingo Irmler, Institute of Pathology, Friedrich Schiller University Jena, wildtype rats could transfer disease to naïve rats. APC's might thus due Germany to lower ROS production, fail to suppress arthritogenic T cells. We found Marion Hückel, Institute of Pathology, Friedrich Schiller University that the extracellular (ec) redox status of T cells is important in determin- Jena, Germany ing disease severity. Both rats and mice expressing mutated Ncf1 have Jutta Simon, Institute of Pathology, Friedrich Schiller University Jena, higher ec redox levels of T cells, probably due to the lack of ROS. In T cell Germany proliferation assays with mouse APC’s and hybridoma T cells that were Mieczyslaw Gajda, Institute of Pathology, Friedrich Schiller University reduced with glutathione, it was shown that reduced T cells proliferate Jena, Germany better. Reduction of APC’s did not infl uence proliferation, indicating that Peter K Petrow, Institute of Pathology, Friedrich Schiller University reduction of T cells determined the effect. This was confi rmed in vivo: glu- Jena, Germany tathion reduced T cells from immunized wildtype rats injected in mutated Alexander Scheffold, German Rheumatism Research Centre Berlin, donor rats induced disease, in contrast to non-reduced T cells from the Germany same source. APC’s from wildtype or Ncf1 mutated mice evoked similar Andreas Radbruch, German Rheumatism Research Centre Berlin, Ger- levels of T cell proliferation indicating that the effect on T cell reduction many takes place earlier than at antigen presentation. To investigate the role of Rolf Bräuer, Institute of Pathology, Friedrich Schiller University Jena, thymic selection, a thymustransplantation from wildtype to Ncf1 mutated Germany rats was done and resulted in lower disease after immunization in contrast to control transplanted rats. These rats showed decreased T cell redox lev- In human rheumatoid arthritis (RA) interactions of immune cells with resi- els and increased ability to produce ROS. These results suggest that the dent cells (especially synovial fi broblasts) play a crucial role in the initia- redox status of T cells is determined by APC’s via ROS production and tion of infl ammatory and destructive processes in affected joints. Besides that this controls T cell autoreactivity. Extracellular redox levels may be macrophages, T-helper (Th) cells are predominant in cell infi ltrates, with Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 117 a feasible therapeutic target and we are currently testing several strategies kine, interleukin-4 (IL-4) on the OSM-induced MMP-13, aggrecanase and in these models. TGF-β1-stimulated TIMP-3 gene expression. METHODS: Bovine articular chondrocytes were pretreated either with IL- 4 alone for 60 min or stimulated further for 24 h with OSM or TGF-β. Total 5007 RNA was analyzed by Northern blot analysis for the MMP-13, ADAMTS- 4 and TIMP-3 levels. Phosphorylated levels of different signaling proteins HLA-DRB1 association in Saudi Rheumatoid Arthritis patients were analyzed by Western blotting. RESULTS: OSM induced and IL-4 suppressed MMP-13 and ADAMTS-4 Ramiz Al- Swailem, Department of Medicine, Armed Forces Hospital, gene expression. IL-4 inhibited basal and OSM-induced TIMP-3 RNA ex- Riyadh, Saudi Arabia pression. IL-4 slightly reduced OSM-stimulated phosphorylation of extra- Hannan Al-Rayes, Department of Medicine, Armed Forces Hospital, cellular signal-regulated kinases (ERKs), protein 38 (p38), c-Jun N-termi- Riyadh, Saudi Arabia nal kinase (JNK) and STAT-1. IL-4 also down-regulated TGF-β1-induced Samia Sobki, Department of Medicine, Armed Forces Hospital, Riyadh, TIMP-3 gene expression and reduced the phosphorylation of Smad2 and Saudi Arabia JNK without affecting that of ERK and p38. Misbahul Arfi n, Research Center, Armed Forces Hospital, Riyadh, Saudi CONCLUSIONS: IL-4 antagonizes the actions of OSM and TGF-β1. The Arabia observed cartilage-protective effects of IL-4 in arthritis animal models Mohammed Tariq, Research Center, Armed Forces Hospital, Riyadh, may be due to its coordinate inhibition of MMPs and ADAMTS-4 expres- Saudi Arabia sion. However, suppression of TIMP-3 by IL-4 suggests some caution for using IL-4 as antiinfl ammatory and cartilage protective therapy. Objective: Association of HLA-DRB1 alleles with rheumatoid arthritis and its implication in the aetiopathogenesis are found to be different in various ethnic groups. This study aimed to investigate HLA-DRB1 alleles 5009 in Saudi patients with rheumatoid arthritis (RA). Methods: The DRB1 region of major histocompatibility complex was Suppressor of Cytokine Signaling 3 (SOCS3) Negatively Regulates screened by polymerase chain reaction/ sequence specifi c primers (PCR/ Innate and Adaptive Immune Mechanisms in Acute IL-1-dependent SSP) in 70 RA patients and compared with 70 matched healthy controls. Infl ammatory Arthritis Results: Signifi cant differences were found in the frequencies of DRB1 *04 (33 vs 14, P=0.0007, RR= 3.56, EF= 0.5), DRB1 *08 (15 vs 1, Peter KK Wong, Reid Rheumatology Laboratory, Walter and Eliza Hall P=0.00012, RR= 18.81, EF= 0.8), DRB1 *10 (12 vs 5, P=0.0 7, RR= 2.68, Institute of Medical Research, Melbourne, Australia EF= 0.4) between RA patients and controls. Molecular subtyping of the Paul J Egan, Reid Rheumatology Laboratory, Walter and Eliza Hall most prevalent allele DRB1 *04 resulted into DRB1 *0401, *0401, *0402, Institute of Medical Research, Melbourne, Australia *040301, *040302, *0404, *0405, *0406, *0408, *0410, *0414, *0424 in Ben A Croker, Cancer & Haematology Division, Walter and Eliza Hall the patients group as compared to *0401, *0402, *040301. *0405, *0410 Institute of Medical Research, Melbourne, Australia in controls. Results indicted a positive association of RA with DRB1 Kristy O'Donnell, Reid Rheumatology Laboratory, Walter and Eliza Hall *0405 with signifi cant relative risk. A signifi cant decrease in frequency of Institute of Medical Research, Melbourne, Australia DRB1 *0403 (RR<0.25) in RA patients as compared to controls indicated Warren S Alexander, Cancer & Haematology Division, Walter and Eliza a negative association. Hall Institute of Medical Research, Melbourne, Australia Conclusion: These results of increased frequency of HLA-DRBI in RA Andrew W Roberts, Cancer & Haematology Division, Walter and Eliza patients suggested that DRB1 *04 could be susceptible to RA in Saudis Hall Institute of Medical Research, Melbourne, Australia like Americans, French, Spanish, Thai and Turkish. Among DRB1 *04, Ian P Wicks, Reid Rheumatology Laboratory, Walter and Eliza Hall subtype DRB1 *0405 showed positive association while DRB1 *0403 a Institute of Medical Research, Melbourne, Australia negative association with RA similar to Malay and Chinese RA patients. Purpose: To determine the role of endogenous SOCS3 in acute infl amma- tory arthritis. 5008 Methods: A conditional gene-targeting strategy allowed generation of viable mice Interleukin-4 Antagonizes Oncostatin M and Transforming Growth lacking SOCS3 in hematopoietic cells (SOCS3-/Dvav mice) by using the Factor Beta-Induced Responses in Articular Chondrocytes vav promoter to drive the Cre recombinase (1). Acute arthritis was induced by intra-articular injection of methylated bovine serum albumin (mBSA) Mohammed El MABROUK, Department of Medicine and Research Cen- into knee joints followed by subcutaneous IL-1 for 3 consecutive days. tre of CHUM Notre-Dame Hospital, Montreal Quebec, Canada Joints were harvested at day 7 to assess histologic severity of arthritis. Se- Hamid Yaqoob Qureshi, Department of Medicine and Research Centre of verity of mBSA/IL-1-induced arthritis was compared to that in WT mice. CHUM Notre-Dame Hospital, Montreal Quebec, Canada Serum levels of IL-6 and G-CSF were determined by ELISA. Draining Wen Qing Li, Department of Medicine and Research Centre of CHUM and non-draining LN T cells were stimulated with mBSA and pulsed with Notre-Dame Hospital, Montreal Quebec, Canada [3H]-thymidine. Supernatants were assayed for IL-17 by ELISA. LN cells, Judith Sylvester, Department of Medicine and Research Centre of CHUM splenocytes and synoviocytes from arthritic knee joints were stained for Notre-Dame Hospital, Montreal Quebec, Canada FACS analysis. BM macrophages were stimulated with IL-1 and superna- Muhammad Zafarullah, Department of Medicine and Research Centre of tants assayed for IL-6 by ELISA. CHUM Notre-Dame Hospital, Montreal Quebec, Canada Results: SOCS3-/Dvav (KO) mice had increased histological se- verity of mBSA/IL-1-induced arthritis (mean score KO:20±0.6 PURPOSE: The proinfl ammatory-antiinfl ammatory cytokines balance reg- vs WT:11.3±0.7;p<0.01) with enhanced bone destruction and in- ulates the matrix metalloproteinases (MMPs)/ aggrecanases (ADAMRS’s) creased periosteal osteoclasts (no. osteoclasts/joint KO:21.5±3.5 vs and tissue inhibitors of metalloproteinases (TIMPs)levels. Oncostatin M WT:1.3±0.9;p<0.05). Arthritic KO mice had increased neutrophils in (OSM) synergizes with other cytokines to stimulate cartilage catabolism in the synovium (CD11b+GR-1+ cells KO:44% vs WT:20%), peripheral arthritis by inducing MMPs. TGF-β1 stimulates cartilage remodeling and blood (KO:5.7±1.2 x103 cells/ml vs WT:0.8±0.2;p<0.001) and spleen promotes infl ammation. OSM and TGF-β1 also induce TIMP-3, an inhibi- (CD11b+GR-1+ cells KO:40% vs WT:10%). Arthritic KO mice had elevat- tor of aggrecanases and tumor necrosis factor converting enzyme (TACE), ed serum levels of G-CSF (KO:2215±480 pg/ml vs WT:1178±100;p<0.05) the principal proteases involved in arthritic infl ammation and cartilage and IL-6 (KO:50.4±19 pg/ml vs WT:2.6±2.6;p<0.05). KO T cells had en- degradation. We investigated the effects of the anti-infl ammatory cyto- hanced antigen-induced proliferation in draining LN (KO:132280±2935 cpm vs WT:1290±433;p<0.001) and non-draining LN (KO:20752±2190 S 118 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) cpm vs WT:737±148;p<0.001), and produced more IL-17 (KO:130±10 pg/ Purpose: Molecular fragments, such as glycosaminglycan peptides (GAG- ml vs WT:not detectable). KO macrophages had increased IL-6 production P) and polypeptides, derived from the breakdown of joint cartilage in os- following IL-1 stimulation (KO:938±31 ng/ml vs WT:195±77;p<0.05). teoarthritis and rheumatoid arthritis (RA) are antigenic and can stimulate Conclusion: These data provide direct in vivo evidence that endogenous an autoimmune responses and local infl ammation. Oral tolerisation of SOCS3 in hematopoietic cells is a critical negative regulator of multiple arthritic animals or RA patients with one of these cartilage components, cell types orchestrating infl ammatory arthritis. type II collagen (Col-II), has been shown to ameliorate the disease but References: similar studies on GAG-P have not been reported. Our earlier studies (1) Croker, BA et al. Immunity 2004; 20:1-20. had shown that Peptacan, a novel GAG-P complex isolated from tracheal cartilage was active prophylactically in a lapine model of infl ammatory arthritis. In the present report we confi rm this activity in rat models of 5010 arthritis but also demonstrate Peptacan's ability to prevent disease onset by oral tolerization. Activated protein C attenuates the production of matrix Methods: For comparative purposes we examined the anti-arthritic effects metalloproteinase-9 in monocytes from rheumatoid arthritic patients of Peptacan in two rat models of arthritis relative to three well known anti- via endothelial protein C receptor and inhibition of NF-kB arthritic agents, chondroitin sulfate (ChS), glucosamine (Gln) and bovine Col-II. Collagen-induced arthritis (CIA) and adjuvant-induced arthritis Meilang Xue, Sutton Arthritis Research Laboratory, University of Sydney (AIA) were established in Wistar rats (6-8 per group) using standard meth- at Royal North Shore Hospital, Australia ods. All drugs were administered orally (10-200mg/kg) either for seven David Campbell, Sutton Arthritis Research Laboratory, University of days prior to disease induction (tolerogenic protocol) or continuously for Sydney at Royal North Shore Hospital 15 days after injecting the arthritogen (prophylactic/therapeutic protocol). Lyn March, Department of Rheumatology, University of Sydney at Royal Joint swelling and arthritis scores were determined on days 15-18 and his- North Shore Hospital, Australia tological sections of joint tissues were assessed post-necropsy. Philip Sambrook, Department of Rheumatology, University of Sydney at Results: Peptacan at 20 mg/kg was active in preventing arthritis, syno- Royal North Shore Hospital, Australia vial infl ammation and joint cartilage breakdown in the rat CIA using the Chris Jackson, Sutton Arthritis Research Laboratory, University of Syd- tolerogenic or prophylactic protocols but only effective prophylactically in ney at Royal North Shore Hospital the AIA at 100mg/kg. These fi ndings were in contrast to the glycosamino- glycan, ChS and Gln which at doses up to 200mg/kg were inactive in both Purpose: To investigate the role of activated protein C (APC), a natural arthritis models. Col-II was also tolerogenic at 10mg/kg in the CIA, model anti-coagulant that plays a key role in preventing infl ammation, in the but unlike Peptacan it was also arthritogenic when injected systemically. regulation of matrix metalloproteinase(MMP)-9 production and nuclear Conclusion: This study has identifi ed the potential of Peptacan as an anti- factor (NF)-kB activity in monocytes isolated from rheumatoid arthritic arthritic agent that could be used to delay the onset of disease as well as (RA) patients and in a monocyte cell line. Methods: Human monocytes slow its rate of progression. isolated from peripheral blood from RA patients and Mono Mac6 cells, were cultured in RPMI 1640 with 1% MMP-free serum (basal medium) for 4 hours and then transferred to fresh basal medium and treated with re- 5012 combinant APC, endothelial protein C receptor (EPCR), MMP-9, protein C, tumor necrosis factor (TNF)-alpha, lipopolysaccharide (LPS) and NF- Rheumatoid arthritis disease severity is correlated with the genetic kB activation inhibitor. Culture supernatants were collected for zymogra- functional variants and circulating levels of Macrophage migration phy. Cells were harvested for reverse transcriptional PCR, western blot or Inhibitory Factor electrophoretic mobility shift assay (EMSA). Results: In both cell types, APC markedly reduced the production of MMP-9, whereas its precursor, Timothy R Radstake, Radboud University Nijmegen Medical Center, The protein C, had no effect on MMP-9. Interestingly, EPCR was expressed by Netherlands unstimulated monocytes from RA patients and was dose-dependently el- Fred C Sweep, Radboud University Nijmegen Medical Center, The evated in response to APC. Using a blocking antibody to EPCR we showed Netherlands that APC’s inhibition of MMP-9 was EPCR-dependent. Furthermore, our Paco Welsing, Radboud University Nijmegen Medical Center, The evidence indicated that APC inhibited MMP-9 production by blocking NF- Netherlands kB in RA monocytes and Mac 6 cells. First, an inhibitor of NF-kB reduced Barbara Franke, Radboud University Nijmegen Medical Center, The the production of MMP-9. Second, APC directly and dose-dependently Netherlands reduced NF-kB activity. Conclusions: This study has shown that APC pre- Sita H Vermeulen, Radboud University Nijmegen Medical Center, The vents MMP-9 production in monocytes by binding to the cell surface re- Netherlands ceptor, EPCR, and subsequently inhibiting intracellular activation of NF- Thierry Calandra, Centre Hospitalier Universitaire Vaudois, Switzerland kB. Our results suggest for the fi rst time that APC may be benefi cial in the Rachelle Donn, Arthritis Research Campaign Epidemiology Unit6, prevention of infl ammation and joint destruction in RA by blocking both United Kingdom MMP-9 and NF-kB activity. Piet L van Riel, Radboud University Nijmegen Medical Center, The Netherlands

5011 Objective: To study whether the genetic variants of the macrophage migra- tion inhibitory factor (MIF), MIF-173G>C and MIF-CATT5-8 alleles, are Arthritic disease suppression by oral tolerization with associated with disease severity and circulating MIF levels in patients with glycosaminoglycan peptide complexes derived from the cartilage rheumatoid arthritis (RA). extracellular matrix Methods: Genotyping was performed in RA patients participating in an early RA inception cohort and healthy controls. We compared demograph- Peter Ghosh, Institute of Nutraceutical Research, PO Box 35, Brookvale, ic data, disease activity and outcome measurements between patients with NSW 2100, Australia or without the MIF variants. Genotyping of the MIF-173G>C and MIF- Susan Shimmon, Institute of Nutraceutical Research, Box 35, Brookvale, CATT5-8 polymorphism was performed as described by Donn et al. An NSW 2100, Australia ELISA for human MIF was developed as described by Grebenschikov et Michael Whitehouse, Department of Medicine, University of Queensland, al. Allele and genotype distribution of the MIF-173G>C and MIF-CATT5- Brisbane , Q 4120, Australia 8 polymorphisms were tested with the Chi-square test. Multiple regression analysis was performed to assess the independence of the MIF functional genetic variants as riskfactors for radiological joint damage. Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 119

Results: 277 Caucasian RA patients and 277 healthy individuals were gen- is to determine the IL-18 protein concentrations in normal individuals and otyped for the MIF polymorphisms. Frequencies of genotypes and hap- lupus patients with different genotypes. Methods:Peripheral venous blood lotypes in patients and controls were similar. No statistically signifi cant samples were obtained from 61 patients with systemic lupus erythemato- differences in demographic or clinical features were observed between RA sus (SLE) and 33 healthy controls. Genomic DNA was extracted. Sequence patients carrying the MIF-173C and/or the MIF-CATT7 allele. In contrast, specifi c primer PCR and restriction fragment length polymorphism (RFLP) radiological joint damage was signifi cantly higher in patients carrying analysis were used to genotype the DNA samples for SNP-607. Levels of risk alleles of the MIF-173G>C or the MIF-CATT5-8 functional vari- bioactive IL-18 in plasma were measured using a commercial sandwich ants. Multivariate regression analysis revealed that the MIF-173CC and enzyme-linked immunosorbent assay. Results: Plasma Interleukin 18 con- MIF-CATT7/7 genotypes and carriership of one MIF-173C allele were centrations were signifi cantly higher in SLE patients than in control sub- independent prognostic variables. Carriership of the MIF-173C allele (P = jects. (p<0.05) . In patients group, the mean of the IL-18 concentrations for 0.002) or MIF-CATT7 (P = 0.004) allele was associated with signifi cantly 8 patients who had AA genotype at position -607 was 72.3pg/ml while for higher circulating MIF levels and were, in turn, correlated with levels of the 20 patients who had AC genotype was 222.1pg/ml. Those 33 patients radiological joint damage (r=0.64, P = 0.001). with CC genotype had a mean of 204.9pg/ml. Signifi cant difference was Conclusion: The MIF polymorphisms are not associated with RA suscep- observed in the three subgroups (p=0.013). In control group, 6 subjects tibility but are associated with high levels of radiological joint damage. with AA genotype at SNP-607 had signifi cantly lower IL-18 protein con- High circulating MIF levels correlate strongly with radiological joint dam- centrations when compared to the 10 subjects with AC genotype and 17 age suggesting that MIF expression is genetically determined and can be with CC genotype (p=0.007). The means for three subgroups are 63.7pg/ used as a novel prognostic tool in RA. ml, 162.4pg/ml and 115.1pg/ml respectively. Conclusion: The IL-18 pro- moter gene polymorphism SNP-607 C allele may result in the enhanced productions of IL-18 protein in SLE and normal individuals. 5013

Interleukin-18 promoter gene polymorphisms in Chinese patients 5015 with systemic lupus erythematosus : Association with CC genotype at position -607 Tweaking infl ammation: Targeting Tweak/Fn14 pathway in Rheumatoid Arthrisits Qian Xu, National University of Singapore, Singapore SK Tin, Singapore General Hospital, Singapore Timothy S Zheng, Biogen Idec, Inc., United States SP Sivalingam, National University of Singapore, Singapore Julian Thumboo, Singapore General Hospital, Singapore The TNF superfamily member Tweak and and its receptor Fn14 constitute Dow Rhong Koh, National University of Singapore, Singapore a noval pathway that regulates tissue infl ammatory responses following Kok Yong Fong, National University of Singapore, Singapore General physical and pathological injuries. In the current study, we demonstrated Hospital, Singapore that the macrophage-derived Tweak can contribute to joint tissue destruc- tion by two distinct mechanisms. First, Tweak can amplify the infl am- Aims: Interleukin-18 is a proinfl ammatory cytokine, which is postulated matory response through induction of cytokine/chemokine production in to play a role in systemic lupus erythematosus (SLE). Two single nucleo- Fn14-expressing resident cells in the joints such as the synovial fi broblasts tide polymorphisms (SNPs) in the IL-18 promoter gene region were found and chondrocytes, as well as altering the properties of local vasculature. to infl uence the quantitative expression of IL-18 protein. The aim of this Second, Tweak can directly trigger joint tissue damage through induction study is to determine whether IL-18 promoter gene polymorphisms are of MMPs from chondrocytes and by inhibiting regenerative response of associated with SLE. Methods: Eighty-two Chinese SLE patients and resident osteoid progenitor populations. Using HFLS culture as a model 295 Chinese healthy individuals were recruited. Genomic DNA was ex- system, we further showed that Tweak and TNF can synergize with each tracted from peripheral venous blood. Sequence specifi c primer PCR and other in driving the production of arthritogenic mediators such as IL-6, restriction fragment length polymorphism (RFLP) analysis were used to IL-8 and RANTES. Finally, we demonstrated that anti-Tweak blocking genotype the DNA samples for SNP-137 and SNP-607. The following antibodies are effi cacious in reducing disease severity in collagen-induced genotypes were obtained: SNP(-607) AA, AC, CC and SNP(-137) GG, arthritis in both mouse and rat without affecting the adaptive immune re- GC, CC. Results: The frequency of SNP-607/CC genotype was signifi - sponses, providing proof-of-principle for Tweak inhibition as a potential cantly higher in SLE patients (n= 30, frequency is 0.37) when compared to novel therapeutic strategy in human RA. control individuals (n=64, frequency is 0.22) (Pc < 0.05). Genotype SNP- 607/AC was signifi cantly decreased in SLE patients compared to control group (frequencies are 0.40 and 0.66 respectively, Pc < 0.05). Conclusion: 5016 SNP-607/CC genotypes of the IL-18 promoter gene polymorphisms is as- sociated with systemic lupus erythematosus. Markers of endothelial cells in chronically infl amed human tissues

Jim F Middleton, Endocube S.A.S., Prologue Biotech -BP-700, Rue Pier- 5014 re et Marie Curie, 31319 Labege Cedex,, France Laure Americh, Endocube S.A.S., Prologue Biotech -BP-700, Rue Pierre Enhanced production of Interleukin 18 protein is associated et Marie Curie, 31319 Labege Cedex,, France with SNP -607 C allele in normal individuals and systemic lupus Regis Gayon, Endocube S.A.S., Prologue Biotech -BP-700, Rue Pierre et erythematosus patients Marie Curie, 31319 Labege Cedex,, France Denis Julien, Endocube S.A.S., Prologue Biotech -BP-700, Rue Pierre et Qian Xu, National Unversity of Singapore, Singapore Marie Curie, 31319 Labege Cedex,, France SK Tin, Singapore General Hospital, Singapore Luc Aguilar, Endocube S.A.S., Prologue Biotech -BP-700, Rue Pierre et Julian Thumboo, Singapore General Hospital, Singapore Marie Curie, 31319 Labege Cedex,, France Kok Yong Fong, National University of Singapore, Singapore General Francois Amalric, Endocube S.A.S., Prologue Biotech -BP-700, Rue Hospital, Singapore Pierre et Marie Curie, 31319 Labege Cedex. Laboratoire de Biologie Vasculaire,, France Aim: Interleukin 18 (IL-18) is a proinfl ammatory cytokine, which strongly Jean-Philippe Girard, Endocube S.A.S., Prologue Biotech -BP-700, Rue induce IFN-γproduction. Two single nucleotide polymorphisms were de- Pierre et Marie Curie, 31319 Labege Cedex. Laboratoire de Biologie tected in the promoter region and previous studies have indicated one of Vasculaire, E, France the polymorphisms is associated with SLE. The aim of the present study S 120 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Endothelial cells play a central role in chronic infl ammation. For example free drug is only transiently effective, Micromethason leads to long-lasting they express adhesion molecules and present chemokines leading to en- suppression of arthritis. A 10-fold dose reduction is achieved by a lipo- hanced leukocyte recruitment into tissues. Numerous markers of endo- somal encapsulation of DxM-P. Micromethason does not only suppress thelial cells have been reported but there has been a lack of comparative clinical signs of arthritis, but also histological signs of joint infl ammation data on their specifi city. The present study compared the specifi city of and destruction. seven endothelial cell markers in the rheumatoid synovium and the colon of patients with Crohn’s disease. These markers were: the sulfated epitope MECA-79, the Duffy antigen receptor for chemokines (DARC), von Wil- 5018 lebrand factor, CD31 (PECAM-1), CD34, CD105 (endoglin) and CD146. Using immunohistochemistry the markers showing an entirely endothe- Cyclooxygenase-2 (COX-2) Activity is Regulated by Proteolysis lial cell distribution were: MECA-79, DARC and von Willebrand factor. MECA-79, which recognizes sulfated ligands for leukocyte adhesion re- Arturo Mancini, Department of Anatomy and Cell Biology, McGill Uni- ceptor L-selectin (CD62L), was selective for a subset of venules in highly versity, Canada infl amed tissue and was present in rheumatoid but not control osteoarthritic QingWen He, Royal Victoria Hospital, McGill University Health Centre, synovia. DARC was also specifi c for venules but had a more widespread Canada distribution than MECA-79, and was present in rheumatoid and control John A Di Battista, Department of Medicine, McGill University, Canada synovia. The other markers all labelled endothelial cells in venules, arte- rioles and capillaries. However they also localized to other cell types. For Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in induced example, CD34 stained fi broblasts, CD146 occurred in the pericytes and prostanoid biosynthesis. We uncovered a novel control mechanism in- smooth muscle cells of vessel walls and CD31 and CD105 labeled a broad volving limited proteolysis of the COX-2 protein that regulates intrinsic range of cell types. cyclooxygenase/peroxidase activities and ultimately the biosynthesis Of interest to histopathologists and cell biologists MECA-79, DARC and of prostaglandin E2 (PGE2). Interleukin-1ß (rhIL-1ß)-induced COX-2 von Willebrand factor were the best markers of endothelial cells, whereas protein synthesis peaked at 4 h and declined gradually accompanied by markers such as CD31, CD34, CD105 and CD146 were less specifi c. increased COX-2 proteolysis into immunoreactive fragments (i.e., 66, 42- 44, 34-36 and 28 kDa). Cleavage positions within COX-2 were confi ned to the catalytic domain as confi rmed by mass spectrometry (MALTI-TOF). 5017 Increased PGE2 release (i.e., COX-2 activity) was observed coincident with the timing and the degree of COX-2 proteolysis with regression anal- Micromethason - a liposomal glucocorticoid for intravenous ysis confi rming a linear relationship (R=0.96). Pharmacological inhibition treatment of rat antigen-induced arthritis (AIA) of induced COX-2 activity by NSAIDS abrogated proteolysis. ß-secretase and cysteine/calpain protease inhibitors also suppressed proteolysis, an ef- Una Rauchhaus, novosom AG, Halle, Germany fect accompanied by markedly reduced COX-2 activity and PGE2 release. Reiner Schulte, Experimental Rheumatology Unit, Friedrich Schiller Our results suggest that nascent COX-2 is initially in an enzymatically University, Jena, Germany latent form and must be activated by limited proteolysis for full biosyn- Raimund W Kinne, Experimental Rheumatology Unit, Friedrich Schiller thetic activity. University, Jena, Germany Rolf Bräuer, Institute of Pathology, Friedrich Schiller University, Jena, Germany 5019 Steffen Panzner, novosom AG, Halle, Germany Small molecule peptides directed at dual targets: sPLA2-MMP dual Background inhibitors as potential therapeutic option for arthritis and cancer Glucocorticoids are well established drugs for the treatment of infl amma- tory diseases. But they have a number of side effects, especially when used Gopalakrishnakone Ponnampalam, National University of Singapore, at high dosage. Singapore Liposomes can effectively target macrophages which are activated at sites Maung-Maung Thwin, Singapore of infl ammation and are thus tested as promising carriers. By encapsula- Kazuki Sato, Japan tion of dexamethasone-phosphate (DxM-P) in liposomes it is possible to avoid side effects and to enhance local therapeutic activity. The present study was aimed to design small molecule peptides with dual Methods inhibition properties against secretory phospholipase A2 (sPLA2) and Liposomes were prepared by fi lm extrusion method. matrix metalloproteinases (MMPs). This dual peptide inhibitor is a mu- For the in vivo studies we used the AIA model in rats. The arthritis was tant of the optimized anti-infl ammatory peptide P-NT.II that has proven induced by intraarticular injection of the antigen into the right knee joint. potential to suppress the infl ammatory and bone damaging components The left knee joint was used as a healthy control. Either free DxM-P or of rheumatoid arthritis (RA). Using solid-phase combinatorial chemistry, Micromethason was injected intravenously into rats 6, 24, and 48 hours P-NT.II variants were designed and examined for inhibition of sPLA2, a post induction. Effi cacy of treatment was evaluated by measuring the joint key enzyme involved in the infl ammatory pathway, and MMPs that are diameter of both knee joints and histopathological analysis. involved in the remodeling and degradation of the extracellular matrix in Results RA and cancer. 3H-labelled E coli membrane assay, BIOMOL AK-404 Whereas a rebound of arthritis was observed already two days following QuantiZyme Assay system and gelatin zymography were used respec- successful suppression of infl ammation with free drug, Micromethason tively for assaying sPLA2, collagenase and gelatinase enzyme activities. led to a long-lasting suppression of arthritic signs throughout the whole Quantitative Real Time RT-PCR and Western blotting were used for gene experiment and protein expression analyses. Among the peptide mutants examined, Additional a successful, complete suppression of arthritis throughout the the homodimer displayed potent inhibition against the enzyme activity of 21 day experimental period was also achieved only by 0,37mg/kg liposo- human recombinant synovial hrsPLA2 and hrMMP-1. The homodimeric mal DxM-P (10fold reduced dose). peptide markedly suppressed the enzyme activity of MMP-1, secreted from Microscopically analysis shows the high therapeutic potential of Micro- IL-1 β -stimulated rabbit synovial fi broblasts, while its inhibition of other methason - it almost completely suppressed signs of joint infl ammation secreted proMMP-2, MMP-2, proMMP-9 and MMP-9 was less marked. and destruction. mRNA and protein expression of MMP-1, MMP-2, MMP-9 and sPLA2 Conclusion were found to be signifi cantly decreased after treatment of the IL-1β -stim- Systemic intravenous application of water-soluble DxM-P in the liposome- ulated rabbit synovial fi broblasts with the dimerized peptide in culture. encapsulated form is highly effi cacious in suppressing rat AIA. Whereas The peptide acts against the gelatinases mainly through downregulation of Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 121 the expression of genes, while its action against collagenase 1 (MMP-1) on clinical or experimental primary infection in the absence of anti-vi- is through suppression of gene expression combined with enzyme inhibi- ral activity. Murine antigen-induced arthritis (AIA) was induced in C57bl tion. This optimized peptide analogue presents strong in vitro evidence mice by sensitisation and i.a. challenge with mBSA. diBGA and its anomer for control and modulation of enzyme activity and/or transcription of both diABGA were synthesised and anomeric ratios determined by NMR. Both MMP and sPLA2, and provides new therapeutic options in the treatment diBGA and the anomer diABGA (10uL, 1uM) i.a. prevented AIA at 24 of arthritis and cancer. hours (-0.32+/-0.06 & -0.47+/-0.06mm). di-ABGA at 100mg/kg reduced AIA when administered i.v. (-0.20+/-0.09mm, p<0.05) and i.p. (-0.34+/- 0.13mm, p<0.05), but is hydrolysed p.o. (0.24+/-0.08mm NS). Spleen 5020 cells were cultured from biozzi mice sensitised to mBSA and challenged in vitro. Cells were treated for 24 hours with or without drugs (1nM-100uM) Selective Activation of Mast Cells in Rheumatoid Synovial Tissue with cyclopsporin as positive control. Cell viabilities were confi rmed at Results in Production of TNF-alpha and IL-1beta each concentration using the MTT assay. diABGA (60% p<0.05, Emax 100uM) signifi cantly inhibited proliferation. diBGA inhibited IL2 (Emax Charlotta Sandler, Department of Medicine, Division of Rheumatology, 0.1nM, p<0.05), IFN (1uM, p<0.001), and IL10 (10uM, p<0.05)synthesis, Helsinki University Central Hospital, Finland whilst diABGA inhibted IL2 (0.01uM, p<0.05) and induced a mild NS Ken A Lindstedt, Wihuri Research Institute, Finland increase in IL10. diGA is a novel endogenous molecule that may play a Timo Juutilainen, Department of Orthopedic Surgery, Helsinki University role in the regulation of the immune response. The anomers may possess Central Hospital, Finland different properties and may provide a basis for a novel series of immuno- Petri T Kovanen, Wihuri Research Institute, Finland modulatory and anti-rheumatic agents. Kari K Eklund, Department of Medicine, Division of Rheumatology, 1)Roberts SM, Collins JE, Severin GA. Safe anti-infl ammatory therapy for Helsinki University Central Hospital, Finland feline herpesvirus conjunctivitis/keratitis. ACVO Conference, Scottsdale 1990. Background: In addition to allergic diseases, mast cells have been recently Funding & supply of diBGA and diABGA: Diosamine Development Corp, implicated as important effector cells in chronic infl ammation and par- 280 Madison Av, Suite 122, NY10016. ticularly in the pathogenesis of rheumatoid arthritis (RA). Mast cells are present in the synovial tissue and many of the proinfl ammatory mediators also produced by activated mast cells, such as TNF-alpha, play a pivotal 5022 role in the pathogenesis of RA. Aim: To evaluate the role of mast cells in the pathogenesis of RA, we GRC3886 - a selective PDE4 inhibitor with potential effect in have studied the effect of selective activation of synovial mast cells on the Rheumatoid arthritis production of TNF-alpha and IL-1beta in cultured human synovial tissue explants. Srinivas Gullapalli, Glenmark Research Centre, Plot:A-607, TTC indus- Methods: Synovial tissue was obtained during surgery from 15 RA pa- trial Area, MIDC, Mahape, Navi Mumbai-400709, India, India tients. The synovial tissue was cut into small pieces and synovial mast cells Vikas Karande, Glenmark Research Centre, Plot:A-607, TTC industrial were activated by adding anti-human-IgE antibodies. After 24 h the levels Area, MIDC, Mahape, Navi Mumbai-400709, India, India of TNF-alpha and IL-1beta were measured in the tissue culture media and Dipak Amrutkar, Glenmark Research Centre, Plot:A-607, TTC industrial the mRNA expression in the synovial tissue was determined. Tissue distri- Area, MIDC, Mahape, Navi Mumbai-400709, India, India bution of IL-1beta was studied using immunohistochemistry. Halina Offner, Oregon Health and Science University, Portland, Oregon, Results: Selective activation of synovial tissue mast cells by anti-IgE in- OR, USA, United States duced a signifi cant increase in the production of TNF-alpha (0.49 +/- 0.88 Shridhar Narayanan, Glenmark Research Centre, Plot:A-607, TTC vs. 4.56 +/- 3.18 pg/mg wet tissue, p = 0.00037)) and IL-1beta (0.058 industrial Area, MIDC, Mahape, Navi Mumbai-400709, India, India +/- 0.032 vs. 2.55 +/- 1.98 pg/mg wet tissue, p = 0.013) from explants to culture media during the 24 h culture. An elevation in mRNA levels of Rheumatoid arthritis (RA) is a chronic disabling disease involving infl am- TNF-alpha and IL-1beta was also observed after synovial mast cell activa- mation of synovial membranes of the joint, accompanied by erosion of tion. Double labeling immunohistochemistry showed that mast cell activa- cartilage and bone. The role of tumor necrosis factor-alpha (TNF-alpha), tion induces IL-1beta expression particularly in CD68 positive synovial a proinfl ammatory cytokine has been well established in the pathogen- macrophages. esis of RA. The involvement of phosphodiesterase IV (PDE IV) in the Conclusions: The obtained results strengthen the present concept that mast regulation of TNF-alpha is vindicated by the ability of rolipram to inhibit cells are involved in the pathogenesis of RA as their activation induced TNF-alpha production and ameliorate collagen-induced arthritis (CIA) in production and secretion of two key proinfl ammatory cytokines, TNF-al- rodents. GRC3886 is a novel PDE4 inhibitor with an IC50 value of 1.4 nM pha and IL-1beta in synovial tissue. Thus, mast cells could be considered for PDE4 enzyme inhibition and >7000 fold selectivity over other PDE as potential targets for novel antirheumatic treatments. (PDE 1 to 11) isoforms. GRC3886 inhibited lipopolysaccharide (LPS) - in- duced serum TNF-alpha production with an ED50 of 0.52 mg/kg (p.o.) in Sprague-Dawley (SD) rats. GRC3886 (0.1 - 10 mg/kg, p.o.) dose depend- 5021 ently reversed Freund’s Complete Adjuvant (FCA) - induced acute hyper- algesia in SD rats with an ED50 of 0.85 mg/kg (p.o.). It also produced Inhibition of antigen-induced arthritis by diglucosylamine, an signifi cant amelioration of CIA in DBA/1J mice at a dose 10 mg/kg (p.o.) immunomodulator identifed from spleen comparable to that produced by valdecoxib at 1 mg/kg (p.o.). Our data suggests that GRC-3886, a potent selective PDE IV inhibitor that could be Christopher Bolton, Wlliam Harvey Research Institute, United Kingdom a promising candidate for the treatment of rheumatoid arthritis. Seema Trivedi, Wlliam Harvey Research Institute, United Kingdom Ella Johnson, Wlliam Harvey Research Institute, United Kingdom Elizabeth G Wood, Wlliam Harvey Research Institute, United Kingdom 5023 Michael P Seed, Wlliam Harvey Research Institute, United Kingdom Role of macrophage migration inhibitory factor in development of Extracts of spleen have been identifi ed to modulate immune responses to spontaneous autoimmune disease in MRL/lpr mice antigen and to prevent anaphylaxis. Chemical isolation and purifi cation, TLC and NMR analysis has identifi ed di-B,B, glucosylamine (diBGA, Alberta Y Hoi, Monash University, Australia Diosamine) as a principle. diBGA (batch I-19) ameliorated secondary Eric F Morand, Monash University, Australia activation of clinical feline herpetic keratitis (1), whilst having no effect S 122 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Michael J Hickey, Centre for Infl ammatory Disease, Monash University, Australia 5025

Macrophage migration inhibitory factor (MIF) is a broad-spectrum proin- The in vivo biology of the TGF-b superfamily cytokine MIC-1 and its fl ammatory cytokine implicated in many immune-infl ammatory phenom- role in mouse models of rheumatoid arthritis ena such as rheumatoid arthritis and atheroma. MRL/lpr mice develop a spontaneous autoimmune disease, which greatly Heiko Johnen, Centre for Immunology, St. Vincent's Hospital and Univer- resemble human systemic lupus erythematosus. Our aim was to assess sity of New South Wales, Sydney, Australia, Australia the role of MIF in disease development in lupus-prone MRL/lpr mice. In Tamara Kuffner, Centre for Immunology, St. Vincent's Hospital and Uni- MRL/lpr mice, MIF was expressed at high levels in the kidney, where it versity of New South Wales, Sydney, Australia, Australia was most highly expressed in tubular epithelial cells. Furthermore, renal Andrew D Cook, Arthritis and Infl ammation Research Centre, Depart- MIF expression was signifi cantly elevated in the MRL/lpr mice relative to ment of Medicine, Royal Melbourne Hospital, and CRC for Chronic non-diseased control MRL+/+, mice at 12 and 16 weeks, timepoints which Infl ammatory, Australia predate the onset of proteinuria. To examine the effect of MIF on lupus Emma L Braine, Arthritis and Infl ammation Research Centre, Depart- development, we generated MRL/lpr mice with a targeted disruption of ment of Medicine, Royal Melbourne Hospital, and CRC for Chronic the MIF gene by eight generations of backcrossing MIF-defi cient B6.129S Infl ammatory, Australia mice with MRL/lpr mice, and compared them to MIF+/+MRL/lpr lit- John A Hamilton, Arthritis and Infl ammation Research Centre, Depart- termates. MIF-defi cient MRL/lpr mice exhibited signifi cantly reduced ment of Medicine, Royal Melbourne Hospital, and CRC for Chronic incidence of proteinuria (25%) compared to MIF+/+ littermates (100%) Infl ammatory, Australia (p<0.001). Proteinuria severity and skin lesion incidence were also reduced Samuel N Breit, Centre for Immunology, St. Vincent's Hospital and Uni- in MIF-defi cient MRL/lpr mice relative to MIF+/+ mice. Finally MIF-de- versity of New South Wales, Sydney, Australia, Australia fi cient MRL/lpr mice demonstrated signifi cantly improved survival, with median survival of 33 weeks compared to 22 weeks in MIF+/+ littermates MIC-1 (macrophage inhibitory cytokine) is a divergent member of the (p<0.05, female mice). These data suggest MIF may play an important TGF-b superfamily, fi rst cloned by us on the basis of increased expression role in the pathogenesis of systemic infl ammation in lupus-prone mice, during macrophage activation. As well as by macrophages, MIC-1 is pro- and for the fi rst time, suggest that inhibition of MIF may be a therapeutic duced in large amounts by placental trophoblasts many cancers and cancer target in SLE. cell lines. MIC-1 is detected in the serum of all subjects and serum levels are elevated in pregnancy, chronic infl ammatory processes such as RA or atherosclerosis, or in cancer. Variation in serum MIC-1 levels is a strong 5024 independent predictor of the risk of cardiovascular events such as heart at- tack. Tumor production of MIC-1 results in elevation of serum MIC-1 lev- Expression of infl ammatory and fi brotic genes in Scleroderma and els especially in patients with prostate, pancreatic, breast or colon cancer, Morphea The MIC-1 promotor is a target for the p53 tumor suppressor gene. The biological function of MIC-1 is not fully understood, however it has been Wendy A Gold, Centre for Immunology, St Vincents Hospital, Sydney, suggested to be proapoptotic, antitumorigenic and to modulate tumor cell Australia or macrophage function in an autocrine or paracrine manner and thus regu- Kristina Warton, Centre for Immunology, St Vincents Hospital, Sydney, late infl ammatory and malignant processes. Australia To help defi ne the role of MIC-1 in infl ammatory diseases like arthritis, Natasha C Foster, Centre for Immunology, St Vincents Hospital, Sydney, we have generated transgenic mice (C57/B6) that overexpress MIC-1 un- Australia der the control of the c-fms (CSF-1 receptor) promotor and constitutively Keith K Stanley, Centre for Immunology, St Vincents Hospital, Sydney, produce large amounts of MIC-1, which is detectable in macrophages and Australia serum. These mice are healthy and breed normally. Transgenic animals are equal- Scleroderma is a fi brotic disease characterised by endothelial cell infl am- ly susceptible as wild type controls to immune complex-mediated arthritis mation, microvascular damage, organ fi brosis and autoimmune abnormali- induced by the passive transfer of serum from arthritic mice (K/B x N se- ties. It is divided into two clinical entities-localized and systemic. Local- rum transfer model). However, transgenic animals are resistant to collagen ized scleroderma such as morphea, affects only the skin whereas systemic induced arthritis, compared with WT controls. scleroderma is a multi-system disease involving the skin, blood vessels These fi ndings suggest an important role of MIC-1 in the regulation of and other major organs. Purpose: It is yet unknown how endothelial cells induction of humoral or cellular immunity involved in the pathogenesis of are involved in the pathogenesis of scleroderma. Therefore our aim was infl ammatory diseases like arthritis. to identify (1) if endothelial cell mediated infl ammation occurs at an early stage of the disease and (2) if this infl ammatory state correlates with the expression of infl ammatory genes in endothelial cells. Methods: 4mm skin 5026 punch biopsies were taken from morphea and scleroderma patients from three clinically different areas : an affected area (mimicking late stage of MIF enhancement of RA FLS survival is associated with p21 disease), the leading edge of the affected area (mimicking early or initiat- induction and nuclear localisation ing stage) and an unaffected area (control or no disease). The RNA was extracted and RT-PCR was performed. Results: High expression of E-se- Elliott Taranto, Monash University, Australia lectin, as measured by RT-PCR, indicated high levels of endothelial cell Jin R Xue, Australia mediated infl ammation in affected areas of morphea patients. In contrast, Derek Lacey, Australia low E-selectin expression was found in the affected areas of scleroderma Paul Hutchinson, Austria patients. In addition we found that two novel genes, NLF1 and NLF2, Eric F Morand, Monash Institute of Medical Research, Monash Univer- which were recently discovered during a genome wide micro array study, sity, Australia and identifi ed to be endothelial cell specifi c and upregulated by infl amma- Michelle Leech, Centre for Infl ammatory Diseases, Monash University tion in vitro, were also upregulated in affected areas of morphea patients. Department of Medicine,Monash Institute of Medical Research, Australia Conclusion: Endothelial cell mediated infl ammation, as measured by E- selectin and NLF1 and NLF2, occurs in the affected areas of morphea pa- BACKGROUND & AIMS: RA is associated with expansion of synovial tients but not in the affected areas of systemic scleroderma patients. This tissue which promotes joint destruction. The proinfl ammatory cytokine suggests a possible role of the novel genes in scleroderma and the presence MIF is overexpressed in RA cells and tissues compared with OA. Recently of endothelial cell mediated infl ammation in the early stages of morphea. MIF has been shown to modulate expression of the tumour suppressor Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 123 gene p53. p21 is a downstream target of p53 and induces G1 cell cycle arrest in response to cellular stress. Recent studies have suggested uncon- 5028 ventional new roles for p21 in cell survival. This study aims to examine the capacity of MIF to modulate the expression and cellular localisation Vascular abnormalities are present in recent onset rheumatoid of p21, and to assess whether this correlates with changes in proliferation arthritis / apoptosis. METHODS: Expression of p21 was examined in FLS and murine dermal Suad M A Hannawi, Centre for Immunology and Cancer Research and fi broblasts (MDF) by permeabilisation fl ow-cytometry, western blotting, Department of Medicine, University of Queensland, Princess Alexandra immunofl uorescence and RT-PCR. Proliferation was analysed using 3H/ Hospital, Br, Australia thymidine incorporation. Apoptosis was examined using cytometric detec- Thomas Marwick, Centre for Immunology and Cancer Research and tion of annexinV/PI labelling. Department of Medicine, University of Queensland, Princess Alexandra RESULTS: Increased p21 protein expression was observed in cells ex- Hospital, Br, Australia pressing endogenous MIF including RA FLS and wtMDFs. This was con- Ranjeny Thomas, Department of Medicine, University of Queensland, trasted with signifi cantly lower levels of p21 expression in OA FLS and Australia MIF-/-MDF respectively. Prominent nuclear localisation was observed in cells expressing MIF. rMIF treatment of cells resulted in increased p21 ex- Back ground and purpose pression and nuclear localisation. RA FLS and wtMDF exhibited reduced Rheumatoid arthritis (RA) has a persistently high level of infl ammation, apoptosis, and increased proliferation compared with OA FLS and MIF-/- associated with reduced life expectancy principally due to cardiovascular MDF. rMIF Treatment of wtMDF further reduced levels of apoptosis. disease. The development of B-mode ultrasound allows non-invasive in- vestigation of early atherosclerotic changes through estimation of the ca- CONCLUSIONS: MIF induces expression and nuclear localisation of p21 rotid intimal-medial thickness (IMT). IMT has been shown to be increased which is associated with a hypoapoptotic phenotype. Increased p21 ex- in patients with established RA compared with controls, even when ac- pression may therefore contribute to synovial expansion in RA by enhanc- counting for usual cardiovascular risk factors. To determine whether in- ing cell survival. creased atherosclerotic disease is already evident at presentation of RA, we studied carotid IMT in RA patients presenting within 12 months of symptom onset. 5027 Methods Ultrasound scans were performed on 22 RA patients and 65 control sub- NF-kappaB1 (p50) is a critical requirement for CD4 T cell function jects without cardiovascular or rheumatological diseases. The RA patients in acute infl ammatory arthritis were recruited in an early RA clinic setting, and were all diagnosed with defi nite RA with symptoms of less than 12 months. The right and left Ian K Campbell, The Walter and Eliza Hall Institute of Medical Research, common carotid arteries were examined in anterior, lateral, and posterior Australia planes. Average IMT was calculated in each individual. Kristy O'Donnell, The Walter and Eliza Hall Institute of Medical Re- Results search, Australia Average IMT was signifi cantly thicker in RA patients than the control sub- Anne-Marie van Nieuwenhuijze, The Walter and Eliza Hall Institute of jects. Results Medical Research, Australia were 0.66 ± 0.14 mm (0.44-0.87 mm) in the RA patients, and 0.56 mm Steve Gerondakis, Australia ± 0.07 mm (0.45-0.78 mm) in the control group (P = 0.006).In male RA Ian P Wicks, The Walter and Eliza Hall Institute of Medical Research, patients mean IMT (0.7 ± 0.13 mm, range 0.47-0.87 mm) was signifi cantly Australia higher than male controls (0.57 ± 0.07 mm, range 0.49-0.72 mm, p=0.001). There was no signifi cant difference in IMT of female RA patients when Purpose: The Rel/NF-kappaB transcription factor family is critical in regu- compared to female controls (p=0.09). lating genes that contribute to the joint infl ammation and tissue destruction Conclusion of rheumatoid arthritis. We previously reported that the Rel/NF-kappaB We show for the fi rst time that patients, especially males, presenting with transcription factors have distinct roles in a model of acute arthritis that is early RA already have an increased atherosclerotic burden. Since small dependent on IL-1 and CD4 T cells. Mice defi cient in NF-kappaB1 (p50-/-) elevations in CRP predate onset of RA (Nielen et al, Arthritis Rheum 2004, were protected from disease whereas c-Rel-defi cient mice were not. Here, 50:2423-27), the data suggest that the accelerated atherogenic process we investigate the function of NF-kappaB1 in the acute arthritis model. which characterizes RA might precede disease onset. Methods: Acute arthritis was induced in mice by intra-articular injection into the knee with methylated BSA followed by 3 daily sub-cutaneous injections of IL-1. Joints were examined histologically on day 7. Bone 5029 marrow (BM) chimeric mice were created by injection of wild-type (WT) or p50-/- BM into lethally irradiated p50-/- or WT recipient mice, respec- Transforming Growth Factor Beta Inhibits Osteoclast Formation Via tively. CD4 RAG-chimeric mice were made by injection of CD4 T cells The Down Regulation Of c-Fos (WT, p50-/- or IL-1R-/-) into RAG-/- mice. Results: BM chimeric mice surprisingly showed that NF-kappaB1 is re- Roya Lari, CRC for Chronic Infl ammatory Diseases, University of Mel- quired by BM-derived cells but not by stromal cells for induction of ar- bourne, Australia thritis. To further identify which cells require p50 in the immune response, Peter Kitchener, The University of Melbourne, Australia mixed lymphocyte reactions were performed. p50-/- antigen presenting John A Hamilton, CRC for Chronic Infl ammatory Diseases, University of cells (APC) had normal function whereas p50-/- CD4 T cells were defi - Melbourne, Australia cient in the proliferative response to WT APC. To specifi cally evaluate the function of p50-/- CD4 T cells in the acute arthritis model, CD4 RAG Bone loss is one of the major pathological symptoms of infl ammatory ar- chimeric mice were created. p50-/- CD4 T cells could not transfer disease thritis, and is the consequence of osteoclast induction, differentiation and susceptibility to lymphocyte-defi cient RAG-/- mice, whereas WT and IL-1 activation. Transforming growth factor-β (TGF-β) is an abundant growth receptor-defi cient CD4 T cells could. Finally, disease susceptibility could factor in bone that has a variety of immunoregulatory and anti-infl amma- be restored to p50-/- mice by transfer of WT CD4 T cells. We are currently tory activities. To gain better understanding of the role of TGF-β in osteo- investigating p50-/- CD4 T cell function and survival. clastogenesis, we have investigated direct effects of TGF-β on osteoclast Conclusions: NF-kappaB1 is required by CD4 T cells for induction of IL-1 differentiation by M-CSF and RANKL from bone marrow derived macro- dependent acute arthritis. This pathway does not involve the IL-1 receptor phages in vitro. TGF-β signifi cantly suppressed the proliferation of osteo- on T cells. clast precursors (p <0.01) and also inhibited their development from bone S 124 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) marrow precursors in a dose dependent manner. At the high concentration Objective. To investigate the effect of ribbon (circular) type NFkB decoy (10 ng/ml), TGF-β reduced the number of TRAP+ cells (p<0.01) and also ODN (RNODN) on induction and activity of osteoclasts. down regulated expression of the osteoclastic genes CTR and Cath K, as Methods. We collected bone marrow cells from the femur of rats and non measured by Q-PCR. Although, TGF-β did not affect expression of the adherent cells were incubated with RANKL (100ng/ml) and M-CSF(20ng/ RANK and c-fms genes, it suppressed the induction of the c-fos gene (an ml). First, transfection effi ciency into osteoclasts, resistance to exonucle- essential transcription factor for osteoclast differentiation) by M-CSF and ase, binding activity of decoy to NFkB were examined. Next, to examine RANKL. GM-CSF, IL-10 and interferon receptor 1and 2 knock out mice the effect of RNODN on induction and activity of osteoclasts, cells were were also used to investigate the involvement of these factors in the in- incubated with or without decoy and TRAP staining was performed on day hibitory effect of TGF-β. No signifi cant effects were observed with cells 7 and the number of TRAP positive multinucleated cells were counted. To from any of these mice compared with those from wild type mice. Our study the effect of RNODN on the bone resorbing activity of mature osteo- data suggests that TGF-β directly act as a negative regulatory factor of clasts, we performed the bone-resorbing assay. Non adherent bone marrow osteoclast differentiation by two different mechanisms. Firstly, by reduc- cells were cultured on Osteologic Bone Cell Culture System. After the ing the number of osteoclast precursors and secondly by down regulation mature osteoclasts induction, (on day 5) the cells were incubated with de- of c-Fos expression. coy and on day 10 calcifi ed matrix resorption area on each disc was mea- sured. Finally, RNODN was injected into the ankle joints of CIA(Collagen Induced Arthritis) rats and 4 weeks later joint destruction and osteoclast 5030 activity were examined. Results: RNODN showed about 80% of transfer effi ciency into osteoclasts. Plasma and Synovial Fluid Cytokines in Juvenile Arthritis (JIA) It was almost same compared to double stranded phosphorothioated NFkB decoy ODN (PNODN). RNODN showed higher resistance to exonuclease Anthea M Downs, PathCentre, Perth, Australia and higher binding activity to NFkB. The total number of TRAP positive Kevin Murray, Princess Margaret Hospital for Children, Australia multinucleated cells was signifi cantly decreased in RNODN treated group. The average calcifi ed matrix resorbed area was signifi cantly decreased JIA is a chronic infl ammatory disease of the joints with onset of symptoms in RNODN treated group. Histological study of the ankle joints showed before 16 years. JIA has been divided into 7 disease groups to facilitate signifi cant suppression of joint destruction and osteoclast induction in treatment and provide a framework for communication and research. JIA is RNODN injected rats. characterised by infi ltration of activated leucocytes into the synovium and Conclusion: These data suggest the inhibitory effect of ribbon type NFkB the associated production of infl ammatory cytokines. Contrasting cytokine decoy ODN on induction and activity of osteoclasts. profi les have been reported in the SF in different JIA subtypes and may be of assistance in classifi cation. Purpose:Pro-infl ammatory cytokines IL-1b, IL-6 and TNFa and anti-in- 5032 fl ammatory cytokine IL-10 were measured in matched SF and plasma from children with JIA to look for associations with disease subtype and Antiinfl ammatory activity of novel dibenzo(e,h)azulene compounds severity. for the treatment of rheumatoid arthritis Cohort:12 females, 6 males (mean 7.3 yrs). 9 oligoarthritis, 3 ext. oligoar- thritis, 2 polyarthritis, 2 systemic arthritis and 2 ERA. SF and peripheral Barbara Stanic, PLIVA Research Institute Ltd., Zagreb, Croatia blood samples were collected at presentation at the rheumatology clinic Dijana Pesic, PLIVA Research Institute Ltd., Zagreb, Croatia with a swollen knee joint. Ivana Ozimec-Landek, PLIVA Research Institute Ltd., Zagreb, Croatia Results: IL-10 was not detectable in any SFs. Plasma TNFa and IL-1b Rudolf Trojko, PLIVA Research Institute Ltd., Zagreb, Croatia levels were highly correlated with each other and between SF and plasma. Renata Rupcic, PLIVA Research Institute Ltd., Zagreb, Croatia IL-6 levels were higher in SF than plasma suggesting a higher rate of pro- Marina Modric, PLIVA Research Institute Ltd., Zagreb, Croatia duction in the synovium. No clear correlations between cytokine levels Milan Mesic, PLIVA Research Institute Ltd., Zagreb, Croatia and JIA subtype were found. JIA patients were divided into severe (sys- Mladen Mercep, PLIVA Research Institute Ltd., Zagreb, Croatia temic, poly) and mild disease (oligo, ext. oligo, ERA) All four patients with severe disease had high IL-6 levels and 3/4 expressed IL-10. The four Purpose: Discovery of effective, orally acting, small molecular weight in- patients with the lowest levels of all four cytokines had mild disease. hibitors of TNFalpha production for the treatment of RA and other diseases Conclusions: Patterns of TNF and IL-1 levels suggests co-regulated ex- in which TNFalpha plays important role in pathogenesis. pression. Plasma and SF TNF and IL-1 levels were highly correlated Methods: Synthesis of new dibenzo(e,h)azulenes and their testing in sever- therefore expression of these cytokines in the joint could be monitored al infl ammatory models (LPS induced TNFalpha production, LPS induced by a blood test. High IL-6 levels in the SFs concurs with previous reports shock, writhing and hot plate models of analgesia, carrageenan induced and implicates activated monocytes as crucial infl ammatory mediators in paw edema and adjuvant induced arthritis model). the synovium. Although elevated cytokine production is clearly associated Results: Members of different classes of dibenzo(e,h)azulenes inhibit TN- with JIA, great variability exists between individuals limiting clinical use. Falpha production in vitro in low micromolar range. Selected compounds Serial measurements of cytokine levels within an individual may indicate administered per-orally showed potent inhibition of LPS induced TN- or predict infl ammatory activity. Further studies are required to verify the Falpha secretion in vivo. They also exhibit excellent activity in different usefulness of cytokine measurements in disease monitoring in JIA. acute infl ammatory models such as LPS induced shock and carrageenan induced paw edema. Activity of some dibenzo(e,h)azulene compounds was confi rmed in adjuvant induced arthritis model in rats when they had 5031 been dosed perorally in established phase of the disease. Effects included not only decrease in degree of infl ammation but also remarkable attenua- Inhibitory effect of Ribbon (Circular) type NFkB decoy tion of degenerative processes reversing histopathological changes that are oligodeoxynucleotides on osteoclastogenesis characteristic for RA. In addition, these compounds have analgesic activ- ity comparable to that of acetylsalicylic acid when tested in the writhing Yasuo Kunugiza, Osaka University, Japan model of analgesia. Tetsuya Tomita, Osaka University, Japan Conclusions: Good toxicology profi le, lack of genotoxicity and good safe- Naruya Tomita, Osaka University, Japan ty profi le indicate that dibenzo(e,h)azulene class of orally available TN- Ryuichi Morishita, Osaka University, Japan Falpha inhibitors could bring disease modifying activity and symptomatic Hideki Yoshikawa, Osaka University, Japan relief to RA patients. Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 125

comprising of lymphocytes, occasional plasma cells ,few macrophages, 5033 mast cells. Scanty/ Sparse in12/20 cases ,moderate in 8/20.(b)Immunohis tochemistry- All lymphocytes expressed CD4,CD8,CD11a,11b,11c,CD18 The induction of apoptosis may contribute to the immuno- and L- selectin. Intense expression of ICAM-1,VCAM-1 by valvular en- modulatory actions of 15 deoxy Δ 12-14 PGJ2 in T lymphocyte driven dothelial cells. Stronger intensity noted in atrial appendage. No immuno- infl ammation in vivo reactivity with CD19.Conclusion: Rheumatic valvulitis appears to be the consequence of extravasation of T lymphocytes via the coordination of Seema G Trivedi, Experimental Pathology Group, William Harvey Re- L- selectin, Integrins and ICAM-1,VCAM-1 ,as can be envisaged. search Institute, United Kingdom Paul R Colville-Nash, SW Thames, Institute of Renal Research, United Kingdom 5035 Micheal P Seed, Experimental Pathology Group, William Harvey Re- search Institute, United Kingdom Biological basis of non-response to TNF alpha neutralization in RA patient subsets: Identifi cation via biosimulation of immunological 12-14 The cyclopentenone prostaglandin 15 deoxy Δ PGJ2 (15d-PGJ2) is as- networks sociated with the remission and resolution of infl ammation. Infl ammation resulting from Type IV delayed type hypersensitivity (DTH) is induced by Saroja Ramanujan, Entelos, Inc, United States T- lymphocytes, and forms an important pathological feature of chronic Herbert Struemper, Entelos, Inc, United States infl ammatory diseases such as Rheumatoid arthritis. Antigen induced ar- Lisl KM Shoda, Entelos, Inc, United States thritis (AIA) is based on a DTH response to the protein methylated BSA Nadine A Defranoux, Entelos, Inc, United States (metBSA) and joint infl ammation is regulated by the cellular activity of Didier A Scherrer, Entelos, Inc, United States the draining lymph nodes. Previously, intra-articular 15d-PGJ2 ameliorated AIA with an inhibition in joint NF-κB activity and increased production of Approximately one third of RA patients fail to achieve acceptable clinical the anti-infl ammatory cytokine IL-10 within the draining lymph node. In response to TNF-alpha(TNFa)neutralizing therapies. Furthermore, bio- vitro treatment with 15d-PGJ2, time and dose dependently reduced NF-κB logical processes leading to a clinically reported decoupling of improve- activation and subsequently induced apoptosis in antigen stimulated lymph ment in infl ammation and cartilage degradation in response to TNFa neu- node cells. Actions of 15d-PGJ2 in vitro were parallel to those within the tralization remain unclear as well as pathophysiologic mechanisms that arthritic joint. Given that apoptotic cells may be anti-infl ammatory and distinguish responders from non-responders. We have developed and used pro-resolving, it was hypothesised that the local induction of apoptosis may a dynamic computational model to identify potential mechanisms underly- account for the 15d-PGJ2 induced increase in lymph node IL-10. Apoptotic ing non-response to TNFa blockade in RA patients. Entelos® Rheuma- or control thymocytes were injected intra-articularly into sensitised mice toid Arthritis PhysioLab® platform, simulates biological processes in a prior to the onset of arthritis. Interestingly at the peak of AIA, lymph node rheumatoid joint and predicts synovial hyperplasia and cartilage degrada- cells (LNCs) from mice that had received apoptotic cells intra-articularly, tion in virtual patients. Our fi ndings indicate that synovial hyperplasia and displayed elevated antigen specifi c IL-10 production when compared to cartilage degradation are sensitive to variations in TNFa levels due to dif- LNCs from mice that had received control cells. LNC cytokine production ferential partitioning of TNFa between the synovial and cartilage tissues. measured throughout the course of AIA showed elevated levels of Th1 like This differential sensitivity allows for decoupling of infl ammatory and cytokines IL-2 and IFNγ at onset, whilst the resolution of infl ammation cartilage responses to TNFa neutralization. A virtual patient representing a was preceded by increased IL-10 production. Taken together, these data typical, anti-TNFa responder exhibits elevated synovial TNFa levels and suggest that local inhibition of NF-κB and induction of apoptosis induced experiences signifi cant (>20%) improvement in hyperplasia and cartilage by 15d-PGJ2 may act to increase IL-10 production by LNCs in vivo, and degradation upon effi cient neutralization of TNFa. In contrast, interpatient thereby promote the resolution of T lymphocyte driven infl ammation. variation in synovial TNFa activity prior to treatment and in the effi ciency of neutralization via therapy are found to result in three distinct patterns of non-response, characterized by negligible improvement in either hy- 5034 perplasia and/or cartilage degradation. We have identifi ed key variations in additional disease-regulating pathways that account for consistent pre- Phenotypic Analysis of Infi ltrating Cells and Expression of treatment disease activity in non-responders with different TNFa profi les Endothelial Cells in Rheumatic Heart Disease and also infl uence the predicted response to other RA therapies. Findings are consistent with clinical studies of response to TNFa neutralization and Shivali Gupta, Post Graduate Institute Of Medical Education And could aid in the identifi cation of non-responders and alternate treatment Research,Chandigarh, India strategies. Harpreet Vohra, Post Graduate Institute Of Medical Education And Research,Chandiga, India Kim Vaiphei, Post Graduate Institute Of Medical Education And 5036 Research,Chandiga, India Arun Kumar Aggarwal, Post Graduate Institute Of Medical Education Increased Toll-Like Receptor and CD16 Expression in Monocytes And Research,Chandiga, India during Acute Exacerbations of RA and effect on Therapy Sandeep Singh Rana, Post Graduate Institute Of Medical Education And Research,Chandiga, India Kumar Visvanathan, CRC for Chronic Infl ammatory Diseases, Dept of Medicine, Royal Melbourne Hospital, Australia Since valvular injury is most serious consequence of rheumatic carditis, Violeta Bogdanoska, CRC for Chronic Infl ammatory Diseases, Dept of understanding of pathogenic mechanisms in valvular infl ammation is cru- Medicine, Royal Melbourne Hospital, Australia cial to know the basis of Rheumatic Heart Disease. Emma Braine, CRC for Chronic Infl ammatory Diseases, Dept of Medici- Aims: To delineate the repertoire of infi ltrating lymphocytes and expres- ne, Royal Melbourne Hospital, Australia sion of adhesion molecules in mitral valve and atrial appendage.Materials: Amanda L Turner, CRC for Chronic Infl ammatory Diseases, Dept of Surgically resected specimens of 20 mitral valves and 5 atrial appendages Medicine, Royal Melbourne Hospital, Australia from follow-up cases of rheumatic heart disease .Methods:Immunohis- Narelle Skinner, CRC for Chronic Infl ammatory Diseases, Dept of Medi- tochemistry using fresh tissue, with monoclonal anti CD4,CD8,CD19, cine, Royal Melbourne Hospital, Australia CD18,CD11a ,CD11b, CD11c,L-selectin,VCAM-1 and ICAM- John A Hamilton, CRC for Chronic Infl ammatory Diseases, Dept of 1.Results:(a)Histology- All had variable degree of fi brosis and vasculari- Medicine, Royal Melbourne Hospital, Australia sation. There was mild to moderate degree of infl ammatory cell infi ltration S 126 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Andrew D Cook, CRC for Chronic Infl ammatory Diseases, Dept of Medi- that ERK1/2, Bax and Bcl-xL are important regulators of infl ammatory cell cine, Royal Melbourne Hospital, Australia death and survival in vivo and may provide novel therapeutic targets for the treatment of infl ammatory diseases. Background: There is considerable evidence that RA is a systemic disease with autoimmune characteristics and that monocytes and the innate im- mune system play a major role. Using human peripheral blood and sy- 5038 novial fl uid we have defi ned the response of the innate immune system during an acute exacerbations and documented changes to this response Nitric Oxide and antioxidants as regulators of collagen biosynthesis: after treatment. relevance to systemic sclerosis Methods: Patients with acute fl ares of RA were followed longitudinally. TLR2 and TLR4 and CD16 expression on CD14 +ve peripheral blood Richard K Bruckdorfer, University College London, United Kingdom mononuclear cells (PBMCs) were measured by fl ow cytometry using Audrey Dooley, University College London, United Kingdom anti-CD14, anti-CD16, anti-TLR2 and anti-TLR4 monoclonal antibodies. Edward Truelove, University College London, United Kingdom CD16 and TLR expression was reassessed in 3-6 weeks. Extensive clinical Sylvia Y Low, University College London, United Kingdom data and scales of activity were administered at each visit. Beirong Gao, University College London, United Kingdom Results: TLR2 expression was signifi cantly increased in RA patients with David Abraham, University College London, United Kingdom acute exacerbations compared with quiescent disease (P=0.001) and these were elevated above that of OA patients. TLR4 expression was signifi - Purpose. Excess collagen biosynthesis or decreased breakdown contributes cantly decreased in RA patients with acute exacerbations compared with to the accumulation of collagen in skin of patients with systemic sclerosis quiescent disease (P=0.01) and these were lower than that of OA patients. (SSc), an infl ammatory disorder of uncertain aetiology. NO is known to Patients treated with TNF- inhibitors demonstrated normalising of their inhibit collagen biosynthesis but the mechanism is unclear. NO reacts with TLR2 and TLR4 levels. The percentage of CD14dimCD16+ monocytes the free radical superoxide anion to form peroxynitrite, the effect of which expanded in patients with acute exacerbations of RA and decreased with on collagen biosynthesis is unknown. Ascorbate is an essential vitamin for effective therapy. During acute exacerbations, TLR2 and TLR4 levels on biosynthesis of collagen. The fl avanoid epigallocatechin gallate (EGCG) is CD14dimCD16+ monocytes were elevated as compared to CD14+CD16- an inhibitor of oxidation by peroxynitrite. The roles of nitric oxide and per- monocytes. oxynitrite and of dietary antioxidants (ascorbate and EGCG) on collagen Conclusions: Patients with RA have a marked upregulation in TLR 2 activ- type 1 production and expression of genes related to collagen biosynthesis ity on CD14+ monocytes with acute RA fl ares. This upregulation is ame- (CTGF, VEGF and MMP-1) were investigated in human dermal fi broblast liorated with TNF- inhibitor therapy. TLR4 is downregulated on CD14+ AG1518 (AG) and primary cell lines from normal and SSc skin. monocytes during fl ares and therapy increases TLR4 expression towards Methods. Fibroblasts were incubated (16h) with sodium nitroprusside normal levels. The CD14dimCD16+ monocyte sub-population should ma- (SNP) or with other NO donors and with peroxynitrite in the presence jor changes during acute fl ares and in response to therapy. and absence of antioxidants. Collagen secretion was measured by ELISA. Gene expression was analysed by RT-PCR. Results. SNP inhibited collagen secretion in AG cells in a concentration- 5037 dependent manner, reducing it to 16.7 ± 4.6% of controls at 0.5mM SNP. It did inhibit in fi broblasts of affected SSc skin, but was effective in normal Induction or inhibition of apoptosis-modulating proteins regulate the human skin primary fi broblasts as were other slow-releasing NO donors. infl ammatory response - implications for NSAID treatment Ascorbic acid, but not EGCG, reversed SNP action on collagen biosyn- thesis. ECGC enhanced the inhibition by NO. Peroxynitrite also inhibited Deborah A Sawatzky, Centre for Infl ammation Research, University of collagen secretion,, with a reduction to 8.4 ± 0.9% of controls at 1mM Edinburgh, United Kingdom (p<0.05), but also reduced cell protein levels. SNP (0.25 mM) enhanced Derek A Willoughby, St Bart's and the London School of Medicine and MMP-1 gene expression in AG cells. Dentistry, United Kingdom Conclusions. NO plays a regulatory role in collagen biosynthesis, possi- Paul R Colville-Nash, St Bart's and the London School of Medicine and bly by stimulation of collagen breakdown. Peroxynitrite may also inhibits Dentistry, United Kingdom but is less potent than NO. There is a loss of this regulatory role of NO in Adriano G Rossi, Centre for Infl ammation Research, University of Edin- systemic sclerosis. burgh, United Kingdom

The balance between cell survival and apoptosis is critical in terms of 5039 regulating the infl ammatory response. This is highly regulated involv- ing several checkpoints that subsequently control the onset and resolu- A Novel Oral Inhibitor of CSF-1R With Anti-Infl ammatory Activity: tion of infl ammation. We investigated the role of key molecules of the Proof-of-Principle Study in Collagen-Induced Arthritis mitogen-activated protein kinase (MAPK) signaling pathway (ERK1/2) and the apoptosis-regulating Bcl-2 family members (Bcl-xL and Bax) in Carl L Manthey, Johnson & Johnson, Pharmaceutical Research Division, the resolution phase of an in vivo rat carrageenan-induced pleurisy model. United States The specifi c ERK1/2 inhibitor PD98059 enhanced the resolution of in- Carl R Illig, Johnson & Johnson, Pharmaceutical Research Division, fl ammation by inducing neutrophil apoptosis, whereas the MEK1/2 inhibi- United States tor U0126 had no effect. Conversely, preventing apoptosis of neutrophils Sanath Meegalla, Johnson & Johnson, Pharmaceutical Research Divisi- in vivo with a specifi c inhibitor of Bax (V5) exacerbated infl ammation on, United States and delayed the resolution phase. Apigenin, non-specifi c fl avonoid inhibi- Mark J Wall, Johnson & Johnson, Pharmaceutical Research Division, tor of ERK1/2 and COX-2, augmented infl ammation, providing evidence United States that inhibition of the pro-apoptotic prostacyclins can delay the resolution Jinsheng Chen, Johnson & Johnson, Pharmaceutical Research Division, of infl ammation. Specifi cally, we show that the COX-2 inhibitors, indo- United States methacin and NS-398, downregulated Bax and upregulated pro-survival Carol F Franks, Johnson & Johnson, Pharmaceutical Research Division,

Bcl-xL expression on infl ammatory cells. Importantly, Bcl-xL upregulation United States could be prevented with pro-resolving cyclopentanone prostaglandin 15de Marge A Chaikin, Johnson & Johnson, Pharmaceutical Research Divisi- 12,14 oxyD prostaglandin J2 pre-treatment. The prolonged survival of infl am- on, United States matory cells combined with reduction of the anti-infl ammatory cytokine Bruce E Tomczuk, Johnson & Johnson, Pharmaceutical Research Divisi-

TGF-beta1 may be contributing mechanisms whereby COX inhibition de- on, United States lays the resolution of infl ammation. In conclusion, our evidence shows Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 127

A CSF-TNF feed-forward cycle has been suggested to drive infl amma- tion in rheumatoid arthritis. Herein we provide the fi rst report of the 5041 pharmacological potential of an orally active inhibitor of CSF-1R. JNJ- 27301937 inhibited CSF-1R kinase autophosphorylation activity with a Ki Antiarthritic Activity of 2-Methoxyestradiol (2ME2) in a Rat of 0.8 nM, and blocked the proliferation of murine bone marrow-derived Adjuvant-Induced Model of Rheumatoid Arthritis macrophages with an IC50 of 2.2 nM. JNJ-27301937 demonstrated good selectivity for FMS vs a panel of kinases. Selectivity for CSF-1R signal- Anthony M Treston, EntreMed, Inc., United States ing was also evident in dosed mice; JNJ-27301937 blocked CSF-1 but Steven J Strawn, EntreMed, Inc., United States not LPS-induced phosphorylation of p44-MAPK. Further, JNJ-27301937 William E Fogler, EntreMed, Inc., United States (30 mg/kg) induced specifi c depletion of a macrophage population known Stacy M Plum, EntreMed, Inc., United States to be CSF-1-dependent. To assess the therapeutic potential of inhibiting CSF-1R in arthritic disease, JNJ-27301937 was evaluated in a collagen- PURPOSE: 2-Methoxyestradiol (2ME2) has antiangiogenic, antiprolifera- induced model of arthritis in B10RIII mice. Oral daily bid doses of 30 tive and proapoptotic activity. Angiogenesis and proliferation are promi- and 5 mg/kg JNJ-27301937 reduced the overall progression of clinical nent features of chronic infl ammatory conditions including rheumatoid scores by 65 and 35%, respectively. Histological examination revealed arthritis (RA), suggesting 2ME2 may have therapeutic activity in these reduced destruction of bone and cartilage. JNJ-27301937 did not suppress conditions. the humoral immune response to type II collagen, but did reduce by 68% METHODS: Initial studies showed 2ME2 results in inhibition of clinical serum amyloid A, a systemic biomarker of infl ammation. Macrophages severity in the Arthrogen CIA® RA model. Histomorphometrics of hind were confi rmed as the cellular target of JNJ-27301937 as these cells were paw sections showed dose-dependent decreases in synovial infl ammation, depleted selectively from the joints of treated mice. We conclude that cartilage damage, pannus, osteoclasts, and bone resorption. Hind paws inhibition of CSF-1R provides a novel mechanism for targeting disease from mice treated with 100 mg/kg 2ME2 showed abrogation of articular progression in infl ammatory arthritis. cartilage damage, as well as marginal pannus formation and bone resorp- tion. Here we evaluate 2ME2 in an adjuvant arthritis model. Lewis rats im- munized in the base of the tail with complete Freund’s adjuvant containing 5040 10mg/ml heat-killed M. tuberculosis developed severe polyarticular arthri- tis by day 10. Cohorts (n=8) were dosed with 100, 10 or 1 mg/kg 2ME2 Serum Interferon-Inducible Protein 10 (IP-10) Levels Correlate with or vehicle po for 11 days, starting 10 days after immunization. Reference Disease Activity in Systemic Lupus Erythematosus groups included 0.1 mg/kg methotrexate and 3 mg/kg Enbrel® . All paws were scored daily for clinical severity (erythema, swelling, loss of use and Kok Ooi Kong, Singapore passive movement) and cumulative arthritic scores were calculated. Bernard YH Thong, Singapore RESULTS: Methotrexate and Enbrel® reduced cumulative arthritic score Tsui Yee Lian, Singapore by 18% (p=0.029) and 23% (p=0.004) resp. 2ME2 (100mg/kg) decreased Hiok Hee Chng, Singapore cumulative arthritic score by 29% (p=0.001) compared to vehicle control. Yew Kuang Cheng, Singapore On day 21 joints from hind paws were processed for H&E, safranin O Cheng Lay Teh, Singapore and TRAP. These are being evaluated histomorphometrically for synovial Bernard PL Leung, Singapore infl ammation, articular cartilage damage, pannus formation, osteoclast Hwee Siew Howe, Tan Tock Seng Hospital, Singapore number and bone resorption. CONCLUSION: The current study demonstrates antiarthritic activity of Purpose: We sought to determine serum IP-10 levels in a large cohort 2ME2 in a treatment model of adjuvant-induced arthritis and suggests that of SLE patients to assess its relationship with disease activity and organ 2ME2 could be a novel therapeutic agent for RA. manifestations, as recent small studies of SLE have reported elevated se- rum IP-10 levels. Methods: 774 serum specimens were collected from 424 SLE ( 1991 revised ACR criteria)patients and 76 unrelated healthy 5042 controls with informed consent. Patients were 80% Chinese, 13% Malay, and 7% Indian and other ethnicity, with mean age 40.1±12.6 (mean±SD) Shortcomings of the collagen-induced arthritis (CIA) in rodents for years, mean disease duration 112±93 months (disease duration less than evaluating new/old therapies 3 years in 22.4%), and female: male ratio 10:1. Disease activity was as- sessed and categorized using SLAM-R scores into inactive (<=5), mild Michael W Whitehouse, Dept Medicine, University of Queensland, (>5-10), moderate (>10-15) and severe (>15)groups. Serum IP-10 levels Brisbane, Australia were determined using ELISA and the correlation with disease activity and levels of anti-dsDNA antibody, C3, C4 and C-reactive protein analyzed. Purpose Results: 74.0% had inactive, 20.0% mild, 3.9% moderate and 2.1% severe To compare CIA with adjuvant-induced arthritis (AIA) in rats for respon- disease according to SLAM-R (median score 2,interquantile range,IQR: siveness to establish clinical therapies and evaluating new/traditional treat- 1 - 4). 60.4% of patients had hematological, 31.1%renal, 17.7% mucocu- ments. taneous and 4.5% nervous system involvement respectively. IP-10 levels Background were elevated in all SLE patients compared to controls (73.1 [IQR: 29.0 - Currently the favoured animal model for rheumatoid arthritis is CIA in 187.7] pg/ml vs. 22.1[IQR: 9.4 - 38.1] pg/ml, respectively; p<0.0001), and mice - a ‘messy’ disease usually requiring a) complete Freund’s adjuvant correlated signifi cantly with SLAM-R scores ([rho] =0.19, p=0.0001) and b) a later shock treatment e.g. LPS to elicit IL-12 etc. and c) considerable disease activity categories (inactive: 74.0 [IQR: 29.0 - 150.9] pg/ ml, mild: time i.e. more than 5 weeks. By contrast CIA in rats can be swift (only 16 88.6 [IQR: 38.4 - 190.7] pg/ml, moderate 174.1 [IQR: 96.6 - 319.5] pg/ml, days) and needs only incomplete adjuvants i.e. no mycobacteria. and severe: 936.7 [IQR: 68.8 - 1865.2] pg/ml, p<0.001), levels of anti- The absence of mycobacterial and LPS insults reduces the overall burden dsDNA antibodies, complement,CRP and the ESR. Patients with active of disease in the rat CIA, seemingly providing a cleaner backdrop for drug haematological(p=0.0013) and mucocutaneous (p=0.02) manifestations studies. had higher median serum levels of IP-10 than those without. Methods Conclusion: Serum IP-10 levels are markedly increased in SLE, correlate On Day-0 female Wistar rats were inoculated in their tailbase with a) with disease activity, and are a potential predictive marker of activity type-II collagen and incomplete Freund’s adjuvant or b) heat-killed in SLE. M.tuberculosis dispersed in squalane, to induce CIA or AIA respectively. Where necessary animals developing CIA or AIA were co-housed, to elim- inate local environmental variations. Results S 128 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Some drugs/test agents given prophylactically (from day-0) gave discor- tion, recombinant IFN-alpha but not IFN-gamma induced IDO-dependent dant responses including (i) suppressing AIA but either amplifying or hav- suppression of T cell proliferation. In this study, we show that genetic dis- ing no effect (-) on CIA development: examples include Na aurothiomalate ruption and pharmacologic inhibition of IDO function blocked IFN-alpha (at 6 mg/kg alt.days s.c.), DMSO (at 1.5 g/kg), colloidal silver p.o. (-); (ii) production by DCs after B7 ligation. However, DCs from IDO-defi cient not affecting AIA but amplifying CIA e.g. some brands of olive, fl axseed mice produced IFN-alpha following CpG stimulation and infl uenza virus and fi sh oils, glucosamine .HCl/HSO4; and occasionally (iii) suppressing infection. These data show that IDO-dependent control of IFN-alpha pro- CIA but not affecting AIA e.g. yet other brands of olive and fi sh oils. duction following B7 ligation is a critical step that infl uences development Conclusions of DCs with potent T cell regulatory function. 1. The move to Political Correctness restricting/forbidding studies with AIA may be highly retrogressive for therapeutic discoveries. 2. Switching to, or relying on, CIA may lead to rejection of some benign 5045 nutritional supports (or even classical chrysotherapy) that have proved valuable for treating human arthritis. A fundamental bimodal role neuropeptide Y1 receptor in infl ammatory responses

5043 Julie Wheway, Garvan Institute of Medical Research, Australia Rebecca Newton, Garvan Institute of Medical Research, Australia Measles antibody in different age groups of children in Shiraz (One Herbert Herzog, Garvan Institute of Medical Research, Australia Year Study) Fabienne Mackay, Garvan Institute of Medical Research, Australia

Abdollah Karimi, Shaheed Beheshti University,Pediatric InfectiousRes- Neuropeptide Y (NPY), a 36aa peptide distributed throughout the body has earch Center, Iran been implicated in the regulation of several physiological processes such Abdolvahab Alborzi, Alborzi infectious research Center,Shiraz, Iran as energy balance, feeding, reproduction and anxiety. NPY actions are me- diated by 5 GPCRs, designated as Y1, Y2, Y4, Y5 and y6. We assessed the Due to vaccination the infection with measles has declined remarkably. role of the NPY Y1 receptor in immune responses using Y1 defi cient mice However due to the waning phenomenon (secondary vaccine failure) there (Y1-/-). Y1-/- T cells are hyper-responsive to activation, and trigger severe have been reports from limited epidemics in older ages. Therefore, this colitis after transfer into lymphopenic mice. Paradoxically, T cell differen- study was undertaken to determine the level of antibody in different age tiation to Th1 T cells in Y1-/- mice appears to be defective as lower IgG2a groups in order to indentify the most the most appropriate age after the age and IL-12p40 levels are detected in their serum. Response of these mice of 15 months at wich the 2 nd vaccine could be administerd. was tested in two Th1 models of infl ammation; delayed-type hypersensi- Method-Seven different age groups, a) 6 month old. B) 9 months old. C) tivity (DTH) and dextran sulfate sodium (DSS)-induced colitis. Y1-/- mice 14 month old. D) 18 month old. e) 6 year old. F) 10 year old & g)15 year were signifi cantly protected against disease in both models and this cor- old. Were selected by random clustered method and serum samples were responded with a reduction in Th1 cytokines, T cell proliferation and Ag- obtained for detection of measles antibody. The data were interpreted us- specifi c IgG2a antibodies. This defect was due to functionally impaired ing Chi square test. The presence of the antibody was determined using APCs. Macrophages isolated from Y1-/- mice produce less IL-12p40 and MORBILO lgG (RADIM ) kits and by ELISA. Six months old children TNF following stimulation with TLR ligands. Furthermore, Y1-defi cient had not received vaccination. The 9 months old samples were obtained just DCs have impaired antigen uptake and less stimulatory function in a mixed before measles vaccination the 14 months old group had received a single lymphocyte reaction (MLR). Bone marrow chimeric mice indicate the vaccine at the age of 9 month. those with the age of 18 months had 2 vac- inhibition in Th1 infl ammation is immune cell intrinsic and further studies cinations at the age of 9 and 15.those with the age of 15 years had also an using conditional knockout mice are underway. additional vaccine received about 3 to 9 months prior to the study. These results demonstrate a fundamental bimodal role for the Y1 receptor Result- On the 6 month, 10 % are positive. In the 9 month, all are nega- in the immune system, serving as a strong negative regulator on T cells, but tive. On the 14 month, 52.9% are positive. On the 18 month, group 89.4% also as a key activator of APC function. Our fi ndings uncovered new mo- are positive. On the 6 years, 60.8% are positive. There was a signifi cant lecular mechanism that may potentially explain aspects of stress-induced difference between the positive cases of each 2 age groups ( p <0.0001). immuno-suppression. Conclusion-Due to signifi cant waning of antibody an additional measles vaccine in Iran is recommended. 5046

5044 Storage time and leucocyte burden increase adhesion of red blood cells for transfusion to vascular endothelium Interferon-alpha Production following B7 Ligation is Dependent on Functional Indoleamine 2,3-Dioxygenase Angela M Anniss, Research Unit, Australian Red Cross Blood Service, Victoria, Australia Anna K Manlapat, Medical College of Georgia, United States Kath A Patton, Research Unit, Australian Red Cross Blood Service, Babak Baban, Medical College of Georgia, United States Victoria, Australia Phillip R Chandler, Medical College of Georgia, United States Rosemary L Sparrow, Research Unit, Australian Red Cross Blood Ser- Andrew L Mellor, Medical College of Georgia, United States vice, Victoria, Australia

Dendritic cells (DCs) can both promote and suppress T cell-mediated im- Adherence of red blood cells (RBCs) to vascular endothelium impairs munity. Manipulation of DC functions can provide multiple opportuni- blood fl ow and decreases oxygen delivery. Although RBCs may be stored ties for therapeutic intervention to enhance or regulate immunity and to for up to 42 days prior to transfusion under current blood banking guide- prevent or cure human diseases such as cancer, infectious diseases, and lines, little is known of how changes to RBCs during storage may affect autoimmune diseases as well as to suppress transplant rejection. Ligation their adherence properties. The aim of this study was to monitor adherence of surface B7 (CD80/CD86) molecules with soluble CTLA4 (CTLA4-Ig) of stored RBCs to vascular endothelium under conditions of continuous induces DCs to express the immunosuppressive enzyme indoleamine 2,3- fl ow in vitro. Specifi cally the infl uence of RBC storage time and leucocyte dioxygenase (IDO). Recently, we have shown that DCs produce interferon burden of stored RBC preparations was investigated. (IFN)-alpha as early as 30 minutes following B7 ligation. Moreover, sig- Human umbilical vein endothelial cells (ECs) were grown to confl uence on naling via IFN-alpha receptors (IFNAR) was essential for activation of the coverslips. Non-leucocyte-reduced, buffy-coat-reduced and leucocyte-fi l- transcription factor STAT1, while IFN-gamma signaling was not. In addi- tered RBC products were prepared according to standard blood bank pro- Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 129 cedures. RBC samples were collected at weekly time points until product Zheng Yang, University of New South Wales, Australia expiry and perfused across an EC monolayer using a parallel fl ow chamber Wei-Xing Yan, University of New South Wales, Australia mounted to an inverted microscope. Perfusion of RBCs was controlled for Hong Cai, University of New South Wales, Australia shear stress and temperature. RBC-EC interactions were recorded using a Nicodemus Tedla, University of New South Wales, Australia digital camera attached to the microscope. Michael Perry, University of New South Wales, Australia The number of RBCs adhering to the unactivated EC layer progressively John Hunt, University of New South Wales, Australia increased with product storage time in all products. RBCs from products Prue Hart, Telethon Institute for Child Health Research, Australia stored for 28 and 42 days were signifi cantly more adherent than fresher cells. RBCs from products containing leucocytes were also signifi cantly Purpose: The S100 calcium-binding protein S100A12 (A12) is expressed more adherent to the EC layer on days 28 and 42 of storage than RBCs in neutrophils and upregulated in macrophages by TNF. Expression is seen from leucocyte-reduced products. in parasitic infections and many chronic infl ammatory diseases such as Our fi ndings indicate that product storage time and leucocyte burden in- IBD and RA. A12 activates binds the receptor for advanced glycation end crease the adhesion of RBCs to an EC layer. These results may lead to products (RAGE) and is chemotactic for monocytes. We considered that greater understanding of the interaction of transfused RBCs with recipient the acute infl ammation initiated by A12 in mice may be mast cell (MC) endothelium and the biological consequences of this adherence. To add to mediated. these results, we are currently examining how stored RBCs for transfusion Methods: Human and murine MC were activated in vitro with A12 and may interact with an activated EC layer such as would occur when infec- mediator release, and cytokines induced (assessed with the Bioplex sys- tion, infl ammation and/or tissue damage is prevalent. tem), determined. Intravital microscopy was used to assess A12 effects on the microcirculation. MC-stabilised/-defi cient mice confi rmed the role of MC. 5047 Results: Mechanisms of non-classical MC activation are unclear. A12 pro- voked degranulation of mucosal and tissue MC and activated MC in vitro Calcitonin peptides are produced by human adipocytes during and in vivo. A12- changes in the microcirculation: oedema, leukocyte roll- infl ammation and exert autocrine/paracrine effects ing, adhesion and transmigration, and leukocyte recruitment i.p. were MC- dependent. A12 amplifi ed IgE-mediated responses and A12 was found in Philippe Linscheid, Switzerland macrophages in asthmatic, but not normal lung. A12-stimulated MC gen- Dalma Seboek, University Hospital Basel, Switzerland erated a different cytokine profi le to IgE-cross-linked MC, with high levels Ulrich Keller, University Hospital Basel, Switzerland of IL-6 and IL-8 and moderate MCP-1 and MIP-1beta. Beat Muller, University Hospital Basel, Switzerland Conclusions: A12 may amplify conditions such as acute asthma, often Purpose To characterize novel adipose tissue cytokine- and endotoxin-in- common in viral infections and associated with neutrophilic infl ammation, duced secretion products related to human infl ammatory diseases. to cause more severe disease. However the cytokine profi le generated by Methods In human cell models (adipose tissue biopsies, explanted ma- A12 strongly indicates a role in innate/Th1-mediated responses. A12 may ture adipocytes, mesenchymal stem cell (MSC)- and preadipocyte-derived be an important mediator of MC activation in host defence against infec- adipocytes, peripheral blood-derived monocytes (PBMCs)), mRNAs were tion and in chronic cell activation in infl ammatory disease. detected by RT-PCR and quantifi ed by real-time PCR. Immuno-detectable peptides were analyzed in culture supernatants. Lipolysis and insulin-me- diated glucose uptake were assessed upon specifi c treatments. 5049 Results mRNAs of several members of the calcitonin gene family of pep- tides including calcitonin (CT), CT gene-related peptide (CGRP) I, CGRP The role of melatonin on scarring in an incisional model of dermal II and adrenomedullin (ADM) but not amylin were detected in adipose wound healing in rats tissue obtained from infected patients. Accordingly, the same mRNA ex- pression pattern was found in all in vitro adipocyte models upon IL-1β Kamali Pugazhenthi, Department of Pharmacology and Toxicology, and endotoxin administration as well as in adipocytes co-cultured with University of Otago, New Zealand activated PBMC-derived macrophages. Procalcitonin (ProCT), CGRP and Mohit Kapoor, Department of Pharmacology and Toxicology, University ADM were released into the culture medium. In contrast to ADM, ProCT of Otago, New Zealand and CGRP expressions were not observed in cytokine- and endotoxin-ac- Paula Young, Department of Pharmacology and Toxicology, University of tivated preadipocytes, MSC, PBMC and in human cell lines. IFNγ co-ad- Otago, New Zealand ministration potently blocked IL-1β- and endotoxin-induced ProCT and Andrew Clarkson, Department of Pharmacology and Toxicology, Univer- CGRP, but augmented ADM production. Expression studies in the pres- sity of Otago, New Zealand ence of cycloheximide suggest the existence of a CT gene transcription Irene Hall, Department of Pharmacology and Toxicology, University of inhibitor with high turnover rate. Exogenous CGRP and ADM adminis- Otago, New Zealand tration induced CT, CGRP I and CGRP II mRNAs and dose-dependently Ian Appleton, Department of Pharmacology and Toxicology, University (>10-10M) enhanced lipolysis. A trend towards reduced insulin-mediated of Otago, New Zealand glucose uptake was observed in the presence of ADM and CGRP. Conclusions CT gene expression is not restricted to specialized neuro-en- The hormone melatonin is known for its anti-oncotic, anti-infl ammatory docrine cells during infl ammatory conditions. CT peptides exert paracrine/ and immunomodulatory effects. However, its role in wound healing has autocrine metabolic and positive feedback regulatory effects in human not been established. Therefore, in this study we determined the effects mature adipocytes, but not in undifferentiated precursor cells. Since viral of melatonin on scarring using a full thickness incisional model of wound infections are, among others, accompanied by IFNγ release, the inhibiting healing in rats. Wound healing was initiated in male Sprague Dawley rats effect of IFNγ on infl ammatory ProCT production might explain the clini- 250±25g (n=6/group/time point). Four 1cm full thickness dermal incisions cal observation of diminished increase in serum ProCT in viral as com- were made on the dorsum of each rat. The treatment and control groups pared to bacterial infections. received 1.2mg/kg melatonin or saline intradermally, at 11pm (to comply with the rats daily melatonin rhythm), 24 hours prior and for a further 7

days postwounding. The rats were sacrifi ced by CO2 exposure on days 0, 5048 1, 3, 7, 14, and 21, at 2pm (the time of maximal endogenous melatonin lev- els), and biopsies extracted. Homogenates of biopsies were assessed for ar- S100A12 and mast cell activation in infl ammation ginase activity, nitric oxide synthase (NOS) activity and nitrite levels. Im- munohistochemical studies to assess melatonin receptor expression were Carolyn L Geczy, University of New South Wales, Australia also carried out. A blind histological analysis of scarring was performed S 130 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) using van Gieson’s collagen stain to assess the maturity and orientation 2004, 27: 113-9). Microarray analysis of apoptosis-related genes in epithe- of collagen fi bres within the scar. Melatonin receptors were expressed on lial cells exposed to proinfl ammatory cytokines suggested that inhibitor of the epidermis and macrophages on day 1 in both the treated and the con- apoptosis protein 2 (cIAP2) expression was upregulated. The aim was to trol wounds. Thereafter the expression decreased post day 3. The blind characterize the pathways involved in regulation of apoptosis susceptibil- study showed that melatonin treatment signifi cantly improved scarring on ity during cytokine stimulation of colonic epithelial cells. Methods: Colon- day 21. Melatonin treatment also signifi cantly (P<0.01) increased arginase ic epithelial HT29 cells were stimulated with 1pM tumor necrosis factor- activity on day 1 and iNOS activity was signifi cantly decreased during alpha (TNF-alpha) and 1pM interferon-gamma for 1 - 24 hours. RT-PCR day 1 (P<0.01) and day 3 (P<0.01), followed by a signifi cant increase in and Western blotting was used to determine expression of cIAP2, and the iNOS activity on day 7, compared to controls. Arginase generates praline, nucleosome inhibitor MG132 was used to inhibit nuclear factor kappaB the building block for collagen synthesis. Melatonin treatment increased (NF-kappaB) activation. NF-kappaB activation was assessed by EMSA- arginase activity and thereby collagen synthesis from day 1. It has been technique, and cell death was determined by the LDH-release assay. Re- reported that increases in NO production, prolongs the infl ammatory phase sults: cIAP2 expression was induced by cytokine stimulation and inhibited of wound healing. Melatonin signifi cantly (P<0.01) decreased NOS ac- by MG132 treatment. Inhibition of NF-kappaB-pathway activation greatly tivity which resulted in improved scarring. We conclude that melatonin increases cell death responses to TNF-alpha and Fas-stimulation. Conclu- improves scarring and that this effect is, inpart, mediated by modulation of sion: It is for the fi rst time reported that cytokine stimulation of colonic NOS and arginase activity. epithelial cells induces cIAP2 expression, possibly through activation of NF-kappaB. NF-kappaB-activation provides a cytoprotective stimulus in colonic epithelial cells which seems to be linked to cIAP2 expression. This 5050 pathway might be activated in regenerative colonic epithelial cells in IBD, suggesting a cytoprotective effect of cIAP2 in colonic epithelial cells. Cyclosporine A-like Immunomodulatory Benefi ts of Polysaccharopeptide (PSP) Derived from Coriolus Versicolor in Activated Human T-cells 5052

Cheuk Lun Lee, The University of Hong Kong, Hong Kong Role of reactive oxygen species in butyric acid-induced T cell Wai Hung Sit, The University of Hong Kong, Hong Kong apoptosis Jennifer Man Fan Wan, The University of Hong Kong, China Tomoko Kurita-Ochiai, Department of Microbiol. and Immunol., Nihon Cyclosporin A (CsA), an potent inhibitor of T cell proliferation, is use- Univ. Sch. of Dent. at Matsudo, Japan ful for treating autoimmune and infl ammatory disease and graft rejection, Tomomi Hashizume, Department of Microbiol. and Immunol., Nihon however, associated with many side effects. Polysaccharide peptide (PSP), Univ. Sch. of Dent. at Matsudo, Japan derived from the Chinese medicinal mushroom Coriolus versicolor, is a Kuniyasu Ochiai, Department of Microbiol. , Nihon Univ. Sch. of Den- neutracuetical currently used as immunomodulatory and anticancer adju- tistry, Japan vant. We have shown that PSP was able to enhance and exhibit cyclospo- Masafumi Yamamoto, Department of Microbiol. and Immunol., Nihon rine-like activities on the ex vivo PHA-activated human peripheral blood Univ. Sch. of Dent. at Matsudo, Japan mononuclear cells (PBMCs) but protected them to undergo cell death. Flow cytometry, Western Blot, ELISA and RT-PCR measurements showed that Emerging evidences indicate that bacterial modulation of apoptosis is an the immunosuppressive mechanisms of PSP were similar to that of CsA. important part of pathogenesis in infections. We have previously shown PSP represented an immunomodulator with primary inhibitory effects on that the antioxidant N-acetyl-L-cysteine (NAC) decreased intracellular interlukin-2 expression, production of cytotoxic T-cell, natural killer cells ROS and prevented butyric acid-induced T cell apoptosis. To determine and gamma-interferon. Together, the reduction on the TH-1 polarization the mechanism by which NAC afforded such protection, we investigated of cytokines led to altered T-lymphocyte function. Both IFN-gamma and the contribution of reactive oxygen species (ROS) in mitochondrial sig- IL-2 signaling pathways down-regulated by PSP appeared to be at the tran- naling pathways and caspase activation, and of antioxidant enzymes such scription level via the suppression of Janus kinase (JAK) and the signal as Zn-SOD, Mn-SOD and HO-1 in butyric acid-induced T cell apoptosis. transducers and activator of transcription (STAT), respectively. CsA was Increases of cytochrome c, apoptosis-inducing factor (AIF), and second unable to protect PHA-induced cell death while PSP induced Go/G1 cell mitochondria-derived activator of caspases protein (Smac) release from cycle arrest, alternated cyclin-cdk complex and inhibited the expression of mitochondria into the cytosol by butyric acid were dose-dependently re- the death receptor Fas (CD95) to protect the survival of the T-cells. Collec- duced by pretreatment with NAC. NAC also downregulated Bcl-2- and tively our results reveal novel aspects of the immunoregulatory properties Bcl-XL-suppression and Bax- and Bad-activation by butyric acid. Further- of PSP and suggest that this medicinal fungal extract could be potentially more, caspase-3, -9, and especially -8 activities were dose dependently developed as a specifi c immunosuppressive agent for clinical applications reduced by pretreatment with NAC. However, expressions of Zn-SOD, such as organ transplantation and other infl ammatory diseases, especially Mn-SOD and HO-1 were not changed after treatment of cells with butyric when PSP does not enhance the apoptotic pathways of the human T-cells. acid. Taken together, our results indicate that butyric acid induces mito- chondrial dysfunction and apoptosis in human Jurkat T cells through a ROS-dependent mechanism. This work was supported by a Grnat-in-aid 5051 from Tsuchiya culture promotion foundation and by a scientifi c research Grnt-in-aid (20130594) from the ministry of Education, Science, Sports Cytokine stimulation of colonic epithelial cells induces inhibitor of and Culture of Japan. apoptosis protein expression

Jakob B Seidelin, Herlev Hospital, University of Copenhagen, Denmark 5053 Ben Vainer, Herlev Hospital, University of Copenhagen, Denmark Lars Andresen, Herlev Hospital, University of Copenhagen, Denmark Does splenic hyperphagism contribute to the development of severe Ole Haagen Nielsen, Herlev Hospital, University of Copenhagen, Den- malarial anaemia? mark Krystal J Evans, The Walter and Eliza Hall Institute of Medical Research, Purpose: Infl ammatory bowel disease (IBD) is characterized by mucosal Australia infl ammation that results in ulceration and subsequent regeneration of the Diana S Hansen, The Walter and Eliza Hall Institute of Medical Re- epithelium. Regenerative epithelium is characterized by a high prolifera- search, Australia tion rate and decreased susceptibility to cytotoxic mediators (Cytokine Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 131

Sara R Prickett, The Walter and ELiza Hall Institute of Medical Re- The number of circulating CD10- neutrophils increased at a steady rate search, Australia during hypothermic cardiopulmonary bypass (0.11 ± 0.02 x 109/L/3min) Louis Schofi eld, The Walter and Eliza Hall Institute of Medical Research, and re-warming (0.15 ± 0.04 x 109/L/3min). With the onset of myocar- Australia dial reperfusion the rate of increase in circulating neutrophil numbers in- creased signifi cantly for both CD10- (from 0.15 ± 0.04 to 0.91 ± 0.25 x Severe malarial anaemia (SMA) is the most frequent life-threatening com- 109/L/3min, p<0.0001) and CD10+ (from 0.33 ± 0.10 to 1.14 ± 0.31 x plication of malaria infection, and is thought to contribute to the majority 109/L/3min, p<0.0001) subpopulations. of malarial deaths worldwide. To explore the mechanisms of pathogen- Conclusions esis we utilised a novel semi-immune murine model of SMA in which Immature CD10- neutrophils represent 17-35% of the circulating neu- haemoglobin (Hb) levels fell to 43% of baseline levels in the presence of trophil population during cardiac surgery. Steady intra-operative release low parasitemias which peaked at 1.1% infection of circulating erythro- of neutrophils from the bone marrow was observed during hypothermic cytes. In vivo biotinylation analysis of the circulating blood compartment cardiopulmonary bypass, and reperfusion was much more potent than re- revealed that anaemia arose from accelerated erythrocyte turnover, with warming as a stimulus to bone marrow neutrophil release during cardiac the labelled cell population experiencing 50% clearance from circulation surgery. by day 4 post-labelling. Clearance from circulation continued and the la- belled population was reduced to less than 1% of circulating cells by 8 days post-labelling, indicating that the entire blood compartment had been 5055 turned over in approximately one week. Splenomegaly developed during the course of the anaemic crisis with a 6.7 fold increase in the spleen-to- Collagen type I signaling reduces the expression and the function of mouse mass ratio on the day of peak anaemia as compared to non-infected human receptor activator of nuclear factor -κB ligand (RANKL) in T controls and the resolution of splenomegaly correlated with the recovery lymphocytes from anaemia. The number of nucleated cells in the spleen increased over the development of anaemia, peaking at 1.4 x109 cells. The kinetics of Steve Gendron, Centre de Recherche en Rhumatologie et Immunologie, infi ltration of F4/80+ macrophages into the spleen correlated with the on- Université Laval, Canada set and duration of anaemia with splenic F4/80+ macrophages accounting Estelle Chamoux, Centre de Recherche en Rhumatologie et Immunologie, for 25% of nucleated spleen cells at the peak of the anaemic crisis. The Université Laval, Canada severity of anaemia was signifi cantly alleviated by depletion of either host Julie Couture, Centre de Recherche en Rhumatologie et Immunologie, phagocytic cells or CD4+ T lymphocytes, with Hb levels being 1.4 fold Université Laval, Canada higher in animals depleted of these immune effector cells at the point of Fawzi Aoudjit, Canada anaemic crisis as compared to controls. Severe malarial anaemia in the semi-immune mouse model may thus arise from a hyperphagocytic syn- PURPOSE: The mechanisms by which β1 integrins modulate T cell func- drome involving the infi ltration and activation of splenic macrophages tions are still poorly defi ned. We have previously reported that signaling under the regulation of CD4+ T lymphocytes. via the Collagen type I (Coll I) receptor, α2β1 integrin, inhibited Fas-Li- gand expression and protected Jurkat T cells from activation-induced cell death (AICD). In this study, we examined whether Coll I signaling in T 5054 cells may also modulate the expression of the human receptor activator of nuclear factor-κB ligand (RANKL); a recently identifi ed TNF family Bone marrow neutrophil mobilization in response to re-warming and member, which has important functions in osteoclastogenesis, cell survival reperfusion following cardiac surgery and apoptosis. METHODS: Jurkat T cell line and primary activated T cells have been used. RANKL expression was assessed by RT-PCR for mRNA Yishay Orr, Centre for Vascular Research,University of New South Wales, and fl ow cytometry for surface expression of the protein. RANKL function Australia was studied by its capacity to induce T cell apoptosis measured by annexin Jude M Taylor, Centre for Vascular Research,University of New South V binding and fl ow cytometry as well as by its capacity to induce TRAP Wales, Australia activity in RAW264.7 macrophage cell line. RESULTS: Our results show Paul G Bannon, Department of Cardiothoracic Surgery, Royal Prince that in both Jurkat T cells and human primary T cells, Collagen I signaling Alfred Hospital, Australia signifi cantly reduces activation-induced RANKL expression by 50-60%. Carolyn Geczy, Infl ammatory Diseases Research Unit, University of New We also found that RANKL participates in doxorubicin-induced apoptosis South Wales, Australia of Jurkat T cells, and in this respect, Collagen I protected Jurkat T cells Leonard Kritharides, Centre for Vascular Research,University of New from doxorubicin-induced apoptosis by inhibiting doxorubicin-induced South Wales, Australia RANKL expression. Moreover, we show that T cells activated in the pres- ence of Collagen I have lost their ability to stimulate formation of osteo- Introduction clast-like cells from RAW 264.7. CONCLUSION: Together, our results Cardiac surgery provokes a systemic neutrophilia which is predominantly suggest that by limiting the production of RANKL, Coll I signaling may attributed to the release of neutrophils from the bone marrow. Hypother- have a protective role against osteoclast development and subsequently mia is understood to suppress and re-warming to stimulate, neutrophil bone loss during immune responses, and can contribute to the resistance bone marrow release. However, the precise contribution of the various of leukemic T cells to chemotherapy. As such, this study emphasizes the operative stimuli such as reperfusion, to total circulating neutrophilia, and importance of Coll I signaling in the control of T cell functions. bone marrow neutrophil release in particular, is unclear. We have investi- gated the extent to which bone marrow neutrophil release is regulated by hypothermia, re-warming and reperfusion using fl ow cytometry to quan- 5056 tify less mature, bone marrow-derived CD10- circulating neutrophils. Methods Prevention of experimental autoimmune diabetes with complete Peripheral blood samples (total n=13/patient) were taken from patients Freund's adjuvant in mice - role of nitric oxide (n=10) undergoing cardiac surgery with cardiopulmonary bypass at regu- lar 3 minute intervals during the stages of hypothermia, re-warming, and Milos Markovic, Institute of Microbiology and Immunology, School of myocardial reperfusion. Total, CD10+ (mature) and CD10- (immature) Medicine, University of Belgrade, Yugoslavia neutrophil counts were determined, and the rate of bone marrow neutrophil Djordje Miljkovic, Institute for Biological Research "Sinisa Stankovic", release was calculated as the increment in blood CD10- neutrophils per 3 Belgrade, Yugoslavia minute interval during each stage of sampling. Vladimir Trajkovic, Institute of Microbiology and Immunology, School of Results Medicine, University of Belgrade S 132 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Marija Mostarica Stojkovic, Institute of Microbiology and Immunology, In conclusion, this study shows that the retinoid atRA has the potential School of Medicine, University of Belgrade, Yugoslavia to increase the expression of IL-1b and its receptor, IL-1RI, in vascular Stanislava Stosic-Grujicic, Institute for Biological Research "Sinisa cells and at the same time decrease the expression of IL-1ra. This study Stankovic", Belgrade, Yugoslavia shows that the retinoid atRA may play a proinfl ammatory role in vascular diseases such as atherosclerosis. The protective effect of the pre-treatment with mycobacterial preparations such as complete Freund's adjuvant (CFA) in various experimental models of autoimmune diseases is well known. Our aim was to investigate the 5058 ability of CFA to confer resistance to experimental autoimmune diabetes induced by multiple low doses of streptozotocin (MLD-SZ) in CBA/H Manipulation of immune cell migration and pro-infl ammatory mice. Single intraperitoneal injection of CFA one week prior to MLD-SZ cytokine expression by Orf virus-encoded virulence factors had a protective effect, as evaluated by plasma glucose levels. In addi- tion, the suppression of disesase was transferred with splenocytes from Lyn M Wise, Virus Research Unit, Department of Microbiology and the CFA-treated animals when given to naïve syngenic recipients together Immunology, University of Otago., New Zealand with the fi rst dose of streptozotocin. In order to explore the mechanisms Catherine A McCaughan, Virus Research Unit, Department of Microbio- underlying the observed protection, mononuclear spleen cells and peri- logy and Immunology, University of Otago., New Zealand toneal macrophages were tested for production of pro-infl ammatory me- Zabeen I Lateef, Virus Research Unit, Department of Microbiology and diators with presumed role in the pathogenesis of diabetes, such as nitric Immunology, University of Otago., New Zealand oxide (NO), oxygen radicals and IL-17, as assessed by Griess reaction, Marie K Inder, Virus Research Unit, Department of Microbiology and NBT test and ELISA, respectively. Cells taken immediately before disease Immunology, University of Otago., New Zealand induction from the CFA-treated animals were, after LPS stimulation, more Andrew A Mercer, Virus Research Unit, Department of Microbiology and potent producers of NO and oxygen radicals compared to cells from non- Immunology, University of Otago., New Zealand treated animals. Interestingly, mononuclear spleen cells of treated animals Stephen B Fleming, Virus Research Unit, Department of Microbiology also produced higher amounts of IL-17. Similar results were obtained with and Immunology, University of Otago., New Zealand cells taken from the animals two weeks after the disease induction. To test whether NO, which has also immunoregulatory functions, is mediator Orf virus (ORFV), a parapoxvirus, causes a highly contagious, pustular of protection induced by CFA, we used aminoguanidine, fairly specifi c skin disease in sheep and is readily transmitted to humans. The ability of inhibitor of inducible NO synthase (iNOS). The multiple intraperitoneal ORFV to survive in the hostile environment of the infected host is criti- administration of aminoguanidine from the day of CFA injection until last cally linked to its expression of multiple virulence factors that manipulate dose of streptozotocin completely abolished the protective effect of CFA. host responses to infection. These factors include homologs of cellular in- Taken together, our data give new insight to the mechanisms of suppres- terleukin-10 (IL-10) and vascular endothelial growth factor (VEGF) and a sion and suggest important role of iNOS and consequent production of NO chemokine-binding protein (CBP). ORFV IL-10 is functionally similar to in the induction of resistance to diabetes by CFA treatment. cellular IL-10 in that it has suppressive effects on infl ammation and Th1 effector function but is a co-stimulator of Th2 lymphocytes, mast cells and B cells. ORFV VEGF recognises VEGF receptor-2 (VEGFR-2), the mi- 5057 togenic receptor for endothelial cells but does not bind VEGFR-1, which regulates haematopoietic cell activation and migration. ORFV CBP binds Induction of IL-1b and IL-1 receptor is associated with reduction infl ammatory CC-chemokines and lymphotactin, which are chemoattrac- of IL-1 receptor antagonist in aortic smooth muscle cells after tants for monocytes, T cells, B cells and neutrophils, but not homeostatic stimulation with all-trans retinoic acid chemokines. We hypothesise that these virally-encoded factors specifi - cally target the infl ammatory and type 1 immune response so as to pro- Ken Jatta, Division of biomedicine, department of caring sciences, Uni- mote ORFV infection. In this study, the abilities of the viral IL-10, VEGF versity of örebro, Örebro, Sweden and CBP to regulate migration and pro-infl ammatory cytokine expression Dick Wågsäter, Division of biomedicine, department of caring sciences, in human THP-1 monocytes were examined by in vitro analyses. ORFV University of örebro, Örebro, Sweden IL-10 inhibited pro-infl ammatory cytokine production in LPS-stimulated Jan Dimberg, Department of natural sciences and biomedicine, Universi- THP-1 cells. ORFV VEGF differed from VEGF-A in that it did not induce ty college of health sciences, Jönköping, Sweden THP-1 migration, which is consistent with its inability to bind VEGFR- Allan Sirsjö, Division of biomedicine, department of caring sciences, 1. It did however stimulate expression of pro-infl ammatory cytokines in University of örebro, Örebro, Sweden THP-1 cells but with a lower potency than VEGF-A. ORFV CBP inhibited CC-chemokine-induced migration of THP-1 cells. The anti-infl ammatory Local immunoregulatory processes in normal vascular biology and patho- activities of the viral CBP and IL-10 and the reduced potency of the viral genesis are mediated partially by the production and response of cytokines VEGF in these assays emphasise the important role the infl ammatory re- by vessel wall cells. The proinfl ammatory cytokine interleukin-1beta sponse plays in controlling ORFV infections. (IL-1b) and the IL-1 receptor antagonist (IL-1ra) are expressed by ath- erosclerotic plaques and may be linked with infl ammatory mechanisms of atherogenesis. The effects of IL-1 are regulated through IL-1 receptor (IL- 5059 1R), which results in a transcriptional induced expression of different cyto- kines, such has TNF-a and IFN-g, and other infl ammatory genes. Existing Short-chain fatty acid, metabolic by-product of periodontopathic evidence shows that retinoids and their ligands are involved in infl amma- bacteria induces apoptosis in gingival fi broblasts isolated from tory mechanisms and that they are found in atherosclerotic plaques. patients with adult periodontitis To determine the effects of the retinoid all-trans retinoic acid (atRA) on IL- 1b, IL-1ra and IL-1RI regulation in vascular cells, aortic smooth muscle Kuniyasu Ochiai, Department of Microbiology, Nihon University, School cells (AOSMC) were stimulated with 1mM atRA. Protein levels in total of Dentistry, Japan cell lysates and media were analysed by ELISA or western blot and mRNA Naoto Suzuki, Department of Biochemistry, Nihon University, School of levels were analysed by real-time RT-PCR. Dentistry, Japan The results show that atRA signifi cantly increases IL-1b in AOSMC. Both Kichibee Otsuka, Department of Biochemistry, Nihon University, School protein and mRNA levels of IL-1RI were also increased in AOSMC by of Dentistry, Japan atRA. Whilst, on other hand the expression of IL-1ra was decreased by Tomoko Kurita-Ochiai, Department of Microbiology and Immunology, atRA in AOSMC. Nihon University, School of Dentistry at Matsudo, Japan Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 133

We have previously demonstrated that butyric acid, an extracellular me- Birgit Bockemuehl, Roche Palo Alto, United States tabolite from periodontopathic bacteria, induces cytotoxicity and apop- Laura Garvin-Queen, Roche Palo Alto, United States tosis in murine- and human T- and B- cells but that gingival fi broblasts Paula N Belloni, Roche Palo Alto, United States (GF) isolated from periodontally healthy human are resistant to butyric Chiradath Satjawatcharaphong, Roche Palo Alto, United States acid-induced apoptosis. In this study, we have isolated fi broblasts from infl amed-periodontal lesions of adult periodontitis patients and examined Background: Retinoic acid (RA), the active metabolite of vitamin A, regu- their susceptibility to apoptosis induced by butyric acid. Butyric acid sig- lates cellular differentiation by activating RA nuclear receptors (RAR). nifi cantly suppressed the viability of infl amed gingival fi broblasts (IGF) In the immune system, RA modulates immune responses, particularly by in a concentration-dependent manner. We observed the apoptotic change favoring Th2 type responses, however the mechanism for this remains such as chromatin condensation and hypodiploid nuclei in IGF treated largely unknown. We sought to elucidate the role retinoic acid receptor- with butyric acid. Incubation of IGF with butyric acid for 20 h resulted alpha (RAR-alpha) in Th2 cell differentiation and Th2 type immune re- in the typical ladder pattern of DNA fragmentation and mitochondrial sponses in vivo that are of importance in pathological situations such as injury. Furthermore, butyric acid-induced apoptosis in IGF was partially allergic asthma. reduced by the inhibitors for initiator caspases (caspase-8 and -9) and ef- Methods: RAR expression was analyzed during Th1 and Th2 cell differ- fecter caspases (caspase-3, -6, and -7). These results suggest that butyric entiation using DO11.10 TCR transgenic T cells. The effect of RAR-alpha acid, one of the main short-chai fatty acids produced by periodontopathic antagonism on Th cell differentiation was evaluated with a highly selective bacteria and one that easily penetrates the oral mucosa, can highly induce inhibitor (RO3308966) in several models, including the TCR transgenic fi broblast-apoptosis followed by destruction of gingival tissues in infl amed T cell system in vitro, in splenocytes from ovalbumin (OVA)-sensitized periodontal lesions. Balb/c mice ex-vivo, as well as in vivo in a murine model of allergic asth- ma where pulmonary eosinophilia, mucus production and Th2 cytokines were quantitated. 5060 Results: RAR-alpha expression increased during Th2 cell differentiation and this was paralleled by increases in GATA3 expression. This was not Immunomodulatory responses by allogeneic mononuclear cells to observed in Th1 cells. RAR-alpha inhibition increased IFN-gamma pro- stored RBC transfusion products duction and Th1 differentiation from naïve Th cells. In differentiated cells, RAR-alpha inhibition increased IFN-gamma production in Th1 cells and Katherine A Patton, Australian Red Cross Blood Service - Victoria, reduced IL-4 production in Th2 cells. When splenocytes from OVA-sen- Australia sitized mice were stimulated with antigen in vitro, RAR-alpha inhibition Geraldine Healey, Australian Red Cross Blood Service - Victoria, Aus- also resulted in increased IFN-gamma and decreased IL-4 production. In tralia a mouse model of asthma, inhibition of RAR-alpha resulted in reduced Rosemary L Sparrow, Australian Red Cross Blood Service - Victoria, allergen-induced lung IL-4, IL-5 and IL-13 levels, accompanied by inhibi- Australia tion of eosinophilia and lung mucus production. Conclusions: This study supports a role for RAR-alpha in the develop- Red blood cell (RBC) transfusion has been implicated in certain adverse ment of Th2 type immune responses and suggests that RAR-alpha inhibi- patient outcomes. Immunomodulation may play a central role. The aim of tion may provide therapeutic benefi t in asthma. this project was to determine the ability of supernatants and cellular frac- tions of routine RBC products to modulate the immune response of alloge- neic mononuclear cells (MNCs). In particular the effects of RBC product 5062 storage and the presence of white blood cells (WBCs) carried over during routine preparation of RBC products were investigated. Immunomodulating effects of milk-derived extract XP-828L RBC products were prepared according to standard blood bank proce- dures. Three types of RBC products were studied: non-WBC-reduced, R Drouin, Advitech, Canada buffy-coat-reduced and WBC-fi ltered RBCs. Samples were collected from G Labbe, Advitect, Canada RBC products on day 1 and at fortnightly time-points until product expiry P E Poubelle, Centre de Recherche en Rheumatologie & Immunologie, (day 42). Samples were centrifuged to obtain supernatant and cellular frac- Canada tions. On the day of sample collection, MNCs were prepared from ABO- C Juneau, Advitech, Canada compatible normal allogeneic donors. Induction of CD11b on allogeneic monocytes following incubation with RBC supernatants or cellular frac- XP-828L is a patented extract of bovine lactoserum obtained from sweet tions was determined by fl ow cytometry. Cytokine release was determined whey by physico-chemical treatments and containing growth factors by incubating MNCs with RBC supernatants for 4-16 h at 37oC and culture (TGFb-2, TGFb-1 & IGF-I) and Ig G. XP-828L has immune effects in supernatants were assessed by ELISA or cytokine-antibody microarrays. vitro by modulating pro-infl ammatory Th1 cytokines. This study evalu- Supernatant and cellular fraction from non-WBC-reduced RBC products ates the ability of XP-828L to decrease the proliferation of ConA-activated induced expression of CD11b on allogeneic monocytes. Buffy-coat-re- lymphocytes from murine spleens and their production of IL-2 & IFN-g. duced and WBC-fi ltered RBC products had minimal effect on monocyte Murine lymphocytes (1.25 x 106 cells/ml) were treated with ConA (1.25 CD11b expression. Cytokine release from MNCs incubated with RBC μg/ml) for 72 hours. Alamar blueÒ was added 48 hours following the supernatant suggested a tenuous balance between proinfl ammatory and start of incubation in order to measure ConA-induced lymphocytes prolif- immunosuppressive responses. All RBC product types induced cytokine eration. XP-828L (50, 100, 500 & 1000 μg/ml), added to the cells for the release from MNCs. whole incubation period, dose-dependently decreased 1) the lymphocytes The results from this study indicate that stored RBC products can modulate proliferation by 17, 23, 39 and 50%, respectively, 2) the production of normal allogeneic MNCs. Both proinfl ammatory and immunosuppressive IL-2 by 39, 54, 54, 80 %, respectively, and of IFN-g by 36, 50, 50, 74%, responses were evident. WBCs in RBC products appear to favour a proin- respectively. In conclusion, these results suggest that XP-828L could have fl ammatory response by MNCs. immunomodulatory functions. This confers to XP-828L, a nutraceuticals, unique properties to address immune-mediated chronic infl ammatory dis- eases such as psoriasis, infl ammatory bowel diseases and related arthropa- 5061 thies.

Retinoic Acid Receptor-alpha Regulates Th2 Cell Differentiation in T cells and Th2 Type Immune Responses In Vivo

Karim Dabbagh, Roche Palo Alto, United States S 134 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Results: Spermine and spermidine decreased the mean fl uorescent intensi- 5063 ties (MFIs) of CD11a in the lymphocyte light scattered region, but not in the monocyte region. The effects of spermidine seem to be weaker than Potent and Selective Antagonists of Vanilloid Receptor 1 spermine, as spermidine concentration in PBMCs cultured with spermi- dine increased beyond clinical settings. Among 42 healthy male volun- Young-Ho Park, AmorePacifi c R&D Center, Korea teers, whole blood spermine levels inversely correlated with the CD11a Young-Ger Suh, College of Pharmacy, Seoul National University, Korea MFIs of cells in the lymphocyte region (r = -0.48, p = 0.001), but not Uhtaek Oh, College of Pharmacy, Seoul National University, Korea with those in the monocyte region. The suppression of the CD11a MFIs Hee-Doo Kim, College of Pharmacy, Sookmyung Women's University, by spermine was observed in a dose- and time-dependent manner and Korea found non-specifi cally on all cell subsets we tested (CD3+, CD4+, CD8+, Hyeung-geun Park, College of Pharmacy, Seoul National University, CD19+, CD45RA+, CD45RO+, CD4+CD45RA+, CD4+CD45RO+, Korea CD8+CD45RA+, CD8+CD45RO+). The expression of adhesion mol- Yeon Su Jeong, AmorePacifi c R&D Center, Korea ecules other than CD11a and CD18 was not decreased by the polyamines. Jin Kyu Choi, AmorePacifi c R&D Center, Korea Conclusions: Spermine suppresses the expression of LFA-1 nonspecifi - Song Seok Shin, AmorePacifi c R&D Center, Korea cally on all cell subsets in the lymphocyte region. Since LFA-1 is one of the most important molecules involved in infl ammatory processes, we Vanilloid receptor-1 (VR1), a member of the transient receptor potential postulate spermine as natural anti-infl ammatory substances. (TRP) ion channel family, is a polymodal nociceptor, responding to ther- mal stimuli, pH, vanilloids, or lipoxygenase products such as anandamide and 12-HPETE. Although the medicinal chemistry goal to exploit vanilloid 5065 agonists as therapeutic products has not yet been achieved, several lines of recent evidence substantially points to a therapeutic potential of VR1 an- Macrolide anti-infl ammatory activity in lipopolysaccharide (LPS) tagonists in pain, neurogenic infl ammation and so forth. We have identifi ed induced infl ammation models several classes of potent and selective VR1 antagonists devoid of vanilloid moiety. The effects of VR1 antagonists were examined with radioactive Vanesa Ivetic Tkalcevic, PLIVA Research Institute Ltd., Croatia ion fl ux experiments, and were subsequently tested in single channel patch Berislav Bosnjak, PLIVA Research Institute Ltd., Croatia clamp assay in cultured rat DRG neurons. In the calcium infl ux and single Ognjen Culic, PLIVA Research Institute Ltd., Croatia channel assay, VR1 antagonistic potencies of some compounds were far Vesna Erakovic, PLIVA Research Institute Ltd., Croatia higher than that of capsazepine with no agonistic stimulation per se. In ac- Boska Hrvacic, PLIVA Research Institute Ltd., Croatia cordance with the action mechanism, VR1 antagonists withdrawn from our research program were free from the undesired excitatory side effects. The Purpose. Macrolides, apart from their antibacterial activity, suppress pro- analgesic activities of the compounds were evaluated in the phenyl-p-ben- infl ammatory cytokine production and migration of infl ammatory cells. zoquinone (PBQ)-induced writhing test in mice. VR1 antagonists showed Therefore, we tested antiinfl ammatory activity of 14-membered macro- unexpectedly potent analgesic activities to suppress writhing responses. lides erythromycin (ERY) and clarithromycin (CLA) and 15-membered Modulatory effect toward tetradecanoyl phorbol acetate (TPA)-induced macrolide azithromycin (AZI), on LPS induced plasma tumor necrosis edema and myeloperoxidase (MPO) accumulation in mouse ear was also factor alpha (TNFα) production and lung neutrophilia. determined to confi rm anti-infl ammatory effect of VR1 antagonists. Topi- Methods. Experiments were performed on BALB/cJ mice. For LPS in- cal administration of VR1 antagonists exhibited potent anti-infl ammatory duced plasma TNFα production, mice were treated intraperitoneally (i.p.) potential in both ear weight increase and MPO activity comparable to with macrolides (15mg/kg) 30min prior to i.p. LPS injection. 120min af- those of indomethacin. The presently available pharmacological/toxico- ter treatment plasma was sampled to determine TNFα concentration by logical profi le of VR1 antagonists strongly suggests their wide therapeutic ELISA. In LPS induced lung neutrophilia, treatment with macrolides potential. (5mg/mouse i.p.) was performed 2h prior to intranasal LPS challenge. Bronchoalveolar lavage and lung sampling were performed 24h after LPS challenge. 5064 Results. In LPS induced plasma TNFα production, CLA signifi cantly re- duced TNFα concentration. Further, in LPS induced lung neutrophilia, Spermine, a natural human polyamine, suppresses LFA-1 expression CLA treatment signifi cantly decreased total cell number, percentage of on human lymphocyte neutrophils and TNFα concentration in bronchoalveolar lavage fl uid (BALF), as well as infl ammatory cell infi ltration into lung tissue. ERY ex- Kuniyasu Soda, Omiya Medical Center, Jichi Medical School, Japan hibited an effect similar to CLA: decreased percentage of neutrophils and Yoshihiko Kano, Omiya Medical Center, Jichi Medical School, Japan interleukin (IL-)6 and TNFα concentration in BALF, and accumulation of Takeshi Nakamura, Omiya Medical Center, Jichi Medical School, Japan granulocytes in lung tissue. AZI signifi cantly reduced IL-6 concentration Masaaki Saitoh, Omiya Medical Center, Jichi Medical School, Japan and markedly, although not signifi cantly, decreased total cell number in Masanobu Kawakami, Omiya Medical Center, Jichi Medical School, BALF. Japan Conclusions. Our results suggest that macrolides could differ in their acute Fumio Konishi, Omiya Medical Center, Jichi Medical School, Japan antiinfl ammatory activity: after single dose administration, 14-membered macrolides seem to be more effective than 15-membered. Further detailed Purpose: LFA-1 (lymphocyte function-associated antigen-1) is one of the testing in chronic models is needed. Namely, unique pharmacokinetic/ac- most important molecules involved in infl ammatory processes. Recently, cumulation properties of some macrolides (especially AZI) could contrib- we have found that human natural polyamines, spermine and spermidine, ute in much greater extent to their antiinfl ammatory activity in chronic suppress the expression of LFA-1 (CD11a and CD18) on human periph- infl ammation models. eral blood mononuclear cells (PBMCs). We further examine the effects of polyamine on the expression of CD11a on various subsets. Methods: PBMCs from healthy volunteers were cultured in RPMI-1640 5066 medium supplemented with 10% human serum. Freshly prepared sperm- ine, spermidine, or putrescine was added to the PBMCs at various con- Discovery of Orally Active JNK Inhibitors centrations, and the effects on the expression of adhesion molecules was estimated by fl ow cytometry. The polyamine concentrations in cell lysate Yoshitaka Satoh, Celgene, United States and whole blood were determined by HPLC. Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 135

It is becoming clear that Jun N-terminal kinases play major roles in human The transcription factor, nuclear factor (NF)-kB is well known as the cen- diseases such as arthritis, asthma, infl ammatory bowel disease, ischemia- tral for lung infl ammation in asthma, but the mechanism of NF-kB acti- reperfusion injury, CNS ailments such as epilepsy, stroke, and Parkinson’s vation is unknown. Induction of mast cell degranulation in normal mice disease, and cancer. Starting from a screening hit, SP600125, we identifi ed failed to induce NF-kB activation in the lungs. Furthermore, in a murime a series of 3,5-disubstituted indazoles as highly potent, selective inhibi- model of asthma, NF-kB activation occurred to a similar extent in mast tors of JNK. Structural optimization leading to the most potent series as cell-defi cient W/Wv mice compared to their littermates. Single i.t. injec- well as effi cacy of lead JNK inhibitors in several animal models will be tion with either anti-chicken gamma globulin IgG1 mAb or anti-OVA IgG discussed. Abs, followed by airway challenge with the specifi c antigen, was capable of inducing NF-kB activation in the airway epithelial cells as well as the expression of NF-kB-dependent infl ammatory proteins. The IgG-immune 5067 complex (IC)-induced activation of NF-kB resulted in the development of airway hyperresponsiveness, but failed to induce other cardinal features of Identifi cation of Potent and Selective MMP-13 Inhibitors asthma such as airway obstruction, an increase in the airway levels of Th2 cytokines, and airway eosinophilia. These data indicate that IgG-IC, but Wei Li,a* Yonghan Hu,a Jianchang Li,a Thomas S. Rush III,a Dianne not mast cells/IgE Abs, plays an important role in NF-kB activation in the DeVincentis,a Xuemei Du,b Jennifer R. Thomason,a Jason S. Xiang,a airway epithelial cells, and would help delineate the exact roles of IgG-IC- Jerauld S. Skotnicki,b Steve Tam,a and Jeremy I. Levin b induced NF-kB activation in the pathogenesis of asthma. a Department of Chemical and Screening Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140,USA bDepartment of Che- mical and Screening Sciences, Wyeth Research, 401 North Middletown 5069 Road, Pearl River, NY 10965,USA Characteristics of a new large animal preclinical model of human MMP-13 (matrix metalloprotainase-13, collagenase-3) is a member of a allergic asthma based on house dust mite family of zinc-containing enzymes that are involved in the degradation and remodeling of extracellular matrix proteins. Exploration of the thera- Robert J Bischof, The University of Melbourne, Australia peutic potential for small molecule inhibitors of these enzymes to treat Kenneth J Snibson, Australia various pathologies has been continuing for more than two decades, and Emmanuel Koumoundouros, Australia many drug candidates generated from this approach have been advanced Els N Meeusen, Australia to clinical trails. Human MMP-13 was fi rst discovered and cloned in hu- man breast carcinoma and was later found expressed by primary and trans- House dust mite (HDM) is the most common allergen associated with hu- formed chondrocytes derived from joint tissue. It is inducible by IL-1. The man allergic asthma. We recently developed a new large animal (sheep) expression of MMP-13 in osteoarthritic cartilage and its activity against model of human asthma based on sensitisation to HDM allergen (Bischof type II collagen suggest that this enzyme plays a signifi cant role in carti- et. al., 2003, Clin. Exp. Allergy 33: 367-375). Following airway challenge lage collagen degradation. with HDM, the pathological features observed in this sheep asthma model Early preclinical testing of nonselective MMP inhibitors has shown that closely resemble those of human asthmatic disease, including specifi c IgE these inhibitors are able to prevent the destruction of cartilage in some responses, increased mucus production and a profound lung tissue and animal models of osteoarthritis. However, most clinical trials of broad BAL eosinophilia (10-33% in BAL). Airway challenge with HDM in aller- spectrum MMP inhibitors have been plagued by dose-limiting toxicity in gic (high IgE) sheep is accompanied by an increase in airways resistance the form of musculoskeletal side effects (MSS). A number of theories have post-challenge. We have also demonstrated the effi cacy of various human been proposed for the cause of MSS, including the inhibition of MMP- anti-asthma therapies such as inhaled corticosteroids and oral leukotriene 1, or MMP-14, or sheddases. As a result, interest in more selective MMP- antagonists in reducing the asthmatic responses observed in the HDM 13 inhibitors has increased. sheep asthma model. This relevant and improved large animal model of We now report a promising series of biphenyl 4-sulfonamide carbox- human asthma will facilitate the preclinical screening of drug candidates ylic acids that are potent and selective inhibitors of MMP-13 that spare for experimentation and registration, and can be used for the discovery of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Agg1 novel drug targets. and TACE. By varying the substituents on the benzofuran portion of the molecule, we have been able to modulate the potency and selectivity of this series of inhibitors. 5070 X-Ray data for MMP-13 was examined and computer modeling analyses of the MMP S1’ subsites were performed to enable structure-based design. ELB353, a new selective PDE4-inhibitor with a high safety margin Subtle differences in a loop region of the MMPs have been exploited by and anti-infl ammatory effects in rat and ferret lungs incorporating substituents at the 3- and 4-positions (R1 and R2) of the 4’- benzofuran, affording enhanced selectivity. Hildegard Kuss, elbion AG, Pharmacology, Radebeul, Germany, Germa- SAR of over 40 compounds will be presented. Synthesis of these com- ny pounds will also be briefl y discussed. Joachim Hoppmann, elbion AG, Pharmacology, Radebeul, Germany, Germany Ute Egerland, elbion AG, Molecular Biology, Radebeul, Germany, 5068 Germany Norbert Hoefgen, elbion AG, Chemistry, Radebeul, Germany, Germany Mechanism for NF-kB activation in asthma: Key role of IgG immune Thomas Kronbach, elbion AG, Radebeul, Germany, Germany complex Chris Rundfeldt, elbion AG, Pharmacology, Radebeul, Germany, Ger- many Hern Ku Lee, Department of Immunology and Research Center for Allergic Immune Diseases, Chonbuk National University Medical School, Introduction: In an effort to develop a new phosphodiesterase type 4 Korea (PDE4) inhibitor with superior tolerability, ELB353, a 7 azaindole deriva- Young Suk Kim, Korea tive, was selected. The compound is a potent and selective PDE4 inhibitor

Young Man Lee, Korea (IC50 PMNL: 2.1 nM) with a low affi nity to the rolipram binding site. Here Nam In Kang, Korea the therapeutic effect and the tolerability was assessed. Methods: Lung neutrophilia was induced by exposing Lewis rats or ferrets to a lipopolysaccharide (LPS) aerosol. Two hours prior to LPS-exposure S 136 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

ELB353 was given to rats orally while ferrets were dosed intragastrically Aspirin and most non-steroidal anti-infl ammatory drugs (NSAIDs) cause with a catheter under slight anaesthesia. The compound was suspended severe and prolonged bronchospasm in patients with aspirin intolerant in 10% PEG300 and 90% watery tylose. Neutrophilia in bronchoalveolar asthma (AIA). It is believed that NSAIDs shunt the metabolism of ara- lavage fl uid was assessed 6 hours after LPS-exposure. Emesis and CNS chidonic acid to the lipoxygenase pathway by inhibiting cyclooxygenase side effects were observed in separate groups of ferrets. and hence leading to an enhanced synthesis of cysteinyl leukotrienes (cys- Results: Treatment of ferrets with single doses of ELB353 in the range 0.1- LT). Although the number of activated mast cells is increased in AIA pa-

10 mg/kg po caused a dose-dependent inhibition of neutrophilia (ID50: 0.83 tients, the contribution of these cells to the increased cys-LT detected in mg/kg). In Lewis rats neutrophilia is reduced at the doses of 0.1 and 1.0 AIA patients is not defi ned. We hence compared the release of cys-LT in mg/kg po, by 46 and 80 %, respectively. Rofl umilast, orally administered mast cells cultured from normal subjects and asthmatic patients with or at the same doses and time interval inhibits lung neutrophila by 48 and without aspirin sensitivity. CD34+ progenitor cells were isolated from 83%. Both, emesis and CNS side effects limit the therapeutic application fresh peripheral blood of subjects who had given prior consent and mature of currently known PDE4 inhibitors. However, no emesis and notable CNS mast cells were obtained after culturing these progenitors with IL-3, IL-6 side effects could be induced after oral/intragastrical administration of up and SCF for 12 weeks. The amount of cys-LT released was detected by to 60 mg/kg ELB353 a commercial ELISA kit. The basal spontaneous release of cys-LT were Conclusion: The data indicate that ELB353 is a potent and tolerable new similar in the three groups of subjects, being 0.8±0.2, 1,1±0.5 and 1.4±0.5 drug candidate. The effi cacy is comparable to rofl umilast. The compound ng/106 cells respectively for normal, aspirin tolerant asthma (ATA) and is excel-lently tolerated and no CNS related side effects could be observed. AIA subjects. Upon immunological activation with anti-IgE, mast cells Based on the long duration of action a once daily treatment of infl amma- from AIA patients produced signifi cantly more cys-LT (6.9±2.1 ng/106 tory diseases in-cluding COPD is targeted. cells, after corrected for basal release) than in other two groups (2.3±0.8 Supported by the EU fund for reg. dev. (EFRE) and by the Free State of and 1.9±0.5 ng/106 cells respectively). The elevated anti-IgE induced cys- Saxony, SAB 8093 LT release in AIA patients was suppressed by increasing concentration of

PGE2 (0.01 to 1 μM) but no suppression of cys-LT release were observed in the other two groups. Our results hence suggest that mast cells in AIA

5071 patients intrinsically over-produce cys-LT and endogenous PGE2 normally suppresses such excessive cys-LT production. Consequently reduction of

Modulation of pulmonary allergic response by bradykinin PGE2 production in AIA patients after ingestion of NSAIDs removes the antagonists endogenous control of cys-LT overproduction and hence resulting in aspi- rin sensitive asthma attack. Luciana M C Vasquez-Pinto, Univesity of Sao Paulo, Brazil Pierre Sirois, Universite de Sherbrooke, Canada Sonia Jancar, University of Sao Paulo, Brazil 5073

The airway remodeling that occurs in asthma involves collagen deposi- A potential therapeutic role for the fi larial nematode secreted tion, thickening of basement membrane and other changes that affects lung product, ES-62 in Asthma function. There is evidence for eosinophils and bradykinin (Bk) involve- ment. In this study we investigated the effect of Bk receptor antagonists on Dorothy E Kean, University of Glasgow, United Kingdom eosinophil migration/activation, peribronchovascular collagen deposition, Alirio J Melendez, Singapore TGF-? and VEGF production in allergic lung infl ammation. Mice were Charles McSharry, United Kingdom immunized with OVA/alum, 2 ip injections one week apart, and challenged Margaret M Harnett, United Kingdom with aerosolized OVA on days 14 and 21. Bronchoalveolar (BAL) cells William Harnett, United Kingdom were harvested at days 22 and 28, cytocentrifuged and stained with Hema3 or OsO4 for cells or lipid bodies count, respectively. VEGF and TGF-? Asthma is generally accepted as refl ecting a polarisation of the immune levels in BAL were measured by ELISA and localized in the tissue, as well response towards a TH2 phenotype. In developing countries, however, the as bradykinin B1 receptor, by immunohistochemistry. Collagen was mea- majority of fi larial nematode-infected individuals demonstrate a TH2 im- sured by digital image analysis in lung slices stained with Sirius Red. The munological phenotype yet do not show an increased incidence of asthma. bradykinin B1(R954) or B2 (HOE140) receptor antagonists were given It has therefore been proposed that immunomodulatory products secreted ip before each aerosol challenge. It was found that BAL eosinophilia and by the parasites suppress an infl ammatory phenotype. ES-62, a phosphor- number of lipid bodies/eosinophil was signifi cantly increased by HOE140 ylcholine-containing glycoprotein, is one such product. We have therefore and reduced by R954. The peribronchovascular collagen and BAL VEGF investigated the prophylactic and therapeutic effects of ES-62 on the ov- levels were increased at day 28 compared to control group and treatment albumin model of murine asthma airway infl ammation and found ES-62 with the antagonists had no effect. BAL TGF? levels were increased by to signifi cantly attenuate the pulmonary infl ammation in sensitised mice. both HOE140 and R954 at day 28. TGF-? was localized in the vessel In particular, ES-62 caused a reduction in lung eosinophilia and associ- muscular layer, VEGF in bronchial epithelial cells and B1 receptor in the ated infl ammatory cytokines. In addition, in prophylactic regimes, ES-62 endothelium. These results show that, in this model of asthma, collagen caused a selective reduction in both Ova- and total IgE levels. Finally, deposition does not correlate with eosinophils infi ltration. Moreover, the we have also found ES-62 to inhibit Fc(epsilon)R1-induced release of the Bk constitutive receptors down regulate eosinophil infi ltration/activation mediators (hexosaminidase, PGE2, LTB4 and infl ammatory cytokines) of whereas the inducible receptor has a pro-infl ammatory effect allergy from human mast cells. ES-62 acts by blocking key Fc(epsilon)R1- coupled signal transduction pathways and ultimately inhibiting NF- (kappa)B activation. 5072

PGE2 suppresses over-production of cysteinyl leukotrienes in aspirin 5074 intolerant asthma patients Defi cient Sputum Anti-Protease Defenses in Neutrophilic and Hang Yung A Lau, Department of Pharmacology, Chinese University of Eosinophilic Asthma Hong Kong, Hong Kong Xian-song Wang, Department of Pharmacology, Chinese University of Jodie L Simpson, School of Medical Practice and Population Health, Hong Kong, Hong Kong The University of Newcastle and Department of Respiratory and Sleep Young Yuen A Wu, Department of Medicine, The University of Hong Medicine,, Australia Kong, Hong Kong Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 137

Peter G Gibson, School of Medical Practice and Population Health, The University of Newcastle and Department of Respiratory and Sleep 5076 Medicine,, Australia Enhanced IL-8 Release from Neutrophils in Non-Eosinophilic Proteolytic enzyme release in the absence of adequate anti-protease de- Asthma fence leads to tissue destruction and may contribute to airway remodel- ing in asthma. Aim: To examine the role of proteolytic enzymes and their Katherine J Baines, School of Biomedical Sciences, The University of inhibitors in the infl ammatory subtypes of asthma. Methods: Subjects with Newcastle, NSW and Department of Respiratory and Sleep Medicine, asthma (n=93) were classifi ed by induced sputum cell counts as: neutro- Hunter Medica, Australia philic (NA, neutrophils ≥ 61%), eosinophilic (EA, eosinophils ≥1%)or Noreen V Bell, Department of Respiratory and Sleep Medicine, Hunter paucigranulocytic (PGA, eosinophils <1% and neutrophils <61%). Sub- Medical Research Institute, John Hunter Hospital, NSW 2305., Australia jects with asthma and healthy controls (n=42) underwent sputum induction. Jodie L Simpson, Department of Respiratory and Sleep Medicine, Hunter Sputum supernatant was assayed for active neutrophil elastase (NE), total Medical Research Institute, John Hunter Hospital, NSW 2305 and School and active MMP-9, alpha-1-antitrypsin, secretory leucoprotease inhibitor of M, Australia (SLPI), and TIMP-1. Results: Asthma with neutrophilic infl ammation had Rodney J Scott, School of Biomedical Sciences, The University of New- increased neutrophil elastase, MMP-9 bound to TIMP-1 and alpha1 anti- castle, NSW., Australia trypsin. The NE was active but 99% of the MMP-9 was inactive and bound Michael J Boyle, Department of Immunology and Infectious Disease, to TIMP-1. In contrast, eosinophilic asthma was associated with increased John Hunter Hospital, NSW 2305., Australia active MMP-9. There was also active MMP-9, but not neutrophil elastase, Peter G Gibson, School of Medical Practice and Population Health, The in paucigranulocytic asthma. Conclusion: There is differential proteolytic University of Newcastle, NSW and Department of Respiratory and Sleep activity in asthma. Active MMP-9 is a feature of asthma, especially with Medi, Australia eosinophilic infl ammation. MMP-9 activity is inhibited in asthma with neutrophilic infl ammation, where elastase activity is the dominant proteo- Background: IL-8 is a chemoattractant involved in the migration of neu- lytic enzyme. Anti-protease defenses are defi cient or impaired in subjects trophils from the blood to tissue sites of infl ammation. Non-Eosinophilic with asthma leading to increased protease activity. Asthma (NEA) is a recently described phenotype defi ned as the persistence of asthma symptoms in the absence of eosinophilia. Increased neutrophil counts and sputum IL-8 are characteristics of NEA. Aim: To investigate 5075 the release of IL-8 from blood and sputum granulocytes in subjects with NEA and healthy controls. Methods: Subjects with NEA (n=4) and healthy Antigen doses used for intranasal challenge differently affects airway controls (n=4) underwent sputum induction and blood collection. Sputum infl ammation and airway hyperresponsiveness and peripheral blood granulocytes were isolated using magnetic cell sepa- ration with CD16 microbeads. Each cell fraction (105cells/ml) was cul- Berislav Bosnjak, PLIVA Research Institute Ltd., Croatia tured in RPMI 1640 1% FCS with or without LPS stimulation (100ng/ml). Vanesa Ivetic Tkalcevic, PLIVA Research Institute Ltd., Croatia Cells and cell culture supernatants were collected at 24hrs and IL-8 protein Daniela Perovic, PLIVA Research Institute Ltd., Croatia was measured using ELISA. Results: Peripheral blood neutrophils iso- Zeljko Ferencic, PLIVA Research Institute Ltd., Croatia lated from subjects with NEA spontaneously released signifi cantly higher Miroslava Dominis Kramaric, PLIVA Research Institute Ltd., Croatia amounts of IL-8 when compared to healthy controls (p=0.02). There was Ognjen Culic, PLIVA Research Institute Ltd., Croatia no difference in IL-8 release from unstimulated blood eosinophils in the Vesna Erakovic, PLIVA Research Institute Ltd., Croatia different groups. In NEA, peripheral blood eosinophils and neutrophils Boska Hrvacic, PLIVA Research Institute Ltd., Croatia responded to LPS in a similar manner to healthy controls. Unstimulated sputum neutrophils released signifi cantly higher amounts of IL-8 than pe- Purpose. Airway infl ammation (AI) and hyperresponsiveness (AHR) are ripheral blood neutrophils (p=0.02) but were refractory to LPS stimula- two asthma hallmarks. AHR is believed to be a result of AI induced chang- tion. Conclusions: In NEA, circulating neutrophils actively release higher es in lung tissue. We hypothesized that intranasal challenge of mice with amounts of IL-8. The increased airway IL-8 release compared to systemic varying antigen doses could affect differently AI and AHR, and therefore IL-8 release from unstimulated neutrophils demonstrates the increase in could imply which AI components are involved in AHR development. activity of neutrophils once they have migrated to the tissue. Constant ex- Methods. Ovalbumin (OVA) sensitized BALB/cJ male mice were chal- posure of the airways to LPS may explain the unresponsiveness of airway lenged intranasally with either of two OVA doses (25 or 100 mg/mice). cells to LPS in this in vitro setting. AHR to methacholine was measured non-invasively 24h after challenge and bronchoalveolar lavage was performed 24 or 48h after challenge. Lungs were isolated and either infl ammatory infi ltrate was histopathologi- 5077 cally scored (24 and 48h after challenge) or gene expression analysis was done using DNA microarrays (Agilent; 24h after challenge). The response of ex vivo rhinovirus infection of alveolar macrophages Results. There is a correlation of AHR development to antigen dose used from asthmatic and non-asthmatic subjects for challenge: the larger OVA dose induced more prominent AHR. In con- trast to AHR, applied antigen doses were not correlated to accumulation Brian G Oliver, Department of Pharmacology, University of Sydney, of eosinophils, major infl ammatory cell type involved in asthma, either in Australia bronchoalveolar lavage fl uid or in lung tissue. Subsequent DNA microar- Nick J King, Department of Pathology, University of Sydney, Australia ray analysis determined that only larger antigen dose increased expression Judy L Black, Department of Pharmacology, University of Sydney, of two chemokines, macrophage chemoattractant protein 1 (MCP-1) and Australia secreted phosphoprotein 1 (Spp1). Janette K Burgess, Department of Pharmacology, University of Sydney, Conclusions. Altogether, two different doses of antigen used for challenge Australia differently affected eosinophilic AI and AHR, indicating that AHR is more Peter R Johnson, Department of Pharmacology, University of Sydney, sensitive to effects of antigen dose compared to eosinophilic component of Australia infl ammation. It is possible that increased expression of chemokines MCP- Melissa Baraket, Department of Pharmacology, University of Sydney, 1 and Spp1 determined after challenge with larger antigen dose correlates Australia to AHR, most probably through effects of these chemokines on cells that Sebastian L Johnston, Department of Respiratory Medicine, National express their receptors, such as T-lymphocytes and macrophages. Heart and Lung Institute, Imperial College London, United Kingdom Sam Lim, ANZAC research institute, University of Sydney, Australia S 138 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Purpose model to examine some of the structural and functional relationships in- Viral respiratory infection is the most common cause of asthma exacerba- volved in airway remodelling. tions, with viruses being identifi ed in approximately 80% of exacerbations, of which Rhinovirus (RV) is the most common virus type. It is likely that RV-induced infl ammatory mediators initiate asthma exacerbations. 5079 Alveolar macrophages (AM) are the most numerous immune reactive cell type found in the airway lumen and are the principal source of many lung Requirement of fatty acid binding protein aP2 in the pathogenesis of cytokines. The aim of this study was to examine RV induced IL-6, IL-8 allergic airway infl ammation and TNF-alpha from AM obtained from allergic asthmatic donors in com- parison to non-allergic non-asthmatic donors. Bennett Shum, Arthritis and Asthma Research Program, Garvan Institute Methods of Medical Research, and CRC for Asthma, Australia, Australia AM obtained by bronchoalveolar lavage from 20 atopic asthmatics (mean Charles R Mackay, Arthritis and Asthma Research Program, Garvan

% predicted FEV1 81.4) and 9 normal healthy volunteers (mean% predict- Institute of Medical Research, and CRC for Asthma, Australia, Australia ed FEV1 94) were exposed to 1 MOI of either infectious or UV inactivated Cem Z Gorgun, Division of Biological Sciences and Department of RV serotype 16. Cytokine release into the cell free tissue culture medium Genetics and Complex Diseases, Harvard School of Public Health, MA, was assayed by ELISA. United States Results Melinda J Frost, Arthritis and Asthma Research Program, Garvan Insti- Only infectious RV induced cytokine release. RV induced TNF-alpha tute of Medical Research, and CRC for Asthma, Australia, Australia in AM derived from both asthmatic (P<0.01, n=20) and non-asthmatic Rakesh K Kumar, School of Medical Sciences, The University of New (p<0.05, n=9) volunteers, however, there was no difference in induction South Wales, Australia, Australia between the two groups. Interestingly, a negative correlation between air- Gokhan S Hotamisligil, Division of Biological Sciences and Department way obstruction (FEV1/FVC %) and rhinovirus-induced TNF was found of Genetics and Complex Diseases, Harvard School of Public Health, in the asthmatic subjects (r= -0.4987, p<0.05, n=20, Pearson correlation). MA, United States RV induced IL-6 in AM derived only from asthmatic donors (p<0.01, Michael S Rolph, Arthritis and Asthma Research Program, Garvan Insti- n=20). No induction in IL-8 protein was found in either cell type. tute of Medical Research, and CRC for Asthma, Australia, Australia Conclusions For the fi st time the response of AM from asthmatic subjects to RV has Using Affymetrix oligonucleotide arrays we have established for the fi rst been examined. Within the asthmatic cohort, disease severity negatively time that the adipocyte fatty acid binding protein aP2 as one of the most correlated with RV-induced TNF-alpha release (i.e. ↑ severity ↓ TNF- strongly regulated genes in human bronchial epithelium (HBE) after stim- alpha), possibly due to persistent severe airways infl ammation. Overall ulation by the Th2 cytokines IL-4 and IL-13. aP2 expression had previ- intrinsic differences in the response of airway cells from asthmatic and ously thought to be exclusive to adipocytes and macrophages. In HBE, non-asthmatic donors were observed, potentially providing a mechanistic we show that aP2 is also signifi cantly down-regulated by the Th1 cytokine basis behind RV induced asthma exacerbation. IFN-gamma. Upregulation by Th2 and downregulation by Th1 cytokines strongly implicates aP2 as a participant in allergy associated disease. Fol- lowing these in vitro fi ndings, we found airway epithelium of mice with 5078 ovalbumin-induced allergic airway infl ammation had markedly increased aP2 expression compared to normal mice. Importantly, aP2 defi cient mice A novel large animal model for airway remodelling and chronic were protected from several key features of allergic airway infl ammation, asthma including reduced airway eosinophilia, peribronchovascular infl ammation and pulmonary allergic cytokine production. Finally, we investigated the Ken J Snibson, University of Melbourne, Australia mechanism for this, and have linked aP2 as a regulator of PPAR-gamma Emmanuel Koumoundouros, Australia activity, a nuclear receptor with well documented anti-infl ammatory activ- Rob J Bischof, University of Melbourne, Australia ity. Taken together, our data represents a paradigm shift for the role of aP2 Els N Meeusen, Australia beyond simple fatty acid transportation in adipocytes, to airway epithelial function with relevance to asthma. It is now recognized that long-term functional and structural changes to lung tissues (airway remodelling) in chronic asthmatics causes signifi cant increases in morbidity. To investigate some of the mechanisms involved 5080 in airway remodeling, we have developed a large animal model based on repeated lung challenges to sheep sensitized to house dust mite (HDM). Neonatal chlamydial infection inhibits the development of allergic House dust mite was administered as either an aerosol or infusion on a airway infl ammation in adult mice weekly basis over a six month period. A morphometric analysis of stained histological sections of lung tissues revealed that a number of sheep Jay C Horvat, The University of Newcastle & Hunter Medical Research showed statistically signifi cant increases in peribronchiolar collagen and Institute, Australia smooth muscle in HDM-challenged compartments compared with con- Julie A Preston, The University of Newcastle & Hunter Medical Research trols. Increased numbers of mast cells were present in HDM-challenged Institute, Australia lung compartments compared with untreated control lung compartments. Nicole G Newcombe, The University of Newcastle & Hunter Medical Immunohistochemistry using a panel of cell surface markers, revealed that Research Institute, Australia there were elevated numbers of CD4, CD5 and gamma/delta lymphocytes Angela L Ferguson, Australia and IgE-positive mast cells in HDM challenged lung compartments com- Peter G Gibson, The Hunter Medical Research Institute, Australia pared to untreated control compartments. An analysis of lung mechanics Kenneth W Beagley, The University of Newcastle & Hunter Medical showed that these sheep had deteriorating lung function in that baseline Research Institute, Australia airway resistance increased two-fold over the challenge period, and this Paul S Foster, The University of Newcastle & Hunter Medical Research increase could not be reversed with bronchodilator aerosols. The early Institute, Australia asthmatic response (i.e. bronchoconstriction induced in the fi rst hour after Philip M Hansbro, The University of Newcastle & Hunter Medical an individual HDM challenge) became progressively increased in a group Research Institute, Australia of responder sheep over the duration of the repeated challenge regime. A L Wade, The University of Newcastle & Hunter Medical Research Airway hyperesponsiveness to the bronchoconstrictor carbachol was also Institute signifi cantly increased in sheep challenged with HDM compared to control G Kaiko, The University of Newcastle & Hunter Medical Research sheep. These results show that sheep provide for an appropriate animal Institute Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 139

The effects of early life bacterial infection on the development of the im- mune system remain unknown. There is an association between chlamyd- 5082 ial infection and asthma in adults but the association in childhood is con- troversial as is the role of early life infection on predisposition to disease. Dexamethasone inhibits chitinase expression in allergic airway We aimed to determine the effect of a neonatal chlamydial infection on the infl ammation in mice development of allergic airway infl ammation (AAI) in response to the Th2 inducing antigen (ovalbumin (OVA)) in adulthood. WS Fred Wong, National University of Singapore, Singapore Models of neonatal chlamydial lung infection (fi rst 24 hrs of life) and AAI Jing Zhao, National University of Singapore, Singapore in adults (6 weeks-old) that are representative of human disease were es- tablished in BALB/c mice. Neonates were given a chlamydial lung infec- Asthma is a chronic lung disorder characterized by airway infl ammation, tion at 6 weeks exposed to OVA inhalation or intra-peritoneal injection. airway hyperresponsiveness and airway remodeling. The mechanisms Mice were challenged intra-nasally with OVA at 9 weeks and features of underlying the pathogenesis of asthma are still not fully understood. The AAI were characterised and compared with uninfected controls that were present study investigated the effects of dexamethasone on the global pro- exposed to OVA. tein expression in bronchoalveolar lavage (BAL) fl uid from mice with After infection mild chlamydial lung disease was evident (days 10 & 15; allergic airway infl ammation using proteomics approach. BALB/c mice increased chlamydial numbers and histopathology in the lung accompa- sensitized and challenged with ovalbumin (OVA) developed airway eosin- nied by reduced growth rate). Infection resolved and mouse growth rates ophilia, mucus hypersecretion and elevation of immunoglobulin E (IgE). recovered by 30 days. Neonatal infection resulted in a signifi cant decrease Dexamethasone, given intratracheally, dose-dependently inhibited OVA- in circulating and pulmonary eosinophils and mucous cell hyperplasia in induced airway infl ammation. BAL fl uid proteins were resolved by 2-di- response to OVA sensitisation and challenge in adulthood, compared to mensional (2-D) gel electrophoresis and identifi ed by MALDI-TOF mass mice that were not infected. These effects were associated with a shift spectrometry. Allergic airway infl ammation increased expression of a fam- from T helper cell-type (Th) 2 to Th1 responses. However, lung function ily of chitinases (acidic mammalian chitinase (AMCase), Ym1 and Ym2), in adulthood was impaired by infection in early life and did not improve a recently described family of pro-infl ammatory mediators; lungkine, a the deleterious effects on lung function that resulted from sensitisation and chemokine for neutrophils; gob-5, a protein that mediates mucus secre- challenge to OVA. tion; and surfactant protein-D (SP-D), a C-type lectin capable of modulat- Thus, a chlamydial infection in early life attenuates the development of ing infl ammatory responses. Dexamethasone signifi cantly modulated the eosinophilia and mucous cell hyperplasia, but does not have benefi cial ef- optical density of 26 protein spots in the 2-D gels. Of particular interests fects on decreased lung function in allergic airway infl ammation in adult- are the down-regulatory effects of dexamethasone on BAL fl uid levels of hood. These cellular effects are associated with a shift from Th2 to Th1 chitinases, lungkine, gob-5 and SP-D. Inhibition of each of these proteins immune responses to OVA. identifi ed may contribute to the anti-infl ammatory effect of dexametha- sone in asthma. In summary, proteomics is a powerful tool in unraveling novel mechanisms of action of clinically useful drugs in the treatment of 5081 allergic airway infl ammation. We have identifi ed novel anti-infl ammatory mechanisms of action of dexamethasone in asthma. Proteomic Analysis of TNF-α-Stimulated Normal Human Lung Fibroblasts 5083 WP Liao, National University of Singapore, Singapore H Zhu, National University of Singapore, Singapore Mast cells activated via the FcεR1 and TL4 receptors the regulatory Jasmine HP Chan, National University of Singapore, Singapore protein A20 WS Fred Wong, National University of Singapore, Singapore Mary Sisavanh, CRC for Asthma, Arthritis and Infl ammation research The infl ammatory and remodeling processes that underlie asthma result group, Garvan Institue, NSW, Australia from a highly complex interaction between various cell types and cyto- Sue M Liu, CRC for Asthma, Arthritis and Infl ammation research group, kines. Apart from infl ammatory cells, resident cells such as fi broblasts are Garvan Institue, NSW, Australia also proposed to play critical roles in infl ammation and tissue repair. Al- Shane T Grey, Garvan Institue, NSW, Australia though asthma is perceived as a disease with a particular profi le of Th2 Michael S Rolph, CRC for Asthma, Arthritis and Infl ammation research cytokines, increasing evidence indicates that TNF-alpha, a classical Th1 group, Garvan Institue, NSW, Australia cytokine, is also associated with the infl ammatory response that character- izes human asthma. Total proteins from both cell lysates and supernatants Mast cells are critical effectors of the immune response against pathogens. between normal human lung fi broblasts (NHLFs) and TNF-alpha-stimu- Mast cells also play a central role in allergic infl ammatory response, such as lated NHLFs were separated by two-dimensional (2-DE) gel electropho- asthma, and recently, have been found to be essential for the development resis, and proteins with signifi cant changes were subsequently identifi ed of autoimmune arthritis. In contrast to other leukocytes, which undergo by MALDI-TOF mass spectrometry and MS/MS. RT-PCR was further ap- apoptosis after activation, mast cells can recover and be re-activated. The plied to confi rm the proteomics fi ndings. Several proteins were found to be process of restoring basal activity of mast cells must involve coordinated markedly induced by TNF-alpha. These included cell protective proteins: expression of regulatory elements, which has not been widely explored in antiviral MxA GTPase, ubiquitin-like protein ISG-15, serine proteinase mast cells. This study aims to identify pivotal genes involved in regulating inhibitor PAI-2, and anti-oxidant protein MnSOD; and potential airway mast cell activation. Using Affymetrix U133 microarrays, we analysed remodeling proteins: prolyl hydroxylase (PH) alpha subunit, lysyl hy- human cord blood-derived mast cell gene expression and have identifi ed droxylase 2, isoform a (LH2a) and matrix metalloproteinase 1 (MMP-1). A20 as a gene highly up-regulated following IgE receptor cross-linking. Especially, a novel protein (NP) was identifi ed to be induced in fi broblasts Up-regulation of A20 expression was also confi rmed in murine bone mar- in response to TNF-alpha, whose induction was blocked by interferon row-derived mast cells activated either by FcεR1 cross-linking or LPS us- regulatory factor 1 (IRF-1) antisense oligodeoxynucleotide (ODN), sug- ing real-time PCR. A20 has been described as a key negative regulator of gesting its induction is dependent on IRF-1. Using ODN targeting at the NF-kB activation, as well as an inhibitor of TNF-induced apoptosis. More NP resulted in inhibition of TNF-alpha-induced downstream infl ammatory recent studies have shown that A20 is an important negative regulator of targets VCAM-1 and IL-6, indicating NP may be important in TNF-al- LPS-induced cytokine production in macrophages. By over-expressing pha-stimulated infl ammatory response. These results indicate that Th1 A20 in primary mast cells, we found that A20 signifi cantly inhibits NF-kB cytokine TNF-alpha, which is increased in asthmatic airway, can induce activity. Furthermore, A20 also blocked TNF-a promoter activity, which major alterations in global protein expression in NHLFs leading to airway is known to contain NF-kB elements. We found TNF-a, MIP-2 and IL-6 infl ammation and remodeling. mRNA expression following FcεR1 cross-linking and LPS-activation is S 140 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) decreased in mast cells overexpressing A20 by real-time PCR. This data cretion was measured by sandwich ELISA. These were reduced to only suggests that A20 is a negative regulator of mast cell cytokine production. 0.7% and 2.8%, respectively, of control levels, when activated PBMC were treated with 10-6M Dex. Secretion of IL-5 and IL-13 were inhibited to a similar very marked extent, as was secretion of the key Th1 cytokine 5084 IFN-g. By contrast, secretion of the key Th1 cytokine IL-4 was not nearly as markedly inhibited by 10-6M Dex, and was only reduced to 31% of con- Altered pulmonary compartmentalization of activin A and its binding trol values. These results indicate that IL-9 production is very markedly protein follistatin during acute allergic asthma: a kinetic analysis inhibited by Dex, as are IL-5 and IL-13, and that the benefi cial effects of glucocorticoids in the treatment of allergic diseases may in part be caused Charles L Hardy, Monash University, Australia by inhibition of secretion of these cytokines. By contrast, IL-4 secretion, Anne E O'Connor, Monash University, Australia although inhibited by Dex, is not nearly as markedly affected. This may Jun Yao, Monash University, Australia explain published observations that glucocorticoid treatment can lead to Kimberly Sebire, Monash University, Australia the skewing of immune responses towards the Th2 phenotype. David M de Kretser, Monash University, Australia Jennifer M Rolland, Monash University, Australia David J Phillips, Monash University, Australia 5086 Robyn E O'Hehir, Monash University & Alfred Hospital, Australia GM-CSF is required for pulmonary eosinophil infi ltration, IL-5 Asthma is a chronic disease of the airways characterized by infl ammation, production and CCR3 expression in a murine model of allergic airway obstruction and bronchial hyperreactivity. TGF-β and related cyto- asthma kines are implicated in asthma pathogenesis via roles in immunomodula- tion and airway wall remodeling. Activins are members of the TGF-beta Yung-Chang Su, Arthritis and Asthma Research Program, Garvan Institu- superfamily, with diverse effects on cellular differentiation, organogenesis te of Medical Research, Australia and infl ammation. The biological effects of activin A are neutralized by Michael S Rolph, Arthritis and Asthma Research Program, Garvan Insti- a soluble binding protein, follistatin. Although recent fi ndings suggest a tute of Medical Research, Australia role for activin A in asthma, detailed kinetic analysis of activin A and fol- William A Sewell, Arthritis and Asthma Research Program, Garvan Insti- listatin compartmentalization during acute experimental allergic asthma tute of Medical Research, Australia has not been performed. This was examined in the context of key markers of the pulmonary allergic immune response in ovalbumin sensitized and Granulocyte-macrophage colony stimulating-factor (GM-CSF) plays a role challenged mice. Activin A and follistatin concentrations in bronchoal- in the growth, development, and maturation of bone marrow hemopoietic veolar lavage fl uid (BALF) peaked after 3 to 4 challenges. This response cells into mature blood cells. It has also been proposed that GM-CSF may coincided with maximal airway eosinophil numbers, and IL-4 and IL-5 be involved in potentiating the function of infl ammatory cells participat- production in lung-draining lymph nodes. Strikingly, these changes were ing in allergic infl ammation. In our study, GM-CSF knockout (KO) mice paralleled by a loss of activin A and follistatin localization by airway epi- were used to investigate the role of GM-CSF in a murine model of allergic thelial cells with simultaneous metaplasia of these cells to a mucus-secret- asthma. In the lungs, eosinophils were seen in the perivascular areas but ing phenotype. Although BALF activin A and follistatin concentrations, not in the peribronchial areas of allergic KO mice, compared to the allergic airways eosinophilia and Th2 cytokine production had returned to baseline wild-type (WT) mice in which eosinophilia appeared in both areas. IL-5 10 days after the fi nal challenge, there was a sustained loss of activin A and production in the lung tissue and bronchoalveolar lavage fl uid was reduced follistatin localization by airway epithelium. Collectively these fi ndings in allergic KO mice, compared to allergic WT mice. However, IgE and suggest that airway epithelium is a major store of activin A and follistatin IL-4 were produced at similar levels in KO and WT mice. The chemokine in the normal lung. In response to Th2-type infl ammatory stimuli these eotaxin, and its ligand, CCR3, which is expressed on several cell types in- cells undergo metaplasia and release activin A and follistatin into the sur- volved in allergic infl ammation, were studied using real-time PCR. CCR3 rounding tissue. was signifi cantly reduced in allergic KO mice, compared to allergic WT mice, whereas eotaxin expression was at similar levels in allergic KO and WT mice. Our fi ndings revealed that GM-CSF is essential to mediate the 5085 eosinophil infi ltration, the production of IL-5 and the expression of CCR3 in allergic pulmonary infl ammation. Inhibition of IL-9 production by dexamethasone and comparison with other Th2 cytokines 5087 William A Sewell, Garvan Institute of Medical Research, NSW, Australia Lauren E Holz, Garvan Institute of Medical Research, NSW, Australia Sterolides - a new class of potent anti-infl ammatory compounds Kristoffer P Jakobsen, Garvan Institute of Medical Research, NSW, Australia Mladen Mercep, PLIVA Research Institute Ltd., Zagreb, Croatia Francoise Cormont, Ludwig Institute of Cancer Research, Brussels, Linda Tomaskovic, PLIVA Research Institute Ltd., Zagreb, Croatia Belgium Boska Hrvacic, PLIVA Research Institute Ltd., Zagreb, Croatia Jacques Van Snick, Ludwig Institute of Cancer Research, Brussels, Stribor Markovic, PLIVA Research Institute Ltd., Zagreb, Croatia Belgium Oresta Makaruha Stegic, PLIVA Research Institute Ltd., Zagreb, Croatia Visnja Poljak, PLIVA Research Institute Ltd., Zagreb, Croatia The effects of IL-9 include stimulation of mucus production, mast cell Marijana Komac, PLIVA Research Institute Ltd., Zagreb, Croatia proliferation, eosinophil function, and IgE secretion, which are all features Milan Mesic, PLIVA Research Institute Ltd., Zagreb, Croatia of allergic infl ammation and parasite infestation. IL-9 is recognized as a cytokine produced by Th2 cells, which also produce IL-4, IL-5 and IL- Purpose: Steroids are the most potent anti-infl ammatory drugs and are a 13. Glucocorticoids have potent anti-infl ammatory effects, and are widely mainstay of asthma therapy. However, their use is associated with a num- used in the treatment of allergic conditions. We examined the effect of the ber of unwanted side activities. The aim was to generate new class of anti- glucocorticoid dexamethasone (Dex) on IL-9 mRNA expression and pro- infl ammatory molecules that will have anti-infl ammatory activity similar tein secretion, and compared the effects on IL-9 with those on other Th2 to that of standard steroids but would lack their typical side effects. cytokines. Peripheral blood mononuclear cells (PBMC) were prepared Methods and results: We created a novel class of anti-infl ammatory com- from healthy human volunteers and activated with OKT3. IL-9 mRNA pounds by synthesizing stable steroid-macrolide conjugates - "sterolides". abundance was measured by quantitative RT-PCR, and IL-9 protein se- Idea was to combine property of macrolides to preferentially accumulate in Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 141 immune cells, especially in phagocyte cells (peripheral blood mononuclear old, 20-25g body weight. Sixty mice were divided into three groups, each cells, peritoneal and alveolar macrophages) with potent anti-infl ammatory group had ten C/EBPβ+/+ mice and ten C/EBPβ -/- mice. Mice were anes- activity comparable or superior to classic steroids. These molecules were thetized by inhalation of isofl urane, and 1x105 cfu Staph aureus, 1x106 profi led in vitro and tested in a number of infl ammatory models in vivo. cfu E.coli and 1x106 cfu Candida albicans, suspended in 100uL endotoxin- They accumulate in infl ammatory and epithelial cells both in vitro and in free PBS, was intratracheal administead into each group of mice. Six hours vivo. Sterolides show activity similar to that of standard steroids in oval- later, mice were anesthetized with phenobarbital, using sterile technique, bumine induced eosinophilia in mice and rats. They have excellent anti- the lungs were removed, and pulverized with Dounce homogenizer. Lung infl ammatory activity in croton oil induced ear edema and TNBS induced homogenates were diluted in saline, spread onto Luria broth(LB) ampi- colitis in rats. Unlike classic steroids, these molecules had no effect on cillin plates, and incubated. The Staph aureus group were incubated for thymus size, blood glucose or corticosterone levels in rats. 16 hours at the room temperature, the E.coli. group were incubated for Conclusions: Sterolides are a new class of potent anti-infl ammatory com- 16 hours at 37ºC and the Candida albicans group were incuabated for 48 pounds with excellent safety profi le and could present a new therapeutic hours at 30ºC. Bacterial pellets of LB plates were counted respectively. option in the treatment of asthma-allergy and COPD. Results: Bacterial pellets counts of the three groups from C/EBPβ+/+ mice were 39± 8, 43±11,17±7 respectively, while were 162±36, 229±43,182±81 from C/EBPβ-/- mice,respectively.There were signifi cantly differences be- 5088 tween C/EBPβ+/+ mice and C/EBPβ-/- mice(P<0.01,P<0.01,P<0.001). Conclusion:These results indicate that C/EBPβ has the effect of inhibiting CCAAT/EBPB Inhabits Lung Infl ammation in Mice bacterial growth, and C/EBPβ defi ciency mice may predispose to infl am- mation. Present study suggests that C/EBPβ protected against endotoxin- Yan Dong, Dept. of Pediatrics, Tangshan Maternity and Children's induced lung infl ammation. This may play an important role in protecting Hospital, China against lung infl ammation arising from bacterial infection. Dong Bao Pang, Dept. of Pediatrics, Tangshan Maternity and Children's Hospital, China 5090 Purpose:The CCAAT enhancer binding protein(C/EBP) family of tran- scriptional factors contains six members of C/EBP,C/EBP and C/EBP etc. Functional Genomics of Macrophage Differentiation A lot of studies had identifi ed that C/EBP and C/EBP are more involved in infl ammation and immunity of the function. Hang C Dinh, University of Melbourne, Australia Methods: Sixty mice were used for the current studies: C/EBP knock in Glen M Scholz, University of Melbourne, Australia mice(C/EBP +/+) and C/EBP knockout mice(C/EBP -/-), 5-6 week-old, 20- John A Hamilton, University of Melbourne, Australia 25g body weight. Sixty mice were divided into three groups, each group had ten C/EBP +/+ mice and ten C/EBP -/- mice. Mice were anesthetized Macrophages are thought to have a central role in the development of by inhalation of isofl urane, and 1x105 Staph aureus, 1x106 cfu E.coli and chronic infl ammatory diseases such as rheumatoid arthritis. Understand- 1x106 cfu Candida albicans, suspended in 100uL endotoxin-free PBS, was ing the various aspects of macrophage development - that is, their survival, intratracheal administead into each group of mice. Six hours later, mice proliferation, differentiation and activation, are therefore of relevance to were anesthetized with phenobarbital, using sterile technique, the lungs understanding chronic infl ammatory disease development and progres- were removed, and pulverized with Dounce homogenizer. Lung homoge- sion. nates were diluted in saline, spread onto Luria broth(LB) ampicillin plates, Many aspects of macrophage development and function, including differ- and incubated. The Staph aureus group were incubated for 16 hours at the entiation, are regulated by colony-stimulating factor 1 (CSF-1; also known room temperature, the E.coli. group were incubated for 16 hours at 37ºC as macrophage CSF). The differentiation of macrophages has been stud- and the Candida albicans group were incuabated for 48 hours at 30ºC. ied at the gene expression level, leading to the discovery of a number of Bacterial pellets of LB plates were counted respectively. genes important to this process. This study is focused on identifying genes Results: The results show in following chart. Bacterial pellets counts of the involved in macrophage differentiation at the global transcriptomic level, three experiment groups mice. rather on a gene-by-gene basis, as was the case with previous studies. A C/EBP -/- C/EBP+/+ P value CSF-1-induced macrophage differentiation system was developed and val- Staph aureus 162+36 39+ 8 P<0.01 idated, and microarray technology has been employed to investigate differ- E.coli. 229+43 43+11 P<0.01 ential gene expression in this system. Subsequent bioinformatics analysis Candida albicans 182+81 17+7 P<0.001 revealed a list of differentially expressed genes, and also provided insight Conclusions: From the chat above, we can see that bacterial counts from into the pathways and networks involved in macrophage differentiation. C/EBP +/+ than C/EBP-/- mice, these results indicate bacterial growth, and C/EBP defi ciency mice may predispose to infl ammation. Present study suggests that C/EBP protected against endotoxin- induced lung infl amma- 5091 tion. This may play an important role in protecting against lung infl amma- tion arising from bacterial infection. Proteomics of splenic B cells from CD38 knockout mice

Neng Zhang, National University of Singapore, Singapore 5089 Hua Zhu, National University of Singapore, Singapore Wai-Shiu Fred Wong, National University of Singapore, Singapore CCAAT/EBPβ Inhabits Lung Infl ammation in Mice Chan Fong Chang, National University of Singapore, Singapore

Yan Dong, Dept. of Pediatrics, Tangshan Maternity and Children's CD38 is a ~45-kDa type II transmembrane glycoprotein expressed in many Hospital, China tissues, including spleen, thymus, brain and etc. CD38 is initially identifi ed Dong Bao Pang, Dept. of Pediatrics, Tangshan Maternity and Children's as an ectoenzyme capable of catalyzing beta-NAD+ to cyclic ADP-ribose Hospital, China (cADPR), a potent modulator of Ca2+-induced Ca2+-release (CICR) via ryanodine-sensitive Ca2+ channel. CD38 also displays activity as a cell Objective:This study was undertaken to assess whether increased levels of surface receptor in lymphocytes and triggers a variety of responses includ- CCAAT enhancer binding protein(C/EBPβ) would diminish infl ammation ing: cell proliferation, rescue from apoptosis, protein tyrosine phosphory- following intratracheal endotoxin exposure. lation, intracellular calcium mobilization, etc. Previous studies have shown Methods: Sixty mice were used for the current studies: C/EBPβ knockin that CD38 controls neutrophil chemotaxis to bacterial chemoattractants mice(C/EBP β+/+) and C/EBPβ knockout mice(C/EBPβ -/-), 5-6 week- through its production of cyclic ADP-ribose, and acts as a critical regula- S 142 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) tor of infl ammation and innate immune responses. CD38-defi cient mice also exhibit marked defi ciencies in antibody responses to T-cell-dependent 5093 protein antigens, suggesting that CD38 may play an important role in vivo in regulating humoral immune responses. The purpose of the present study Nectin-like protein 2 defi nes a subset of T-cell zone dendritic cells and is to determine differential protein expression in splenic B cells from wild is a ligand for class-I restricted T-cell associated molecule type and CD38 knockout mice by proteomic approach. Novel information on the changes in protein profi les in CD38-defi cient mice may provide new Laurent Galibert, Amgen, United States insights into the functional roles of CD38 in B cells. Mouse splenic B cells Geoff S Diemer, Amgen, United States were isolated using magnetic assisted cell sorting (MACS) and its purity Zhi Liu, Amgen, United States was ascertained by fl ow cytometry. Purifi ed splenic B cells were subjected Richard S Johnson, Amgen, United States to two-dimensional gel electrophoresis (2DE) and differentially expressed Jeffery L Smith, Amgen, United States protein spots were analysed by MALDI-TOF mass spectrometry. The dif- Martin F Wolfson, Amgen, United States ferential expression status of the candidate proteins were further analysed Raymond M Koelling, Amgen, United States by RT-PCR and Western blotting. A total of ~15 differentially expressed Wei Yan, Amgen, United States proteins were identifi ed in the preliminary studies and their identities are consistent with a role of CD38 in cell survival pathway and as an immu- Dendritic cells (DCs)1 are a phenotypically and functionally heterogenous noregulatory molecule. population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell pan- ning approach using antibody phage display technology to identify cell- 5092 surface epitopes specifi cally expressed on human blood BDCA3+ DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated which rec- The use of Biacore for rapid screening and ranking of Fab hits for ognizes a conserved surface antigen expressed on both human BDCA3+ lead selection DCs and mouse CD8a+ DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF or Igsf4), an ad- Mieke Steukers, Dyax s.a., Belgium hesion molecule involved in tumor suppression, synapse formation and Jean-Michel Schaus, Dyax s.a., Belgium spermatogenesis. Thus, Necl2 defi nes a specialized subset of DCs in both Reinhoud van Gool, Dyax s.a., Belgium mouse and human. We further show that Necl2 binds Class-I Restricted T- Anne Hoyoux, Dyax s.a., Belgium cell Associated Molecule (CRTAM), a receptor primarily expressed on ac- Pascale Richalet, BIAsolution Stockholm, Sweden tivated cytotoxic lymphocytes. When present on antigen presenting cells, Daniel J Sexton, Dyax Corp., United States Necl2 regulates IL22 expression by activated CD8+ T cells. We propose Andrew E Nixon, Dyax Corp., United States that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell Marc Vanhove, Dyax s.a., Belgium interactions both within and outside of the immune system.

Purpose Dyax uses its Fab-on-phage display libraries to identify fully human an- 5094 tibodies that bind with high affi nity and specifi city to drug targets. A key step in the library selection and subsequent screening process is the early Investigations into the Genetic Control of the Colonic Microfl ora in characterization of Fab hits to identify leads for further study. Here we Healthy Individuals and Patients with Infl ammatory Bowel Disease describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened as crude periplasmic extracts and Jessica A Stewart, Wakefi eld Gastroenterology Research Institute, New ranked based on their affi nity against the target. Zealand Methods Vinton S Chadwick, Wakefi eld Gastroenterology Research Institute, New The principle of our screening assay was as follows. (i) Clones of interest Zealand were expressed as Fabs in E. coli and periplasmic extracts were prepared. Alan Murray, IVABS, Massey University, New Zealand (ii) Fab concentration in the periplasmic extracts was measured by running the samples over a very high density Protein A chip under conditions of The role of colonic fl ora in immune disorders such as IBD is the focus of full Mass Transfer Limitation. (iii) Periplasmic extracts were run over the much research. Evidence suggests that the microfl ora acts as an environ- target-coated chip and the affi nities of the clones were computed from the mental trigger for infl ammation in predisposed individuals. binding curves. The colonic microfl ora is largely uncultured therefore we have used Results PCR-TTGE to target 16SrRNA genes to produce a population snapshot. Twenty-two clones isolated from a selection campaign on Tie-1, a receptor We studied host genetic control over the fl ora in healthy controls and in kinase involved in angiogenesis, were screened as described above and IBD. Faecal samples were analysed from 13 identical and 7 fraternal twin affi nities were computed from the sensorgrams. The Fab fragments were sets, and 24 unrelated individuals, and from 3 groups of patients 1) IBD then expressed in E. coli, purifi ed from the periplasmic extracts and sub- (CD n=9, UC n=10) 2) healthy controls (n=10) 3) control disease groups jected to a detailed Biacore characterization. The affi nities measured with (n=40). crude and purifi ed preparations of Fab were in very good agreement, the Identical twins microfl ora was very similar (median = 82%) with less simi- difference in KD being less than 2-fold for most clones. larity in non-identical twins (median = 68%). Unrelated individuals had Conclusions the least with a median = 45%. Differences were signifi cant (p<0.037). In Our assay takes advantage of our ability to measure Fab concentration in fraternal twins a negative correlation was found between similarity and crude bacterial extracts with Biacore using very high density Protein A twin's age (p=0.022). chips. The procedure allows the screening of up to 100 clones per week, Signifi cant differences were found between healthy controls and both UC and it is very useful for selecting high-affi nity Fabs from large batches and CD (p=0.000001 and p=0.03). A signifi cant difference was also pres- obtained after library selection or affi nity maturation. ent between CD and UC (p=0.00004). All control disease groups were signifi cantly different to UC(p≤0.005), and CD was signifi cant different to diverticula(p=0.04). Differences were characterised by less similarity in IBD.

All infant twins had high similarity, however increasingly divergent pop- ulations emerged in fraternal twins with age, suggesting the host exerts a degree of genetic control over the developing fl ora. IBD patients may Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 143 share immune based genetic determinants for IBD, and these may affect the composition of the fl ora resulting in higher levels of similarity com- 5097 pared to controls. However IBD groups demonstrated signifi cantly less similarity compared to controls. Therefore there are not common genetic In vitro characterisation of an alveolar macrophage-like determinants for IBD that infl uence microfl ora composition. Alternatively infl ammatory cell model an infl uence may be overwhelmed by disease factors such as the gut’s in- fl ammatory milieu. Anne Kjerrström, Pfi zer Global Research and Development, United Kingdom Jessica Watson, Pfi zer Global Research and Development, United King- 5095 dom Wai Liu, Pfi zer Global Research and Development, United Kingdom Gene expression studies on phospholipase A2 induced pulmonary Gary P Salmon, Pfi zer Global Research and Development, United infl ammation and fl uid accumulation Kingdom

Kandiah Jeyaseelan, Department of Biochemistry, Faculty of Medicine, Purpose: Macrophages are one of the major cell types that contribute to National University of Singapore, Singapore the chronic infl ammation seen in chronic obstructive pulmonary disease Charmian DN Cher, Department of Biochemistry, Faculty of Medicine, (COPD) through elevated production of pro-infl ammatory mediators like National University of Singapore, Singapore MCP-1, GRO-a, IL- 6, IL-8, TNF-a, and MMP-9. We have characterised Arunmozhiarasi Armugam, Department of Biochemistry, Faculty of three differentiated macrophage-like cell systems to develop an in vitro Medicine, National University of Singapore, Singapore model of pro-infl ammatory macrophage function. Siaw C Chai, Department of Biochemistry, Faculty of Medicine, National Methods: Monocyte-derived macrophages (MDM) were differentiated University of Singapore, Singapore from peripheral blood for 8 days. Two monocytic cell lines were dif- Marelyn W Coghlan, Deaprtment of Physiology, Faculty of Medicine, ferentiated for 8 days using a variety of different stimuli. Expression of Monash University, Clayton 3800, Australia macrophage-associated antigens was assessed over time of differentiation. On day 9, the cells were challenged with LPS from E. coli or peptido- The mechanisms of venom phospholipase A2 induced infl ammation and glycan (PGN) from S. aureus for 18 hours before cytokine and MMP-9 fl uid accumulation have been investigated by studying the gene expres- release was measured. Primary human alveolar macrophages (AM) were sion profi les of lungs from rats treated intravenously and intratracheally obtained from UK Human Tissue Bank, and were characterised as above. with neutral phospholipase A2 from Naja sputatrix, using oligonucleotide Pharmacological modulation of cytokine and MMP-9 production was fur- microarrays. We found that the expression of infl ammatory cytokines and ther assessed using reference glucocorticoids, PDE4 inhibitors and p38 related genes was signifi cantly up-regulated while interestingly, the ex- inhibitors. pression of water-transporting membrane channels such as aquaporins and Results: Only one of the monocytic cell lines, THP-1, and MDM exhib- the Na+/K+-ATPase pump was decreased. These observations not only ited increased cell surface expression of macrophage scavenger receptor-1, shed light on the pathways activated by venom phospholipase A2 during MMP-12, and calprotectin, similar to AM staining, during the course of pulmonary infl ammation and consequently on the causes of respiratory differentiation. High levels of cytokine production were seen from both failure upon snake envenomation but also provide a clue to the mecha- MDM and AM after LPS or PGN challenge, while differentiated THP-1 nism by which endogenous phospholipase A2 could mediate infl ammation and U937 cells had low to undetectable levels of most pro-infl ammatory in conditions such as allergy and rheumatoid arthritis. mediators measured. MMP-9 activity could not be detected in any of the three cell systems at day 0, while high levels could be detected in MDM at day 8 and 9. 5096 Conclusions: MDM exhibit similar in vitro pro-infl ammatory responses as AM and thus represent an attractive cell model for studying the role Novel macrophage-restricted gene involved in infl ammation of macrophages in infl ammatory diseases. Pharmacological modulation of pro-infl ammatory function of these cells will be presented. Vera M Ripoll, CRC for Chronic Infl ammatory Diseases, Institute for Molecular Biosciences. University of Queensland, Australia Timothy Ravasi, CRC for Chronic Infl ammatory Diseases, Institute for 5098 Molecular Biosciences. University of Queensland, Australia David A Hume, CRC for Chronic Infl ammatory Diseases, Institute for The incidence of copd and how the coomunity people responses to Molecular Biosciences. University of Queensland, Australia smoking habits

Macrophages are extraordinarily versatile cells with many distinctive Shambhu D Joshi, Nepal Medical College and Teaching Hospital, Nepal functions in both innate and acquired immunity, such as phagocytosis, Shyam K Bk, Nepal Medical College and Teaching Hospital, Nepal antigen presentation and secretion of cytokines. Macrophage function is Naresh Pandit, Nepal Medical College and Teaching Hospital, Nepal mediated by the coordinate and inducible expression of specifi c gene sets Rajan P Bhandari, Community Youth HIV/AIDS Intervention Network, in response to extracellular signals. The characterisation of genes and gene Nepal families involved in the complex networks that control macrophage dif- ferentiation and function will enable the understanding of the regulatory ISSUES: Nepal has one of the high smoking rate in the world. most of the mechanisms that govern macrophage function in health or disease. This women than men smokes non fi ltered cigarette.they come to the hospital in study used a microarray approach to investigate the macrophage transcrip- the advanced stage of disease. tome. A large set of genes was found to be restricted to the macrophage lin- OBJECTIVE: age. These included genes with known functions in macrophage biology, To study the relationship between smoking index and the incidence of and also many novel genes. Interestingly, one of the novel genes (Gpnmb) COPD to persuade smokers quit smoking among community people and may possess antinfl ammatory activity. Preliminary data demonstrated hospital patients. that Gpnmb overexpression in the macrophage-like cell line, RAW 264.7 METHODS: COPD was diagnosed based on family inquiring, question- resulted in a two-fold reduction in LPS-dependent cytokine production. naire and pulmonary function test. The smoking index was calculated by Furthermore, Gpmnb mutant mice have a particular macrophage proin- the average cigarettes a day times the years of smoking. The data were fl ammatory phenotype. The function of this gene and its specifi c role in analysed and edited with the help of EPI info program. macrophage biology and infl ammation is currently under investigation. RESULTS: 822 cases(account for 24.6% of investigated subjects) of COPD due to smoking were identifi ed, and 624 cases were found (account S 144 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005) for 40.4% of investegated subjects) to have a history of both smoking and Results: In comparison to placebo, azithromycin resulted in an early tran- chronic airway infl ammation. The higher the smoking index, the higher the sient increase in serum nitrites plus nitrates (day 3), associated with a ten- incidence of COPD (above 40%), and the more severe their lung function dency towards an increase in the blood neutrophil oxidative burst to phor- imparement. The smoking was 71.6% of the cause in COPD patients. bol myristic acetate. Subsequently, prolonged decreases in blood leukocyte CONCLUSIONS: The incidence of COPD associated with smoking is and platelet counts, serum acute phase proteins (including C reactive pro- higher in nepal than that in the developed countries. The incidence of tein) and soluble E-selectin and blood neutrophil lactoferrin concentra- COPD in population with long smoking history is not invariable, while it tions and a transient decrease in serum interleukin-8 were observed. Blood is gradually rising with increasing smoking index. neutrophil glutathione peroxidase activity showed a prolonged increase after azithromycin treatment. Conclusions: While the initial neutrophil stimulating response to azithro- 5099 mycin seen in healthy volunteers was not obvious in COPD patients, de- layed potential anti-infl ammatory effects were observed, as in the earlier Treatment Of Pseudomonas aeruginosa In Patients W ith study. The data suggest that long-term treatment with azithromycin may Pneumoniaith be of benefi t in COPD.

Fariba Fayaz, Microbiology Department,Faculty of Medicine,Shaheed Beheshti University Of Medical Siences, Iran 5101 Aliakbar Soleimanirahbar, Iran Chronic exposure of cigarette smoke (CS) induced signifi cant Purpose:The present study focused on the antibiotic treatment of Pseudo- changes in lung mechanics in C57BL/6 mice monas aeruginosa as an organism with many potent infl ammatory factors, in patients with pneumonia.Some of these factors are elastase ,with a tissue Harbans Lal, Department of Biological Sciences, AstraZeneca R&D damaging proteolytic activity, exotoxin A,with protien synthesis inhibition Lund, Sweden and exotoxin S,with its effect on cell mediated immunity. Kerstin Thim, Department of Biological Sciences, AstraZeneca R&D Methods:Diagnosis of pneumonia caused by Pseudomonas aeruginosa was Lund, Sweden made in 50 patients ,treated in Loghman Hospital.Sputum culture and an- Maria Lindahl, Department of Biological Sciences, AstraZeneca R&D tibiotic sensivity test were done for all the isolated bacteria.For isolation Lund, Sweden of Pseudomonas aeruginosa ,differential and specifi c bacterial media were Naila Svitacheva, Department of Biological Sciences, AstraZeneca R&D used.Antibiotics such as imipenem,aztreonam, ceftazidime, ciprofl oxacin, Lund, Sweden amikacin, piperacilline, tazobactem, meropenem, clavulanic acid were ap- Kjell Ekholm, Department of Biological Sciences, AstraZeneca R&D plied in two combined and monobacterial therapy in 30 days.Combined Lund, Sweden antibacterial therapy was applied in 50% of patients and monotherapy in Catharina Zackrisson, Department of Biological Sciences, AstraZeneca the rest. R&D Lund, Sweden Result:Nosocomial pneumonia was diagnosed in 10(20%) patients.In 80% Charlott Brunmark, Department of Biological Sciences, AstraZeneca coexisting lung diseases ,were present,mainly COPD and bronchiectasis. R&D Lund, Sweden Strains of pseudomonas aeruginosa were susceptible mostly to imipenem, Per-Ola Önnervik, Department of Biological Sciences, AstraZeneca meropenem,aztreonam,amikacin and ceftazidime.Treatment was success- R&D Lund, Sweden ful in 30(60%) of patients .10 patients died because of lung cancer and COPD. We have examined the lung mechanics in mice after chronic exposure to Conclusion:The result of this research shows that treatment of Pseudomonas cigarette smoke(CS). Female C57BL/6 mice (10-12weeks old) were ex- aeruginosa with combination of aminoglycosides or fl uroquinolones,plus posed to CS mixed with a continuous fl ow of fresh air for 50 min, twice one of remaining antipseudomonal antibiotics, can be successful.Treat- daily (5 days/week for 12 weeks). ment usually fails in patients with severe lung diseases. During the last week of smoke exposure, respiratory patterns were stud- ies by unrestrained wholebody plethysmography (EMMS, UK). Dynamic

lung resistance (Rdyn), dynamic lung compliance (Cdyn), tissue elastance

5100 (Htis), tissue resistance (Gtis) and Newtonian resistance (Rn) were measured 16 hrs after the last smoke exposure in anaesthetised-ventilated animals Azithromycin in the short-term treatment of COPD using the FlexiVent system (SCIREQ, Canada). The animals were sacri- fi ced and infl ammatory cells and mediators were measured in bronchoal- Michael J Parnham, PLIVA Research Institute Ltd, Zagreb, Croatia veolar lavage (BAL) fl uid and lung tissues were assessed for histopathol- Vesna Erakovic, PLIVA Research Institute Ltd, Zagreb, Croatia ogy. Urine desmosine levels were assayed by LC-MS methodology.

Ognjen Culic, PLIVA Research Institute Ltd, Zagreb, Croatia Results showed a signifi cant increase in Rdyn(1.5 ± 0.04 vs.1.3 ± 0.03

Vesna Munic, PLIVA Research Institute Ltd, Zagreb, Croatia cmH2O.s/ml), Rn(0.3 ± 0.01 vs. 0.21 ± 0.01 cmH2O.s/ml), Gtis(6.4 ± 0.3 vs.

Ines Glojnaric, PLIVA Research Institute Ltd, Zagreb, Croatia 5.4 ± 0.08 cm.H2O/ml), Htis(35 ± 1.5 vs. 30 ± 0.8 cmH2O/ml) and breathing Ivana Cepelak, University Dept of Medical Biochemistry, Zagreb, Croatia frequency (340 ±13 vs.294 ±17 breaths/min), but a signifi cant decrease in

Sanja Popovic-Grle, Hospital for Pulmonary Diseases Jordanavac, Cdyn(0.029 ± 0.001 vs.0.035 ± 0.001 ml/cmH2O) in CS-exposed mice vs. Zagreb, Croatia control mice (mean ± SEM, n= 9-10, P<0.01). Chronic CS exposure in Zoran Manojlovic, Diagnostic Polyclinic Nematova, Zagreb, Croatia mice also induced a signifi cant (P< 0.05) increase in number of neutrophils and infl ammatory mediators (GM-CSF, IL1-alpha, TNF-alpha and IL-6) Purpose: Macrolide antibiotics exert a variety of anti-infl ammatory effects. in BAL fl uid. In addition, aggregates of macrophages and an increased We have previously found that azithromycin (500 mg for 3 days) causes number of mucus cells were seen in the lung tissue of CS animals. Fur- a biphasic stimulation then prolonged inhibition of neutrophil function in thermore, desmosine levels in urine were signifi cantly higher in CS vs. healthy volunteers (Culic et al, 2002). We have now further explored the control animals. anti-infl ammatory potential of azithromycin in COPD patients. These results indicate that chronic exposure to CS induces a local infl am- Methods: Patients with moderate COPD were treated orally with azithro- mation, signifi cantly increases urine desmosine levels and affects several mycin (500 mg, n = 16) or placebo (n = 8) once daily for 3 days in a ran- parameters of lung function in mice. domized, double blind design, to compare effects on infl ammation markers with those seen in the previous study in healthy volunteers. A battery of tests was made on serum, blood neutrophils and induced sputum on days 1 (baseline), 3, 4, 11, 18 and 32. Infl amm. res. Supplement 2 (2005) Posters: Sunday 21 August 2005 S 145

(5.5mg/L, range 0.8-11). We have screened a viral sample using SELDI- 5102 ToF and identifi ed serum proteins elevated during the acute phase of an exacerbation. Conclusions: In this pilot study, CRP was elevated in all Associations of (-1562C/T) polymorphism of MMP9 with risk and infective exacerbations, however it did not discriminate between viral or clinical features of COPD bacterial aetiologies. Our preliminary SELDI-ToF screen identifi ed novel markers that may be predictive of a viral AECOPD. In order to confi rm E Yanchina, Pavlov' State Medical University, Russia these fi ndings a larger study of similar design is currently ongoing. These T Ivtchik, Pavlov' State Medical University, Russia studies may lead to better diagnosis of AECOPD and hence result in more N Khodzhayants, Pavlov' State Medical University, Russia appropriate treatments. N Yakovleva, Pavlov' State Medical University, Russia

Genetic polymorphism of proteinases implicated in proteolytic destruc- 5104 tion of lung parenchyma may be important factor in development of in- fl ammation related lung injury, which results in emphysema. (-1562 C/T) An acute model of ultrafi ne carbon black-induced infl ammation in polymorphism of MMP9 gene is signifi cantly associated with elevated mice level of gene expression. AIM of study were to investigate I) frequency of MMP9 (-1562C/T) in Russian COPD patients, II) a combined effects Helinor Johnston, Novartis Institute of Biomedical Research, United of imbalance in MMP9 and xenobiotic detoxycation systems (GSTM1 Kingdom and GSTT1 null genotypes) and III) associations with clinical features of Daniel Wyss, Novartis Institute of Biomedical Research, United Kingdom COPD. It had examined 72 COPD patients, 38 patients with chronic cough Clare Blaydon, Novartis Institute of Biomedical Research, United King- but without airway obstruction (CNB) and 39 heavy smokers without his- dom tory of chronic bronchitis (Control). Genotypes had determined by PCR- Gillian Kinnear, Novartis Institute of Biomedical Research, United RFLP. RESULTS I)The differences in distribution of MMP9 CT genotype Kingdom in COPD, CNB and Control groups were not signifi cant (32%, 23% and Christopher S Stevenson, Novartis Institute of Biomedical Research, 21%). II)However, we observed a combined effect GSTM1 0/0 and MMP9 United Kingdom CT conferred 8-fold higher risk of COPD. (14% vs. 3%; OR 7.7, CI95% 1.1-53.3, P=0.05) Similar analysis in the group with severe COPD in com- Particulate air pollution can contribute to and exacerbate pre-existing re- pare to Control showed 10-fold increase in the risk for COPD in carri- spiratory disease conditions, such as asthma and COPD. The carbon core ers of the two mutant genotypes (21% vs. 3%, OR 9.9, CI95% 1.3-70.0, of air pollution particles accounts for 50% of its mass, with the ultrafi ne P=0.015). III) In the COPD the more of carriers of CT genotype had a component being predominantly implicated in its pathogenicity. Our aims severe form of disease at the age of till 45 years (38% vs. 14%, χ2=3.59, were to develop a model of ultrafi ne carbon black (ufCB)-induced infl am- p=0.058. Retrospective analysis in 3 groups of COPD patients aged 35-45, mation in mice to study the changes due solely to inert particle inhalation. 46-55 and 56-65 years showed more fast decline of lung function in car- The response to ufCB in 3 strains of mice (c57, Balb/c, and A/J) were in- riers of GSTM1 0/0 in compare to GSTM1+. Detected differences were vestigated and compared. The effect of a number of anti-infl ammatory and signifi cant in a group aged 56-65 years: FEV1 (% of Rred.) 31.87 vs. anti-oxidant compounds were also assessed in this model to validate its use 44.66 (p=0.050) and FVC 56.23 vs. 73.25 (p=0.056). In CNB group has for pharmacological profi ling. Mice were administered (i.n.) an ufCB sus- showed signifi cant differences of RV and TLC in carriers of MMP9 CC pension in saline (0.05% Tween-20). For drug studies, budesonide (BUD) versus MMP9 CT genotypes (RV 108.80 vs 95.50, p=0.05; и TLC 117.20 (0.3-3 mg/kg, i.n. or p.o.), N-acetylcysteine (100-1000 mg/kg p.o.), or vs 81.00, p=0.05). rofl umilast (1-10 mg/kg p.o.) were dosed 1h prior to ufCB delivery and Our results suggested that MMP9 CT genotype is associated with a risk of culled 24h later. ufCB induced dose- and time-dependent infl ammatory severe and early-onset form of COPD. It may be associated with a charac- changes in all 3 strains characterised by increased neutrophil infi ltration ter of remodeling of lung and decrease of lung function in patients with a peaking 24h post-exposure. Differential staining revealed that macro- chronic bronchitis. phages were fi lled with ufCB, but did not increase in the BAL fl uid at any time. In addition, IL-1b and KC were increased in dose- and time-depen- dent manners, peaking 6h post-exposure. The Balb/c and A/J strains were 5103 the most responsive to ufCB and the Balb/c strain was chosen to further characterise this model. BUD delivered i.n. decreased the ufCB-induced Identifying Systemic Markers in the Melbourne COPD Exacerbation neutrophil infi ltration and IL-1b levels, but not KC production. Prelimi- Cohort nary studies using BUD suggest that IL-1b may be important mediating the infl ammatory response elicited by ufCB, but further studies are needed be- Steven Bozinovski, University of Melbourne, Australia fore defi nitive conclusions on KC’s role can be drawn. These data suggest Anastasia Hutchinson, University of Melbourne, Australia that this model may be useful for investigating the infl ammation induced Michelle Thompson, Royal Melbourne Hopital, Australia by inert, ultrafi ne carbon particles and for testing the anti-infl ammatory David Smallwood, Royal Melbourne Hopital, Australia effects of test compounds. Jim Black, Royal Melbourne Hospital, Australia Ross Vlahos, University of Melbourne, Australia Louis Irving, Royal Melbourne Hopital, Australia 5105 P Anderson, University of Melbourne, Australia A chronic model of cigarette smoke-induced lung injury in rat - the Aim: To establish the utility of C-reactive protein (CRP) as a marker for beginning of COPD-like changes acute exacerabations of COPD (AECOPD) and to identify novel serum biomarkers that reliably discriminate between non-infective, viral or bac- Christopher S Stevenson, Novartis Institute of Biomedical Research, terial causes. Methods: In this study thirty two subjects with moderate/ United Kingdom severe COPD were enrolled. In response to AECOPD symptoms serum Cerys Docx, Novartis Institute of Biomedical Research, United Kingdom samples were collected and clinical diagnostics performed. Stable samples June Giddings, Novartis Institute of Biomedical Research, United King- were also collected upon resolution of symptoms. CRP levels were as- dom sessed by ELISA and serum subjected to SELDI-ToF proteomics for iden- Phillip R Cooper, National Heart and Lung Institute, United Kingdom tifi cation of novel biomarkers. Results: Twelve subjects exacerbated (three Richard G Sturton, National Heart and Lung Institute, United Kingdom bacterial and one viral detected). Mean CRP levels were signifi cantly el- Paul Whittaker, Novartis Institute of Biomedical Research, United evated (p=0.04) during an AECOPD (9.6mg/L, range 0.2 -17.5) vs. Stable Kingdom S 146 Posters: Sunday 21 August 2005 Infl amm. res. Supplement 2 (2005)

Christine Charman, Novartis Institute of Biomedical Research, United were isolated in cases while Adenovirus (1) was identifi ed in a control. A Kingdom viral symptom score of at least 3 symptoms was 80% sensitive and 94% Keith D Butler, Novartis Institute of Biomedical Research, United King- specifi c for predicting the detection of virus on PCR (OR 10.5, 95%CI dom 1.64-110.6, p = 0.0022). "Fever and Chills" had the highest predictive val- ue for virus (OR 7.42, 95%CI 1.20-77.85). Exacerbations in which a virus No effective therapies exist for treating COPD due partly to the poor un- was detected were associated with raised CRP = mean 51 mg/L and CRP derstanding of the infl ammatory mechanisms involved in its pathogenesis. on day 1 was predictive of maximum exacerbation severity, (OR 1.02, The aims of this study were to gain insights into the temporal gene ex- 95%CI 1.009 -1.04; p = 0.002). pression, infl ammatory, and pathological changes occurring after chronic Conclusion: This study demonstrates that the presence of virus in upper exposure to cigarette smoke (CS) to try and understand how the patho- airway secretions is strongly associated with the development of COPD physiology of COPD develops. Male, Sprague-Dawley rats were exposed exacerbations. Patients with moderate to severe COPD were 17 times more to whole-body CS from 4x2R1 Research Cigarettes daily for 1d-8m. One likely to have a virus identifi ed than their time-matched controls. These group was exposed for 6m followed by 2m with no exposure to study the data support the causative role of viruses in exacerbations of COPD. effects of cessation. Results were compared with time-matched air-ex- posed sham animals (n=8/timepoint). Using microarray analysis, a "tran- scriptional shift" was observed between smoked and sham animals starting at 1m. The greatest changes occurred in genes associated with oxidant me- tabolism and infl ammation. At the biochemical level, the most signifi cant changes were the increased expression of the rat GRO homologues and IL- 1b in BALF and lung tissue. Macrophages, lymphocytes, and neutrophils were all signifi cantly increased and formed lesions around terminal bron- chioli/alveolar sacs which became more pronounced over time. There was a trend towards an increase in collagen staining and an increase in smooth muscle density around the small airways at 2m (p<0.05). By 8m there was also a signifi cant increase in the mean linear intercept of the alveolar spac- es of the smoke exposed animals (p<0.05). Goblet cell metaplasia and mu- cus hypersecretion were also prominent features of this model (p<0.001). In the smoking cessation group, the infl ammation was still present, but the mucus production returned to control levels. These signifi cant patholo- gies did not translate into decreased lung function in the smoke-exposed rats, but were associated with increased sensitivity to carbachol. Thus, the changes which occur in this model may refl ect and provide insights into the early changes which occur in the pathogeneisis of COPD.

5106

The Melbourne Longitudinal COPD Exacerbation Cohort- A case controlled study highlighting the role of viruses in infl ammation.

Anastasia Hutchinson, University of Melbourne, Australia Michelle Thompson, Royal Melbourne Hopital, Australia Steve Bozinovski, CRC for Chronic Infl ammatory Diseases, The Universi- ty of Melbourne, Australia A Ghimire, University of Melbourne, Australia Jim Black, Royal Melbourne Hospital, Australia C Brand, Royal Melbourne Hospital, Australia David Smallwood, Royal Melbourne Hopital, Australia Ross Vlahos, CRC for Chronic Infl ammatory Diseases, The University of Melbourne, Australia Gary P Anderson, CRC for Chronic Infl ammatory Diseases, The Univer- sity of Melbourne, Australia Louis Irving, Royal Melbourne Hopital, Australia

Aim: To determine if viral infections are more common in cases with an exacerbation of COPD than in controls with stable COPD. Methods: Eighty-seven subjects (mean age 73 yrs, range 57-85 yrs) with COPD (mean FEV1 45%) and at least one admission in the last 24 months were enrolled. At baseline and each exacerbation sputum culture, nose & throat swabs for viral multiplex PCR and atypical serology were obtained. For each exacerbation, case control samples were collected from a stable subject randomly selected from within the cohort. Clinical measures of in- fl ammation (c-reactive protein (CRP), white cell count (WCC) and eryth- rocyte sedimentation rate (ESR)) were obtained during stable COPD and on day 1 and 30 of each exacerbation.. Results: PCR at baseline revealed viruses in 2 subjects (adenovirus and RSV). There were 83 exacerbations over 68 weeks of surveillance in 2003-4. There were 18 discordant case control pairs. In 17 pairs only the case had virus detected and in 1 pair only control had virus. (Odds ratio (OR)=17,95%CI 2.7-710,p = 0.0001). Infl uenza A (3) and Rhinovirus (14) Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 147

and 7 after injection of 300 ng (p24 value) LV-Luc into the knees of na- 6001 ïve C57Bl/6 mice. Thereafter, expression slowly decreased to 40% of the peak value at day 21. The observed profi le was quite distinct from the Subchronic allergic contact dermatitis in pigs: a new model for fast and short-lasting expression seen with adenoviruses. LV-GFP and LV- profi ling topical anti-infl ammatory drugs IL-1Ra were used to study the localisation of synovial transduction using fl uorescence microscopy and immunostaining. The LV-mediated transgene Hermann Fahrngruber, Novartis Institutes for BioMedical Research expression was localized in the lining but also in the deeper layers of the Vienna, Austria synovium that adenoviruses could not target. Equal amounts of transgene Elisabeth Kowalsky, Novartis Institutes for BioMedical Research Vienna, expression was measured when LV were injected into arthritic knees. This Austria is remarkable as the adenoviral synovial transduction was markedly re- Josef G Meingassner, Novartis Institutes for BioMedical Research Vien- duced (-85%) under the same arthritic condition. Next, the therapeutic ef- na, Austria fi cacy of LV-mediated gene transfer was tested. For this, LV were injected in the knees of collagen-type II immunized DBA1/J mice 1 day after the Available models for testing effi cacy of topical agents in T cell-mediated booster (day 21). Arthritis (day 31) was ameliorated (0.55 ± 0.37) by the infl ammatory skin conditions such as atopic dermatitis (AD) or psoriasis LV-mIL-1Ra treatment as compared to the LV-Luc treated knees (1.55 ± have limited predictability, because skin permeability and/or the course of 0.53). Notably, histology showed clear protection of bone destruction in the experimentally induced infl ammation in animals differ substantially CIA by LV-IL-1Ra. Here, we showed that lentiviral-mediated synovial from the human situation. Here we present a new model of subchronic al- transduction is feasible in naïve and arthritic joints, and when used for IL- lergic contact dermatitis (ACD) in pigs that shows a high degree of correla- 1Ra gene therapy is effi cacious for treatment of arthritis. tion with clinical effi cacy in AD patients when testing various anti-infl am- matory drugs. Sensitized young domestic pigs were challenged with 0.2% 2,4-dinitrofl uorobenzene at test sites on both sides of the dorso-lateral back 6004 on days 1, 3 , 6, 8 and 10. The test sites were treated with the active formu- lations (or contra-laterally with placebos) twice on days 6, 8 and 10 (2 and The Phosphodiesterase 4 (PDE4) inhibitor ELB353 is a potent 6 hrs after challenge), followed by clinical (and/or biophysical) examina- inhibitor of pro-infl ammatory cytokines in vitro tion and scoring of the lesions on days 7, 9 and 11. Additionally, biopsies taken after the last treatment were examined. In comparison with untreated Christin Galetzka, elbion AG, Department of Molecular Biology & test sites, Protopic® 0.1% (tacrolimus) inhibited infl ammation by 77%, Screening, Germany Protopic® 0.03% by 49%, Alfason® (0.1% hydrocortisone-17-butyrate) Ute Egerland, elbion AG, Department of Molecular Biology & Screening, by 53% and Hydroderm® (1.0% hydrocortisone acetate) by 15%. Infl am- Germany matory cell infi ltration (T cells) was similarly reduced. The degree of anti- Norbert Höfgen, elbion AG, Department of Chemistry, Germany infl ammatory activity of the compounds in this model closely mirrors the Thorsten Hage, elbion AG, Department of Molecular Biology & Scree- relative effi cacy reported in AD patients. The model is stringent because ning, Germany treatment is started when pronounced infl ammation is established. This ACD model appears, therefore, to be valuable for assessing the clinical Introduction: PDE4 plays an important role in modulating the activity of a potential of new compounds for the treatment of AD and, possibly, other variety of cells that are involved in the pathophysiology of infl ammatory T- cell mediated skin diseases. processes like asthma and chronic obstructive pulmonary disease (COPD) and is the predominant PDE isoenzyme expressed in human leukocytes, neutrophiles and monocytes. Here we describe the anti-infl ammatory pro- 6003 fi le of ELB353, a novel and selective PDE4 inhibitor which has shown excellent tolerability in vivo and is an ideal drug candidate for once daily Local lentiviral gene transfer of IL-1Ra ameliorates collagen-induced treatment. arthritis Methods and Results: ELB353 inhibits PDE4 catalytic activity in human polymorphonuclear leukocytes (PMNLs) with high potency and selectivity Fons AJ van de Loo, Rheumatology Research and Advanced Thera- (IC50 = 2.1 nM, other phosphodiesterases are inhibited at >290 fold higher peutics, Department of Rheumatology, Radboud University Nijmegen concentrations). In comparison, the affi nity towards the high affi nity rolip- Medical Centre, The Netherlands ram binding site (HARBS) is reduced by a factor of more than 28-fold. In Ruben L Smeets, Radboud University, The Netherlands cell-based assays, ELB353 attenuated production of tumor necrosis fac- Laure M Delavallee, Rheumatology Research and Advanced Thera- tor alpha (TNF-alpha) upon stimulation with anti-CD3/CD28 or LPS in peutics, Department of Rheumatology, Radboud University Nijmegen peripheral blood mononuclear cells (PBMCs) with an IC50 value of 26.8 Medical Centre, The Netherlands nM. These results were further confi rmed in isolated human monocytes Onno J Arntz, Rheumatology Research and Advanced Therapeutics, and monocytic cell line U937, where ELB353 elevates intracellular cAMP Department of Rheumatology, Radboud University Nijmegen Medical levels upon stimulation with prostaglandin E2 (PGE2). The compound Centre, The Netherlands was deeply characterized in a multiplex ELISA system based on luminex Miranda B Bennink, Rheumatology Research and Advanced Thera- technology, demonstrating that ELB353 also potently inhibits pro-infl am- peutics, Department of Rheumatology, Radboud University Nijmegen matory cytokines like IL-1 beta, IL-8, IL-12, TNF-alpha and INF-gamma Medical Centre, The Netherlands as well as T-cell cytokines like IL-2, IL-4, IL-5 and IL-13. Wim B van den Berg, Rheumatology Research and Advanced Thera- Conclusion: Taken together, these results emphasise the therapeutic po- peutics, Department of Rheumatology, Radboud University Nijmegen tential of ELB353 for the treatment of infl ammatory diseases like asthma, Medical Centre, The Netherlands COPD and atopic dermatitis. This work has been supported by the EU fund for reg. dev. (EFRE) and by The infl amed joints in RA patients are gene therapeutic target sites to the Free State of Saxony, SAB 8093. achieve local production of potent anti-infl ammatory biologicals and the challenge is to obtain long-term transgene expression. Hence, HIV-based self-inactivating lentiviruses (LV) were used and their effi cacy was tested 6005 in collagen-induced arthritis (CIA). The cDNA of murine IL-1Ra was cloned into the RRLSIN plasmid (D. Trono, Geneva, Switzerland), and Ahr locus involvement in lung carcinogenesis and infl ammatory VSV-G pseudotyped LV were produced. In this study, LV encoding for response regulation in genetically selected AIRmax and AIRmin mice fi refl y luciferase (Luc) or green fl uorescent protein (GFP) were used as a control. Luciferase expression in the synovium peaked between day 5 Vinicius R C Souza, Instituto Butantan, Brazil S 148 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Wafa H Cabrera, Instituto Butantan, Brazil Marcelo De Franco, Instituto Butantan, Brazil 6007 Nancy Starobinas, Instituto Butantan, Brazil Orlando G Ribeiro, Instituto Butantan, Brazil A potent CSF-1 Receptor tyrosine kinase inhibitor targets survival Olga M Ibañez, Instituto Butantan, Brazil and effector functions in murine macrophage populations

Mice genetically selected for maximal (AIRmax) local acute infl amma- Katharine M Irvine, Institute for Molecular Bioscience, University of tory response (AIR) were resistant and those selected for minimal response Queensland, Australia (AIRmin) were susceptible to lung carcinogenesis induced by the intraper- Christopher J Burns, Cytopia, Pty Ltd, Australia itoneal injection of urethane, or dimethyl hydrazine (DMH) or by repeated Andrew F Wilks, Cytopia, Pty Ltd, Australia epicutaneous applications of dimethylbenzo[a])anthracene (DMBA). Xiangyong Bu, Cytopia, Pty Ltd, Australia Herein these mice were treated with one i.m. application in the hind leg Michael F Harte, Cytopia, Pty Ltd, Australia of 0,3 mg 3-methylcholanthrene (MCA) in sesame oil. Tumor appearance Stephen Su, Cytopia, Pty Ltd, Australia and 50% incidence occurred 3 to 4 weeks earlier in AIRmin than in AIR- David A Hume, Institute for Molecular Bioscience, University of Queens- max, but at 29 weeks about 90% of all mice developed fi brosarcomas at the land, Australia site of MCA injection. Nevertheless, at the time of the sacrifi ce multiple Matthew J Sweet, Institute for Molecular Bioscience, University of lung adenomas and large lung adenocarcinomas were found in AIRmin Queensland, Australia mice only. The aryl hydrocarbon receptor (Ahr) plays an important role in the metabolism of aromatic hydrocarbon carcinogens (PAHs) like MCA CSF-1 regulates macrophage differentiation, survival and function, and and DMBA. An allelic polymorphism was found in the Ahr gene between is an attractive therapeutic target for chronic infl ammation and malignant inbred mouse lines which correlated to variations in binding affi nity of the diseases. Here we identify a compound, CYC10268, which is a potent and receptor to PAHs and in carcinogenesis susceptibility. Accordingly, all selective inhibitor of CSF-1R action. In in vitro kinase assays, CYC10268 AIRmax are homozygous for the low affi nity Ahr allele conferring resis- was active in the 10 nM range and showed selectivity over other kinases tance to carcinogenesis whereas all AIRmin bear the high affi nity related such as Abl (>100 fold) and Kit (~10 fold). CYC10268 blocked survival Ahr allele. This allelic segregation in the two lines further indicates Ahr mediated by CSF-1R in primary murine bone marrow derived macro- as a genetic marker or a gene target involved in infl ammatory response phages (BMM) and in the factor-dependent cell-line, Ba/F3 in which the control. This was confi rmed by a linkage analysis or the co-inheritance of CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regu- Ahr parental genotypes with the degree of acute infl ammation in a hetero- lated signalling (Akt, ERK-1/2), gene expression (urokinase plasminogen geneous F2 (AIRmax X AIRmin) intercross population. activator, toll-like receptor 9 and Apolipoprotein E) and priming of LPS- inducible cytokine production in BMM. In thioglycollate-elicited perito- neal macrophages (TEPM), which survive in the absence of exogenous 6006 CSF-1, CYC10268 impaired LPS-induced cytokine production and regu- lated expression of known CSF-1 target genes. These observations support ABX10-0031, a High Affi nity Fully Human Anti-IL-1b Monoclonal the conclusion that TEPM are CSF-1-autocrine and that CSF-1 plays a Antibody for the Treatment of Infl ammatory Diseases central role in macrophage effector functions during infl ammation. CSF- 1R inhibitors such as CYC10268 provide a powerful tool to dissect the role Raffaella Faggioni, Abgenix Inc., United States of the CSF-1/CSF-1R signalling system in a range of biological systems Larry L Green, Abgenix Inc., United States and have potential for a number of therapeutic applications. Xiao Feng, Abgenix Inc., United States Orit Foord, Abgenix Inc., United States Scott L Klakamp, Abgenix Inc., United States 6008 Qiaojuan J Su, Abgenix Inc., United States Mary Haak-Frendscho, Abgenix Inc., United States Antibacterial and Antiinfl ammatory Activity of Rubia tinctoria Giorgio Senaldi, Abgenix Inc., United States Extract on Burn Wound Infections

Interleukin-1b (IL-1b) is a pro-infl ammatory cytokine that plays a major Fariba Fayaz, Shaheed Beheshti Univ.Medical of Sciences, Iran role in a wide range of infl ammatory diseases. IL-1 receptor antagonist Sorrayya Saremi, Shaheed Beheshti Unv.Medical Of Sciences, Iran (anakinra) is approved for the treatment of rheumatoid arthritis and is in clinical studies for other infl ammatory indications. The therapeutic effi - Purpose:This project was designed to evaluate the antibacterial and anti- cacy of anakinra may be limited by its biological and pharmacokinetic infl ammatory activity of extract of the herbal plant ,Rubia tinctoria(R.t) properties. Anakinra must compete with IL-1 at the level of multiple ubiq- on bacteria causing wound infections and infl ammation in patients with uitous receptors, and it has rapid clearance. Targeting IL-1b with a spe- burn injuries. cifi c antibody may offer a more advantageous mechanism of action and an Methods:Escherichia coli,Pseudomonads aeruginosa and Staphylococ- improved pharmacokinetic profi le compared to anakinra. ABX10-0031 is cus areus were isolated from burn wounds of 30 patients at Burn Cen- a high-affi nity (KD = 300 fM), fully human IgG4 mAb that neutralizes IL- ter Motahari Hospital, with total surface area burn(TSAB) of 10 to more 1b in vitro with high potency, and inhibits IL-1b-induced IL-6 production than 50%.These bacteria are known as microorganisms with many potent by MRC-5 cells and IL-8 production in whole blood. In mice, ABX10- infl ammatory factors such as endotoxin, exotoxinA and S and proteo- 0031 inhibits IL-1b-induced IL-6 production. ABX10-0031 has potencies lytic enzymes.Extracts of the whole aerial plant parts were dissolved in superior to anakinra, in vitro [MRC-5 cells: IC50 (pM) 4 ± 0 vs 77 ± 22; Dmso,membrane fi lter (pore size 0.47 micrometer),sterilized and tested for whole blood: IC50 (pM) 135 ± 17 vs 984 ± 223] and in vivo [mice: IC50 antimicrobial activity using the agar diffusion method.Sterile 6mm diam- (pm/mouse) 5 ± 3 vs 222 ± 37]. Because the concentration of IL-1b used eter fi lter paper disc were imprignated with 200-400microgram of the ster- in the in vitro assays was antigen limiting ([IL-1b] > KD), the actual po- ile plant extract and placed in duplicates on to nutrient agar plates ,which tency of ABX10-0031 may be higher. Blockade of IL-1b with a mAb is a spread with 0.2ml bacteria cultures(10^8)cells/ml).For comparative pur- valid approach to the neutralization of IL-1b function, and may be a useful poses standard tetracycline(50microgram disc),gentamycin(10microgram therapeutic approach to infl ammatory diseases. disc),chloramphenicol(25microgram disc) and ampicillin(10microgramdi sc)were included in the disc assay. Results:As the diameter of the disc was 6mm, inhibition zones less than 7mm were not evaluated.The aqueous extract of R.t showed a good (>8mm)of inhibition against both gram positive and gram negative bacte- Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 149 ria.The concentrations of 400 microgram of the extracts tested are compa- sine kinase receptor. We examined in the African Green Monkey (AGM) rable to the standard pure antibiotics. the ability of recombinant human SCF (rhSCF) to induce mast cell activa- Conclutions:According to previous researches, R.t had been used in tra- tion. The fi rst step was to ascertain whether activities of rhSCF and hu- ditional medicine,specially for its antiinfl ammatory activity.In our re- man c-kit antibodies were conserved in hematopoietic cells derived from search concentrations of 4mg of R.t extract was effective on the isolated the AGM. Flow cytometric analysis showed that human c-kit antibodies, bacteria,and showed detectable antimicrobial activity,that comes to the anti-CD-117 and SR-1, bound to bone marrow (BM) cells from the AGM. result of supressing the potent infl ammatory factors of these bacteria rhSCF is required for GM-CFC derived colony formation from AGM (BM) with maximal effects at 1 ng rhSCF/mL. GM-CFC derived colony formation was inhibited by SR-1, thus confi rming the activity of rhSCF 6009 and SR-1 on AGM c-kit expressing BM cells. These data suggested that the AGM may be a suitable model to study SCF effects on mast cells. Interleukin-17 - a missing piece of the diabetes infl ammatory puzzle Repeated systemic SCF dosing has been reported to induce dermal mast cell degranulation in human and mice. In the AGM, systemic dosing with Djordje Miljkovic, Institute for Biological Research, Yugoslavia rhSCF (100 ug/kg i.v.) resulted in a Cmax of ~80 ug/ml @ 4h. rhSCF up Ivana Cvetkovic, Institute for Biological Research, Yugoslavia to 300 ug/kg i.v. or s.c. led to no signifi cant changes in serum histamine, Miljana Momcilovic, Institute for Biological Research, Yugoslavia TNF, IL-1b or IL-6 compared to controls during the post-dosing sampling Danijela Maksimovic-Ivanic, Institute for Biological Research, Yugosla- time points (15 min up to 24h). A single intradermal dosing of rhSCF up via to 100 ug/site did not elicit a weal and fl are response up to 2h post-chal- Stanislava Stosic-Grujicic, Institute for Biological Research, Yugoslavia lenge. Consistent with this data, rhSCF at concentrations up to 100 ug/ml Vladimir Trajkovic, Institute of Microbiology and Immunology, School of did not degranulate human mast cells in culture. Taken together, the data Medicine, University of Belgrade, Yugoslavia suggest that hematopoietic cells from the AGM are dependent on SCF for growth and survival, and that activation of mast cells in vivo may require Interleukin-17 (IL-17), a pro-infl ammatory cytokine produced by ac- repeated dosing. Sustained levels of SCF acting as an autocoid may be a tivated T cells, has been shown relevant for the pathogenesis of several pathophysiological mechanism contributing to mast cell diseases. autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, and infl ammatory bowel diseases. As its role in autoimmune diabetes has not been elucidated so far, we have induced diabetes in CBA mice using 6011 multiple low doses of streptozotocin, and analyzed IL-17 concentration in both sera and peritoneal cells' supernatants. It was shown that IL-17 lev- In vitro semi-quantitative determination of human gamma- els were higher in streptozotocin-treated animals, comparing to untreated interferon-mRNA Level by RT-PCR controls, indicating that IL-17 could be of importance for the pathogenesis of the disease. As nitric oxide (NO) produced by pancreatic beta cells has Alireza Zamani, Hamadan University of medical sciences,, Iran been implicated in infl ammatory destruction of pancreatic islets, the infl u- Siavash Sadeghian, Hamadan University of medical sciences, Iran ence of IL-17 on NO production in mouse beta cells was investigated. Jalil Tavakkol Afshari, BuAli Research Institute, Iran Mouse insulinoma - MIN6 cells and primary islets of pancreas were used Ebrahim Nasiri, Hamadan University of medical sciences, Iran for the experiments. Both recombinant and T cell-derived IL-17 stimulated beta cells to produce NO through up-regulation of inducible NO synthase The functionally distinct repertoire of secreted cytokines of Th1(T-helper)/ (iNOS)-gene expression, as deduced by Griess reaction and RT-PCR, re- Th2 cells has been shown to play an important role in the pathogenesis of spectively. The observed infl uence was, at least partly, mediated through diseases. Effector Th1 cells secrete predominantly IFN- γ and IL-2 and p38 mitogen activated protein kinase signaling, as concluded through the regulate cell-mediated immunity against intracellular pathogens. use of p38 signaling inhibitor - SB203580, and determination of p38 ac- The expression of IFN- γ -mRNA was studied using semiquantitative RT- tivation by Western blot. Importantly, the observed NO production cor- PCR. Lymphocytes were stimulated by PHA (1μg/106cell/ml) in culture related with, and was shown responsible for the reduction of beta cell vi- medium (incubation time: 0,4,8,12,24,48 and72 hours) and total RNA ability in our experimental setting, as determined by MTT assay. Thus, we extracted and cDNA synthesized. IFN-γ-mRNA was detected by the RT- propose that IL-17 produced by activated infi ltrating T cells could provoke PCR (reverse transcription-polymerase chain reaction) and semi-quantita- islets' destruction through NO-dependent mechanism. It seems reasonable tive-RT-PCR methods, in which sequences (273bp) between two oligo- that IL-17 neutralization could be a promising strategy for the treatment nucleotide primers (chosen from two exons of the IFN- γ gene sequences) of diabetes. were amplifi ed using a heat-stable DNA polymerase. In semi-quantitative RT-PCR we used a serial dilution (1/2,1/4…) of cDNA in order to deter- mine the titer of cDNA for that will give visible band in 2% agarose gel 6010 electrophoresis. Results show that after 4h incubation express highest level of IFN- γ - The Effects Of Stem Cell Factor (Mast Cell Growth Factor) mRNA and it is stable until 24 hour after that. Then it falls to baseline Following Acute Dosing In African Green Monkeys level. In order to study of IFN- γ gene expression kinetic compare results with Robert Briddell, Weston Sutherland, Brent Kern, Graham Molineux, other studies show that RT-PCR detection of IFN- γ level is more sensitive Shamin Summer, Sharon Mu, Todd Covey, Chris Saris, Violeta Yu, than other methods like ELISA and for maximum expression of IFN- γ Dave Powers, Ramak Pourvasei, Mark Ma, Rohini Deshpande, Nessa gene 4h incubation of lymphocytes with PHA is suffi cient. Hawkins, Dao Kemp, Kristi Daris, Frank Martin, Gary Elliot, Jilin Sun, Keywords: RT-PCR, semi-quantitative-RT-PCR. IFN-γ, PHA activated Ester Trueblood, Ray Koelling, Xuxia Zhang, Andrea Itano, Gordon Ng lymphocytes Depts of Hematology, HTS, PKDM, Medical Sciences and Infl ammation, Amgen Inc, Thousand Oaks, CA, USA. 6012 Mast cells are recognized as key effector cells in allergic disorders and are a major source of mediators such as histamine, tryptase, cytokines Molecular cloning & expression of human gamma interferon full and growth factors suggesting that mast cells may play a signifi cant role cDNA gene in CHO cells in chronic infl ammatory diseases. Mast cell development, survival, and function are critically regulated by stem cell factor (SCF) also known as Alireza Zamani, Hamedan university of medical sciences, Iran mast cell growth factor, kit or steel ligand, a pleiotrophic growth factor , Mohammad yousef Alikhani, Hamedan university of medical sciences, whose biological activities are mediated via the proto-oncogene c-kit tyro- Iran, S 150 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Ebrahim Nasiri, Hamedan university of medical sciences, Iran Jalil Tavakkol Afshari, Mashad university of medical sciences, Iran 6014

Background: Gamma interferon is mostly secreted by activated lympho- IL-10 mediated anti-infl ammatory effects on human monocytes occur cytes (TCD4+, TCD8+ and NK). This cytokine has immunomodulatory, via a SOCS3-independent mechanism anticancer and antimicrobial effects and is important in prophylaxis, di- agnosis and treatment of many diseases. Objective: The purpose of this Cecilia M Prele, Telethon Institute for Child Health Research, Australia project was to clone human IFN-γ full cDNA and express in CHO cell line. April Keith-Magee, Telethon Institute for Child Health Research, Methods: we isolated pooled lymphocytes from normal individual and ac- Australia tivated by PHA in the cell culture medium. After 4 hours of activation, to- Monika Murcha, Telethon Institute for Child Health Research, Australia tal RNA was extracted and fi rst strand cDNA synthesized. cDNA amplifi ed Prue H Hart, Telethon Institute for Child Health Research, Australia by PCR. Nested-PCR was performed by primers containing EcoRI&NotI sites. This fragement and PcDNA3.1 vector were cut by EcoRI&NotI and SOCS3 is a negative regulator of the JAK/STAT pathway. SOCS3 expres- ligated again. The construction(pcDNA3.1-IFN-γ) was transformed into sion is induced by IL-10 in mouse infl ammatory cell lines and human E.coli (strain: DH5α) by CaCl2 and selected by selective medium which neutrophils, macrophages and monocytes. The aim of this study was to contained ampicillin. after confi rming the correct sequence by PCR and determine the role of SOCS3 in regulating the anti-infl ammatory effects sequence analysis. Stable expression of the pcDNA3.1-IFN-γ construct of IL-10 on human monocytes. Monocytes were isolated by centrifugal was achieved by performing a calcium phosphate-mediated transfection elutriation from whole blood and infected with an adenoviral vector ex- into CHO cells(8×96=768wells), followed by clonal selection of stable pressing SOCS3 (AdV-SOCS3), control vector (AdV-GFP) or were left drug (G418) resistant clones by LDA assay. Results&Conclusion: in pres- untreated. AdV-SOCS3 monocytes were either pre-incubated for 24 h with ence of ampicillin 33 bacterial colonies were grown. but only 6 colonies 10 ng/ml IL-10 (transcriptional regulation), or treated simultaneously with had inserted sequence. 47 drug resistant clones out of total CHO cells(768 1 or 10 ng/ml IL-10 and LPS (a model of post-translational control by IL- wells) were grown but ELISA test showed only 5 clones were producing 10). FACS analysis demonstrated infection effi ciencies of 45 ± 5 % and 45 more than 100 ng/ml/106 cells. The maximum production was the 143 ± 4 % (n=6, mean ± SEM) of monocytes expressing AdV-GFP and AdV- ng/ml/106 cells.this clone was selected and propagated. SOCS3, at MOI of 10 and 100 respectively. Western blot analysis showed Keywords: RT-PCR, IFN-γ cloning, IFN-γ expression, CHO increased SOCS3 protein expression in AdV-SOCS3 infected cells. TNF production was increased in monocytes co-stimulated with interferon gamma (IFN) and LPS, however, a signifi cant reduction in TNF produc- 6013 tion was observed in AdV-SOCS3 over-expressing monocytes confi rming functional regulation of IFN by over-expressed SOCS3. The level of AdV- Role of proinfl ammatory cytokines IL-17 and IL-18 in acute SOCS3 infection had no effect on the percentage suppression of TNF pro- pancreatitis duction by IL-10. AdV-SOCS3 alone also had no effect on LPS-induced TNF production by monocytes. We observed 42 ± 8 % (n=3, 2 h) and 86 ± Serge Chooklin, Medical University, Lviv, Ukraine 2 % (n=4, 24 h) suppression of TNF production by monocytes following Andre Perejaslov, Medical University? Lviv, Ukraine the simultaneous administration of LPS and IL-10. Incubation of AdV- SOCS3 monocytes with 1 or 10 ng/ml IL-10 had no synergistic effect on Introduction. Enhanced expression of cytokines often determined the se- the percentage suppression observed, implying that SOCS3 together with vere course of acute pancreatitis, which characterized by the development other signals induced by low dose IL-10 do not control LPS-induced TNF of multiple organ dysfunction syndrome. IL-17 and IL-18 may play an production. These data suggest that IL-10-mediated suppression of LPS- important role in acute pancreatitis. induced TNF production occurs via a SOCS3-independent mechanism. Methods. Plasma levels of IL-17 and IL-18, using ELISA technique, were studied in 87 patients with acute pancreatitis. According the Atlanta crite- rion the mild pancreatitis was established in 56 patients and 31 had necro- 6015 tizing pancreatitis. Results. The elevated IL-17 plasma levels were noted in all patients with Effects of equine CCL11 on eosinophil function acute pancreatitis, besides that, the highest levels were in patients with established severe pancreatitis. The IL-17 levels clear correlated with the Marie C Weston, The Royal Veterinary College, United Kingdom quantity of total leukocyte, polymorphonuclear leukocytes and hemo- M Fernanda Sepulveda, The Royal Veterinary College, United Kingdom globin level at the time of admission. The highest levels of IL-18 were Margaret E Collins, United Kingdom observed in patients with necrotizing pancreatitis. During fi rst week the Fiona M Cunningham, The Royal Veterinary College, United Kingdom levels of IL-18 gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased start- Equine CCL11 has been shown to cause equine eosinophil migration and ing from the third day. The clear strong correlation was observed between superoxide production and a shape change in human eosinophils (Benarafa IL-18 and quantity of circulating granulocytes in patients with necrotizing et al. 2002). As CCL11 in supernatant from transfected insect cells was pancreatitis. There is a clear correlation between IL-18 levels and bilirubin used for this study, the aim of the present work was to examine the proper- and alkaline phosphatase levels. The clear correlation between IL-18 and ties of the purifi ed chemokine. glucose plasma levels was documented. Following incubation of eosinophils from normal horses with CCL11, the Conclusion. Hematologic effects of IL-17 may connect with its ability polymerised form of actin was measured by fl ow cytometry using fl uores- induced the GM-CSF expression which also enhanced in severe pancre- cently labelled phalloidin. Migration was determined in a 96 well plate atitis. The development of liver disorders accompanied with the enhanced chemotaxis assay using 8μm pore membranes and superoxide production expression of IL-18 that can aggravate acute liver injury. The IL-18 may quantitated by the reduction of cytochrome C (cyt C). play an important role in the development of metabolic disturbances in acute pancreatitis. Equine CCL11 induced rapid actin polymerisation, a maximal increase of 143 ± 22% being observed within 10 sec of stimulation by 500ng/ml CCL11 (p<0.001 versus un-stimulated cells; 1-way ANOVA, n=3). CCL11 also caused migration, a maximal response being observed at 50ng/ml (mi- gration index of 5.2 ± 1.1; p< 0.05, 1-way ANOVA, n=5). In contrast to previous fi ndings, no signifi cant superoxide production was detected (2.1 ± 1 and 2.2 ± 0.9 nmol cyt C/106 cells for 500ng/ml CCL11 and un-stimulated cells, respectively, n=4). Pre-incubation for 30 min with Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 151

0.05nM hIL-5 primed histamine induced superoxide production (p<0.05, Grant McFadden, University of Western Ontario; Robarts Research 3-way ANOVA) but had no signifi cant effect on the response to CCL11 Institute, Canada (4.9 ± 2.4 and 6.0 ± 0.2 nmol cyt C/106 cells for 500ng/ml CCL11 -/+ IL- Alexandra R Lucas, Robarts Research Institute, Canada 5, respectively, n=3). Equine CCL11 induces cytoskeletal reorganisation and migration of equine Background: Chemokines play an essential role in the innate immune eosinophils but, unlike the human chemokine, does not appear to stimulate response leading to the development of infl ammatory diseases, such as superoxide production. atherosclerosis, by controlling direction of infl ammatory cell migration and activation. Thus, inhibition of chemokine responses is important in Benarafa C, Collins ME, Hamblin AS, Sabroe I, Cunningham FM (2002). developing potential therapies. Chemokine modulating proteins (CMPs) Characterisation of the biological activity of recombinant equine eotaxin represent a large DNA virus immune evasion mechanism, interacting with in vitro. Cytokine 19, 27-30 chemokines to disrupt chemokine gradient formation and infl ammatory cell activation. The CMPs M-T1 and M-T7 from myxomavirus, and M3 from g68 herpesvirus have been shown to reduce cell invasion and plaque 6016 growth in animal models. M-T7 is believed to disrupt chemokine gradient formation through interaction with the glycosaminoglycan (GAG) binding IL-6 and its soluble receptor stimulates MCP-1 and sICAM-1 domain of chemokines. This action is unique and only partially under- secretion in human myoblasts and drives monocyte chemotaxis stood. We have postulated that mutations of M-T7 will aid in the identi- fi cation of inhibitory peptide sequences and chemokine binding domains Mariapaola Marino, Institute of General Pathology, Catholic University, responsible for anti-infl ammatory activity. Methods & Results: M-T7 Rome, Italy C-terminal deletion mutants have been prepared in a baculovirus expres- Flavia Scuderi, Institute of General Pathology, Catholic University, sion system. The mutants will be tested with CC and CXC chemokines Rome, Italy in a membrane fl uidity assay for cell activation and a mouse peritoneal Carlo Provenzano, Institute of General Pathology, Catholic University, monocyte migration assay. M-T7 signifi cantly reduced both monocyte Rome, Italy migration and activation in vitro and in vivo. A signifi cant decrease in Stefan Rose-John, Institute of Biochemistry, University of Kiel, Germany monocyte migration was detected in mouse ascites in response to MCP-1 Emanuela Bartoccioni, Institute of General Pathology, Catholic Univer- activation. M-T7 was also able to signifi cantly reduce MCP-1-mediated sity, Rome, Italy THP-1 cell activation. Deletion mutants have been engineered and are in preparation for analysis in these models. Conclusions: M-T7, a unique C- Purpose: In local infl ammatory response IL-6, upon binding to soluble IL- terminal CMP, alters cell migration and activation into peritoneal ascites 6Ralpha (trans-signaling), acts as a switch regulating the transition from and modifi es monocyte activation in vitro. Full-length M-T7 virus has IL-8(neutrophil-recruiting) to MCP-1(monocyte-recruiting) secretion by been created using baculovirus and deletion mutants are in development. endothelial cells. This mechanism could make virtually all cells sensitive Protein production of M-T7 and its mutants will begin once all viruses to IL-6/sIL-6Ralpha. Our purpose was to demonstrate that human myo- have been made. blasts can use trans-signaling to attract monocytes by evaluating MCP-1 secretion and chemotaxis. Methods: We incubated primary human myoblasts with Hyper-IL-6 6018 (HyIL-6, recombinant form of IL-6/sIL-6Ralpha), sgp130Fc as inhibitor and rIL-1beta as infl ammatory stimulus. MCP-1 and sICAM-1 secretion Immunomodulatory impact of the A2A adenosine receptor on the was measured by ELISA, membrane ICAM-1 and gp130 were evaluated profi le of chemokines produced by neutrophils by FACS, while transcription of MCP-1, IL-8, IL-6, ICAM-1 and gp130 mRNA by RTPCR. Chemotaxis was studied by migration of human mono- Mireille St-Onge, Laval University, Canada cyte THP-1 cells. Shaun R McColl, University of Adelaide, Australia Results: Myoblasts respond to HyIL-6 stimulation with an increase in Andrée-Anne Dussault, Laval University, Canada MCP-1 and ICAM-1 mRNA, an effect that is inhibited by sgp130Fc. The Cynthia Lafl amme, Laval University, Canada increase in MCP-1 and ICAM-1 mRNA parallels an increase in MCP-1 Line Bouchard, Laval University, Canada and sICAM-1 protein in supernatants, while membrane ICAM-1 level does Jean Boulanger, Laval University, Canada not change. Following HyIL-6 stimulation no IL-8, and very low levels of Marc Pouliot, Laval University, Canada IL-6, are produced; if HyIL-6 stimulation follows IL-1beta prestimulation, there is an increase in IL-6 and ICAM-1 mRNA, paralleled by an increase In toll-like receptor-4-stimulated human neutrophils, engagement of the in membrane and sICAM-1. MCP-1 released by HyIL-6-stimulated myo- adenosine A2A receptor (A2AR) selectively prevented the expression and blasts is biologically active as shown by chemotaxis assay. HyIL-6 treat- release of TNF-alpha, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2alpha/ ment does not modify gp130 basal mRNA expression or protein level. CXL2 and MIP-3alpha/CCL20. Little effect was observed on IL-1beta and Conclusions: These data demonstrated that in infl ammatory milieu human IL-8. These results were corroborated in vivo, using the murine air pouch myoblats can use locally secreted IL-6 and trans-signaling to target mono- model of infl ammation. In mice lacking the A2A receptor, neutrophils that cyte chemotaxis through MCP-1 gradient. Moreover, we can depict a sce- migrated into the air pouch 4h following LPS injection expressed higher nario in which locally produced IL-6 can further increase the IL1beta-in- mRNA levels of TNF-alpha, MIP-1alpha and MIP-1beta than neutrophils duced ICAM-1 expression while the sICAM-1 induced by IL-6 alone can from wild type mice. When comparing the expression of selected infl am- behave as a negative regulator through lymphocyte adhesion antagonism. matory genes in mononuclear cells present in the air pouch 72h after LPS injection, we found that expression of IL-1beta, TNF-alpha, IL-6 and MCP-2 was higher in A2AR knock out mice. In addition to highlight neu- 6017 trophils as an early and pivotal target for mediating adenosine anti-infl am- matory activities, these results identify TNF-alpha and the MIP chemokine Altering Monocyte Responses Through Chemokine Modulation family as gene products which expression is pivotally modulated by acti- vation of A2AR in TLR-activated human neutrophils. Early modulation of Dana L McIvor, Robarts Research Institute, Canada their production by infl ammatory neutrophils may be of signifi cance in the Kasinath Viswanathan, Robarts Research Institute, Canada way adenosine limits local and systemic infl ammation. John W Barrett, University of Western Ontario, Canada This work is supported by grants from the Canadian Institutes of Health Shannon M Clark, Robarts Research Institute, Canada Research (CIHR), The Arthritis Society (TAS) and the Canadian Arthritis Erbin Dai, Robarts Research Institute, Canada Network (CAN). Liying Liu, Robarts Research Institute, Canada S 152 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

IL-23. Interestingly, M-Mf produced higher levels of the anti-infl amma- 6019 tory cytokine IL-10, and CCL2 (MCP-1). Flow cytometry revealed both Mf populations to be positive for the myeloid markers Mac-1, F4/80, and Monocyte chemokine release during the acute infl ammatory response c-Fms (M-CSFR). Expression of the Toll-like receptors, TLR2 and TLR4, to coronary artery bypass surgery were similar on both Mf populations and could not explain the enhanced secretory capability of GM-Mf. Of particular interest, ‘priming’ of M-Mf Yishay Orr, Centre for Vascular Research, University of New South with GM-CSF prior to LPS stimulation led to enhanced release of TNFa, Wales, Australia IL-12 and NO. GM-Mf and M-Mf from IL-10-/- mice displayed compa- Jude M Taylor, Centre for Vascular Research, University of New South rable release of IL-6 and NO after LPS stimulation, suggesting that the Wales, Australia amount of IL-10 secreted may, in part, explain the enhanced proinfl amma- Paul G Bannon, Department of Cardiothoracic Surgery, Royal Prince tory nature of GM-Mf. From the current study, it is clear that GM-Mf and Alfred Hospital, Australia M-Mf represent alternate functional states (or belong to a range of diverse Carolyn Geczy, Infl ammatory Diseases Research Unit, University of New functional states) with GM-Mf adopting a proinfl ammatory, and M-Mf an South Wales, Australia anti-infl ammatory, phenotype. These fi ndings reaffi rm the role of GM-CSF Leonard Kritharides, Centre for Vascular Research, University of New as a proinfl ammatory cytokine. South Wales, Australia

Background 6021 The systemic acute infl ammatory response following both coronary artery bypass (CABG) and non-cardiac thoracic surgery (NCTS) is qualitatively The generation of large numbers of M-CSF and GM-CSF directed remarkably similar with respect to infl ammatory markers and cytokines human macrophage-lineage populations such as CRP, IL-1, IL-6 and TNF-α. However, CABG demonstrates a much higher incidence of organ dysfunction than NCTS, and shows sus- Kerrie J Way, The University of Melbourne, Australia tained monocyte activation. As monocyte activation is likely to have a role Martin Keene, The University of Melbourne, Australia in later infl ammatory and thrombotic sequelae of CABG, we have investi- Felix IL Clanchy, The University of Melbourne, Australia gated the monocyte chemokine response to CABG and NCTS. John A Hamilton, The University of Melbourne, Australia Methods Patients (n=6) undergoing CABG and NTCS (thoracotomy and pulmo- M-CSF (CSF-1) and GM-CSF control macrophage lineage survival, pro- nary lobectomy, n=6) had intra-operative and post operative blood sam- liferation and differentiation. A supply of large numbers of human cells ples taken. Systemic cytokines (Interleukin (IL)-1β, IL-6, IL-10, TNF-α), to study these processes is needed. For this purpose the single addition of Granulocyte-Colony Stimulating Factor (G-CSF), cortisol and chemokines each of these cytokines to human precursor cells is not suffi cient. There- (GRO-α, IL-8, SDF-1α, MCP-1 and MIP-1β) were measured using either a fore, we investigated the effects of M- and GM-CSF on human CD34+ customized Bio-plex Multiplex Cytokine Assay or standard ELISAs. cell expansion and differentiation in the presence of cytokine combina- Results tions (stem cell factor or SCF, IL-3, IL-6). G-CSF mobilised CD34 stem IL-6, IL-8, IL-10 and G-CSF increased signifi cantly following surgery in cells (>90% pure) were cultured in serum-free defi ned medium on low-at- both groups, but were higher after CABG (eg. IL-6: 1101.1 ± 397.6 pg/ml tachment plates. Inclusion of M-CSF induced a 60-70 fold cell expansion CABG vs 112.6 ± 12.5 pg/ml NCTS). Cortisol levels were higher intra- by Day 12-13, compared to a 30-40 fold increase in its absence. Addition operatively in the NCTS group for the same degree of operative trauma of GM-CSF induced a 500-fold increase in number. Cytospin histology at (p=0.05 unpaired t test) however post-operative levels were similar in the Day 12 revealed that M-CSF promoted differentiation of blast-like cells two groups. NCTS failed to induce a change in plasma chemokine levels into monocytic and larger macrophage-like cells. By Day 21 the M-CSF whereas SDF-1α, MCP-1 and MIP-1β increased (SDF-1α 2 fold, MCP-1 expanded cell population was positive for the following markers: CD34 5.4 fold and MIP-1 β 19.25 fold increases at peak concentrations, p<0.001 <1%, CD14 38%, CD11b 30%, CD64 29%, and c-Fms 30%, as determined compared with baseline) in CABG patients, peaking during myocardial by FACS. In contrast, expansion in GM-CSF produced fewer macrophage- reperfusion following aortic cross-clamp release. like cells - smaller, more immature cells were present. Conclusion As part of these studies, cell proliferation and adherence (differentiation) These results confi rm that cardiac surgery with cardiopulmonary bypass were assessed following culture of Day-7 expanded cells on bacteriologi- represents a potent stimulus for systemic chemokine release. The release cal plates in base medium containing M-CSF alone, or with various com- of chemokines specifi c for monocyte recruitment and activation may play binations of the cytokines. After 2-weeks the greatest expansion and the a role in the sustained monocyte activation following CABG. largest number of adherent cells were observed in the presence of IL-3 and SCF. The inclusion of GM-CSF in place of M-CSF produced a similar pattern of non-adherent cell growth; however little to no adherence of cells 6020 was observed after 2 weeks indicating a difference in phenotype. These studies highlight differences in the effects of M- and GM-CSF on CD34 Functional heterogeneity of GM-CSF- and M-CSF-derived cell expansion, and the generated populations offer potential for commit- macrophages ment toward the macrophage, dendritic or osteoclastic lineage.

Andrew J Fleetwood, CRC for Chronic Infl ammatory DiseasesDepart- ment of Medicine, RMH, University of Melbourne, Australia 6022 Andrew D Cook, CRC for Chronic Infl ammatory DiseasesDepartment of Medicine, RMH, University of Melbourne, Australia In vitro and in vivo evaluations of IL-6 cytokine subfamily John A Hamilton, CRC for Chronic Infl ammatory DiseasesDepartment of antagonists for the treatment of infl ammatory arthritis Medicine, RMH, University of Melbourne, Australia Jalal Jazayeri, Monash University, Australia Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical Warren Raye, Australia regulator implicated in generating macrophage (Mf) heterogeneity seen in Graeme J Carroll, Australia various tissues, infl ammatory sites and disease states. In the current study, GM-CSF-derived Mf (GM-Mf) were compared with M-CSF-derived Mf Although the aetiology of Rheumatoid Arthritis (RA) is not fully under- (M-Mf), derived from in vitro murine bone marrow culture. In response stood, laboratory and clinical evidence suggest that pro-infl ammatory to LPS stimulation, GM-Mf were capable of signifi cantly greater release cytokines such as Interleukin-6 (IL-6), IL-11, leukemia inhibitory factor of the infl ammatory cytokines TNFa, IL-6, nitric oxide (NO), IL-12 and (LIF), Oncostatin M (OSM), IL-1 α & β and tumour necrosis factor α Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 153

(TNFα) play very important roles in disease pathogenesis. Utilising site- monocytes. It is also proposed that PM cells maintain macrophage popula- directed mutagenesis several groups have generated mutant forms of LIF tions via proliferation in tissue, particularly at sites of infl ammation. The (i.e. LIF05 and MH35D), which have no agonist activity, but act in vitro PM population may also contribute to osteoclasts in the normal state and as effective antagonists for LIF. In our laboratory, LIF05 and MH35 were in rheumatoid arthritis. The aim of this research was to phenotype the PM found to inhibit the pro-catabolic effects of LIF on pig cartilage explant based on cell surface markers. DNA synthesis and proliferation kinetics cultures and inhibit the proliferation of BAF3 cells. At a concentration of studies were used to explore the rate and scale of monocyte proliferation. 100 ng/ml, MH35 produced a 72% reduction in the recombinant human Monocytes were then sorted into populations according to the level of ex- LIF (rhLIF) stimulated proteoglycan release. However in their monomeric pression of cell surface antigens and cultured at low cell concentrations forms high concentrations of LIF05 and MH35 are required. To overcome to minimise the effect of endogenous M-CSF production. Populations this problem, the LIF05 and MH35 gene fragments were fused to the Fc of interest included CD14med/loCD16+, CD14+CD13hi/lo, CD14+CD11bhi/lo, portion of the human immunoglobulin G (hIgG). The advantages of these CD14+CD33hi/lo and CD14+CD64hi/lo. Proliferation was determined by the constructs include possible increased avidity for multivalent ligands due to incorporation of nucleotide analogue, bromodeoxyuridine (BrdU) or ob- the resulting bivalency of dimeric fusion proteins, a longer serum half-life, served with CFSE labelling. CFSE labelled monocytes were able to be lower toxicity; and increased drug stability. The overall aim of the project sorted based into different generations for further studies. is therefore to (i) further investigate whether the antagonists have the ca- pacity to abrogate the effects of LIF and OSM in vitro, by quantifying the mRNA levels of cartilage degrading enzymes (metalloproteinases and col- 6025 lagenases); MMP1, MMP3, ADAM-TS5 and tissue inhibitors metallopro- teinases (TIMPs) from RNA samples isolated from chondrocytes (primary Anti-infl ammatory Properties of Serum IgA: Comparisons with and cell lines) cultured in the presence of LIF and OSM with and without IVIG the antagonists and (ii) to explore, in vivo, whether the antagonists can at- tenuate arthritis in the collagen induced form of arthritis (CIA) models. Anthea M Downs, PathCentre Perth, Australia Wendy N Erber, Australia

6023 Serum IgA defi ciency is associated with autoimmune infl ammatory dis- eases (AID) and IgA defi ciency at mucous membranes contributes signifi - CD34+ stem cell derived macrophages cantly to morbidity and mortality in GVHD. IVIG is a category 1 indication for treating these diseases. The majority of Felix IL Clanchy, Department of Medicine (RMH), The University of IVIG preparations, including Intragam (Australia), contain predominantly Melbourne, Australia IgG with trace amounts of IgM and IgA. entaglobulin (Germany) contains John A Hamilton, Department of Medicine (RMH), The University of 38mg/ml IgG, 6mg/ml IgM and 6mg/ml IgA and infusion twice weekly for Melbourne, Australia 3 weeks has been shown to restore serum IgA levels to the normal range. IgM/gA-supplemented preparations such as Pentaglobulin may have im- Large, pure cultures of murine bone marrow macrophages, created from proved effi cacy in AID. femur-harvested marrow and cultured in the presence of M-CSF, are a con- Purpose: Purifi ed serum IgA and IVIG (Intragam) were added to whole venient means to study this cell type. Human differentiation studies are blood cultures to compared their effects on activated monocyte surface limited as human marrow yields few macrophages via this technique. A antigen expression and intracellular cytokine production. system for generating human stem cell-derived macrophages would pro- Methods: Blood was collected from healthy volunteers and cultured with vide a means to understand and control this differentiation from an early RPMI. Monocytes were stimulated with LPS+IFNg, in the presence of developmental stage and also to study signal transduction. IgA or IVIG. Cultures were incubated for 4-20 hours and expression of G-CSF mobilised blood was collected by aphaeresis and CD34+ stem monocyte surface antigens and intracellular cytokines were measured by cells isolated using immuno-magnetic selection. The cells were cultured fl ow cytometry. in serum-free media with differing cytokines and on various substrates Results: CD64 expression was up-regulated by IgG but not IgA. CD89 was to induce proliferation and differentiation along the macrophage lineage up-regulated by in IgA and IgG. TNFa was present in 65% of monocytes pathway. Terminally differentiated macrophages were enriched via adher- stimulated with LPS+ IFNg. Co-incubation with 1mg/ml IgA reduced ence, then phenotyped and found to be phagocytic and macrophage-like in TNFa expresssion to 9.5% of monocytes. Incubation of LPS+IFNg-stimu- morphology and by FACS profi le (CD14medCD11bhiCD64+). The course lated monocytes with IgG at doses up to 5mg/ml had no effect on TNFa of development and lineage commitment were measured by cell prolifera- production. Similar results were obtained for IL-6 production. Co-incuba- tion, histology/morphology, adhesion and cell surface markers. Differenc- tion with IgA and IgG stimulated basal IL-10 production. Binding studies es in these characteristics can be attributed to differentiation or activation detected specifi c antibodies against LPS in IgA but not IgG. depending on the cell culture milieu. Conclusions: IgA defi ciency is associated with AID and GVHD in which dysregulated cytokine production is implicated. The minimum concentra- tion of IgA required for inhibition of monocyte cytokine production in this 6024 in vitro study equates to the lower limit of the normal range for serum IgA. If one of the roles of circulating IgA is to modulate cytokine production, The nature of in vitro monocyte proliferation IgA supplementation maybe of benefi t in treating these diseases. Inactiva- tion of LPS by specifi c binding to IgA may account for the reduction in Felix IL Clanchy, Department of Medicine (RMH), The University of infl ammatory cytokine production. Melbourne, Australia John A Hamilton, Department of Medicine (RMH), The University of Melbourne, Australia 6026

Monocytes are heterogeneous in terms of function, size and surface marker TIMP-2- and Anti-SDF-1 Antibody-loaded Biodegradable Polymeric expression but discrete subsets have been identifi ed with specialised func- Films Inhibit Post-surgical Adhesion Formation in Rats tions. These subpopulations may also be representative of maturational states. The proliferative potential of a monoctye/macrophage lineage cell Johanne D Cashman, ARC Pharmaceuticals Inc., Canada decreases as it differentiates although a subpopulation of both monocytes Christopher M Springate, ARC Pharmaceuticals Inc., Canada and macrophages are thought to retain a portion of this potential. It is proposed that the proliferating monocyte (PM) subpopulation migrates Purpose. Adhesions occur when peritoneal trauma, ischemia or infection from bone marrow into the blood and differentiates therein to more mature initiate an infl ammatory cascade that culminates in the formation of dense S 154 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

fi brous bands between affected organs. Cytokines regulate leukocyte re- Ailsa Frew, St Vincent's Institute, Australia cruitment and activation at sites of infl ammation and may therefore play a Nicos A Nicola, Walter and Eliza Hall Institute, Australia key role in adhesion formation. In this study a number of cytokines were Douglas J Hilton, Walter and Eliza Hall Institute, Australia loaded into biodegradable polymeric fi lms to determine if sustained local Warren S Alexander, Walter and Eliza Hall Institute, Australia release of these agents at the site of injury could affect post-operative ad- Robyn Starr, St Vincent's Institute, Australia hesion development in an in vivo model of surgical adhesions. Methods. The anti-adhesion properties of cytokines TGF-β1, TIMP-2, and Suppressor of cytokine signalling (SOCS) proteins regulate the intensi- CD40 ligand, as well as protease inhibitor MG125 and anti-SDF-1 anti- ty, duration and quality of responses to cytokine. Previous studies have body were investigated by application of hyaluronic acid fi lms containing shown SOCS1 and SOCS3 to have similar functions in vitro, but the phe- the agent at the site of surgical injury in a total of 36 rats using the caecal notype of mice lacking each of these inhibitors is quite different. We have sidewall surgical adhesion model. Control animals received no fi lm (sham) previously established that SOCS1 is a key regulator of T cell homeostasis or a control (no drug) fi lm. The animals were sacrifi ced seven days after by regulating signals triggered by gc-dependent cytokines. The role of treatment and an adhesion score was determined based on the strength and SOCS3 in the immune system has proved more diffi cult to defi ne as mice extent of adhesions. Potential toxicity was determined by changes in white lacking SOCS3 die during embryogenesis. Using the Cre-loxP approach, blood cell count, animal weight and surgical incision thickness. Statistical we have generated mice lacking SOCS3 specifi cally in T and NKT cells. analysis was carried out using Student’s t-test and p values less than 0.05 These mice are healthy and thymic development appears normal. Lymph were considered signifi cant. nodes, however, are enlarged and the number of peripheral lymphocytes Results. The application of control fi lms as well as fi lms loaded with TGF- is increased. In response to an anti-CD3 stimulus, SOCS3-defi cient T β1, TIMP-2, CD40 ligand, MG125 and anti-SDF-1 antibody inhibited cells show greater IL-2 production and proliferation than wildtype cells. adhesion formation compared to sham treated animals. In addition, a sig- Co-stimulation of SOCS3-defi cient T cells through CD28 ligation does nifi cantly greater reduction in adhesions was observed using TIMP-2- or not substantially augment the response, suggesting that full activation of anti-SDF-1 antibody-loaded fi lms compared to control fi lms. No toxicity T cells proceeds largely independently of co-stimulation when SOCS3 is was observed in any of the treatment groups. absent. These data suggest that SOCS3 modulates T cell activation and, Conclusions. These results suggest the potential use of TIMP-2- or anti- like the closely related SOCS1, SOCS3 may be an important regulator of SDF-1 antibody-loaded biodegradable polymeric fi lms for the inhibition T cell behaviour and homeostasis. of post-surgical adhesion formation. 6029 6027 Macrophage migration inhibitory factor regulation of glucocorticoid Periodontal Therapy: A Novel Acute Infl ammatory Model sensitivity

Francesco D'Aiuto, Eastman Dental Institute and Hospital, University Daniel Aeberli, Monash University, Monash Medical Centre, Clayton, College London, United Kingdom Melbourne, Australia Mohamed Parkar, Eastman Dental Institute and Hospital, University Yuan Yang, Monash University, Monash Medical Centre, Clayton, Mel- College London, United Kingdom bourne, Australia Maurizio S Tonetti, Connecticut Health Science Centre, Farmington, CT, Jin Xue, Monash University, Monash Medical Centre, Clayton, Melbour- United States ne, Australia Lanie Santos, Australia Purpose. Understanding mechanisms involved in acute infl ammation may Eric F Morand, Monash Institute of Medical Research, Monash Univer- provide valuable information for individual risk prediction and in identify- sity, Australia ing novel therapeutic targets. We therefore aimed at developing a novel infl ammatory in-vivo model of Objective: Glucocorticoids (GC) are widely used in the treatment of in- acute infl ammation. fl ammatory diseases but are associated with signifi cant dose-dependent Methods. 55 otherwise healthy subjects suffering from severe periodontitis side-effects. Macrophage migration inhibitory factor (MIF) is a pro-infl am- were included in this investigation. They received intensive periodontal matory cytokine induced by GC which antagonises their effects through as therapy within a 6-hr period in a controlled clinical research environment. yet unknown mechanisms. We investigated the role of MIF in the regula- Blood samples were taken from each individual at baseline and 1 and 7 and tion of GC responsiveness, and the effect of MIF on p38 MAP kinase, 30 days after treatment. Circulating concentrations of a series of infl amma- the GC-inducible MAP kinase antagonist protein MAP kinase phosphatase tory mediators (TNF-alpha, IL-6, CRP and Fibrinogen) were assessed by (MKP)-1, and IkB. high-sensitivity immuno assays at the end of the study. Differential blood Methods: Peritoneal macrophages from wildtype (WT) and MIF -/- mice counts were also measured by standard laboratory procedures. were treated with LPS 100ng/ml and dexamethasone (DEX) 0-7 to 10-10 Results. TNF-alpha levels were signifi cantly raised after 1 day of therapy M. Soluble TNF alpha was measured by ELISA. Expression of phospho- (P<0.01) whereas IL-6 (P<0.01), CRP (P<0.001) and Fibrinogen (P<0.001) p38 MAP kinase, MKP-1, and IkB alpha were measured using Western concentrations peaked 24hrs after and returned to baseline values one blotting. month following therapy. We also observed mild neutrophilia, monocyto- Results: We report MIF-dependent regulation of GC responsiveness. Com- sis and lymphopenia after day 1 from the stimulus (periodontal therapy). pared to WT, LPS stimulated MIF-/- macrophage TNF was 3-fold more Conclusions.These results suggest that intensive periodontal therapy in- sensitive to DEX (MIF-/- cell IC50 = 1.25nM, WT = 3.98 nM, P<0.0001). duces a one week acute infl ammatory response of moderate magnitude. LPS-induced phosphorylation of p38 MAPK was impaired in MIF -/- cells Periodontal therapy is therefore proposed as a novel and reliable non drug- compared to WT (p=0.01). In contrast, no signifi cant difference in IkB induced in-vivo model of acute infl ammation. degradation was found. Expression of MKP1 was higher in MIF-/- both basally (p<0.05) and in response to DEX 10-9 M (p<0.05). Induction of MKP-1 was associated with dephosphorylation of phospho-p38. 6028 Conclusions: These data demonstrate that MIF regulates GC sensitivity of macrophage TNF production, and suggest that MKP-1 may play an im- Regulation of T cell function by SOCS3 portant role in the MIF regulated GC sensitivity. Increased sensitivity to GC in the absence of MIF suggests the possibility of MIF antagonism as a Christine Brender, St Vincent's Institute, Australia steroid-sparing therapy. Brendan Jenkins, Ludwig Institute for Cancer Research, Australia Joel Fletcher, St Vincent's Institute, Australia Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 155

Osteoclasts are terminally differentiated cells that arise from a haemato- 6030 poietic stem cell lineage, which also gives rise to monocytes and macro- phages. Osteoclast differentiation and regulation of this process to main- Identifi cation of a novel enhancer in the fi rst intron of Macrophage tain bone homeostasis are central to the understanding of the pathogenesis Migration Inhibitory Factor (MIF) and treatment of bone diseases, such as osteoporosis. In vitro, osteoclast formation from bone marrow macrophages is induced by RANKL (recep- Elaine Beaulieu, Universiy of Manchester, United Kingdom tor activator of David W Ray, Universiy of Manchester, United Kingdom NF-kappa B ligand) in the presence of M-CSF (macrophage colony stimu- Andrew D McMaster, Universiy of Manchester, United Kingdom lating factor). Osteoclastogenesis is markedly enhanced in bone mar- May Tassabehji, Universiy of Manchester, United Kingdom row macrophages from IFNAR1-/- and IFNAR2-/- mice and results in Rachelle Donn, Universiy of Manchester, United Kingdom increased number of multinucleated cells positive for osteoclast marker, TRAP (tartrate-resistant acid phosphatase). Consequently, the mutant mice Background: Macrophage migration inhibitory factor (MIF) is a pleiotro- develop osteoporotic phenotype, characterised by reduced bone density. pic pro-infl ammatory cytokine. The MIF gene is genetically associated These fi ndings suggest that the IFN alpha/beta system is critical for the with rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis and sev- negative feedback regulation of osteoclastogenesis and that RANKL sig- eral other infl ammatory diseases. The balance between MIF activity and naling is essential for the induction of osteoclast differentiation. endogenous glucocorticoid production appear central to the pathogenesis of chronic infl ammatory diseases. Aim: To identify important transcriptional elements across the MIF gene 6032 locus. Methods: DNase I hypersensitivity assay was utilised to identify sites of High-throughput cell-based assays for the measurement of chromatin remodelling across a 10.2 Kb region encompassing the entire infl ammation MIF gene (846bp), 3 Kb 5’ and 6 Kb 3’. Functional analysis of the hyper- sensitive site was carried out via reporter assays using a simple heterolo- Liming Yu, General Electric Global Research, United States gous viral promoter (SV40) in combination with the well characterised Stewart Abbot, General Electric Global Research, United States SV40 enhancer as a positive control. Transient transfections were per- Christoph Hergersberg, General Electric Global Research, United States formed in human cell types relevant to infl ammation: a T-lymphoblast cell Anton Beletskii, General Electric Global Research, United States line (CEMC7A) and a monocytic cell line (THP-1). Cells were transfected in biological triplicates by electroporation and incubated for 24h. Cells Infl ammation is a complex phenomenon involving multiple cellular pro- were then lysed and a luciferase assay carried out to determine transcrip- cesses. Infl ammation assays currently used to screen small molecules or tional activity. Fold induction was determined relative to the SV40 pro- siRNAs are diffi cult to perform at a scale required to screen large libraries moter control using Anova (2 tailed) on log transformed values. and commonly measure cellular changes that are distant surrogates of the Results: A hypersensitive site centred on the fi rst intron of MIF was found processes of interest. We have developed novel high-throughput cell-based in CEMC7A cells. The isolated intron was shown to have signifi cant en- functional assays of cell migration, phagocytosis, and modifi ed low-den- hancer activity in both cell types (CEMC7A >3 fold induction, p < 0.0001 : sity lipoprotein (LDL) uptake in a 96 and 384 well formats. These assays THP-1 > 3 fold induction p < 0.0001). As for a classical enhancer the effect rely on the combination of novel fl uorescent reporter systems and high was observed to be independent of orientation. throughput automated fl uorescence microscopy. In addition we have de- Discussion: We have identifi ed a novel MIF gene regulatory region within veloped dedicated image processing algorithms that allow detailed quan- the fi rst intron of MIF. Bioinformatic analysis of the MIF intron has been titative analysis of the assays. The resulting high-content readout allows used to identify a limited number of potential transcription factors respon- assessment of functional changes in immune cell activation, migration sible for the enhancer effect observed. These are currently being tested in and morphology. Phagocytosis and cell migration assays were validated vitro. Such transcription factors are natural targets for understanding the with known modulators of these processes and demonstrated acceptable transcriptional control of MIF gene expression and could lead to novel Z’ values of 0.2-0.5 and defi ned inhibitory concentrations consistent with ways of regulating infl ammatory diseases for which MIF is known to play published values. We have investigated the sensitivity and robustness of a central role. Acetylated-LDL (AcLDL) uptake assays by conducting a limited screen of 640 members of a Library Of Pharmaceutically Active Compounds (LOPAC) for inhibition of AcLDL uptake by macrophages. Hits identifi ed 6031 by the assay confi rmed several compounds known to modify LDL uptake. The limited screen also uncovered several previously uncharacterized Factors involved in bone metabolism and osteoclast function modulators of AcLDL uptake and further functional validation is ongo- ing. Collectively, these assays and observations offer better understanding Durda Pavasovic, Centre for Functional Genomics and Human Disease, of how different pathways may contribute to infl ammatory responses and Monash Institute of Medical Research, Monash University, Clayton VIC demonstrate the utility of functional cell-based screens of infl ammation. 3800, Australia Roya Lari, Cooperative Research Centre for Chronic Infl ammatory Diseases, Department of Medicine, The University of Melbourne, Royal 6033 Melbo, Australia John A Hamilton, Cooperative Research Centre for Chronic Infl amma- Caffeine metabolites and other methylxanthines exerting anti- tory Diseases, Department of Medicine, The University of Melbourne, infl ammatory properties Royal Melbo, Australia Paul J Hertzog, Centre for Functional Genomics and Human Disease, Harald JJ Moonen, Department of Health Risk Analysis and Toxicology, Monash Institute of Medical Research, Monash University, Clayton VIC Maastricht University, The Netherlands 3800, Australia Liesbeth Geraets, Department of Pharmacology and Toxicology, Maastricht University, The Netherlands Bone remodeling is a tightly regulated process that couples resorption of Aalt Bast, Department of Pharmacology and Toxicology, Maastricht old bone by osteoclasts and the deposition of new bone by osteoblasts. University, The Netherlands An imbalance between bone formation and bone resorption can result in Emiel FM Wouters, Department of Pulmonology, Academic Hospital various metabolic bone diseases, such as rheumatoid arthritis and osteo- Maastricht, The Netherlands porosis. Geja J Hageman, Department of Health Risk Analysis and Toxicology, Maastricht University, The Netherlands S 156 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Background: The nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP- Michael J Hickey, Monash University, Australia 1) is believed to play an important role in infl ammation, by co-activating the transcription factor NF-kB and the resulting NF-κB mediated gene Macrophage migration inhibitory factor (MIF) is a pro-infl ammatory cyto- expression. Therefore, inhibition of PARP-1 activation is hypothesized to kine with an important role in the development of infl ammatory diseases reduce the NF-κB mediated infl ammatory response. Increased infl amma- including septic shock and rheumatoid arthritis. Recent observations have tion and production of reactive oxygen species (ROS) are characteristic shown that in the absence of MIF, leukocyte recruitment during infl amma- for chronic infl ammatory diseases such as diabetes and chronic obstructive tory responses is reduced. However, it is not known if this is due to the pulmonary disease (COPD). Damage to tissues and cells imposed by ROS ability of MIF to directly induce leukocyte-endothelial cell interactions. contribute to complications such as vascular dysfunction and a decreased Therefore, the aim of this study was to determine the direct effects of re- function of vital organs. Recently, several natural food-grade PARP-1 in- combinant MIF (rMIF) on leukocyte-endothelial cell interactions in the hibitors were identifi ed in our lab. microvasculature. To determine if MIF was capable of mediating a rapid Purpose: To determine whether certain natural food-grade compounds ex- response in leukocyte-endothelial cell interactions, rMIF was superfused ert anti-infl ammatory properties in human endothelial cells, and in an ex- over the cremaster muscle of C57BL/6 mice at various doses for 60 min- vivo assay with human whole blood. utes. Intravital microscopy was used to assess leukocyte rolling, adhesion Methods: Cell cultures and whole blood cultures were exposed to endo- and emigration in post-capillary venules. Acute treatment of the cremaster toxin (LPS) during 16 hours at 37 °C, with or without pre-incubation with with rMIF had no affect on leukocyte-endothelial cell interactions. To single methylxanthines or combinations of methylxanthines, including liv- determine if MIF could mediate delayed microvascular responses, we ad- er metabolites of caffeine (1-100 μM). Levels of pro-infl ammatory cyto- ministered rMIF via intrascrotal injection to the cremaster muscle. This kines (IL-6, TNF-α) were subsequently determined in cell culture medium resulted in marked and signifi cant increases in leukocyte adhesion and or blood plasma using ELISA. emigration relative to saline-treated mice. Immunohistochemical assess- Results: Pre-incubation with several methylxanthines (e.g. 1,7-dimethyl- ment of rMIF-treated cremaster muscles demonstrated the recruited leu- xanthine, theophylline) or combinations of methylxanthines led to a de- kocytes were predominantly CD68+, indicating selective recruitment of crease in pro-infl ammatory cytokine levels upon exposure to LPS in both monocyte/macrophage lineage cells. rMIF-induced recruitment was not cell cultures and whole blood. detected in P-selectin-/- mice, indicating rMIF-induced adhesion was P- Conclusion: Caffeine liver metabolites and other methylxanthines are able selectin dependent. In contrast, treatment with anti-CD18 function block- to decrease the LPS-induced pro-infl ammatory cytokine production in both ing antibodies had no effect. These fi ndings indicate that MIF is a direct endothelial cells and whole blood cultures, suggesting that infl ammatory inducer of monocyte-lineage leukocyte recruitment, and that this process processes, as occur in chronic infl ammatory diseases, could be attenuated is dependent on P-selectin but not CD18. by natural food-grade compounds. 6036 6034 Tactile allodynia induced by PAF and glutamate is mediated through TNF-alpha is a Survival Factor for RAW264.7 Macrophage Cells, NO/cyclic GMP /G-kinase cascade in spinal cord Antagonising Apoptosis Through Activation of NF-kappaB Pathways Toshihiro Dohi, Department of Dental Pharmacology, Hiroshima Univer- Susan Z Lo, University of Western Australia, Australia sity Graduate School of Biomedical Sciences, Japan Jay H Steer, University of Western Australia, Australia Katsuya Morita, Department of Dental Pharmacology, Hiroshima Uni- David A Joyce, University of Western Australia, Australia versity Graduate School of Biomedical Sciences, Japan Md Joynal Abdin, Department of Dental Pharmacology, Hiroshima Uni- Tumour necrosis factor-alpha (TNF-alpha) is a macrophage product that versity Graduate School of Biomedical Sciences, Japan activates the death-domain receptor-induced pathway to apoptosis in many Norimitsu Morioka, Department of Dental Pharmacology, Hiroshima cell types. In RAW264.7 murine macrophages themselves, however, TNF- University Graduate School of Biomedical Sciences, Japan alpha promotes survival by suppressing the spontaneous rate of apoptosis Tomoya Kitayama, Department of Dental Pharmacology, Hiroshima and suppressing apoptosis induced by interference with DNA replication University Graduate School of Biomedical Sciences, Japan (etoposide or cisplatin) or through activation of the mitochondrial-linked apoptosis pathway with the mitochondrial proton ionophore, CCCP. This Platelet-activating factor (PAF) is released from neutrophils and other in- is apparent as a reduced rate of Annexin-V positivity in fl ow cytometry, fl ammation-related cells in spinal cord injury and is a signaling molecule and is accompanied by reduced activity of caspase-3, the terminus of the triggering the infl ammatory events and tissue damage in the injury. The caspase cascade. development of chronic pain following spinal cord injury is a signifi cant TNF-alpha activates signalling in the NF-kappaB pathways, as well as problem that is often refractory to a majority of clinical interventions. We activating the death-domain initiated pathways. Blocking activity in the have previously reported that intrathecally administered PAF produced hy- NF-kappaB pathway blocks the protective effects of TNF-alpha, implying peralgesia and tactile allodynia in mice and these noxious events can be that it is essential for survival. NF-kappaB activation does not exert its blocked by antagonists for PAF receptors, ATP P2X receptors or NMDA pro-survival effect through preventing p53 activation: both etoposide and receptors (Pain 111, 351-359, 2004). cisplatin activate p53 through their actions on DNA replication, but TNF- In the present study, the role of NO and cGMP in PAF- and glutamate- alpha does not prevent either the accumulation of p53 or p53-induced gene induced tactile allodynia in spinal cord was studied in mice. Tactile allo- transcription. Experiments to date point to a single point of intersection dynia induced by intrathecal injection of PAF and glutamate was blocked between the pro-survival NF-kappaB pathways and the death pathways by MK801, an NMDA antagonist. Intrathecal injection of 5-amino-3-mo- initiated by etoposide, cisplatin and CCCP, at or close to the point of mito- epholinyl-1,2,3-oxadiazolium chloride (SIN-1), a NO donor and 8-pCPT- chondrial pore transition and cytochrome c release. cGMP, a membrane-permeable and phosphodiesterase stable analog of cGMP, produced potent tactile allodynia. PAF- and glutamate-induced allodynia was blocked by the pretreatment with 7-nitroindazole (7-NI), a 6035 NO synthase inhibitor. 7-NI did not affect the allodynia induced by SIN-1 and 8-pCPT-cGMP. Pretreatment with carboxy-PTIO and hemoglobin, NO Macrophage Migration Inhibitory Factor (MIF) Induces the scavengers, ODQ and NS 2028, soluble guanylate cyclase inhibitors pro- Recruitment of Monocytes in the Cremasteric Microvasculature tected from the induction of allodynia by PAF, glutamate and SIN-1 but not by 8-pCPT-cGMP. Rp-8-pCPT-cGMPS, a G-kinase inhibitor blocked the Julia L Gregory, Monash University, Australia induction of allodynia by PAF and glutamate, SIN-1 and 8-pCPT-cGMP. Eric F Morand, Monash University, Australia These results suggest that the cascade of the production of NO and the fol- Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 157 lowing activation of soluble guanylate cyclase and cyclic GMP/G-kinase In murine macrophage RAW264.7 cells and in human A549 lung epithelial plays key roles for the transduction of the noxious signaling for tactile cells, exposure to each of these toxins results in increased mitochondrial allodynia induced by PAF and glutamate in spinal cord. electron reduction of O2 to the superoxide radical (O2-). We demonstrate this using O2-activate fl uorophores both at the single cell level (fl ow cy- tometry) and in cell culture. 6037 A sharp reduction of NF-kappaB-induced transcription occurs with each toxin and in both cell types. Paradoxically, this is associated with in- Characterization of the Human Oncostatin M (OSM) Receptort Sub- creased, rather than reduced binding of nuclear NF-kappaB factors (p65/ Unit, OSMBR p50 heterodimer, primarily) to DNA, implying that the mitochondrial tox- ins are acting at the level of NF-kappaB-induced transcription, rather than Kirrily A O'Hara, Asthma & Allergy Research Institute; Centre for Asth- at the level of NF-kappaB factor cytosol-nuclear translocation, the classi- ma, Allergy and Respiratory Research, University of Western Australia; cal means of NF-kappaB signal activation. Wes, Australia We have investigated several potential mitochondria - nuclear signals that Mary-Anne Kedda, Asthma & Allergy Research Institute, Centre for may mediate this pathway interaction. The redox-cycling O2- generator, Health Research, Queensland University of Technology, Australia DMNQ, increases NF-kappaB signaling, implying that the mitochondrial Mirjana Fogel-Petrovic, Asthma & Allergy Research Institute; School of signal is not transmitted through O2- . Nor does it seem to be a conse- Medicine and Pharmacology, University of Western Australia,, Australia quence of mitochondrial Ca2+ release, because the effects are generally Philip J Thompson, Asthma & Allergy Research Institute; Centre for opposite to those predicted from experimentally raising or lowering intra- Asthma, Allergy and Respiratory Research, University of Western Austra- cellular Ca2+ concentration. Activation of a caspase cascade, following lia;, Australia mitochondrial pore transition (a possible consequence of mitochondrial Lawrence J Abraham, Western Australian Institute for Medical Research reactive oxygen generation) and cytochrome C release is under investiga- and UWA Centre for Medical Research; UWA School of Biomedical and tion. Chemical Sc Darryl A Knight, Asthma & Allergy Research Institute; James Hogg Icapture Centre, St Pauls Hospital Vancouver. 6039

Introduction: OSM is an IL-6 family cytokine, which displays biological Cannabinoids Inhibit Cartilage Degradation Induced by Cytokines activities that suggest it is a key mediator in the wound repair response after infl ammatory injury. OSM signals through a receptor complex of Kim D Rainsford, Sheffi eld Hallam University, United Kingdom OSMBR/gp130. Since little is known about the regulation of OSMBR Estery C Mbvundula, Sheffi eld Hallam University, United Kingdom expression, the structure of the OSMBR promoter region was investi- Rowena A Bunning, Sheffi eld Hallam University, United Kingdom gated. Methods & Results: A 2.6 kb fragment incorporating the putative OSMBR promoter region was cloned into the promoter-less luciferase Cannabinoids have analgesic, anti-infl ammatory and immunosuppressive vector and nested deletions generated. Deletion clones and full-length effects and reduce joint damage in animal models of arthritis (Malfait et al., constructs were transiently transfected into HFL-1 cells and the effect of 2000). We recently showed that synthetic cannabinoids protect cartilage these constructs on promoter activity determined using a Luciferase As- from IL-1-induced extracellular matrix degradation in vitro (Mbvundula say. Transfections with the full length promoter construct resulted in a 20 et al., 2005). This suggests a potential role for cannabinoids in controlling fold increase in luciferase activity compared to the control vector. With cartilage destruction in arthritis. Here, we further explored the mechanisms the full-length promoter construct activity being 100%, transfection of de- of action of these drugs on cartilage destruction and determined if CB1 or letion constructs into HFL-1 cells resulted in a 13% to 160% change in CB2 receptors are present on chondrocytes. luciferase activity. Analysis of the transcriptional start site of the OSMBR Effects of Win-55,212-2 were studied on IL-1-induced NO and PGE-2 gene (using RNA ligase-mediated rapid amplifi cation of 5' prime cDNA production in bovine articular chondrocytes and the expression of induc- ends (RLM-RACE)) indicates the presence of two transcripts in HFL-1 ible NOS, COX-2, CB receptors. The effect of this cannabinoid was also cells. Conclusions: These results show that the cloned fragment contains a studied on IL-1-stimulated collagen breakdown in explant cultures. Bovine functional promoter of the OSMBR gene. Nested deletion analysis and 5' articular chondrocytes maintained as primary cultures and were stimulated RLM-RACE analysis indicate that important regulatory regions are pres- with IL-1; (100 U/ml; 0.06nM) to produce NO and PGE-2 in the pres- ent in the proximal promoter fragment. In HFL-1 cells two transcripts of ence or absence of 1-10uM Win-55,212-2. NO production was measured the OSMBR gene are present. as nitrite using the Griess reagent and PGE-2 using EIA. Expression of Supported by the Asthma & Allergy Research Institute, The CRC for Asth- iNOS and COX-2 as well as cannabinoid receptors in chondrocytes was ma and the University of Western Australia. studied by Western blotting. Collagen breakdown was studied in bovine Key Words: Oncostatin M (OSM), Oncostatin M receptor (OSMBR), Fi- nasal cartilage explant cultures, cultured for 21 days and stimulated to re- broblasts, Wound repair response sorb with IL-1 (500 U/ml; 0.3 nM) in the presence or absence of 5-10 uM Win-55,212-2. Win-55,212-2 <10 uM, inhibited NO and PGE-2 production and collagen 6038 breakdown and also inhibited expression of iNOS and COX-2. Chondro- cytes appeared to constitutively express cannabinoid receptors CB1 and Abrupt Cessation of NF-kappaB Signaling and NF-kappaB-Induced CB2. Cannabinoids may inhibit iNOS and COX, thus reducing NO and Gene Transcription on After Interference with Mitochondrial PGE2 production, possibly by acting through the cannabinoid receptors, Electron Transport or Mitochondrial Proton Gradient CB1 and/or CB2. References: Tindaro M Giardina, University of Western Australia, Australia Malfait AM et al. (2000) Proc Natl Acad Sci; 97(17):9561-6. Jay H Steer, University of Western Australia, Australia Mbvundula EC et al. (2005)Biochem Pharmacol; 69(4):635-40. David A Joyce, University of Western Australia, Australia

Rotenone, a toxin implicated human Parkinson’s disease, blocks mito- 6040 chondrial electron transport at Complex I. Antimycin A blocks electron delivery through Complex III. The protonophore, CCCP, removes the pro- Beta-Arrestin Expression and Function in Macrophages ton-motive force responsible for mitochondrial ATP synthesis through the F1/F0 ATP’ase in the inner mitochondrial membrane. Jane E Lattin, Institute for Molecular Bioscience, University of Queens- land, St Lucia, QLD 4072., Australia S 158 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Lovisa M Blomberg, Institute for Molecular Bioscience, University of induced by adenosine. However, the cells treated with the selective A3 Queensland, St Lucia, QLD 4072., Australia adenosine receptor antagonist VUF5574 showed a general decrease of ad- Norelle L Daly, Institute for Molecular Bioscience, University of Queens- enosine induced potentiation as compared with the cells without antagonist land, St Lucia, QLD 4072., Australia treatment. Results from the current study indicate the existence of A2A David J Craik, Institute for Molecular Bioscience, University of Queens- and A3 adenosine receptors on the rat peritoneal mast cells. Although an land, St Lucia, QLD 4072., Australia overall potentiation of anti-IgE induced histamine release is achieved by Jodie A Robinson, Institute for Molecular Bioscience, University of activating the A3 adenosine receptor, simultaneous activation of the A2A Queensland, St Lucia, QLD 4072., Australia adenosine receptor provides an endogenous modulation on the level of Stuart Kellie, Institute for Molecular Bioscience, University of Queens- A3-mediated potentiation. land, St Lucia, QLD 4072., Australia David A Hume, Institute for Molecular Bioscience, University of Queens- land, St Lucia, QLD 4072., Australia 6042 Matt J Sweet, Institute for Molecular Bioscience, University of Queens- land, St Lucia, QLD 4072., Australia Chemotactic factors activate the Nuclear Factor kappa-B (NF-kB) and COX-2 through ERK1/2 and p38 MAPK, but not PI3K, in HL- G-protein coupled receptors (GPCRs) and their regulatory molecules play 60-derived neutrophilic cells important roles in infl ammation and immune regulation. Beta-arrestin 1 and Beta-arrestin 2 regulate GPCR signaling by promoting receptor desen- María A Hidalgo, Universidad Austral de Chile, Chile sitisation and internalisation. Beta-arrestins may also regulate macrophage Loreto J Igor, Universidad Austral de Chile, Chile signalling in a GPCR-independent manner through mediation of other sig- Juan L Hancke, Universidad Austral de Chile, Chile nalling pathways due to their ability to act as adapter molecules in the Ilona I Concha, Universidad Austral de Chile, Chile assembly of signalling complexes and by regulating the intracellular com- Rafael A Burgos, Universidad Austral de Chile, Chile partmentalisation of these complexes. We show here that murine primary macrophages constitutively express elevated levels of Beta-arrestin 1 and The neutrophils are the fi rst line of defense of the organism against infec- 2 mRNA in comparison to other cell types. Expression of Beta-arrestin 2 tions, however, the sustained activation may contribute to the genesis of mRNA in affected joints of mice undergoing experimental arthritis was infl ammatory process. The neutrophils are activated by chemotactic fac- also elevated. Beta-arrestin 1 and 2 mRNA and protein levels were also tors such as platelet activating factor (PAF) and formyl-methionyl-leucyl- regulated in response to both lipopolysaccharide (LPS) and colony-stimu- phenylalanine (fMLP). PAF and fMLP bind to a receptor linked to G-pro- lating factor 1 (CSF-1) in primary bone marrow macrophages (BMM) and tein, and activate p38 and ERK1/2 MAPK. The MAPK participate in the thioglycollate-elicited peritoneal macrophages (TEPM). Beta-arrestin 2 activation of NF-kB in other cells. The NF-kB has an important role in contains a binding domain for members of the JNK family, kinases which infl ammation, regulate the production of infl ammatory mediators as pros- are important in signalling pathways involved in the activation and sur- taglandins and COX-2 expression in endothelial cells and macrophages. vival of macrophages. Administration of a membrane permeable HIV TAT Therefore, to know the intracellular signaling pathways that participate in peptide linked to the Jun kinase (JNK) binding domain of Beta-arrestin 2 the COX-2 expression and NF-kB activation in neutrophils is important to (tat-arr2) triggered growth factor independent survival of BMM compared asses new potential drugs. to control peptides with mutations in the JNK docking domain. These In the present work, NF-kB activation by western blot and electropho- studies suggest that Beta-arrestins are likely to have a role in macrophage retic mobility shift assay (EMSA) in HL-60-derived neutrophilic cells is activation and survival, and further studies on the role of Beta-arrestins shown. The COX-2 expression, and phosphorylation of p38, ERK1/2 and in macrophage biology may highlight avenues for development of novel PI3K is analyzed by western blot. The NF-kB is activated after 30 min of therapeutics which modulate infl ammation and immune function. stimulation with PAF (100 nM) or fMLP (100 nM). The p38, ERK1/2 and PI3K proteins are rapidly (2 min) activated by PAF and fMLP. The U0126 inhibitor reduced the NF-kB activation induced by PAF or fMLP, and the 6041 SB203580 inhibitor reduced the NF-kB activation induced by fMLP. The LY294002 inhibitor did not interfere in the NF-kB activation. PAF or Pharmacological characterisation of mast cell adenosine receptors fMLP induced the COX-2 expression after 2 hours, and U0126 and an- drographolide, a new NF-kB inhibitor, reduced this effect. PAF and fMLP Hang Yung A Lau, Department of Pharmacology, Chinese University of also induce changes in intracellular pH. The U0126 reduced the alcaliniza- Hong Kong, Hong Kong tion, but the LY294002 and SB203580 reduced the acidifi cation. Lai Lok K Wong, Department of Pharmacology, Chinese University of In conclusion, PAF and fMLP activate NF-kB through of the ERK1/2 and Hong Kong, Hong Kong p38 MAPK, and increase the COX-2 expression through of the ERK1/2 and NF-kB. On the other hand, PI3K has an important role in intracellular Adenosine has been demonstrated to induce bronchoconstriction in asth- acidifi cation. matic patient and it is believed to be partly due to potentiation of infl am- Support: Conicyt AT-4040024; Fondef D01I1024 matory mediators from mast cells. Adenosine mediates its effects through four different subtypes of adenosine receptors (A1, A2A, A2B and A3) which all belong to the G protein coupled receptor family. This study 6043 characterized the adenosine receptor subtypes on rat peritoneal mast cells by investigating the effects of specifi c ligands of different adenosine re- Expression of substance P and hemokinin-1, and neurokinin-1 ceptors on the cells. Purifi ed peritoneal mast cells from ovalbumin sen- receptor, the receptor for these peptides, in acute pancreatitis sitized Sprague-Dawley rats were activated by anti-IgE and the degree of activation was quantifi ed by measuring the level of histamine released. Madhav Bhatia, National University of Singapore, Singapore Histamine assay was performed spectrofl uorometrically. Adenosine sig- Hon Yen Lau, National University of Singapore, Singapore nifi cantly increased anti-IgE induced histamine release by 41±8% at 10 μM. The A1 agonists CCPA and the A3 agonist Cl-MECA also showed Neurokinin-1 receptor (NK-1R) and its ligands, substance P (SP) and he- a dose-depending potentiation (29%±11% and 75%±21% potentiation at mokinin-1 play an important role in the pathogenesis of some, but not all, 10μM respectively). In order to further characterise adenosine receptors infl ammatory conditions. We have previously shown a key role of SP and on rat mast cells, different adenosine receptor antagonists were prein- NK-1R in the pathogenesis of acute pancreatitis and associated lung injury. cubated with the cells before the addition of the agonist, adenosine and In the current study, we investigated pancreatic and lung mRNA expres- anti-IgE. The cells treated with selective A2A adenosine receptor antago- sion of NK-1R, preprotachykinin-A gene (PPT-A gene that codes for SP) nist ZM241385 showed a general increase in the percentage potentiation and preprotachykinin-C gene (PPT-C gene that codes for hemokinin-1) in Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 159 acute pancreatitis in mice. Acute pancreatitis was induced in mice by in- agonist, respectively). The activity of COX-2 was assessed by measuring traperitoneal injections of caerulein (50 μg/kg/h) for 10 hours. The mRNA the production of 6-keto-PGF1alpha in the presense of exogenous arachi- expression of NK-1R, PPT-A and PPT-C was investigated using reverse- donic acids (10 microM; 10 min) using enzyme immunoassay. The expres- transcription polymerase chain reaction. SP concentrations in pancreas and sion of COX isoform protein was detected by immunoblot using specifi c lung were determined using ELISA. In the pancreas, up-regulation of NK- antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC 1R (5-fold) and PPT-A (3-fold) mRNA was observed after the induction treated with VEGF (0.01 to 50 ng/ml), COX-2 protein, but not COX-1, and of acute pancreatitis. SP levels in the pancreas were elevated by 2-fold. In COX activity were increased in a dose dependent manner. The increased the lung, similar results were obtained with an increase in NK-1R mRNA COX-2 protein and activity in response to VEGF (10 ng/ml) was syner- (3-fold), PPT-A mRNA (3-fold) and SP levels (2-fold). PPT-C gene was gised by PGE2 (0.003 to 3 microM). Interestingly, foskolin and butaprost found to be expressed in the lung but not in the pancreas. PPT-C mRNA synergised the increased COX-2 protein and activity in HUVEC treated expression in the lungs was increased by 4-fold upon induction of acute with VEGF plus PGE2 while SQ22536 and PD98059 inhibit the increased pancreatitis. These results show that an important component of neurogen- COX-2 protein and activity in HUVEC treated with VEGF plus PGE2. ic infl ammation in acute pancreatitis is pancreatic and lung upregulation of SC19220 and sulprostone did not effect the increased COX-2 protein and PPT-A and NK-1R genes. Moreover, upregulation of the PPT-C gene that activity in HUVEC treated with VEGF plus PGE2. Thus, PGE2 upregu- codes for hemokinin-1 suggests a potential role for this peptide in second- lated the induction of COX-2 in HUVEC treated with VEGF via cAMP ary injury, but not pancreatic injury in acute pancreatitis. through EP2 receptor and MEK kinase.

6044 6046

Effect of nitric oxide on apoptosis in human granulose cell cultures Cyclic ADP-ribose is an intracellular messenger for fMLP-induced intracellular Ca2+ rise and migration of human neutrophils Ebrahim Nasiri, Hamedan university of medical sciences, Iran Mohsen Pourghasem, Babol University of Medical Sciences, Iran Toshihiro Dohi, Department of Dental Pharmacology, Hiroshima Univer- Iraj Amiri, Hamedan University of Medical Sciences, Iran sity Graduate School of Biomedical Sciences, Japan Alireza Zamani, Hamedan university of medical sciences, Iran Katsuya Morita, Department of Dental Pharmacology, Hiroshima Uni- versity Graduate School of Biomedical Sciences, Japan Background: Recent evidence suggested that nitric oxide (NO) acts as an Norimitsu Morioka, Department of Dental Pharmacology, Hiroshima important factor in a variety of physiological and pathological roles, in- University Graduate School of Biomedical Sciences, Japan cluding reproductive function. The purpose of the present study was to in- Tomoya Kitayama, Department of Dental Pharmacology, Hiroshima vestigated whether NO might signifi cantly induce any change in apoptosis University Graduate School of Biomedical Sciences, Japan in cultured human granuose cells. Material and Methods: The granulose cells (GC) obtained from women Although an increase in cytosolic free Ca2+concentration ([Ca2+]i) is a key taking part in an in vitro fertillization (IVF) program, were cultured for 48 signal neutrophil functions, the mechanisms for regulation of [Ca2+]i is hour then add the NO generator, DETA/NO (1mM), immediately and after unclear. Cyclic ADP-ribose (cADPR) has powerful action of mobiliza- 2+ on hour culture respectively. Apoptosis of granulose cells was evaluated by tion Ca from endoplasmic reticulum, independently from IP3-mediated DNA fragmentation detect by in situ terminal deoxynucloetidyl Transfer- Ca2+ release and is a candidate as a second messenger of many agonists, ase-mediated dUTP nick end labeling (TUNEL). thereby modulating a wide number of calcium-mediated cellular functions.

Result: Nitric oxide was signifi cantly increased apoptotic index after one Neutrophils from IP3 receptor knockout mice still keep normal migration, hour in human granulose cell culture (p 0.05), but it had no signifi cantef- suggesting other pathway than IP3-mediated Ca2+ mobilization may be fect on apoptotic index in immediately culture. involved in the migration. The present study examined the regulation by Conclusinons: These results suggest that, apoptosis of human granulose cADPR of fMLP-induced changes of Ca2+ dynamics and its involvement cells is mediated by DETA/NO. Which there is a direct relation with the in migration of human neutrophils. duration of the effect. cADPR induced Ca2+ release from digitonin-permeabilized neutrophils Keywords: Nitric oxide / apoptosis /granulose cell and the release was blocked by 8Br-cADPR, an antagonist of cADPR and immunosuppressants FK506 and rapamycin. FMLP induced bipha- sic [Ca2+]i rise in intact neutrophils; the initial sharp rise and following 6045 sustained rise. 8Br-cADPR, FK506, rapamycin and anti-CD38 antibody reduced fMLP-induced [Ca2+]i rise. b-NAD+ added into the medium in- PGE2 upregulated COX-2 expressed in VEGF treated endothelial creased [Ca2+]i and this rise was inhibited by 8Br-cADPR, immunosup- cells via EP2 receptor and MEK kinase pressants and anti-CD38 antibody. fMLP-induced sustained [Ca2+]i rise were inhibited by NADase and nucleoside transporter inhibitors such as Pravit Akarasereenont, Department of Pharmacology, Faculty of Medici- inosine, uridine and NBMPR. Migration of neutrophils is inhibited by the ne Siriraj Hospital, Thailand removal of extracellular Ca2+ or by the addition with 8Br-cADPR, FK506 Kitirat Techatraisak, Department of Obstetrics and Gynecology, Faculty and rapamycin, NADase, nucleoside transporter inhibitors and anti-CD38 of Medicine Siriraj Hospital, Thailand antibody. mRNA of ENT1, ENT2, CNT2, CNT3 are expressed by RT-PCR Athiwat Thaworn, Department of Pharmacology, Faculty of Medicine in neutrophils. These results suggest that cADPR is synthesized extracel- Siriraj Hospital, Thailand lulary by CD38 in response to fMLP stimulation and transported into the Sirikul Chotewuttakorn, Department of Pharmacology, Faculty of Medi- cells through nucleoside transporter and mobilizes Ca2+ through FK506 cine Siriraj Hospital, Thailand binding protein-dependent process. This process may play important roles for fMLP-induced Ca2+signaling and migration in neutrophils. Vascular endothelial growth factor (VEGF) has been shown to play im- portant roles in infl ammation while mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a 6047 variety of signalling molecules. Here, we have investigated the linkage between PGE2, COX-2 signalling and the MAPK cascade in human um- Optimal proliferation of bone marrow-derived macrophages requires bilical vein endothelial cells (HUVEC) activated with VEGF and PGE2 adhesion by using pharmacological agents such as PD98059 (specifi c inhibitors of MEK enzyme), SQ22536, foskolin, SC19220, butaprost and sulprostone Caryn L Elsegood, University of Melbourne, Australia (cAMP inhibitor, cAMP activator, EP1 antagonist, EP2 agonist and EP3 John A Hamilton, University of Melbourne, Australia S 160 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Adhesion via integrins is required for the survival and proliferation of many cells in response to growth factors. However, the role of adhesion in 6049 growth factor-dependent survival and proliferation of macrophages has not been examined in detail. In this study, we have utilised CSF-1(M-CSF)- Suppressor of Cytokine Signalling (SOCS)4 and SOCS5 negatively dependent bone marrow-derived macrophages (BMM) to look at the role regulate IL-4 and IL-13 signalling in vitro: A functional analysis of of adhesion in the survival and proliferation of macrophages in response SOCS4/5 protein domains to the CSF-1. We have found that adhesion is required for optimal CSF- 1-dependent proliferation. In contrast, adhesion is not required for CSF-1- Ruth E Columbus, The Walter and Eliza Hall Institute of Medical Re- dependent survival of BMM. BMM express a number of beta 1 and beta search, Australia 2 integrins, as well as alpha V beta 5 integrin. Proliferation was examined Tracy A Wilson, The Walter and Eliza Hall Institute of Medical Research, on specifi c extracellular matrices (VCAM-1, denatured collagen, laminin, Australia ICAM-1, ICAM-2, vitronectin) which bind to specifi c beta 1 integrins, Kirsten R Palmer, The Walter and Eliza Hall Institute of Medical Re- beta 2 integrins or alpha V beta 5 integrin. Optimal proliferation occurred search, Australia on matrices which bound to alpha 4 beta 1, alpha 5 beta 1, or alpha V Naomi S Sprigg, The Walter and Eliza Hall Institute of Medical Research, beta 5 integrin. We then examined the signalling pathways which may be Australia responsible for the adhesion-dependent optimal CSF-1-induced prolifera- Warrren S Alexander, The Walter and Eliza Hall Institute of Medical tion. Phosphatidyl inositol-3 kinase (PI-3 kinase) and ERK activation in Research, Australia response to CSF-1 was not adhesion-dependent. In contrast, the optimal Nicos A Nicola, The Walter and Eliza Hall Institute of Medical Research, tyrosine phosphorylation of a number of molecules including Pyk2 and Australia paxillin in response to CSF-1 stimulation required adhesion. In conclusion, Sandra E Nicholson, The Walter and Eliza Hall Institute of Medical we have found that CSF-1-dependent BMM require adhesion via alpha 4 Research, Australia beta 1, alpha 5 beta 1, or alpha V beta 5 integrin for optimal proliferation. The optimal CSF-1-induced proliferation of BMM correlates with signal- Negative regulation of cytokine signalling can occur through a family of ling via Pyk2 and paxillin, but not that via PI-3 kinase or ERK. proteins known as the Suppressors of Cytokine Signalling (SOCS). These proteins contain a 40 amino acid sequence of homology, the SOCS box, which recruits an E3-ubiquitin ligase to target proteins for proteosomal 6048 degradation. Little is known regarding the functional roles of two closely related SOCS proteins, SOCS4 and SOCS5. However a report by Seki et Regulation of Syntaxin 7 expression and function in macrophages by al.(1) demonstrates SOCS5 association with the IL-4Ra1 and suggests a CSF-1 role for SOCS5 as a regulator of Th1/Th2 differentiation. Our initial stud- ies suggest that genetic deletion of SOCS5 does not perturb the Th1/Th2 Adrian Achuthan, CRC for Chronic Infl ammatory Diseases, Department axis(2). To investigate this further we have utilised a Stat6-reporter assay of Medicine, Royal Melbourne Hospital, The University of Melbourne, to delineate the functional domains of SOCS5 required for inhibition of IL- Australia 4 and IL-13 signalling and have shown that this requires the N-terminus, Jamie Lopez, Garvan Institute of Medical Research, Australia and a functional SH2 domain and SOCS box. Interestingly, point muta- Matthew Sweet, CRC for Chronic Infl ammatory Diseases, Institute for tions in the putative SOCS5 kinase inhibitory region “KIR” are suffi cient Molecular Bioscience, University of Queensland, Australia to abrogate SOCS5 action, suggesting that analogous to SOCS1 and 3, David James, Garvan Institute of Medical Research, Australia SOCS5 may act to inhibit JAK1 kinase activity. In contrast, SOCS4 only John Hamilton, CRC for Chronic Infl ammatory Diseases, Department partially inhibits IL-4- and IL-13-induced Stat6 activation at low protein of Medicine, Royal Melbourne Hospital, The University of Melbourne, levels and Stat6 activation is enhanced as the amount of SOCS4 increases. Australia The SOCS4 N-terminus and SH2 domain, but not the SOCS box, are re- Glen Scholz, CRC for Chronic Infl ammatory Diseases, Department of quired for inhibition of signalling. While a physiological role for SOCS4 Medicine, Royal Melbourne Hospital, The University of Melbourne, or SOCS5 has yet to be elucidated, this in vitro data suggests that SOCS4 Australia and SOCS5 may regulate Th1 differentiation. 1. Seki, Y et al, PNAS 99(20):13003-13008, 2002 Colony stimulating factor 1 (CSF-1) regulates the differentiation, prolif- 2. Brender, C et al, MCB 24(13):6094-6103, 2004 eration and survival of macrophages. CSF-1 has also been shown to play a role in governing some of the immune functions of macrophages (e.g. phagocytosis and cytokine secretion). However, the mechanisms by which 6050 CSF-1 governs phagocytosis and cytokine secretion are poorly understood. Phagocytosis and cytokine secretion involves a number of vesicle mem- The effect of IL-6 and sIL-6R on liver progenitor oval cell biology brane fusion events, processes in which SNARE proteins have been shown to play crucial roles. The SNARE protein Syntaxin 7 has been reported Vance B Matthews, Laboratory for Cancer Medicine, Western Australian previously to be involved in phagocytosis by macrophages. However, Institute for Medical Research, Perth, Australia while initial studies suggested a role for Syntaxin 7 in the early stages of Stefan Rose-John, Biochemistry Department, Christian Albrechts Univer- endocytosis/phagocytosis, recent studies have reported that it is localised sity, Kiel, Germany to late endosome/lysosomes in macrophages. George C Yeoh, Laboratory for Cancer Medicine, Western Australian We report here that CSF-1 up-regulates Syntaxin 7 gene expression in pri- Institute for Medical Research, Perth, Australia mary mouse bone marrow-derived macrophages, and that this leads to an increase in the levels of Syntaxin 7 protein. Signifi cantly, CSF-1 also in- In some situations when liver is chronically damaged, lost liver cells are duces the rapid phosphorylation of Syntaxin 7. Surprisingly though, Syn- replaced by the expansion and differentiation of liver progenitor oval taxin 7 undergoes serine rather than tyrosine phosphorylation in response cells. Little is known about the signals which initiate and maintain this to CSF-1 stimulation. The CSF-1-induced phosphorylation of Syntaxin 7 response which restores the liver mass. Liver progenitor oval cells have was found to be dependent on PI 3-kinase activity. The effects of CSF-1 attracted increased interest as they are implicated in tumorigenesis. We and PI 3-kinase inhibitors on the subcellular localisation of Syntaxin 7 in have developed a model of chronic liver disease which involves placing macrophages will be presented and discussed. mice on a carcinogenic choline defi cient, ethionine supplemented (CDE) diet. Initially, a putative role of IL-6 in oval cell biology was suggested by its elevation in mice placed on this diet and an impaired oval cell response in IL-6 knock-out mice. IL-6 mediates its effect through the membrane bound IL-6R. Cells that express gp130, but do not express the membrane Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 161 bound IL-6 receptor (IL-6R) may be activated by IL-6 and the agonistic soluble IL-6 receptor (sIL-6R). This form of activation has wide impli- 6052 cations as gp130 is expressed by all body cells. To determine effects of the sIL-6R on oval cells, we have utilised the human IL-6/sIL-6R fusion Ligand-Specifi c, Differential Utilaization of MAP Kinase Pathways protein called hyper-IL-6. We have shown that IL-6 and hyper IL-6 ad- by TLR2 ministration increases CDE induced oval cell migration and proliferation in vivo and promoted the release of TNF-alpha and IFN-gamma which Elizabeth M Long, University of Calgary, Canada have also been implicated in oval cell proliferation. Using murine oval Steve M Robbins, University of Calgary, Canada cell lines, we demonstrated that IL-6 directly activates and induces prolif- eration; however, hyper IL-6 was considerably more active. This may be Toll-like receptors (TLRs) are germline-encoded receptors that recognize due to the higher level of gp130 expression than IL-6R in oval cells. The pathogen associated molecular patterns (PAMPs), which are evolutionarily antagonistic soluble gp130Fc inhibited proliferation due to hyper IL-6. In conserved amongst infectious agents. Stimulation of these receptors leads addition, human IL-6 and hyper-IL-6 induced IL-6 production suggesting to the induction of an infl ammatory response and the development of an an autocrine mechanism. Potentially, inhibition of IL-6 and sIL-6R signal- antigen-specifi c adaptive immune response. The mammalian TLR-family ling may be used to minimise oval cell numbers and reduce cancer in some is comprised of at least 10 members that recognize specifi c PAMPs. Al- liver pathologies. though all TLRs share common cytoplasmic signaling domains, evidence suggests that the cellular response to individual TLR stimulation is specifi c and presumably tailored to individual PAMP/TLR interactions. Interest- 6051 ingly, we have evidence to support the idea that not only is there specifi c- ity in signaling from different TLR’s, but distinct ligands for individual Structural basis of the differential proinfl ammatory signaling of type TLR’s also send the pro-infl ammatory signal in unique ways. We used I interferons through IFNAR1 and IFNAR2 receptors global microarray technology to examine the transcription response of bone marrow derived macrophages from wild-type animals when exposed Nicole A de Weerd, Centre for Functional Genomics and Human Disease, to different TLR2 ligands. We were able to observe subtle differences in Monash Institute of Medical Research, Monash University, Clayton, the transcriptional profi les activated by distinct TLR2 ligands and we were Victoria., Australia able to verify that these responses were dependent upon TLR2. Intrigu- Jodee A Gould, Centre for Functional Genomics and Human Disease, ingly, we have been able to observe, using MAP Kinase pathway specifi c Monash Institute of Medical Research, Monash University, Clayton, inhibitors, that the signals that are sent to activate genes that are commonly Victoria., Australia up-regulated in response to the distinct TLR2 ligands show a differential Jennifer E Fenner, Centre for Functional Genomics and Human Disease, requirement for the MAP Kinase pathways. We are furthering this work Monash Institute of Medical Research, Monash University, Clayton, to evaluate the exact differences in the utilization of specifi c MAP Kinase Victoria., Australia components, such as the MAP(3) Kinases as well as specifi c MAP Kinase Bernadette Scott, Centre for Functional Genomics and Human Disease, scaffolding proteins. We believe that due to the evolutionary requirement Monash Institute of Medical Research, Monash University, Clayton, of these pathways to send an immediate ‘danger’ signal in response to Victoria., Australia pathogen invasion, this system has adapted to utilize multiple distinct path- Paul J Hertzog, Centre for Functional Genomics and Human Disease, ways to achieve the activation of infl ammatory cells. Monash Institute of Medical Research, Monash University, Clayton, Victoria., Australia 6053 The type I interferons (IFNs) are a family of cytokines with antiviral, anti-proliferative and immunomodulatory effects. Their proinfl ammatory The effect of tissue type-plasminogen activator deletion and fi brin actions have been recently highlighted by their involvement in toll-like re- deposition on macrophage traffi cking in peritoneal infl ammation ceptor responses. All type I IFNs exhibit their pleiotropic effects by bind- ing to and signaling through the same multicomponent receptor; IFNAR1 Christine M Massa, The University of Melbourne and CRC for Chronic and IFNAR2. Due to their participation in the pathogenesis of infl amma- Infl ammatory Diseases, Australia tion and autoimmune diseases there is great interest in the determining the Ross Vlahos, The University of Melbourne, Australia mechanism of IFN/IFNAR interactions. Emma L Braine, The University of Melbourne and CRC for Chronic Infl ammatory Diseases, Australia We have used knockout mouse models to attempt to understand the mecha- Amanda L Turner, The University of Melbourne and CRC for Chronic nism of type I IFN/IFNAR interactions. We demonstrate that mice with a Infl ammatory Diseases, Australia null mutation in either receptor chain exhibit distinctive phenotypes. IF- John A Hamilton, The University of Melbourne and CRC for Chronic NAR1-/- and IFNAR2-/- mice are more susceptible to viral infection than Infl ammatory Diseases, Australia WT mice, with IFNAR1-/- mice being the most susceptible. Furthermore, Andrew D Cook, The University of Melbourne and CRC for Chronic IFNAR2-/- mice showed enlarged thymi, reduced apoptosis in thymo- Infl ammatory Diseases, Australia cytes, and increased thymic cellularity. IFNAR2-/- mice showed other- wise normal myeloid and haemopoietic cell development. In contrast, IF- Fibrin deposition is thought to be a result of an altered balance between NAR1-/- mice showed normal thymus development and cellular content, coagulation and fi brinolysis. The plasminogen activators (PAs), tissue- but abnormal myeloid lineage cells. These results show a difference in type (tPA) and urokinase (uPA), are involved in cleavage of plasminogen the phenotype of IFNAR1-/- and IFNAR2-/- mice and thereby imply that to plasmin. While the PA/plasmin system is involved in fi brinolysis, we signaling still occurs in these mice. We have demonstrated that JAK/STAT have shown differing roles for tPA and uPA in collagen-induced arthritis. signaling does not occur in either IFNAR-defi cient line, but gene profi ling The aim of this study was to investigate the roles of the individual PAs, experiments demonstrate that novel sets of genes are activated by IFNa/b uPA and tPA, in thioglycolate (TM)-induced peritonitis. uPA-/- and tPA-/- treatment of IFNAR1-/- and IFNAR2-/- cells. These data indicate that each mice were injected intraperitoneally with TM. There was no difference in receptor component can mediate a different set of signals, independently the TM-elicited exudates response from uPA-/- and uPA+/+ mice. In con- of the other receptor chain. Our current studies are addressing the mecha- trast, 4 day TM-exudates from tPA-/- mice contained signifi cantly fewer nism whereby IFNa/b interact independently with either receptor chain. Mac-1hi macrophages compared with those from tPA+/+ mice. Examina- This has important implications for differentiating the specifi c proinfl am- tion of the peritoneal wall revealed increased numbers of macrophages matory signals elicited by IFNs and for the development of therapeutic and fi brin(ogen) staining in tPA-/- mice. The defect in the infl ammatory antagonists. response in tPA-/- mice could be corrected by administration of the fi brino- lytic agents, tPA or plasmin, prior to harvesting TM-elicited exudates. This S 162 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005) led to an increase in the numbers of recoverable Mac-1hi macrophages Aspirin, naproxen, indomethacin and sulfasalazine are among common- from the peritoneal cavity of tPA-/- mice, such that numbers were now ly used non-steroidal antiinfl ammatory drugs (NSAIDs) for treatment similar to that seen in tPA+/+ mice. These results suggest that tPA is the of various infl ammatory diseases. Side effects of these drugs have been PA controlling fi brinolysis in this infl ammatory model, and in its absence, clearly demonstrated, however; the toxicity of these drugs on the fetus macrophages become trapped in the cavity wall adhering to the accumu- and placenta tissues is not well defi ned. In this study we tested these drugs lated fi brin(ogen). on a human placental trophoblast cell line (choriocarcinoma JAR) on cell adhesion, proliferation, cytotoxicity and induction of cell death after drugs treatments at various concentrations. With MTT assays, we demonstrated 6054 that all of drug treatments (0-400 μg/mL) resulted in decrease of cell adhe- sion in 3 hours after treatments in a dose dependent fashion (p<0.05). At The fi brinogen gene polymorphism, plasma levels of fi brinogen and 72 hours of drug treatments, relative cell number is decreased dramatically chemokines involved in vascular infl ammation in patients survived in a dose dependent manner compared to the non-treatment control groups an ischemic stroke (p<0.05). At highest dose tested in this experiment, the relative cell num- ber was decreased more than 75 percents. 50% inhibitory concentrations Olga V Sirotkina, Petersburg Nuclear Physics Institute RAS, Russia (IC50) on cell proliferation were determined as 76.25, 20.3, 4.8, and 24.4 Kirill A Syssoev, Saint-Petersburg Pavlov State Medical University, μg/mL in aspirin, naproxen, indomethacin and sulfasalazine, respectively. Russia In addition, the inhibition of cell proliferation was accompanied with in- Alla V Merculova, Hospital “Mariinskaya”, Russia creased cytotoxicity (LDH: lactate dehydrogenase assays) in treated group Maria L Chukhlovina, Saint-Petersburg StatePediatric Medical Academy, compared to the controls (p<0.05). Further analysis using DNA laddering Russia techniques, we demonstrated that the decrease of cell proliferation in this cancer cell line is also mediated by the induction of cell death secondary Hemostatic balance depends on complex interaction among endothelial to inhibition of cell adhesion. Findings in this study have shown the cyto- cells, blood cells and coagulation-fi brinolitic system. Cytokines play im- toxic effect of NSAIDs on human placental trophoblastic cancer cell line portant role in such interaction. Chemokines eotaxin and RANTES might survival and this could be benefi cial to an effective administration of these be involved in vascular infl ammation. drugs and future study. The aim of present study was to evaluate impact of the beta-fi brinogen gene mutation and infl ammation in plasma level of fi brinogen and devel- opment of predisposition to ischemic stroke (IS). 6056 For this purpose we investigated allele distribution of the genetic variant G/A (-455) in the beta-fi brinogen gene (FGB) by PCR-RFLP method in 56 Septic arthritis caused by kingella kingae individuals (mean age 41±1) with IS and in control group of 320 healthy subjects (mean age 41±0.4) showing no history of cerebrovascular diseas- Gita Eslami, Shaheed Beheshti University,Pediatric InfectiousResearch es. The levels of the mRNA of eotaxin, eotaxin-2 and RANTES were ana- Center, Iran lyzed by semiquantitive PCR in small group of IS patients (15 subjects). Fatemeh Fallah, Shaheed Beheshti University, Iran The plasma level of fi brinogen was analyzed by standard laboratory test. Jalal Mardaneh, Shaheed Beheshti University, Iran The mRNA levels of studied infl ammatory markers were correlated with each other - eotaxin and eotaxin-2 (R=0.7, p=0.002), eotaxin-2 and RAN- Objective: Kingella kingae is a short gram-negative rod that is a normal TES (R=0.56, p=0.03). It was found tendency to correlation between part of the oral fl ora rarely causes septic arthritis. We report a case of mRNA level of RANTES and plasma level of fi brinogen (R=0.45, p=0.1). documented arthritis caused by kingella kingea in Imam Hossein Hospital The plasma level of fi brinogen in IS patients was strongly depend on G/A Tehran- Iran. An eight - year- old boy referred to hospital with high fever, (-455) FGB: carriers of GG genotype had lowest concentration of fi brino- Pain and swelling in knee joint and past medical history revealed minor gen - 2.71±0.12 g/l, carriers of GA had medium level - 3.46±0.22 g/l and thalassemia. carriers of AA genotype had highest level - 4.10±0.25 g/l (p=0.01). The Materials & Methods: aspirated synovial fl uid from knee joint by specialist G/A (-455) FGB was also associated with increased levels of eotaxin-2 doctor and sample transported to trypticase soy broth and thioglycollate and RANTES mRNA in IS patients: 1.03±0.52 % and 2.05±0.53 % in car- broth mediums. Culture incubated in 37oC during 24 hours. Subsequent- riers of A-allele and non-carriers, respectively, for eotaxin-2 mRNA level ly prepared smear for gram staining and sample plated on to blood agar, (p=0.08) and 2.67±2.67 % and 7.61±2.28 % in carriers of A-allele and chocolate agar, macconkey agar and incubated in 37oC during 24h. The non-carriers, respectively, for RANTES (p=0.1). In IS patients the ten- genus and species of bacteria determined by diagnostic tests. dency to prevalence of the G/A (-455) FGB compared with control group Results: the gram staining showed gram- negative and plump rods that were observed: 57% and 48%, respectively, were carriers of the A-allele tend to occur in pairs and short chains. Culture was positive for kingella (p=0.1). kingae. It was susceptible to B-lactam antibiotics. Our fi ndings display that G/A (-455) FGB and vascular infl ammation Conclusion: this organism rarely causes systemic disease in children and might be risk factors for IS in patients before 45 years old. It is necessary even when defense mechanisms are impaired. Kingella kingae is fastidi- include anti-infl ammation therapy in anti-stroke treatment. ous microbe and its culture is often diffi cult therefore it is important to use enriched medium to inoculate joint fl uid. Key words: septic arthritis, kingella kingae 6055

Cytotoxic effect of NSAIDs on human choriocarcinoma cell line 6057

Athip Nilkaeo, Department of Microbiology, Faculty of Science, Prince of Involvement of matrix metalloproteinase-9 in platelet-activating Songkla University, Thailand factor-induced experimental tumor metastasis Suthinee Bhuvanath, Department of Microbiology, Faculty of Science, Prince of Songkla University, Thailand Hyun-Mi Ko, Department of Biological Sciences, The Institute of Basic Jutarat Mo-tae, Department of Microbiology, Faculty of Science, Prince Sciences, Chonnam National University, Kwangju 500-757, Republic of of Songkla University, Thailand K, Korea Walailak Kanchanapin, Department of Microbiology, Faculty of Science, Bongnam Jung, Department of Biological Sciences, The Institute of Basic Prince of Songkla University, Thailand Sciences, Chonnam National University, Kwangju 500-757, Republic of K, Korea Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 163

Jee-Hae Kang, Department of Biological Sciences, The Institute of Basic Sciences, Chonnam National University, Kwangju 500-757, Republic of 6059 K, Korea So-Hee Kim, Department of Biological Sciences, The Institute of Basic Human natural polyamine modulates cellular functions closely Sciences, Chonnam National University, Kwangju 500-757, Republic of related to the LFA-1 expression and the TNF production K, Korea Hern-Ku Lee, Department of Immunology and Research Center for Takeshi Nakamura, Department of Surgery and Integrated Medicine, Allergic Immune Diseases, Chonbuk National University Medical School, Omiya Medical Center, Jichi Medical School, Japan Chonju 5, Korea Kuniyasu Soda, Department of Surgery and Integrated Medicine, Omiya Suhn-Young Im, Department of Biological Sciences, The Institute of Basic Medical Center, Jichi Medical School, Japan Sciences, Chonnam National University, Kwangju 500-757, Republic of Yoshihiko Kano, Department of Surgery and Integrated Medicine, Omiya K, Korea Medical Center, Jichi Medical School, Japan Masaaki Saitoh, Department of Surgery and Integrated Medicine, Omiya In this study, we have investigated the role of matrix metalloproteinase Medical Center, Jichi Medical School, Japan (MMP)-9 in platelet-activating factor (PAF)-induced experimental pulmo- Masanobu Kawakami, Department of Surgery and Integrated Medicine, nary metastasis of murine melanoma cell, B16F10. Administration of PAF Omiya Medical Center, Jichi Medical School, Japan enhanced pulmonary metastasis of B16F10 about 3.5-fold, and this effect Fumio Konishi, Department of Surgery and Integrated Medicine, Omiya of PAF was signifi cantly inhibited by nuclear factor (NF)-kappaB inhibi- Medical Center, Jichi Medical School, Japan tor, such as antisense oligodeoxynucleotide against p65 subunit of NF-kap- paB or antioxidant, N-acetyl-L-cysteine. Administration of PAF enhanced Purpose: Cell adhesion and production of cytotoxic cytokines are impor- the expression mRNA, protein synthesis, and activity of MMP-9 in the tant for infl ammatory processes and cancer immunity. We have found that lungs, all of which were inhibited by pretreatment of NF-kappaB inhibi- human natural polyamines, spermine and spermidine, decrease adhesion tors. Immunohistochemistry analysis revealed that MMP-9 expression was capacity of non-stimulated peripheral blood mononuclear cells (PBMCs) observed in the distal airway epithelium. MMP inhibitor, 1,10-phenanth- to the plastic plate and inhibit the production of tumor necrosis factor roline or anti-MMP-9 antibody reduced the PAF-induced enhanced lung (TNF) upon stimulation with LPS. We also have found that the polyamine colonies in a dose-dependent manner. These data indicate that induction concentration-dependent decrease in adhesion capacity is accompanied by of MMP-9 in the lungs in a NF-kappaB dependent way plays an important the decrease in the expression of CD11a. In order to evaluate the effects role in PAF-induced experimental tumor metastasis of polyamine on cellular functions related to LFA-1 (CD11a) expression and TNF production, we further investigate whether the changes in blood polyamine levels affect cellular immunities among healthy volunteers and 6058 cancer patients. Methods: The expression of CD11a on PBMCs was estimated by fl ow Inverse genetic predisposition to lung and colon carcinogenesis cytometry, and blood polyamine levels were measured by HPLC. As the in mouse lines phenotypically selected on the basis of acute LFA-1 expression and TNF production are closely related to the cytotoxic infl ammatory responsiveness activities of lymphokine-activated killer (LAK) cells, we measured LAK activities among patients suffering from various cancers before and after Roberto F Di Pace, Instituto Butantan, Brazil surgery. Solange Massa, Instituto Butantan, Brazil Results: Among 42 healthy male volunteers, blood spermine levels in- Orlando G Ribeiro, Instituto Butantan, Brazil versely correlated with the fl uorescent intensities of CD11a on lymphocyte Wafa H Cabrera, Instituto Butantan, Brazil (r = -0.48, p = 0.001). Polyamine concentrations among patients suffer- Olga M Ibañez, Instituto Butantan, Brazil ing various cancers were higher than those of age-matched healthy volun- teers. Among patients (n = 9) whose polyamine levels were higher than the Colorectal and lung cancer development was studied in two mouse lines maximum value in the healthy volunteers, tumor extirpation signifi cantly AIRmax and AIRmin, which thoroughly differ in the ability to produce decreased blood polyamine levels (73 %), and increased LAK activities acute infl ammatory reactions (AIR) in different tissues and in the colon. (299 %) and fl uorescent intensities of CD11a (118%). The mice received 2 or 7 weekly doses ip of 1,2 Dimethylhidrazine (DMH, Conclusion: Polyamine is suggested as being one of the factors that pro- 60 or 185mg/kg bw). AIRmax were more susceptible, with 3-fold more co- mote suppressed cellular functions closely related to LFA-1 expression lonic aberrant crypt foci (ACF) at 60 to 150 days after DMH. At 300 to 475 and TNF production. days, adenocarcinomas were also found in higher numbers and committing larger intestinal areas in AIRmax mice. Conversely,at this time,several and large lung adenocarcinomas were found in AIRmin mice only,pointing out 6060 to an organ specifi c pattern of susceptibility in these two mouse lines. A sub-group of mice that received two DMH doses was treated with Nime- The 'Study of Prostatitis' Microbial Agents In Men sulide starting 24h before the fi rst dose and during 60 days.This treatment almost completely inhibited the appearance of ACF in the colon in both Hossein Goudarzi, Shaheed Beheshti University, Iran lines. Secretory phospholipase 2 (PLA2s) gene polymorphism is found to Ahad Zargarizadeh, Iran be associated to colon cancer susceptibility in the mouse. Accordingly to the observed phenotypes, the mutated allele whose product is a non func- Objective: Becterial prostatitis is an infl ammatory process of prostate tional enzyme was found in AIRmin mice, whereas the wild type allele was gland which occurs in a wide range age of men. This process is seen in fi xed in AIRmax. AIRmax were previously shown to be resistant and AIR- acute and chronic forms, and results is disturbance in urination. min susceptible to urethane lung carcinogenesis. In urethane treated mice, Method: This study was done in Tajrish Shohada hospital on 186 patients Nimesulide increased the tumor incidence in AIRmax but not in AIRmin. according to chinical symptoms and prostatitis diagnostic. The results evidence that the relationship between infl ammation and can- Patients were introduced to laboratotry urologist of detecting of pathogen. cer largely depends on the target organ. The high infl ammatory reactivity So, urethtal discharge , prostatic fl uid and urin sample after massage , were of AIRmax mice might both condition the propensity to colon cancer and bacteriologically analyzed . the resistance to lung cancer development. The opposite was observed in Result: In this study, 31 (16.6%) patients in prostatic fl uid ,12 (6.5%) in AIRmin mice. Given this unique pattern of resistance and susceptibility to urethral discharges and 20 (13%) in urin culture after massage , had posi- tumorigenesis in association with AIRmax and AIRmin infl ammatory re- tive cultures. activity, these mice provide a powerful model system for the identifi cation of common genetic modifi ers of infl ammation and cancer. S 164 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Results of urethral discharge culture and urin culture after massage , had BACKGROUND: Cancer of the oral and esophagus are common in Ne- signifi cant (P<0.005) correlation . But this correlation was not obtained pal. The risk factors predisposing to cancer in Nepalese patients are not between results of prostatic fl uid and urethral discharge cultures. known. A posit,ve correlation(P<0.005) was obtained between prostatic fl uid cul- OBJECTIVES: To determine the role of smoking, alcohol and their com- ture and WBC counts in dirrect examination of prostatic fl uid isolated bination, and diet factors in the etiology of cancer of the oral and esopha- bacteria from prostatic fl uid culture were 14(45.1%) Gram negative and gus. 17(59.9%) Gram positive bacteria. METHODS: Risk factors like alcohol consumption, smoking, tobacco From this bacteria , Ecoli (29%) was more frequently isolatied and coagu- chewing, and pre-illness diet details in 90 patients with cancer of the oral lase negative Staph (25.8%) , coagulase positive staph (12.9%) Entrococci and esophagus were compared with those in age- and sex-matched con- (9.9%) haemolytic Streptococci (6.4%), Klebsiella (3.2%1), Pseudomonas trol subjects. the data were analysed and edited with the help of EPI info (3.2%) and Neiseria gonorae (3.2%). program. All Bacteria isolated from prostatic fl uid were done for antibiogram sus- RESULTS: The risk for oral and esophageal cancer was 3.5 times higher ceptibility by disk agar diffusion. with alcohol consumption with smoked food, 2.5 times higher for tobacco Susceptibility of gram negative bacteria to 15 antibiotics were the same users, and 2.8 times higher each for betel nut chewers and smokers. The and Pseudomonas aeroginosa was resistant to all antibiotics. calculated odds ratio for the social habits and diet factors(smoked foods) Susceptibility of gram positive bacteria to 15 antibiotics was different and was signifi cant amongst cases of oral & esophagus cancer. patients comes only they were similar in susceptible to Vancomycine and ciprofl oxancine. to the hospital in advance stage and most of the they have infl ammation In one case, isolated coagulase negative staph was resistant to all antibiot- at the site. ics. CONCLUSION: Alcoholism,smoked food, smoking, and chewing of to- bacco are factors predisposing to oral and esophageal cancer in Nepal. Inspite of poverty, illiteracy and work load most of the people takes local 6061 made alcohol, unfi ltered cigarette smoking and chewing of tobacco with slaked lime. Inhibition of cyclooxygenases does not explain the anti-nociceptive effects of paracetamol and dipyrone in a rat model of infl ammatory pain 6063

Janetti N Francischi, Federal University of Minas Gerais, Brazil Induction of NO Production by V-ATPase Inhibitors and Possible Dorothéa S França, Federal University of Minas Gerais, Brazil Participation of NO in V-ATPase Inhibitor-Induced Decrease in Y S Bakhle, Imperial College - School of Medicine, United Kingdom Growth and Survival of RAW 264.7 Cells

Paracetamol and dipyrone are anti-pyretic analgesics with only weak in- JangJa Hong, Graduate School of Pharmaceutical Sciences, Tohoku hibition of prostaglandin (PG) biosynthesis via the cyclo-oxygenase iso- University, Japan forms, COX-1 or COX-2. However, both have been shown to be more Yasuhiro Nakano, Graduate School of Pharmaceutical Sciences, Tohoku effective against the recently identifi ed isoform of cyclo-oxygenase, COX- University, Japan 3, associated with nervous tissue (Simmons et al, 2004). In rat paws (n= Aya Yokomakura, Graduate School of Pharmaceutical Sciences, Tohoku 4-6 throughout) injected with carrageenan (CG, 250μg /paw), the ensu- University, Japan ing mechanical hyperalgesia was measured over 4h and is given below as Kenji Ishihara, Graduate School of Pharmaceutical Sciences, Tohoku the area under the curve of nociceptive threshold vs.time. Hyperalgesia is University, Japan shown a negative value as the threshold is lowered. Hyperalgesia to CG OkPyo Zee, Graduate School of Pharmacy, Sungkyunkwan University, alone (-337.2 ± 18.9 units) was abolished by indomethacin (2 mg/kg; 47 ± Korea 62 units) or piroxicam (3 mg/kg; -32 ± 36 units), given s.c. 30 min before Kazuo Ohuchi, Graduate School of Pharmaceutical Sciences, Tohoku CG. This hyperalgesia was also reduced by paracetamol (60 mg/kg; -162 University, Japan ± 32.8 units) or dipyrone (60 mg/kg; -82 ± 14.2 units), given orally 30 min before CG and a higher dose of paracetamol or dipyrone (180 mg/kg) We have reported that apicularen A, a cytostatic macroloide isolated from produced a state of hypoalgesia (threshold increased above normal; 150 ± a variety of the myxobacterial genus Chondromyces, induces apoptosis

16 units or 200 ± 12 units, respectively). Exogenous PGE2 (200 ng/paw), in the mouse leukemic monocyte cell line RAW 264.7 (Hong et al., J. rather than the endogenous PGs generated by CG, induced a similar degree Pharm. Pharmcol., 55:1299-1306 (2003)) and in human promyelocytic of hyperalgesia over 4 h (- 207 ± 51 units). In this infl ammatory model, leukemia cell line HL-60 (Hong et al., Planta Med., 71:306-312 (2005)). indomethacin is not anti-nociceptive (Sachs et al, 2004) but the hypoalge- In addition, we have reported that apicularen A induces NO production sic response to dipyrone (180 mg/kg; 139 ± 28.5 units) was still observed. and the apicularen A-induced NO partially participates in the apicularen From this result and the hypoalgesic responses, we conclude that, in these A-induced apoptosis in RAW 264.7 cells (Hong et al., J. Pharmacol. Exp. models of infl ammatory pain, paracetamol Ther., 312:968-977 (2005)). Recently, as we have clarifi ed that apicularen and dipyrone are anti-nociceptive by mechanisms that do not involve in- A inhibits V-ATPase determined by proton transport using microsome hibition of PG biosynthesis at the infl ammatory site, by any of COX-1, vesicles, we speculated that V-ATPase inhibitors might commonly induce COX-2 or COX-3 isoforms. NO production and concomitantly produced NO might participate in the Simmons et al (2004) Pharm Rev 56 387-437 V-ATPase inhibitor-induced apoptosis. Sachs et al (2004) Proc Natl Acad Sci 101 3680-3685 To analyze the mechanism of action of V-ATPase inhibitors, effects of

Support: CNPq, CAPES. the other V-ATPase inhibitors such as bafi lomycin A1 and concanamycin A on NO production and the role of concomitantly produced NO in the V- ATPase inhibitor-induced decrease in growth and survival of RAW 264.7 6062 cell were examined.

RAW 264.7 cells were incubated in the presence of bafi lomycin A1 or Risk factors for oral and oesophageal cancer in developing country: concanamycin A, and nitrite concentrations in the conditioned medium a hospital based case control study were determined using Griess reagent. Levels of iNOS mRNA and its protein were determined by RT-PCR and Western blotting, respectively. Shambhu D Joshi, Nepal Medical College and Teaching Hospital, Nepal Cell growth and survival were determined by MTT method. Shyam K Bk, Nepal Medical College and Teaching Hospital, Nepal Bafi lomycin A1 and concanamycin A at 10 to 100 nM induced NO pro- Naresh Pandit, Nepal Medical College and Teaching Hospital, Nepal duction through the expression of iNOS mRNA and its protein, and cell growth and survival were decreased. The NO donor SNAP further low- Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 165 ered the V-ATPase inhibitor-induced decrease in MTT response and the the mechanism of sustained dermatitis in NC mice induced by repeated NO scavenger carboxy-PTIO alleviated it partially. The Na+,K+-ATPase challenge with chemical antigen. inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce [Methods] 1) Dermatitis was induced by repeated topical application with NO production. These fi ndings suggest that the V-ATPase inhibitors com- dinitrofl uorobenzene (DNFB) to the ears of NC and C3H mice 5 times at monly induce NO production and the concomitantly produced NO par- 7-day intervals and evaluated by measuring ear thickness. 2) Production ticipates in part in the V-ATPase inhibitor-induced decrease in cell growth of Th1/Th2 cytokines were examined ex vivo; CD4+ cells from draining and survival. lymph node were stimulated with anti-TCR and anti-CD28 antibodies, and IL-4 and IFN-gamma in the supernatant were quantifi ed by ELISA. Also, IL-12 and IL-18 in the skin lesions were measured by ELISA. 3) To exam- 6064 ine the involvement of IL-12, antibody to IL-12 was administered iv twice a week starting at 6 weeks of the experiment. Chymase enhances angiogenesis via the activation of pro matrix [Results] Topical application of DNFB increased skin thickness in C3H metalloproteinase-2 in hamster hindlimb ischemia model as well as NC mice, and the response was enlarged by repeating the chal- lenge. Though the skin reaction in C3H mice was ameliorated when the Michiko Muramatsu, Dept. of Pharmacology, Osaka Medical College, painting was discontinued after the 5th challenge, the reaction in NC mice Japan was sustained at least for 3 weeks. Analyses of cytokine production by Kazuyoshi Kirimura, Environ. Biol. Res. Ctr. (BILIS), Japan CD4+ cells revealed that NC but not C3H mice fail to induce IL-4, where- Shinji Takai, Dept. of Pharmacology, Osaka Medical College, Japan as the level of IFN-gamma in NC mice is equivalent to that of C3H mice. Denan Jin, Dept. of Pharmacology, Osaka Medical College, Japan In addition, NC mice highly expressed IL-12, a cytokine preventing Th2 Mizuo Miyazaki, Dept. of Pharmacology, Osaka Medical College, Japan response, while C3H mice did not. Administration of anti-IL-12 antibody reduced duration of dermatitis in DNFB-treated NC mice. These results Chymase, a chymotrypsin-like serine protease stored in mast cell gran- suggest that IL-12 plays a role in the persistent dermatitis in NC mice, and ules, recognizes angiotensin I (AI) as a physiological substrate and is in- that the action might be mediated by the decrease in IL-4 production. volved in pathogenesis of vascular hypertrophy and myocardial infarction [Conclusion] IL-12 may be important in chronicity of allergic dermatitis. through angiotensin II (AII) production. Previously, we have demonstrated that chymase induces angiogenesis in hamster sponge model. However, chymase-induced angiogenesis was inhibited to only 50 % by AII type 1 6066 receptor antagonist, suggesting that alternative substrates except AI might be involved in this angiogenesis. In this study, we investigated the possi- Indolin-2-ones with High in vivo Effi cacy in a Model for Multiple bility that chymase induces angiogenesis via the activation of pro MMP-2 Sclerosis as an alternative substrate. In addition we demonstrated the blood fl ow improvement following angiogenesis by chymase in hamster hindlimb Laëtitia Bouérat, LEO Pharma A/S, Industriparken 55, DK2750 Balle- ischemia model. Activation of pro MMP-2 by chymase was examined us- rup, Denmark, Denmark ing human fi brosarcoma cells (HT-1080), which secretes an abundance of Jef Fensholdt, LEO Pharma A/S, Industriparken 55, DK2750 Ballerup, MMP-2. Male Syrian hamster underwent surgery to induce ischemia by Denmark, Denmark ligation of left femoral artery. A week after the surgery, purifi ed hamster Xifu Liang, LEO Pharma A/S, Industriparken 55, DK2750 Ballerup, chymase (40 mU/head) was injected into the femoral once every three Denmark, Denmark days. Blood fl ow was measured by using a laser Doppler perfusion imager. Sophie Havez, LEO Pharma A/S, Industriparken 55, DK2750 Ballerup, Four weeks after the surgery, microvessel density and MMP-2 activity in Denmark, Denmark excised femoral muscles were analyzed. The lean blood fl ow of hindlimb Simon F Nielsen, LEO Pharma A/S, Industriparken 55, DK2750 Balle- by ligation was slightly recovered during experimental period. Chymase rup, Denmark, Denmark increased the paw blood fl ow about 1.4 times as compared to the control. Jens R Hansen, LEO Pharma A/S, Industriparken 55, DK2750 Ballerup, The microvessel density in chymase-treated femoral muscle increased sig- Denmark, Denmark nifi cantly (30.1±1.4 to 45.0±2.0 numbers /fi eld). Furthermore, activation Simon Bolvig, LEO Pharma A/S, Industriparken 55, DK2750 Ballerup, of pro MMP-2 was observed in parallel with the increment of angiogenesis Denmark, Denmark by chymase. Chymase induced angiogenesis was suppressed by a chymase Christina Andersson, LEO Pharma A/S, Industriparken 55, DK2750 inhibitor (BCEAB; 100 mg/kg/day, p.o.). Both AII production and pro Ballerup, Denmark, Denmark MMP-2 activation contributes to angiogenesis by chymase. And chymase may be a new candidate for regenerative therapy in ischemic diseases. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. Existing treatments have limited effi cacy on attack fre- quency and on slowing down disease progression. Screening low molecu- 6065 lar weight compounds with potential to fulfi ll the unmet clinical needs within MS, we surprisingly found the known VEGF-R2 inhibitor SU5416 Repeated Topical Challenge with Chemical Antigen Elicits Sustained and several analogues of the indolin-2-one family to be highly effi cacious Dermatitis in NC/Nga Mice in Specifi c-pathogen Free Condition in the EAE model, an in vivo model for Multiple Sclerosis. Up to 98% inhibition of the clinical score was obtained when mice were dosed daily Yoshiaki Tomimori, Daiichi Suntory Pharma Co., Ltd., Biomedical Re- for 21 days with SU5416 (50 mg/kg). The indolin-2-one family has pri- search Laboratories, Japan marily been investigated by Sugen as VEGF-R2 inhibitors exhibiting anti- Yoshitaka Tanaka, Daiichi Suntory Pharma Co., Ltd., Biomedical Re- angiogenesis activity for the treatment of cancer. When testing SU5416 search Laboratories, Japan and its analogues, we did not fi nd any correlation between in vivo effi cacy Megumi Goto, Daiichi Suntory Pharma Co., Ltd., Biomedical Research in the EAE model and in vitro potency in the VEGF-R2 assay. However, Laboratories, Japan our results suggest that the in vivo effi cacy of the indolin-2-one family Yoshiaki Fukuda, Daiichi Suntory Pharma Co., Ltd., Biomedical Re- in the EAE model might be related to the inhibition of the pro-infl am- search Laboratories, Japan matory cytokine IL-2. The IL-2 PBMC assay was then successfully used as a screening assay to fi nd other types of indolin-2-ones exhibiting high [Purpose] NC/Nga (NC) mice are known to develop dermatitis sponta- in vivo activity. Remarkably, good p.o. effi cacy was obtained by prepar- neously in air-uncontrolled circumstance but not in specifi c-pathogen ing prodrugs of the indolin-2-one family. The mechanism of action needs free (SPF) condition. The mice also develop sustained dermatitis follow- further elucidation. ing repeated painting with chemical antigen in SPF condition. To study pathogenesis of chronic dermatitis such as atopic eczema, we examined S 166 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

tors involved in CYP3A regulation including HNF4, HNF3 & C/EBP. 6067 The activities of transcription factors involved in infl ammatory signalling pathways in livers of tumour-bearing mice were also examined by EMSA Poverty of Infl ammation and Immunity in Neonatal Skin or Western blots. Functional NFkB was increased, consistent with an in- fl ammatory response that could mediate induction of acute phase response Greg M Woods, University of Tasmania, Australia genes, or conversely, be involved in the repression of drug metabolism. Bernadette M Bellette, University of Tasmania, Australia These fi ndings give insights into molecular mechanisms underlying the Georgina Kalidomos, University of Tasmania, Australia ability of tumour-derived infl ammation to reprogram hepatic gene expres- H Konrad Muller, University of Tasmania, Australia sion. The frequent involvement of CYP3A4 in metabolism of anti-cancer agents makes this enzyme an important target for studies aimed at the de- It is long established that there is a poor infl ammatory response in neonatal sign of anti-infl ammatory interventions to normalise drug clearance and skin. Accompanying this is the development of tolerance, rather than im- hence, improve chemotherapy. munity, which occurs when antigen is applied to neonatal skin. This proj- 1)Slaviero et al 2003, Infl ammatory response: An unrecognised source of ect evaluated cellular aspects within the neonatal skin environment that variability in pharmacokinetics of cancer chemotherapy. Lancet Oncology may account for these events. Mast cells are critical to the development of 4:224 an effective infl ammatory response and analysis of neonatal skin revealed a surprisingly high number of histamine expressing mast cells. The epider- mis is be much thicker at this time with greater keratinisation compared 6069 to adult skin. Analysis of Langerhans cells in neonatal skin showed that they had a reduced ability to migrate following antigen exposure, but they CC Chemokine CCL-1 and its receptor modulate CC chemokine could internalise and proteolyse antigens but were ineffective in present- CCL - 2 (MCP-1) gene expression in HeLa cells ing this antigen on the surface of the cell in association with MHC-II. This may have been a consequence of an alteration in the antigen uptake Nasreen S Haque, Barnard College, Columbia University, United States pathway. In neonatal Langerhans cells there was a preference to utilise Lauren Tal, United States fl uid phase, rather than receptor mediated, uptake. The uptake of antigen Diana Huang, United States by these neonatal Langerhans cells appeared to be designed to remove the antigen without inducing an immune response. In fact, tolerance arises The CC chemokine, CCL-1 induces a variety of biological responses in with the generation of regulatory T cells. many cell types, namely infl ammation and chemotaxis . We have previ- It is concluded that cells critical to the development of infl ammation and ously shown that the CC chemokine CCL-1 and its receptor CCR8 reg- the induction of immunity, (eg. mast cells and Langerhans cells) appear ulate cell chemotaxis in endothelial,vascular smooth muscle and cancer to have altered functions in the neonatal skin environment. The result cells. In the present study we examined the role of CCL-1 and its receptor is reduced infl ammation, reduced antigen presentation and hence reduced CCR8 in the regulation of gene expression in human cervical carcinoma immunity. This will protect the developing neonatal environment from (HeLa) cells by microarray analysis and targeted siRNA inhibition. HeLa damaging infl ammation and immune injury. The neonatal environment cells have been widely used as a model for studies on infl ammation and is also generates tolerance rather than immunity, a favourable outcome con- known to express chemokine receptors. Our preliminary results show that sidering the extent of cellular remodelling and structural events required HeLa cells constitutively express CCR8 and this expression of CCR8 is for effective development of the skin. stimulated by CCL-1. Also, CCL1 induces HeLa cell chemotaxis. Further, as demonstrated by Affymetrix human microarray data analysis, CCL-1 regulates numerous genes with over two-fold induction rate. CCL-1 regu- 6068 lated genes are involved in angiogenesis, apoptosis, oxidative stress and chemotaxis. Interestingly CCL1 induces expression of another CC che- Analysis of transcription factors involved in repression of hepatic mokine CCL-2 (MCP-1) a potent regulator of monocyte/macrophage che- drug metabolism associated with tumour derived infl ammation motaxis and invasion in various disease states. This stimulation of CCL-2 by CCL-1 was further validated by RT-PCR analysis, which correlated Marina Kacevska, Department of Pharmacology, University of Sydney, exactly with microarray data. In corollary, targeted CCR8 siRNA inhibi- Australia tion down regulates CCL-2 expression further substantiating our fi ndings. Kellie Charles, Molecular Pharmacology, Westmead Millennium Institu- It is known that CCL2 protects human neuronal cells from tat-induced te, Australia apoptosis and CCL1 is anti-apoptotic for thymic and leukemia cells. In Chris Liddle, Molecular Pharmacology, Westmead Millennium Institute, the present study genes involved in oxidative stress are inhibited and those Australia involved in protection against apoptosis are upregulated suggesting a com- Stephen Clarke, Cancer Pharmacology Unit, ANZAC Research Institute, mon pathway for the two CC chemokines. Close examination of the role Australia of CCL-1/CCR8 pathway in the regulation of CCL-2 and other angiogenic Graham Robertson, Cancer Pharmacology Unit, ANZAC Research and/or apoptotic factors will provide important insights into vascular cell Institute, Australia pathologies.

Tumour-derived infl ammation is associated with reduced cytotoxic drug metabolism mediated by cytochrome P450 CYP3A4(1). This is a major 6070 concern as cancer patients with elevated infl ammatory markers experience extreme toxicity in response to chemotherapy. To study the link between Anti-infl ammatory activity in vitro and in vivo of the protein infl ammatory cytokines & repression of hepatic drug metabolism, we farnesyltransferase inhibitor, tipifarnib examined transcription factors in the livers of mice bearing a xenograft tumour, with a focus on nuclear receptors PXR and CAR responsible for Anne M Fourie, Alza/Johnson & Johnson PRD, United States inductive drug interactions involving CYP enzymes and drug transport- Xiaohua Xue, Alza/Johnson & Johnson PRD, United States ers. EHS sarcoma explants were injected into the hind limb of transgenic Jing-Feng Huang, Alza/Johnson & Johnson PRD, United States CYP3A4/lacZ mice and grown for 14-18 days. The potent mouse PXR Kuei-Tai A Lai, Alza/Johnson & Johnson PRD, United States activator, PCN or the CAR ligand, TCPOBOP were then administered Yin Gu, Alza/Johnson & Johnson PRD, United States 3 days before harvest. Assessment of induction of the human CYP3A4 Lars Karlsson, Alza/Johnson & Johnson PRD, United States transgene showed that PXR & CAR activities are markedly impaired in livers of tumour-bearing mice. Gel shift analysis (EMSA) on liver nuclear The farnesyltransferase inhibitor (FTI) tipifarnib (ZARNESTRA®, extracts profi led the DNA binding capability of various transcription fac- R115777) has demonstrated clinical responses in adults with refractory Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 167 and relapsed acute leukemias. The links between infl ammation and cancer, and the observations that certain FTIs inhibit NF-kappa B activation, sug- 6072 gested that tipifarnib might inhibit production of infl ammatory mediators. We tested the effects of tipifarnib on LPS-induced infl ammation in a human Treatment with antileukinate, a CXCR2 chemokine receptor leukemia cell line, THP-1. Tipifarnib showed inhibition of LPS-induced antagonist protects mice against acute pancreatitis and associated transcription of MCP-1, IL-1beta, STAT-1 and MMP-9. Inhibition of LPS- lung injury induced secretion of IL-1beta, MMP-9, IL-6, MCP-1 and TNF-alpha was also observed for THP-1 cells cultured in the presence of tipifarnib. How- Madhav Bhatia, National University of Singapore, Singapore ever, tipifarnib did not inhibit LPS-induced IL-8 transcription or secretion. Akhil Hegde, National University of Singapore, Singapore Signifi cant inhibition of LPS-induced TNF-alpha by tipifarnib was also observed in human PBMC. In a murine model of LPS-induced infl am- Acute pancreatitis is a common, and as yet incurable, clinical condition, mation, pre-treatment with tipifarnib resulted in signifi cant inhibition of the incidence of which has been increasing over recent years. Chemokines TNF-alpha production, as well as signifi cant inhibition of MIP-1 alpha, IL- are believed to play a key role in the pathogenesis of acute pancreatitis. 6, IL-1beta, and MCP-1 production, but not IL-8. Thus, tipifarnib showed We have earlier shown that treatment with a neutralising antibody against global inhibition of LPS-induced infl ammation in vitro and in vivo, While CINC, a CXC chemokine, protects rats against acute pancreatitis associ- FTIs have been shown to inhibit NF-kappa B activation, the difference in ated lung injury. The hexapeptide antileukinate (Ac-RRWWCR-NH2) is a the potency of inhibition by tipifarnib for LPS-induced TNF-alpha versus potent inhibitor of binding of CXC chemokines to the receptors (CXCR2). IL-8 suggests the mechanism is more complex. This data may have impli- This study aims to evaluate the effect of treatment with antileukinate on cations for therapeutic use of FTIs, and in particular, tipifarnib, in cancer acute pancreatitis and the associated lung injury in mice. Acute pancreatitis and infl ammatory diseases. was induced in adult male swiss mice by hourly intraperitoneal injections of caerulein (50 ug/kg/h) for (up to) 10 hours. Antileukinate (52.63 mg/kg, s.c.) was administered to mice either 30 min before (prophylactic) or 1 6071 h after (therapeutic) starting caerulein injections. Severity of acute pan- creatitis was determined by measuring plasma amylase, pancreatic water Annexin 1 Peptide Protects against Focal Cerebral Ischaemia- content, pancreatic myeloperoxidase (MPO) activity and histological ex- Reperfusion Injury in Mice amination of lung sections. A rise in lung MPO activity and histological evidence of lung injury of lung sections were used as criteria for pancre- Felicity N Gavins, WILLIAM HARVEY RESEARCH INSTITUTE, United atitis-associated lung injury. Treatment with antileukinate protected mice Kingdom against acute pancreatitis and associated lung injury, showing thereby that Niel Granger, LSUHSC, United States anti-chemokine therapy may be of value in this condition. mauro Perretti, WILLIAM HARVEY RESEARCH INSTITUTE, United Kingdom 6073 Brain ischaemia continues to be a leading cause of death in the world. Using a model of cerebral ischaemia-reperfusion (IR) and intravital mi- CXC chemokines modulate the mononuclear cell infi ltration induced croscopy in this project, we set out to address the potential modulatory by angiotensin II effects of the anti-infl ammatory mediator, annexin 1. In C57BL/6 mice, middle cerebral artery occlusion (MCAO) was induced for 1 h followed Yafa Naim Abu Nabah, Department of Pharmacology, Faculty of Medici- by 24 h reperfusion. At this time point, neurological defi cit and infarct ne, Valencia, Spain volume were monitored, together with the extent and characteristics of Rossana Estelles, Department of Pharmacology, Faculty of Medicine, the leukocyte-endothelial cell interactions (LEI) as monitored in cortical Valencia, Spain venules by intravital microscopy. Sham mice showed little LEIs in cere- Teresa Mateo, Department of Pharmacology, Faculty of Medicine, bral vessels. Permanent focal cerebral IR induced increases in LEI. The Valencia, Spain number of adherent leukocytes was signifi cantly increased in MCAO and Henry M Sarau, Glaxo Smith Kline, PA, USA, United States 24 h reperfused animals compared to their sham counterparts (622±116 vs. Peter J Jose, Leukocyte Biology Section, Imperial College London, 0cells, P<0.05, n = 6 mice/group). Upon administration of the annexin 1 N- United Kingdom terminus-derived peptide, Ac2-26 (100 μg or 33 nmol) at the start of reper- Maria J Sanz, Department of Pharmacology, Faculty of Medicine, fusion, and subsequent administrations at 6 h and 18 h, the infarct volume Valencia, Spain was signifi cantly reduced (28±3% vs. 39±3% in vehicle-treated animals; P<0.05, n=6 mice/group). Leukocyte adhesion in cerebral venules was The development of vascular lesions in atherosclerosis is associated with signifi cantly inhibited upon Ac2-26 administration compared to vehicle- increased levels of angiotensin II (AII) and selected CC chemokines lead- treated mice (260±122 vs. 622±116. P<0.05, n = 6 mice/group). The FPR ing to mononuclear leukocyte infi ltration. Because CXC chemokine syn- antagonist Boc2 was inactive when given to IR treated mice, but prevented thesis is also demonstrable in atherosclerosis, this study has investigated the inhibitory effect of Ac2-26 (n=6 mice/group. P<0.05 vs. peptide alone). the effects of a CXCR2 antagonist, SB-517785-M, on AII-induced mono- These results demonstrate that a bioactive peptide of the anti-infl ammatory nuclear cell recruitment in the rat. AII (1 nM, 5ml i.p.) induced neutro- protein annexin 1 produces signifi cant cerebral protection, which appears phil and mononuclear cell accumulation in the peritoneal cavity that was to involve a receptor of the FPR family (i.e. Boc sensitive). These results maximal at 4 and 8h respectively, these events being preceded by CXC help provide a basis for understanding the molecular mechanism respon- and CC chemokine synthesis peaking at 1 and 4h respectively. Pretreat- sible for the annexin 1 effects, which may provide therapeutic potential in ment (1h) with SB-517785-M (25 mg/kg p.o.) inhibited both neutrophil cerebral ischaemia. (94% inhibition at 4h) and mononuclear cell (60% inhibition at 8h) accu- This work was supported by the British Heart Foundation UK Junior Fel- mulation. SB-517785-M did not affect CXC chemokine levels (CINC/KC lowship (FS/03/100/16326) and MIP-2) but reduced the AII-induced CC chemokines that contribute to mononuclear cell infi ltration: MCP-1, RANTES and MIP-1alpha were reduced by 66, 63 and 77% respectively. Intravital microscopy 4h after the i.p. injection of AII to measure earlier responses within the rat mesenteric microcirculation revealed signifi cant arteriolar leukocyte (predominantly mononuclear cell) adhesion and venular leukocyte (neutrophil and mono- nuclear cell) rolling, adhesion and emigration. SB-517785-M pretreatment decreased these responses by 85, 92, 100 and 100% respectively. Thus, mononuclear leukocyte infi ltration elicited by AII appears to be partly me- S 168 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005) diated by a CXC chemokine-dependent CC chemokine release. Therefore production of the cytokines IL-1, IL-6, or the chemokines IL-8, MIP-1α CXCR2 antagonists may reduce the onset and progression of atheroscle- and RANTES. The present study aimed to defi ne the role of MCP-1 in rotic lesions. acute pancreatitis by the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit signifi cantly reduced MCP-1 levels in the pancreas. 6074 Also, this treatment signifi cantly protected mice against acute pancreatitis as determined by hyperamylasemia neutrophil sequestration in the pan- Regulation of neutrophil traffi cking by leukotrienes and platelet- creas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis activating factor in dermal infl ammation on histological examination of pancreas sections. These results show an important role of MCP-1 in the pathogenesis of acute pancreatitis and sug- Leila Hamdan, Faculty of Pharmacy, Université de Montréal, Québec, gest that anti MCP-1 strategies may be of potential therapeutic value in the Canada treatment of acute pancreatitis. Caroline Bélanger, Faculty of Pharmacy, Université de Montréal, Qué- bec, Canada Pierre Borgeat, Centre de recherche en Rhumatologie et Immunologie, 6076 Centre de recherche du CHUL (CHUQ), Sainte-Foy, Québec, Canada

Sylvie Marleau, Faculty of Pharmacy, Université de Montréal, Québec, Inhibition of Prostaglandin F2α release by selective cyclooxygenase-2 Canada inhibitors accounts for a reduced leukocyte migration in rats

The present study aims to investigate the role of leukotrienes (LTs) and Gustavo B Menezes, Federal University of Minas Gerais, Brazil platelet-activating factor (PAF) on polymorphonuclear neutrophils (PMNs) Janetti N Francischi, Federal University of Minas Gerais, Brazil traffi cking to dermal infl ammatory sites using selective receptor antago- nists. Rabbits were pretreated orally with BIIL 284 (0,1 mg/kg), a BLT1 PURPOSE: To investigate whether selective cyclooxygenase 2 (COX 2) receptor antagonist, and/or WEB 2086 (10 mg/kg), a PAF receptor antago- inhibitors would play a role in a model of leukocyte migration in rats. nist, 2 hours before the intradermal injections of agonists (50 μl/site). The METHODS: E. coli lypopolysaccharide (LPS; serotype 0111B4) or pros- agents under investigation included LTB4 (500 pmol/site), PAF (2 nmol/ taglandin (PGE2 and PGF2α) were intraperitoneally injected at time zero site), TNFα (10 pmol/site), Il-8 (10 pmol/site), zymosan activated plasma in adult female Holtzman rats. The animals (n=3-10/group) were previ- (ZAP, 1%) and fMLP (50 pmol/site). Thirty minutes before sacrifi ce, the ously treated (½ h before) by subcutaneous route with unspecifi c (indo- rabbits received an i.v. injection of 125I-BSA (1 μCi/kg) to assess vascular methacin, piroxicam), selective COX 1 (SC560) or COX 2 (celecoxib, permeability. Following a 2 hours accumulation period, PMN migration rofecoxib, SC236) inhibitors and compared with their respective controls. elicited by LTB4 was inhibited by 50 ± 5 % (P < 0.001 versus controls) Total (cells/ mm3) and specifi c (%) cell numbers were obtained under with BIIL 284, by 28 ± 5 % (P < 0.01) with WEB 2086, and by 64 ± 4 % (P optical microscopy on samples from peritoneal lavage fl uid collected at < 0.001) with both drugs. PAF-elicited PMN migration was reduced by 29 different time intervals over 24 h. RESULTS: Compared with controls, ± 6 % (P <0.01) with BIIL 284, by 60 ± 6 % (P < 0.001) with WEB 2086, LPS induced a dose (0.03 - 3 mcg/cavity) and time-dependent increase and by 70 ± 6 % (P < 0.001) with both drugs. Interestingly, BIIL 284 and in leukocyte number (C=12.0±1.3; LPS 0.3=19,0±2.5, ANOVA, p<0.05), WEB 2086 exerted additive inhibitory effects on PMN migration elicited which was related predominantly to neutrophil (50 %) and to a lesser ex- by chemically unrelated agonists. Indeed, TNFα-elicited PMNs migration tent to macrophage increase. Only the animals treated with selective COX was inhibited by 29 ± 7 % (P < 0.01) and 22 ± 7 % (P < 0.01) by BIIL 284 2 inhibitors and indomethacin showed a signifi cant reduction in leukocyte and WEB 2086 respectively, whereas both drugs add an additive effect numbers following 6 h of LPS administration (0.3 mcg/cavity). To fur- (53 ± 7 %, P < 0.001). Similar results were observed for IL-8. BIIL 284 ther verify whether prostaglandins would be involved in leukocyte mi- and WEB 2086 inhibited IL-8-elicited PMNs accumulation by 52 ± 8 % gration, prostaglandins E2 and F2α (1- 1000 ng/cavity) were injected into (P < 0.001), whereas BIIL 284 and WEB 2086 alone add inhibitory effects the peritoneum and the cell number was compared with vehicle-injected of 36 ± 6 % (P < 0.01) and 12 ± 7 %, respectively. Cooperative effects of animals. Only PGF2α was able to induce neutrophil increase (C=2.75±0.09; drugs were also observed for fMLP and ZAP-induced PMNs accumulation PGF2α(100ng)=13.35±2,78) following 3 h of injection. In addition, intra- as well as for agonists-induced local oedema. Our results support that LTs peritoneal reposition of PGF2α restored the abrogated leukocyte response and PAF are key regulators of PMNs traffi cking to infl ammatory sites. to LPS shown by animals pretreated with rofecoxib. CONCLUSION: Our

Supported by the Canadian Institutes of Health Research. data suggests that COX 2, through PGF2α release, is the important isoform for neutrophil recruitment in the rat model of LPS-induced infl ammation. SUPPORT: CNPq, CAPES. 6075

Treatment with bindarit, a specifi c MCP-1 synthesis blocker, protects 6077 mice against acute pancreatitis Adhesion molecules in acute pancreatitis Madhav Bhatia, National University of Singapore, Singapore Raina D Ramnath, National University of Singapore, Singapore Serge N Chooklin, Medical University, Lviv, Ukraine Lakshmi Chevali, National University of Singapore, Singapore Andre A Perejaslov, Medical University, Lviv, Ukraine Angelo Guglielmotti, ACRAF SpA, Italy Ihor Y Bihalskyy, Medical University, Ukraine

Acute pancreatitis is a common, and as yet incurable, clinical condition, The development of organ dysfunction often accompanies the early stage the incidence of which has been increasing over recent years. Chemokines of necrotizing pancreatitis. It is believed to be a consequence of the devel- are believed to play a key role in the pathogenesis of acute pancreatitis. opment of systemic infl ammatory response syndrome, which is character- We have earlier shown that pancreatic acinar cells produce the chemo- ized by the activation and interaction of leukocytes and endothelial cells, kine MCP-1 in response to caerulein hyperstimulation, demonstrating that and enhanced expression of proinfl ammatory cytokines. The role of the acinar-derived MCP-1 is an early mediator of infl ammation in acute pan- endothelium in the human acute pancreatitis is still unclear. creatitis. Blocking chemokine production or action is a major target for Applying the ELISA technique, plasma levels of soluble E-selectin (sE- pharmacological intervention in a variety of infl ammatory diseases, such selectin) and sICAM-1 were studied in 87 patients with acute pancreatitis. as acute pancreatitis. Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-inda- Thirty-one of them had a severe and 56 - a mild course of disease. In this zol-3yl]methoxy]propanoic acid) has been shown to preferentially inhibit study enrolled patients who admitted to clinic within 48 hours after disease MCP-1 production in vitro in monocytes and in vivo, without affecting the onset. The control group compiled 10 healthy volunteers. Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 169

Already after admission the increased levels of adhesion molecules were graft as well as in the neo intima in eighteen sheep. These cells were shown noted in all patients. By that, the highest levels were observed in patients to produce collagen and α-SMA at 1, 2 and 4 weeks. These cells then un- with severe acute pancreatitis (sICAM-1 - 186.9±14.6 ng/ml vs 141.0±20.0 derwent immunohistochemical analysis and were found to express a set of ng/ml; sE-selectin - 159.7±17.9 ng/ml vs 94.5±9.8 ng/ml). During fi rst markers unique to the fi brocyte (CD34, CD45, Vimentin and α-SMA) and seven days plasma levels of sE-selectin and sICAM-1 practically did not also to double stain for CD34 and α-SMA. change in patients with necrotizing pancreatitis, while in patients with the Conclusions-These observations demonstrate the contribution of the fi bro- mild pancreatitis its levels gradually decreased starting from the third day. cyte to IH. The VSMC mass in IH is partially derived from a haematopoi- There was a clear correlation between adhesion molecules levels, hemato- etic circulating progenitor cell that acquires mesenchymal features as it crite and hemoglobin levels in patients with severe pancreatitis. Within the matures at the site of injury. group of patients with necrotizing pancreatitis the relationship between sE-selectin, sICAM-1 and severity of hemodynamic compromise was es- tablished. 6080 The intensity of endothelial activation, measured by adhesion molecules concentration, considered to be key features of systemic infl ammation in Mechanisms Involved On Inhibition Of Neutrophil Recruitment By severe pancreatitis. Chronic Blockage Of Nitric Oxide Synthesis

Cristina Bichels Hebeda, University of São Paulo, Brazil 6078 Suzana Beatriz Veríssimo Mello, University of São Paulo, Brazil Sandra Helena Poliselli Farsky, Cidade University, Brazil Carrageenan induced cell-migration in the air pouch; a pharmacodynamic model to evaluate CCR1 antagonists Introduction: We previously showed that nonspecifi c chronicle inhibition of NO synthases inhibits neutrophil migration to infl ammatory site, de- Anna Ridderstad Wollberg, AstraZeneca R&D Södertälje, Bioscience, pendent, at least in part, on impairment on leukocyte-endothelial interac- SE-15185 Södertälje, Sweden tions and on decreasing of L-selectin expression (Farsky et al., Infl amm. Res. 53:442-52, 2004). Now, we are investigating the role of the treatment Chronic disease models, such as experimental autoimmune encephalo- on secretion of glucocorticoids and on endothelial adhesion molecules myelitis, EAE, are labor intensive and not suitable for extensive in vivo expressions. Methods: Male Wistar rats were treated with L-NAME or screening of drug candidates. Therefore we aimed to establish a rapid D-NAME (20 mg/kg/day, oral route, during two weeks). Circulating NO pharmacodynamic model to evaluate chemokine receptor antagonists in and corticosterone were measured by gaseous phase chemiluminescence vivo. The carrageenan induced air pouch model is a well-established in and radioimmunoassay, respectively. Expressions of P-selectin, ICAM- vivo infl ammation / migration model. At different time intervals following 1 and PECAM-1 on endothelium of postcapillary venules from cremas- carrageenan injection into the air pouch infi ltrating cells can be recovered, ter muscle were evaluated by immunohistochemstry using FITC-labeled quantifi ed and analysed for phenotypic cell markers by fl ow cytometry. monoclonal antibodies. Results: The effectiveness of L-NAME treatment It has previously been reported that antagonizing CCR1 in vivo affects was accomplished by reduction of about 50% on circulating NO. Corticos- migration of neutrophils into the air pouch. Therefore, we employed this terone levels in serum was not modifi ed by the treatment, as well as, the model to establish a plasma concentration vs response relationship for a expressions of adhesion molecules, in both basal or stimulated conditions. small molecule CCR1 antagonist in mice to calculate an EC50. Similar Conclusions: Data obtained suggest that inhibition on neutrophil recruit- experiments were carried out in the rat. Administration of a CCR1 antago- ment induced by chronicle blockage of NO synthesis is not depending on nist, with a Ki of 933nM for the rat CCR1, inhibited the neutrophil migra- glucocorticoid secretion or inhibition of endothelial adhesion molecules. tion in vivo to the rat air pouch and also directly inhibited the carrageenan Financial Support: FAPESP (grant 02/02659-5), CNPq. induced MCP-1 production in the air pouch. In conclusion, this model, - both in the rat and the mouse, was found to be very useful in evaluating CCR1 antagonists in vivo. 6081

6079 In vivo expression of CD11b and oxidative burst production in healthy subjects and patients with early renal failure The Circulating Progenitor in Intimal Hyperplasia: Could it be the Fibrocyte? Elham Dadfar, Department of Medicine, Division of Clinical Immunology & Transfusion medicine, Karolinska University Hospital, Sweden Ramon L Varcoe, Westmead, Australia Stefan H Jacobson, Department of Nephrology, Danderyds Hospital, Heather J Medbury, Westmead, Australia Sweden Ann K Guiffre, Westmead, Australia Joachim Lundahl, Department of Medicine, Division of Clinical Immuno- Mauro Vicaretti, Westmead, Australia logy & Transfusion medicine, Karolinska University Hospital, Sweden John P Fletcher, Westmead, Australia The aim of present study was to evaluate the impact of early state of renal Background-Experimental animal studies have shown that the intimal hy- failure on oxygen radical production and CD11b expression in peripheral perplasia (IH) responsible for occlusion after successful revascularization and transmigrated human monocytes (mo). procedures may be partially caused by a bone marrow derived cell that mi- Methods: Two skin blisters were raised in 10 patients (pts) with renal fail- grates to the site of vascular injury. Concurrent studies have demonstrated ure and 12 healthy subjects (hs). Blister exudates were collected at time 0 an extensive role in wound healing for the circulating fi brocyte. We aimed (unstimulated). In order to induce diversity in the intensity of infl amma- to trace the path of the circulating cell that contributes to IH and determine tion (infl .), the blisters were challenged with buffer (intermediate infl .) or if it is the fi brocyte. autologus serum (intense infl .). The CD11b expression and the oxidative Methods and Results-We established an in vitro model whereby purifi ed burst (H2O2 production) were measured by fl ow cytometry, in peripheral monocytes from six healthy human volunteers were cultured into fi bro- and in vivo transmigrated cells. cytes. These cells were morphometrically similar to the vascular smooth Results: The CD11b expression on mo increased upon transmigration muscle cell (VSMC) found in IH and expressed alpha-smooth muscle actin to the infl ammatory site, from peripheral circ, in pts and hs (p<0.05 and (α-SMA) as well as CD34, CD45 and Collagen I (Col I), markers indica- p<0.05). Upon fMLP stimulation the CD11b expression was signifi cantly tive of the fi brocyte. In an in vivo ovine carotid artery synthetic patch graft upregulated in all samples (p<0.05). Peripheral and transmigrated mo had model CFSE labelled circulating leukocytes were observed throughout the the same ability to upregulate CD11b upon fMLP stimulation, and the pts S 170 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005) had the same capacity to upregulate CD11b as hs. Unstimulated trans- isolated with similar frequencies from the lesions of the patients with in- migrated mo had a lower H2O2 production compared to peripheral mo fective endocarditis. Thus the adhesive characteristics of A. defectiva to (p<0.05). Both mo increased their H2O2 production upon fMLP and PMA endothelial cells could differ, and that may account in part for the virulence stimulation (p<0.05 and p<0.05). However, transmigrated mo at the site of of A. defectiva in infective endocarditis. To assess the potential pathogenic interstitial infl . had a lower H2O2 response upon PMA stimuli, compared mechanism, in this study, we examined the adhesive ability of A. defectiva to peripheral mo (p<0.05). Peripheral mo from pts had equal H2O2 pro- to cultured human umbilical vein endothelial cells (HUVEC) and investi- duction as hs. In addition, patients had a higher H2O2 production at the gated A. defectiva-induced cytokine production and leukocyte adhesion site of unstimulated blister (p<0.05). molecule expression. The results indicated that the adhesive ability of A. In concl., in vivo transmigrated mo have a higher basal CD11b expression defectiva to HUVEC was signifi cantly higher than those of G. adiacens, compared to peripheral mo. However, upon stimulation, transmigrated mo G. balaenopterae, G. elegans, G. paraadiacens and Streptococcus sangui- upregulates CD11b to the same extend as peripheral. The oxidative burst nis. The adhesive abilities of A. defectiva to immobilized fi bronectin and in mo decreases upon transmigration but is increased to normal level upon vitronectin were also higher than those of the oral streptococci tested, sug- further stimuli. Mo from pts with early renal failure has not developed the gesting that the relatively higher adhesive ability to endothelial cells could dysfunctional features as observed in later stage of renal failure. be ascribable in part to its higher adhesive ability to the extracellular ma- trix proteins. Furthermore, A. defectiva induced HUVEC to produce IL-8 and TNFa with NF-kB activation, and subsequently to express E-selectin and ICAM-1 molecules on the cell surfaces. These results suggest that A. defectiva entering into bloodstreams could attach to endothelial cells with 6082 a higher frequency than other related bacterial species, and could induce endothelial cells to produce infl ammatory cytokines and to express leuko- The Interaction of Macrophage Migration Inhibitory Factor (MIF) cyte adhesion molecules, which may account for the potential pathogenic and Glucocorticoids in Regulating Leukocyte Traffi cking traits of A. defectiva in infective endocarditis.

Julia L Gregory, Monash University, Australia Michelle T Leech, Monash University, Australia 6084 Eric F Morand, Monash University, Australia Michael J Hickey, Monash University, Australia Macrophage migration inhibitory factor (MIF) rapidly activates the Akt pathway in a PI3K- and JAB1/CSN5-dependent manner: Macrophage migration inhibitory factor (MIF) has been demonstrated to Implications for MIF signaling in infl ammation and cancer promote leukocyte recruitment induced by LPS. This may be due to the ability of MIF to inhibit the effects of endogenous glucocorticoids (GC). Hongqi Lue, Department of Biochemistry and Molecular Cell Biology, The aim of this study was to determine whether MIF infl uences the effects University Hospital RWTH Aachen, Germany of GC on leukocyte-endothelial cell interactions in vivo. Endogenous GC Michael Thiele, Department of Biochemistry and Molecular Cell Biology, were inhibited with the GC receptor antagonist, RU486. Intravital mi- University Hospital RWTH Aachen, Germany croscopy was used to assess leukocyte rolling, adhesion and emigration in Günter Fingerle-Rowson, Department of Hematology and Oncology, post-capillary venules. MIF-/- mice exhibited reduced LPS-induced leu- University Hospital Cologne, Germany kocyte adhesion and emigration compared to wildtype mice. RU486 in- Richard Bucala, Department of Medicine and Pathology, Yale University, creased LPS-induced leukocyte adhesion and emigration in both wildtype School of Medicine, United States and MIF-/- mice, indicating that endogenous GC exert an inhibitory effect Jürgen Bernhagen, Department of Biochemistry and Molecular Cell on leukocyte traffi cking independent of the presence of MIF. Correspond- Biology, University Hospital RWTH Aachen, Germany ingly, reductions in leukocyte adhesion and emigration in MIF-/- mice per- sisted regardless of GC antagonism with RU486. The interaction between Discovered 40 years ago as an ill-defi ned T lymphocyte immune activity, MIF and exogenous GC was assessed by treating wildtype and MIF-/- mice MIF is now known as a potent and broadly expressed infl ammatory cyto- with dexamethasone (DEX). DEX reduced LPS-induced leukocyte roll- kine. Numerous studies have established that MIF is a pivotal mediator ing, adhesion and emigration in wildtype mice to levels similar to those of acute and chronic infl ammatory diseases. For example, MIF is one of observed in MIF-/- mice not treated with DEX. In MIF-/- mice, adhesion the few novel promising target proteins for the treatment of septic shock and emigration were not additionally inhibited by DEX. In contrast leuko- (Riedemann et al., Nat. Med. 2003). Similarly, MIF is of critical impor- cyte rolling was additionally inhibited by DEX in MIF-/- mice. Together, tance in the pathogenesis of rheumatoid arthritis (Morand & Leech, Front these results suggest that endogenous MIF and GC reciprocally regulate Biosci. 2005). Although MIF has been implicated in the regulation of cell leukocyte-endothelial cell interactions. However, the effects of MIF and proliferation, apoptosis and infl ammatory gene expression, the precise sig- GC on leukocyte-endothelial cell interactions are independent. naling and regulatory mechanisms by which MIF controls these processes are largely unknown. MIF inhibits p53-dependent apoptosis and this event has been linked to synovial infl ammation and cancer. However, it has not 6083 been studied whether MIF can infl uence the anti-apoptotic and pro-prolif- erative Akt pathway. We demonstrate that exogenous MIF (rMIF) rapidly The adhesive ability of Abiotrophia defectiva to endothelial cells and activates the phosphorylation of Akt in fi broblasts. Phosphorylation of Akt A. defectiva-induced cytokine production and leukocyte adhesion by MIF is associated with Akt activation, as immunoprecipitated Akt from molecule expression MIF-treated fi broblasts phosphorylates the substrate GSK3. Activation of Akt can also be induced in MIF KO mouse embryo fi broblasts (MEF) by Shihoko Tajika, Iwate Med.Univ., Japan treatment with rMIF or secreted MIF from wildtype MEFs, demonstrating Minoru Sasaki, Iwate Med.Univ., Japan that Akt activation is due to paracrine MIF signaling. We fi nd that MIF- Yuko Ohara-Nemoto, Iwate Med.Univ., Japan induced activation of Akt is dependent on the activation of PI3K, an up- Chiharu Yamaura, Iwate Med.Univ., Japan stream activator of Akt, as no MIF-induced Akt activation is seen in the Shigenobu Kimura, Iwate Med.Univ., Japan presence of the PI3K inhibitors LY294002 and wortmannin. MIF-induced Akt signaling is also dependent on the signalosome component and MIF Abiotrophia defectiva as well as the related bacteria of Granuricatella binding protein JAB1/CSN, as JAB1-knock down by siRNA leads to a species is one of the nutritionally variant oral streptococci, and can cause marked inhibition of PI3K/Akt activation by MIF. This points towards infective endocarditis. Although the frequency of oral colonization of A. an intriguing molecular link between extracellularly induced MIF signal defectiva was much lower than that of G. adiacens in healthy volunteers transduction processes and cellular JAB1 or MIF/JAB1 complexes. As (11.8 and 87.1%, respectively), it was reported that these two species were phosphorylation of Akt is connected to the inhibition of apoptosis through Infl amm. res. Supplement 2 (2005) Posters: Monday 22 August 2005 S 171 an early phosphorylation of Bad, we conclude that activation of Akt signal- Mohammad JahanSepas, Shaheed Beheshti Univ, Iran ing by MIF could represent a mechanism by which MIF inhibits apoptosis Hossein Goudarzi, Shaheed Beheshti Univ., Iran in infl ammation and cancer. The Prevalence and incidence of the NTM (Non Tuberculosis Mycobacte- rium) are diffi cult to characterize infection due to the NTM rarely reported 6085 to public health departments.They affect immune-competent, immune- comprised persons and who has been affected of disasters especially chil- Gelatinase B participates in cellular infi ltration during experimental dren .The aim of this study was isolation of mycobacterial agents, espe- acute peritonitis induced by zymosan in mice cially atypical species from dermal lesions of children in Bam's earthquake and determination of their antibiotic resistance. Elzbieta Kolaczkowska, Department of Evolutionary Immunobiology, Matherial&Methods-In this descriptive study, 88 children of Bam's Jagiellonian University, Krakow , Poland earthquake who settled in camps have studied. The samples from lesion Magdalena Chadzinska, Department of Evolutionary Immunobiology, transported to laboratory for isolation of mycobacterial agents .After Jagiellonian University, Krakow, Poland decontamination ,staining ,they had been cultured in Lewenstein -jensen Anna Scislowska-Czarnecka, Department of Physiotherapy, Faculty of medium .After isolation of mycobacterium by differential test such as Anatomy, Academy of Physical Education, Krakow, Poland niasin,nitrate,arylsulfatase,catalase and urease…,their antibiotic resistance Barbara Plytycz, Department of Evolutionary Immunobiology, Jagiello- were studied. nian University, Krakow, Poland Results-Among 88 samples 3 mycobacterium isolated (3.4%)They were Ghislain Opdenakker, Rega Institute for Medical Research, University of 2 M.chelonae(66.7%) and one M.scrofulaceum (33.3%) .In antimyco- Leuven , Belgium bacterial susceptibility test,M.chelonae were resistant to isoniasid(INH), Bernd Arnold, German Cancer Research Center, Heidelberg , Streotomycin(SM) Rifampin(RMP) and ssensitive to Ethambutol (EMB Germany ),Kanamycin(Km),Ethionamid (ThA0,M.scrofulaceum was resistant to INH,SM,RMP,EMB,THA and only sensitive to Kanamycin. Background & Aim Coclusion:Since atypical mycobacteria exist in soil, isolation of them Cellular infi ltration into a site of injury/infection is a distinctive feature from person especially children after a disaster such as earth quake is of any infl ammatory response. The process is well described in a mod- important . el of acute zymosan-induced peritonitis in mice but the mechanisms by Key word-dermal lesion,mycobacterium , earth quake which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. On the other hand, gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte trans- 6087 migration through endothelia during infl ammation remains controversial. Metalloproteinases (MMP) are enzymes involved in degradation of basal Pharmacological and biochemical analysis of anti-infl ammatory membrane and extracellular matrix macromolecules. Thus the aim of this response of latex of Calotropis procera study was to evaluate the role of gelatinase B (MMP-9) in the process of leukocyte infi ltration during murine zymosan-induced peritoneal infl am- Soneera Arya, Department of Pharmacology, All India Institute of Medi- mation. cal Sciences, New Delhi- 110 029, India Methods V L Kumar, Department of Pharmacology, All India Institute of Medical Peritonits was induced by i.p. zymosan administration into Balb/c mice Sciences, New Delhi- 110 029, India pre-treated with a specifi c peptide inhibitor of gelatinases CTTHWGFTLC (INH) or into gelatinase B-defi cient mice (MMP-9-/-) and their respective Calotropis procera (Ait). R.Br. (Asclepiadaceae), a wild growing plant ex- controls. Additionally, some Balb/c mice were i.p. injected with rm pro- hibits pleiotropic effects and various parts of the plant have been used as MMP-9. Levels of MMP-9 were analyzed by zymography, Activity Assay folk medicine for the treatment of numerous diseases. The white milky and Western blot. Levels of TIMP-1, TNF-a, KC and IL-10 were measured latex obtained from the aerial parts of the plant has been demonstrated to by ELISAs. possess potent anti-infl ammatory activity, however the mechanisms under- Results & Discussion lying this effect remain unknown. In the present study we have carried out Zymosan administration induced synthesis/release of MMP-9 as it was the pharmacological and biochemical characterization of the anti-infl am- detected in the peritoneal fl uid and plasma of Balb/c mice, along with its matory activity of the dry latex (DL) and its methanolic extract (MeDL) at natural inhibitor TIMP-1. This was observed at the time of neutrophil infi l- oral doses ranging from 50-1000 mg/kg using various animal models of in- tration into the peritoneum (6 hrs) as well as during monocyte/macrophage fl ammation. The anti-infl ammatory potential of DL and MeDL was evalu- infl ux (24 hrs). When peritonitis was induced in either MMP-9-/- mice or ated against rat paw oedema formation induced by early and late mediators INH-treated animals the neutrophil infi ltration was impaired at 6 hrs while of infl ammation. Both DL and MeDL when administered orally 1h prior neither cytokine levels nor migratory properties of the leukocytes were to the sub-plantar injection of phlogistic agents signifi cantly inhibited the changed. 24 hrs post zymosan a signifi cant increase in neutrophil levels oedema induced by carrageenin, histamine, compound 48/80, bradykinin was observed in MMP-9-/- animals, but not in the INH-treated mice, in and PGE2 while only marginal inhibition was observed against serotonin. comparison to their respective controls. Moreover, i.p. injection of rm pro- The inhibitory effects of latex were comparable with antihistaminic drug MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the chlorpheniramine and more pronounced than bradykinin antagonist brady- fi ndings indicate that gelatinase B participates in neutrophil transmigra- zide and standard anti-infl ammatory drug phenylbutazone. The anti-in- tion; however, its function can be partially compensated by other mecha- fl ammatory potential of latex was further substantiated by the pronounced nisms. decrease of the cellular infi ltration and subcutaneous oedema observed on histological analysis in carrageenin induced paw oedema model. Both DL and MeDL signifi cantly decreased nitric oxide (NO), tumor necrosis fac- 6086 tor-α (TNF- α) myleoperoxidase (MPO) and vascular permeability in the carrageenin induced paw oedema. Further both these extracts also exhib- The Isolation of Mycobacterial agents from Children in Bam's ited antioxidant activity and produced a signifi cant decrease in the tissue Earthquake levels of malondialdehyde (MDA) and increase in the antioxidant levels of glutathione, SOD and catalase in the carrageenin induced paw oedema Fatemeh Fallah, Shaheed Beheshti University,Pediatric InfectiousRes- model. (AS is SRF of CSIR, Delhi) earch Center, Iran Gita Eslami, Shaheed Beheshti University, Iran Mostafa Sharifi an, Shaheed Beheshti University, Iran S 172 Posters: Monday 22 August 2005 Infl amm. res. Supplement 2 (2005)

Results: 6088 Multiple whitish nodules were seen on cut surface of liver tissue. Micro- scopically there was patchy liver cell necrosis associated with infl amma- Harpagophytum procumbens (Devil’s claw): A possible natural anti- tion. Microbiological examination revealed the presence of Gram-negative infl ammatory agent (An Experimental study) bacilli. Bacterial cultures and Antibiotic sensitivity test were done. Follow- ing these tests other axolotls were treated with Ceftriaxone. Ibrahim H Younos, Clinical Pharmacology department, Faculty of medi- Conclusion: cine, Minufi ya university, Egypt Infection with gram negative bacilli leading to acute liver cell necrosis of Mohamed I Ahmed, Clinical Pharmacology department, Faculty of medi- axolotls could be successfully treated with Ceftriaxone. cine, Minufi ya university, Egypt Mohamed I Afi fi , Clinical Pharmacology department, Faculty of medici- ne, Minufi ya university, Egypt

Extract of harpagophytum procumbens (devil`s claw) have become the fo- cus of research as a potential therapeutic agent in the treatment of rheumat- ic arthritis and pain due to its favourable side effects profi le compared to synthetic alternatives.Harpagophytum procumbens (devil's claw) is often used in the supportive treatment of infl ammatory and degenerative diseases of the skeletal system. Devil's claw-treated patients had low incidence of side effects. This superior safety of treatment is very valuable, especially in view of that in chronic diseases mandatory long duration of therapy. Different investigations evaluated the anti-infl ammatory and analgesic ef- fects of harpagophytum procumbens with inconsistent and contradictory results.The aim of this work was to investigate the effect of harpagophy- tum procumbens on both acute and chronic infl ammatory processes in rats and pain responses in mice. Also, safety on gastric and duodenal mucosa is evaluated histopathologically.Eighty (80) rats of both sexes weighing 150-200 grams each and twenty four (24) mice of both sexes weighing 25- 30 grams each, were used in this work. These animals were classifi ed for induction of the different experimental model. The acute model of infl am- mation includes carrageenan-induced rat backpaw edema test. The chronic model of infl ammation includes cotton pellet-induced granuloma test and complete adjuvant-induced arthritis test. The analgesic model includes writhing test. Histo-pathological assessment of gastric & duodenal mucosa for the effect of harpagophytum procumbens comparable to the effect of indomethacin was done in the Adjuvant-induced arthritis group.It can be concluded that harpagophytum procumbens is a new herbal agent having reasonable anti-infl ammatory effect compared to indomethacin, as a stan- dard anti-infl ammatory agent and reasonable analgesic effect compared to acetyl salicylic acid,as a standard analgesic agent without harmful effect on gastric and duodenal mucosa.

6089

Liver cell necrosis of Axolotls following an infection, associated with infl ammation

Mangala Gunatilake, Dept. of Physiology, Faculty of Medicine, Universi- ty of Colombo, Sri Lanka Ramani Jayalath, National Zoological Gardens, Dehiwela., Sri Lanka Dilani Lokuhetty, Dept. of Pathology, Faculty of Medicine, University of Colombo, Sri Lanka Jeniffer Perera, Dept. of Microbiology, Faculty of Medicine, University of Colombo, Sri Lanka

Introduction : Axolotl is an amphibian vertebrate, which belongs to Ambystomatidae family. Six healthy axolotls were brought to the National Zoological Gar- dens of Sri Lanka from UK in early 2004 in order to introduce this spe- cies to the locals. They were kept in a separate tank and fed with chopped earthworms. Method : After few weeks, one axolotl died without having any external lesions ex- cept bloating. Sections from the liver were taken for pathological and mi- crobiological examination. Two days later another axolotl was found dead. Superfi cial parts of the skin of this axolotl were scaling off. Liver sections were preserved in formal saline for examination in order to identify the cause of the death and to decide the measures to prevent the remaining four axolotls falling into the same fate. Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 173

Unstimulated Anx-1-/- cells exhibited increased basal levels of IL-6 7001 mRNA and protein compared to WT cells. In concert with this, these cells exhibited increased basal levels of phosphorylated p38, JNK and ERK1/2 Platelet-activating factor causes shock via phosphatidylinositol 3- MAPKs, IL-1-inducible p38 activity was also increased in Anx-1-/- cells. kinase and constitutive nitric oxide synthase activation Blocking either p38 or JNK pathways inhibited the increase in basal and IL-1-induced IL-6 release but not mRNA in Anx-1-/- cells. Dexametha- Anje Cauwels, Ghent University and VIB, Belgium sone inhibited IL-6 release induced by IL-1 in WT and Anx-1-/- cells, but Peter Brouckaert, Ghent University and VIB, Belgium Anx-1-/- cells were less sensitive to dexamethasone than WT cells. The inhibitory effect of dexamethasone on NF?B activity was intact in Anx- Purpose: Platelet-activating factor (PAF) is an important phospholipid 1-/- cells. MAP kinase-phosphatase-1 (MKP-1), a GC-induced negative modulator of infl ammation. In pathological conditions, PAF has been im- regulator of MAPK activation, was signifi cantly upregulated by dexa- plicated in the hypotension and cardiac dysfunctions occurring in various methasone in WT cells, but this effect of dexamethasone was impaired in cardiovascular shock syndromes. However, the contribution of nitric oxide Anx-1-/- cells. Treatment with Anx-1 N-terminal peptide restored MKP-1 (NO) and the signaling events downstream of the PAF receptor that direct expression and inhibited MAPK activities in Anx-1-/- cells. its shock-inducing effect are still ambiguous. Additionally, it is generally In summary, these data demonstrates that Anx-1 is an endogenous inhibi- accepted that transcriptionally regulated inducible NOS (iNOS) is respon- tory regulator of MAPK activation and IL-6 expression. Anx-1 is required sible for the production of excessive NO triggering and mediating shock. for GC regulation of MKP-1. Therapeutic manipulation of annexin I could Methods and Results: To elucidate PAF-mediated signaling in vivo, we provide GC-mimicking effects in infl ammatory disease. studied PAF shock in conscious mice. Surprisingly, the hyperacute PAF- induced shock entirely depended on NO, produced not by the inducible iNOS but by the so-called constitutive endothelial eNOS, rapidly activated 7003 via the (alternative) phosphatidylinositol-3-kinase pathway. Nevertheless, PAF shock was only partially prevented by methylene blue, implying that Pathogenicity of Candida albicans is associated with inactivation of this NO-induced shock is only partly mediated by soluble guanylate cy- cytosolic phospholipase A2 in the kidneys clase (sGC), commonly regarded as the principal vasorelaxing negotia- tor of NO. Pretreatment with specifi c K+ channel inhibitors indicated the Sung Jun Park, Chonnam National University, Korea importance of especially ATP-regulated (KATP) K+ channels, as well as Jung-Hwa Choi, Chonnam National University, Korea large- and small-conductance Ca2+-dependent K+ channels. The combi- Eun Kyoung Choi, Chonnam National University, Korea nation of sGC inhibition with K+ channel toxins revealed a signifi cant Hyun-Mi Ko, Chonnam National University, Korea additive protective effect only in case of combined sGC and KATP inhibi- Kyoung-Jin Kim, Chonnam National University, Korea tion. Su-Ji Han, Pusan National University, Korea Conclusions: We conclude that PAF causes rapid NO release and subse- Suhn-Young Im, Lab. of Immunology, Korea quent NO-dependent lethal shock via a mechanism that entirely depends on phosphatidylinositol-3-kinase-induced eNOS activation, providing evi- Renal failure is a major cause of death in systemic candidiasis in an im- dence for a new, shock-inducing function for eNOS in vivo. Downstream, munocompromised host. In studying the mechanisms underlying kidney NO-dependent PAF shock is mediated only partly via sGC. We also found susceptibility to candida infection, we previously reported that the reduced an important role for K+ channels, especially KATP channels, which we production of cytokines (i.e. TNF) via platelet-activating factor (PAF)- propose to be the target for the sGC-independent NO activation. induced activation of NF-kB renders the organ susceptible to the fungal burden. In this study, we investigated the possibility that pathogenic C. albicans may evade clearance and perhaps even multiply by inhibiting 7002 elements in the signaling pathway that leads to the release of PAF. The fungal burden of pathogenic C. albicans in the kidneys was 104 - 105 fold Annexin-I Regulates IL-6 Expression Via MAP Kinases higher than that of a nonpathogenic strain. PAF-induced early activation of NF-kB and the expression of TNF mRNA were observed in the kid- Yuan H Yang, Monash Institute of Medical Research, Monash University, neys of mice infected with nonpathogenic, but not pathogenic, strains of C. Australia albicans. Impairment of PAF-mediated early NF-kB activation following Ling Toh, Centre for Infl ammatory Diseases, Monash University Depart- infection with pathogenic C. albicans was associated with inactivation of ment of Medicine,Monash Institute of Medical Research, Australia the enzyme cytosolic phospholipase A2 (cPLA2), which is responsible for

Colin D Clyne, Prince Henry's Institute of Medical Research, Australia the release and/or synthesis of PAF, suggesting that either cPLA2 itself or Michelle Leech, Centre for Infl ammatory Diseases, Monash University an upstream initiator of activation of the enzyme is affected by pathogenic Department of Medicine,Monash Institute of Medical Research, Australia C. albicans. Collectively, these fi ndings indicate that C. albicans appears Jin Xue, Centre for Infl ammatory Diseases, Monash University Depart- to exert its pathogenicity through impairing production of anti-candidal ment of Medicine,Monash Institute of Medical Research, Australia cytokines by inactivating cPLA2 activity. This novel mechanism provides April Dacumos, Centre for Infl ammatory Diseases, Monash University insight into understanding, and perhaps a target for the treatment of patho- Department of Medicine,Monash Institute of Medical Research, Australia genic C. albicans. Laveena Sharma, Centre for Infl ammatory Diseases, Monash University Department of Medicine,Monash Institute of Medical Research, Australia Eric Morand, Centre for Infl ammatory Diseases, Monash University 7004 Department of Medicine,Monash Institute of Medical Research, Australia Topical Decoy Annexin I (Anx-1) is a mediator of the anti-infl ammatory actions of glu- cocorticoids (GC). The mechanisms of annexin I anti-infl ammatory effects Rolf O Ehrhardt, Corgentech.Inc, United States are not known. We investigated the role of annexin I in the regulation of Maya Dajee, Corgentech.Inc, United States the proinfl ammatory cytokine, IL-6. Tony Muchamuel, Corgentech.Inc, United States Lung fi broblast cell lines derived from Anx-1-/- and wild-type (WT) mice Brian Shryver, Corgentech.Inc, United States were treated with dexamethasone (10-7 M) and/or IL-1 (1ng/ml). IL-6 Laszlo Komuves, Corgentech.Inc, United States mRNA and protein was measured using real-time PCR and ELISA. Mito- Jennifer Alleman-Sposeto, Corgentech.Inc, United States gen-activated protein kinase (MAPK) and NF-kappaB pathway activation Aung Oo, Corgentech.Inc, United States was studied. Leslie M McEvoy, Corgentech.Inc, United States S 174 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

Atopic Dermatitis (AD) release of intra-cellular contents is undesirable as it could initiate auto- immunity. Therefore, we have tested the role of complement inhibitor, C4b-binding protein (C4BP) in limiting the pro-infl ammatory potential of 7005 necrotic cells. We found that C4BP binds strongly to necrotic cells, irrespective of the cell Macrophage Migration Inhibitory Factor in T Cell Activation in type used or method of induction. Following binding of the C4BP-protein Antigen-Induced Arthritis S (PS) complex to necrotic cells, C4BP-PS inhibits complement activation on the surface of these cells. In addition we found C4BP to bind DNA via Leilani L Santos, Monash Institute of Medical Research, Monash Univer- its alpha-chains. By using deletion mutants and point mutants of C4BP we sity, Australia have mapped the binding site for DNA to a patch of positively charged Pam H Hall, Monash Institute of Medical Research, Monash University, amino acids mainly on the second complement control domain (CCP) of Australia the C4BP alpha-chain (KD 190 nM). By binding to DNA, C4BP limits April Dacumos, Monash Institute of Medical Research, Monash Univer- DNA release from necrotic cells and inhibits complement activation by sity, Australia DNA in solution. C4BP-necrotic cell interactions also occur in vivo as ne- Eric F Morand, Monash Institute of Medical Research, Monash Univer- crotic areas of arteriosclerotic plaques and of various cancers stain strongly sity, Australia positive for C4BP. Using a co-culture system in which macrophages were incubated with necrotic cells in the presence of serum we found that C4BP PURPOSE: MIF is a pro-infl ammatory cytokine implicated in rheumatoid limits the release of TNF-alpha from macrophages. arthritis (RA). The role of MIF in T cell activation remains undefi ned. In conclusion: C4BP limits the infl ammatory potential of necrotic cells by Antigen-induced arthritis (AIA) is a T cell mediated model of RA requir- inhibiting complement activation and limiting DNA release. ing MIF for its full expression. We investigated the role of MIF in T cell activation in AIA. METHODS: AIA was induced by intra-articular injection of methylated 7007 bovine serum albumin (mBSA) in pre-immunized mice. Synovitis, joint space exudate, cartilage degradation and bone damage were assessed by Effects of p38 MAP kinase inhibition are highly species dependant histological scoring of Safranin-O-stained knee joints. T cell activation was assessed by examination of footpad delayed type hypersensitivity Tine Skak-Nielsen, LEO-Pharma, Dept. of Biochemistry & Cell Biology, (DTH), antigen and mitogen (PHA)-induced splenocyte proliferation, and Denmark antigen-induced cytokine production, activation marker expression, and Lene Jensen, LEO-Pharma, Dept of Pharmacology, Denmark MAP kinase and AKT activation. Marianne S Fjording, LEO-Pharma, Dept. of Biochemistry & Cell RESULTS: MIF -/- mice had signifi cantly reduced arthritis as evidenced Biology, Denmark by lower scores for synovitis (p<0.001), joint space exudate (p<0.001), and Mogens W Madsen, LEO-Pharma, Dept. of Biochemistry & Cell Biology, cartilage degradation (p<0.03). Bone damage was similar in both groups. Denmark Footpad DTH reactions were signifi cantly reduced in MIF -/- mice com- pared to WT (p<0.001). MIF -/- splenocytes exhibited signifi cantly lower Confl icting data regarding anti-infl ammatory activity of p38 MAP kinase basal (p<0.001), antigen-specifi c (p<0.00001) and mitogen-stimulated inhibitors in mouse models of infl ammation led us to investigate the ef- (p<0.05) proliferation compared to WT. In addition, MIF -/- splenocytes fect of p38 MAP kinase inhibitors in peripheral blood mononuclear cell had signifi cantly lower basal IL-10 (p<0.05) and IFN-gamma (p<0.05) (PBMC) preparations from different animal species. production than WT cells but comparable antigen-induced IL-10 and IFN- Using PBMC isolated from mouse, rat, pig, monkey and human blood, gamma. MIF -/- cells exhibited diminished CD54 expression and ERK the effect of different p38 MAP kinase inhibitors and of dexamethasone phosphorylation. AKT activation was similar in MIF -/- and WT cells. as control was investigated on lipopolysaccharide induced cytokine secre- CONCLUSION: These data confi rm a pivotal role for MIF in arthritis. tion. MIF defi ciency is associated with impairment of DTH, antigen-induced The inhibition of TNF-a secretion by the p38 MAP kinase inhibitor T cell proliferation, ERK MAP kinase phosphorylation and CD54 expres- EO1428 occurred at much lower (x100) concentrations in human PBMC sion, despite intact IFN-gamma production and AKT activation. These than in mouse, rat, porcine and monkey PBMC. The same species differ- fi ndings suggest an intrinsic abnormality in cellular activation in the ab- ence was seen with three other p38 MAP kinase inhibitors (SB203580, sence of MIF. VX745 and a pyrazolyl urea compound from Bayer) all with different chemical background. In some species, the secretion of IL-1b was not in- hibited, but rather increased by p38 MAP kinase inhibitors. 7006 In conclusion, the potency of p38 MAP kinase inhibitors was much higher in human PBMC than in other animal species. Prediction of ef- C4b-binding protein binds to necrotic cells and DNA, limiting DNA fi cacy and toxicity of p38 MAP kinase inhibitors in humans is hampered release and complement activation by the species variation demonstrated in this study.

Leendert A Trouw, Lund University, Dept. of Laboratory Medicin, Clini- cal Chemistry, Sweden 7008 Sara C Nilsson, Lund University, Dept. of Laboratory Medicin, Clinical Chemistry, Sweden Signaling Mechanism of TGF-b in Regulation of Renal Infl ammation Isabel Goncalves, Lund University, Dept. of Medical Sciences, Sweden Göran Landberg, Lund University, Dept. of Laboratory Medicin, Patho- Hui Y Lan, Baylor College of Medicine, United States logy, Sweden Wansheng Wang, Baylor College of Medicine, United States Anna M Blom, Lund University, Dept. of Laboratory Medicin, Clinical Xiao R Huang, Baylor College of Medicine, United States Chemistry, Sweden Allen G Li, Oregon Health & Science University, United States Fang Liu, Baylor College of Medicine, United States Following cell death, via apoptosis or necrosis, cells should be taken up Luan D Truong, Baylor College of Medicine, United States to prevent release of intra-cellular content. Several molecules, including Xiao J Wang, Oregon Health & Science University, United States initiation molecules of the complement system, are involved in marking dead cells for uptake. As a consequence of binding of these molecules, Signaling Mechanism of TGF-b in Regulation of Renal Infl ammation. complement activation takes place, which when uncontrolled would lead to infl ammation. The combination of a pro-infl ammatory environment and Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 175

Lan HY, Wang W, Huang XR, Li AG, Liu F, Truong LD, Wang XJ. Dept. of Medicine, Baylor College of Medicine, Houston, TX, and Dept. of Der- 7010 matology, Oregon Health & Science University, Portland, OR, USA. TGF-b plays a critical role in regulation of immune and infl ammatory re- Possibility of cross talk between nitric oxide and hydrogen sulphide sponse, however, its signaling mechanisms remains largely unclear. This synthesis in a mouse model of endotoxic shock study reported that mice that overexpress latent TGF-b1 on skin were pro- tected against renal infl ammation in a model of obstructive kidney disease Ling Li, National University of Singapore, Singapore and investigated the signaling mechanism of TGF-b1 in inhibition of renal Philip K Moore, National University of Singapore, Singapore infl ammation in vivo and in vitro. While wild-type mice developed severe renal infl ammation, including massive T cell and macrophage infi ltration Both nitric oxide (NO) and hydrogen sulphide (H2S) are naturally occur- and marked upregulation of IL-1b, TNF-a, and ICAM-1 (all P<0.001) after ring vasodilator gases. Lipopolysaccharide (LPS) administration upregu- renal obstruction at day 7, renal infl ammation was prevented in transgenic lates inducible nitric oxide synthase (iNOS) expression causing increased mice with an increase in latent TGF-b1 in circulation (a 10-fold increase) production of NO. We have recently shown that the biosynthesis of H2S and renal tissues (a 2.5-fold increase). Inhibition of renal infl ammation in is increased in tissues from LPS-injected (i.e. endotoxin-shocked) mice transgenic mice was also associated with a marked upregulation of renal probably due to upregulation of the H2S synthesizing enzyme, cystathio- Smad7 and IkBa (an inhibitor of NF.kB) and a suppression of NF.kB ac- nine-g-lyase (CSE) expression. We have now assessed the effect of DL- tivation in the diseased kidney (all p< 0.01), suggesting the importance of propargylglycine (PAG, CSE inhibitor) on plasma H2S and NO (measured TGF-b-NF.kB crosstalk signaling pathway in regulating renal infl amma- as nitrate/nitrite, NOx) concentration as well as H2S biosynthesis (from tion. This was tested in vivo and in vitro by overexpressing Smad7. Gene added L-cysteine, ex vivo) from LPS-injected mice. Administration of transfer of Smad7 into the kidney was able to substantially inhibit NF.kB E. coli LPS (10-20 mg/kg, i.p.) resulted in a time- and dose-dependent activation and renal infl ammation in a rat obstructive kidney disease. In increase in both plasma H2S and NOx concentrations. Pre-treatment of vitro overexpression of Smad7 in kidney tubular cell line upregulated animals with PAG (50 mg/kg, i.p. 1 h before LPS) signifi cantly (P<0.05) IkBa directly in a time- and dose-dependent manner, thereby inhibiting reduced plasma H2S (and liver H2S formation from added L-cysteine) NF.kB activation and NF.kB-driven infl ammatory response. Thus, latent and NOx concentration. PAG did not alter plasma NOx levels in control TGF-b may have protective role in renal infl ammation. Smad7-mediated (saline-injected) animals. Inclusion of L-N(G) nitroarginine methyl ester inhibition of NF.kB activation via the induction of IkBa may be the central (L-NAME, 1 mM) in the incubate did not affect the in vitro conversion of mechanism by which TGF-b prevents renal infl ammation. L-cysteine to H2S in either liver or kidney homogenates from control (i.e. saline-injected) animals but signifi cantly (P<0.05) augmented H2S forma- tion in homogenates prepared from LPS-treated animals. These results 7009 show that LPS injection increases the formation of both NO and H2S. The fi nding that PAG pre-treatment reduces plasma NOx levels in LPS-in- H2S is a novel mediator of haemorrhagic shock in the rat jected animals and that L-NAME promotes H2S formation in tissues from LPS-injected animals suggests the possibility of cross-talk in this model Pamela YY Mok, National University of Singapore, Singapore of endotoxic shock. Such cross talk may have an important bearing on the Philip K Moore, National University of Singapore, Singapore potential therapeutic application of drugs which inhibit the formation of NO/H2S in this (and perhaps also other infl ammatory) conditions. Infl ammation as a result of haemorrhagic shock contributes to organ injury and dysfunction. We have previously shown that H2S production is in- creased in liver and kidney following haemorrhagic shock in the anaesthe- 7011 tized rat. We suggest therefore that H2S plays a role haemorrhagic shock. In the present experiments, Sprague-Dawley rats (male, 200-250g) were Nitrofl urbiprofen reduces formation of pro-infl ammatory hydrogen subjected to haemorrhagic shock (mean arterial blood pressure reduced sulphide in lipopopolysaccharide-treated rat to 35-40 mm Hg) for 3 h. Animals were divided into 3 treatment groups. Control animals received saline (1 ml/kg, i.v.) 1.5 h after blood loss. Other Farhana Anuar, National University of Singapore, Singapore animals received PAG (50 mg/kg, i.v., irreversible inhibitor of cysthathio- Matthew Whiteman, National University of Singapore, Singapore nine-g-lyase) 0.5 h before (prophylactic) or 1.5 h after (therapeutic) onset Jia Ling Siau, National University of Singapore, Singapore of shock. Blood samples (1 ml) were taken before and 3 h after shock. Siew Hwa Chu, National University of Singapore, Singapore After 3 h, animals were killed and organs harvested for biochemical analy- Philip K Moore, National University of Singapore, Singapore sis. In a separate survival experiment, animals were divided into 2 treat- ment groups i.e. saline-injected and PAG (50 mg/kg)-injected 1 h after Hydrogen sulphide (H2S) is a naturally occurring vasodilator gas. The bio- onset of shock) and subjected to 3h haemorrhagic shock total. Survival synthesis of H2S is increased in tissues from lipopolysaccharide (LPS)-in- was monitored for up to 48h. Haemorrhagic shock caused an increase in jected (endotoxin-shocked) rats most likely due to upregulation of the H2S plasma infl ammatory cytokine (TNF-a and IL-1b) concentrations which synthesizing enzymes, cystathionine-g- lyase (CSE) and cystathionine-b- was reduced by PAG treatment. Plasma alanine aminotransferase (ALT) synthetase (CBS). Nitrofl urbiprofen (NOF) is a nitric oxide (NO) releasing activity was also elevated (P<0.05) in haemorrhagic shock, suggestive of derivative of fl urbiprofen with enhanced anti-infl ammatory activity and liver injury. The increase in plasma ALT activity was inhibited by prophy- reduced gastrointestinal toxicity. The mechanism of the augmented anti- lactic administration of PAG. H2S production by liver homogenates was infl ammatory effect of NOF is, as yet, not clear. In this report, we describe inhibited (P<0.05) by PAG administered either prophylactically or thera- the effect of NOF (and fl urbiprofen) on H2S metabolism in LPS-injected peutically. PAG administration after onset of shock signifi cantly (P<0.05) rats. Administration of LPS (10 mg/kg, i.p.) resulted (6 h) in an increase increased survival time. Thus, increased H2S production in liver is associ- in plasma H2S and NO (measured as nitrate/nitrite) concentrations. LPS ated with liver damage in haemorrhagic shock. Inhibition of H2S biosyn- treatment also elevated the biosynthesis of H2S from added cysteine (in thesis reduces the extent of infl ammation and thereby promote survival the presence of pyridoxal phosphate as cofactor) and upregulated CSE and after haemorrhagic shock. CBS mRNA in liver and kidney homogenates measured ex vivo. Inducible nitric oxide synthase (iNOS) was also signifi cantly upregulated in these tissues. Flurbiprofen (21 mg/kg, i.p.) pre-treatment did not affect plasma H2S concentration, liver or kidney H2S biosynthesis from cysteine or CSE/CBS mRNA expression in LPS-injected animals. In contrast, nitro- fl urbiprofen (3-30 mg/kg, i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated rise in formation of H2S from cysteine (liver and kidney) coupled with reduced CSE and CBS mRNA expres- S 176 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) sion in these tissues. Nitrofl urbiprofen (but not fl urbiprofen) also dose-de- was revealed by Western blotting (activated caspases are cleaved in well pendently reduced the LPS-mediated increase in plasma TNF-a and IL-1b defi ned fragments). Results: mRNA and protein levels of Caspases 1 and 5 concentration. These results suggest that nitrofl urbiprofen downregulates are elevated more than a 3 fold in UC colonic epithelial cells (p<0.02). Un- the biosynthesis of pro-infl ammatory H2S and suggests that such an ef- stimulated epithelial cell lines have low expression levels and no activa- fect may contribute to the augmented anti-infl ammatory activity of this tion of caspase 1. Cytokine stimulation rapidly activates caspase 1 within compound. one hour, and it remains activated for 24 hours. Cytokine stimulation also increases caspase 1 mRNA and protein expression after 24 hours. The ac- tive caspase 1 is secreted to the media after 24 hours. Conclusion: For 7012 the fi rst time it is reported that members of the infl ammasome cytokine processor are elevated in colonic epithelial cells from UC. From cell lines The role of mitochondria in wound healing. An in vivo and in vitro studies we propose a mechanism of activation of this pathway: The kinetic study on the mechanisms of action of the anti-scarring effects of studies suggest that the caspases are rapidly activated directly and later epicatechin gallate induced by transcription. Since the infl ammasome could be a target for pharmacological modifi cation, inhibition of the infl ammasome might be Mohit B Kapoor, Department of Pharmacology and Toxicology, Universi- benefi cial in controlling UC. ty of Otago, New Zealand Ivan A Sammut, Department of Pharmacology and Toxicology, University of Otago, New Zealand 7014 Andrew N Clarkson, Department of Pharmacology and Toxicology, Uni- versity of Otago, New Zealand Smad3 signaling plays an important role in keloid pathogenesis via Irene Hall, Department of Pharmacology and Toxicology, University of epithelial-mesenchymal interactions Otago, New Zealand Ian Appleton, Department of Pharmacology and Toxicology, University Thang T Phan, Department of Surgery, National University of Singapore, of Otago, New Zealand Singapore Ivor J Lim, Department of Surgery, National University of Singapore, During healing the removal of infl ammatory cells and production of the Singapore avascular scar is via apoptosis. Mitochondria are pivotal in mediating this Oliver Aalami, Department of Surgery, Stanford University, United States process. However, the role of mitochondria in wound healing have not Florence Lorget, Department of Growth, Development and Anatomy, been established. We have recently shown that epicatechin gallate (ECG), University of California, San Francisco, United States signifi cantly improves wound healing and scar formation. The aims of this Audrey Khoo, Department of Surgery, National University of Singapore, study were therefore three fold: 1) to establish the role of mitochondrial Singapore enzyme activity during repair; 2) to determine the effects of ECG on mi- Kim E Tan, Department of Orthopedic Surgery, National University of tochondrial enzymes during healing and 3) to establish the mechanisms of Singapore, Singapore action of ECG both in vivo and in vitro (using LPS stimulated J774 ma- Anan Mukhopadhaya, Department of Pharmacy, National University of rophages). In this study, a signifi cant decrease in all mitochondrial enzyme Singapore, Singapore activities, except complex-II-III, were observed at days 1 and 3 during the Micheal T Longaker, Department of Surgery, Stanford University, United reparative process. These in vivo effects were mirrored in vitro using LPS States stimulated macrophages. ECG treatment therefore restored the complex activities to near normal levels both in vivo and in the in vitro studies. Smad signaling plays important roles in developmental and cancer biology The generation of free radicals early in repair and after LPS stimulation in as well as in fi bropathogenesis. Its role in keloid biology is not known. macrophages may cause mitochondrial damage, with resultant decrease in Epithelial-mesenchymal interactions, originally described in normal skin, complex enzyme activity. Thus the benefi cial effects of ECG during heal- have recently been established to play a signifi cant role in keloid patho- ing may be due to preservation of mitochondrial energetics. genesis, and demonstrate the important infl uence of keratinocyte paracrine factor signaling on fi broblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. 7013 Normal fi broblasts (NF) and keloid fi broblasts (KF) were co-cultured in serum-free medium with normal keratinocytes (NK) or keloid keratino- Proinfl ammatory caspases are activated by cytokine stimulation of cytes (KK) for 5 days, after which fi broblast cell lysates were subjected to colonic epithelial cells in ulcerative colitis Western blot and immuno-precipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild Jakob B Seidelin, Herlev Hospital, University of Copenhagen, Denmark type (wt), Smad2-null (Smad2-/-) and Smad3-null (Smad3-/-) mouse em- Laura Ferrero, Herlev Hospital, University of Copenhagen, Denmark bryonic fi broblasts (MEF) were assayed for cell proliferation and collagen Camilla L Christensen, Herlev Hospital, University of Copenhagen, production post serum-free coculture with KK or exposure to conditioned Denmark media collected from serum-free KK/KF coculture. Ole Haagen Nielsen, Herlev Hospital, University of Copenhagen, Den- Compared to normal skin, keloids expressed high basal levels of TGF-βR1 mark and R2, Smad2, 3 and 4 and phopho-Smad2. Upregulation of TGF-βR1 and R2, Smad3 and p-Smad2 was observed in KF cocultured with KK, Purpose: Recent research has shown that cytokine processing and acti- together with enhanced Smad3 phosphorylation and Smad2/3/4 binding vation of interleukin-1β (IL-1β) and IL-18 are controlled by a protein complex production. When MEF-wt, MEF-Smad2-/- or MEF-Smad3-/- multimer designated the infl ammasome. The infl ammasome activates the were cocultured with KK or exposed to KK/KF coculture conditioned me- infl ammatory caspases 1 and 5 which cleaves and activates the cytokines. dia, enhanced proliferation and collagen production was seen in MEF-wt Ulcerative colitis (UC) is a chronic infl ammatory disease of the colon, and and -Smad2-/-, but not in MEF-Smad3-/- cells. there is growing evidence that epithelial cells might play an active role in The activation of Smad signaling, importantly that of Smad3, appears to the infl ammatory process. The aim was to reveal whether the infl amma- be one facet of the complex epithelial-mesenchymal interactions in ke- some is induced in colonic epithelial cells in vitro and in vivo. Methods: loid pathogenesis, resulting in active KF proliferation and collagen-ECM Colonic epithelial cells from patients with UC and control subjects were production in coculture with KK. This fi nding suggests the suppression of isolated. Colonic cell lines (HT29, Caco-2, and DLD-1) were stimulated Smad signaling as a novel approach in keloid therapy. with 1pM tumor necrosis factor-alpha and 1pM interferon-gamma for 1 - 24 hours. Expression of caspases 1 and 5 was analysed by PCR and Western blotting, and secretion by ELISA-technique. Caspase activation Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 177

in their levels in both tissues. These fi ndings suggest SP mediates the lo- 7015 cal and systemic infl ammatory responses in AP (partially) by inducing the release of chemokines. The role of Stem Cell Factor and Fibroblast Growth Factor-2 in keloid pathogenesis 7017 Thang T Phan, Department of Surgery, National University of Singapore, Singapore TNF-alpha stimulated neutrophils release membrane-bound BAFF Ivor J Lim, Department of Surgery, National University of Singapore, Singapore Lakhvir K Assi, University of Birmingham, United Kingdom Anan Mukhopadhyay, Department of Pharmacy, National University of Caroline Gordon, University of Birmingham, United Kingdom Singapore, Singapore Mike Salmon, University of Birmingham, United Kingdom Man Yi Wong, Department of Pharmacy, National University of Singapo- Janet M Lord, University of Birmingham, United Kingdom re, Singapore Dagmar Scheel-Toellner, University of Birmingham, United Kingdom Sui Yung Chan, Department of Pharmacy, National University of Singa- pore, Singapore B cell activating (BAFF) is a novel member of the tumour necrosis Audrey Khoo, Department of Surgery, National University of Singapore, factor(TNF) superfamily of ligands. It is essential for B cell survival and Singapore maturation, and is implicated in the pathogenesis of autoimmune infl am- matory diseases such as systemic lupus erythematosus (SLE) and rheuma- Keloid scars manifest from alterations in the normal wound healing pro- toid arthritis. Mice transgenic for BAFF develop an autoimmune disease cess. They cause considerable aesthetic and functional problems. How- similar to SLE and circulating levels of BAFF protein are elevated in a ever, due to a lack of understanding on the pathogenesis of keloid scars, subset of SLE patients. BAFF is produced by cells of the myeloid origin treatment options for them remain limited and unsatisfactory. This project including monocytes, dendritic cells and macrophages, however recent re- determines the expression profi les of Stem Cell Factor (SCF) & Fibroblast ports have shown that fi broblasts and neutrophils can also produce BAFF. Growth Factor 2 (FGF-2) in keloid tissue and keloid derived cell cultures. Neutrophils constitute the fi rst line of defence against infectious agents This generates preliminary data to gauge if SCF and FGF-2 play a role in and are the most abundant leukocyte in the blood. Therefore BAFF pro- keloid pathogenesis. Three models are used in this project. The normal duced by these cells may enhance the survival of B cells in SLE patients. skin and keloid tissue extract was used to determine the overall difference Using a BAFF specifi c ELISA, we were able to identify some SLE pa- in the expression of SCF and FGF-2 by all the components of the tissue. tients who had high levels of BAFF in their sera, but neutrophils isolated The differential expression of SCF and FGF-2 in normal and keloid fi bro- from these patients did not show increased levels of BAFF production blasts under serum stimulation or coculture with keratinocytes was further when compared to control individuals. However, using fl ow cytometric studies. The results show that both SCF and FGF-2 are elevated in keloid analysis, we were able to identify a novel mechanism of BAFF release tissue and increased in fi broblasts after serum stimulation. In addition, the from neutrophils. We report that BAFF can be released from the surface of secretion of SCF by keloid fi broblasts into conditioned media is increased neutrophils in response to the proinfl ammatory cytokine, TNF-alpha. The in co-culture. The persistently elevated levels of SCF may cause mast cell mechanism of BAFF release required the stimulation of neutrophils by hyperplasia, leading to chronic infl ammation and excessive extracellular TNF-alpha, which activates a furin-like convertase dependent mechanism matrix deposition, which are two major characteristics of keloid scars. of direct BAFF shedding from the neutrophil membrane. In certain SLE Higher FGF-2 expression is also detected in the cell lysates of the keloid patients, serum levels of TNF-alpha positively correlated with BAFF and fi broblasts and keloid keratinocytes in the co-culture. Since FGF-2 is a sera taken from these patients led to a rapid release of BAFF from control powerful mitogen, elevation of FGF-2 levels leads to increased prolifera- neutrophils. Therefore, although no difference was observed in the level tion of fi broblasts, a hallmark of keloid scars. The results suggest that SCF of BAFF production by neutrophils from SLE or control individuals, we and FGF-2 are likely to be involved in the pathogenesis of keloid scars and propose that the BAFF shedding mechanism may be accelerated in certain warrant for further studies to be done. SLE patients, due to the increased presence of TNF-alpha in their sera.

7016 7018

Effect of neurokinin 1 receptor antagonist treatment on pancreatitis- The Frequency Of TB Pericarditis In PatientsWith Pericardits- One induced increase in chemokine levels Year Study

Madhav Bhatia, National University of Singapore, Singapore Fatemeh Fallah, Shaheed Beheshti University,Pediatric InfectiousRes- Sun Jia, National University of Singapore, Singapore earch Center, Iran Gita Eslami, Shaheed Beheshti University, Iran The current study aims to test the hypothesis that substance P, acting via Hossein Goudarzi, Shaheed Beheshti University, Iran neurokinin-1 receptor (NK-1R), amplifi es the severity of acute pancreati- Shahla Zabihian, Modarres Hospital, Iran tis and associated lung injury by inducing the release of chemokines. To test the hypothesis, we examined the effect of pharmacological interven- Abstrasct: tion against NK-1 receptor using CP-96,345, selective NK-1R antago- Objective-Although during the last decades the prevalence of Tb has nist, on pancreas and lung levels of three chemokines, namely monocyte declained and its prognosis has changed dramatically owing to chemo- chemotactic protein (MCP)-1, regulated on activation, T cell expressed therapy cases of Tb pericarditis can still beobserved.Dffi culties in early and secreted (RANTES) and macrophage infl ammatory protein (MIP)-2 and accurate diagnosis probably contributed to this phenomenon and may in caerulein-induced acute pancreatitis in mice. The severity of pancre- deliy diagnosis and therapy or lead to antituberculous chemotherapy for atitis was evaluated by quantitating plasma amylase activity, pancreatic idiopathic pericarditis. myeloperoxidase (MPO) activity, and pancreatic edema and by examin- Patients&Methods- we studied patients in whom a diagnosis of Tb peri- ing morphological changes in pancreas. The severity of pancreatitis-as- carditis was made by following Adenosin Deaminase Activity and other sociated lung injury was evaluated by quantitating lung MPO activity and procedures. by examining morphological changes in lung. It was found that MCP-1, -We now report our experience in a descriptive group of 30 cases(19 RANTES, and MIP-2 levels were increased in pancreas and lung tissues in men,11women) ,aged 12-76 years admitted to the hospital for acute mice treated with caerulein. CP-96,345 administrated either half an hour pericardial disease.The diagnosis of Tb pericarditis was made by the before or one hour after the fi rst caerulein injection attenuated the increase followingstudies:Culture on Lowenstein-Jensen media,Ziehlneelsen S 178 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) stain,measurement of ADA of pericardial fl uid,biopsy of pericardial tissue Thang T Phan, National University of Singapore, Singapore and evaluation of tuberculin test. Results 13 patients were with Tb pericarditis.All of them had clinical Tb Purpose: Skin fi brotic disorders such as keloid are characterized by an ex- manifestation .The culture of pericardial fl uid in 6 patients(46%),the stain- cessive production of extracellular matrix (ECM) and understood to devel- ing in 3 cases(23%),the culture of pericardial tissue in 6 patients(46%) op under the infl uence of certain growth factors. Connective tissue growth were positive.The caseating granuloma in 46% of cases was identifi ed. factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in Ten case (77%) had positive PPD reaction.All of them with increased various fi brotic disorders and induced in fi broblasts after the activation ADA (>45u/l)were reported.Conclusion-Therefore measurement of level with transforming growth factor-β (TGF-β). of ADA may prove a good screening for Tb pericarditis. Method: To better understand the mechanism of persistent fi brosis seen in Keywords-Pericarditis-Tuberculosis-Diagnostic methods-Adenosine De- keloid, we evaluate the protein expression of CTGF from keloid tissues (in aminase Activity vivo model) and keloid derived cell cultures (in vitro model) comparing against normal skin tissues. To further defi ne the kinetics of CTGF expres- sion during the different stages of wound healing, two in vitro models were 7019 employed to investigate if CTGF plays a role in the early and late phases of wound healing. For this purpose, we utilized a serum stimulation model to The role of Activin system in Keloid Pathogenesis mimic the early phases of wound healing. Next, a serum-free two chamber co-culture model was used to study epithelial-mesenchymal interactions Anandaroop Mukhopadhyay, Singapore between keloid and normal keratinocytes with keloid and normal fi bro- Audrey Khoo, Department of Surgery, National University of Singapore, blast. Western blotting was employed primarily for this experiment. Singapore Results: We report high expression of CTGF in keloid tissues as compared David Phillips, Australia to normal skin tissues. Cells subjected to serum stimulation did not show Sabin Werner, Switzerland any signifi cant expression of CTGF. Interestingly, we found a decrease Sui Y Chan, Singapore expression of CTGF in keloid co-culture system as compared to normal Ivor J Lim, Department of Surgery, National University of Singapore, co-culture cell lysates. In conditioned medium we obtained an opposite Singapore result suggesting that in keloid co-culture, most of the CTGF (active form) Thang T Phan, Department of Surgery, National University of Singapore, are being secreted out of the cell therefore resulting in a decrease expres- Singapore sion in keloid co-culture cell lysates. Conclusion: Our data suggested that CTGF may be involved mainly in the Purpose: Keloid scars represent a pathological response to cutaneous in- late phase of wound healing and the active form of CTGF are being re- jury. Many growth factors have been found to play a crucial role in keloid leased in keloid co-culture system therefore allowing to cause a persistent pathogenesis. Activin A, a dimeric protein and a member of the TGF-β fi brosis in keloid pathogenesis. super family has been shown to regulate various aspects of cell growth and differentiation in many tissues. In vitro studies suggest a role of Activin A in the repair of mesenchyme and possibly also the epithelium, thus a 7021 disruption in the control of Activin expression could lead to tissue fi brosis. Our aim is to study the role of Activin system in keloid pathogenesis. The Important role of IL-18 system in Keloid and Scar Pathogenesis Methods: Normal and Keloid fi broblasts were treated with rhActivn A (100 ng /ml) and TGF-β1 (1 ng/ml, 10 ng/ml) and subjected to MTT Vinh D Do, National University of Singapore, Singapore Assay. The expression of extracellular matrix proteins by normal and ke- Audrey Khoo, National University of Singapore, Singapore loid fi broblasts under activin A and follistatin stimulation was assessed Mukhopadhyay Anandaroop, National University of Singapore, Singa- by Western blotting. Conditioned media, from non co-culture and co-cul- pore ture of normal and keloid fi broblasts and keratinocytes, were subjected to Alan Porter, Institute of Molecular and Cellular Biology, Singapore ELISA to quantify Activin A and Follistatin concentrations respectively. Ivor J Lim, National University of Singapore, Singapore RNase protection assay was performed to investigate the Activin A mRNA Thang T Phan, National University of Singapore, Singapore expression in Normal, Hypertrophic and Keloid tissue. Normal and keloid tissue were analyzed to investigate localization of Activin A and Follistatin Purpose: Keloids are benign skin “tumors” that are characterized by over- by immunostaining. proliferation of fi broblasts and over-production of extracellular matrix Results: rhActivin A increased the proliferation of normal and keloid fi bro- components. To date, the role of growth factors and various cytokines in blasts signifi cantly. Activin A up-regulated collagen, fi bronectin, α-SMA the pathogenesis of these lesions has not been fully characterized. Inter- and PCNA expression in normal and keloid fi broblasts. ELISA demon- leukin-18 (IL-18) is an important proinfl ammatory cytokine that has been strated a higher concentration of Activin A and Follistatin in conditioned found in a number of fi brotic autoimmune diseases and multiorgan sys- media from keloid fi broblasts. A similar pattern was observed for secreted temic diseases. The aim of the present study was to assess the expression Follistatin levels in keratinocytes. However Activin levels were higher in of IL-18 protein in normal control and keloid derived cell lines subjected normal keratinocyte conditioned media as compared to keloid keratino- to growth factor stimulation, serum stimulation and those which have been cytes. In addition, an increased mRNA expression was observed in Ke- co-culture. loid and Hypertrophic scar tissues. Immunostaining of keloid and normal Methods: A serum-free two chamber co-culture model was used whereby tissues showed an increased localization of Activin A in the supra basal keloid and normal keratinocyte were co-cultured with keloid and normal epithelium of keloid tissue as compared to normal tissue. fi broblast in serum-free media with non co-cultured samples as controls. Conclusion: The results underscore the importance of Activin system in Additionally, different strains of normal and keloid fi broblasts treated with keloid biology and suggest a key role of Activin A in keloid pathogenesis. 10% serum, activin, TGF-β1 and follistatin to study effect of serum and the growth factors on IL-18 expression. Results: Increased levels of IL-18 were detected in keloid tissues as com- 7020 pared to normal skin controls. A similar pattern of expression was observed in Keloid derived cells as compared to normal cells in both non co-culture The role of Connective Tissue Growth Factor (CTGF) in the biology and co-culture systems. Interestingly, we found a biased expression of of epithelial-mesenchymal interactions of keloid pathogenesis IL-18 in keratinocytes as compared to fi broblasts. These results were af- fi rmed by immunohistochemical staining which showed higher expression Anandaroop Mukhopadhyay, Singapore of IL-18 in epidermis of keloid tissues compared to that of normal skin Audrey Khoo, National University of Singapore, Singapore tissues. Cells subjected to serum stimulation and growth factor stimula- Ivor J Lim, National University of Singapore, Singapore Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 179 tion (Activin, follistatin, TGF-β) did not show any signifi cant expression subjects. Modulation by pro-infl ammatory mediators indicate that NURR1 of IL-18. induction represents a convergence point of distinct signalling pathways. Conclusion Aim: To determine if NURR1 is a molecular target of methotrexate (MTX) These expression profi les of IL-18 indicate its signifi cant role in patho- action in human infl ammatory joint disease & to elucidate mechanisms genesis of keloids. involved. Methods: Immunocytochemistry determined effects of MTX treatment on NURR1 expression in vivo using synovial tissue obtained before & during 7022 treatment from patients (n=10) with active disease who were beginning MTX therapy. Northern blot analysis measured dose effect of MTX on en- Inhibition of Classical Protein Kinase C Isoenzymes Down-Regulates dogenous, pro-infl ammatory mediator- & growth factor-induced NURR1 LPS-Induced Expression of Inducible Nitric Oxide Synthase in mRNA levels in primary human synoviocytes & microvascular endothelial Macrophages cells. Western blot analysis of nuclear extracts determined NURR1 protein levels in response to MTX. The consequence of adenosine receptor an- Tiina Salonen, The Immunopharmacological Research Group, University tagonists on MTX action was examined by northern blot analysis. of Tampere Medical School and Tampere University Hospital, Finland Results: MTX signifi cantly suppresses expression of NURR1 in vivo in Hannu Kankaanranta, The Immunopharmacological Research Group, patients with active PsA (n=10, p=0.005) who were prescribed low-dose University of Tampere Medical School and Tampere University Hospital, MTX. Reduction in NURR1 levels correlated with changes in clinical & Finland laboratory parameters. We demonstrate that MTX selectively modulates Raimo Tuominen, The Division of Pharmacology and Toxicology, Univer- NURR1 levels induced by infl ammatory stimuli & growth factors in resi- sity of Helsinki Faculty of Pharmacy, Finland dent cells of synovial tissue. In primary human synoviocytes & microvas- Eeva Moilanen, The Immunopharmacological Research Group, Universi- cular endothelial cells we observe dose-dependent differential effects of ty of Tampere Medical School and Tampere University Hospital, Finland MTX on endogenous & inducible NURR1 levels. At pharmacologically relevant low doses, MTX potently inhibits induced NURR1 mRNA & pro- Proinfl ammatory cytokines and bacterial products trigger inducible nitric tein levels. We confi rm that adenosine can mimic the differential effects oxide synthase (iNOS) expression and nitric oxide (NO) production in of MTX on NURR1 expression & inhibitory effects of low-dose MTX on infl ammatory and tissue cells. In infl ammation, NO mediates proinfl am- NURR1 activation are mediated via adenosine receptor A2a. matory and destructive effects. Protein kinase C (PKC) is a family of 12 Conclusion: In human infl ammatory joint disease MTX targets NURR1 serine-threonine protein kinase isoenzymes involved in signal transduc- via adenosine receptor mediated responses. tion pathways related to infl ammatory responses. In the present study we investigated the role of three classical PKC isoenzymes (alpha, betaI and betaII) in the regulation of LPS-induced iNOS protein expression and NO 7024 production in murine J774 macrophages. The activation of classical PKC isoenzymes (alpha, betaI and betaII) was Successful CD40 Knock Down using Smarticles® for Topical studied by isoenzyme translocation from the cytosolic fraction to the Delivery - The Case for an Antisense Based IBD drug membrane fraction by Western blot. All three isoenzymes were activated after 10 min incubation with 100 nM phorbol myristate acetate (PMA). Stefan Fankhaenel, novosom AG, Halle, Germany However, incubation with 1 μM PMA for 6 h down-regulated all three Andreas Wagner, Department of Cardiovascular Physiology, University isoenzymes, which was seen as a total loss of the isoenzyme in both cell of Göttingen, Germany fractions. Una Rauchhau, novosom AG, Halle, Germany LPS induced iNOS protein expression and NO production in J774 cells. Markus Hecker, Department of Cardiovascular Physiology, University of Activation of PKC by 100 nM PMA enhanced LPS-induced iNOS protein Göttingen, Germany expression and NO production as measured by Western blot and Griess Steffen Panzner, novosom AG, Halle, Germany method, respectively. PKC inhibitors RO318220 (non-selective PKC in- hibitor) and GÖ6976 (selective inhibitor of classical isoenzymes) inhib- Background ited LPS-induced iNOS protein expression and NO production in a dose CD40/CD154 interactions play a pivotal role in the development of chron- dependent manner as did 6 h preincubation with 1 μM PMA which down- ic infl ammatory bowel diseases (IBD). Disruption of CD40 signalling has regulates PKC expression. PKC inhibitors caused a marked reduction of the potential to be therapeutically useful. iNOS mRNA expression, but they had no effect on iNOS mRNA half-life We are able to show for the fi rst time the using of a validated CD40 ODN, as measured by RT-PCR or NF-kB activation as measured by EMSA. formulated in Smarticles® liposomes nov-058 as a therapeutic moiety in These results suggest that classical PKC isoenzymes alpha, betaI and/or an infl ammatory model for IBD. betaII are involved in the regulation of iNOS protein expression and NO Methods production in activated macrophages by an NF-kB-independent manner. Charge reversible liposomes (nov-058) were made by fi lm extrusion meth- od. The TNBS induced colitis was induced by a single intra-colonic applica- 7023 tion of TNBS in 35% EtOH in rats. The mixture was given with a catheter into the descending colon. The nov-058 formulated CD40 ASO and also Modulation of orphan receptor NURR1 expression by methotrexate formulated scrambled ASO were administered by intra-colonic injection in human infl ammatory joint disease involves adenosine receptor 4 hours prior to and/or 3 days post colitis induction. The used dose was mediated responses 13,5μg ASO/kg bodyweight. After 7 days colonic infl ammation and dam- age was assessed macroscopically. Jennifer A Ralph, University College Dublin, Ireland Results Alice N McEvoy, University College Dublin, Ireland With a single injection of nov-058 formulated CD40 ASO in a very low David Kane, St. Vincents University Hospital, Ireland amount of 13,5 μg/kg bodyweight either 4 hours prior to or 3 days post Barry Bresnihan, St. Vincents University Hospital, Ireland colitis induction, most severe signs of damage to the colon were signifi - Oliver FitzGerald, St. Vincents University Hospital, Ireland cantly reduced. Repeat treatment with CD40 ASO at both time points did Evelyn P Murphy, University College Dublin, Ireland not improve the macroscopic score while nov-058 formulated scrambled control ASO, irrespective of the time of application, failed to exert a thera- Introduction: Markedly enhanced NURR1 expression is present in synovi- peutic effect. um of patients with rheumatoid & psoriatic arthritis compared with normal Conclusion S 180 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

The in vivo functionality of a specifi c antisense against rat CD40 was rat paws. Mechanical hyperalgesia (in grams) was measured by Randall- checked by using the Smarticles® liposomes as drug delivery system. We Selitto method, from 0 to 6 h of control (vehicle) and carrageenan-treated here for the fi rst time show that the local application of nov-058 formulat- animals. Maximal hyperalgesia (C= -10 ± 3.2; CG= -66 ± 4.0) occurred at ed CD40 ASO is highly effi cacious in suppressing TNBS-induced colitis, 3h-post injections. Electrodes of 2 cm² applied to the hindpaws at the site whereas scrambled ASO shows no effect. As a perspective of these results of infl ammation allowed to delivery for 20 min both LF and HF TENS Smarticles® liposomes are a general tool for a delivery of ASO and there- in CG and control-injected animals. Both LF and HF inhibited by 100% fore the usage of Smarticles® will be from now on state of the art for the CG-induced hyperalgesia. Naltrexone-treated (3 mg/kg; sc) in relation to delivery of ASO for a wide range of infl ammatory diseases. control animals showed a complete reversion of LF but not HF TENS. Animals made tolerant to morphine (7 days, 20mg/kg/day; sc) also did not respond to LF-TENS, whereas they maintained the response to HF TENS. 7025 We confi rm and expand previous results from our laboratory (Resende et al. 2004) showing that endogenous opioids via mu-receptors are involved Prostaglandin E2 (PGE2) Antagonizes MEKK-1-induced in infl ammed rat paws treated with LF TENS. Collagenase-1 (MMP-1) Gene Expression by a p53-mediated Support: PRPq/UFMG, CNPq, CAPES Suppression of c/EBPß/AP-1 Transactivation of the MMP-1 promoter in Human Synovial Fibroblasts (HSF) 7027 Wissam H Faour, Ottawa Research Institute, University of Ottawa, Canada In vivo activity of potent MEK inhibitors in two models of Arturo Mancini, Department of Anatomy and Cell Biology, McGill Uni- infl ammatory disease versity, Canada QingWen He, McGill University Health Centre, Canada Patrice A Lee, Array BioPharma, United States John A Di Battista, Department of Medicine, McGill University, Canada Eli Wallace, Array BioPharma Alison M Bendele, United States Cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins have been Tracy M Pheneger, Array BioPharma, United States linked to cell survival, malignant transformation and autonomous hyper Jed Pheneger, Array BioPharma, United States proliferation. We examined the regulation of the tumour suppressor gene p53 and p53 target genes by prostaglandin E2 (PGE2) in human synovial The ERK pathway is activated in infl ammatory conditions such as rheu- fi broblasts (HSF). At 80% receptor occupancy, PGE2 induced a time de- matoid arthritis and infl ammatory bowel disease. The goals of the present pendent increase in p53 Ser 15 phosphorylation, a process mediated by studies were to evaluate modulation of the ERK pathway in vivo, through p38 MAP kinase as SB202190 (p38 inhibitor) blocked the response. Over the use selective inhibitors of the mitogen-activated kinase (MEK). Colla- expression of a constitutively activated MKK3 construct mimicked the ef- gen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) models in fects of PGE2 in terms of Ser15 phosphorylation, an effect reversed by rats were used to determine effi cacy in the subacute infl ammation setting. SB202190. PGE2 up-regulated p53RE(GADD45)-Luciferase reporter Body weight and ankle diameter were used to monitor disease progression activity in transiently transfected SN7 cells (HSF cell strain), an effect in both models. In CIA studies, rats with established disease, induced by compromised by over expression of a dominant-negative mutant of p53 (G injections of Type II collagen, were enrolled and treated with AR00241389 to A, bp 1017). Induction of the reporter was also observed when wt p53 for 6 days. In AIA model, disease was induced by an injection of a lipoi- was co-transfected under PGE2 stimulation or with a constitutively active dal amine in FCA on day 0, with AR000241389 treatment beginning on p53S15E expression construct. Addition of excess p53S15A expression day 8 and continuing for 7 days. Doses of AR00241389 evaluated in both plasmid abrogated PGE2-dependent p53 reporter activation. PGE2 inhibi- models were 1, 3, 10 or 30 mg/kg, PO, QD. An indomethacin (INDO)- tion of steady-state levels of MEKK1-induced MMP-1 mRNA was mim- induced IBD model in rats was used to further evaluate the activity of icked by over expression of p53S15E or wt p53 in the presence of PGE2. MEK inhibitors in infl ammation. Rats received INDO on days-1 and 0 Such inhibitory effects could be nullifi ed with the appropriate additions of (7.5 mg/kg, SC). A MEK inhibitor was dosed at 1, 3, 10 and 30 mg/kg, either p53 dnm or p53S15A. MEKK1-induced MMP-1 promoter (-2900 PO, QD for 4 consecutive days. Endpoints: body weight, gut weight and bp) driven Luciferase activity appeared to be dependent on a c/EBP-NF- gross score at necropsy and histopathology. AR00241389 reduced disease kB like enhancer site (-2013 to -1990 bp) transactivated, in large measure, severity in a dose-related manner in the CIA model. The ED50 with re- by c/EBPß as judged by point mutation analysis of the MMP-1 promoter spect to ankle diameter was <5 mg/kg; disease regression was seen at 10 and gel shift competition analysis. PGE2 alone, over expression of wt p53 mg/kg. AR00241389 (10 mg/kg) was superior to both positive controls, with PGE2, or the addition of p53S15E to the plasmid mix, abolished the indomethacin (1 mg/kg, PO, QD) and Enbrel® (10 mg/kg, SC, d1, 4). Im- MEKK1-induced MMP-1 promoter Luciferase activity. Our data provide provements in ankle histopathology were seen, with signifi cant inhibition the fi rst evidence that COX-2 is directly involved in the regulation of p53 at 1 mg/kg. In AIA, the ankle diameter ED50 was <3 mg/kg, with near and, by extension p53 target genes, through the pleiotropic actions of pros- 100% inhibition at 30 mg/kg. The latter group also had a superior reduc- taglandins. tion in paw diameter when compared to the positive control, methotrexate (0.075 mg/kg, PO, QD, d0-14) group. In IBD models, the MEK inhibitor signifi cantly reduced gut weight, severity score and improved histology. 7026 The latter included reduced infl ammation and necrosis of the affected tis- sue. We have shown that potent, selective inhibitors of MEK are active in Reduced analgesic effect of Transcutaneous Electrical Nerve in vivo models of infl ammation. This activity is dose-related, potent and Stimulation (TENS) in morphine-tolerant rats extensive.

Heloisa H Gonçalves, Federal University of Minas Gerais, Brazil George S Sabino, Federal University of Minas Gerais, Brazil 7028 Janetti N Francischi, Federal University of Minas Gerais, Brazil Marcos A Resende, Federal University of Minas Gerais, Brazil IL-10 inhibition of LPS-induced TNF production by human monocytes is STAT3-independent TENS is a routine non-invasive treatment used in Physiotherapy practice for pain acute and chronic relief. The aim of this study was to investigate Cecilia M Prele, Telethon Institute for Child Health Research, Australia the effects of low (LF; 10 Hz) and high frequency (HF; 130 Hz) TENS in April Keith-Magee, Telethon Institute for Child Health Research, morphine-tolerant rats. Hyperalgesia was induced by intraplantar injec- Australia tion of lambda-carrageenan (CG; 250 mg/paw) in adult male Holtzman Monika Murcha, Telethon Institute for Child Health Research, Australia Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 181

Prue H Hart, Telethon Institute for Child Health Research, Australia 7030 Studies using macrophages from STAT3 knockout mice have suggested that STAT3 is required for IL-10-mediated suppression of LPS-induced TNF Signal Transduction in Human Neutrophils: Role of Calcium- TNF production. Subsequent reports in both mouse and human models Sensing Soluble Adenylyl Cyclase propose a STAT3-independent mechanism of action of IL-10. Thus, the role of STAT3 in IL-10 mediated anti-infl ammatory actions is unresolved. Hyunsil Han, SK-Biopharmaceuticals, United States The effect of adenoviral dominant negative STAT3 (AdV-STAT3D) ex- pression on the IL-10-mediated suppression of LPS-induced TNF was in- Through chemical screening we identifi ed a pyrazolone that reversibly vestigated. Monocytes were isolated by centrifugal elutriation from whole blocked activation of phagocyte oxidase (phox) in human neutrophils in blood and infected with an adenoviral vector expressing wildtype STAT3 response to tumor necrosis factor (TNF) or formylated peptide. The pyr- (AdV-STAT3 wt), a dominant negative STAT3 (AdV-STAT3D), a control azolone spared activation of phox by phorbol ester or bacteria; bacterial vector (AdV-GFP) or were left untreated. Viral-infected monocytes were killing; TNF-induced granule exocytosis and phox assembly; and endo- treated simultaneously with 10 ng/ml IL-10 and 500 ng/ml LPS, and TNF thelial transmigration. We traced the pyrazole’s mechanism of action to levels measured in tissue culture supernatants after 2 and 24 h. The abil- inhibition of TNF-induced intracellular Ca2+ elevations, and identifi ed a ity of AdV-STAT3D to suppress STAT3 activation in COS-7 cells was non-transmembrane (“soluble”) adenylyl cyclase (sAC) in neutrophils as confi rmed using a STAT3-specifi c luciferase reporter construct. FACS a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazo- analysis demonstrated infection effi ciencies of 59 ± 2 %, 73 ± 4 % and lone’s effect on phox. Both compounds blocked TNF-induced activation of 47 ± 4 % (n=5, mean ± SEM) of monocytes expressing AdV-STAT3 wt, Rap1A, a phox-associated GTPase regulated by cAMP. Thus TNF turns on AdV-STAT3D and AdV-GFP at MOI of 100, 100 and 10, respectively. We phox through Ca2+-triggered, sAC-dependent activation of Rap1A. This observed 54 % (n=2, 2 h) and 89 ± 4 % (n=3, 24 h) suppression of TNF pathway may offer opportunities to suppress oxidative damage during in- production by monocytes following treatment with LPS and IL-10. Over- fl ammation without blocking antimicrobial function. expression of AdV-STAT3 wt and AdV-STAT3D had no effect on the level of TNF induced by LPS, or on IL-10-mediated suppression. Western blot analysis showed increased STAT3 protein expression in AdV-STAT3 wt 7031 and AdV-STAT3D-infected cells and high levels of STAT3 phosphoryla- tion following stimulation with LPS and IL-10. In summary, over-expres- The Paradox of H5N1 Avian Infl uenza Virus Infection in Primary sion of STAT3 wt or a dominant negative STAT3 does not appear to alter Blood macrophages: TNF-alpha Hyper-induction and a Delay in the suppressive effects of IL-10 on TNF production by human monocytes Apoptosis and macrophages suggesting the presence of a STAT3-independent mecha- nism. Davy CW Lee, The University of Hong Kong, Hong Kong Chris KP Mok, The University of Hong Kong, Hong Kong CY Cheung, The University of Hong Kong, Hong Kong 7029 Anna HY Law, The University of Hong Kong, Hong Kong Malik Peiris, The University of Hong Kong, Hong Kong Nitric Oxide Synthase Expression in Human Mast Cells Allan SY Lau, The University of Hong Kong, Hong Kong

Kwok Ho Yip, Department of Pharmacology, Chinese University of Hong Infl uenza A subtype H5N1 (H5N1/97) virus was the fi rst avian infl uenza Kong, Hong Kong virus documented to infect humans causing mortality of >30%. The precur- Hang Yung A Lau, Department of Pharmacology, Chinese University of sor H5N1 viruses continued to reassort leading to a widespread outbreak Hong Kong, Hong Kong with transmission human in SE Asia in 2004. We previously demonstrated Yu Huang, Department of Physiology, Chinese University of Hong Kong, that in contrast to human infl uenza A virus subtypes including H1N1 or Hong Kong H3N2, the H5N1/97 virus hyper-induces proinfl ammatory cytokines, in primary human blood macrophages, thus contributing to the unusual dis- Nitric oxide is a substantial regulatory molecule generated by different ease severity. To delineate the disease mechanisms, we analyzed the role cells during allergic and infl ammatory responses through conversion of of NF-κB and mitogen-activated protein kinases (MAPK) in the TNF-α L-arginine by nitric oxide synthase (NOS). NOS expression has been dysregulation. Primary macrophages were infected with the infl uenza demonstrated in rodent peritoneal mast cells and the human mast cell line, H5N1/97 or H1N1 viruses and harvested at different times for analysis. HMC-1. However, little is known about NOS expression in human tissue Here, we showed that the H5N1/97 or H1N1 viruses did not differ in mast cells. Human cultured mast cells (HCMC) derived from CD34+ pro- the activation of NF-κB as determined by electrophoretic mobility shift genitor cells are known to resemble the MCT mast cells in human lung. In assay. Consistent with this, the levels of degradation of IκB-α, in western the current study, NOS mRNA expression and regulation in HCMC were analysis, were reduced simultaneously in the H5N1/97 or H1N1-infected investigated. CD34+ progenitor cells were isolated from human buffy coats macrophages. However, H5N1/97, but not H1N1 virus, strongly activates and mature HCMC were obtained after incubating these progenitors with p38 MAPK and ERK1/2 by Western analysis. Kinase inhibitors of p38 IL-3, IL-6 and SCF for 12 weeks. Results from reverse transcription-poly- MAPK, but not ERK1/2, signifi cantly reduced the H5N1/97-induced merase chain reaction (RT-PCR) showed that in basal non-stimulated state, TNF-α expression in macrophages determined by real-time RT-PCR and mRNA of the three NOS isoforms were not found in the mast cells. How- ELISA. Surprisingly, a delay in the onset of apoptosis was observed in ever, increased levels of iNOS mRNA were observed when HCMC were macrophages after H5N1 infection, as compared to H1N1 virus. Promi- sensitized with human myeloma IgE or incubated overnight with a mix- nent chromatin condensation by electron microscopy and caspase activa- ture of pro-infl ammatory cytokines (TNF-α, IFN-γ and IL-1β). Expression tion by western blotting were found in the H1N1-infected cells. Taken of NOS proteins was detected by double immunofl uorescence technique together, our fi ndings showed the differential activation of p38 MAPK and in combination with confocal laser scanning microscopy and confi rmed survival in H5N1-infected macrophages, compared to H1N1 virus, may the PCR fi ndings. In conclusion, we demonstrated that although human contribute to TNF-α hyper-induction. These results provide insights into cultured mast cells had no basal expression of NOS, iNOS mRNA and the pathogenesis of H5N1 disease and rationales for therapeutic develop- protein expression were increased subsequent to exposing these cells to ment. (Supported by HK Research Grants Council) infl ammatory conditions where IgE antibodies and various pro-infl amma- tory cytokines are abundant. This suggested that human mast cells could be a possible source of nitric oxide during infl ammation and modulation of iNOS expression may be benefi cial in the treatment of allergic and infl am- matory diseases. S 182 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

that specifi cally disrupt Jun dimers of the transcription factor complex 7032 AP1. This screen identifi ed specifi c peptide inhibitors of c-jun dimerisa- tion, and these inhibitors are currently being developed as potential neuro- M-CSF, oil-in-water emulsion, and oxidized LDL induce glucose protective peptides using both in vitro and in vivo models of stroke. uptake via distinct mechanisms in macrophages The use of these natural peptides allows for specifi c knockouts of indi- vidual protein functions, rather than ablation of all the activities of a given Margaret Chang, Department of Medicine, Royal Melbourne Hospital, target protein. Using specifi c peptides targeted to individual protein inter- and Cooperative Research Centre for Chronic Infl ammatory Diseases, actions will be important to be able to fully understand the implications of University, Australia each interaction identifi ed within the infl ammatory response, and also in Caryn L Elsegood, Department of Medicine, Royal Melbourne Hospital, the development of effective and specifi c drugs aimed at disrupting critical and Cooperative Research Centre for Chronic Infl ammatory Diseases, protein interactions and reducing the problem of infl ammatory disease. University, Australia John A Hamilton, Department of Medicine, Royal Melbourne Hospital, and Cooperative Research Centre for Chronic Infl ammatory Diseases, 7034 University, Australia Regulation of intracellular prostaglandin biosynthetic pathways

Our laboratory has previously shown that M-CSF (CSF-1), GM-CSF, and by "outside-in" secreted phospholipase A2-dependent signalling in concanavalin A induce survival and/or proliferation as well as glucose fi broblast-like synoviocytes transport in bone marrow-derived macrophages (BMM) (1). We have also shown that poorly degraded particulates, such as oil-in-water emulsion Kieran F Scott, St Vincent's Hospital Clinical School, The University of (Em) and oxidized LDL (oxLDL), enhance macrophage survival (2-3). In New South Wales, Australia this study, we have investigated the effect of M-CSF-1, Em and oxLDL Pei-Wen Lei, St Vincent's Hospital Clinical School, The University of New on glucose transport in BMM. M-CSF induced an acute increase in glu- South Wales, Australia cose transport, as measured by the uptake of 3H-2-deoxyglucose, with Lawrence K Lee, School of Medical Sciences, The University of New maximal rate at ~20-30 min post stimulation. Treatment with Em induced South Wales, Australia similar upregulation of glucose transport, but the maximal rate was at- Matthew J Bidgood, St Vincent's Hospital Clinical School, The University tained at ~60 min. In contrast, oxLDL-induced glucose uptake peaked at of New South Wales, Australia ~30 min, although the absolute rate of uptake was lower compared to that Megan Taberner, St Vincent's Hospital Clinical School, The University of induced by M-CSF. We also examined the signalling cascades that regulate New South Wales, Australia the glucose transport using a panel of pharmacological inhibitors. M-CSF- Caroline Salom, St Vincent's Hospital Clinical School, The University of mediated glucose uptake was found to be PI3-kinase and PLC-gamma- New South Wales, Australia dependent. Oxidized LDL-mediated glucose uptake was found to be PI-3 Brett G Courtenay, Australia kinse- and PKC-dependent. In contrast, Em-stimulated glucose uptake was Katherine J Bryant, Australian Proteome Analysis Facility (UNSW node), not dependent upon PI-3 kinase but rather was PLC-gamma- and partially Australia p38MAPK-dependent. In conclusion, M-CSF, Em, and ox-LDL can in- W Bret Church, The University of New South Wales, Australia duce glucose transport in macrophages with distinct temporal patterns and Garry G Graham, The University of New South Wales, Australia via different signalling pathways. H P McNeill, The University of New South Wales, Australia References

1. Hamilton JA et al (1988) J. Cell Physiol. 134:405-12 Secreted phospholipase A2 (sPLA2) enzymes regulate cytokine-mediated 2. Hamilton JA et al (2000) J. Leukoc. Biol. 67:226-32 infl ammatory pathways in vitro and in vivo by undefi ned mechanisms.

3. Hamilton JA et al (1999) Arterioscler. Thromb. Vasc. Biol. 19:98-105 Exogenous addition of one secreted phospholipase A2, (sPLA2-IIA) to cultured fi broblast-like synoviocytes (FLSs) from rheumatoid arthritis pa- tients, at concentrations found in synovial fl uid, enhances tumour necrosis

7033 factor (TNF)-stimulated prostaglandin E2 (PGE2) production via upregula- tion of NF-kB-dependent cyclooxygenase (COX-2) protein levels (Bid-

Using Nature's Repertoire of Protein Scaffolds to block good, M.J. et al., J. Immunol. 165: 2790-2797, 2000). In contrast, sPLA2-

Infl ammation- A novel path to specifi c therapeutics? IIA alone, upregulates COX-2 protein, without increasing PGE2.

Here we show, with an "activity-dead" sPLA2-IIA mutant, H48Q, that

Mark W Fear, Telethon Institute for Child Health Research, Australia upregulation of TNF-dependent PGE2 in FLSs occurs independently of

Darcelle N Dixon, Telethon Institute for Chidl Health Research, Australia sPLA2-IIA enzyme function. PGE2, but not COX-2, upregulation is depen-

Nadia MD Milech, Telethon Institute for Child Health Research, dent on cPLA2-a activity because selective pyrrolidine cPLA2-a inhibitors

Australia abrogate PGE2 production without affecting COX-2. Unlike TNF, sPLA2- Vanessa S Cull, Telethon Institute for Child Health Research, Australia IIA alone does not activate nuclear factor-kB (NF-kB) as measured by gel- Paul M Watt, Phylogica, Australia shift assays, Western blots for nuclear NF-kB subunits and degradation

of cytosolic IkB subunits. sPLA2-IIA does not activate phosphorylation

The JNK/Jun signalling pathway is an important component of cytokine- of p38 mitogen activated protein kinase (MAPK), while sPLA2-IIA and driven chronic infl ammatory disease. The value of targeting this pathway TNF both activate the ERK MAPK pathway. Activation of ERK suggests with biologicals to reduce inappropriate infl ammatory response has clearly that sPLA2-IIA has broader effects on cell function than the regulation of been demonstrated, for example with TNF-? inhibitors. However, an effec- prostaglandin production alone. tive cure for chronic infl ammatory diseases remains elusive. Provision of arachidonic acid to COX-2 in the presence of sPLA2-IIA

We have investigated the use of peptides as potential therapeutic agents in is thus mediated exclusively by cPLA2-a, while upregulation of COX-2 the infl ammatory response. Since random screening of small (largely un- is dependent on sPLA2-IIA-mediated signal transduction in FLSs. This structured) peptide libraries has been unsuccessful, we developed a novel mechanism is in contrast to proliferation induced by sPLA2-IIA enzyme peptide library, designated the Phylomer library. This library contains both activity-dependent activation of cPLA2-a in prostate cancer cells (Sved, randomly encoded peptides and structured peptides derived from natural P, Scott, K.F. et al. Cancer Res. 64, 6934-6940, 2004). Exogenous sPLA2-

ORFs of diverse bacterial genomes. Due to the inherent redundancy and IIA therefore modulates cell function through indirectly regulating cPLA2- limited number of protein folds in nature, the natural ORFs from this di- a and/or COX enzymes by distinct context-dependent mechanisms. verse library provide a rich source of peptides that interact specifi cally and with high affi nity to human proteins. To target infl ammation and validate this novel approach, the Phylomer library was screened to identify peptides Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 183

a potentiated NF-kappaB response. Importantly, Mal failed to undergo 7035 LPS-induced degradation in SOCS-1-defi cient macrophages. Targeted degradation of Mal by SOCS-1 therefore regulates TLR-mediated activa- Transcriptional regulation of cyclooxygenase-2 (COX-2) in hypoxia tion of NF-kappaB, causing a temporary refractoriness to prolonged TLR2 and TLR4 activation and chronic infl ammatory responses as evidenced by Rebecca J Cook-Johnson, Rheumatology Unit, Royal Adelaide Hospital, the susceptibility of SOCS-1-defi cient mice to chronic LPS-induced in- Australia fl ammation. Dan Peet, School of Molecular Biosciences, University of Adelaide, Australia Jenny Gamble, Human Immunology, Hanson Institute, Australia 7037 Leslie Cleland, Rheumatology Unit, Royal Adelaide Hospital, Australia Michael James, Rheumatology Unit, Royal Adelaide Hospital, Australia Induction of Bax-dependent mitochondrial permeabilization and caspase-independent apoptotic cell death by the neutrophil We have demonstrated that joints with effusions are hypoxic and that hy- infl ammatory oxidant hypochlorous acid poxia can upregulate COX-2 expression in monocytes, synoviocytes and endothelial cells. Hypoxia inducible factors (HIFs) have been shown to be Matt Whiteman, National University of Singapore, Singapore involved in the hypoxia-mediated transcriptional regulation of genes such Jeremy PE Spencer, University of Reading, United Kingdom as VEGF and erythropoietin. We identifi ed a putative HIF binding site, Jeffrey S Armstrong, National University of Singapore, Singapore Hypoxia Response Element (HRE), in the COX-2 promoter region. Aim: To examine the transcriptional regulation of COX-2 in hypoxia with refer- At sites of chronic infl ammation, such as in the infl amed rheumatoid joint, ence to HIF involvement. Methods: A COX-2 promoter (-922bp from the activated neutrophils release hydrogen peroxide (H2O2) and the enzyme transcriptional start site) luciferase reporter was produced (COX2-922). myeloperoxidase catalyzing the formation of hypochlorous acid (HOCl). Human Umbilical Vein Endothelial Cells (HUVEC) were transfected us- 3-chlorotyrosine, a marker HOCl has been observed in synovial fl uid ing Effectene (Qiagen) at 60% confl uency and incubated for 8h. Lucifer- proteins from rheumatoid arthritis patients. However the mechanisms of ase activity was determined using a Dual-Luciferase reporter assay system HOCl-induced cytotxicity are unknown. We have determined the molecu- (Promega). Results: The COX2-922 construct transiently transfected lar mechanisms by which HOCl induced cell death in human mesenchy- into HUVEC responded to hypoxia with an approximate 2-fold increase mal progenitor cells differentiated into a chondrogenic lineage as a model in luciferase activity. When the COX-2 promoter-reporter construct was of human cartilage cells. Using confocal microscopy, fl ow cytometric and co-transfected with expression vectors for normoxia-stable HIF-1alpha or western blotting methods, we show that HOCl induced rapid Bax con- HIF-2alpha an increase in luciferase expression was achieved in normoxic formational change, mitochondrial permeability and release of intra-mito- conditions. A consistent fi nding was greater luciferase expression when chondrial pro-apoptotic proteins (cytochrome c, SMAC, AIF and EndoG) the construct was co-transfected with HIF-2alpha rather then HIF-1alpha. which resulted in nuclear translocation of AIF and EndoG. siRNA-medi- When a putative hypoxia response element (HRE) was mutated within the ated Bax knockdown completely prevented mitochondrial permeability, COX-2 promoter, responsiveness to hypoxia and to HIFs was lost. Con- release of intra-mitochondrial pro-apoptotic proteins and cell death. Simi- clusions: The results suggest hypoxic induction of COX-2 is due, in part at larly siRNA-mediated knockdown of AIF or EndoG signifi cantly, but not least, to HIF binding to a HRE in the COX-2 promoter region. completely, inhibited HOCl-mediated cell death. Although chondrocyte death exhibited several biochemical markers of apoptosis there was no evidence for caspase involvement either by western blotting, fl uorescence 7036 activity assays or using caspase inhibitors to prevent cell death. In vitro exposure of recombinant human caspases resulted in complete inhibition Suppressor of cytokine signaling (SOCS)-1 negatively regulates Toll- of caspase activity and the addition of HOCl to staurosporine-treated chon- like receptor signaling by targeting Mal for degradation drocytes resulted in substantial inhibition of caspase-2, -3/7, -6, -8 and-9 activity further suggesting caspase-independence. Our results show, for the Ashley Mansell, Monash Institute of Medical Research, Monash Univer- fi rst time, that HOCl induces Bax-dependent mitochondrial permeability sity, Australia leading to caspase-independent cell death by a process mediated by but not Rosie Smith, Monash Institute of Medical Research, Monash University, exclusive to an AIF / EndoG-dependent pathway. Australia Pearl Grey, Trinity College Dublin, Ireland Jen Fenner, Monash Institute of Medical Research, Monash University, 7038 Australia Douglas Hilton, Walter and Eliza Hall Institute, Australia PAC-1 phosphatase is a key positive regulator of infl ammatory Luke AJ O'Neill, Trinity College Dublin, Ireland responses Paul J Hertzog, Monash Institute of Medical Research, Monash Univer- sity, Australia Kate L Jeffrey, The Garvan Institute of Medical Research, Australia Tilman Brummer, The Garvan Institute of Medical Research, Australia Toll-like receptors initiate the innate immune response to pathogens via Michael Rolph, The Garvan Institute of Medical Research, Australia their ability to activate NF-kappaB and induce a strong pro-infl ammatory Christian Rommel, Serono Pharmaceutical Research Institute, Switzer- response. Therefore, it is necessary that TLR signaling requires negative land regulation to prevent excessive infl ammatory damage to the host. Mal/TI- Montserrat Camps, Serono Pharmaceutical Research Institute, Switzer- RAP, a member of the TIR-domain containing adapter proteins, has been land shown to have a critical role in the pro-infl ammatory response in recogni- Steve Gerondakis, Australia tion of microbial pathogens by TLR2 and TLR4. We recently described a Charles R Mackay, The Garvan Institute of Medical Research, Australia novel role for Mal in TLR-mediated signaling in regulating NF-kappaB- dependent gene transcription via an interaction with TRAF6. Mitogen-activated protein kinases (MAPKs) facilitate numerous cellular Concurrent with previous studies of SOCS-1 function, SOCS-1 acts as an processes, including immune cell effector function. MAPKs are activated E3 ligase to mediate polyubiquitination of Mal via two N-terminal lysines by dual phosphorylation of a conserved Thr-Xxx-Tyr-motif. Dual-speci- thus targeting it for degradation via the 26S proteasome, and that Mal deg- fi city phosphatases (DUSPs) that dephosphorylate MAPK are thought to radation is dependent upon tyrosine phosphorylation. Furthermore, mu- be negative regulators to control MAPK activity. Many members of the tation of these N-terminal Lys residues rendered Mal unable to undergo DUSP family have been identifi ed but few have demonstrated functional SOCS-1-mediated degradation, and when overexpressed in cells, induced roles in vivo. A comprehensive Genechip analysis of human leukocyte S 184 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) types identifi ed PAC-1 (Phosphatase of Activated Cells, DUSP2) as one of Metalloproteinases are abundant enzymes in crotaline and viperine snake the most up-regulated transcripts during early activation of immune cells. venoms and are involved in local and systemic toxic effects of these ven- We generated Pac-1-/- mice and found that PAC-1 defi ciency in mast cells oms. BaP1, a 22,7 kDa metalloproteinase with weak hemorrhagic activity, and macrophages led to an unexpected impairment of p38 and ERK MAPK was purifi ed from the venom of Bothrops asper. In this study we examined activation, reduced Elk-1 transactivation and decreased production of pro- the effect of BaP1 on release of NO from macrophages and the mechanisms infl ammatory mediators. Moreover reconstitution of Pac-1 into Pac-1-/- involved in this effect. Macrophages obtained 96h after intraperitoneal in- cells rescued Elk-1 activity and cytokine-producing ability and this was jection of thioglycollate into male Swiss mice were incubated with select- dependent on the phosphatase activity of PAC-1. Moreover, Pac-1-/- mice ed concentrations of BaP1 (6.25 - 200 micrograms/ml) or RPMI (control). were protected in a mast cell-dependent model of infl ammatory arthritis. NO concentrations in cell culture supernatants and exudates from animals Thus PAC-1 phosphatase serves as a key regulator of infl ammatory cell injected with BaP1 (0.1mg/Kg) were mensured by Griess method and signaling and mediator production, and its transcriptional regulation repre- chemiluminescence, respectively. Inducible NO synthase (iNOs) protein sents an important mechanism for control of infl ammatory responses. and mRNA expression were analysed by Western blot and RT-PCR in cell homogenates, respectively. BaP1 (6,25 - 50 micrograms/ml) signifi cantly increased the release of NO from macrophages, maximum at concentra- 7039 tion of 12.5 micrograms/ml. This effect was inhibited in the presence of 1 micromolar L-NAME and aminoguanidine and abrogated by incubation PhosFlow Detection of STAT5 Phosphorylation Following Antigen or of BaP1 with 2mM EDTA. Moreover, increased iNOs mRNA expression Cytokine Administration In Vivo was detected 1h and 3h after incubation of macrophages with BaP1, and increased iNOs protein expression ocurred from 3h up to 24h after in- Sharon L Wannberg-Anderson, Amgen, United States cubation. These fi ndings demonstrate the ability of BaP1 to induce the Todd Covey, Amgen, United States release of NO from macrophages. This effect is related to an increased Sara C Smelt, Amgen, United States expression of inducible NO synthase, and BaP1-induced increase on iNOs gene expression appears to be the primary mechanism. Furthermore, pro- Jak3 is required for cytokine signaling via the common-gamma chain (IL- teolytic activity of BaP1 is relevant for its effect on production of NO by 2, 4, 7, 9, 15, 21) and is critical for lymphocyte differentiation, survival macrophages. and function. Because Jak3 has limited tissue distribution, a lack of con- stitutive activation and is evident in immune activation, a highly specifi c Jak3 inhibitor could prove to be a desirable immunosuppressive agent. 7041 Development of an inhibitor that inhibits Jak3 but not the closely related

Jak2 is paramount, as inhibition of Jak2 would interfere with red blood Effects of phospholipases A2 (PLA2s) isolated from Bothrops asper cell production. venom on expression of cyclooxygenases and release of prostanoids IL2 mediates STAT5 phosphorylation via Jak3 whereas GM-CSF medi- ates STAT5 phosphorylation via Jak2. To determine in vivo STAT5 phos- Catarina F Teixeira, Butantan Institute, Brazil phorylation via Jak2 or Jak3, mice were treated with different doses of IL2 Vanessa Moreira, Butantan Institute, Brazil or GM-CSF. STAT5 phosphorylation was measured in peripheral blood Stella R Zamuner, Buntatan Institute, Brazil monocytes and lymphocytes by PhosFlow. Juliana P Zuliani, Buntatan Institute, Brazil José M Gutiérrez, Clodomiro Picado Institute, Costa Rica Following IL2 administration, mouse peripheral blood CD4 lymphocytes exhibited a dose-dependent and time-dependent increase in STAT5 phos- PLA2s are major components of Viperidade snake venoms. Two myotoxins phorylation whereas CD11b monocytes did not. Following GM-CSF ad- with PLA2 structure were isolated from Bothrops snake venom: MT-II an ministration, an increase in STAT5 phosphorylation was detected in CD11b inactive Lys 49 PLA2and MT-III an active Asp 49 PLA2. Despite differ- peripheral monocytes but not lymphocytes. By treating mice with either ences in enzymatic activity both evoke infl ammatory events. In this study IL2 or GM-CSF, differentiation between STAT5-mediated phosphoryla- the effects of MT-II and MT-III on expression of COX-1 and COX-2 and tion via Jak2 or Jak3 was possible. We tested this model using a Jak3 in- release of prostanoids were evaluated in vivo and in vitro. Male Swiss hibitory compound (compound X) that inhibited Jak2 with a lower EC50. mice were i.p. injected with both PLA2PLA2s (2 μg/g) or sterile saline This model enabled the assessment of the relative potency of compound X (controls). At selected time intervals after these injections, peritoneal to inhibit Jak2- vs Jak3-mediated STAT5 phosphorylation. washes were performed for determination of PGE2and PG2concentrations The technology was further developed to enable detection of antigen-spe- by EIA and expression of COX-1 and -2 proteins by western blot in leu- cifi c STAT5 phosphorylation in T lymphocytes using the DO11.10 adop- kocyte homogenates. Results showed that MT-II and MT-III i.p. injec- tive transfer model. Ovalbumin immunization of BALB/c mice containing tion induced expression of COX-2 but not COX-1 in peritoneal leuko- DO11.10 T cells resulted in STAT5 phosphorylation that was inhibited in cytes from 1 h up to 24 h. At this same time interval the exudate levels of mice treated with immunomodulatory drugs. PGE2and PGD2were signifi cantly increased in comparison to controls. In These acute models serve as useful tools to confi rm selectivity and mecha- vitro incubation of mice peritoneal resident macrophages (1x106cells) with nism of action of immunomodulatory drugs in vivo. both venom PLA2s (6,5 μg/mL) resulted in increased release of PGE2and

PGD2and expression of COX-2 but not of COX-1 by these cells from 3 up to 6 h of incubation. These data demonstrate the ability of snake venom

7040 PLA2s to induce expression of COX-2 and release of prostanoids at the site of infl ammation. These effects may be at least in part due to a direct Nitric oxide release induced by BaP1, a snake venom action of both myotoxins on macrophages. Since MT-II lacks enzymatic metalloproteinase requires expression of the inducible NO synthase activity, the catalytic domain of some snake secretory PLA2s may not be gene by elicited macrophages relevant for their stimulating effects on metabolism of arachidonic acid via cyclooxygenases. Cristina Maria Fernandes, Butantan Institute, Brazil Financial Support: FAPESP and CNPq - Brasil. Silvia Fernanda Zamunér, Butantan Institute, Brazil Eduardo Cava Leanza, University of Sao Paulo, Brazil Estella Bevilacqua, University of Sao Paulo, Brazil José Maria Gutiérrez, Clodomiro Picado Institute, Costa Rica Juliana Pavan Zuliani, Butantan Institute, Brazil Catarina Fátima Teixeira, Butantan Institute, Brazil Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 185

our results point to a prominent role for free heme on the acute and chronic 7042 infl ammatory responses associated to hemolytic episodes.

Drotrecogin alfa (activated) attenuates the secretory phospholipase A2 group IIA expression in human aortic smooth muscle cells 7045

Mario Menschikowski, Technical University of Dresden, Medical Faculty Neutrophil Interaction with Adenovirus Vectors During the Innate Carl Gustav Carus, Institute of Clinical Chemistry and Laboratory Immune Response Limit Viral Persistence In Vivo Medicine, Germany Albert Hagelgans, Technical University of Dresden, Medical Faculty Matthew J Cotter, University of Calgary, Canada Carl Gustav Carus, Institute of Clinical Chemistry and Laboratory Daniel A Muruve, University of Calgary, Canada Medicine, Germany Gabriele Siegert, Technical University of Dresden, Medical Faculty Carl Neutrophils are phagocytic leukocytes that represent one of the fi rst lines Gustav Carus, Institute of Clinical Chemistry and Laboratory Medicine, of defense during infection, and are hypothesized to play a signifi cant Germany role in the innate immune response against adenovirus vectors. To study the role of neutrophils in limiting the persistence of adenovirus vectors Secretory phospholipase A2 group IIA (sPLA2-IIA) is thought to play a in vivo, we isolated tissue-recruited neutrophils by negative immunomag- crucial role in the initiation and progression of infl ammation. Therefore, netic separation from infl amed mouse liver 1-3 hours following systemic pharmacological restriction of the synthesis and secretion of sPLA2-IIA administration of the fi rst generation adenovirus vector, AdLuc. Neutro- is assumed to be benefi cial in the treatment of infl ammatory diseases such phils isolated in this manner were 95 % pure as determined by fl ow cytom- as sepsis and septic shock. In this study we investigated the effect of re- etry and more than 97 % viable by propidium iodide staining. Compared combinant human activated protein C (APC; drotrecogin alfa) both on to circulating cells, tissue-recruited neutrophils expressed high levels of the sPLA2-IIA expression and on the NF-κB and C/EBP-β DNA binding CD11b and were transcriptionally activated expressing multiple chemo- activity in human aorta smooth muscle cells (HASMC) stimulated with kine mRNA transcripts including MIP-1b, MIP-2 and IP-10. Depletion of IFN-γ. The cell-associated and the amounts of sPLA2-IIA protein released neutrophils prior to the administration of AdLuc resulted in a more than into the cell medium as well as the activity of NF-κB and C/EBP-β in 50% decrease in total liver chemokine mRNA expression. Following the nuclear extracts were measured using ELISA-based technique. The data systemic administration of Cy2-labelled AdLuc, isolated neutrophils con- have shown that APC (2 - 320 nM) dose-dependently decreased the IFN- tained a subpopulation of Cy2-high cells. PCR analysis of isolated neutro- γ-induced synthesis and secretion of sPLA2-IIA in HASMC. Simultane- phil lysates detected a signifi cant amount of adenovirus vector genomes, ously, the NF-κB and C/EBP-β DNA-binding activities in HASMC were confi rming that liver-recruited neutrophils interact with and take up adeno- diminished after exposure of cells to APC. Ethyl pyruvate and caffeic acid virus vectors. Neutrophil depletion improved vector persistence of AdLuc phenethyl ester known as anti-infl ammatory compounds with NF-κB- at 24 hours confi rming that neutrophils play an important role in the innate blocking activity also inhibited the sPLA2-IIA expression in HASMC at clearance of adenovirus vectors. This work describes for the fi rst time a concentrations of 5-10 mM and 25 μM, respectively. Conclusions: The signifi cant interaction between neutrophils and adenovirus vectors in vivo IFN-γ-induced synthesis and secretion of sPLA2-IIA in HASMC is effec- and provides an increased understanding of the innate immune response tively be reduced by APC which may explain the benefi cial effect of APC against these agents. treatment of patients having severe sepsis or septic shock. 7046 7043 Treatment with H2S-releasing derivative of diclofenac reduces Proinfl ammatory and antiapoptotic effects of heme: a possible link infl ammation in carrageenan-induced hindpaw oedema between acute and chronic infl ammatory responses during hemolytic episodes Madhav Bhatia, National University of Singapore, Singapore Jenab N Sidhapuriwala, National University of Singapore, Singapore Maria Augusta Arruda, Universidade do Estado do Rio de Janeiro, Brazil Anna Sparatore, Istituto di Chimica Farmaceutica e Tossicologica, Italy Pedro B de Souza, Universidade do Estado do Rio de Janeiro, Brazil Philip K Moore, National University of Singapore, Singapore Adriano Rossi, University of Edinburgh Medical School, United Kingdom Aurelio V Graça-Souza, Universidade Federal do Rio de Janeiro, Brazil Hydrogen sulfi de (H2S) is a naturally occurring gas, which has been shown Christina Barja-Fidalgo, Universidade do Estado do Rio de Janeiro, to be a potent vasodilator. Diclofenac is a nonsteroidal anti-infl ammatory Brazil drug (NSAID) and has been shown to have anti-infl ammatory, analgesic, and antipyretic activity. ACS15 is an H2S-releasing derivative of diclof- Sickle cell disease, malaria, ischaemia-reperfusion and other pathologi- enac. Little is known about its effectiveness as an anti-infl ammatory drug. cal states of increased hemolysis result in high levels of free heme in cir- In this report, we describe the effect of diclofenac and its H2S-releasing culation (up to 20 μM). These events are often associated with an acute derivative on carrageenan-induced hindpaw oedema in the rat. Intraplan- infl ammatory response that usually leads to the development of chronic in- tar injection of carrageenan (150 μl, 2 % w/v; n=10 rats in each group) fl ammation. We have previously shown that free heme (1-20 μM) is a pro- resulted in hindpaw oedema (increase in hindpaw weight: 1.03±0.05 c.f. infl ammatory molecule, able to promote neutrophil migration both in vivo 0.04±0.03 in saline treated rats). Pre-treatment with ACS15 (11.89 mg/kg), and in vitro, actin polymerization, O2- production, PKC activation, IL-8 but not diclofenac (7.5 mg/kg - mole per mole equivalent dose), signifi - expression. and delays human neutrophil spontaneous apoptosis. These ef- cantly reduced hindpaw oedema in the rat (increase in hindpaw weight: fects depend on protein synthesis and NADPH oxidase-generated ROS, 0.68±0.06; p<0.05 vs. vehicle control). Also increase in hindpaw myelo- require heme oxygenase (HO) activity, and involve PI3K, ERK and NF- peroxidase activity was signifi cantly attenuated as a result of pre-treatment kB pathways (Graça-Souza et al. 2002; Arruda et al. 2004). In this work with ACS15, but not diclofenac (MPO activity - fold increase over control: we report that heme-induced delay of neutrophil apoptosis correlates with vehicle: 2.24±0.7; ACS15: 1.21±0.4; p<0.05). These results show for the the prevention of mitochondria transmembrane potential dissipation, in a fi rst time that ACS15, an H2S-releasing derivative of the NSAID diclof- mechanism that depends on ROS and HO activity. ERK, PI3K and NF-kB enac, acts as an anti-infl ammatory drug in carrageenan-induced hindpaw pathways have also been shown to be involved on heme protective effects. oedema even at doses at which diclofenac is not effective. The maintenance of mitochondria transmembrane potential is probably due to heme inhibition of Bax insertion into mitochondria, which seems to be closely related to an increase on Bcl-XL / Bad ratio. Taken together, S 186 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

or up-regulation of CD86, despite producing a strong response to single- 7047 stranded 1668. The lack of response to E. coli DNA appears to be due to a combination of weak responses to long and double-stranded DNA. Up- Heat Shock Protein 10 Modulates Production of Proinfl ammatory take of longer oligonucleotides (44nt) by splenic B-cells was much lower Mediators than the uptake of 1668 (22nt) indicating that restricted access to TLR9 may be responsible for the decreased immunostimulatory activity of long Barbara J Johnson, CBio Limited, Australia DNA molecules. In contrast to the results with purifi ed B-cells, B-cells Caroline Dobbin, CBio Limited, Australia in a mixed splenocyte population could up-regulate CD86 in response to Tatjana Banovic, Queensland Institute of Medical Research, Australia the presence of E. coli DNA. This data suggests that the B-cell response Thuy T Le, Queensland Institute of Medical Research, Australia to bacterial DNA is dependent on another cell type, either for production Chris Howard, CBio Limited, Australia of cytokines allowing for B cell recognition of E. coli DNA, or for partial Flor de Maria L Flores, CBio Limited, Australia digestion of E. coli DNA. Geoff R Hill, Queensland Institute of Medical Research, Australia Andreas Suhrbier, Queensland Institute of Medical Research, Australia 7049 Heat shock protein 10 (Hsp10 or Cpn10) is a highly conserved mitochon- drial chaperone playing an essential role in protein folding. However, Regulation and function of toll-like receptor 9 in macrophages Hsp10 has also been shown to have a number of extracellular immu- nomodulatory activities. We have shown that purifi ed recombinant hu- Matthew J Sweet, Institute for Molecular Bioscience and CRC for Chro- man Hsp10 incubated with cells in vitro (i) reduced lipopolysaccharide nic Infl ammatory Diseases, University of Qld, Australia (LPS)- or Pam3Cys-induced nuclear factor-kB activation, (ii) inhibited Kate Schroder, Institute for Molecular Bioscience and CRC for Chronic LPS-induced tumour necrosis factor-alpha (TNF-a), interleukin-6 (IL-6) Infl ammatory Diseases, University of Qld, Australia and RANTES secretion from murine splenocytes, the macrophage cell David P Sester, Institute for Molecular Bioscience, University of Qld, line RAW264.7, and human peripheral blood mononuclear cells, and (iii) Australia increased interleukin-10 (IL-10) secretion from the same cells. Treatment Angela Trieu, Institute for Molecular Bioscience and CRC for Chronic of mice with Hsp10 delayed mortality from graft-versus-host disease, and Infl ammatory Diseases, University of Qld, Australia in an endotoxemia model, Hsp10 reduced LPS-induced serum TNF-a and Michael Rehli, Department of Hematology and Oncology, University of RANTES production, and elevated serum IL-10 levels. Regensburg, Regensburg, Germany During phase I clinical trials in healthy volunteers (phase 1a) or in sub- Kumar Visvanathan, CRC for Chronic Infl ammatory Diseases, Depart- jects with multiple sclerosis (phase 1b), peripheral blood mononuclear ment of Medicine, Royal, Australia cells (PBMC) were isolated eight hours following intravenous infusion Katryn J Stacey, Institute for Molecular Bioscience and CRC for Chronic with Hsp10 or placebo. When stimulated in vitro with LPS, PBMC from Infl ammatory Diseases, University of Qld, Australia Hsp10-infused subjects produced signifi cantly lower levels of proinfl am- David A Hume, Institute for Molecular Bioscience and CRC for Chronic matory mediators, in a dose-responsive manner, as compared with both Infl ammatory Diseases, University of Qld, Australia pre-infusion PBMC, and PBMC from placebo-infused subjects. We pro- pose, therefore, that Hsp10 may have therapeutic potential in the modula- Toll-like receptor (TLR) 9 is required for responses to bacterial CpG-con- tion of certain infl ammatory diseases. taining DNA. In contrast to a number of other TLRs, TLR9 is expressed Our current focus of research is understanding the mechanism of action at low levels in macrophage populations. The major macrophage growth whereby Hsp10 interacts with Toll-like receptors (TLR) to modulate the factor, macrophage colony stimulating factor (M-CSF/CSF-1) is expressed quality of signal induced by specifi c TLR ligands. constitutively in vivo and regulates a number of macrophage effector func- tions. We show here that CSF-1 downregulates expression of TLR9 and responses to CpG DNA in murine bone marrow-derived macrophages 7048 (BMM). Accordingly, antagonism of CSF-1 action in vivo enhanced re- sponses to CpG DNA. CSF-1 also downregulated TLR9 expression in hu- Purifi ed splenic B-cells do not respond to E. coli DNA man monocytes. In murine macrophages, the effect of CSF-1 on TLR9 expression is likely to be mediated via interferon regulator factor (IRF) Tara L Roberts, Institute for Molecular Bioscience; CRC for chronic 8; an IRF binding site exists immediately upstream of the transcription Infl ammatory Disease, Australia start site in the mouse TLR9 promoter and IRF8 mRNA expression was Jasmyn A Dunn, Institute for Molecular Bioscience; CRC for chronic downregulated by CSF-1 in BMM. Further, TLR9 mRNA levels were sub- Infl ammatory Disease, Australia stantially reduced in BMM from IRF8 gene targeted mice. Although ex- Matthew J Sweet, Institute for Molecular Bioscience; CRC for chronic pressed at low levels in CSF-1 replete murine macrophages, TLR9 mRNA Infl ammatory Disease, Australia was induced by bacterial products such as LPS. LPS triggered the rapid David A Hume, Institute for Molecular Bioscience; CRC for chronic loss of cell surface CSF-1R and CSF-1 responsiveness in BMM. This an- Infl ammatory Disease, Australia tagonism of CSF-1 action by LPS induced expression of TLR9, as well as Petar Lenert, 3Department of Internal Medicine, Carver College of other CSF-1-repressed genes that were identifi ed by micro-array analysis. Medicine, The University of Iowa, United States Hence, CSF-1 acts to prevent constitutive expression of TLR9 and co-reg- Katryn J Stacey, Institute for Molecular Bioscience; CRC for chronic ulated genes in macrophages and bacterial challenge induces expression Infl ammatory Disease, Australia of this class of genes by interfering with the CSF-1 signal. This study, as well as identifying a novel mechanism of LPS-inducible gene expression Cells of the innate immune system respond to the presence of bacterial in macrophages, suggests that CSF-1 antagonism may enhance the thera- DNA by production of pro-infl ammatory cytokines and up-regulation of peutic potential of CpG DNA. co-stimulatory molecules. Bacterial DNA is recognised by the immune sys- tem due to the presence of unmethylated CG dinucleotides (CpG motifs). It is currently accepted in the literature that dendritic cells, macrophages 7050 and B-cells respond directly to the presence of bacterial DNA via TLR9. Unexpectedly we found that the immature mouse B-cell line, WEHI231, PU.1 and ICSBP regulate the tlr9 promoter in mouse macrophages did not produce IL-6 in response to either E. coli DNA or double-stranded CpG-containing oligonucleotide 1668, but did respond to single-stranded Kate Schroder, Institute for Molecular Bioscience, University of Queens- 1668. Additionally, we showed that purifi ed murine splenic B-cells did land, Australia; CRC for Chronic Infl ammatory Diseases, Australia, not respond to the presence of E. coli DNA, either with IL-6 production Australia Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 187

Katharine M Irvine, Institute for Molecular Bioscience, University of mast cell histamine release. When mice were given a single oral dose of Queensland, Australia, Australia Gleevec(TM) at 10, 30, or 100 mg/kg prior to the SCF challenge, hista- Kristian Biron, Institute for Molecular Bioscience, University of Queens- mine release was inhibited at the highest dose. However, the same dos- land, Australia; CRC for Chronic Infl ammatory Diseases, Australia, ing regimen did not inhibit IgE-induced histamine release, indicating that Australia while a single dose of Gleevec(TM) specifi cally blocked c-kit, it had no ef- Monika Lichtinger, Abt. für Hämatologie und Internistische Onkologie, fect on the FceR-mediated activation pathway. To determine if cumulative Klinikum der Universität Regensburg, Germany dosing could affect mast cell survival, mice were given Gleevec(TM) for 7 Timothy Ravasi, Institute for Molecular Bioscience, University of days at 40 mg/kg twice a day. Similar to the single injection, Gleevec(TM) Queensland, Australia; CRC for Chronic Infl ammatory Diseases, Austra- given 7 days blocked SCF-induced histamine release, but not IgE-medi- lia, Australia ated histamine release. Histological analysis of skin samples indicated no Michael Rehli, Department of Hematology and Oncology, University of statistical difference in mast cell numbers from those in skin samples of Regensburg, Regensburg, Germany untreated control animals. These data indicate that although Gleevec(TM) Matthew J Sweet, Institute for Molecular Bioscience, University of did inhibit the ability of mast cells to respond to SCF, a longer period of Queensland, Australia; CRC for Chronic Infl ammatory Diseases, Austra- continuous inhibition of SCF/c-kit signaling may be necessary to impact lia, Australia mast cell survival and IgE-dependent allergic responses, perhaps via mast David A Hume, Institute for Molecular Bioscience, University of Queens- cell apoptosis. land, Australia; CRC for Chronic Infl ammatory Diseases, Australia, Australia 7052 Macrophages are activated by unmethylated CpG motifs present in bacte- rial DNA via Toll-like receptor 9 (TLR9). Both interferon-gamma (IFN- Cellular microenvironment: A defi ning role for TLR4 location and gamma) and lipopolysaccharide (LPS) can synergise with CpG DNA to expression in vivo enhance pro-infl ammatory gene expression and effector function in murine macrophages. In this study, LPS and IFN-gamma upregulated tlr9 mRNA Angela M Fonceca, School of Paediatrics and Child Health, The Univer- in murine bone marrow-derived macrophages (BMM). The ability of LPS sity of Western Australia, Australia and IFN-gamma to induce tlr9 expression in BMM was dependent upon Erika N Sutanto, Department of Respiratory Medicine, Princess Marga- the presence of the growth factor, colony stimulating factor-1 (CSF-1) that ret Hospital for Children, Perth, Western Australia, Australia suppressed tlr9 expression. However, there were clear mechanistic differ- Anthony Kicic, Department of Respiratory Medicine, Princess Margaret ences in tlr9 mRNA induction by LPS and IFN-gamma. LPS stimulation Hospital for Children, Perth, Western Australia, Australia caused a rapid removal the CSF-1 receptor (CSF-1R) from the cell surface Darryl A Knight, Department of Pharmacology and Therapeutics, Uni- and thereby blocked CSF1-mediated transcriptional repression, resulting versity of British Columbia, Canada in indirect induction of tlr9 expression. By contrast, IFN-gamma did not Stephen M Stick, Department of Respiratory Medicine, Princess Marga- dramatically regulate cell surface CSF-1R expression, but induced tlr9 ex- ret Hospital for Children, Perth, Western Australia, Australia pression by promoter activation. The murine tlr9 promoter was identifi ed, cloned and promoter elements necessary for gene regulation determined. Introduction: Airway epithelium may be an interface for innate and adap- A 100 bp proximal promoter was suffi cient to confer basal and IFN-gam- tive immunity, due to its ability to secrete or induce cytokine secretion in ma-inducible expression in the murine macrophage cell line RAW264.7. response to proinfl ammatory stimulation via Toll-like receptor 4 (TLR4). A composite PU.1/interferon regulatory factor (IRF) binding site overlap- This study assesses TLR4 co-localisation to the Golgi apparatus in the ping the major transcription start site was found to be a key mediator of airway epithelium in vitro and translocation to the surface in vivo due constitutive and IFN-gamma-inducible tlr9 expression in murine macro- to extracellular signals. Methods: Transformed human bronchial epithe- phages. The lack of conservation of this IRF site in the human promoter, lial (16HBE), adult primary bronchial epithelial (NHBE) and bronchial and the striking differences in promoter architecture between mice and epithelial cells obtained from healthy, asthmatic, atopic healthy and atopic humans, may partially account for the divergent patterns of tlr9 expression asthmatic phenotypes via trans-laryngeal, non-bronchoscopic brushings of apparent in the myeloid compartment between these species. the tracheal mucosa (<16yrs) including childrens epithelial cells in culture (CHBE) were stained for TLR4 and Golgi apparatus. Extracellular signal infl uence on TLR4 location was assessed via fl ow cytometry using IFN-g 7051 to prime NHBE cells. qPCR for TLR4 was completed on all the cell types used. Results: Intracellular co-localised staining of TLR4 to the Golgi seen Effects of imatinib mesylate, Gleevec(TM), on stem cell factor- in 16HBE and CHBE cells. IFN-g primed cells illustrated translocation of mediated and IgE-mediated mast cell degranulation TLR4 to the cell surface after 6hrs. Greater staining intensity evident in asthmatic and atopic phenotypes compared to non-atopic healthy epithe- Xuxia Zhang, Amgen, Inc., United States lial cells. TLR4 mRNA expression signifi cantly increased in atopic CHBE Gordon Ng, Amgen, Inc., United States cells. Conclusions: Differential location and expression of TLR4 in asth- Zhang Dawei, Amgen, Inc., United States matic and atopic bronchial epithelial cells illustrates a defi ning infl uence of Gore Anu, Amgen, Inc., United States cellular microenvironment in innate immune function. Trueblood Esther, Amgen, Inc., United Kingdom Itano Andrea, Amgen, Inc., United States 7053 Many studies support a key role for mast cells and their secretory products in the pathophysiology of allergic infl ammatory diseases. This premise Cryptolepine hydrochloride: a potent anti-mycobacterial underlies efforts to develop stabilizers of mast cells and antagonists of mast cell mediators that are involved in these responses. Another potential Fatemeh Fallah, Shaheed Beheshti University,Pediatric InfectiousRes- strategy is to deplete or disable mast cells by inhibiting signaling of the earch Center, Iran tyrosine kinase receptor c-kit, the receptor for stem cell factor (SCF), upon Gita Eslami, Shaheed Beheshti University, Iran which mast cells are dependent for their survival. Gleevec(TM) (imatinib Simon Gibbones, The School of Pharmacy, University of London, United mesylate, STI-571), an inhibitor of bcr-abl, PDGFR and c-kit, profoundly Kingdom blocks the SCF-dependent growth of human cultured mast cells in vitro. Colin W Wright, Bradford University, United Kingdom To elucidate the role of c-kit in mast cell function and survival in vivo, we studied the ability of Gleevec(TM) to block an acute systemic dose of SCF The activity of cryptolepine hydrochloride, a salt of the main indolo- in mice. One subcutaneous dose of 10 mg/kg SCF induced measureable quinoline alkaloid from the West African medicinal plant Cryptolepis san- S 188 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) guinolenta, was assessed against the fast growing mycobacterial species investigate the role of PPTA gene products in sepsis. PPTA-/- mice with Mycobacterium fortuitum, which has recently been shown to be of use in BALB/c background and PPTA+/+ BALB/c mice (20-23g) were randomly the evaluation of anti-tubercular drugs. The low minimum inhibitory con- assigned to control, sham operated and cecal ligation and puncture (CLP) centration (MIC) of this compound (16 μg/mL) prompted further evalua- groups and operated with 22G needle for 2 punctures for the induction of tion against other fast growing mycobacteria namely, M. phlei, M. aurum, polymicrobial sepsis. The onset of mortality and disease progression be- M. smegmatis, M. bovis BCG and M. abcessus and MICs ranged from tween PPTA-/- and PPTA+/+ mice were assessed at different time points. 2-32 μg/ml for these species. The strong activity of this agent, the need for The criteria for the severity of sepsis were alveolar congestion, oedema and new antibiotics with activity against Mycobacterium tuberculosis, coupled infl ammation, bacterial counts in blood, myeloperoxidase activity (an indi- with the ethnobotanical use of C. sanguinolenta extracts to treat infections, cator of neutrophil sequestration) in lungs, liver and kidneys. The survival highlight the potential of the cryptolepine template for development of rate in PPTA-/- mice with CLP-induced sepsis was signifi cantly higher anti-mycobacterial agents. than in PPTA+/+ mice. Sham operated mice remained healthy. Histopatho- Keywords: Mycobacterium; antibacterial; cryptolepine; Cryptolepis san- logical examination showed higher alveolar congestion, higher oedema, guinolenta. and cellular infi ltrates in lung, liver and kidney in PPTA+/+ mice than the PPTA-/- mice. Bacterial counts in blood, myeloperoxidase activity and the total and differential peripheral blood cell counts showed a signifi cant pro- 7054 tection in the PPTA-/- mice. These results show that PPT-A gene products play a key role in the pathogenesis of polymicrobial sepsis. Involvement of PAF receptor in the phagocytosis of apoptotic and necrotic cells by murine macrophages 7056 Soraya Imon Oliveira, Sao Paulo University (USP), Brazil Patricia Dias Fernandes, Rio de Janeiro Federal University (UFRJ), Clinical improvement proved Anti-infl ammatory properties of Herbs Brazil Sonia Jancar, Sao Paulo University (USP), Brazil Eladevi M Shah, Ayurved Consultancy UK Ltd., United Kingdom

In this study we investigated the possible participation of the Platelet Purpose: Develop non-steroidal treatments for Itchy skin conditions such Activating Factor receptor (PAFR) on the phagocytosis of apoptotic and as adult-Eczema, elderly eczema (eczema and lichen simplex on the sheen), necrotic thymocytes by murine peritoneal macrophages.The effect of infantile eczema, neurodermatitis, psoriasis,Lichen planus. Methods: No phagocytosis on LPS induced Nitric Oxide Synthase (iNOS) and Cycloox- difference in Age, sex, or race. All patients c/o of itchy skin are chosen. ygenase-2 (COX-2) expression was also investigated.Apoptosis of murine In 90% of patients they had past history of long term use of steroids. I thymocytes was induced by gamma radiation and necrosis by heating at considered more precizely the effects on the skin (1)associated signs and 60oC.Thioglycolate elicited murine peritoneal macrophages were co-cul- symptoms such as hypertension, side effects of medicines for hyperten- tivated with normal (t), apoptotic(ta) or necrotic (tn) thymocytes (1:2) in sion, (2)Acrodermatitis a) and/ or planter psoriasis in hyperthyroidism b) the presence or absence of different concentrations of PAFR antagonists, menopause HRT overusage c) contraceptive pills overusage (3) overuse WEB2170 or CV3988 for 2h,37oC,5%CO2.Phagocytosis was evaluated of alchohol triggeing psoriasis (4) Cosmetic jwellary ,nickel sensitisation by counting 200 cells to determine the percentage of phagocytosing mac- rubber sensitisation in adults (5) rubber sensitisation in Infants(bulley in rophages and the average number of phagocytosed thymocytes per mac- the mouth) lack of proper education of mothers. I prepared nonsteroidal rophage.The phagocytic index (PI) was calculated by multiplying these natural creams containing Water dried extracts of seeds of purple fl aeban two variants.The effect of these altered cells on LPS induced iNOS and plant suspended in clerifi ed butter. Given to all category of Itchy skin pa- COX-2 expression was quantifi ed by Immunoblot.The results obtained tients apprximately 5000 patients over a period of my 17 years in the UK. showed that: a) phagocytosis of ta and tn was greater than of t; b) PAFR One elderly lady suffering from erythroderma and intertrigal infection She antagonists inhibited phagocytosis of ta and tn but not of t; c) phagocytosis recovered in three months time with this treatments. Results: Itching sub- of ta and tn,but not of t, increased LPS-induced COX-2 expression, but sided in three days time in most of the patients and rash started to disappear not iNOS; d) PAFR antagonists treatment inhibited the increase on COX-2 in three weeks. Complete clearance three months in psoriatic pateints, expression after ta and tn phagocytosis.These data indicate that the PAF three-four months for eczema patients and fi ve months for lichen planus receptor is involved in the phagocytosis of apoptotic and necrotic cells, patients. For neurodermatitis six weeks treatment. Coclusion: Ghee acts as and in the increased COX-2 expression induced by the phagocytosis of a most powerful soothing agent. Ghee provides anti-infl ammatory proper- these altered cells. ties. Ghee acts as a preservative also. It do not allow fungus to grow in the cream. The herb-purple fl aeban Centratherum anthelminticum the major herb in the cream and in the oral treatments. (It is proved in the laboratory 7055 inhibition of proliferation of the keratinocytes.) antiproliferative proper- ties, anti infl ammatory properties, antiallergic properties. Preprotachykinin-A (PPTA) gene products play a critical role in the pathogenesis of sepsis 7057 Madhav Bhatia, National University of Singapore, Singapore Padmam Puneet, National University of Singapore, Singapore Probiotics activate distinct signaling pathways involve in host- Akhil Hegde, National University of Singapore, Singapore microbe interaction in intestinal epithelial cell Siaw Wei Ng, National University of Singapore, Singapore Hon Yen Lau, National University of Singapore, Singapore Shugui Wang, Microbiology department, Faculty of Medicine, National Shabbir Moochhala, National University of Singapore, Singapore university of Singapore, Singapore Yuan kun Lee, Microbiology department, Faculty of Medicine, National Despite recent progress in the management of septic patients as well as the university of Singapore, Singapore understanding of basic pathophysiological mechanism of sepsis, the high mortality rate due to sepsis, septic shock and multiple organ failure has Background & Aims: Intestine epithelial cells serve as a barrier between not been signifi cantly reduced in the past two decades. Sepsis is a clinical our body and the bacteria present in intestine. The continuous epitheli- syndrome defi ned as the presence of systemic infl ammatory response syn- al microbial cross-talk is mediated in part by Toll-like receptor (TLRs). drome associated with a confi rmed infectious process. Preprotachykinin-A However, the mechanism how the immune system tells the pathogens (PPTA) gene products substance P and neurokinin-A have been shown to from probiotics has not been fully understood. We therefore analyzed ex- play an important role in neurogenic infl ammation. This study aimed to pression and regulation of TLRs and cytokine in colonic human cell line Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 189

(Caco-2, HCT116 and HT29). Methods: Expression of the genes of TLRs (1,2,3,4,5,9),Tumor necrosis factor receptor-associated factor 6 (TRAF6), 7059 interleukin (IL)-8, IL-4, IL-10, and TGF-βwere assessed before and after stimulation with probiotics and pathogen by reverse-transcription poly- Tpl2 is an important mediator of extracellular signal regulated merase chain reaction. Secreted IL-8, IL-4, IL-10 and TGF-β were deter- kinase (Erk) signaling downstream of diverse toll like receptors mined using ELISA. Results and Discussion: IL-8 protein production was (TLRs) in mouse bone marrow derived macrophages (BMDMs) down regulated to about 20% after exposure of the cell line to probiotics. However their mRNA level did not change signifi cantly even with a high Ashish Banerjee, The Walter and Eliza Hall Institute of Medical Re- concentration of probiotics(108CFU/ml). This implies that the regulation search, Australia on the IL-8 protein production might involve post-transcriptional pathway. Martin McMahon, United States Interestingly, TGF-β, which is important in infl ammation disease recovery, Steve Gerondakis, Australia was up regulated signifi cantly by Lactobacillus especially Lactobacillus Plantanum (L1) in Caoco-2 cell line both on protein level and gene level, Activation of ERK in wild-type BMDMs in response to LPS stimulation but was up regulated only at a high concentration (108CUF/ml) in HCT116 was shown to depend on the MAP3K, Tpl2. nfkb1-/- BMDMs fail to acti- cell line. We also found that most of the probiotics especially Lactobacillus vate ERK in response to LPS as a result of markedly reduced levels of Tpl2 para paracasei (L16) up regulated TLR9 gene expression by 1.5 to 5 folds kinase, the stability of which is dependent on p105 NF-kB1. These obser- in Caco-2 cell line but down regulated the TLR2, TLR3 and TRAF6 gene vations prompted us to determine if Tpl2 is required for ERK activation expression in all the cell lines. Interestingly, up regulation of TLR9 and downstream of all TLRs or whether it is specifi c to TLR4. BMDMs from TGF-β gene expression showed linear correlation. These results imply C57BL/6 and nfkb1-/- mice were used as a model to study MAP kinase that TLR9 signaling is essential in mediating the anti-infl ammatory effect signaling pathways activated by TLRs. Following treatment with diverse of probiotics and thus activate Th3 to release disease recovery cytokine. TLR ligands, activation of the ERK, JNK and p38 pathways was analyzed Conclusion: Probiotics were involved in the regulation of the TLRs ex- by western blotting and immune complex kinase assays. Expression of pression and subsequent cytokine production. The regulation was dose and downstream targets like IL-10 and Cox2 was analysed by ELISAs and strain dependent. western blotting respectively. Our data suggests that in BMDMs, ERK is activated via the Tpl2/Mek1 pathway upon engaging TLR2, TLR3, TLR7 or TLR9, but as shown previously for TLR4, the activation of ERK but not 7058 JNK1/2 or p38 is affected in nfkb1-/- BMDMs stimulated with ligands for these TLRs. This ERK activation defect in nfkb1-/- BMDMs was shown Anti-infl ammatory Viral Serpin, Serp-1, Associates With and to result in the defective induction of downstream targets like IL-10 and Modulates Monocyte Adhesion in part through Differential Cox2. Although expression of a conditionally active version of another Expression of the Actin Binding Protein Filamin-b MAP3K, Raf in BMDMs could by-pass the TLR dependent ERK activa- tion defect in nfkb1-/- macrophages, it was unable to restore IL-10 or Cox2 Jokubas Richardson, Robarts Research Institute, Canada expression in nfkb1-/- BMDMs to wild-type levels. Collectively, our data Kasinath Viswanathan, Robarts Research Institute, Canada indicate that the transcriptional induction of genes encoding cytokines and Babajide Togonu-Bickersteth, Robarts Research Institute, Canada immune mediators by TLR stimuli depends on the complex interplay be- Pracha Vatsya, Robarts Research Institute, Canada tween NF-kB and MAP kinase regulated transcription factors Alexandra R Lucas, Robarts Research Institute, Canada

Introduction: Adhesion and migration of mononuclear cells play criti- 7060 cal roles in the pathogenesis of atherosclerosis. Urokinase-type-activator (uPA) and its receptor uPAR promote proteolysis, migration and activation Down regulation of IRAK-4 expression in response to TLR activation of cells in the vasculature. A myxoma viral serpin, Serp-1, known to inhibit in primary mouse macrophages uPA, tPA, plasmin and factor Xa in vitro and infl ammation in vivo acts through uPAR. Serp-1 effectively attenuates cellular adhesion and migra- Dominic F De Nardo, Department of Medicine and CRC for Chronic In- tion of THP-1 cells and platelets, among others. Microarray analysis of fl ammatory Diseases, The University of Melbourne, Parkville, Australia, human monocytic THP-1 cells treated with Serp-1 demonstrated clearly Australia elevated expression of fi lamin-b, an actin-binding integrin-interacting-pro- John A Hamilton, Department of Medicine and CRC for Chronic In- tein that modulates cell migration. The aim of the present study is to deter- fl ammatory Diseases, The University of Melbourne, Parkville, Australia, mine whether Serp-1 directly associates with cell surface receptors and to Australia identify the role of increased fi lamin expression in adhesion and migration. Glen M Scholz, Department of Medicine and CRC for Chronic Infl amm- Methods: Serp-1 was labeled with FITC and tested for association with atory Diseases, The University of Melbourne, Parkville, Australia, monocytes using confocal microscopy and spectrofl uorometry. Molecular Australia interaction of Serp-1 with serine proteases uPA, tPA and thrombin in the presence of uPAR was analyzed using western blot analysis. Filamin-b The innate immune system is the fi rst line of defense against infection gene expression was quantifi ed using real time-PCR in THP-1 cells and by microbial pathogens and relies greatly on the expression of Toll-like co-localization of fi lamin-b to uPAR was studied using confocal micros- receptors (TLRs) by immune cells (e.g. macrophages). Members of the copy. Results: Serp-1 showed a clear association with THP-1 monocytes TLR family are able to recognise foreign microbes by virtue of highly and Jurkat T cells. Serp-1 complexed with uPA, tPA and thrombin but no conserved pathogen-associated molecular patterns (PAMPs). For example, heterotrimeric complexes during uPA treatment were observed. Real Time- TLR4 recognises lipopolysaccharide, while TLR9 is a receptor for CpG PCR analysis of THP-1 cells revealed that Serp-1 increased expression of DNA. Recognition of TLRs by PAMPs triggers the activation of intra- fi lamin-b and this expression was nullifi ed when a blocking antibody to cellular signalling pathways that are critical for induction of the immune uPAR was used. In Serp-1 treated THP-1 cells uPAR and fi lamin-b co-lo- response. Interleukin-1 receptor associated kinases (IRAKs) are key me- calized. Conclusion: Filamin-b gene expression was increased with Serp-1 diators of TLR signaling. Specifi cally, activation of IRAK-1 and IRAK-4 treatment in monocytes through a uPAR dependent mechanism. In Serp-1 leads to the nuclear translocation of NF-κB and expression of NF-κB tar- treated cells, uPAR co-localized with fi lamin-b demonstrating that Serp-1 get genes (e.g. TNFα and IL-1). IRAK-4 plays a paramount role in TLR reduces cellular adhesion and migration. signalling as mice defi cient in IRAK-4 are highly susceptible to bacterial infection and resistant to lipopolysaccharide-induced septic shock. Despite the importance of IRAK-4 in TLR signalling, its regulation is still poorly understood. We have found that activation of TLRs in mouse bone marrow-derived macrophages (BMM) leads to a rapid decrease in S 190 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) levels of IRAK-4 mRNA. Our fi nding that the half-life of IRAK-4 mRNA In addition, we investigated the association between gene polymorphisms transcripts is unaffected by TLR ligands indicates that activation of TLRs in TLR2 and TLR4 for association with BD. leads to a decrease in IRAK-4 gene expression. Notably, the half-life of Methods: Immunohistochemical analysis was used to investigate the ex- IRAK-4 protein in BMM is reduced upon TLR activation. Thus activa- pression of TLR 2, 4, 6, & 9 in mouth ulcer biopsies from BD patients tion of TLRs leads to both a decrease in IRAK-4 gene expression and the and compared with aphthous ulcer pyogenic granuloma and lichen planus degradation of IRAK-4 protein. The effects of various pro-infl ammatory biopsies as disease controls and healthy oral mucosa. To determine the na- and anti-infl ammatory cytokines on IRAK-4 gene and protein expression ture of cells expressing the TLRs, dual staining with CD56 and CD68 was in BMM will be presented and discussed. performed. Single nucleotide polymorphisms in TLR2 (C677T: G753A) and TLR4 (A299G: T399C) were analysed in 82 UK patients with BD and compared to ethnic controls. 7061 Results: BD lesions express TLR2, TLR4, TLR6 and TLR9, and show greater expression of all TLR compared to lichen planus and normal tis- Methylated Bovine Serum Albumin induces macrophage survival in sue. By comparison, BD lesions show similar expression of all TLR to a TLR4- dependent manner pyogenic granuloma. TLR expression appears to be mainly on infi ltrating cells in lesions. Double staining has supported expression on CD68+ mac- Paul J Egan, Reid Rheumatology laboratory, Division of Autoimmunity rophages. No association was seen between any of the SNP tested and BD. and Transplantation, The Walter and Eliza Hall Institute of Medical Rese, For TLR2 no patient expressed 677T, and only 3 (4%) expressed 753A, for Australia TLR4 3 (4%) 299G, and none expressed 399C. Ian P Wicks, Reid Rheumatology laboratory, Division of Autoimmunity Conclusion: TLR expression was raised compared to normal tissue, how- and Transplantation, The Walter and Eliza Hall Institute of Medical Rese, ever it was similar in biopsies from other infl ammatory conditions. Al- Australia though it is possible that each condition is characterised by different cells expressing TLR at present there is no characteristic pattern of TLR ex- Intra-articular injection of methylated bovine serum albumin, in combina- pression in BD. Moreover, there was no association between SNP in TLR tion with subcutaneous administration of interleukin-1 induces an acute genes and BD. This data suggests that TLR are functional in BD lesions mono-articular infl ammatory arthritis in mice that is dependent of the pres- and not directly causative in the persistence of infl ammation. ence of synovial macrophages. The mechanisms by which mBSA contrib- utes to the induction of infl ammatory arthritis, however, are unclear. We hypothesise that mBSA uptake by macrophages is pro-infl ammatory and 7063 contributes to macrophage activation in vivo. In order to investigate this, we stimulated bone marrow- derived macrophages with mBSA. Macro- Toll-like receptors expression in human cornea from patients with phages bound FITC-labelled mBSA, with approximately 70% of macro- stromal keratitis phages binding mBSA within 30 minutes. Macrophages were unable to bind FITC-labelled BSA, however, demonstrating that methylation of Graham R Wallace, University of Birmingham, United Kingdom BSA was necessary for uptake. Although stimulation of macrophages with Philip I Murray, University of Birmingham, United Kingdom mBSA did not induce the production of infl ammatory cytokines, as mea- Rebecca Lone, University of Birmingham, United Kingdom sured by a cytokine array, mBSA signifi cantly increased the viability of John Curnow, University of Birmingham, United Kingdom macrophages following 24 hours of culture. The percentage of viable cells Hazel Williams, University of Birmingham, United Kingdom increased from 70% following stimulation with BSA to greater than 90% Saaeha Rauz, United Kingdom following stimulation with mBSA. Enhanced survival of macrophages in response to mBSA was dependent on Toll-Like Receptor 4 (TLR4), Purpose: The cornea is protected from pathogens by several physical bar- since mBSA was unable to enhance the survival of macrophages derived riers that are breached in microbial keratitis. As Toll-like receptors (TLR) from C3H/HeJ mice. Binding of mBSA-FITC to C3H/HeJ macrophages, recognise pathogen-derived products we evaluated the expression of TLR however, was similar to the control strain, C3H/HeN, demonstrating that in human corneal tissues taken from infected and non-infected eyes.. TLR4 was not acting as a ligand for mBSA. These results demonstrate Methods: Immunohistochemical analysis was used to investigate the ex- that mBSA is stimulatory to macrophages in a TLR4-dependent manner pression of TLR 2, 4, 6, & 9 in corneal buttons taken during penetrating and that, in combination with IL-1 stimulation, may provide arthritogenic keratoplasty from patients with severe acute and chronic microbial kerati- stimuli to synovial macrophages. tis secondary to gram positive and gram negative bacteria, and herpes sim- plex virus. Normal corneal sections from enucleated globes were used as controls. To determine the nature of cells expressing the TLR, dual stain- 7062 ing with CD68, and methyl-green pyronin staining was performed. Results: TLR2, 4, 6 and 9 were expressed in epithelial, stromal and en- Toll-like receptor (TLR) expression in mouth ulcer biopsies from dothelial cells in infected corneal buttons. The majority of the staining patients with Behcet’s disease appeared to be on infi ltrating cells, including macrophages, plasma cells, neutrophils located in the peripheral cornea and at the site of infection. By Graham R Wallace, University of Birmingham, United Kingdom comparison, there is no expression of TLR in the central cornea with the Omar Durrani, University of Birmingham, United Kingdom exception of TLR4 and TLR9 within the endothelial cells. John Curnow, University of Birmingham, United Kingdom Conclusions: The TLR+ cells in infected corneae appear to be blood-borne Hazel Williams, University of Birmingham, United Kingdom rather than resident, as there was no expression in normal corneal tissue. John Hamburger, University of Birmingham, United Kingdom Although it is therefore not clear how pathogens are initially recognised, Farida Fortune, Queen Mary's, London, United Kingdom it suggests that lack of TLR expression in the normal cornea may aid im- Robert Vaughan, King's, Guy's and St Thomas', London, United Kingdom mune privilege. Philip I Murray, University of Birmingham, United Kingdom

Background: Behcet’s disease (BD) is characterised by oral and genital 7064 ulceration, ocular, joint, skin and CNS manifestations. Although the aetiol- ogy of BD is unclear a bacterial or viral infection of mucosal surfaces has Impaired Toll-Like Receptor Expression in Chronic Hepatitis B: Role been implicated. As TLR recognise pathogen derived products we sought of the Pre-core protein in Immunoevasion to determine whether there are any defects in TLR expression in BD. We analysed buccal ulcer biopsies from patients with BD for TLR expression. Kumar Visvanathan, Dept of Medicine, Royal Melbourne Hospital, Mur- doch Children's Research Institiute, Australia Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 191

Narelle Skinner, Dept of Medicine, Royal Melbourne Hospital, Murdoch Children's Research Institiute, Australia 7066 Stephen M Riordan, The Prince of Wales Hospital and University of NSW, Australia Blockade of CSF-1 action enhances CpG DNA responses in vivo T Sozzi, Victorian Infectious Diseases Laboratory and University of Melbourne, Australia Angela Trieu, Institute for Molecular Biosciences, Australia Ros Edwards, Victorian Infectious Diseases Laboratory and University of Steve Cronau, Institute for Molecular Biosciences, Australia Melbourne, Australia Tara Roberts, Institute for Molecular Biosciences, Australia Alexander Thompson, Victorian Infectious Diseases Laboratory and Jane Lattin, Institute for Molecular Biosciences, Australia University of Melbourne, Australia Ross Vlahos, University of Melbourne, Australia Stephen Locarnini, Victorian Infectious Diseases Laboratory and Univer- Katryn J Stacey, Institute for Molecular Biosciences, Australia sity of Melbourne, Australia David A Hume, Institute for Molecular Biosciences, Australia Matthew J Sweet, Institute for Molecular Biosciences, Australia Background: Mechanisms by which hepatitis B virus (HBV) establishes persistent infection remain unclear. In particular, expression of Toll-like Critical to the innate response is the discrimination between self and non- receptors (TLR’s), increasingly recognised as critically involved in the in- self by a series of genetically encoded pattern recognition receptors (PRRs) nate immune response to bacterial and viral pathogens, has not been inves- which recognize pathogen- associated molecular patterns (PAMPs) of tigated. Methods: TLR2 and TLR4 expression on Hepatocytes from fresh pathogens. PAMPs are recognised by a set of thirteen pattern recognition liver biopsies of patients with chronic HBV was measured by fl ow cytome- receptors called Toll like receptors (TLRs). TLR9 is the receptor that is try using anti-CD14 and anti-TLR2 and anti-TLR4 monoclonal antibodies. required for bacterial CpG-containing DNA responses. CSF-1 is a hemo- Hepatic cell lines including HepG2 cells were infected with recombinant poietic growth factor, which is constitutively present in the circulation. It HBV baculovirus expressing wildtype HBV, precore stop codon (G1896A) regulates the survival, differentiation and proliferation of macrophages. mutant HBV or the mock baculovirus control and grown for 7 days prior We have shown that CSF-1 suppresses TLR9 mRNA levels and CpG DNA to harvesting and staining for subsequent fl ow cytometry. The TLR2 and responses in macrophages, but the signifi cance in vivo is unknown. In this TLR4 expression on HepG2 was also measured by fl ow cytometry and study, we assessed the role of CSF-1 in regulating CpG DNA responses analysed for specifi c mRNA levels. TLR2 and TLR4 expression on HepG2 in vivo. BALB/c mice were challenged with an α-CSF-1R antibody that was measured by fl ow cytometry Results: TLR2 expression (expressed as antagonises CSF-1 action. Cytokine production and gene expression af- a ratio to control results) was signifi cantly reduced in chronic hepatitis B ter CpG DNA challenge was monitored. This model will help determine patients compared with controls. TLR2 expression normalised in each of whether antagonism of CSF-1 down modulation of TLR9 will improve the the 5 lamivudine-treated chronic hepatitis B patients in whom HBV DNA action of CpG DNA as a vaccine adjuvant. The effect of CSF-1 regulation became undetectable. Down regulation of TLR2 expression was found to of TLR9 gene expression may also be important during infections. Mac- occur in HepG2 cell cultures infected with wild type HBV (HBeAg-posi- rophages from wildtype and TLR9 knockout mice were challenged with tive). The level of TLR4 expression did not change. In contrast, there was Pseudomonas aeuriginosa and the effect on bacterial survival and cytokine an increase in TLR2 and TLR4 expression in the HepG-2 cells infected production was monitored. These studies will enhance our understanding with pre-core mutant (HBeAg-negative) HBV. TLR2 and TLR4 mRNA of TLR9 gene regulation and function. analysis confi rmed these fi ndings. Conclusions: Chronic HBV down-reg- ulates expression of TLR2 on hepatocytes, This downregulation can be demonstrated in HepG2 with wildtype virus but not in cells infected with 7067 the pre-core mutant. This study highlights the importance of the precore protein in the innate response to HBV and suggests that this virus-induced Defective response to Toll-like receptors 3 and 4 ligands by activated defect in innate immunity may contribute to the development of persistent monocytes in HCV infection infection. Maria C Villacres, Keck School of Medicine, University of Southern California, United States 7065 Wenbo Du, Keck School of Medicine, University of Southern California, United States How does the innate immune system recognize allergic stimuli Andrea Kovacs, Keck School of Medicine, University of Southern Califor- nia, United States Michael J de Veer, Centre for Animal Biotechnology, Australia Paul D Sheean, Centre for Animal Biotechnology, Australia Innate immunity has a critical role in initiation of protective responses Els Meeusen, Centre for Animal Biotechnology, Australia against pathogens. We investigated the IL-1beta, IL-6, IL-10, IL-12 and TNF-alpha response by antigen-presenting cells of subjects infected with Currently there is little information regarding how pathogens that induce a HCV or HIV and subjects with simultaneous HCV and HIV infection in type 2 response interact with the host innate immune system, and wheth- the Women Interagency HIV Study (WIHS) cohort. IL-6 response to the er they stimulate or suppress innate immune type pathways such as toll TLR 3 ligand poly I:C and the TLR 4 ligand LPS was signifi cantly reduced receptor activation. We are interested in how the innate immune system in women with HCV infection, both as single infection and in co-infection recognises stimuli that normally induce a type-2 or allergic immune re- with HIV. In addition, a signifi cant frequency of women with HCV infec- sponse characterized by induction of IL-4 or IL-5 and IgE. We are utilizing tion or co-infection had high spontaneous secretion of IL-6, suggesting both murine and ovine models to elucidate what molecular patterns may pre-existing cell activation as a factor mediating reduced responses to TLR be present in allergic stimuli with a particular focus on house dust mite 3 and TLR 4 stimulation. Moreover, in some HCV-infected women, the and helminth parasites. We have developed assays to detect the induction spontaneous secretion of cytokines was quantitatively similar or higher of type-2 responses by innate cell populations and aim to fractionate and to the secretion in cultures stimulated with TLR 3 and TLR 4 ligands. characterize the relevant molecules responsible for driving the innate al- Depletion experiments and intracellular staining identifi ed monocytes as lergic response. the cell population spontaneously secreting cytokines and also the cells responding to TLR 3 and TLR 4 stimulation. These results highlight a functional defect in antigen-presenting cells of women with HCV infection or co-infection. In women with existing HIV co-infection, decreased func- tion of antigen-presenting cells implies another mechanism contributing to immune dysfunction in addition to the known defect on CD4 function associated with HIV infection. S 192 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

7068 7070

Streptozotocin-induced diabetes mellitus in the rat is associated with The role of cyclooxygenase-1 and -2 in the rat orofacial formalin test enhanced biosynthesis of hydrogen sulphide Satyanarayana SV Padi, Pharmacology Division, University Institute Yusuf M Ali, National University of Singapore, Singapore of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, Benny KH Tan, National University of Singapore, Singapore INDIA, India Philip K Moore, National University of Singapore, Singapore Pattipati S Naidu, Pharmacology Division, University Institute of Phar- maceutical Sciences, Panjab University, Chandigarh-160014, INDIA, Hydrogen sulphide (H2S) is a naturally occurring gas which dilates blood India vessels by opening K-ATP channels. Some anti-diabetic drugs (e.g. gliben- Shrinivas K Kulkarni, Pharmacology Division, University Institute of clamide) block K-ATP channels thereby preventing the vasodilator action Pharmaceutical Sciences, Panjab University, Chandigarh-160014, of H2S and suggesting that H2S may play a part in the aetiology of diabe- INDIA, India tes mellitus and/or the vascular complications thereof. To investigate this hypothesis, rats (180-220g) were administered vehicle (10mmol/l citrate Objective: Prostaglandins (PGs) are known to play an important role in buffer, pH 4.5, control), streptozotocin (STZ, 65mg/kg, i.p., diabetic) or nociceptive transmission and are produced by cyclooxygenase (COX), streptozotocin (65mg/kg, i.p. with daily s.c. injections of insulin lente, 8 which catalyzes the conversion of arachidonic acid. Two forms of COX IU/kg). After 9 days, animals were killed and plasma/tissues were harvest- have been identifi ed i.e. COX-1 and -2, which are constitutively expressed ed. Plasma glucose was elevated in STZ-diabetic rats (417.6±26.7 mg/ml in brain. The role of COX isoforms is well understood in acute and chronic cf 138.9±5.5 mg/ml, P<0.05) which effect was partially reversed by insu- pain, however, their role in orofacial pain is not clear. Thus the present lin administration 299.6±69.0 mg/ml, P<0.05). Plasma H2S concentration study was performed to investigate effects of selective COX-1, -2 or non- was not different between control (40.32 ± 1.26uM) and diabetic (38.61 ± selective COX-1/2 inhibitors to address the relative role COX isoforms in 0.63uM). In contrast, H2S formation (from added cysteine) was signifi - orofacial pain of trigeminal origin. Methods: Orofacial pain was induced cantly elevated in homogenates of pancreas and liver (but not kidney) of by subcutaneous injection of a 50 μl of 5% formalin into the vibrissa pad diabetic animals. Insulin treatment of STZ-injected rats reversed the rise in and the time spent grooming, rubbing, and/or scratching the facial region H2S synthesising activity in liver and pancreas. Both CSE and CBS (H2S proximal to the injection site were recorded for fi fteen successive 3-min in- synthesizing enzymes) mRNA was expressed in liver from control ani- tervals for each animal. To defi ne the role of COX isoforms in nociceptive mals. Expression of both enzymes was signifi cantly increased in diabetic transmission, we examined the effect of intraperitoneal and local adminis- rats which effect was prevented in the liver by insulin pretreatment. Previ- tration (pretreatment) of resveratrol, a selective COX-1 inhibitor, naprox- ous reports have revealed upregulation of liver (but not kidney) CBS and en, a nonselective COX-1/2 inhibitor, or rofecoxib, a selective COX-2 CSE in STZ-induced diabetic rats. However, this is the fi rst study which inhibitor on the rat orofacial formalin test. Results: The orofacial formalin has attempted to correlate such metabolic changes with altered tissue H2S responses exhibited two distinct phases with early (0-9 min) and continu- biosynthesis. We propose that disordered metabolism of H2S may play a ous prolonged (10-45 min) responses. Pretreatment with resveratrol (3-30 role in diabetes mellitus. mg//kg, i.p., 100 and 300 mg/rat) or naproxen (10-100 mg/kg, i.p., 100 and 300 mg/rat) signifi cantly depressed agitation behavior during late phase of the rat formalin test. In contrast, administration of rofecoxib (1-10 mg/kg, 7069 i.p., 30 and 100 mg/rat) had no effect on the agitation behavior in the rat formalin test. Conclusion: These data demonstrate that PGs synthesized by Effect of PDE5 inhibitors in diabetic neuropathy COX-1, but not COX-2 are involved in nociceptive transmission and play an important role during the rat orofacial formalin test and suggest that a Shrinivas K Kulkarni, University Institute of Pharmaceutical Sciences, COX-1 selective inhibitor may produce analgesic effect on the orofacial Panjab University, Chandigarh-160014, INDIA, India pain state in a clinical situation. Chandrashekhar S Patil, Panacea Biotec Ltd., Lalru, Punjab, INDIA, India VijayPal Singh, University Institute of Pharmaceutical Sciences, Panjab 7071 University, Chandigarh-160014, INDIA, India Effect of protein kinase C inhibitors in attenuation of thermal Diabetic neuropathy is one of the most frequent peripheral neuropathies hyperalgesia in STZ-induced diabetic mice associated with hyperalgesia and hyperesthesia. Besides alteration in the levels of neurotransmitter, alteration in the neuronal nitric oxide synthase Anjaneyulu Muragundla, Pharmacology Division, University Institute of (nNOS) is a key factor in the pathogenesis of diabetic neuropathy. The Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India, present study was aimed to evaluate the role of PDE5 inhibitor on nocicep- India tion in streptozotocin-induced diabetes in animal models of nociception Kanwaljit Chopra, Pharmacology Division, University Institute of (writhing assay in mice and paw hyperalgesia test in rats). Diabetic ani- Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India, mals showed a signifi cant decrease in pain threshold as compared to non- India diabetic animals in both the test, indicating diabetes induced hyperalgesia in mice and rats. PDE5 inhibitor, sildenafi l signifi cantly increased the pain Neuropathic pain is an important complication of diabetes and elevated threshold in both diabetic and non-diabetic animals. However, L-NAME, a protein kinase C activity has been reported in the vascular and neural com- non-specifi c NOS inhibitor and methylene blue (MB), a guanylate cyclase plications of diabetes. Thus, the aim of the present study was to explore an- inhibitor blocked the antinociceptive effect. The per se administration of tinociceptive effect of selective PKC inhibitor, GF109203X and its combi- L-NAME or MB augmented the hyperalgesic response in diabetic animals nation with natural antioxidant, quercetin with PKC inhibiting properties, with little or no effect in non-diabetic, indicating the alteration of NO- in attenuation hyperalgesia in streptozotocin (STZ)-induced diabetic mice. cGMP pathway in diabetes. The results in the present study demonstrate Diabetes was induced in mice by single intraperitoneal injection of STZ the decreased cGMP content in diabetic animals, which may be due to (200 mg/kg). After 4 weeks of STZ injection, diabetic mice were subjected down regulation of nNOS activity, suggesting the decreased nNOS-cGMP to tail-immersion and hot plate assay. Diabetic mice exhibited a signifi - system may play a crucial role in the pathogenesis of diabetic neuropathy. cant hyperalgesia as compared with control mice. GF109203X (0.3, 1 and 10 mg/kg, i.p) injected into diabetic mice produced signifi cant antinoci- ceptive effect in both the tail-immersion and hot-plate assays. Similarly, quercetin (50, 100 and 200 mg/kg, i.p) also produced dose dependent an- Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 193 tinociception in both the tail-immersion and hot-plate assays. The percent- Yousef Al-Abed, Laboratory of Medicinal Chemistry, North Shore Long age maximum possible effect produced by GF109203X and quercetin was Island Jewish Health System, United States observed at 60 min. The antinociceptive effect of GF109203X in diabetic mice was potentiated by quercetin in both test systems. The results of the A novel isoxazolyne compound, 3-phenyl-4,5-dihydro-5-isoxazoleacetic present study suggest that quercetin treatment potentiates antinociceptive acid is a recently developed drug designed for the treatment of those dis- effect of GF109203X in diabetes. Thus protein kinase C contributes to the eases which may be alleviated by the inhibition of TNF-alpha. Having in development of hyperalgesia in experimental diabetes; GF109203X is a mind the role for TNF-alpha in the pathogenesis of type 1 diabetes (T1D), candidate for further study in treatment of diabetic neuropathy. The results present study was conducted to investigate possible therapeutic value of also suggest that concomitant intake of quercetin along with GF109203X 3-phenyl-4,5-dihydro-5-isoxazoleacetic acid in the animal model of the may useful in the treatment of diabetic neuropathy. disease. Experimental T1D was induced in susceptible CBA/H mice by multiple low doses of streptozotocin (MLD-STZ, 5x40 mg/kg/day for 5 days). Prophylactic 12 day treatment with 3-phenyl-4,5-dihydro-5-isoxa- 7072 zoleacetic acid (0.5 mg/mouse/day), markedly reduced histopathological changes in the islets of pancreas and suppressed the development of hy- Neutralization of Macrophage Migration Inhibitory Factor activity perglycemia. Moreover, benefi cial effect of the drug was obtained even reduces local infl ammation during Type 1 diabetes development when it was administered under a therapeutic regime starting from the last of the 5 injections of STZ. Ex vivo analysis, performed during early Ivana Cvetkovic, Institute for Biological Research disease development revealed that the observed antidiabetogenic effect Danijela Maksimovic-Ivanic, Institute for Biological Research of 3-phenyl-4,5-dihydro-5-isoxazoleacetic acid could be attributed to the Djordje Miljkovic, Institute for Biological Research, Yugoslavia reduced proliferation and adhesion of islet Ag reactive lymphocytes, as Ferdinando Nicoletti, Department of Biomedical Sciences, University of well as to down-regulation of TNF-alpha, IL-1beta and inducible nitric Catania, Italy oxide synthase (iNOS)-mediated NO. Moreover, direct inhibitory effect Yousef Al-Abed, Laboratory of Medicinal Chemistry, North Shore Long of 3-phenyl-4,5-dihydro-5-isoxazoleacetic acid on TNF-alpha production Island Jewish Health System, United States was shown by using IFN-gamma + IL-1beta stimulated MIN6 and RIN-5F Stanislava Stosic-Grujicic, Institute for Biological Research insulinoma cells. However, neither IFN-gamma nor IL-10 was affected by the drug treatment. Therefore, 3-phenyl-4,5-dihydro-5-isoxazoleacetic Type 1 diabetes (T1D), an infl ammatory disorder of autoimmune origin, acid tends to favor the inhibition of several pro-infl ammatory and destruc- develops as a consequence of pancreatic beta cell destruction mediated tive factors, thus interfering with both infl ammation and tissue destruction. by various pro-infl ammatory mediators. Recent investigations suggest that 3-phenyl-4,5-dihydro-5-isoxazoleacetic acid may thus be considered as a pro-infl ammatory cytokine, macrophage migration inhibitory factor (MIF) candidate drug in developing a new therapeutic strategy for pharmacologi- plays an essential role in the pathogenesis of T1D, and that neutralization cal intervention against T-cell-mediated injury of endocrine pancreas. of MIF could prevent the development of the disease. We have previously demonstrated that in vivo MIF neutralization by a novel pharmacological inhibitor, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxasole acetic acid 7074 methyl ester (ISO-1) down-regulates the infl ammatory autoimmune pro- cess triggered in genetically susceptible strains of mice by multiple low Anti-hyperglycemic effect of latex of Calotropis procera in alloxan doses of streptozotocin (STZ). Since in STZ-induced T1D nitric oxide induced diabetes (NO) has been implicated in tissue destruction, the aim of the present study was to explore in more detail the immunomodulatory potential of ISO-1, Vijay L Kumar, Department of Pharmacology, All India Institute of Medi- with special emphasis on local NO-mediated infl ammatory pathway. Im- cal Sciences, Ansari Nagar, New Delhi - 110 029, India munohistochemical studies of pancreata performed in early disease de- Sanjeev Roy, Department of Pharmacology, All India Institute of Medical velopment phase revealed that ISO-1 reduced the expression of IL-18, a Sciences, Ansari Nagar, New Delhi - 110 029, India cytokine that drives IFN-gamma production. Similarly, pro-infl ammatory Raman Sehgal, Department of Pharmacology, All India Institute of Medi- cytokine, IL-1beta was also reduced by ISO-1 treatment. It is well estab- cal Sciences, Ansari Nagar, New Delhi - 110 029, India lished that IFN-gamma and IL-1beta induce NO production in infi ltrating Biswa M Padhy, Department of Pharmacology, All India Institute of macrophages as well as in beta cells thereby promoting beta cell death. Medical Sciences, Ansari Nagar, New Delhi - 110 029, India In accordance with down-regulation of pro-infl ammatory cytokine expres- sion, pancreatic islets of ISO-1-treated animals had lower expression of The latex of Calotropis procera, a wild growing plant possesses multifari- inducible nitric oxide synthase, an enzyme which catalyses NO produc- ous properties and has been used in traditional medicinal system for the tion. These results suggest that benefi cial effects of ISO-1 in T1D involve treatment of various diseases. It has been shown to possess free radical down-regulation of deleterious local iNOS pathway thus protecting pan- scavenging, antioxidant and potent anti-infl ammatory properties in various creata from the infl ammatory autoimmune attack. This study identifi es a experimental models. In view of the infl ammatory changes as well as oxi- new therapeutic strategy for prevention and/or treatment of T1D based on dative stress associated with the pathogenesis of diabetes mellitus and its selective inhibition of MIF activity by a novel chemical ISO-1. complications, we have evaluated the latex of this plant for its anti-hyper- glycemic and anti-oxidant properties in rat model of diabetes. Diabetes was induced in rats by a single intra-peritoneal injection of alloxan (150mg/kg) 7073 and the protective effect of daily oral dose of 100 and 400mg/kg of dried latex was evaluated for a period of 30 days. Blood glucose, body weight Anti-diabetogenic effect of 3-phenyl-4,5-dihydro-5-isoxazoleacetic and water consumption were recorded on day 0, 3, 7, 31 and hepatic gly- acid cogen and anti-oxidant parameters namely SOD, Catalase, GSH and lipid peroxidation were measured on day 31 after sacrifi cing the animals. Treat- Stanislava Stosic-Grujicic, Institute for Biological Research ment with DL produced a dose dependent decrease in blood glucose in Ivana Cvetkovic, Institute for Biological Research diabetic rats from 266.7 ± 41.3mg/dl to 136.8 ± 19.3 and 98.7 ± 10.8mg/dl Ljubica Harhaji, Institute for Biological Research, Yugoslavia at 100 and 400mg/kg doses (p<0.01). It also produced a dose dependent Dusan Popadic, Institute of Microbiology and Immunology, School of increase in hepatic glycogen levels from 3.3 ± 1.4mg/g to 7.5 ± 1.3mg/g Medicine, Yugoslavia and 14.4 ± 0.5mg/g tissue. DL afforded protection against oxidative stress Danijela Maksimovic-Ivanic, Institute for Biological Research and increased the levels of SOD, Catalase, and GSH and brought down Gianni Garotta, Ganial Immunotherapeutics, Delaware, United States the levels of TBARS. Further, it also prevented the loss in body weight Ferdinando Nicoletti, Department of Biomedical Sciences, University of in diabetic rats and brought down the daily water consumption to values Catania, Italy comparable to normal rats. The effi cacy of DL as anti-oxidant and as an S 194 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) anti-hyperglycemic agent was comparable to standard anti-diabetic drug, ERK 1/2 signaling involves activation of phospholipase C (PLC)/calcium glibenclamide. signaling, cAMP-dependent protein kinase A (PKA) and type 1 Ang II re- ceptors (AT1). Rat MCs were cultured to 80% confl uence and starved for 24 h before they were treated with vehicle, glucagon (1 nM), high glu- 7075 cose (30 mM), or Ang II (1 nM). Glucagon, high glucose and Ang II in- creased [3H]thymidine incorporation by 46%, 53% and 41%, respectively C-reactive protein is associated with adiposity in women but not in (p<0.01). The proliferative effects of glucagon were blocked by the spe- men in an Australian Aboriginal community cifi c glucagon antagonist, [Des-His1-Glu9] glucagon (1 μM), while those of high glucose or Ang II were inhibited by the ACE inhibitor captopril Tomer Shemesh, Menzies School of Health Research, Institute of Advan- (10 μM) or the AT1 receptor antagonist losartan (10 μM), respectively. The ced Studies, Charles Darwin University, Darwin NT, Australia effects of glucagon on MC proliferation were associated with two-fold in- Kevin G Rowley, The University of Melbourne, Dept. of Medicine, St. creases in MAP kinase ERK 1/2 phosphorylation (p-ERK 1/2) 5 min after Vincent’s Hospital, Fitzroy Vic, Australia glucagon stimulation (p<0.01). Total ERK1/2 was not affected by gluca- Alicia Jenkins, The University of Melbourne, Dept. of Medicine, St. gon. Glucagon-stimulated ERK 1/2 phosphorylation was blocked by [Des- Vincent’s Hospital, Fitzroy Vic, Australia His1-Glu9] glucagon, captopril, or losartan. Furthermore, pretreatment of Kerin O'Dea, Menzies School of Health Research, Darwin NT, Australia MCs with U73122 (1 μM), a selective PLC inhibitor, or with H89 (1 μM), a selective PKA antagonist, signifi cantly attenuated ERK 1/2 phosphory- Background lation induced by glucagon. These results suggest that glucagon binds to Median life expectancy in Aboriginal Australians is 20 years less than all- specifi c receptors to increase MC proliferation by stimulating MAP kinase Australian life expectancy. The premature adult mortality is largely due to ERK1/2 phosphorylation and that these effects of glucagon involve PLC-, cardiovascular disease (CVD). Atherosclerosis is an infl ammatory process, cAMP-dependent PKA- and AT1 receptor-activated signaling cascades. and high (>3.0 mg/L) circulating levels of C-reactive protein (CRP) has been proposed to identify people at high CVD risk. CRP is upregulated by pro-infl ammatory cytokines, including those released from adipose tissue. 7077 Circulating CRP levels have been shown to correlate with body fat, which is implicated in the development of diabetes and its complications. This Mercury Exposure among Residents of a building blacke relationship has not been evaluated in Indigenous Australians. Methods Abdollah Karimi, Shaheed Beheshti University,Pediatric InfectiousRes- In a cross-sectional study of 379 adults in a geographically-isolated coast- earch Center, Iran al Aboriginal community we tested the relationship between serum CRP, Abdolvahab Alborzi, Alborzi infectious research Center,Shiraz, Iran measured by nephelometry with measures of adiposity and body fat dis- tribution. Exposure to mercury can cause serious multiorgan damage affecting the Results central nervous system, kidneys, liver, lungs, spleen, bone marrow, and Median age (age range) was 35 (15-76) years and 54% were women. skin. At the end of the summer of 1999, the accidental leakage of 4 liters Mean (95% CI) CRP concentration was 3.7 (3.4, 4.1) mg/L. 59% of of mercury from a container into the waterway canals resulted in mass study participants had CRP levels >3.0 mg/L and only 11% had low CVD exposure to elemental mercury among the residents of a building block of risk CRP levels <1.0 mg/L. Compared to men, women had higher mean a residential area of the city of shiraz, in the south of Iran. One hundred and (95% CI) BMI 23.2 (22.5, 23.8) vs. 21.6 (20.8, 22.4) kg/m2 for women eleven individuals who experienced exposure to elemental mercury were and men respectively) and waist circumference (86.6 vs. 82.4 cm; both investigated. Twenty four hour measurement of the urine mercury level P<0.05) but similar waist-to-hip ratio (0.93 vs. 0.92; P=0.80) and CRP revealed a toxic level of more then 2 mg/L in 6 children and 3 adults ( in- levels (P=0.107). CRP levels increased signifi cantly across BMI and waist cluding a pregnant woman).despite normal physical and laboratory (CBC, quartiles in women (both P<0.001) but not in men (P=0.452 and P=0.118 renal and liver function testes , and urinalysis ) fi ndings, dimercaprol was respectively). prescribed. One month later during the course of the follow-up the urine Discussion mercury level in 6 patients, including the pregnant woman from the same High CRP levels are common in Indigenous Australians, and consistent family , was found to be again at a toxic level . The pregnant mother from with their recognized high premature CVD mortality. CRP was associated the same family aborted her fetus; however, due to the lack of equipment with measures of body fat and abdominal adiposity in women but not in for measuring the serum mercury level, it was not possible to confi rm the men. Other underlying factors, such as infection, dietary quality and psy- relation between the mercury toxicity and the abortion. This family had chosocial stress may promote chronic infl ammation. kept mercury in their kitchen against health workers’ instructions. The at- tractive physical and chemical properties of mercury could explain the continuity of exposure and poisoning in these 6 cases. It is concluded that 7076 prophylactic therapy in the presence of an asymptomatic state in exposed residents, is effective in preventing the development of signs and symp- Glucagon Induces Glomerular Mesangial Cell Proliferation toms, though instruction of high- risk cases is the best way to combat it. and MAP Kinase ERK 1/2 Phosphorylation by Interacting with 2002 elsevier science Phospholipase C, Protein Kinase A, and Angiotensin II Key Words: mercury; poisoning; toxiclevel; DMSA: dimercaprol Xiao C Li, Henry Ford Hospital, Detroit, MI 48202, United States Oscar A Carretero, Henry Ford Hospital, Detroit, MI 48202, United States 7078 Yuan Shao, Henry Ford Hospital, Detroit, MI 48202, United States Jia L Zhuo, Henry Ford Hospital, Detroit, MI 48202, United States The kynurenine pathway of tryptophan metabolism: multiple roles in the pathogenesis of cerebral malaria Glucagon plays a critical role in maintaining blood and tissue glucose ho- meostasis. Inappropriate increases in serum glucagon relative to insulin Nicholas H Hunt, Molecular Immunopathology Unit, University of may lead to the development of insulin resistance and target organ dam- Sydney, Australia ages in type 2 diabetes. We here tested the hypotheses that: a) glucagon in- Latifu Sanni, Molecular Immunopathology Unit, University of Sydney, duces proliferation of glomerular mesangial cells (MCs) through glucagon Australia receptor-activated mitogen-activated protein (MAP) kinase ERK 1/2 sig- Helen J Ball, Molecular Immunopathology Unit, University of Sydney, naling in synergy with high glucose and Ang II; and b) glucagon-induced Australia Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 195

Anna M Hansen, Molecular Immunopathology Unit, University of Syd- increases in size of capsaicin fl are (t(1,33)=3.65, p=0.001) with a trend ney, Australia to improvement in time to initial fl are response (t(1,34)=-1.86, p=0.07). Jenny Miu, Molecular Immunopathology Unit, University of Sydney, No changes were evident in the placebo group. The responders, where the Australia time to initial fl are response (p=0.02, r=0.64) and size of capsaicin fl are Silvia Weiser, Molecular Immunopathology Unit, University of Sydney, (p=0.00 r=1.0) improved signifi cantly after LF-SNS, were signifi cantly Australia younger, with a shorter duration of diabetes. Conclusions- Sensory nerve Roland Stocker, University of New South Wales, Australia function signifi cantly declines with age and diabetes. LF-SNS improves Isabelle M Medana, University of Oxford, United Kingdom nerve function in DPN. Targeting toward likely responders may deliver greatest benefi t from short-term therapy. Testing optimal application in Cerebral malaria (CM) is a life-threatening complication of Plasmodium others seems warranted. falciparum infection. A murine model, P. berghei ANKA (PbA) infection in CBA or C57 mice, mimics many features of the human disease (Hunt & Grau, Trends Immunol 24, 491, 2003). 7080 At the stage when PbA-infected mice exhibit the characteristics of CM, the activity of the rate-limiting enzyme of the kynurenine pathway of tryp- Angiogenesis: PECAM-1 as a marker of neuroprotection following tophan metabolism, indoleamine 2,3-dioxygenase (IDO), in the brain was hypoxia-ischaemia increased 40-fold compared to uninfected mice, and expression was con- fi ned to the endothelium. The ratio between the levels of two products of Andrew N Clarkson, Department of Pharmacology and Toxicology, Uni- this pathway, the neuroexcitotoxin quinolinic acid (QA) and its antagonist versity of Otago, New Zealand kynurenic acid (KA), in the brain also was signifi cantly increased. In the Hanzhong Liu, Department of Pharmacology and Toxicology, University cerebrospinal fl uid (CSF) of 261 Vietnamese adults with severe malaria of Otago, New Zealand (80% with defi ned CM), the mean QA level and QA:KA ratio were sig- Fredericke Schiborra, Department of Pharmacology and Toxicology, nifi cantly increased relative to UK controls. In 83 African children with University of Otago, New Zealand CM, the mean CSF QA level was increased compared to UK controls but David M Jackson, Department of Pharmacology and Toxicology, Univer- the mean QA:KA ratio was not. In both studies, increased CSF QA was sity of Otago, New Zealand statistically associated with fatal outcome. Ian Appleton, Department of Pharmacology and Toxicology, University Interferon-g (IFNg) is the major physiological inducer of IDO expression, of Otago, New Zealand and this cytokine is essential for the pathogenesis of experimental CM. IFNg-/- mice were protected against CM and their brain IDO activity did Angiogenesis due to an ischaemic episode resides both within and around not increase during PbA infection. the infracted tissue. The growth of new and protection of existing blood Activation of the kynurenine pathway in the brain in malaria may be pri- vessels following ischaemia may in part contribute to a better outcome with marily a host protective response, since metabolites have antioxidant, vas- an increase in oxygen and nutrient delivery to injured and compromised cular and anti-microbial actions in other systems. IDO activation in other tissues. Previous evidence has shown that CD31 is a marker of endothelial situations is important in immunomodulation. Furthermore, the kynuren- cells, which would implicate CD31 as a putative marker of angiogenesis ine pathway yields NAD, which becomes depleted in brain tissue in mu- and hence a marker of neurodegeneration. The present study utilized a rine CM. Distortion of brain kynurenine metabolism in CM might lead to modifi ed ‘Levine’ rat-pup model of hypoxia-ischaemia (HI) in order to alterations in the balance between QA and KA levels, which in turn could assess whether CD31 is a marker for neurodegeneration / neuroprotec- be a signifi cant factor in the pathogenesis of the disease. tion. Assessment of CD31 staining 7-days post-HI revealed a signifi cant increase in angiogenesis compared to non-intervention controls (P<0.001). Treatment with the GABAA receptor modulator, clomethiazole (CMZ; 7079 414mg/kg/day via osmotic minipumps), signifi cantly decreased the level of angiogenesis compared to HI + saline treatment (P<0.001) back to non- Sensory nerves and diabetic peripheral neuropathy: basic intervention control levels. Conversely, treatment with the non-selective assessments and a novel treatment nitric oxide synthase (NOS) inhibitor, L-NAME (5mg/kg/day), resulted in a marked decrease in angiogenesis compared to non-intervention con- Rajna Ogrin, National Ageing Research Institute, University of Melbour- trols (P<0.001). In our hands, L-NAME was shown to exacerbate the size ne, Australia of the ischaemic lesion (P<0.001), whilst CMZ was previously shown to Peteris J Darzins, Monash Ageing Research Centre, Monash University, provide lasting protection. The extent of cerebral ischaemia was shown Kingston Centre, Australia to be dependent of the changes in NOS activity levels. Treatment with Zeinab Khalil, National Ageing Research Institute, University of Mel- CMZ has been shown to increase NOS levels compared to HI + saline, bourne, Australia whilst L-NAME halted the HI-induce increase in NOS activity (P0.001). We conclude that CD31 may be used as a marker of neurodegeneration / This study examined changes in sensory nerve activity with age and diabe- neuroprotection for conditions of cerebral ischaemia. tes and assessed the effect of a novel treatment to improve sensory nerve function in diabetic peripheral neuropathy (DPN). First, we examined the effect of age and diabetes on sensory nerve function via assessing electri- 7081 cal cutaneous perception threshold (ECPT) in young, healthy old and old participants with diabetes. ECPT’s were higher in the old healthy and the The Study of CSF in Children With Meningitis -One Year at One old diabetes groups in all threshold values compared to the young group. Pediatric’s Hospital At 5Hz, the observed differences were statistically signifi cant in both old healthy and old diabetes groups, with greater signifi cance in the diabe- Fatemeh Fallah, Pediatric Infectious Research Center ,Shaheed Beheshti tes group. At 250 and 2000Hz the difference between the young and old University, Iran diabetes groups reached statistical signifi cance. Second, we assessed the Gita Eslami, Shaheed Beheshti University, Iran effect of Low Frequency Sensory Nerve Stimulation (LF-SNS) (Interna- Colin W Wright, Bradford University, United Kingdom tional Patent Application Number PCT/AU2004/001079: (Khalil, Z.)) ad- Hossein Goudarzi, Shaheed Beheshti Univ, Iran ministered for 12 weeks in a randomised, placebo controlled, double blind Farzauneh Jadali, Pediatric Infectious Research Center, Iran trial in people with DPN (35 active and 31 placebo). Between group’s comparisons showed the intervention group had trends to improvement in Purpose-Meningitis is the most dangerous disease in children and remains size of fl are response to capsaicin and time to respond to capsaicin. With- irreversible mental returdatio in them.The specifi c causes of meningitis in subject comparisons in the active LF-SNS group showed signifi cant S 196 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) can be divided in to two major diagnostic groups bacterial or viral men- Hiroyuki Tanaka, Japan ingitis . Patients& Methods-In this study we want to fi nd the prevalence of Bru- Asthma is a chronic infl ammatory disease characterized by variable bron- cellosis and TB meningitis in children that admitted to pediatric hospitals chial obstruction, hyperresponsiveness, and by tissue damage known as (Mofi d &Markaz Tebbi and Aliasghar) in Tehran & Shahryar in Karaj in airway remodeling. In the present study we demonstrate that interleukin one year .In this descriptive study the CSF of children with meningitis (IL)-5 plays an obligatory role in the airway remodeling observed in ex- is taken and send for counting of WBC &protein and glucose . Micro- perimental asthma. BALB/c mice sensitized by intraperitoneal injections biological and biochemical analysis and counting cells were done in 500 of ovalbumin and exposed daily to aerosol of ovalbumin for up to 3 wk, CSF samples from meningitis children from pediatric hospitals in Tehran develop eosinophilic infi ltration of the bronchi and subepithelial and peri- in one year. bronchial fi brosis. The lesions are associated with increased amounts of Results-We resulted 36 CSF samples were culture positive (7.2%) for such hydroxyproline in the lungs and elevated levels of eosinophils and trans- as gram positive bacteria (66.6%) and gram negative bacteria(30.5%). forming growth factor (TGF)-β1 in the bronchoalveolar lavage fl uid. After Two CSF samples were positive culture for mycobacterium bovis(5.6% 1 wk of allergen challenge, TGF-β is mainly produced by eosinophils ac- of total meningitis).Also two samples were positive culture for Brucella cumulated in the peribronchial and perivascular lesions. At a later stage abortus(5.6%). of the disease, the main source of TGF-β is myofi broblasts, identifi ed by Conclusion-The identifi cation of bacterial meningitis especially Tb and α-smooth muscle actin mAb. We show that all these lesions, including fi - Brucella meningitis, and treatment of them is very important in endemic brosis, are abolished in sensitized and allergen-exposed IL-5 receptor-null countries such as Iran.Many kind of microorganism cause meningitis,so mice, whereas they are markedly accentuated in IL-5 transgenic animals. we suggest applying sensitive methods to determine them especially my- More importantly, treatment of wild-type mice with neutralizing anti-IL-5 cobacterium and Brucella in diagnostic laboratory . antibody, administered before each allergen challenge, almost completely Keywords-Bacterial Meningitis-children-Tuberculosis-Brucellosis prevented subepithelial and peribronchial fi brosis. These fi ndings demon- strated that eosinophils are involved in allergen-induced subepithelial and peribronchial fi brosis probably by producing a fi brogenic factor, TGF-β1. 7082

Autoantibodies to brain tissue-specifi c proteins - prognostic markers 7084 of ischemic stroke Effect of lipoxygenase enzyme inhibition in radicular pain induced Natalya Yu Ruleva, Russian Cardiology Research Complex MH RF, by nucleus pulposus in rats Russia Tatyana K Lukova, Russian Cardiology Research Complex MH RF, Vijay P Singh, University Institute of Pharmaceutical Sciences, Panjab Russia University, Chandigarh 160 014, India Alexandr V Chugunov, Russian State Medical University, Russia Chandrashekhar S Patil, University Institute of Pharmaceutical Sciences, Pavel R Kamchatnov, Russian State Medical University, Russia Panjab University, Chandigarh 160 014, India Sergey Ph Dugin, Russian Cardiology Research Complex MH RF, Russia Shrinivas K Kulkarni, University Institute of Pharmaceutical Sciences, Michail Yu Martynov, Russian State Medical University, Russia Panjab University, Chandigarh 160 014, India Evgeniy I Gusev, Russian State Medical University, Russia Arachidonic acid metabolites, prostaglandins and leukotrienes are detected BACKGROUND. Activation of infl ammation and the release of brain tis- in clinical cases of herniated nucleus pulposus. However, little is known sue-spesifi c proteins (BTSP) in acute carotic ischemic stroke (CIS) is well about their role in the associated symptoms like radicular pain. The pres- established. The lesser is known about the induction of autoimmunity to ent study was aimed to examine the role of leukotrienes in animal model BTSP and its relation to the prognosis. METHODS. Samples of serum of hyperalgesia induced by application of autologus nucleus pulposus to were obtained from 27 patients with CIS (15 males; mean age - 64±6 sciatic nerve in the rats. Hyperalgesia was assessed employing noxious years). Plasma levels of autoantibodies (Aab) to neuron-specifi c enolase mechanical and thermal stimuli. Zileuton, a 5-lipoxygenase inhibitor dose (NSE), protein S100β, glial fi brillar acid protein (GFAP) were determined dependently (25-100 mg/kg, po) and indomethacin (2mg/kg, po), a non on the days 1-2 , 7 and 21 after stroke (ELISA). First group (n=15) had selective cyclooxygenase inhibitor signifi cantly (p<0.05) decreased me- good recovery on the day 21, death or absence of recovery (increase in chanical as well as thermal hyperalgesia on postoperative days 3,5, and European Stroke Scale - ESS, lesser then 5 points) were in the 2nd group 7 as compared to nucleus pulposus group. Further, co-administration of (n=12). zileuton (25mg/kg, po) with indomethacin (2mg/kg, po) enhanced anti-hy- RESULTS. The level of anti-NSE and anti-S100β Aab was stabile, where- peralgesic effect in both the paradigms as compared to effect per se. as concentration of anti-GFAP Aab was higher in the 1st groupe on the The present study thus suggested that leukotrienes as well as prostaglan- 1st (2,98±0,45 and 1,29±0,08 r.u.; p<0,005) and the 7th days (2,65±0,42 dins might play a signifi cant role in hyperalgesia induced by autologus nu- and 1,31±0,12 r.u.; p<0,05). On the day 21st the level of anti-GFAP Aab cleus pulposus in rats. The results suggested that dual inhibition approach decreased comparing with the starting (p<0,05) and was unsignifi cantly of 5-lipoxygenase and cyclooxygenase enzymes may prove benefi cial in higher, than in the 2nd group. In 5 patients from 1st group with low such conditions. basal anti-GFAP Aab (<2,0 r.u.), it increased on the 7th day (1,57±0,31 and 1,87±0,22 r.u.; χ2=12,3, p=0,015). The anti-GFAP Aab level on the day 7 correlayted with the ESS score on the day 21 (r=0,510; p=0,019). 7085 CONCLUSION. The increased level of anti-GFAP Aab in the fi rst week of CIS is positively associated with the good prognosis, but its physiological Neuroprotective effects of (-)-epigallocatechin gallate in the acute signifi cance is still unclear. treatment of cerebral ischaemia

Rosanna M Rahman, Department of Pharmacology and Toxicology, 7083 University of Otago, New Zealand Shiva M Nair, Department of Pharmacology and Toxicology, University Role of interleukin-5 and eosinophils in allergen-induced airway of Otago, New Zealand remodeling in mice Stephen C Helps, Department of Medical Biochemistry, School of Medici- ne, Flinders University, Australia Hiroichi Nagai, Gifu Pharmaceutical University, Japan Odette M Shaw, Department of Pharmacology and Toxicology, University Naoki Inagaki, Japan of Otago, New Zealand Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 197

Neil R Sims, Department of Medical Biochemistry, School of Medicine, centrated on the pivotal infl ammatory enzymes, arginase and nitric oxide Flinders University, Australia synthase (NOS), and its product, nitric oxide which breaks down to nitrite. Rhonda J Rosengren, Department of Pharmacology and Toxicology, A post-hoc Mann Whitney U test showed a signifi cant (P<0.05) decrease University of Otago, New Zealand in infarct volume in the melatonin group (46±8mm3) compared to controls Ian Appleton, Department of Pharmacology and Toxicology, University (155±33mm3). No signifi cant effects on arginase and NOS activity were of Otago, New Zealand observed. However, melatonin caused a signifi cant (P<0.001) decrease of nitrite levels. This study clearly demonstrated that melatonin is a key Stroke is a leading cause of mortality and morbidity worldwide. Currently, mediator in infl ammation-induced neurodegeneration. We conclude that there are no neuroprotective drugs approved for the acute treatment of the neuroprotective effects of melatonin are, in part, due to its free radical ischaemic stroke. Catechins are dietary polyphenolic compounds, which scavenging effects on nitric oxide. have proven themselves (in vitro and in vivo) to be potent antioxidants, free radical scavengers, as well as exhibiting anti-infl ammatory capabili- ties. Catechins have also shown neuroprotective effects following gluta- 7087 mate toxicity and most recently, in animal models of cerebral ischaemia. (-)-Epigallocatechin gallate (EGCG) is the most abundant catechin in Spatial distribution of COX-1, COX-2 and iNOS enzymes in the rat dietary consumption (~60%) and is arguably the most active polyphenol brain following hypoxia-ischaemia responsible for the majority of the benefi cial health effects elicited by green tea extracts. This study examined the neuroprotective effects and Odette M Shaw, Department of Pharmacology and Toxicology, University potential hepatotoxicity of EGCG in a rat model of transient focal cere- of Otago, New Zealand bral ischaemia. Male Sprague-Dawley rats (265-295g) were treated with Rosanna Rahman, Department of Pharmacology and Toxicology, Univer- either 50mg/kg of EGCG or saline i.p., immediately post-ischaemia and sity of Otago, New Zealand every day thereafter, in the fi lament insertion, middle cerebral artery oc- Andrew N Clarkson, Department of Pharmacology and Toxicology, Uni- clusion model of stroke. Animals were sacrifi ced 72h post-ischaemia and versity of Otago, New Zealand 2,3,5-triphenyltetrazolium chloride staining was used to quantify neuronal Brad A Sutherland, Department of Pharmacology and Toxicology, Uni- infarction. Plasma alanine aminotransferase (ALT) activity was used to versity of Otago, New Zealand determine hepatotoxicity. Spleen, kidney, liver and testes wet weights were Shiva M Nair, Department of Pharmacology and Toxicology, University also recorded. Total infarct volume was signifi cantly (P<0.05) reduced in of Otago, New Zealand the EGCG treated group as compared to controls. Analysis of the mean Ian Appleton, Department of Pharmacology and Toxicology, University infarct area showed signifi cant (P<0.05) decreases in slices 6 & 7 in the of Otago, New Zealand EGCG treated group. No signifi cant differences were found in ALT activ- ity or organ wet weights between treatment groups. Our fi ndings, in part, Following an ischaemic event there is an upregulation of the inducible validate and extend previous observations illustrating that 50mg/kg i.p. isoform of cyclooxygenase (COX-2) mRNA in the brain. Other studies EGCG is non-toxic and neuroprotective. However, we also found that have shown that there is an upregulation in the hippocampus and cortex of EGCG treatment appreciably increased (>50%) the risk of haemorrhagic COX-2 expressing cells in response to noxious stimuli. It has also been conversions. We therefore conclude that 50mg/kg EGCG is not a viable observed that there is a concomitant increase in COX-2 mRNA and the intervention for the acute treatment of cerebral ischaemia, as it is likely to inducible isoform of nitric oxide synthase (iNOS) expression in animal increase the risk of an intracerebral haemorrhage. models of stroke. The aim of this study was to determine the spatial and cellular distribution of COX-1 (the constitutive isoform of COX), COX-2 and iNOS proteins in the hypoxia-ischaemia (HI) model of neurodegenera- 7086 tion. HI was induced using a modifi ed “Levine” model by permanently ligating the left common carotid artery, followed 2 hours later by 1 hour The mechanism of the neuroprotective effects of melatonin in stroke- of hypoxia. Animals were sacrifi ced 3 days post HI. The distribution of induced brain damage COX-1 and COX-2 in the brain was determined by immunohistochem- istry. The colocalisation studies of COX-2 and iNOS were determined Shiva M Nair, Department of Pharmacology and Toxicology, University by double immunofl uorescence labelling. COX-1, in both control and HI of Otago, New Zealand brains, was uniformly expressed throughout all brain cells and regions. Rosanna Rahman, Department of Pharmacology and Toxicology, Univer- In the HI sections, COX-2 positive astrocytes, neuroglia, activated mi- sity of Otago, New Zealand croglia and hippocampal neurons were localised throughout the ispilateral Ian Appleton, Department of Pharmacology and Toxicology, University hemisphere. Furthermore, infi ltrating macrophages within the ischaemic of Otago, New Zealand penumbra were immunolabelled for COX-2. The double immunolabelling studies demonstrated high levels of COX-2 expression in the nucleus of One of the major goals for acute therapy in ischaemic stroke is to reca- cells which were colocalised with iNOS. From this we can determine that nalise the cerebral artery as soon as possible. However, the ensuing re- COX-2 expressing cells are mainly immune cells and distributed widely perfusion leads to an increase in free radicals, thereby exacerbating fur- throughout the ipsilateral hemisphere. COX-1 was distributed throughout ther neurodegeneration by oxidative stress. Currently, the only clinically the whole brain with no regional or cellular variations. COX-2 and iNOS approved therapeutic intervention for acute ischaemic stroke only target were colocalised within the ipsilateral hemisphere demonstrating the po- reperfusion of the ischaemic region, but do not otherwise aid in neuropro- tential neurodegenerative relationship between these two enzymes. Based tection. Melatonin, a hormone synthesized by the pineal gland, has been on our previous work, we have shown that COX-2 can have both pro-in- implicated in numerous physiological processes. Recently it has also been fl ammatory and anti-infl ammatory properties. It is therefore possible that shown to have neuroprotective effects against acute focal cerebral isch- during the time course of HI, COX-2 up regulation may not be detrimental. aemic damage; possibly by acting as a free radical scavenger. Hitherto, the However, the coexpression of iNOS and COX-2 is more indicative of the mechanisms of these neuroprotective effects have not been determined. greater neurodegenerative potential of the iNOS enzyme. Therefore, this study explored the mechanism(s) by which melatonin acts as an acute neuroprotective agent post stroke. Male Sprague-Dawley rats, 285±15g underwent a 2-hour transient middle cerebral artery occlusion by fi lament insertion. Based on previous observations, rats were treated with 5mg/kg i.p. melatonin or vehicle (5% DMSO in 0.9% saline) for 3 days. Brain damage was assessed histologically at 72 hours post stroke with the use of 2, 3, 5 - triphenlyltetrazolium chloride. As melatonin has previously been shown to exhibit anti-infl ammatory properties, we con- S 198 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

and 11th after ligature fi xation). The animals were then killed by cervical 7088 dislocation, the maxillae excised, fi xed in 4% formaldehyde, and decalci- fi ed in 10% EDTA. The specimens were dehydrated in graded alcohol and Spatial distribution of heme oxygenase isoforms 1 and 2 in the rat embedded in paraffi n for further cutting (4-6mm thickness). The images brain after middle cerebral artery occlusion and hypoxia-ischaemia of the slides were digitalized and special software analyzed alveolar bone and attachment loss as well as the total number of cells. Morphine admin- Brad A Sutherland, Department of Pharmacology and Toxicology, Uni- istration resulted in a signifi cant decrease in the attachment and alveolar versity of Otago, New Zealand bone loss both during disease onset and during its peak. However the same Rosanna Rahman, Department of Pharmacology and Toxicology, Univer- treatment did not account for a decrease in the total number of cells on sity of Otago, New Zealand the tissue surrounding the tooth with ligature. Besides the involvement of Andrew N Clarkson, Department of Pharmacology and Toxicology, Uni- opioid receptors, our results suggest that the benefi cial effect of morphine versity of Otago, New Zealand on PD development does not seem to derive from a direct action on local Odette M Shaw, Department of Pharmacology and Toxicology, University infl ammatory cells. of Otago, New Zealand Support: CNPq, CAPES, FAPEMIG Shiva M Nair, Department of Pharmacology and Toxicology, University of Otago, New Zealand Ian Appleton, Department of Pharmacology and Toxicology, University 7090 of Otago, New Zealand Regulation of infl ammation and autoimmunity in a murine model of Hypoxia-ischaemia (HI) and stroke initiate the activation of infl ammatory multiple sclerosis cascades that induce cell death within the brain. Heme oxygenase (HO), which breaks down heme is activated after an ischaemic event. The aim of Jonathan L McQualter, LaTrobe Univeristy, Australia this study was to determine the spatial distribution of the inducible isoform Thomas Kay, St Vincents Research Institute, Australia (HO-1) and the constitutive isoform (HO-2) in the brain following middle John A Hamilton, Royal Melbourne Hospital, Australia cerebral artery occlusion (MCAO) / HI. MCAO was achieved in 285g Claude CA Bernard, LaTrobe Univeristy, Australia male Sprague-Dawley rats by insertion of an intraluminal fi lament for 2 hours. HI was induced in 26 day old male Wistar rats by ligating the left The pathogenesis of multiple sclerosis (MS) and its experimental auto- common carotid artery and 2 hours later exposing the rats to 8% oxygen immune encephalomyelitis (EAE) animal model involves a co-ordinated for 1 hour. Animals were sacrifi ced 3 days after ischaemia and the spatial immune attack on the CNS which results in massive infl ammation and distribution of HO-1 and HO-2 was determined by immunohistochemistry. local tissue destruction, including demyelination and axon degeneration. In control brains, HO-1 was barely detectable. However, in MCAO and Autoreactive T cells, which are pivotal in the pathogenesis of most or- HI brains, HO-1 expression was present in monocytes and macrophages gan-specifi c autoimmune diseases, are thought to orchestrate this process. in the infarct as well as astrocytes, microglia and oligodendrocytes within However, disruption of the CNS micro-environment, including the local the penumbra. Neuronal populations such as the frontal cortex, caudate cytokine network, and sustained activation of the innate immune system putamen and the hippocampus were also labelled for HO-1 post MCAO is imperative for maintenance of the chronic infl ammatory response and and HI. HO-2 immunolabelling had little variation between control, hence perpetuation of the disease. We report that several key regulators MCAO and HI brains. HO-2 was constitutively expressed in endothelial of granulocyte and macrophage function play crucial roles in autoimmune and smooth muscle cells of most cerebral blood vessels. HO-2 was also CNS infl ammation. We have shown that GM-CSF-defi cient mice are resis- immunopositive in the neurons of many distinct brain regions such as cor- tant to actively-induced and passively transferred disease, associated with tex, hippocampus and thalamus. However, the expression within neuroglia an inability to sustain infl ammation in the CNS. In addition, mice treated was increased after MCAO and HI. Overall, HO-1 expression increased in with an anti-GM-CSF monoclonal antibody or vaccinated against GM- infl ammatory cells and neurons within the penumbra of the insult. HO-2 CSF are resistant to disease, while ablation of GM-CSF during the induc- does not alter its spatial expression in neurons after an ischaemic episode, tion phase has no effect on the disease course. These results suggest that but increases its expression in neuroglia. Therefore, HO-1 is an important there is an absolute requirement for GM-CSF during the effector phase of enzyme in the pathogenesis of ischaemic neurodegeneration. We suggest disease possibly in controlling the supply of monocytes and macrophages that the selective modulation of HO-1 activity could be a viable target to and regulating expression of other pro-infl ammatory molecules, such as prevent cell death within the brain. MCP-1. We also report that G-CSF-defi cient mice showed a two week delay in the onset of clinical symptoms of EAE, associated with a slower advancement of infl ammation into the CNS, even though the autoreactive 7089 response was unaffected, suggesting that G-CSF may be important for the initial recruitment of infl ammatory cells to the CNS and/or breakdown of Effects of morphine on periodontal disease development in rats the blood-brain barrier. These results may provide the basis for develop- ment of effective and more enduring immunotherapies for infl ammatory Cinthia Mara F Pacheco, Federal University of Minas Gerais, Brazil autoimmune diseases, and more specifi cally for MS. Celso M Queiroz Júnior, Federal University of Minas Gerais, Brazil Marcelo V Caliari, Federal University of Minas Gerais, Brazil Orivaldo A Rocha, Federal University of Minas Gerais, Brazil 7091 Janetti N Francischi, Federal University of Minas Gerais, Brazil Activin A in cerebrospinal fl uid is elevated in patients with meningitis Periodontal disease (PD) is a progressive infl ammatory condition of the or cerebral haemorrhages tooth-supporting tissues which derives from a subgingival accumulation of Gram-negative bacteria. Endogenous and exogenous opioids have been Sandra Ebert, University of Gottingen, Germany described as having important interactions with the immune system and David J Phillips, Monash University, Australia the controlling of infl ammation. Therefore the aim of this work was to Peter Jenzewski, University of Gottingen, Germany investigate the role of an opioid agonist, morphine on PD development Anne E O'Connor, Monash Universitry, Australia in rats. PD was induced by placing a sterile silk ligature around the cer- Uwe Michel, University of Gottingen, Germany vix of the second left upper molar. The contralateral right tooth was used as the unligated control side. Morphine (40 mg/kg/day) was administered Activin A is a member of the tranforming growth factor-beta superfamily by subcutaneous route for three consecutive days either at disease onset and is regulated at multiple levels, one of which is by binding to its high (3rd, 4th, and 5th days after ligature fi xation) or close to its peak (9th, 10th, affi nity binding protein, follistatin (FS). These proteins are produced in the Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 199 central nervous system (CNS) and we have previously shown that they are responsive to CNS infl ammation, such that FS is elevated in cerebrospinal 7093 fl uid (CSF) of patients diagnosed with meningitis, and activin is elevated in a rabbit model of meningitis. In this study, we analyzed activin and Involvement of the kynurenine pathway in neuroinfl ammation FS in matched CSF and serum samples from different groups of patients under various infl ammatory and pathological conditions using specifi c Gilles j Guillemin, Centre for Immunology, Australia immunoassays. In healthy persons, serum levels of both activin and FS Karen M Cullen, Anatomy and Histology, Australia were related to age (activin: r=0.42, p<0.0001; FS: r=0.32, p<0.0001). In George A Smythe, Australia CSF, levels of activin were also correlated with age (r=0.64, p<0.0001), Bruce j Brew, Australia whereas in more than 80% of these subjects, FS levels in CSF were below the limit of detection of the assay. Comparison of activin levels in CSF of The kynurenine pathway (KP) is a major route of L-tryptophan catabo- patients with various neurological diseases, including meningitis, chronic lism resulting in the production several neuroactive intermediates. Among infl ammatory CNS diseases, neurodegenerative diseases, malignancies in them, the N-methyl D-aspartate receptor agonist and excitotoxin QUIN is the CNS, hypoxic-ischemic insults, CNS haemorrhages and epilepsy, to perhaps the most important in term of biological activity. Within the brain, their respective age-and sex-matched controls, revealed signifi cantly el- QUIN is mainly produced by activated microglia and infi ltrating macro- evated levels of activin in meningitic (0.14 versus 0.10 ng/ml, p=0.032) phages. QUIN can use different mechanisms, direct or indirect, to exert its and cerebral haemorrhagic patients (0.16 versus 0.12 ng/ml, p=0.032). As neurotoxicity. Depending of its concentration, QUIN can lead to an acute expected, serum levels of both proteins were not affected by any of these neuronal death or to a chronically and progressive neuronal dysfunction diseases examined. by at least four mechanisms. We previously showed that chronic exposure We show for the fi rst time that, in normal subjects, activin and concentra- to QUIN in pathophysiologically appropriate concentrations causes ultra tions in CSF increase with age, but the reasons for this pattern are not well structural changes in cultured human neurons. We demonstrated that QUIN known. Furthermore, activin in CSF is increased in patients with menin- can lead to an amplifi cation of the infl ammatory response within the cen- gitis and cerebral haemorrhages. The latter fi ndings underline a role of tral nervous system through its ability to activate astrocytes and increased activin in acute infl ammatory processes, and extend them to include in- the production of chemokines. Recent fi ndings have shown that the KP fl ammation within the CNS. is one of the major regulatory mechanisms of the immune response. Two theories have been proposed: 1) that tryptophan depletion suppresses T cell proliferation by dramatically depleting the supply of this critical amino 7092 acid; 2) that some downstream KP metabolites act to suppress certain im- mune cells. For KP and QUIN to be involved in the neuropathogenesis of Control of severe malarial pathogenesis by the Natural Killer brain diseases, at least two features must be present: 1) neuroinfl ammation Complex and 2) activated monocytic cells. QUIN has been shown to be involved in several major neurodegenerative diseases including AIDS dementia Diana S Hansen, The Walter and Eliza Hall Institute of Medical Re- complex, stroke, epilepsy, and meningitis. We demonstrated recently that search, Australia QUIN is probably involved in the neuropathogenesis of Alzheimer’s dis- Krystal J Evans, The Walter and Eliza Hall Institute of Medical Research, ease and amyotrophic lateral sclerosis. Australia Nicholas J Bernard, The Walter and Eliza Hall Institute of Medical Research, Australia 7094 Louis Schofi eld, The Walter and Eliza Hall Institute of Medical Research, Australia Leukocyte Infi ltration into the Brains of Lupus-Prone MRL/faslpr Mice: Location and Phenotype of Infi ltrated Cells Both humans and experimental animals affected by severe malaria may suffer several disease syndromes including respiratory distress, metabolic Will G James, Monash Institute of Medical Research, Centre for Infl amm- acidosis, pulmonary oedema, anaemia and cerebral involvement. Pro-in- atory Diseases, Department of Medicine, Monash University, Australia fl ammatory cytokines such as IFN-? and TNF-? and immune system effec- Michael J Hickey, Monash University, Australia tor cells have been shown to contribute to the development of these syn- dromes. We have previously shown that CD1d-restricted NKT cells favour Systemic lupus erythematosus (SLE) is a systemic infl ammatory disease TH2 polarization and resistance to cerebral malaria in BALB/c mice and in which a wide range of organs, including the brain, is targeted by an in- induce early IFN-? production and promote disease in C57BL6 animals. appropriate infl ammatory response. To fully understand the infl ammatory NK and NKT cells from C57BL6 and BALB/c mice differ in expression mechanisms of SLE specifi c to the brain, we examined cerebral infl amma- of loci encoded by the Natural Killer Complex (NKC), located in chromo- tion in an animal model of SLE, the MRL/faslpr mouse. Previously we have some 6. The NKC encodes diverse stimulatory and inhibitory receptors ex- shown that leukocyte-endothelial cell interactions in the cerebral micro- pressed in NK and NKT cells, which determine the magnitude and charac- vasculature of MRL/faslpr mice increase as disease progresses. However, ter of innate immune responses. To study whether the NKC genotype can this study did not elucidate the types of leukocytes recruited to the brain. infl uence the susceptibility to severe malaria, congenic BALB.B6-Cmv1r Therefore the aim of this experiment was to characterise the leukocyte mice, bearing the NKC from C57BL6 background were challenged with populations present in the brains of MRL/faslpr mice during disease devel- P. berghei-ANKA. Unlike resistant BALB/c mice, BALB.B6-Cmv1r ani- opment. Immunohistochemical analysis of brains of MRL/faslpr mice indi- mals developed cerebral malaria and 50 % of the animals died. Histologi- cated that the choroid plexus villi and the surrounding ventricle walls were cal examination of brains from BALB.B6-Cmv1r animals showed cerebral the predominant site of leukocyte accumulation, with minor accumulation vasculature occluded with parasitised erythrocytes and infi ltrating macro- adjacent to meningeal vessels. To identify the types of leukocytes pres- phages and leukocytes. BALB.B6-Cmv1r mice also displayed increased ent, leukocytes were isolated from the brains of MRL/faslpr mice using a pulmonary oedema and signifi cantly higher severe anaemia compared to Percoll density gradient technique and characterised using fl ow cytometry. wild-type controls. BALB.B6-Cmv1r mice produced signifi cantly lower The phenotypes of circulating leukocytes were also characterised. At 16 parasite-specifi c IgG antibodies than BALB/c animals. IgM responses and 20 weeks of age, over 50% of leukocytes present in the brains were T were elevated in BALB.B6-Cmv1r mice at late time points post-infection, cells, comprising approximately equal proportions of CD4+, CD8+ and the suggesting a delayed switch to IgG production. The IFN-?/IL-4 ratio pro- characteristic MRL/faslpr CD4- CD8- (DN) T cells. However, the propor- duced by T cells from infected mice was signifi cantly higher in BALB. tion of infi ltrated DN cells was very low relative to the number of these B6-Cmv1r compared to BALB/c mice. Thus, polymorphism within innate cells in the circulation. MRL/faslpr mice defi cient in adhesion molecules system NKC loci regulates severe malarial pathogenesis and infl uences the P-selectin or ICAM-1 displayed only minor changes in the phenotype TH1/TH2 balance and B cell responses to infection. of infi ltrated T cells. These data show that as the infl ammatory response S 200 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) in these animals progresses, large numbers of lymphocytes infi ltrate the were detected in a neuronal population of the AD hippocampus (CA1) brain. However, the DN T cells characteristic of MRL/faslpr mice are less prone to neurofi brillary tangle formation and in neurons undergoing neu- effective at infi ltrating than CD4+ and CD8+ T cells. rofi brillary degeneration. Whole brain extracts of AD have not previously shown a signifi cant upregulation of QA, however the lesions of AD are microlocal and evolve over time. To confi rm the presence of QA in senile 7095 plaques and provide some quantitative estimates, we microdissected le- sions from AD hippocampus using laser capture microdissection and then Cytokine genotypes establish a role for infl ammation in anti- used gas chromatography-mass spectroscopy for QA detection. QA was retroviral toxic neuropathy (ATN) and predict an individual’s NRTI- enriched in plaque regions compared to hippocampal tissue of undiseased neuropathy risk brain and compared to non-plaque areas of AD tissue. These preliminary results provide a platform on which to assay the evolving presence of QA Patricia Price, University of Western Australia, Australia in the plaque. Our data show that QA is a candidate factor in the complex Kate Cherry, Burnet Institute for Medical Research and Public Health, and multi-factorial cascade of AD neurodegeneration. Tempering the ac- Australia tion of this excitotoxin with kynurenine pathway inhibitors may open a Ann Rosenow, University of Western Australia, Australia therapeutic door for patients in active stages of the disease. Martyn French, Royal Perth Hospital, Australia Steve Wesselingh, Burnet Institute for Medical Research and Public Health, Australia 7097

Sensory neuropathy is one of the commonest problems faced by people The Role of CXCR3 in Murine Cerebral Malaria living with HIV, with a prevalence of 44% in an Australian out-patient clinic. Distal sensory polyneuropathy (DSP) due to HIV itself is clinically Jenny Miu, University of Sydney, Australia indistinguishable from anti-retroviral toxic neuropathy (ATN) associated Andrew J Mitchell, University of Sydney, Australia with exposure to nucleotide reverse transcriptase inhibitors (NRTI), specif- Helen J Ball, University of Sydney, Australia ically d4T, ddI or ddC, as anti-retroviral therapy. This causes dilemmas in Iain L Campbell, University of Sydney, Australia patient management. Histological correlates of DSP in untreated patients Nicholas H Hunt, University of Sydney, Australia include increased numbers and activation of macrophages throughout the peripheral nervous system, but particularly at the distal nerve. Macrophage Cerebral malaria (CM) is a serious complication of Plasmodium falci- activation seen in DSP may also correlate with advancing HIV disease. parum infection, with patients presenting with neurological symptoms that Epidermal nerve fi bre is also lost in ATN, but infl ammation has not been can progress to death. The pathogenesis of CM is not well understood, examined. although it is believed to be largely host-mediated, with cytokines playing As not all individuals exposed to potentially neurotoxic NRTI develop a prominent role. ATN, host genotype can be used to assess the importance of infl ammatory The role of chemokines in CM is less clear, although recent work sug- pathways. We have analysed alleles of cytokine genes in DNA from indi- gests that some chemokine receptors expressed on brain-sequestered leu- viduals in our cohort. Subjects with no ATN despite ≥6 months of d4T, ddI kocytes may be crucial for the development of murine CM. Mice defi cient or ddC (n=28) and those with a defi nite (n=21) or probable (n=19)diagnosis in CCR5 (Belnoue, Blood 101: 4253, 2003), but not CCR2 (Belnoue, In- of ATN (neuropathy onset temporally related to d4T, ddI or ddC) could be fection and Immunity 71: 3648, 2003), are protected against murine CM. distinguished by alleles of TNFA and IL12B (p=0.023 and p=0.047, resp). Furthermore, there is increasing evidence supporting non-immune roles of Alleles of genes encoding IL-1, IL-4 and IL-6 had less pronounced effects. chemokines, such as during the development of the CNS, and in intercel- If confi rmed, these fi ndings will enable assessment an individual’s risk of lular communication. ATN prior to commencing NRTI and so guide the choice of drugs. They The expression of chemokines and their receptors was examined in a mu- will also facilitate an understanding of the cytokines involved and so aid rine CM model. Real-time PCR was used to determine the relative expres- the development of rationale immunotherapeutic strategies. sion of chemokine genes in the brain, with several chemokines and recep- tors found to be differentially expressed in the brains of mice presenting with CM, including CXCR3 and its ligands. Laser capture microdissec- 7096 tion was employed to determine the cellular source of these chemokines. Vessels were separated from parenchymal cells and the mRNA levels of Mass spectroscopic detection of quinolinic acid in microdissected candidate chemokine and chemokine receptor genes were assessed using Alzheimer’s disease plaques real-time PCR. The role of CXCR3 was investigated further with CXCR3-/- mice. A pro- Gilles J Guillemin, Centre for Immunology, Neuroimunnology Depart- portion of mice defi cient in CXCR3 were able to recover from the cerebral ment, St Vincent's Hospital, Sydney, Australia symptoms of murine CM, and did not present with any histopathological Bruce j Brew, Australia features typical of CM. Furthermore, these mice did not up-regulate genes Claire E Noonan, Department of Anatomy and Histology, The Institute thought to be important in the pathogenesis of CM such as CD8 and LT-α, for Biomendical Research, The University of Sydney, Australia as observed in wild-type mice. Toby G Knight, The Institute for Biomendical Research, The University of Chemokines, including CCL5/RANTES and CXCL10/IP-10, as well as Sydney, Australia chemokine receptors such as CXCR3, are up-regulated during murine CM. George Smythe, Biomedical Mass Spectrometry Facility, University of Our results suggest that CXCR3 plays a role in the pathogenesis of murine New South Wales, Australia CM. Karen M Cullen, Anatomy and Histology, Australia

Infl ammation is a prominent feature of Alzheimer’s disease (AD) pathol- 7098 ogy. The kynurenine pathway of tryptophan degradation is activated in in- fl ammation. One of the end products of the pathway, quinolinic acid (QA), Hydrogen Sulphide: A novel anti-infl ammatory neuromodulator? is a potent excitotoxin and is produced by macrophages and microglia. In our study of AD patients, we investigated the presence of QA using immu- Matt Whiteman, National University of Singapore, Singapore nohistochemistry and mass spectroscopy. In AD tissue QA-immunoreac- Madhav Bhatia, National University of Singapore, Singapore tivity was detected in cortical microglia, astrocytes and neurons, with mi- Philip K Moore, National University of Singapore, Singapore croglial and astrocytic expression of QA highest in the perimeter of senile plaques. Interestingly, QA-immunopositive granular neuronal inclusions Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 201

Hydrogen sulphide (H2S) is a cytotoxic gas that has recently been pro- posed as a novel neuromodulator. Endogenous levels of H2S in the brain 7100 range between 50 and 160 microM, and considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, a neuro- Microglial Cell-Associated Activating and Inhibitory Myeloid degenerative and neuroinfl ammatory disease. Elevated levels of levels of Receptors Characterize a Unique Molecular Fingerprint that Defi nes myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) are Blood to CNS Transition and Underlies Maintenance of Microglial elevated in the prefrontal cortex, hippocampal microglia, and neurons of Cell Homeostasis AD patients where they co-localise with beta-amyloid plaque deposition. These proinfl ammatory enzymes catalyse the formation of cytotixic reac- Craig A Murphy, DNAX Research Institute, United States tive chlorine (RCS) species such as hypochlorous acid (HOCl) and nitro- Barbara Joyce Shaikh, DNAX Research Institute, United States gen species (RNS) such as peroxynitrite (ONOO-) in the brain. Recently Joseph H Phillips, DNAX Research Institute, United States 3-chlorotyrosine and 3-nitrotyrosine, bio-markers for RCS and RNS pro- Jonathon D Sedgwick, DNAX Research Institute & Eli Lilly and Compa- duction respectively, were shown to be markedly elevated in hippocampal ny, United States proteins from AD patients. Since H2S, HOCl and ONOO- are important mediators in brain function and disease, we investigated the effects of H2S Despite being of similar lineage, central nervous system (CNS) microglia on HOCl- and ONOO- -mediated damage to bio-molecules and to human are distinct functionally from other macrophage subsets. Signifi cantly, the neuronal cells in culture. H2S signifi cantly inhibited HOCl-and ONOO-- molecular basis of cell-cell interactions regulating function and activation mediated inactivation of alpha(1)-antiproteinase, protein oxidation, nitra- of myeloid lineage cells is now coming into focus. Myeloid regulatory tion and chlorination to a comparable extent to reduced glutathione. H2S surface proteins mediate effects through inhibitory and activating recep- also inhibited HOCl- and ONOO--induced cytotoxicity, oxidative intra- tors, many of which utilize immunoreceptor-tyrosine inhibition (ITIM) cellular protein modifi cation, and lipid peroxidation in human neuroneal or activation (ITAM) motifs. The trigger receptor expressed on myeloid cells. These data suggest that H2S has the potential to act as an inhibitor of cells (TREM) family, amongst others, employ the ITAM-containing RCS and RNS-mediated processes in vivo and that the potential antioxi- DNAX adapter protein (DAP)12 to signal cellular activation. In contrast, dant action of H2S deserves further study, especially in neuroinfl ammatory CD200R, despite the absence of a classic ITIM, is an inhibitor of my- and neurodegenerative diseases where the already very low extracellular eloid function. Here we show that signaling via CD200R also suppresses levels GSH are further depleted by oxidative, nitrosative and chlorinative microglial cell function. Moreover, through gene expression profi ling of stress. adult microglia and other brain macrophages we identify a number of mol- ecules, including novel activating receptors, which characterize a unique molecular fi ngerprint for microglial cells. That these molecules regulate 7099 microglia in situ is likely.

Microglial activation and the staging of Alzheimer plaques: A post- microhaemorrhage rationale 7101

Karen M Cullen, Anatomy and Histology, Australia Altered oxidative burst in peripheral and in vivo transmigrated Claire E Noonan, Department of Anatomy and Histology, The Institute leukocytes in atherosclerotic patients for Biomendical Research, The University of Sydney, Australia Josefi n M Paulsson, Institution of Medicine, department of Clinical Infl ammation is a prominent feature of Alzheimer’s disease (AD). Senile Immunology and Allergy, Karolinska Institute, Sweden plaques contain a myriad of infl ammation-related factors and microglia, Elham Dadfar, Institution of Medicine, department of Clinical Immunolo- which have been attributed to the presence of various species of ß-amyloid gy and Allergy, Karolinska Institute, Sweden (ßA) and to dystrophic neurites. Microglia may also contribute to the pro- Claes Held, Department of Cardiovascular Medicine, Karolinska Univer- cessing of ßA into neurotoxic forms. The differential association of acti- sity Hospital, Sweden vated microglia with various plaque types has generated numerous hypoth- Stefan Jacobson, Department of Nephrology, Karolinska University eses on infl ammation in AD. Our work has shown that senile plaques are Hospital, Sweden the sites of microhaemorrhage and are always located perivascularly. These Joachim Lundahl, Department of Clinical immunology & Transfusion fi ndings allow us to make a direct analysis of the plaque/microhaemorrhage medicine, Karolinska University Hospital, Sweden domain and microglial activation. We assessed formalin-fi xed brain tissue from 10 AD patients, including two with Down syndrome, 5 age-matched Atherosclerosis is characterized by chronic infl ammation and enrichment controls. On 45μm sections of tissue, we used markers for blood vessels of infl ammatory cells in the vessel wall. Events leading to cellular accumu- (collagen IV, UEA lectin, von Willebrand factor), smooth muscle and peri- lation and activation are therefore of special interest. We hypothesized that cytes, microglia and macrophages (ferritin, HLADr), ßamyloid and tau, as leukocyte transmigration and activation in patients with atherosclerosis is well as haem histochemistry to determine the association of microglia with dysregulated, contributing to plaque increase. To test this hypothesis, we microhaemorrhages/plaques. In Nissl-stained sections, plaques were clas- analysed in vivo transmigration of leukocytes and cellular activation after sifi ed according to cellular content. The degree of microglial activation in in vitro stimulation. AD differed dramatically to controls. Although there was a global recruit- We applied the skin blister model to study cells that transmigrate in vivo. ment of cells, the greatest microglial activation was clustered around mi- Blisters are raised by suction and cellular functions are measured the fol- crovessels and corresponded to hypercellular plaque lesions. Morphology lowing morning and after ten hours of incubation with either PBS or au- of the microglia within the plaque domain varied, with enlarged, newly tologous serum, corresponding to intermediate and intense infl ammation. activated phagocytic microglia associated with vascular debris and hyper- Peripheral blood is collected at both time points. Functional aspects were cellular plaques and post-active microglia associated with older, hypocel- measured by the production of oxygen radicals before and after stimula- lular plaques.We hypothesise that the highest level of microglial activation tion. (HLADr-positive with a phagocytic morphology) is related principally to Basal levels of radical production were the same in both patients and con- active plaques, although old lesions sustain hyperplasic cells. Based on trols. However, peripheral monocytes and granulocytes from atherosclero- evidence that the plaque is a microhaemorrhage and that the evolution of sis patients had a signifi cant (p<0,05, Tukey HSD) reduced production of the lesion parallels larger vessel breaches, we suggest that the active lesion oxygen radicals after in vitro stimulation with PMA at both time points. in AD can be identifi ed by the high cellular infi ltrate around capillaries. In vivo transmigrated granulocytes from patients shoved a tendency (non These fi nding may help to identify both the degree of active lesions and the signifi cant) to lower radical production while transmigrated monocytes did rate of progression of the plaque in AD. not differ between the two groups. S 202 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

In this work we demonstrate that peripheral leukocytes from atheroscle- hypercholestoremia in rabbits, the profi le of lipoproteins was determined. rotic patients have a reduced response to exogenous stimuli. However, Group I rabbits were fed on high cholesterol diet and received nimesulide once the cells have transmigrated to sites of infl ammation this difference, (a COX-2 inhibitor) and Group II was fed on high cholesterol diet and re- compared to healthy controls, is restored. Therefore, the relative decrease ceived saline. Blood samples were taken at week 0,2,4,6,8,10,12, and 14 af- in oxygen radical between blood and tissue is lower in atherosclerotic pa- ter feeding the rabbits with high cholesterol diet. TAS and GPX were mea- tients, probably due to the chronic infl ammation. The impact of these dif- sured using commercially available enzyme assay kits. The results show ferences on infl ammatory processes in the endothelial lining needs further that TAS levels in Group I rabbits were 1.87,1.69,1.90,2.09,2.10,1.95,1.88 studies to be delineated. and 2.11 mmol/l of plasma at week 0,2,4,6,8,10,12, and 14 respectively as compared to Group II rabbits with TAS levels of 1.22,1.00,1.18,1.15,1.29, 1.36,1.19 and 1.29 mmol/l of plasma on the same time points. Similarly, in 7102 rabbits of Group I GPx levels were 3564,3425,3625, 3456,3542,3561,3724 and 3245 U/ liter of the blood at week 0,2,4,6,8,10,12, and 14 respectively The Detection of Cytomegalovirus And Helicobacter Pylori compared to 2967,2879,3067,2709,2897,2781,3037 and 2675 U/liter of in Athrosclerotic Plaque in Patients With Coronary Artery the Group II rabbits on corresponding sampling points. These results are Disease(CAD) indicative that TAS and GPx during hypercholestoremia treated rabbits with nimesulide were signifi cantly higher (p< 0.05) as compared to saline- Gita Eslami, Shaheed Beheshti University, Iran treated rabbits on all corresponding points. These results demonstrate that Fatemeh Fallah, Pirc of Shaheed Beheshti University, Iran nimesulide may have antioxidant and cardioprotective effects in athero- Hossein Goudarzi, Shaheed Beheshti University, Iran sclerosis. Bahram Kazemi, Biology Molecular Center, Iran

Cytomegalovirus and Helicobacter pylori can cause persistent and chronic 7104 infections of the organs related to transplant and gastrointestinal tract, respectively. Interleukin-10 Inhibits Infl ammatory Cell Recruitment and It has been suggested that persistent infection arteries specially coronary Intimal Hyperplasia and Enhances Endothelial Recovery after artery with these mircorganisms can contribute to the development of ath- Arterial Injury: Evidence for Two Distinct, Gene Specifi c Molecular erosclerosis .The aims of this study are to determine the presence of cyto- Mechanisms for IL-10 Suppression of Infl ammatory Cytokine megalovirus and H.pylori genome in atherosclerotic plaques by PCR and Synthesis also H.pylori by culture. Method and Patients-In this descriptive study One hundred two (102) pa- Evelyn Bord, Caritas St. Elizabeth's Medical Center, Tufts University tients (71 Male,31 Female) that died for myocardic infarction related to School of Medicine, Boston, USA coronary atherosclerosis were included in the study.After sampling ,they Corinne Luedemann, Caritas St. Elizabeth's Medical Center, Tufts Uni- transported to molecular biology and bacteriology-virology laboratory foe versity School of Medicine, Boston, USA diagnosis of cytomegalovirus and helicobacter pylori. Atsushi Iwakura, Caritas St. Elizabeth's Medical Center, Tufts University Results-Cytomegalovirus genome was found in 18(18%) of 102 specimens School of Medicine, Boston, USA and H.pylori genome was found in 34 (33%) of 102 specimens.H.pylori David Goukassian, Caritas St. Elizabeth's Medical Center, Tufts Univer- just isolated on culture media in one of 34 samples positive by PCR(3%) . sity School of Medicine, Boston, USA Prevalence of known risk factors and their correlation to cytomegalovirus Marcy Silver, Caritas St. Elizabeth's Medical Center, Tufts University in our study revealed LDL more than 160 mg/dl (75%),HDL less than 30 School of Medicine, Boston, USA mg/dl(65%), Hypertention(24%),Diabetes melitus (26%),Smoking(45%) Douglas Losordo, Caritas St. Elizabeth's Medical Center, Tufts University and family history of heart disease(33%). School of Medicine, Boston, USA Prevalence of known risk factors and correlation to helicobacter pylori in Raj Kishore, Caritas St. Elizabeth's Medical Center, Tufts University our study revealed LDL more than 160 mg/dl (70%),HDL less than 30 mg/ School of Medicine, Boston, USA, United States dl (65%), Hypertention (24%),Diabetes melitus (24%),Smoking(44%) and family history of heart disease (40%). Infl ammation plays an essential role in vascular injury and repair. Mono- Seven of atherosclerotic plaques showed coexistence of both of the miro- nuclear phagocytes participate in this process, partly via adhesive inter- organisms genome. actions and secretion of pro-infl ammatory cytokines. These functions are Conclusion-The presence of cytomegalovirus and helicobacter pylori ge- dependent upon new gene expression, which refl ects the infl ammatory cell nome in atherosclerotic plaques supports the idea that they may have a response to stimuli encountered in the arterial environment. Anti-infl am- role in the development of atherosclerosis ,especially in countries where matory cytokine IL-10 suppresses such responses by repressing infl amma- infection is prevalent. tory gene expression. IL-10 knockout mice develop atherosclerosis and exhibit a severe infl ammatory phenotype. The mechanisms of IL-10 sup- pressive action are, however, incompletely characterized. Here we report 7103 that in monocytes IL-10 suppressed LPS-induced mRNA expression of a number of infl ammatory cytokines via at least two distinct, gene spe- Nimesulide Inhibits Oxidative Stress in Hypercholestoremia cifi c mechanisms: mRNA degradation (e.g. TNFa) and reduced transcrip- tion (e.g. chemokine IP-10). Further studies using TNF mRNA sequences Sheikh A Saeed, Dr. Panjwani Center for Molecular Medicine and Drug linked to reporter gene revealed that A+U rich elements (ARE) in the 3’ Research, International Center for Chemical Sciences, University of untranslated region (UTR) are necessary for IL-10-mediated TNF mRNA Karachi, Pakistan destabilization. IL-10 mediated TNF mRNA instability depends on the Sagheer Ahmed, Dr. Panjwani Center for Molecular Medicine and Drug ability of IL-10 to inhibit LPS-induced activation of p38 MAP kinase: Research, International Center for Chemical Sciences, University of inhibition of p38 MAPK abrogated IL-10 sensitivity to TNF. Transcrip- Karachi, Pakistan tional inhibition of IP-10 gene by IL-10, however appears to be indirect and depends upon the inhibition of LPS-induced expression of interferon Oxidized Low Density Lipoproteins (LDL) is believed to migrate across b. Furthermore, IL-10 co-treatment abrogated TNF-induced inhibition of the endothelial membranes into the arterial wall to initiate the processes endothelial cell (EC) proliferation and signifi cantly diminished TNF-in- of ischaemic heart disease. This may be associated with reduced levels of duced binding of monocytes to EC, in vitro. Additionally in in vivo stud- glutathione peroxidase (GPx) and total antioxidant status (TAS). Cyclo- ies, systemic IL-10 treatment of mice following carotid artery denudation oxygenase-2 (COX-2) is expressed by endothelial cells, smooth muscle blunted infl ammatory cell infi ltration, TNF expression and neo-intimal cells, and macrophages in atherosclerosis. Using a model of experimental thickening while signifi cantly accelerating re-endothelialization. Because Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 203

IL-10 function and signaling are important components for control of in- Purpose:Several infl ammation-related markers, or acute phase reactants, fl ammatory responses, our results, by providing a better understanding of have been identifi ed to be associated with atherosclerosis. According to the molecular mechanisms of infl ammatory gene regulation by IL-10, may previous study there is a link between serum bilirubin and atherosclerosis. provide insights necessary to develop strategies for modulating vascular The links of infl ammation markers of atherosclerosis and bilirubin are as- repair and other accelerated arteriopathies, including transplant vasculopa- sessed. thy and vein graft hyperplasia. Methods:We studied 145 individuals with coronary artery disease (n=92) and controls without coronary artery disease (n=53). The whole blood samples were collected. Hs-CRP and bilirubin are assayed to clarify the 7105 concentration-related correlation with coronary artery disease. Results:The average concentration of hs-CRP in the coronary artery dis- Chronic Ethanol Synergistically Exacerbates the Negative Inhibitory ease group is 12.44±4.30 mg/l,whereas in the control group 1.35±0.21 Effects of TNF on Endothelial Cell Growth and Function mg/l. The average concentration of bilirubin in the coronary artery disease group is 0.49±0.21 mg/dl,whereas in the control group 0.96±0.36 mg/dl. Corinne Luedemann, Caritas St. Elizabeth's Medical Center, Tufts Uni- The levels of bilirubin in control patients are statistically higher than those versity School of Medicine of the coronary artery disease group, while the levels of hs-CRP in controls Evelyn Bord, Caritas St. Elizabeth's Medical Center, Tufts University are signifi cantly lower compared to the coronary artery disease group. School of Medicine Conclusions:This study shows that bilirubin may offer benefi cial effects in Gangjian Qin, Caritas St. Elizabeth's Medical Center, Tufts University protecting from atherosclerosis, whereas the correlations between bilirubin School of Medicine and hs-CRP are reverse. Descendant levels of bilirubin appear to be a risk Yan Zhu, Caritas St. Elizabeth's Medical Center, Tufts University School factor of atherosclerosis. The relative risk of bilirubin on atherosclerosis of Medicine needs to be further assessed. David Goukassian, Caritas St. Elizabeth's Medical Center, Tufts Univer- sity School of Medicine Douglas Losordo, Caritas St. Elizabeth's Medical Center, Tufts University 7107 School of Medicine Raj Kishore, Caritas St. Elizabeth's Medical Center, Tufts University The Opposing Roles of Monocytes in atherosclerosis School of Medicine, United States Heather J Medbury, Vascular Biology Research Centre, Surgery, Univer- Despite the reported cardio-protective effects of low alcohol intake, chron- sity of Sydney, Westmead Hospital, Australia ic alcoholism remains a major risk factor in the pathogenesis of coronary Ann K Guiffre, Vascular Biology Research Centre, Surgery, University of artery disease. Dose related bimodal effects of alcohol on cardiovascular Sydney, Westmead Hospital, Australia system might refl ect contrasting infl uences of light vs. heavy alcohol con- Michelle W Williams, Vascular Biology Research Centre, Surgery, Uni- sumption on the vascular endothelium. Chronic ethanol induced damage versity of Sydney, Westmead Hospital, Australia to various organs has been linked to the increased release of TNF, both Mauro Vicaretti, Vascular Biology Research Centre, Surgery, University in humans and in alcohol fed animals. We have previously shown that of Sydney, Westmead Hospital, Australia TNF, expressed at the sites of arterial injury, suppresses re-endothelializa- John P Fletcher, Vascular Biology Research Centre, Surgery, University tion of denuded arteries and inhibits endothelial cell (EC) proliferation in of Sydney, Westmead Hospital, Australia vitro. Here we report that exposure of EC to ethanol alone (25mM/48h) induced TNF mRNA and protein expression, which was substantially The stability of an atherosclerotic plaque is a key-determining factor in the upregulated in agonist (LPS)-treated EC. Nuclear run-on and actinomy- clinical outcome of cardiovascular disease. In this respect, alpha smooth cin D chase assays revealed that the ethanol-mediated increment in TNF muscle cell (SMC) actin positive cells play a crucial role in maintaining expression refl ected increased de novo transcription and not increased plaque stability through the formation of a fi brous cap. Recent evidence mRNA stability. DNA binding assays utilizing oligonucleotides encom- in mouse models suggests that circulating progenitors may be a source passing cis-elements in the TNF promoter showed that ethanol-induced of these cells. We examined 60 human carotid endarterectomy specimens TNF upregulation was AP1 dependent. Furthermore, exposure to ethanol for the presence of progenitor derived cells and investigated in vitro the potentiated and prolonged agonist-induced activation of p38 and JNK possible origin of these cells. CD34 +ve cells were found in the fi brous MAP kinases. Inhibition of JNK kinase by over-expression of dominant cap of 7 samples, specifi cally in regions of healing of a recent rupture. negative JNK1 substantially attenuated ethanol-mediated augmentation in They possessed the typical SMC spindle shape and some of these cells TNF transcription, suggesting modulation of JNK1 signaling as the mech- co-stained for alpha SMC actin. We suggest that these cells are fi brocytes, anism for ethanol-induced increments in TNF expression. Functionally, a blood derived cell known to acquire SMC like functions in other models TNF induced EC dysfunction, including reduced proliferation, migration of wound healing. Consistent with this, they co-localised with TGF beta a and cyclin A expression, were all markedly enhanced in the presence of factor known to promote fi brocyte formation as well as plaque stability. In TNF. Additionally, expression of cyclin D1 was signifi cantly attenuated in contrast there was minimal CD154 present in the fi brocyte area. In vitro cells co-treated with TNF and ethanol while each treatment alone had little we confi rmed that monocytes can transform into fi brocytes, and found that effect on cyclin D1 expression. Taken together, these data indicate that platelets can induce this transformation. This is consistent with the platelet chronic ethanol consumption not only elevates TNF expression but also rich environment upon plaque rupture. We suggest that monocytes play a synergistically exacerbates the inhibitory effects of TNF on EC growth and more crucial role in atherosclerosis than previously realized as they may functions and suggest that chronic ethanol consumption may negatively contribute directly, not just to foam cell formation and therefore plaque in- infl uence post angioplasty re-endothelialization thereby contributing to the stability, but also to fi brocyte formation and hence plaque stability. Under- development of restenosis. standing which factors affect monocyte differentiation down either path- way will elucidate mechanisms by which stable versus unstable plaque are formed. Targeting these mechanisms has the potential to decrease cardio- 7106 vascular events such as myocardial infarction and stroke.

Infl ammation in atherosclerosis

Meng-Jung Chen, Chi-Mei Medical Center, Taiwan Chia-Jung Hsieh, Chung-Hwa Colloge of Medical Technology, Taiwan S 204 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

Jacek Moll, Department of Pediatrics Cardiosurgery, Institute of Polish 7108 Mother’s Health, Lodz, Poland, Poland Maciej Moll, Department of Pediatrics Cardiosurgery, Institute of Polish Analysis of secretory leukocyte protease inhibitor (SLPI) Mother’s Health, Lodz, Poland, Poland concentrations and neutrophil functions in children after Andrzej Sysa, Department of Pediatrics Cardiology of Institute of Polish cardiopulmonary bypass Mother’s Health, Poland Krzysztof Zeman, Department of Pediatrics, Prevention of Cardiology Jarek Pasnik, Department of Pediatrics, Prevention of Cardiology and and Clinical Immunology, Medical University, Poland Clinical Immunology, Medical University, Lodz, Poland, Poland Jadwiga A Moll, Department of Pediatrics Cardiology, Institute of Polish Purpose: Children undergoing cardiac operations for congenital heart dis- Mother’s Health, Lodz, Poland, Poland eases show systemic infl ammatory reaction syndrome that jeopardizes the Lech T Pokoca, Department of Pathophysiology and Clinical Immunolo- postoperative outcome. Matrix metalloproteinase-9 (MMP-9) and metal- gy, Medical University, Lodz, Poland, Poland loproteinase-2 (MMP-2) are enzymes involved in cytokine processing and Jacek Moll, Department of Pediatrics Cardiosurgery, Institute of Polish leukocyte extravasation. These are secreted as a pro-enzyme in response Mother’s Health, Lodz, Poland, Poland to several infl ammatory mediators and are inhibited by tissue inhibitor of Jerzy Arendarczyk, Department of Pediatrics, Prevention of Cardiology metalloproteinase-1 (TIMP-1) and tissue inhibitor of metalloproteinase-2 and Clinical Immunology, Medical University, Lodz, Poland, Poland (TIMP-2). To explore metalloproteinase activation during cardiac surgery Wojciech W Mazurowski, Department of Pediatrics, Prevention of we investigated MMP-9, MMP-2, TIMP-1 and TIMP-2 levels in young Cardiology and Clinical Immunology, Medical University, Lodz, Poland, children during and after surgery. Methods: We measured the dynamic of Poland these enzymes concentrations in peripheral blood. These investigations Andrzej Sysa, Department of Pathophysiology and Clinical Immunology, were carried out in 31 children, aged 6-35 months who were undergoing Medical University, Lodz, Poland, Poland a cardiac operation with cardiopulmonary bypass (CPB). Serum concen- Krzysztof Zeman, Department of Pediatrics, Prevention of Cardiology trations of metalloproteinases (MMPs) and inhibitors were sequentially and Clinical Immunology, Medical University, Lodz, Poland, Poland measured before induction of anesthesia, at the initiation of CPB, after 30 minutes of CPB, at the end of CPB, 24 hours and 72 hours after CPB. Purpose: The nature of the participation of neutrophil in the post cardio- Results: MMP-9 concentration increased at the end of CPB and remained pulmonary bypass (CPB) infl ammatory response is less clear. The aim elevated for a period of 24 hours. The concentration of MMP-9 detected at of our study was to investigate alterations in neutrophil phagocytic ac- the end of CPB positively correlated with time of CPB (r=0.69, p=0.0004). tivity and adhesion molecules expression on these cells in children dur- TIMP-1 and TIMP-2 concentrations decreased signifi cantly after 30 min- ing and after cardiopulmonary bypass. Methods: Twenty four children utes of CPB, remained lowered to the end of CPB, and returned to the start aged 6-33 months with congenital heart disease, scheduled for primary of CPB level after 24 hours. Conclusions: Our data confi rm that MMP-9 corrective surgical repair, were enrolled. Expressions of CD11b adhesion plays an important role in infl ammatory complications after cardiac sur- molecules and Fcγ receptor on neutrophils, phagocytic activity, and secre- gery in children. These fi ndings suggest that kinetics of MMPs concentra- tory leukocyte protease inhibitior (SLPI) were evaluated. Study markers tions in serum after cardiac surgery appear to depend on many factors. We were sequentially measured before, on initiation, and after CPB. Results: demonstrated the link between CPB duration and the MMP-9 concentra- The expression of CD11b molecule on neutrophils was increased signifi - tion. Future studies will determine whether inhibition of MMPs activity cantly at the start of CPB, compared with baseline value (876.5±104.8 diminishes morbidity in children after cardiac surgery. Mean Fluorescence Intensity - MFI vs. 768.1±178.2 MFI; p=0.0047), and The study was supported by the KBN grant no. 2PO5E 081 27 and Medical then slowly decreased with the lowest values detected after the comple- University grant no. 502-15-277. tion of the procedure (689.01±166.7 MFI). The expression of CD11b on neutrophils at the end of CPB inversely correlated with the time of the CPB (r=-0.68, p=0.00059). The expression of CD16 dropped signifi cantly 7110 at the start of CPB compared with baseline value (1164.6±307.3 MFI vs. 1327.4±345.3 MFI; p=0.0007), and remained decreased until the end of A novel beta-oxa polyunsaturated fatty acid down regulates the CPB (814.0±198.1 MFI). We found that the serum concentration of SLPI activation of the IkappaB kinase - NFkappaB pathway, inhibits dropped signifi cantly at the start of CPB compared with baseline level expression of endothelial cell adhesion molecules and depresses (20.35±4.36 ng/mL vs. 33.6±9.8 ng/mL; p=0.003), and remained decreased infl ammation until the end of CPB (14.7±3.6 ng/mL). Conclusions: These fi ndings sug- gest that the characteristics of the neutrophil response to cardiac surgery Antonio Ferrante, Children, Youth and Women's Health Services, Univer- appear to depend on many factors. We demonstrated the link between CPB sity of Adelaide, SA, Australia duration and the adhesion molecule expression on neutrophils. Brenton S Robinson, Children, Youth and Women's Health Services, SA, The study was supported by the KBN grant no. 2PO5E 081 27 and Medical Australia University grant no. 502-15-277. Hubertus PA Jersmann, Children, Youth and Women's Health Services, SA, Australia Hua Huang, Children, Youth and Women's Health Services, SA, Australia 7109 Neil Trout, Flinders University, SA, Australia Deborah A Rathjen, Children, Youth and Women's Health Services, SA, Matrix metalloproteinases and tissue inhibitors of metaloproteinases Australia may be may act as proinfl ammatory markers after cardiopulmonary Christopher J Easton, Australian National University, Canberra, Austra- bypass in children lia Frank S Lee, University of Pennsylvania School of Medicine, Philadel- Jarek Pasnik, Department of Pediatrics, Prevention of Cardiology and phia, United States Clinical Immunology, Medical University, Poland Jadwiga A Moll, Department of Pediatrics Cardiology of Institute of Recently we have synthesised several novel polyunsaturated fatty acids Polish Mother’s Health,, Poland (PUFA) by chemically inserting an oxygen or sulphur atom in the beta- Dorota A Szalowska, Department of Pediatrics, Prevention of Cardiology position of the PUFA. We now demonstrate that these molecules exhibit and Clinical Immunology, Medical University, Poland more selective anti-infl ammatory properties than their natural PUFA coun- Zbigniew Baj, Department of Pathophysiology and Clinical Immunology, terparts. One of these, beta-oxa-23:4n-6, unlike the natural PUFA lacked Medical University,, Poland the ability to stimulate signifi cant oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 205 adhesion to cultured human umbilical vein endothelial cells (HUVEC) and Purpose: Infl ammation contributes to the pathogenesis of atherogenesis. the expression of E-selectin, intercellular adhesion molecule-1, and vascu- S100A12, a member of the S100 calcium binding protein family, mediates lar cell adhesion molecule-1. In addition, beta-oxa-23:4n-6 inhibited acute monocyte/macrophage migration and induces TNF and IL-1 by binding and chronic infl ammatory responses in mice as well as the upregulation the receptor for advanced glycation end products (RAGE). The study was of adhesion molecule expression in arterial endothelium. This action of designed to identify S100A12 in atheroma, and to further explore mecha- the fatty acid was also evident at the level of adhesion molecule mRNA nisms of S100A12 involvement. expression and required a functional 12- but not 5-lipoxygenase or cyclo- Methods: S100A12 expression in diseased and normal arteries was de- oxygenases. The metabolism of beta-oxa-23:4n-6 by HUVEC was mainly tected by immunohistochemistry and in tissue extracts by Western blot. via the 12-lipoxygenase pathway. While the beta-oxa compound did not Human primary PBMC were stimulated with S100A12. Levels of IL-6, affect the activation of MAP kinases by tumor necrosis factor, activation of IL-8, IL-1 and TNF in supernatants were measured by ELISA. Cytokine the IkappaB kinase - NFkappaB pathway was selectively inhibited. These mRNA levels in PBMC were analyzed by real time RT-PCR. novel PUFA could form the basis for a potential new class of research Results: Anti-S100A12 reactivity was present in foam and non-foam cells, tools as well as pharmaceuticals for use in the treatment of infl ammation particularly in CD68+ macrophages and weakly positive in microvascular and atherosclerosis. endothelial cells and some mural smooth muscle cells. RAGE was ex- pressed on cells in atherosclerotic but not non-atherosclerotic areas, and some S100A12-positive cells expressed RAGE. S100A12 (~10 kD) was 7111 detected in plaque extracts by Western blot; anti-S100A12 positive com- ponents with masses of 24, 38, 56 and 71 were also evident. IL-6, IL-8, Modulation of HUVEC:neutrophil interactions by the cholesterol IL-1 and TNF mRNA expression in PBMC increased 2 h after stimulation lowering drug simvastatin with S100A12 and IL-6 and IL-8 increased further at 6 h. Protein levels of these cytokines increased in a dose- and time- dependent manner. IL-6 was Anne M Graham, University of Bradford, United Kingdom produced at higher levels than the other cytokines with maximal 50-fold Kirstie A Eccles, University of Bradford, United Kingdom increases at 24 h. Cytokine induction was partially inhibited by a soluble Susan M Parkin, University of Bradford, US Minor Outlying Islands RAGE antagonist. Shervanthi Homer-Vanniasinkam, Leeds General Infi rmary, United Conclusions: S100A12 is present in atherosclerotic lesions S100A12 is Kingdom chemotactic for monocytes and also induces cytokines, particularly IL-6. Karen E Porter, University of Leeds, United Kingdom As IL-6 is an important inducer of acute phase reactions such as CRP (a predictor of CV risk), S100A12 may contribute to the pathogenesis and Endothelial cell adhesion molecules (CAM) are important in recruitment acceleration of atherosclerosis. of leucocytes during infl ammation and other vascular diseases. Several families of CAMs have been described on endothelial cells, specifi cally P-selectin, E-selectin, ICAMs and VCAMs. Modulation of surface ex- 7113 pression of these CAMs is responsible for recruitment of leucocytes during infl ammation. Inhibitors of HMG-CoA reductase, the statin family, have Endogenous granulocyte macrophage-colony stimulating factor been used in the clinic to lower cholesterol, but it is becoming evident that attenuates development of atherosclerosis in apolipoprotein defi cient they may have additional benefi cial effects within the vascular system. mice Previous studies have shown that statins may modulate expression of ad- hesion molecules but results have been contradictory with some authors Michael Ditiatkovski, Baker Heart Research Institute, Monash Universi- observing reduced expression while others showed upregulation. We have ty, Australia investigated the roles of statins in modulation of P-selectin expression on Ban-Hock Toh, Monash University, Australia human endothelial cells in an in vitro fl ow model. Alex Bobik, Baker Heart Research Institute, Australia HUVEC were pre-treated with a range of concentrations of simvastatin (1-5μM,6h) before treatment with the pro-infl ammatory mediator hista- Granulocyte macrophage-colony stimulating factor (GM-CSF) has fre- mine (5x10-4M,18min) to upregulate cell surface expression of P-selectin. quently been implicated in infl ammatory disorders and is highly expressed HUVEC were then perfused with freshly isolated neutrophils at a fl ow rate in human atherosclerotic lesions. Contrary to its infl ammatory effects ad- of 1.1dynes and alterations in adhesion molecule expression recorded by ministration of pharmacological doses of GM-CSF appear to suppress the video microscopy. Tethering of individual neutrophils to HUVEC were development of atherosclerosis in animals fed a high cholesterol diet with- then quantifi ed. out affecting plasma low-density lipoprotein levels. In the present study Treatment of HUVEC with both 1μM and 5μM simvastatin signifi cantly we investigate how endogenous GM-CSF affected the development of reduced tethering interactions when compared to histamine treated con- atherosclerosis. trol cells, indicating a lack of P-selectin expression on the cell surface. Methods-GM-CSF-/- mice were crossed with ApoE-/- mice to generate Prevention of P-selectin expression on the endothelial cell surface may GM-CSF-/-.ApoE-/- mice and this was confi rmed by genotyping. Male be important for the benefi cial effects of statins in the vasculature. The ApoE-/- and GM-CSF-/-.ApoE-/- mice were placed on a high fat diet at mechanisms by which endothelial cell adhesion molecule expression is 8-weeks of age. Twelve weeks later the mice were culled and their aortas altered by statins will have important implications for vascular biology, collected for histological and immunohistochemical studies. Atheroscle- particularly due to the widespread use of statin therapy as a tool for lower- rotic lesions size was measured at the level of the aortic sinus after staining ing cholesterol. sections for lipids with Oil Red O. Macrophage accumulation was assessed after staining with anti-CD68 whilst endothelial cell numbers within the plaques and expression of VCAM-1 were assessed after staining with anti- 7112 CD31 and anti-VCAM-1 antibodies. Results-GM-CSF-/-.ApoE-/- developed lesions that were on average 50% Roles of S100A12 in atherosclerosis larger in size than those in ApoE-/- mice (P < 0.05). Macrophage cell num- bers were also greater (~100%) in lesions of the GM-CSF-/-.ApoE-/- mice Weixing Yan, School of Medical Sciences, University of NSW, Australia compared to ApoE-/- mice (P < 0.05). Vascularisation of the lesions of Farid Rahimi, Medical Research Institute, Prince Wales Hospital and GM-CSF-/-.ApoE-/- was more extensive with 30% more endothelial cells University of NSW, Australia within the lesions (P < 0.05). VCAM-1 expression was also upregulated Yuri Bobryshev, Prince Wales Hospital and University of NSW, Australia (100%) in lesions of the GM-CSF-/-.ApoE-/- mice (P for difference < Changjie Song, Concord Hospital and University of Sydney, Australia 0.05). Kenneth Hsu, School of Medical Sciences, University of NSW, Australia Carolyn Geczy, School of Medical Scineces, University of NSW, Australia S 206 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005)

Conclusion-We conclude that endogenous GM-CSF suppresses the devel- in atheroma development. To date, no anti-cytokine therapy has been de- opment of atherosclerotic lesions, attenuating macrophage accumulation, veloped for the prevention or treatment of atherosclerosis. We investigated lesion vascularization and expression of adhesion molecules. MIF expression in relation to the development of atherosclerotic lesions in ApoE-/- mice and the effect of a structurally unique orally active MIF antagonist small molecule, COR100140. 7114 Eight week old male ApoE-/- mice were fed a high fat diet for 12 weeks. Lesion development and MIF expression were assessed at 4, 8 and 12 The effects of n-3 polyunsaturated fatty acids on metalloproteinase weeks at the aortic sinus using immunohistochemistry and RT-PCR. expression in THP-1 monocytic cells treated with infl ammatory ApoE-/- mice were also treated with either vehicle or COR100140 given cytokines and PMA by oral gavage. After 8 weeks therapy aortic lesions were examined. Oil Red-O staining was used to assess lesion size, and macrophage and T-cell Xanthe V Scott, School of Biological Sciences, University of East Anglia, numbers were assessed immunohistochemically. Norwich, UK, United Kingdom Normal vessels did not express MIF. MIF expression increased substan- Joanna R Worley, School of Biological Sciences, University of East tially as atherosclerotic lesions increased in size, mainly due to an increase Anglia, Norwich, UK, United Kingdom in the number of MIF-expressing cells. The increase in MIF expression David A Hughes, Institute of Food Research, Norwich Research Park, was most marked between 4 and 8 weeks with no further increase thereaf- Norwich, UK, United Kingdom ter. Treatment with COR100140 reduced lesion size by 50% compared to Mike J Sampson, Bertram Diabetes Research Unit, Norfolk & Norwich vehicle treated mice (P<0.05). Lesion macrophage numbers were also at- University Hospital, Norwich, UK, United Kingdom tenuated by 50% in COR100140 treated mice (P<0.05). COR100140 treat- Jelena Gavrilovic, School of Biological Sciences, University of East ment did not affect CD4 or CD8 T cell numbers in the lesions, the number Anglia, Norwich, UK, United Kingdom of cells expressing GM-CSF or the expression of HSP60. COR1000140 did not affect the elevations in plasma cholesterol induced by high fat diet The chronic infl ammatory disorder, atherosclerosis, involves recruitment We conclude (1) that MIF expression increases substantially during the and trans-endothelial migration of monocytes into the arterial intima. Pop- early development of atherosclerotic lesions and (2) MIF antagonism in- ulations with diets rich in n-3 polyunsaturated fatty acids (PUFAs) have a hibits atherosclerotic lesion development by mechanisms independent of lower risk of developing chronic infl ammatory disorders. Matrix metal- lipid lowering. Orally active small molecule MIF antagonists represent a loproteinases (MMP), ADAMs (A Disintegrin And Metalloproteinase) and potential new class of therapy for atheroma. ADAMTSs (ADAMs with thrombospondin motifs) have been implicated in atherosclerosis. MMPs and ADAMTSs are involved in cleavage of the extracellular matrix. The functions of ADAMs include ectodomain shed- 7116 ding of membrane bound proteins (eg cytokines) and mediating cell-cell interactions. Given that n-3 PUFAs can regulate mRNA expression of cell Biochemical Assessment of Myocardial Injury After Coronary Artery adhesion molecules we investigated the effects of n-3 PUFAs on metal- Surgery : Effects of COX-II inhibition loproteinase (MP) expression in monocytic cells. THP-1 monocytic cells were pre-incubated with n-3 PUFAs for 24 hours Poo-Sing Wong, National University of Singapore, Singapore and then stimulated with TNF-alpha or PMA. We analyzed the relative Atasha Asmat, National University Hospital, Singapore expression of various MMPs and ADAMs expressed in monocytes. Yiong-Huak Chan, National University of Singapore, Singapore TNF-alpha signifi cantly induced up-regulation of ADAM28 which was Chuen-Neng Lee, National University of Singapore, Singapore further enhanced (p<0.05) by n-3 PUFAs whereas signifi cant PMA medi- ated induction of ADAMTS4 was inhibited 2-fold by n-3 PUFAs. Surpris- Objectives ingly we saw an enhancement of PMA induced MMP9 expression by n-3 The endothelium of patients with coronary artery disease shows increased PUFAs (p<0.05). However MMP2 and ADAMs 9, 15 and 17 were not expression of cyclooxygenase-II (COX-II) during coronary artery bypass regulated by n-3 PUFAs at the mRNA level. Our results suggest that n-3 graft surgery (CABG) using cardiopulmonary bypass. Together with sero- PUFAs can selectively regulate gene expression of MMPs and ADAMs tonin this may lead to coronary microvessel spasm, which can contribute in response to cytokine and PMA stimulation. We have previously shown to myocardial ischemia and injury after surgery. MMP9, ADAM28 and ADAMTS4 to be differentially regulated by peroxi- Methods some proliferator-activated receptor (PPAR)-gamma (Worley et al, JBC, We performed a randomized, double-blind, placebo-controlled trial in pa- 2003), a receptor for PUFAs. N-3 PUFAs may induce their effects through tients undergoing isolated CABG to determine whether short-term treat- this pathway. Current investigations are focused on n-3 PUFA effects on ment with a selective COX-II inhibitor, Rofecoxib, given preoperatively MPs at the protein level and on possible mechanisms by which they exert and for 5 days post-operatively, can offer better myocardial protection in these effects. patients undergoing CABG by measuring serial cardiac troponin T (cTnT) Supported by the BHF, UK; Norwich and Norfolk Diabetes Trust; BBSRC, levels. UK. Results The study was powered to recruit 150 consecutive patients undergoing iso- lated CABG. However, the study was terminated prematurely following 7115 the worldwide withdrawal of Rofecoxib. Of the 107 patients recruited, 31 patients were excluded from the fi nal analysis: 9 coronary endarterectomy, MIF is expressed during development of atherosclerotic lesions 4 gastrointestinal bleed, 4 renal impairment, 1 concomitant left ventricu- in ApoE-/- mice and an orally active MIF inhibitor attenuates lar aneurysmectomy, 1 opted for alternative treatment, 2 withdrew from atherosclerosis development study and 10 patients had raised preoperative cTnT, leaving data from 76 patients for analysis. There were highly statistically signifi cant (p < 0.001) Michael Ditiatkovski, Baker Heart Research Institute, Monash Universi- increases in cTnT in both groups at each time point (1, 6, 24 and 48 hours ty, Australia after onset of cardiopulmonary bypass) compared to preoperative levels. Eric Morand, Monash University, Australia cTnT levels were similar at all post- operative time points between the 2 Magdy Iskander, Monash University, Australia groups. Ban-Hock Toh, Monash University, Australia Conclusion Alex Bobik, Baker Heart Research Institute, Australia Our study found no evidence that short-term use of rofecoxib has a myo- cardial protective effect in patients undergoing CABG. More importantly, Microphage migration inhibitory factor (MIF) is a proinfl ammatory cyto- we found no evidence that it has a deleterious effect on the myocardium. kine expressed by vascular cells in atherosclerotic lesions and implicated Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 207

period of 12 weeks. Long-term treatment with ghrelin failed to modulate 7118 the development of oil red-O-stained aortic lesions whereas HEX mod- estly reduced aortic lesions by 28%. In contrast, chronic treatment with Possession of eotaxin (CCL11) 67G>A genotype is associated with EP 80317 was associated with a 51% reduction of lesions. Importantly, higher plasma eotaxin levels in myocardial infarction survivors the effects of GHRPs were shown to be CD36-dependent, inasmuch as no anti-atherosclerotic effects were observed in apoE/CD36 double-defi cient Birgitta Olsen, Sweden mice. At 18 weeks of age, treated mice received 111In-labeled peritoneal Allan Sirsjo, Sweden macrophages from apoE-/- donor mice. Aortic accumulation of labeled Yuri Sheikine, Sweden macrophages was assessed by densitometric analysis of the aortic tree 48 Baback Gharizadeh, United States hours later. Chronic treatment with EP 80317 was associated with a 40 ± Per Eriksson, Sweden 8 % reduction of 111In-labeled macrophages accumulation within lesions Mehran Ghaderi, Örebro University, Sweden compared to 0.9% NaCl-treated mice, suggesting a potential role of EP 80317 in modulating the infl ammatory component of atherosclerosis. Our Several studies suggest that the CC chemokine eotaxin (CCL11) is in- results suggest that GHRPs, as CD36 ligands, might be prototypes for a volved in pathogenesis of allergic asthma and certain infl ammatory dis- novel class of anti-atherosclerotic agents. Supported by Canadian Insti- orders. However the role of eotaxin in atherosclerosis and cardiovascu- tutes of Health Research and Ardana Bioscience. lar diseases is not widely studied. To further understand the function of CCL11, we studied eotaxin plasma levels taken from Swedish patients after myocardial infarction (MI) and compared it with the levels in sex 7120 and age matched healthy control group. Furthermore, the correlation of eotaxin plasma levels was compared in relation to non-conservative amino Model of Myocardial Ischemia and Infarction in the Rabbit acid “Ala to Thr” substitution at position 23 (A23T), which is encoded by a single G to A base substitution at position 67 of the CCL11 gene (67 G>A). Sheikh A Saeed, Dr. Panjwani Center for Molecular Medicine and Drug PyrosequencingÔ was performed to estimate eotaxin genotype frequencies Research, International for Chemical Sciences, University of Karachi, among patients and healthy subjects. Genotyping data from 311 Swed- Karac, Pakistan ish patients and 338 control subjects was available in the following study. Sagheer Ahmed, Dr. Panjwani Center for Molecular Medicine and Drug Commercial ELISA-based method was employed to estimate eotaxin Research, International for Chemical Sciences, University of Karachi, plasma levels in the sera of participating donors. No differences in eotaxin Karac, Pakistan 67 G>A genotype frequencies or eotaxin plasma levels were observed be- Anwar H Gilani, The Aga Khan University Karachi, Pakistan, Pakistan tween patients and control population. Conversely, eotaxin plasma altitude was signifi cantly higher in patients with the G/G genotype compare to the The objective was to devise an animal model of myocardial infarction (MI) respectively control group (p=0.027). Interestingly, patients homozygote against which cardioprotective drugs might be tested. We describe experi- for the G/G genotype had signifi cantly higher eotaxin plasma levels in ence with development and validation of such a model. The rabbit was compare to the patients with G/A genotype (p=0.050). The fact that eotaxin chosen in preference to rodents because its heart and cardiac circulation is elevated only in patients with G/G genotype and not in total population, more closely resemble those of human. Ear veins also provide ready ac- implies that eotaxin gene up-regulation requires additional regulatory fac- cess for intravenous injections and blood sampling in awake, unrestrained tors such as proinfl ammatory cytokines that are expressed in atheroma. rabbits. Chemically induced cardiomyopathy was desired, rather than sur- gical vascular occlusion, to avoid interpretation complications associated with general anesthesia and sutures on the heart. Furthermore, the pos- 7119 sibility of reperfusion with time was considered a potentially important feature. Thus, the cardiovascular system of anaesthetized male rabbits, 1 Growth Hormone-Releasing Peptides: a novel class of anti- to 1.5 kg., was stressed by a single bolus intravenous injection of isoproter- atherosclerotic agents enol HCL, 65 mg/kg. This compound is a nonspecifi c beta adrenergic ago- nist that, at this dose, exerts potent inotropic and chronotropic responses Diala Harb, Faculty of Pharmacy, Université de Montréal, Québec, suffi cient to provoke an MI. The effects of the injection were followed Canada, Canada over several weeks and were evaluated three ways: 1) measurements of Kim Bujold, Faculty of Pharmacy, Université de Montréal, Québec, creatinine kinase isozyme and troponin-I in blood 2) EKG changes indica- Canada, Canada tive of infarcts 3) Cardiac histopathology observed in tissue sections. Maria Febbraio, Department of Cell Biology, Lerner Research Institute, All three indices revealed similarities with effects commonly associated Cleveland Clinic Foundation, Cleveland, OH, USA, United States with MI in humans. The model seems suitable for longitudinal studies Martin G Sirois, Department of Pharmacology, Faculty of Medicine, to evaluate drugs or other treatments that may prevent MI or attenuate its Université de Montréal, Québec, Canada, Canada adverse effects. Andre Tremblay, Obstetrics & Gynecology and Biochemistry, Faculty of Medicine, Université de Montréal, Québec, Canada, Canada Huy Ong, Faculty of Pharmacy, Department of Pharmacology, Faculty of 7121 Medicine, Université de Montréal, Québec, Canada, Canada Sylvie Marleau, Faculty of Pharmacy, Université de Montréal, Québec, Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Promote Canada, Canada Leukocyte Adhesion in the Glomerular Microvasculature

Our recent studies have shown that long-term (12 weeks) treatment with Rain Yu Qing Kwan, Monash Institute of Medical Research, Monash growth hormone-releasing peptides (GHRPs), as ligands of the CD36 type University, Australia B scavenger receptor, shows striking anti-atherosclerotic and hypocholes- Stephen R Holdsworth, Monash Institute of Medical Research, Monash terolemic effects in apoE-defi cient mice (apoE-/-) fed a high fat high cho- University, Australia lesterol (HFHC) diet. Synthetic GHRP analogs such as hexarelin (HEX), in Michael J Hickey, Monash University, Australia addition to binding CD36 on macrophages, also bind to the ghrelin receptor (GHS-R1a). In order to assess the relative contribution of these receptors Anti-neutrophil cytoplasmic antibodies (ANCA) are present in most pa- to macrophage accumulation and fatty streak formation at lesion-proned tients with small vessel vasculitis. The two main antigens recognised by sites, apoE-/- mice have been treated with either ghrelin (320 μg/kg), the ANCA are the polymorphonuclear neutrophil proteins myeloperoxidase endogenous GHS-R1a ligand, HEX (100 μg/kg), a ligand of both CD36 (MPO) and proteinase-3. However the role of ANCA in the etiology of and GHS-R1a, or EP 80317 (300 μg/kg), a selective CD36 ligand, for a the vasculitis and glomerulonephritis which affects these patients is not S 208 Posters: Tuesday 23 August 2005 Infl amm. res. Supplement 2 (2005) known. Recent in vitro evidence suggests that ANCA promote leukocyte Maria Cavasin, Henry Ford Hospital, Detroit, MI 48202, United States adhesion to endothelial cells. However, whether this applies in vivo is not Werner Muller-Esterl, Johann Wolfgang Goethe University at Frankfurt, known. Therefore the aim of this study was to study the effects of ANCA- Germany like antibodies on leukocyte-endothelial cell interactions in the intact mi- Nour Eddine Rhaleb, Henry Ford Hospital, Detroit, MI 48202, United crovasculature. ANCA-like antibodies were generated by immunizing States MPO-/- mice with human or mouse MPO. Indirect immunofl uorescence Oscar A Carretero, Henry Ford Hospital, Detroit, MI 48202, United demonstrated that these anti-MPO sera recognized cytoplasmic antigens States in murine neutrophils. These antibodies were then administered to mice to determine if they alter leukocyte-endothelial cell interactions. Two vas- Angiotensin II (Ang II)-induced renal injury is characterized by severe cular beds were examined: postcapillary venules in the cremaster muscle, tubulo-interstitial infl ammatory and fi brotic responses; however, it is not and glomerular capillaries in the hydronephrotic kidney. Using intravital known whether the injury is associated with increased circulating and renal microscopy, we observed that treatment with anti-MPO serum did not in- infl ammation-associated peptide kinins or induction of B1 and B2 recep- crease the level of leukocyte adhesion in cremasteric postcapillary venules. tors in Ang II-induced hypertension. Two groups of adult male Sprague- In contrast, examination of leukocyte recruitment in glomeruli revealed Dawley rats (n = 8 each) were either not treated (sham) or treated with Ang that anti-MPO serum induced a rapid and signifi cant increase in leukocyte II (80 ng/min) via osmotic minipump for 4 weeks. Compared with sham, adhesion in mice pre-treated with LPS, but not in untreated mice. These systolic blood pressure rose from 120 ± 5 to 222 ± 12 mm Hg in Ang II-in- data demonstrate that ANCA are capable of inducing leukocyte adhesion fused rats (p < 0.01), circulating Ang II from 116 ± 23 to 345 ± 50 fmol/mL in the glomerular microvasculature, under conditions of infl ammatory acti- (p < 0.01), renal ACE binding (measured by quantitative in vitro autoradi- vation. The difference between the effects of anti-MPO in glomerular cap- ography using 125I-351A) from 91 ± 4 to 132 ± 6 dpm/mm2 (p < 0.01) and illaries and cremasteric postcapillary venules suggests that this response is renal Ang II from 180 ± 26 to 396 ± 46 fmol/g (p < 0.01). By contrast, cir- restricted to specifi c locations within the microvasculature. culating (2.5 ± 0.2 vs. 3.2 ± 0.3 fmol/mL) and renal bradykinin-(1-9) levels (263 ± 31 vs. 386 ± 83 fmol/mL) were not signifi cantly altered. However, B2 receptor binding, as measured by quantitative in vitro autoradiography 7122 using 125I-icatibant, was signifi cantly increased in the inner cortex and the entire medulla of the Ang II-infused rats (198 ± 16 vs. 310 ± 25 dpm/ The role of myeloperoxidase in crescentic glomerulonephritis and mm2; p < 0.05). Ang II-infused rats had severe tubulo-interstitial injury, immune responses with widespread formation of tubular cysts, fi brosis, and dilatation in the inner cortex and outer medulla. Immunocytochemistry using anti-human Dragana Odobasic, Monash University, Australia B1 (AS434) receptor antibodies showed strong B1 receptor immunostain- A Richard Kitching, Monash University, Australia ing in most of the injured tubules and fi brotic interstitium compared with Peter G Tipping, Monash University, Australia sham (average of 54 ± 8 B1 receptor-positively stained tubules per tissue Stephen R Holdsworth, Monash University, Australia section vs. none in sham). We concluded that Ang II-induced renal tubulo- interstitial injury is associated with signifi cant induction of kinin B1 and Myeloperoxidase (MPO), an enzyme found inside neutrophils and mono- B2 receptors, which may play an important role in mediating renal infl am- cytes/macrophages, plays an important role in intracellular killing of matory and fi brotic responses in Ang II-induced hypertension. pathogens, but is also involved in tissue damage at sites of infl ammation. The purpose of the current study was to investigate the role of MPO in crescentic anti-glomerular basement membrane (GBM) glomerulonephri- 7124 tis (GN). GN was induced in C57BL/6 and MPO-/- mice by injecting mice with sheep anti-mouse GBM globulin. Heterologous injury (mediated by Up-regulation of the Expression of Pro-apoptotic Molecules neutrophils) was assessed 24 hours after anti-GBM globulin administra- Contributes to Tubulointerstitial Damage in Chronic Renal Disease tion. Autologous phase injury (mediated by T cells and macrophages) and immune responses were assessed 21 days after anti-GBM globulin injec- Simon A Teteris, Department of Medicine, Central and Eastern Clinical tion. Heterologous proteinuria was signifi cantly reduced in MPO-/- mice School, Monash University, AMREP, Alfred Hospital., Australia compared to controls. Autologous injury, as assessed by proteinuria, serum Solomon Menahem, Department of Medicine, Central and Eastern Clini- creatinine and crescent formation, was not different in MPO-/- compared cal School, Monash University, AMREP, Alfred Hospital., Australia to C57BL/6 mice. MPO-/- mice had signifi cantly increased antigen-spe- John Dowling, Department of Anatomical Pathology, Alfred Hospital., cifi c skin delayed type hypersensitivity, as well as enhanced splenic CD4+ Australia T cell proliferation and IFN-gamma production. CD4+ T cell activation Greg Perry, Renal Service, Alfred Hospital., Australia (assessed by CD44 and CD69 expression) and apoptosis (assessed by Robyn Langham, Department of Medicine, Central and Eastern Clinical Annexin-V staining) were not affected in MPO-/- mice. Antigen-specifi c School, Monash University, AMREP, Alfred Hospital., Australia circulating total Ig, as well as IgG isotypes, IgG1 and IgG3, levels were Napier M Thomson, Department of Medicine, Central and Eastern Clini- signfi cantly increased in MPO-/- mice compared to controls. Splenic B cal School, Monash University, AMREP, Alfred Hospital., Australia cell proliferation and apoptosis were not affected in MPO-defi cient mice. Alicia N Stein, Department of Medicine, Central and Eastern Clinical These results demonstrate that MPO has differential roles during the de- School, Monash University, AMREP, Alfred Hospital., Australia velopment of crescentic anti-GBM GN. Reduced heterologous injury in MPO-/- mice sugggests that it plays a pathogenic local role in anti-GBM Aim: The presence of infi ltrating leukocytes and the infl ammatory cyto- GN. In contrast, MPO has a protective role in anti-GBM GN during the kine milieu present in chronic renal disease produces a pro-apoptotic envi- initiation of the nephritogenic immune response by attenuating T cell re- ronment, leading to the loss of intrinsic renal cells and a decrease in renal sponses and antibody production. function. Having previously demonstrated increased apoptosis in various chronic renal diseases, we aimed to identify which apoptotic regulators are altered in chronic renal disease. 7123 Methods: RNA was extracted from snap frozen renal biopsy tissue. Nor- mal renal tissue was obtained from the uninvolved pole of tumour nephrec- Increased Bradykinin B1 and B2 Receptor Expression Is Associated tomies or kidneys unsuitable for transplantation (n=8). Renal biopsies with Intrarenal Tubulo-Interstitial Injury in Angiotensin II-Induced were from patients with Thin Basement Membrane nephropathy (TBMN, Hypertension n=15), IgA nephropathy (IgAN, n=23), Hypertensive nephropathy (HTN, n=10), Cyclosporine A nephropathy (CyAN, n=5), Focal Segmental Glo- Jia L Zhuo, Henry Ford Hospital, Detroit, MI 48202, United States merulosclerosis (FSGS, n=5) or Lupus Nephritis (LN, n=7). Expression of Xiao C Li, Henry Ford Hospital, Detroit, MI 48202, United States the pro-apoptotic molecules Fas, Fas ligand (FasL), Bax, Bad and Bid and Infl amm. res. Supplement 2 (2005) Posters: Tuesday 23 August 2005 S 209 the anti-apoptotic molecules Bcl-2 and Bcl-xL were measured by Real- time PCR and standardised to the housekeeping gene 18S. Results: Expression of Fas, FasL and Bid was up-regulated in all patient groups versus normal controls. Bid was increased between 2-fold in CyAN (p<0.05) and 4-fold in IgAN (p<0.0001); Fas between 2.5-fold in FSGS (p<0.05) and 6-fold in IgAN (p<0.0001) and FasL between 6.5-fold in FSGS (p<0.05) and 29-fold in IgAN (p<0.0001). Bax was up-regulated approximately 2-fold in IgAN and HTN patients only (p<0.005) and Bad was increased 2-fold in IgAN patients only (p<0.01). Expression of these pro-apoptotic molecules was correlated with the degree of tubulointersti- tial injury (p<0.005). Bcl-2 and Bcl-xL mRNA did not differ signifi cantly in patients versus normal controls. Conclusion: Up-regulation of the pro-apoptotic molecules Fas, FasL and Bid, all members of the death receptor pathway, suggests this is a major pathway through which apoptosis is carried out in chronic renal disease.

7125

A Pathogenetic Role for Semaphorins in Renal Injury: CD100 enhances pathogenetic humoral immune responses by promoting T cell-APC interaction

Ming Li, Monash University Department of Medicine, Monash Institute of Medical Research, Australia Kim O'Sullivan, Monash University Department of Medicine, Monash Institute of Medical Research, Australia Lynelle K Jones, Monash University Department of Medicine, Monash Institute of Medical Research, Australia Atsushi Kumanogoh, Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Japan Hitoshi Kikutani, Department of Molecular Immunology, Research Insti- tute for Microbial Diseases, Osaka University, Japan Stephen R Holdsworth, Monash University Department of Medicine, Monash Institute of Medical Research, Australia Richard A Kitching, Monash University Department of Medicine, Monash Institute of Medical Research, Australia

Purpose: CD100, a member of the semaphorin family, acts as a costimula- tory molecule in adaptive immune responses. To understand the poten- tial pathogenetic effects of CD100 in damaging immune responses, we investigated the involvement of CD100 in experimental immune complex glomerulonephritis in which T cell dependent B cell responses mediate glomerular injury. Methods: Immune complex glomerulonephritis model was generated by daily (i.p.) injections of horse spleen apoferritin into CD100+/+ and CD100-/- BALB/c recipients for 14 days. Experiments ended on day 15. Urine, serum, kidneys and spleens were collected and examined for renal injury and immune responses. Results: Mice defi cient in CD100 developed signifi cantly attenuated glo- merular injury with reduced infl ammatory responses in glomeruli. Attenu- ated renal injury was associated with decreased CD4+ T cell activation, cytokine production and Ag-specifi c Ig production. Flow cytometric anal- yses of CD100+/+ splenocytes showed up-regulated CD100 expression on CD4+ T cell and CD86 expression on dendritic cells(DCs) following anti- gen injection. However, in CD100-/- mice, up-regulation of CD86 expres- sion on DC was signifi cantly reduced, suggesting that endogenous CD100 on CD4+ cells switches off CD72 signalling by dendritic cells, so enabling full expression of CD86 and consequent CD4+ cell activation.

Conclusion: CD100 enhances pathogenetic humoral immune responses by promoting T cell-APC interactions, as well as the more convention- ally recognised T cell-B cell interactions. In the absence of endogenous CD100, co-stimulatory signalling was inhibited and CD72 continues to negatively regulate CD4+ T cell priming and subsequent B cell activation leading to the attenuation of renal injury. Inhibitors of CD100 may have the potential to interrupt established pathogenetic immune responses in autoimmune and non-autoimmune renal disease. Young Investigator Award Finalists S 212 Infl amm. res. Supplement 2 (2005)

Introduction

IAIS Young Investigator Award Finalists

The International Association of Infl ammation Societies (IAIS) is a body comprising representatives from major National and Regional Infl ammation societies from around the world. Its aim is to promote and strengthen basic and translational research, as well as education, in the fi eld of Infl ammation and associated diseases. This is achieved, in part, by the IAIS promoting a World Congress on Infl ammation every two years. These meetings, which bring together scientists and clinicians from different disciplines, and from medical, academic and industry contexts, are organised by the member societies and the venue moves for each congress in order to promote a truly global presence.

The IAIS believes that the attendance of young scientists at these meetings is crucial to the future development of infl ammation research as these young scientists will be the Department Heads and Key Opinion Leaders in Infl ammation Research of the future. To encourage and support the attendance of young scientists at these World Congresses, the IAIS organises competition open to Young Scientists, with 6 selected fi nalists receiving Travel Awards of up to $3,000 in 2005. In addition, the winner of the Young Investigator Award receives a fi nancial prize.

The competition is based on the submission of an abstract and mini-paper. An international panel of 6 independent judges, drawn from the various International Infl ammation Societies, then judges these submissions with the 6 highest scoring applicants being invited to present their research orally in a dedicated Young Investigator session at the congress for fi nal judging. Judging criteria for the work are scientifi c merit, novelty of the approach, potential for development of novel anti-infl ammatory agents, and clarity of presentation.

We had over forty applicants for the IAIS Young Investigator Award in 2005, with fi nalists from the Netherlands, England, Japan, USA, and Australia.

As further support for Young Scientists the Committee also awarded travel grants of up to $1,500 to 11 runners up from the submitted entries to enable their work to be presented in Poster sessions at the Congress.

The Committee congratulates all the applicants on the high standard of their research.

IAIS Young Investigator Award Committee (Infl ammation 2005, Melbourne, Australia) Infl amm. res. Supplement 2 (2005) Mini-Paper 1 S 213

IL-23 drives the expansion of pathogenic ThIL-17 cells required for autoimmune inflammation Claire L. Langrish1, Yi Chen1, Wendy M. Blumenschein2, Jeanine Mattson2, Beth Basham3, Jonathan D. Sedgwick1, Terrill McClanahan2, Robert A. Kastelein1, and Daniel J. Cua1. 1Discovery Research, 2Experimental Pathology and Pharmacology, 3Bioinformatics, DNAX Research Inc., 901 California Ave, Palo Alto, CA 94304, USA.

Cytokines play a pivotal role in the establishment and maintenance of autoimmune diseases. Numerous studies have established interleukin-12 (IL-12) as an important factor for the differentiation of naïve T cells into IFN producing T helper 1 (Th1) cells, which are essential for cell mediated immunity and anti-microbial responses. Historically IL-12 driven Th1 cells were also thought to mediate autoimmune diseases, primarily through investigations using IL-12p40-/- mice and IL-12p40 neutralising antibodies. However, mice which lack critical components of the Th1/IFN pathway are highly susceptible to inflammatory autoimmune diseases, thereby questioning whether IFN producing Th1 cells are essential for autoimmune pathogenesis. The discovery of IL-23 at DNAX has prompted re-evaluation of the role of IL-12 in autoimmunity. IL-23 is a heterodimeric cytokine with a unique p19 subunit complexed with a p40 subunit shared with IL-12. Like IL-12, IL-23 also binds to the IL-12R1 chain, however this is complexed with a novel IL-23R chain to form the active IL-23 receptor complex. Although both cytokines share a common p40 subunit, IL-23 displays distinct biological activities from IL-12. By investigating IL-23 deficient p19-/- mice, and careful re-analysis of IL-12 deficient p35-/- mice and IL-12/IL-23 deficient p40-/- mice, IL-23 has now emerged as the key player in chronic inflammatory autoimmune responses. Results IL-23p19-/- deficient mice are resistant to EAE The CNS are normally free of immune cells, but following immunization with strain-specific CNS antigens, such as myelin-oligodendrocyte glycoprotein peptide (MOG35-55), wildtype mice exhibit a large influx of invading immune cells into the CNS prior to disease onset. Although IL-23p19-/- mice are completely resistant to EAE, comparable numbers of immune cells invade the CNS. In vitro stimulation of the CNS infiltrating CD4+ T cells showed an equivalent frequency of MOG-specific IFN+ (Th1) cells in both the IL-23p19-/- and wildtype mice, suggesting that IL- 23 is not required for CNS invasion or for Th1 development. MOG-specific IL-6, IL-17 and TNF production was also found in the wildtype CNS invading CD4+ T cell population. In contrast, CD4+ T cells isolated from IL-23p19-/- mice had reduced IL-6, TNF and undetectable IL-17 production.

WTp19-/- p40-/- p35-/- Ex-vivo analysis of draining lymph node (DLN) cells harvested prior to 8 -/- -/- -/- 6 disease onset from MOG-immunized IL-23p19 , IL-12p35 and IL-12p40 4 IL-17 mice was in agreement with the CNS data (Fig. 1). Ex-vivo analysis following -/- -/- 2 PMA restimulation confirmed that both p35 and p40 mice, which lack IL- 0 12 had defective Th1 development, whereas the p19-/- mice had normal Th1 2 development. Most strikingly, IL-17 producing CD4+ T cells (Th ) were T cells producing cytokine T cells producing cytokine 4 IFN- IL-17 + + -/- -/- 6 absent from mice lacking IL-23 (both IL-23p19 and IL-12p40 ), present in -/- 8 Highly wildtype mice, and actually increased in IL-12p35 mice (specifically lacking Susceptible Resistant Resistant % of CD4 % of CD4 Susceptible IL-12), with the frequency of IL-17 cells directly correlating to the severity of Fig. 1. IL-17 or IFN production from CD4+ DLN cells from MOG-primed mice, and re-stimulated with PMA/Ionomycin EAE disease displayed by each strain.

IL-23 is required for autoantigen-specific IL-17 production by T cells To dissect the mechanism of action of IL-23, we used the passive transfer model of EAE. SJL mice were immunised with the CNS-restricted antigen + PLP139-151, then the antigen-primed CD4 T cells were isolated from the DLNs nine days later. Immediately following ex vivo isolation, PLP-specific CD4+ T

cells producing either IFNTh1cells) or IL-17 (ThIL-17 cells) were detected. In vitro addition of rIL-23 induced expansion of ThIL-17 cells and abolished Th1 cell generation, whereas IL-12 promoted Th1 but abolished ThIL-17 growth (Fig. 2). The dependence of IL-17 production on IL-23 was clear, as neutralization with IL-23p19 severely impaired IL-17 production in these

cultures. In addition neutralisation of IL-12 or IFN also increased ThIL-17 cell numbers. Overall, these results establish the distinct roles of IL-12 and IL-23 on T cell differentiation, and confirm that IL-23 drives the antigen-specific production of IL-17 by a population of activated T cells, distinct from Th1 Figure. 2. CD4+ T cell production of IL-17 or IFN- from PLP-  primed mice cultured with IL-12/IL-23 for 5 days (Mean  1se). cell, which can be down-regulated by IL-12/IFN . S 214 Infl amm. res. Supplement 2 (2005)

IAIS Young Investigator Award Claire L Langrish + IL-17 producing CD4 ThIL-17 cells are highly encephalogenic and induce EAE

3 To compare the capacity of Th1 versus ThIL-17 cell populations to induce EAE, IL-23 CD4+ + IL-12 CD4+ we transferred either IL-23 or IL-12 expanded PLP-specific CD4 cells into 6 2 naïve recipients. Mice that received IL-23 driven ThIL-17 cells (1x10 /mouse) showed severe clinical signs of EAE. However, mice that received an  1 equivalent number of IL-12 driven IFN producing Th1 cells showed no signs EAE score of clinical disease (Fig. 3). In addition, the number of ThIL-17 cells injected 0 directly correlated with the severity of disease observed. These results and the 6101418222630  -/- Days post cell transfer fact that IFN producing cells are found in the EAE resistant IL-23p19 mice suggest that these alternative IL-17 producing ThIL-17 cell population, and not Fig. 3. PLP primed CD4+ T cells driven in culture with IL-23 or IL-12 for 10d prior to transfer (1x106 cells) into naïve mice. IFN producing classical Th1 cells, are important for EAE development.

IL-23 is an effective therapeutic target for autoimmune disease Prophylactic treatment with neutralising IL-23p19 protected mice from A 5  4 developing EAE (Fig. 4A). Therapeutic treatment with IL-23p19 following 3 the initial appearance of EAE in a relapse-remitting model was able to protect 2 from subsequent relapses, thereby confirming the IL-23 pathway as an 1 effective therapeutic target (Fig. 4B). IL-23p19 treatment appeared to Mean Score EAE 0 protect the mice from EAE by suppressing the expansion and invasion of the 818283848 Days after immunization pathogenic ThIL-17 cells into the CNS. Treatment with neutralizing IL-17 B 3 resulted in partial protection from EAE, suggesting that other factors induced 2 by IL-23 including IL-6, IL-17F and TNF are also important in EAE pathogenesis. Indeed, the highly intense disease induced by passive transfer of 1 ThIL-17 cells cannot be blocked by anti-IL-17 antibody treatment of recipient Mean EAE score EAE Mean 0 mice, suggesting that multiple factors produced by these exceptionally 8 18283848 Days after immunization pathogenic ThIL-17 cells must be neutralized prior to inhibiting their actions. In   mIgG1 IL-23p19 contrast, treatment of immunized mice with neutralizing IFN resulted in Fig. 4. PLP-immunized SJL mice treated at either day 0 (A) intensified disease and increased mortality, indicating that IFN is not required or after remission (B) with anti-IL-23 mAbs or mIgG. for EAE but may provide some protective function. Discussion + In these studies, we have identified a novel population of pathogenic CD4 T cells (named ThIL-17) which are critical for autoimmune inflammation. These IL-23 driven T cells are not associated with IFNor IL-4, but are characterized by their production of IL-17, IL-17F, IL-6 and TNF. ThIL-17 cells are highly pathogenic and essential for the establishment of chronic inflammation associated with autoimmunity. Consistent with our EAE data, using IL-23p19-/- mice we have previously demonstrated that IL-23, and not IL-12, is essential for joint autoimmune inflammation. IL- 23p19-/- mice are resistant to collagen induced arthritis (CIA), whereas IL-12p35-/-mice displayed elevated CIA scores, with reduced IFN and increased IL-17 production. Together, our data questions the concept that autoimmunity is Th1-mediated, and instead suggests that the IL-23/IL-17 pathway is the real villain in autoimmune inflammation. IL-17-/- mice are reported to be partially resistant to CIA and EAE, and therapeutic treatment with antagonist anti-IL-17 antibodies can reverse ongoing CIA, consistent with the notion that IL-17 functions during the effector phase of an inflammatory response. The function of IL-23 clearly seems associated with IL-17 production. IL-17 is an important pro-inflammatory effector cytokine, which has been identified in many human autoimmune diseases, including multiple sclerosis, rheumatoid arthritis and psoriasis. Engagement of IL-17 and/or IL-17F on multiple cell types promotes expression of IL-1, IL-6, IL-8, TNF and ICAM-1 which are critical factors for driving inflammation. It is becoming increasing clear that IL-12p40 treatment is highly efficacious IFN-- TNF for inflammatory autoimmune diseases, with clinical trials currently in 12 Th1 IL- progress for psoriasis and inflammatory bowel disease. However, IL-12p40 IL-12R1  IL-12R2 treatment inhibits both the classical IL-12/IFN and this newly described IL- Ag-primed  CD4+ T 23/IL-17 immune pathway. Consequently, IL-12p40 therapy may affect a IL-12R1 IL IL-23R broad range of immune responses and host defence capabilities. Our data -23 IL-17F strongly suggests that specific inhibition of the IL-23/IL-17 immune pathway ThIL-17 TNF IL-17 is sufficient to block organ-specific autoimmune inflammation, and represents IL-6 a preferred approach for the treatment of a range of autoimmune diseases.

All studies were performed at DNAX Research Inc, 901 California Avenue, Palo Alto, CA 94304 USA. CLL present address: Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. JDS present address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA. Infl amm. res. Supplement 2 (2005) Mini-Paper 2 S 215

DIRECTED GP130-MEDIATED SIGNALING DISSOCIATES INFLAMMATION FROM FIBROSIS IN BLEOMYCIN INDUCED LUNG INJURY. 1O’Donoghue RJJ, 2Anderson GP, 2Bozinovski S, 2Jones J, 3Ernst M, 1Knight DA, 1Mutsaers SE. 1Asthma & Allergy Research Inst., Perth Australia; 2University of Melbourne, Melbourne Australia; 3Ludwig Inst. For Cancer Research, Melbourne Australia. Background Immunohistochemistry. Lung tissue sections were Cryptogenic fibrosing alveolitis (CFA), also known stained for proliferation (Ki67), apoptosis (TUNEL), as idiopathic pulmonary fibrosis (IPF), is a T-lymphocytes (CD3), B-lymphocytes (B220) and heterogeneous group of progressive and largely macrophages (F4/80) using a biotin-streptavidin untreatable disorders of unknown aetiology [1]. It is detection system with diaminobenzaldehyde as a believed that IPF is the result of a chronic chromogen. inflammatory processes [2], however current Bone marrow reconstitution. Bone marrow cells were treatments for IPF, aimed at inhibiting inflammation, collected from the femurs of donor mice and recipient are largely ineffective [3]. Recent data from our mice were given two doses of 5.5Gy 3 hours apart laboratory has demonstrated differences in then reconstituted with 2x106 bone marrow cells via interleukin (IL)-6/gp130 mediated signaling between the tail vein. normal lung fibroblasts and lung fibroblasts obtained from patients with IPF [4, 5]. The IL-6 family of Results cytokines is a group of cytokines produced by a Gp130ΔSTAT mice are protected from pulmonary variety of cells in response to inflammatory stimuli. fibrosis. Bleomycin exposed wt animals had These cytokines exert their effects on target cells increased collagen deposition and lung remodeling, through specific cell surface receptors and the shared shown histologically after 30 and 60 days. HPLC signal transducing subunit, gp130 [6]. Upon analysis confirmed the histological observations activation, gp130 initiates intracellular signaling indicating wt animals developed fibrosis 30 and 60 through the STAT1/3 or the ERK1/2 pathway [7]. days post bleomycin. However gp130ΔSTAT mice did This study aimed to examine the role of gp130- not develop fibrosis as there was no increase in lung mediated signaling in the development of pulmonary collagen over controls when examined by HPLC or fibrosis using a bleomycin-induced lung injury histology (Figure 1). model. Mice used in this study expressed a mutated Enhanced inflammation in the airways of gp130ΔSTAT. gp130 molecule directing gp130-mediated signaling The BAL fluid of gp130ΔSTAT had increased numbers ΔSTAT through the ERK1/2 pathway (gp130 ) [8]. of inflammatory cells compared to wt. This increase in inflammatory cells was consistent across all Methods inflammatory cell types. Mice. Bleomycin (0.05U) and control saline was TGF-β is decreased while PGE2 and MMP2/9 are administered intranasally to wild-type (wt) and ΔSTAT ΔSTAT increased in gp130 mice. BAL fluid of mice gp130 mice. Animals were sacrificed by an culled 3 days after bleomycin had lower levels of overdose of Phenobarbital (50mg/kg). bioactive pro-fibrotic TGF-β and higher levels of Histology and morphology. Mouse lungs were anti-fibrotic PGE2 and MMP2/9 compared with wt. perfused with 4% paraformaldehyde and embedded Increased B-lymphocytes in the lungs of gp130ΔSTAT in paraffin 3, 30 and 60 days after bleomycin animals. Immunohistochemistry did not illustrate any μ delivery. Sections (5 m) were stained with significant differences in cell proliferation, apoptosis, haemotoxylin and eosin and Masson’s trichrome. or T-lymphocytes and macrophage numbers within Collagen measurement. Lungs were weighed 21, 30 the lungs of wt and gp130ΔSTAT animals 3, 30 and 60 and 60 days after bleomycin administration. Tissue days after bleomycin. There was a significant increase was hydrolysed and hydroxyproline levels were in B-lymphocytes 3 days after bleomycin in quantified by reverse phase high pressure liquid ΔSTAT gp130 mice compared with wt. chromatography (HPLC). Δ Chimeric mice develop fibrosis. Gp130 STAT animals Bronchoalveolar lavage (BAL) and analysis of BAL Δ wt fluid. BAL was performed 3 days after bleomycin with wild type bone marrow ( STAT ) did not delivery by instilling 4 aliquots of 0.4ml PBS and survive the reconstitution period. Wt animals with wt bone marrow (wtwt) and wt animals with gp130ΔSTAT total and differential cell counts were performed. ΔSTAT The levels of transforming growth factor-beta (TGF- bone marrow (wt ) had an increase in lung weights 21 days after bleomycin delivery. There was β), prostaglandin E2 (PGE2) and matrix no significant difference in the inflammatory cell metalloproteases (MMPs) 2 and 9 were measured in wt the BALF supernatant. number and content of BAL fluid taken from wt and wtΔSTAT animals. S 216 Infl amm. res. Supplement 2 (2005)

signaling pathways. The TGF-β signaling molecule, 60% > SalineData Table-1 550000 Smad2/3, was found to interact with IL-6 signaling molecule STAT3 suggesting this interaction is 4 Hyp 40000 important in the progression from chronic Units inflammation to pulmonary fibrosis [12]. By targeting per 3 30000 2.5% > Saline specific intracellular signaling molecules this study lung 4 suggests that we can preserve the inflammatory x10 unitshyp 2 20000 response required for the resolution of injury and prevent the development of fibrotic diseases 1 10000 associated with chronic inflammation.

0 0 WT sal WT bleo ΔSTAT sal ΔSTAT bleo Sal Bleo Sal Bleo Bibliography 1. ATS, American Thoracic Society. Idiopathic pulmonary wt gp130ΔSTAT fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the Figure 1. Reverse-phase high pressure liquid European Respiratory Society (ERS). Am. J. Respir. Crit. chromatography analysis of lung hydroxyproline Care Med., 2000. 161: p. 646-64. (n=4). Gp130ΔSTAT animals show no increase in lung 2. Mason, R., et al., NHLBI Workshop Summary. collagen after bleomycin delivery as measured by Pharmacological therapy for idiopathic pulmonary lung hydroxyproline content. fibrosis. Past, present, and future. Am J Respir Crit Care Med, 1999. 160(5): p. 1771-1777. 3. Monaghan, H., et al., Prognostic implications of Discussion histologic patterns in multiple surgical lung biopsies from The initial results of this study have demonstrated patients with idiopathic interstitial pneumonias. Chest, that inhibition of gp130-mediated STAT signaling 2004. 125(2): p. 522-526. prevents the development of pulmonary fibrosis in 4. Moodley, Y., et al., Fibroblasts isolated from normal the bleomycin-induced lung injury model. As IL-6 is lungs and those with idiopathic pulmonary fibrosis differ in released during the acute inflammatory response and interleukin-6/gp130-mediated cell signaling and is implicated in chronic inflammatory diseases such proliferation. Am J Pathol., 2003a. 163(1): p. 345-354. as rheumatoid arthritis and IPF [9] we examined 5. Moodley, Y., et al., Inverse effects of interleukin-6 on inflammation within the conducting airways and apoptosis of fibroblasts from pulmonary fibrosis and ΔSTAT normal lungs. Am J Respir Cell Mol Biol, 2003b. 29(4): p. lungs of wt and gp130 mice. The increased 490-498. number of all inflammatory cell types within the 6. Kishimoto, T., et al., Interleukin-6 family of cytokines ΔSTAT conducting airways of gp130 suggested the and gp130. Blood, 1995. 86(4): p. 1243-1254. inflammatory response may confer protection to 7. Heinrich, P., et al., Interleukin-6-type cytokine signalling pulmonary fibrosis. Furthermore, the increase in B- through the gp130/Jak/STAT pathway. Biochem J, 1998. lymphocytes within the lungs implicated these cells 334(2): p. 297-314. specifically in mediating a protective effect on these 8. Ernst, M., et al., Defective gp130-mediated signal mice. However this protective effect is not due to the transducer and activator of transcription (STAT) signaling inflammatory cells alone. Our preliminary results results in degenerative joint disease, gastrointestinal ΔSTAT ulceration, and failure of uterine implantation. J Exp Med, demonstrate that wt animals develop fibrosis 2001. 194(2): p. 189-203. despite having the inflammatory cell genotype of the 9. Pantelidis, P., et al., Analysis of tumor necrosis factor- ΔSTAT gp130 animals. It is possible that the fibrosis alpha, lymphotoxin-alpha, tumor necrosis factor receptor protection in the gp130ΔSTAT animals is due to II, and interleukin-6 polymorphisms in patients with disrupted communication between lung resident idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, cells and blood-borne inflammatory cells. Recent 2001. 163(6): p. 1432-1436. studies have highlighted the importance of this 10. Epperly, M.W., et al., Bone marrow origin of communication in finding that the majority of myofibroblasts in irradiation pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol., 2003. 29(2): p. 213-224. collagen secreting cells in bleomycin and irradiation 11. Hashimoto, N., et al., Bone marrow-derived progenitor models of fibrosis are recruited from a circulating cells in pulmonary fibrosis. J Clin Invest, 2004. 113(2): p. population of cells [10, 11]. The decrease in TGF-β 243-252. signaling and concomitant increases in PGE2 and 12. Becker, C., et al., TGF-beta suppresses tumor MMPs 2 and 9 is indicative of tight regulation progression in colon cancer by inhibition of IL-6 trans- between inflammatory processes and mechanisms signaling. Immunity, 2004. 21(4): p. 491-501. involved in the normal resolution of injured tissue. TGF-β is know to induce fibrosis in a variety of tissue including the lung, recent reports indicate that there is cross-talk between the TGF-β and IL-6 Infl amm. res. Supplement 2 (2005) Mini-Paper 3 S 217

A novel role for Annexin 1 in macrophage phagocytosis Simon Yona1/2, Sigrid Heinsbroek2, Siamon Gordon2, Mauro Perretti1 & Roderick J Flower1. 1William Harvey Research Institute, University of London, London UK, 2Oxford University, Oxford, UK

BACKGROUND. Glucocorticoids (GCs) are still the most powerful anti-inflammatory drugs in clinical use today, fifty years after they were initially developed [1]. GCs regulate the synthesis, phosphorylation and cellular disposition of the anti-inflammatory protein annexin 1 (Anx-A1), leading to the concept that Anx-A1 acts as a ‘second messenger’ of GC action. Human and rodent macrophages (Mφ) contain high levels of Anx-A1 [2], and cellular activation or glucocorticoid treatment augments Anx-A1 protein expression and externalisation in human alveolar and rat peritoneal Mφ as well as in human monocytes [3]. In early experiments, GCs were observed to act as potent inhibitors of eicosanoid release from Mφ [4] and an annexin-like phagocytosis-inhibitory protein released from Mφ treated with GCs was identified by Grasso and his co-workers [5]. Treatment of monocytes and Mφ with exogenous Anx-A1 inhibits cellular activation, as assessed by superoxide generation, prostanoid release and enzyme or cytokine expression [6]. More recently, the Anx-A1 mimetic peptide Ac2-26 was shown to inhibit IgG immuno-complex phagocytosis by mouse peritoneal Mφ, as determined by flow cytometry [7].

To elucidate further the role of Anx-A1 in physiology and pathology we recently generated a line of Anx-A1 null (Anx-A1-/-) mice [8]. Superficially the phenotype of these animals is unexceptional but upon closer examination these mice displayed a heightened sensitivity to both acute [8] and chronic [9] inflammatory stimuli coupled with a reduced sensitivity to GCs. This study examines aspects of Mø phagocytosis which are altered in the Anx-A1-/- mouse and which may relate to the function of Anx-A1 during the inflammatory response.

METHODS. Anx-A1-/- and littermate control (Anx-A1+/+) mice (6-8 weeks old) were killed and bone marrow extracted, washed and cultured for one week in RPMI-1640 supplemented with 10% bovine serum and 15% L929-cell conditioned medium to obtain bone marrow derived Mφ (BMDMφ). To assay Mφ particle uptake, BMDMφ were incubated for 2 h at 37°C with FITC labelled zymosan or Neisseria. Control Cells were co-incubated with cytochalasin D (2 µM) throughout the experiment. Mφs were washed and harvested with PBS containing 10 mM EDTA supplemented with 4 mg/ml lidocaine-HCl prior to fixation with 4% formaldehyde. Fluorescence was analyzed by confocal microscopy and on a FACScan (Becton Dickinson, Mountain View, US) FL-1 channel. The median fluorescence intensity (MFI) of unloaded control cells was subtracted from the mean assay fluorescence and the average determined. To establish a better understanding of events occurring during phagocytosis, BMDMφ were incubated for 1 h at 37°C with FITC-zymosan, phagocytosis of the particles was recorded in real time. In certain experiments Anx-A1 localisation was recorded by confocal microscopy.

RESULTS. Figure 1 demonstrates that Anx-A1-/- BMDMφ (squares) exhibited a reduced uptake of, or association with, all particles tested when compared with the Anx-A1+/+cells (circles). For instance, cell association following 30 min with non-opsonised FITC-zymosan in Anx-A1+/+ cells (panel a) yielded 1944 ±54 MFI relative to 1450±48 MFI in the Anx-A1-/- cells (n=3 in duplicate); this phenomenon was also seen with Nesseria and sheep red blood cells and confirmed in the case of zymosan by real-time microscopy studies (panel b) where un-phagocytosed particles are still evident at 60 min in the Anx-A1-/- cells. In figure 2, BMDMφ were incubated with zymosan for 5 min together with FITC zymosan and stained for Anx-A1 and actin. During the initial stages of phagocytosis Anx-A1 is seen to co-locate with actin in the early phagosome.

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Infl amm. res. Supplement 2 (2005) Mini-Paper 4 S 219

Signaling pathway of TNF-α-induced AQP3 regulation of this protein in connection with acute expression in human gingiva: Implications in the periodontitis (Fig. 1A). In the gingival abscess tissues, AQP3 pathogenesis of periodontitis is found to be expressed in the spinous and lower granular Tancharoen S*, Matsushita K**, Matsuyama T*, Maruyama layers decreasing towards the surface. In addition, relatively I*, Torii M*. Kagoshima University Graduate School of vessels and infiltrate cells were observed in the connective Medical and Dental Science, Kagoshima*, Japan. Johns tissue area (Fig. 1B). Replacement of anti-AQP3 antibody Hopkins University Medical School, Baltimoure, USA**. with normal serum gave no specific labeling. The positive Background staining of AQP1 was strongly expressed under the basement Periodontitis is a disease initiated by overgrowth of membrane which exists between epithelium and connective periodontal bacteria and releasing of tissue-destructive tissue (Fig. 1A). Intensely stained AQP1 on vessels also cytokines, in particular tumor necrosis factor-alpha (TNF-α), appear positively at the periphery of the infiltrated cells (Fig. in conjunction with dramatic changes in fluid homeostasis at 1B). TNF-α positive§ staining was strongly recognized at the the level of the gingival sulcus. In keratinized oral epithelia wide area of connective tissue inflammatory cell infiltrate such as gingiva, a first physical barrier against the invasion of including in the epithelium (Fig. 1A), implying that the cells pathogenic organisms is represented by neutral lipid, and actively participate in the initiation and development of appears that it confers permeability to glycerol as well as to periodontitis. These results suggest high incidence of AQP3, water. AQP1 and TNF-α associated with the gingival inflammation, Specialized protein water channels, termed aquaporins compared to the healthy gingival tissues. (AQPs) are able to support a large volume of water flow in a mammalian tissue with a 10-to-100 fold higher capacity, compared to simple diffusion. AQPs are suspected in numerous pathological condition involving skin, airway inflammation, as well as gastrointestinal disease. The imbalance of ion transport in epithelia is paralleled by respective water movement across cell membranes, and subsequent alterations of cell volumn. AQP3 is implicated in water absorption across human epithelial cell surfaces. Transgenic mouse and mammalian cell models have recently provided clues about the role of AQP3 in normal physiology as well as the pathophysiology of multiple clinical disorders affecting humans. Although numerous analytical studies try to quantify the impact of gene variants on the risk of periodontal disease, our new study found that there must be a certain factor put individuals at higher risk for this rapidly progressing disease. An alteration in mechanism of a water sensor AQP3 We performed § three independent experiments to is a possible candidate for underlying periodontitis. visualize AQP3 protein on the gingival epithelial cell (Ca Methods & Results 9-22) surface membranes by depicting the fluorescence of the We analyzed the various isoforms of the AQPs- like cell population labeled with each AQP antibody and examined immureactivity family localized on human gingival epithelial by FACs analysis. Ca9-22 expressed significant levels of cells both in vitro and in vivo study. The severity of gingival AQP3 (Fig. 2A), AQP 7 and 10 protein on the cell surfaces, tissue inflammation was illustrated by clinical parameters and somewhat lower levels of AQP4 and 5 (data not shown). cellular histologic changes. The intensity of Immunofluorescence assay indicated AQP3 protein expression surface-associated AQP3 positive staining on inflamed on the cell surface (Fig. 2A, arrow head) gingiva was observed in the entire epidermal layer, and a part of infiltrated cells in the connective tissue, indicating an up S 220 Infl amm. res. Supplement 2 (2005)

TNF-§α initiates its actions by binding to its receptor. JNK, p38 MAPK and ERK1/2 activation following TN We found that TNF-§α react with anti-55-kd TNFR, but not stimulation. Phosphorylated JNK and p38 MAPK anti-75-kd TNFR antibodies examined by FACs analysis (Fig. observed at maximal at 7.5 min, and sustained for 30 m 2B, left). This data were confirmed by western blot analysis while the ERK1/2 signal was delay to 15 m revealing clearly that cooperation of the 55-kDa TNF receptor Pharmacological inhibition of p38 MAPK, JNK and ERK (TNF-R 55, Fig. 2B, right) is required for a full blown blocked TNF-§α -induced AQP3 production (Fig. 4A). response to TNF-α. Following addition of proinflammatory cytokine TNF-§α •§AQP3 transcription levels were increased in a The cAMP responsive element binding (CREB) pro time-dependent manner in both epithelial cell lines (Ca9-22 activates the transcription of target genes in response and KB). Few AQP3 positive cells were apparent among diverse stimuli. Phosphorylation of the CREB on serine control cells at time 0. However, exposure of cells to TNF-α by which TNF-α affected AQP3 were examined by wes for more than 4-hour induced major increases in both AQP3 bloting. TNF-α induced CREB phosphorylation at mRNA and protein expression, as confirmed by northern blot beginning of 7.5 min and sustained as long as 240 m and SDS-PAGE analysis, respectively (Fig. 2C). Pretreatment with selective inhibitor of cAMP-depend protein kinase, H-89 attenuated its phosphorylation The intercellular adhesion molecule (ICAM) 1 is an blocked AQP3 expression (Fig. 4B). Ig-like cell adhesion molecule expressed by several cell types, Discussion including human oral epithelial cell lines. It can be induced in Taken together, these results indicate a novel role a cell-specific manner by TNF-α in periodontitis condition. MAPK and CREB phosphorylation in TNF-α induced AQ Small interfering RNA (siRNA) induced remarkable gene expression in human gingival epithelial cells. The ability silencing in mammalian cells, has been shown for great inflammatory cytokines to increase AQP3 expression m promise as a tool to silence the specific genes with high help explain the connection between inflammation, edema specificity. We, therefore, knocked down the aqp-3 gene using contribute to the development of novel treatment AQP3 siRNA follow by additional of TNF-§α. We found that periodontal disease. The relationship between this w silencing expression of AQP3 blocks TNF-§α- §mediated channel protein and its functional role in periodontitis nee induction of ICAM-1 protein, indicating metabolic activities be further investigated. of specific water channel protein in periodontal disease (Fig. 3A and B).

It is well documented that mitogen-activated protein kinases (MAPKs) pathways are thought to be most important in transmitting inflammatory signals, after being triggered by cytokines from the cell surface to the nucleus. We identified Infl amm. res. Supplement 2 (2005) Mini-Paper 5 S 221

Joint damage and DC function during rheumatoid arthritis is strictly controlled by Fc gamma receptor IIb.

TRDJ Radstake, MD, PhD. Department of Rheumatology, Radboud University Nijmegen Medical Centre, The Netherlands.

Background Every immune response that is ignited has to be terminated to prohibit breakthrough of tolerance. Autoimmune diseases potentially result from an imbalance between activating and inhibitory pathways. Rheumatoid arthritis (RA) is such a disease characterized by chronic inflammation and cartilage breakdown of the synovial joints. DCs are the professional antigen presenting cells unrivalled in their capacity to regulate the balance between immunity and tolerance. Nowadays, a large body of evidence points toward the involvement of DCs in synovial inflammation. Antigen uptake by DCs is crucial for the regulation of the immune response for which DC are equipped with several receptors including Fc gamma receptors (Fc
Rs). Besides the uptake of antigens, Fc
Rs are crucial for the recognition of IgG-containing immune complexes (ICs) that are abundantly present in RA patients. In humans, three classes of Fc
Rs can be distinguished, Fc
RI, Fc
RIIa/b and Fc
RIII of which only Fc
RIIb exert inhibitory effects upon ligation. ICs therefore might have a dual role, both activating as well as repressing immune responses, which is ultimately linked to the phase of the inflammatory response and balance between activating and inhibitory Fc
Rs. In the light of such a conceptual framework it is conceivable that functional variants of Fc
Rs have profound effects on DCs function and synovial inflammation. The significance of Fc
Rs in arthritis has been extensively shown using experimental arthritis models. In humans however, the research on genetic variants of Fc
Rs has been limited to the activation Fc
Rs yielding variable results. In RA, alterations of Fc
R expression on macrophages and DCs both in the synovium and circulation have been documented and resulted in an altered cell behavior. Given the hypothesis that RA is associated with a decreased negative feedback of effector immune responses, the fundamental question arose whether Fc
RIIb confers protection to synovial inflammation and subsequent joint damage. The recent characterization of the functional 695T>C (I232T) polymorphism in the transmembrane region of the human inhibitory Fc
RIIb offers a good opportunity for a critical evaluation of this hypothesis and might provide pivotal clues for novel therapeutic strategies.

Methods Genotyping was performed in all RA patients participating in an early RA inception study started in 1985. This study includes only those patients who meet the ACR criteria for RA, have a disease duration < than one year, and have not been treated at presentation. All patients are well characterized and are regularly monitored for disease phenotype, severity and outcome. Healthy controls were recruited from blood donors of the same catchments area. Demographic data such as gender, age at onset and rheumatoid factor, HLA-DR4 and the shared epitope (SE) positivity were included in the analysis. We used the Disease Activity Score (DAS28) to determine the disease course during time. Radiological damage was assessed using the modified Sharp’s score. Genotyping of the 695T>C (I232T) polymorphism in FCGR2B was based on restriction fragment length polymorphism (RFLP) analysis and direct sequencing. We started with a long range PCR as described by Kyogoku et al (Arthritis Rheum 2002;46:1242- 54). Since we were not confident with regard to the specificity of the nested PCR product (sequencing showed evidence of FCGR2C in the samples, which might hamper the identification of homozygous carriers of the variant allele), we performed a nested-over- nested PCR on the purified 863 bp template (after 10.000 times dilution) from carriers of the variant allele with a forward primer that distinguishes between FCGR2B and 2C and the reverse primer used in the nested PCR. Monocyte-derived dendritic cells were cultured using standardized protocols as previously described (Radstake et al 2004). Flowcytometry was performed to determine the expression of Fc
RI (CD64), II (CD32) and III (CD16) and the expression of fluorescent ICs. The antigen uptake capacity was studied using human gamma globulins labeled with FITC. After binding, DCs were resuspended in the presence of 0.4% (w/v) trypan blue, which quenches only extracellular fluorescence. Flow cytometry was used to quantify antigen uptake. DCs from 4 patients and 4 healthy individuals were cultured simultaneously. TNF, IL-6, IL-10 and IL-12 levels were measured in the DC supernatants, using the Bio-Plex system (Bio-Rad Laboratories). The genotype frequencies of the 695T>C polymorphism in FCGR2B was tested for Hardy-Weinberg equilibrium using the standard goodness-of-fit test. Similarity of genotype and allele distribution between patients and controls was tested with Chi square test for 2x2 contingency tables. Differences in the disease characteristics between patients with the T/T and those with the T/C or C/C genotype were analyzed using Student’s t-test or Mann-Whitney U test, where appropriate. P-values of 0.05 and smaller were considered statistically significant. Multiple linear regression analysis was performed to assess whether the Fc
RIIb functional genetic variant is an independent explanatory factor for the level of disease activity and/or extend of radiological joint damage after 3 and 6 years of disease. To this aim a staged approach to model building was used. Based on earlier studies the following prognostic factors (independent variables) were included in the analysis: presence/absence of rheumatoid factor, HLA-DR4, SE and radiological damage at baseline.

Results 246 Caucasian RA patients and 269 healthy individuals were consecutively included in the present study and genotyped for the FCGR2B 695T>C (I232T) polymorphism. The distribution of genotypes and the allele frequencies among RA patients and healthy controls were found to be similar (pooled analysis for T/C and C/C: p=0.29). Neither the patient nor the control group showed significant deviations from Hardy Weinberg Equilibrium (p=0.44 and p=0.66, respectively). Altogether, these results suggest that the FCGR2B 695T>C variant is not associated with RA disease susceptibility. We next investigated the relationship between the FCGR2B 695T>C (I232T) polymorphism and RA disease characteristics. No differences in demographic or clinical features were observed. From the prospective analysis of patient and disease characteristics, data on 150 and 123 patients was available after three and six years of follow-up, respectively. No differences in disease phenotype was observed between the two study groups. Intriguingly however, patients possessing the FCGR2B functional variant used S 222 Infl amm. res. Supplement 2 (2005)

significantly more (and more aggressive) disease-modifying anti-rheumatic drugs (DMARDs), suggesting that these patients suffered from a more aggressive disease phenotype. When looking at radiological joint damage, patients possessing the FCGR2B functional variant were found to have a significantly higher level of joint damage after three (P < 0.0001) and six years (P < 0.0001) of disease follow-up, as Figure 1. Radiological joint damage measured by the modified Sharp method (figure 1). Remarkably, the 695T>C functional variant was not associated with disease features often associated with joint damage, suggesting that FcRIIb variation 150 should be considered as a risk-factor independent of these others. To test the independency of Fc
RIIb Sharp TC TT as a risk factor for radiological damage, we performed a multivariate regression analysis including disease 100 variables that have been shown to be predictive for progression of radiological joint damage. In the model, the FCGR2B 695T>C variant was by far the strongest predictor for radiological damage after three (P < 0.00001) and six (P < 0.0001) years of disease. The genetic variant alone explained 23% and 28% of the 50 radiological damage after three and six years. In accordance with previous studies, the only other significant contributors to this model were the presence of rheumatoid factor (P < 0.005) and the level of

0 radiological damage at inclusion into the study (P = 0.003). Baseline 3 years 6 years

We have recently Figure 2. IC uptake by DCs shown that Fc
R-driven DC function in RA is altered and heavily dependent on the expression of the inhibitory Fc
RIIb (Radstake et al Ann Rheum % cells * Dis 2004). Here we70 studied whether the possession of a FCGR2B 695T>C allele * translates into altered60 DC functional behavior. The uptake of fluorescent ICs by * immature DCs from RA 50 patients expressing one allele of the Fc
RIIb functional variant was already strikingly40 lower (46%, P = 0.0005) after 30 minutes of incubation compared to those30 carrying only wild type alleles (figure 2). After 60 minutes of incubation the20 percentage of fluorescence-positive cells in the wild type samples was 58%  10 6% (mean  SEM). In contrast, only 33%  4% of the immature 0 DCs expressing one 15 30 45 60 allele of the Fc
RIIb functional variant was found to be Time (min)

695 TT genotype 695 CT genotype Figure 3. TNF secretion upon stimulation with IC fluorescence-positive (P < 0.00001). We have shown previously that DCs from most RA patients display TNF-
an inhibitory phenotype upon Fc
R-mediated triggering compared to the activating phenotype displayed 4000 3500 by DCs from healthy controls (Radstake et al Ann Rheum Dis 2004). The production of cytokines by DCs 3000 is an important read-out for DC function. We therefore compared the production of TNF-, IL-6, IL-10 2500 and IL-12 by DCs from RA patients with and without the Fc
RIIb functional variant upon stimulation with 2000 1500 either LPS alone or LPS in combination with ICs. As expected, DCs from all RA patients (n=4) carrying 1000 FCGR2B wild type alleles decreased the production of TNF- (figure 3), IL-6 and IL-12 and increased 500 0 secretion of IL-10. In contrast, DCs from RA patients possessing one allele of the FCGR2B functional TT TC variant (n=4) failed to inhibit this response to LPS. These findings further substantiate that the genetic variant has marked functional consequences for DC function upon IC mediated triggering. Flowcytometry with specific antibodies against Fc
RIIa and Fc
RIIb reveals a similar expression of these Fc
Rs between DC with or without the functional variant. These data suggest that the genetic variant influences the function of Fc
RIIb rather than its surface expression.

Discussion Although the role of activating Fc
Rs in rheumatoid arthritis (RA) has been thoroughly studied, the potential role of the inhibitory Fc
RIIb has not been subjected to similar scrutiny. In this study we provide strong evidence for the functionality of the Fc
RIIb genetic variant. At first, the FCGR2B 695T>C polymorphism is the far most strongest predictive factor for joint damage in RA thus far. Secondly, we demonstrated that the function of DCs is markedly altered in patients carrying the Fc
RIIb variant, resulting in the loss of an inhibitory DC phenotype as seen in those patients carrying the Fc
RIIb wild type. It is well documented that the balance between triggering of activating and inhibitory Fc
Rs determines the outcome of the immune response. Kalergis et al. demonstrated that selective engagement of activation Fc
Rs by ICs led to potent T cell activation whereas stimulation with ICs in the presence of such inhibitory Fc
RIIb resulted in tolerance (Kalergis et al. 2002). Furthermore, Fc
RIIb has been identified as a modulator of peripheral tolerance given the observation that Fc
RIIb mice render susceptible to experimental arthritis and other autoimmune diseases including SLE (Yajima K et 2003 and Bolland et al 2003). On the functional level Li et al. elegantly demonstrated that the Fc
RIIb I232T genetic variant, when co-engaged with the B-cell antigen receptor (BCR), resulted in an enhanced capacity to dephosphorylate tyrosine residues in CD19 and inhibition of the calcium influx (Li et al 2003). We here demonstrate that the genetic alteration does not influence the expression of the receptor. In this light it is tempting to speculate that patients with the Fc
RIIb functional variant have a less functional Fc
RIIb, resulting in a less efficient inhibition of the activation Fc
Rs leading to a chrinic activation of DC and subsequent damage. The exact effect of the function of Fc
RIIb needs to be unravelled. Therefore, we are planning to build in the functional mutation in the Fc
RIIb gene to test the folding, transmembrane expression and function using sophisticated molecular techniques. Taken together, here we provide evidence for the important role of Fc
RIIb in DC function and joint damage in RA, which underscores the rationale to manipulate DC function via Fc
R balances in the treatment of RA and other autoimmune diseases. Infl amm. res. Supplement 2 (2005) Mini-Paper 6 S 223

IMMUNE COMPLEXES ALTER CEREBRAL MICROVESSEL PERMEABILITY: ROLES OF COMPLEMENT AND LEUKOCYTE ADHESION

Karyn J. Lister and Michael J. Hickey BACKGROUND: Immune complexes are highly potent pro-inflammatory mediators, which have been implicated in several autoimmune diseases, particularly systemic lupus erythematosus (SLE). Numerous studies have identified the mechanisms responsible for IC-mediated inflammation in the peripheral microvasculature. These studies have demonstrated that ICs induce rapid changes to the microvasculature by increasing leukocyte recruitment and microvascular permeability. However it is not known whether ICs are capable of inducing similar changes to the cerebral microvasculature. This question is relevant because the brain is among the most commonly affected organs in SLE patients, with ICs regularly detected in the CNS. Studies have shown that mediators of acute inflammation can be much less effective in the brain compared to responses observed in peripheral microvasculatures. The CNS is considered an immuno-privileged site due to the presence of the blood brain barrier (BBB), a structural and functional barrier within the microvasculature, which restricts the entry of macromolecules and immune cells into the CNS. However, despite the refractory nature of the brain to inflammatory stimulation, studies have shown that the cellular and molecular requirements for IC-mediated inflammation are in fact present in the cerebral microvasculature. Therefore the aim of this study was to determine if ICs are capable of inducing responses the cerebral microvasculature. These results reveal for the first time that ICs are capable of inducing alterations in microvascular permeability and leukocyte recruitment in the cerebral microvasculature.

METHODS: Closed Cranial Window Preparation: A craniotomy was performed in the right parietal bone to expose the pial microvasculature. A superfusion chamber was applied containing two ports for continual superfusion of the brain with artificial cerebral spinal fluid (aCSF), and sealed to maintain intracranial pressure.

Reverse Passive Arthus (RPA) Reaction in the Pial Microvasculature: RPA was induced by i.v. injection of 1000 g ovalbumin (OVA) and superfusion of anti-OVA over the pial surface at 5 or 50 g/ml in aCSF. Control animals received OVA but were superfused with non-specific rabbit IgG at the same concentration. Leukocyte recruitment and microvascular permeability responses were assessed at various time points immediately after induction of the RPA response.

Location of IC formation in vivo: Location and timing of IC formation was examined using RPA response with fluorochrome conjugated OVA (Alexa-488) injected i.v. via tail vein and fluorochrome conjugated anti-OVA (Alexa-594) superfused over the brain. The response was visualized using fluorescent IVM. Unfixed 10 µm brain sections were examined using confocal microscopy for co-localisation.

Cerebral Microvascular Permeability: The closed cranial window was prepared and mice received i.v. FITC-dextran-70kDa and aCSF was collected. Arterial blood samples were taken to determine plasma FITC-dextran concentration. The concentration of FITC-derived fluorescence in aCSF and plasma samples was measured on a 96-well microplate reader. Clearance (nl/sec) was determined as a ratio of fluorescence present in aCSF to fluorescence in plasma, multiplied by the flow rate of aCSF across the brain.

Intravital Microscopy (IVM): The pial microvasculature was visualized using intravital microscopy to examine leukocyte-endothelial cell interactions, to analyze rolling flux, (cells/min), velocity (m/sec) and adhesion (stationary cells for 30 sec or longer).

Depletion of Complement: Purified Cobra Venom Factor (CVF) derived from Naja melanoleuca venom and was administered in a dose of 25 g/mouse injected i.p. 24 hrs before surgery.

Leukocyte ß2 (CD18) Integrin Expression Blockade: Mice were treated with mAb anti-CD18 (4mg/kg) immediately prior to the RPA model. S 224 Infl amm. res. Supplement 2 (2005)

IAIS YOUNG INVESTIGATOR AWARD Karyn Lister

Immunohistochemical Identification of C3 Deposition: 10 µm brain sections of the cranial window were fixed in periodate/lysine/paraformaldehyde (PLP) and cryoprotected in 20% PBS and were directly stained with FITC-goat anti-mouse C3.

RESULTS: Location of IC formation during the RPA response: Anti-OVA was localised to perivascular sites within minutes after initiation of the RPA response. Confocal microscopy confirmed co-localisation of OVA and anti- OVA in perivascular regions (Fig.1). Immunohistochemistry revealed C3 deposition in RPA- treated brains. This suggests that ICs form rapidly adjacent to pial microvessels in association with C3 deposition. Figure 1: Co-localisation of OVA-488 and anti-OVA-594 60 min after the initiation of the RPA response

Effects of ICs on the cerebral microvasculature: 0.5 RPA Initiation of the RPA response induced rapid dose- OVA/Rabbit IgG Untreated 0.4 # # # dependent changes in cerebral microvascular # # * # Start RPA * # * permeability, with higher doses increasing leukocyte * * * * 0.3 # adhesion in pial post-capillary venules (Fig. 2) * *

0.2

Cerebral RPA response requires C3: Depletion of C3 Clearance (nl/sec) prevented RPA-induced alterations in microvascular 0.1 permeability and leukocyte adhesion. The absence of C3 staining in brains of CVF-treated mice after the 0.0 RPA response was confirmed using -10-50 5 1015202530354045505560 Time (min) immunohistochemistry. 250 RPA OVA/Rabbit IgG Untreated 200 Inhibiting leukocyte adhesion reduces the RPA # ) response: P-selectin was required for the RPA- 2 Start RPA * induced leukocyte adhesion and microvascular 150 permeability, as both responses were inhibited in P- 100 selectin-/- mice. Similarly, blockade of the leukocyte Adhesion (cells/mm Adhesion # 2-integrin (CD18) inhibited leukocyte adhesion and 50 was associated with reduced microvascular permeability 0 -15 0 15 30 45 60

Time (min) Figure 2:RPA response in the pial microvasculature after administration of OVA and superfusion of 50 g/ml anti-OVA (RPA) or non-specific IgG.

DISCUSSION: In SLE, a wide range of tissues can be affected by inappropriate inflammation, including the brain. The effects on the brain are demonstrated by the high incidence of neurologic complications such as cognitive impairment, seizure and stroke in SLE patients. Furthermore, the presence of ICs has been detected in the CNS of SLE patients. However, the role of the inflammatory response in inducing these complications is poorly understood. Additionally the role of ICs in mediating inflammatory responses in the CNS has not been investigated. These studies are the first to show that ICs, a key effector pathway in SLE, are capable of perturbing the immuno-privileged environment of the cerebral microvasculature, demonstrated by elevated levels of microvascular permeability and increased leukocyte recruitment. This suggests that the IC-mediated inflammatory pathway has the potential as a therapeutic intervention in cerebral SLE. Infl amm. res. Supplement 2 (2005) S 225 Author Index

Önnervik, Per-Ola 5101 Bélanger, Caroline 6074 Bosnjak, Berislav 5065 5075 Aalami, Oliver 7014 Baban, Babak 5044 Bouérat, Laëtitia 6066 Abbot, Stewart 6032 Baines, Katherine J 5076 Bouchard, Line 6018 Abdin, Md Joynal 6036 Baj, Zbigniew 7109 Boulanger, Jean 6018 Abdollahi, Shahla 5002 Bakhle, Y S 6061 Boulday, Gwenola 2038 Abraham, David 5038 Ball, Helen J 7078 7097 Boyle, Michael J 5076 Abraham, Lawrence J 6037 Banerjee, Ashish 7059 Bozinovski, Steve 1017 5106 Achuthan, Adrian 6048 Bannon, Paul G 5054 6019 Bozinovski, Steven 1018 1031 2048 5103 Aeberli, Daniel 6029 Banovic, Tatjana 7047 Bräuer, Rolf 5005 5017 Afi fi , Mohamed I 6088 Bao, Shisan 2018 Bradford, Tessa 3028 Aggarwal, Arun Kumar 5034 Baraket, Melissa 5077 Braine, Emma 5036 Aguilar, Luc 5016 Barja-Fidalgo, Christina 2036 7043 Braine, Emma L 5025 6053 Ahmad, Rasheed 3011 Barrera, Pilar 2047 Brand, C 5106 Ahmed, Mohamed I 6088 Barrett, John W 6017 Breit, Samuel N 5025 Ahmed, Sagheer 7103 7120 Bartoccioni, Emanuela 6016 Brender, Christine 6028 Ahmed, Salahuddin 5003 Bast, Aalt 6033 Bresnihan, Barry 7023 Akarasereenont, Pravit 6045 Beagley, Kenneth W 5080 Brew, Bruce j 7093 7096 Al- Swailem, Ramiz 5007 Beaulieu, Elaine 6030 Briddell, Robert 6010 Al-Abed, Yousef 7072 7073 Beavitt, Sarah-Jane E 3014 Brouckaert, Peter 7001 Al-Rayes, Hannan 5007 Beletskii, Anton 6032 Bruckdorfer, Richard K 5038 Alborzi, Abdolvahab 5043 7077 Bell, Noreen V 5076 Brummer, Tilman 7038 Alexander, Warren S 5009 6028 Bellette, Bernadette M 6067 Brunmark, Charlott 5101 Alexander, Warrren S 6049 Belloni, Paula N 5061 Bryant, Katherine J 7034 Ali, Yusuf M 7068 Bendele, Alison M 7027 Bu, Xiangyong 6007 Alikhani, , Mohammad yousef 6012 Bennink, Miranda B 2037 6003 Bucala, Richard 3034 6084 Alleman-Sposeto, Jennifer 7004 Bernard, Claude CA 7090 Buckton, Jacky 3026 Amalric, Francois 5016 Bernard, Nicholas J 7092 Bujold, Kim 7119 Americh, Laure 5016 Bernhagen, Jürgen 3034 6084 Bunning, Rowena A 6039 Amiri, Iraj 6044 Bertrand, Claude P 3007 Burger-Kentischer, Anke 3034 Amrutkar, Dipak 5022 Bevilacqua, Estella 7040 Burgess, Janette K 5077 Anandaroop, Mukhopadhyay 7021 Bhandari, Rajan P 5098 Burgos, Rafael A 6042 Anderson, Gary P 1017 1018 1031 2048 3014 Bhatia, Madhav 6043 6072 6075 7016 7046 Burns, Christopher J 6007 5106 7055 7098 Butler, Keith D 5105 Anderson, Karen 5004 Bhuvanath, Suthinee 6055 Cabrera, Wafa H 6005 6058 Anderson, P 5103 Bidgood, Matthew J 7034 Cai, Hong 5048 Andersson, Christina 6066 Bihalskyy, Ihor Y 6077 Calandra, Thierry 5012 Andrea, Itano 7051 Biron, Kristian 7050 Caliari, Marcelo V 7089 Andresen, Lars 5051 Bischof, Rob J 5078 Campbell, David 5010 Anniss, Angela M 5046 Bischof, Robert J 5069 Campbell, Iain L 3025 7097 Anu, Gore 7051 Bk, Shyam K 5098 6062 Campbell, Ian K 2017 5027 Anuar, Farhana 7011 Black, Jim 5103 5106 Camps, Montserrat 3015 7038 Anumula, Madhavi 3021 Black, Judy L 5077 Carretero, Oscar A 7076 7123 Aoudjit, Fawzi 5055 Blaydon, Clare 5104 Carroll, Graeme J 6022 Appleton, Ian 5049 7012 7080 7085 7086 Blom, Anna M 7006 Cashman, Johanne D 6026 7087 7088 Blomberg, Lovisa M 6040 Cauwels, Anje 7001 Ardissone, Vittoria 3015 Blumenschein, Wendy M 2044 Cavalcanti, Danielle Maia 2025 Arendarczyk, Jerzy 7108 Bobik, Alex 3032 7113 7115 Cavasin, Maria 7123 Arfi n, Misbahul 5007 Bobrowski, Paul J 5003 Cepelak, Ivana 5100 Armstrong, Jeffrey S 7037 Bobryshev, Yuri 7112 Chadwick, Vinton S 5094 Armugam, Arunmozhiarasi 5095 Bockemuehl, Birgit 5061 Chadzinska, Magdalena 6085 Arndtz, Cora H 5002 Boermann, Otto C 2015 Chai, Siaw C 5095 Arnold, Bernd 6085 Bogdanoska, Violeta 5036 Chaikin, Marge A 5039 Arntz, Onno J 2037 6003 Boichot, Elisabeth 3010 Chamoux, Estelle 5055 Arruda, Maria Augusta 7043 Bolton, Christopher 5021 Chan, Jasmine HP 5081 Arya, Soneera 6087 Bolvig, Simon 6066 Chan, Sui Y 7019 Asadullah, Khusru 2020 Bord, Evelyn 7104 7105 Chan, Sui Yung 7015 Asmat, Atasha 7116 Borelli, Primavera 2025 Chan, Yiong-Huak 7116 Assi, Lakhvir K 7017 Borgeat, Pierre 6074 Chandler, Phillip R 5044 S 226 Infl amm. res. Supplement 2 (2005)

Chang, Chan Fong 5091 Dabbagh, Karim 5061 Eyles, Jo L 2017 Chang, Margaret 7032 Dacumos, April 7002 7005 Faggioni, Raffaella 6006 Charles, Kellie 6068 Dadfar, Elham 6081 7101 Fahmy, Roger 1026 Charman, Christine 5105 Dai, Erbin 1021 3035 6017 Fahrngruber, Hermann 6001 Charreau, Beatrice 2038 Dai, Xu-Ming 3004 Fallah, Fatemeh 6056 6086 7018 7053 7081 Chatterjee-Kishore, Moitreyee 1012 Dajee, Maya 3016 7004 7102 Chaudry, Ataf 3011 Daly, Norelle L 6040 Fankhaenel, Stefan 7024 Chen, Jinsheng 5039 Danylec, Basil 1036 Fantino, Emmanuelle 1024 Chen, Meng-Jung 7106 Darzins, Peteris J 7079 Faour, Wissam H 7025 Chen, Yi 2044 Dawei, Zhang 7051 Farrugia, William 3028 Cheng, TT 4005 De Franco, Marcelo 6005 Farsky, Sandra Helena Poliselli 2025 6080 Cheng, Yew Kuang 5040 De Freitas, Marta S 2036 Fayaz, Fariba 5099 6008 Cher, Charmian DN 5095 de Kretser, David M 5084 Fear, Mark W 7033 Cherry, Kate 7095 De Nardo, Dominic 3029 Febbraio, Maria 7119 Cheung, CY 7031 De Nardo, Dominic F 7060 Feldmann, Marc 2001 Chevali, Lakshmi 6075 de Souza, Pedro B 7043 Feng, Xiao 6006 Chitu, Violeta 3004 de Veer, Michael J 7065 Fenner, Jen 7036 Chng, Hiok Hee 5040 Defranoux, Nadine A 5035 Fenner, Jennifer E 6051 Choi, Eun Kyoung 7003 Delavallee, Laure M 6003 Fensholdt, Jef 6066 Choi, Jin Kyu 5063 van den Berg, Wim B 5002 Ferencic, Zeljko 5075 Choi, Jung-Hwa 7003 Dey, Deben 3021 Ferguson, Angela L 5080 Chooklin, Serge 6013 Di Battista, John A 5018 7025 Fernandes, Cristina Maria 7040 Chooklin, Serge N 6077 di Nezza, Lisa 1036 Fernandes, Patricia Dias 7054 Chopra, Kanwaljit 7071 Di Pace, Roberto F 6058 Ferrante, Antonio 7110 Chotewuttakorn, Sirikul 6045 Diemer, Geoff S 5093 Ferreira, Jorge Mario 2025 Christensen, Camilla L 7013 Dimberg, Jan 5057 Ferrero, Laura 7013 Chu, Siew Hwa 7011 Dinh, Hang C 5090 Fingerle-Rowson, Günter 6084 Chugunov, Alexandr V 7082 Ditiatkovski, Michael 7113 7115 Fitau, Juliette 2038 Chukhlovina, Maria L 6054 Dixon, Darcelle N 7033 FitzGerald, Oliver 7023 Church, W Bret 7034 Do, Vinh D 7021 Fjording, Marianne S 7007 Cirillo, Rocco 3015 Dobbin, Caroline 7047 Fleetwood, Andrew J 6020 Clanchy, Felix IL 6021 6023 6024 Docx, Cerys 5105 Fleming, Stephen B 5058 Clark, Shannon M 6017 Doecke, Wolf-Dietrich 2020 Fletcher, Joel 6028 Clarke, Stephen 2030 6068 Dohi, Toshihiro 6036 6046 Fletcher, John P 6079 7107 Clarkson, Andrew 5049 Dominis Kramaric, Miroslava 5075 Flores, Flor de Maria L 7047 Clarkson, Andrew N 7012 7080 7087 7088 Dong, Yan 5088 5089 Flower, Roderick J 2045 Cleland, Leslie 7035 Donn, Rachelle 5012 6030 Fogel-Petrovic, Mirjana 6037 Clyne, Colin D 7002 Dooley, Audrey 5038 Fogler, William E 5041 Coelho, Ana Lucia J 2036 Dowling, John 7124 Fonceca, Angela M 7052 Coenen, Marieke J 2047 Downs, Anthea M 5030 6025 Fong, Kok Yong 5013 5014 Coghlan, Marelyn W 5095 Drevets, Douglas A 3005 Foord, Orit 6006 Collins, Margaret E 6015 Drouin, R 5062 Fortune, Farida 7062 Columbus, Ruth E 6049 Du, Wenbo 7067 Foster, Natasha C 1013 5024 Colville-Nash, Paul R 1022 5033 5037 Dugar, Sundeep 1027 Foster, Paul S 3020 5080 Commandeur, Zoe A 3020 Dugin, Sergey Ph 7082 Fourie, Anne M 6070 Concha, Ilona I 6042 Dunn, Jasmyn A 7048 França, Dorothéa S 6061 Conway, Theresa M 4006 DuPont, Michelle 5004 Francischi, Janetti N 6061 6076 7026 7089 Cook, Andrew D 2042 5025 5036 6020 6053 Durrani, Omar 7062 Franke, Barbara 2047 5012 Cook-Johnson, Rebecca J 7035 Dussault, Andrée-Anne 6018 Franks, Carol F 5039 Cooney, Gregory J 3020 Easton, Christopher J 7110 French, Martyn 7095 Cooper, Phillip R 5105 Ebert, Sandra 7091 Frew, Ailsa 6028 Cormont, Francoise 5085 Eccles, Kirstie A 7111 Frost, Melinda J 5079 Cotter, Matthew J 7045 Edwards, Ros 7064 Fukuda, Yoshiaki 6065 Coulon, Flora 2038 Egan, Paul J 5009 7061 Fung-Leung, Wai-Ping 3030 Courtenay, Brett G 7034 Egerland, Ute 5070 6004 Gahmberg, Carl G 2015 Couture, Julie 5055 Ehrhardt, Rolf O 3016 7004 Gajda, Mieczyslaw 5005 Covey, Todd 7039 Ekholm, Kjell 5101 Galetzka, Christin 6004 Craik, David J 6040 Eklund, Kari K 5020 Galibert, Laurent 5093 Croker, Ben A 5009 El MABROUK, Mohammed 5008 Gamble, Jenny 7035 Cronau, Steve 7066 El MABROUK, Mohammed 3011 Gao, Beirong 5038 Cross, Lena 3029 Elsegood, Caryn L 6047 7032 Garotta, Gianni 7073 Cua, Daniel J 2044 Emery, P 4005 Garvin-Queen, Laura 5061 Culic, Ognjen 5065 5075 5100 Erakovic, Vesna 5065 5075 5100 Gavins, Felicity N 6071 Cull, Vanessa S 7033 Erber, Wendy N 6025 Gavrilovic, Jelena 7114 Cullen, Karen M 7093 7096 7099 Eriksson, Per 7118 Gayon, Regis 5016 Cunningham, Fiona M 6015 Ernst, Matthias 1018 2028 2048 Geczy, Carolyn 1026 5054 6019 7112 Curnow, John 7062 7063 Eslami, Gita 6056 6086 7018 7053 7081 7102 Geczy, Carolyn L 5048 Cvetkovic, Ivana 6009 7072 7073 Estelles, Rossana 6073 Gelderman, Kyra A 5006 D‘Aiuto, Francesco 6027 Esther, Trueblood 7051 Gendron, Steve 5055 d‘Apice, Anthony J 3037 Evans, Krystal J 5053 7092 Georgiev, Ivan 3034 Infl amm. res. Supplement 2 (2005) S 227

Geraets, Liesbeth 6033 Han, Hyunsil 7030 Huang, Yu 7029 Gerondakis, Steve 5027 7038 7059 Han, Su-Ji 7003 Hughes, David A 7114 Geutskens, Sacha B 3005 Hancke, Juan L 6042 Hultqvist, Malin 5006 Ghaderi, Mehran 7118 Hannawi, Suad M A 5028 Hume, David A 5096 6007 6040 7048 7049 Gharizadeh, Baback 7118 Hanrahan, P 4005 7050 7066 Ghimire, A 5106 Hansbro, Philip M 5080 Hunt, John 5048 Ghosh, Peter 5011 Hansen, Anna M 7078 Hunt, Nicholas H 2018 7078 7097 Giardina, Tindaro M 6038 Hansen, Diana S 5053 7092 Hutchinson, Anastasia 5103 5106 Gibbones, Simon 7053 Hansen, Jens R 6066 Hutchinson, Paul 5026 Gibson, Peter G 5074 5076 5080 Hansen, Michelle J 1017 1031 Huynh, Francois 3011 Giddings, June 5105 Haqqi, Tariq M 5003 Ibañez, Olga M 6005 6058 Gilani, Anwar H 7120 Haque, Nasreen S 6069 Igor, Loreto J 6042 Gilbert, Tom 1036 Harb, Diala 7119 Illig, Carl R 5039 Gilroy, Derek W 1022 Harder, Kenneth W 3014 Im, Suhn-Young 6057 7003 Girard, Jean-Philippe 5016 Hardy, Charles L 5084 Inagaki, Naoki 7083 Giraud, Andrew S 2028 Harhaji, Ljubica 7073 Inder, Marie K 5058 Glojnaric, Ines 5100 Harnett, Margaret M 5073 Inglese, Melissa 1018 Godilot, Paul 4006 Harnett, William 5073 Irmler, Ingo 5005 Goebel, Heike 3034 Hart, Prue 5048 Irvine, Katharine M 6007 7050 Gold, Wendy A 1013 5024 Hart, Prue H 6014 7028 Irving, Louis 5103 5106 Gonçalves, Heloisa H 7026 Harte, Michael F 6007 Ishihara, Kenji 6063 Goncalves, Isabel 7006 Hashimoto, Hideo 1037 Iskander, Magdy 1036 7115 Gordon, Caroline 7017 Hashizume, Tomomi 5052 Itano, Andrea 6010 Gordon, Siamon 2045 Havez, Sophie 6066 Ivetic Tkalcevic, Vanesa 5065 5075 Gorgun, Cem Z 5079 He, QingWen 5018 7025 Ivtchik, T 5102 Goto, Megumi 6065 Healey, Geraldine 5060 Iwakura, Atsushi 7104 Goudarzi, Hossein 6060 6086 7018 7081 7102 Hebeda, Cristina Bichels 6080 Jackson, Chris 5010 Goukassian, David 7104 7105 Hecker, Markus 7024 Jackson, David M 7080 Gould, Jodee A 6051 Hegde, Akhil 6072 7055 Jacobson, Stefan 7101 Graça-Souza, Aurelio V 7043 Heinsbroek, Sigrid 2045 Jacobson, Stefan H 6081 Graham, Anne M 7111 Helal, Edward 2036 Jadali, Farzauneh 7081 Graham, Garry G 7034 Held, Claes 7101 Jaffee, Bruce 5004 Grail, Dianne 1018 Helps, Stephen C 7085 JahanSepas, Mohammad 6086 Granger, Niel 6071 Helsen, Monique M 5002 Jakobsen, Kristoffer P 5085 Green, Larry L 6006 Hergersberg, Christoph 6032 James, David 6048 Gregory, Julia L 6035 6082 Hertzog, Paul 2031 James, Michael 7035 Grey, Pearl 7036 Hertzog, Paul J 6031 6051 7036 James, Will G 7094 Grey, Shane T 5083 Herzog, Herbert 5045 Jancar, Sonia 5071 7054 Griffi ths, John R 2011 Hibbs, Margaret L 3014 Jatta, Ken 5057 Grinnell, Brian W 2040 Hickey, Michael J 2026 2049 5023 6035 6082 Jayalath, Ramani 6089 Gu, Yin 6070 7094 7121 Jazayeri, Jalal 6022 Guénon, Isabelle 3010 Hidalgo, María A 6042 Jeffrey, Kate L 7038 Gualano, Rosa C 1017 1031 Hill, Geoff R 1020 7047 Jenkins, Alicia 7075 Guglielmotti, Angelo 6075 Hilton, Douglas 7036 Jenkins, Brendan 2028 6028 Guiffre, Ann K 6079 7107 Hilton, Douglas J 6028 Jensen, Lene 7007 Guillemin, Gilles j 7093 Hoefgen, Norbert 5070 Jenzewski, Peter 7091 Guillemin, Gilles J 7096 Hogarth, Mark 3028 Jeong, Yeon Su 5063 Gullapalli, Srinivas 5022 Hohlfeld, Reinhard 3023 Jersmann, Hubertus PA 7110 Gunatilake, Mangala 6089 Hoi, Alberta Y 5023 Jeyaseelan, Kandiah 5095 Gupta, Shivali 5034 Holdsworth, Stephen R 7121 7122 7125 Ji, Hong 3015 Gusev, Evgeniy I 7082 Holmberg, Jens 5006 Jia, Sun 7016 Gutiérrez, José M 7041 Holmdahl, Rikard 5006 Jin, Denan 6064 Gutiérrez, José Maria 7040 Holz, Lauren E 5085 Johnen, Heiko 5025 Höfgen, Norbert 6004 Homer-Vanniasinkam, Shervanthi 7111 Johnson, Barbara J 7047 Hückel, Marion 5005 Hong, JangJa 6063 Johnson, Ella 5021 Haagen Nielsen, Ole 5051 7013 Hoppmann, Joachim 5070 Johnson, Peter R 5077 Haak-Frendscho, Mary 6006 Horvat, Jay C 5080 Johnson, Richard S 5093 Hage, Thorsten 6004 Hotamisligil, Gokhan S 5079 Johnston, Helinor 5104 Hagelgans, Albert 7042 Howard, Chris 7047 Johnston, Sebastian L 5077 Hageman, Geja J 6033 Howe, Hwee Siew 5040 Jones, Jessica 1018 2048 Hall, Irene 5049 7012 Howlett, Meegan 2028 Jones, Jessica E 1017 Hall, Pam H 7005 Hoyoux, Anne 5092 Jones, Lynelle K 7125 Hall, Pamela 1036 Hrvacic, Boska 5065 5075 5087 Joosten, Leo AB 2037 5002 Hall, S 4005 Hsieh, Chia-Jung 7106 Jose, Peter J 6073 Hamburger, John 7062 Hsu, Kenneth 7112 Joshi, Shambhu D 5098 6062 Hamdan, Leila 6074 Hu, Yonghan 5067 Joyce, David A 6034 6038 Hamilton, John 6048 Huang, Diana 6069 Judd, Louise M 2028 Hamilton, John A 3006 3029 5025 5029 5036 Huang, Hua 7110 Julien, Denis 5016 5090 6020 6021 6023 6024 6031 6047 6053 Huang, Jing-Feng 6070 Juneau, C 5062 7032 7060 7090 Huang, Xiao R 3039 7008 Jung, Bongnam 6057 S 228 Infl amm. res. Supplement 2 (2005)

Juutilainen, Timo 5020 Kowalsky, Elisabeth 6001 Lichtinger, Monika 7050 Kacevska, Marina 6068 Kremer, J 4005 Liddle, Chris 2030 6068 Kaiko, G 5080 Kritharides, Leonard 5054 6019 Lim, Ivor J 7014 7015 7019 7020 7021 Kairemo, Kalevi 2015 Krohn, Regina 3034 Lim, Sam 5077 Kalidomos, Georgina 6067 Kronbach, Thomas 5070 Lin, HY 4005 Kamchatnov, Pavel R 7082 Kubes, Paul 4001 Lindahl, Maria 5101 Kanchanapin, Walailak 6055 Kuffner, Tamara 5025 Lindstedt, Ken A 5020 Kane, David 7023 Kujawa, Julie 5004 Linscheid, Philippe 5047 Kang, Jee-Hae 6057 Kuligowski, Michael P 2026 Lister, Karyn J 2049 Kang, Nam In 5068 Kulkarni, Shrinivas K 7069 7070 7084 Listopad, Joanna J 2020 Kankaanranta, Hannu 7022 Kumanogoh, Atsushi 7125 Liu, Fang 7008 Kano, Yoshihiko 5064 6059 Kumar, Rakesh K 5079 Liu, Hanzhong 7080 Kapoor, Mohit 5049 Kumar, V L 6087 Liu, Liying 6017 Kapoor, Mohit B 7012 Kumar, Vijay L 7074 Liu, Lying 1021 3035 Karande, Vikas 5022 Kunugiza, Yasuo 1037 5031 Liu, Sue M 5083 Karimi, Abdollah 5043 7077 Kurita-Ochiai, Tomoko 5052 5059 Liu, Wai 5097 Karlsson, Lars 6070 Kuss, Hildegard 5070 Liu, Xiang 2031 Karwa, Amolkumar 2013 Kwan, Rain Yu Qing 7121 Liu, Zhi 5093 Kastelein, Robert A 2044 Labbe, G 5062 Lo, Susan Z 6034 Kaukinen, Sami 2015 Lacey, Derek 5026 Lobb, Roy R 2024 Kawakami, Masanobu 5064 6059 Lafl amme, Cynthia 6018 Locarnini, Stephen 7064 Kay, Thomas 7090 Lagente, Vincent 3007 3010 Lokuhetty, Dilani 6089 Kazemi, Bahram 7102 Lai, Kuei-Tai A 6070 Lone, Rebecca 7063 Kean, Dorothy E 5073 Lal, Harbans 5101 Long, Elizabeth M 6052 Kedda, Mary-Anne 6037 Lan, Hui Y 3039 7008 Longaker, Micheal T 7014 Keene, Martin 6021 Landberg, Göran 7006 Lopez, Jamie 6048 Keith-Magee, April 6014 7028 Langham, Robyn 7124 Lord, Janet M 7017 Keller, Ulrich 5047 Langrish, Claire L 2044 Lorget, Florence 7014 Kellie, Stuart 6040 Lari, Roya 5029 6031 Losordo, Douglas 7104 7105 Kemp, Joanna M 3014 Lateef, Zabeen I 5058 Lotuffo, Celina Maria 2025 Khachigian, Levon M 1026 Lattin, Jane 7066 Low, Sylvia Y 5038 Khalil, Zeinab 7079 Lattin, Jane E 6040 Lucas, Alexandra R 1021 3035 6017 7058 Khodzhayants, N 5102 Lau, Allan SY 7031 Lucet, Isabelle S 1024 Khoo, Audrey 7014 7015 7019 7020 7021 Lau, Hang Yung A 5072 6041 7029 Lue, Hongqi 6084 Kicic, Anthony 7052 Lau, Hon Yen 6043 7055 Luedemann, Corinne 7104 7105 Kikutani, Hitoshi 7125 Laumonnier, Yves 2043 Lukova, Tatyana K 7082 Kim, Hee-Doo 5063 Law, Anna HY 7031 Lundahl, Joachim 6081 7101 Kim, Kyoung-Jin 7003 Lawlor, Kate E 2017 Ma, Mark 6010 Kim, So-Hee 6057 Laybutt, Ross 3020 MacDonald, Kelli P 1020 Kim, Young Suk 5068 Le, Thuy T 7047 Mackay, Charles R 3020 5079 7038 Kimura, Shigenobu 6083 Leanza, Eduardo Cava 7040 Mackay, Fabienne 5045 King, Nick J 5077 Lee, Cheuk Lun 5050 Mackman, Nigel 2039 Kinne, Raimund W 5017 Lee, Chuen-Neng 7116 Madsen, Mogens W 7007 Kinnear, Gillian 1032 5104 Lee, Davy CW 7031 Makaruha Stegic, Oresta 5087 Kirimura, Kazuyoshi 6064 Lee, Frank S 7110 Maksimovic-Ivanic, Danijela 6009 7072 7073 Kirkwood, John M 4006 Lee, Hern Ku 5068 Mancini, Arturo 5018 7025 Kishore, Raj 7104 7105 Lee, Hern-Ku 6057 Manderson, Anthony P 2019 Kitayama, Tomoya 6036 6046 Lee, Lawrence K 7034 Manlapat, Anna K 5044 Kitchener, Peter 5029 Lee, Patrice A 7027 Manojlovic, Zoran 5100 Kitching, A Richard 7122 Lee, Young Man 5068 Manoury, Boris 3010 Kitching, Richard 2026 3038 Lee, Yuan kun 7057 Mansell, Ashley 7036 Kitching, Richard A 7125 Leech, Michelle 5026 7002 Mansour, Tarek S 5067 Kjerrström, Anne 5097 Leech, Michelle T 6082 Manthey, Carl L 5039 Klakamp, Scott L 6006 Leenen, Pieter JM 3005 March, Lyn 5010 Knight, Darryl A 2048 6037 7052 Legaud, Gaelle 2038 Marcinkiewicz, Cezary 2036 Knight, Toby G 7096 Lei, Pei-Wen 7034 Mardaneh, Jalal 6056 Ko, Hyun-Mi 6057 7003 Lenert, Petar 7048 Mariano-Oliveita, Andrea 2036 Koch, Kevin 4006 Leng, Lin 3034 Marino, Mariapaola 6016 Koelling, Raymond M 5093 Leung, Bernard PL 5040 Markovic, Milos 5056 Koh, Dow Rhong 5013 Levin, Jeremy I 5067 Markovic, Stribor 5087 Koivunen, Erkki 2015 Li, Allen G 3039 7008 Markus, Regina Pekelman 2025 Kolaczkowska, Elzbieta 6085 Li, Jianchang 5067 Marleau, Sylvie 6074 7119 Komac, Marijana 5087 Li, Ling 7010 Martin, Frank 6010 Komuves, Laszlo 7004 Li, Ming 7125 Martynov, Michail Yu 7082 Kong, Kok Ooi 5040 Li, Wei 5067 Maruyama, Ikuro 2046 Konishi, Fumio 5064 6059 Li, Wen Qing 5008 Marwick, Thomas 5028 Koon, Henry 4006 Li, Xiao C 7076 7123 Masendycz, Paul 3029 Koumoundouros, Emmanuel 5069 5078 Lian, Tsui Yee 5040 Massa, Christine M 6053 Kovacs, Andrea 7067 Liang, Xifu 6066 Massa, Solange 6058 Kovanen, Petri T 5020 Liao, WP 5081 Mateo, Teresa 6073 Infl amm. res. Supplement 2 (2005) S 229

Matsushima, Kouji 2007 Mostarica Stojkovic, Marija 5056 Oh, Uhtaek 5063 Matsushita, Kenji 2046 Moyer, Richard W 1021 3035 Ohara-Nemoto, Yuko 6083 Matsuyama, Takashi 2046 Muchamuel, Tony 7004 Ohuchi, Kazuo 6063 Matthews, Vance B 6050 Mukhopadhaya, Anan 7014 Okada, Seiji 2010 Mattson, Jeanine 2044 Mukhopadhyay, Anan 7015 Okano, Hideyuki 2010 Mazurowski, Wojciech W 7108 Mukhopadhyay, Anandaroop 7019 7020 Oliveira, Soraya Imon 7054 Mbvundula, Estery C 6039 Muller, Beat 5047 Oliver, Brian G 5077 McCaughan, Catherine A 5058 Muller, H Konrad 6067 Olofsson, Peter 5006 McClanahan, Terrill 2044 Muller-Esterl, Werner 7123 Olsen, Birgitta 7118 McColl, Shaun R 6018 Munic, Vesna 5100 Ong, Huy 7119 McCulloch, Christopher A 1006 Muragundla, Anjaneyulu 7071 Oo, Aung 7004 McEvoy, Alice N 7023 Muramatsu, Michiko 6064 Opdenakker, Ghislain 6085 McEvoy, Leslie M 7004 Murcha, Monika 6014 7028 Oppers-Walgreen, Birgitte 5002 McFadden, Grant 1021 6017 Murphy, Craig A 7100 Orr, Yishay 5054 6019 McIvor, Dana L 6017 Murphy, Evelyn P 7023 Otsuka, Kichibee 5059 McMahon, Martin 7059 Murray, Alan 5094 Oyen, Wim JG 2015 McMaster, Andrew D 6030 Murray, Kevin 5030 Ozimec-Landek, Ivana 5032 McNeill, H P 7034 Murray, Philip I 7062 7063 Pacheco, Cinthia Mara F 7089 McQualter, Jonathan L 7090 Murray, Rachael Z 2014 Padhy, Biswa M 7074 McSharry, Charles 5073 Murthy, Sreekant 2013 Padi, Satyanarayana SV 7070 Medana, Isabelle M 7078 Muruve, Daniel A 7045 Palmer, Kirsten R 6049 Medbury, Heather J 6079 7107 Mutsaers, Steven E 2048 Pandey, Bindu 3021 Meegalla, Sanath 5039 Nénan, Soazig 3010 Pandit, Naresh 5098 6062 Meeusen, Els 7065 Nabbe, Nabbe C 2047 Pang, Dong Bao 5088 5089 Meeusen, Els N 5069 5078 Nag, Abhijeet 3021 Panzner, Steffen 5017 7024 Meingassner, Josef G 6001 Nag, Bishwajit 3021 Papazoglou, Elisabeth 2013 Melendez, Alirio J 5073 Nagai, Hiroichi 7083 Pardo, Annie 3008 Mello, Suzana Beatriz Veríssimo 6080 Naidu, Pattipati S 7070 Park, Hyeung-geun 5063 Mellor, Andrew L 5044 Naim Abu Nabah, Yafa 6073 Park, Ruth A 1017 Menahem, Solomon 7124 Nair, Shiva M 7085 7086 7087 7088 Park, Sung Jun 7003 Menezes, Gustavo B 6076 Nakamura, Masaya 2010 Park, Young-Ho 5063 Menschikowski, Mario 7042 Nakamura, Takeshi 5064 6059 Parkar, Mohamed 6027 Mercep, Mladen 5032 5087 Nakano, Yasuhiro 6063 Parker, Michael W 1023 Mercer, Andrew A 5058 Nandi, Sayan 3004 Parkin, Susan M 7111 Merculova, Alla V 6054 Narayanan, Shridhar 5022 Parmar, Harsukh 2012 Mesic, Milan 5032 5087 Nasiri, Ebrahim 6011 6012 6044 Parnham, Michael J 5100 Michel, Uwe 7091 Nenan, Soazig 3007 Pasnik, Jarek 7108 7109 Middleton, Jim F 5016 Neogi, Partha 3021 Patil, Chandrashekhar S 7069 7084 Mier, James W 4006 Newcombe, Nicole G 5080 Patton, Kath A 5046 Milech, Nadia MD 7033 Newson, Justine 1022 Patton, Katherine A 5060 Miljkovic, Djordje 5056 6009 7072 Newton, Rebecca 5045 Paulsson, Josefi n M 7101 Miller, Mark JS 5003 Ng, Gordon 7051 Pavasovic, Durda 6031 Mitchell, Andrew J 7097 Ng, Gordon Y 6010 Peet, Dan 7035 Miu, Jenny 7078 7097 Ng, Siaw Wei 7055 Peiris, Malik 7031 Miyazaki, Mizuo 6064 Ngo, Karen 3030 Perejaslov, Andre 6013 Mo-tae, Jutarat 6055 Nguyen, Steven 3030 Perejaslov, Andre A 6077 Modric, Marina 5032 Nicholls, Andy 1032 Perera, Jeniffer 6089 Mohammed, M 4006 Nicholson, Sandra E 6049 Perovic, Daniela 5075 Moilanen, Eeva 7022 Nicola, Nicos A 6028 6049 Perretti, mauro 6071 Mok, Chris KP 7031 Nicoletti, Ferdinando 7072 7073 Perretti, Mauro 2045 Mok, Pamela YY 7009 Nielsen, Simon F 6066 Perry, Greg 7124 Molero, Juan-Carlos 3020 Nikolic, Tatjana 3005 Perry, Michael 1026 5048 Molineux, Graham 6010 Nilkaeo, Athip 6055 Pesic, Dijana 5032 Moll, Jacek 7108 7109 Nilsson, Sara C 7006 Petrow, Peter K 5005 Moll, Jadwiga A 7108 7109 Nixon, Andrew E 5092 Phan, Thang T 7014 7015 7019 7020 7021 Moll, Maciej 7109 Noonan, Claire E 7096 7099 Pheneger, Jed 7027 Momcilovic, Miljana 6009 O‘Connor, Anne E 5084 7091 Pheneger, Tracy M 7027 Moochhala, Shabbir 7055 O‘Dea, Kerin 7075 Phillips, David 7019 Moonen, Harald JJ 6033 O‘Donnell, Kristy 5009 5027 Phillips, David J 5084 7091 Moore, Philip K 7009 7010 7011 7046 7068 O‘Donoghue, Robert JJ 2048 Phillips, Joseph H 7100 7098 O‘Hara, Kirrily A 6037 Pierce, Andrew 2011 Morand, Eric 1036 7002 7115 O‘Hehir, Robyn E 5084 Pietersz, Geoffrey A 3028 Morand, Eric F 5023 5026 6029 6035 6082 O‘Neill, Luke A 3001 Pitterle, Kai 2043 7005 O‘Neill, Luke AJ 7036 Pixley, Fiona J 2032 3004 Moreira, Vanessa 7041 O‘Sullivan, Kim 7125 Plum, Stacy M 5041 Moreland, L 4005 Ocain, Tim 5004 Plytycz, Barbara 6085 Morioka, Norimitsu 6036 6046 Ochiai, Kuniyasu 5052 5059 Pokoca, Lech T 7108 Morishita, Ryuichi 1037 5031 Odobasic, Dragana 7122 Poljak, Visnja 5087 Morita, Katsuya 6036 6046 Offner, Halina 5022 Ponnampalam, Gopalakrishnakone 5019 Morris, Edward S 1020 Ogrin, Rajna 7079 Popadic, Dusan 7073 S 230 Infl amm. res. Supplement 2 (2005)

Popovic-Grle, Sanja 5100 Rosenow, Ann 7095 Sharifi an, Mostafa 6086 Porter, Alan 7021 Rossi, Adriano 7043 Sharma, Laveena 2041 7002 Porter, Karen E 7111 Rossi, Adriano G 5037 Sharma, Rohini 2030 Poubelle, P E 5062 Rossjohn, Jamie 1024 Shaw, Odette M 7085 7087 7088 Pouliot, Marc 6018 Rowley, Kevin G 7075 Sheean, Paul D 7065 Pourghasem, Mohsen 6044 Roy, Sanjeev 7074 Sheikine, Yuri 7118 Pourrezaei, Kambiz 2013 Ruckle, Thomas 3015 Shemesh, Tomer 7075 Powell, Maree S 3028 Ruleva, Natalya Yu 7082 Shimmon, Susan 5011 Prele, Cecilia M 6014 7028 Rundfeldt, Chris 5070 Shin, Song Seok 5063 Preston, Julie A 5080 Rupcic, Renata 5032 Shoda, Lisl KM 5035 Price, Patricia 7095 Rush III, Thomas S 5067 Shryver, Brian 7004 Prickett, Sara R 5053 Russell, A 4005 Shum, Bennett 5079 Proietto, Joseph 3019 Sabino, George S 7026 Siau, Jia Ling 7011 Provenzano, Carlo 6016 Sadeghian, Siavash 6011 Sidhapuriwala, Jenab N 7046 Pugazhenthi, Kamali 5049 Saeed, Sheikh A 7103 7120 Siegert, Gabriele 7042 Puneet, Padmam 7055 Saitoh, Masaaki 5064 6059 Silva, Matthew 5004 Qin, Gangjian 7105 Salmon, Gary P 5097 Silver, Marcy 7104 Queiroz Júnior, Celso M 7089 Salmon, Mike 7017 Simmet, Thomas 2043 Qureshi, Hamid Yaqoob 5008 Salom, Caroline 7034 Simon, Jutta 5005 Radbruch, Andreas 5005 Salonen, Tiina 7022 Simpson, Jodie L 5074 5076 Radstake, Timothy R 2047 5012 Sambrook, Philip 5010 Sims, Neil R 7085 Radstake, Timothy RD 5002 Sammut, Ivan A 7012 Singh, Vijay P 7084 Rahimi, Farid 7112 Sampson, Mike J 7114 Singh, VijayPal 7069 Rahman, Rosanna 7086 7087 7088 Sandler, Charlotta 5020 Sirois, Martin G 7119 Rahman, Rosanna M 7085 Sanni, Latifu 7078 Sirois, Pierre 5071 Rainsford, Kim D 6039 Santos, Lanie 6029 Sirotkina, Olga V 6054 Rajakariar, Ravindra 1022 Santos, Leilani L 7005 Sirsjö, Allan 5057 Ralph, Jennifer A 7023 Sanz, Maria J 6073 Sirsjo, Allan 7118 Ramanujan, Saroja 5035 Sarau, Henry M 6073 Sisavanh, Mary 5083 Ramnath, Raina D 6075 Saremi, Sorrayya 6008 Sit, Wai Hung 5050 Ramsland, Paul 3028 Saris, Chris 6010 Sivalingam, SP 5013 Rana, Sandeep Singh 5034 Sasaki, Minoru 6083 Skak-Nielsen, Tine 7007 Ranta, Tanja-Maria 2015 Satjawatcharaphong, Chiradath 5061 Skinner, Narelle 5036 7064 Rao, Navin 3030 Sato, Kazuki 5019 Skotinicki, Jerauld S 5067 Rathjen, Deborah A 7110 Satoh, Yoshitaka 5066 Slaviero, Kellie 2030 Rauchhau, Una 7024 Savinainen, Anneli 5004 Sly, Peter 1016 Rauchhaus, Una 5017 Sawatzky, Deborah A 5037 Smallwood, David 5103 5106 Rauz, Saaeha 7063 Schaus, Jean-Michel 5092 Smeets, Ruben L 2037 6003 Ravasi, Timothy 5096 7050 Scheel-Toellner, Dagmar 7017 Smelt, Sara C 7039 Ray, David W 6030 Scheffold, Alexander 5005 Smith, Duncan 2011 Ray, Keith P 3026 Scherrer, Didier A 5035 Smith, Jeffery L 5093 Raye, Warren 6022 Schiborra, Fredericke 7080 Smith, Rosie 7036 Rehli, Michael 7049 7050 Schofi eld, Louis 5053 7092 Smythe, George 7096 Resende, Marcos A 7026 Scholz, Glen 6048 Smythe, George A 7093 Rhaleb, Nour Eddine 7123 Scholz, Glen M 3029 5090 7060 Snibson, Ken J 5078 Ribeiro, Orlando G 6005 6058 Schopf, Lisa R 5004 Snibson, Kenneth J 5069 Richalet, Pascale 5092 Schroder, Kate 7049 7050 So, Trina 3020 Richardson, Jokubas 7058 Schulte, Reiner 5017 Sobki, Samia 5007 Ridderstad Wollberg, Anna 6078 Schwarz, Matthias K 3015 Soda, Kuniyasu 5064 6059 Rintelen, Felix 3015 Scislowska-Czarnecka, Anna 6085 Soleimanirahbar, Aliakbar 5099 Riordan, Stephen M 7064 Scott, Bernadette 2031 6051 Song, Changjie 7112 Ripoll, Vera M 5096 Scott, Kieran F 7034 Souza, Vinicius R C 6005 Robbins, Steve M 6052 Scott, Rodney J 5076 Sozzi, T 7064 Roberts, Andrew W 5009 Scott, Xanthe V 7114 Sparatore, Anna 7046 Roberts, Tara 7066 Scuderi, Flavia 6016 Sparrow, Rosemary L 5046 5060 Roberts, Tara L 7048 Sebire, Kimberly 5084 Spaull, John 3026 Robertson, Graham 6068 Seboek, Dalma 5047 Spencer, Jeremy PE 7037 Robertson, Graham R 2030 Sedgwick, Jonathon D 2044 7100 Sprigg, Naomi S 6049 Robertson, Michael J 4006 Seed, Michael P 5021 Springate, Christopher M 6026 Robinson, Brenton S 7110 Seed, Micheal P 5033 Squires, Ginette R 3026 Robinson, Bruce WS 2031 Sehgal, Raman 7074 St-Onge, Mireille 6018 Robinson, Jodie A 6040 Seidelin, Jakob B 5051 7013 Stacey, Katryn J 7048 7049 7066 Rocha, Orivaldo A 7089 Senaldi, Giorgio 6006 Stanic, Barbara 5032 Roelofs, Mieke F 5002 Sepulveda, M Fernanda 6015 Stanley, E Richard 2032 3004 Rolland, Jennifer M 5084 Sester, David P 7049 Stanley, Keith K 1013 5024 Rolph, Michael 7038 Sewell, William A 5085 5086 Starobinas, Nancy 6005 Rolph, Michael S 3020 5079 5083 5086 Sexton, Daniel J 5092 Starr, Robyn 6028 Rommel, Christian 3015 7038 Shah, Eladevi M 7056 Steer, Jay H 6034 6038 Rose-John, Stefan 6016 6050 Shaikh, Barbara Joyce 7100 Stein, Alicia N 7124 Rosengren, Rhonda J 7085 Shao, Yuan 7076 Steukers, Mieke 5092 Infl amm. res. Supplement 2 (2005) S 231

Stevenson, Christopher S 1032 5104 5105 Toh, Ban-Hock 7113 7115 de Weerd, Nicole A 6051 Stewart, Jessica A 5094 Toh, Ling 7002 Weiser, Silvia 7078 Stick, Stephen M 7052 Tomai, Mark A 3027 Welsing, Paco 5012 Stocker, Roland 7078 Tomaskovic, Linda 5087 Wenink, Mark 2047 Stosic-Grujicic, Stanislava 5056 6009 7072 Tomczuk, Bruce E 5039 Werner, Sabin 7019 7073 Tomimori, Yoshiaki 6065 Wesselingh, Steve 7095 Stow, Jennifer L 2014 2019 Tomita, Naruya 1037 5031 Westhovens, R 4005 Strawn, Steven J 5041 Tomita, Tetsuya 1037 5031 Weston, Marie C 6015 Struemper, Herbert 5035 Tonetti, Maurizio S 6027 Whetton, Anthony D 2011 Sturton, Richard G 5105 Torii, Mitsuo 2046 Wheway, Julie 5045 Styles, Michelle 1024 Toyama, Yoshiaki 2010 Whitehouse, Michael 5011 Su, Qiaojuan J 6006 Trajkovic, Vladimir 5056 6009 Whitehouse, Michael W 5001 5042 Su, Stephen 6007 Tremblay, Andre 7119 Whiteman, Matt 7037 7098 Su, Yung-Chang 5086 Treston, Anthony M 5041 Whiteman, Matthew 7011 Suh, Young-Ger 5063 Trieu, Angela 7049 7066 Whittaker, Paul 5105 Suhrbier, Andreas 7047 Trivedi, Seema 5021 Wicks, Ian P 2017 5009 5027 7061 Sun, Yuming 1021 Trivedi, Seema G 1022 5033 Wilks, Andrew F 6007 Sutanto, Erika N 7052 Trojko, Rudolf 5032 Wilks, Andrew W 1024 Sutherland, Brad A 7087 7088 Trout, Neil 7110 Williams, Hazel 7062 7063 Suzuki, Naoto 5059 Trouw, Leendert A 7006 Williams, Michelle W 7107 Svitacheva, Naila 5101 Truelove, Edward 5038 Willoughby, Derek A 5037 Sweep, Fred C 5012 Truong, Luan D 7008 Wilson, Tracy A 6049 Sweet, Matt J 6040 Trzaskos, James M 4007 Wines, Bruce D 3028 Sweet, Matthew 6048 Tuominen, Raimo 7022 Wise, Lyn M 5058 Sweet, Matthew J 6007 7048 7049 7050 7066 Turner, Amanda L 5036 6053 Wolfson, Martin F 5093 Sylvester, Judith 3011 5008 Turner, Peter C 1021 3035 Wong, Lai Lok K 6041 Syrovets, Tatiana 2043 Tyagi, Som 2013 Wong, Man Yi 7015 Sysa, Andrzej 7108 7109 Unwin, Richard 2011 Wong, Peter KK 5009 Syssoev, Kirill A 6054 Vainer, Ben 5051 Wong, Poo-Sing 7116 Szalowska, Dorota A 7109 Vaiphei, Kim 5034 Wong, Wai-Shiu Fred 5091 Taberner, Megan 7034 Valtanen, Heli 2015 Wong, WS Fred 5081 5082 Tajika, Shihoko 6083 van de Loo, Fons AJ 2037 6003 Wood, Elizabeth G 5021 Tak, Paul P 3003 van den Berg, Wim B 2037 2047 6003 Woods, Greg M 6067 Takai, Shinji 6064 van Gool, Reinhoud 5092 Worley, Joanna R 7114 Tal, Lauren 6069 van Nieuwenhuijze, Anne-Marie 5027 Wouters, Emiel FM 6033 Tam, Steve 5067 van Riel, Piet L 2047 5012 Wright, Colin W 7053 7081 Tan, Benny KH 7068 van Rooijen, Nico 2031 Wu, Young Yuen A 5072 Tan, Kim E 7014 Van Snick, Jacques 5085 Wyss, Daniel 1032 5104 Tan, PeckSzee 3028 Vanhove, Marc 5092 Xu, Qian 5013 5014 Tanaka, Hiroyuki 7083 Varcoe, Ramon L 6079 Xu, YingHua 2018 Tanaka, Yoshitaka 6065 Vasquez-Pinto, Luciana M C 5071 Xue, Jin 6029 7002 Tancharoen, Salunya 2046 Vatsya, Pracha 7058 Xue, Jin R 5026 Tapley, Peter 1036 Vaughan, Robert 7062 Xue, Meilang 5010 Taranto, Elliott 5026 Veenbergen, Sharon 2037 Xue, Xiaohua 6070 Tariq, Mohammed 5007 Vermeulen, Sita H 5012 Yakovleva, N 5102 Tarlinton, David M 3014 Vicaretti, Mauro 6079 7107 Yamamoto, Masafumi 5052 Tassabehji, May 6030 Villacres, Maria C 7067 Yamaura, Chiharu 6083 Tavakkol Afshari, Jalil 6011 6012 Visvanathan, Kumar 5036 7049 7064 Yan, Wei 5093 Taylor, Jude M 5054 6019 Viswanathan, Kasinath 1021 3035 6017 7058 Yan, Wei-Xing 5048 Techatraisak, Kitirat 6045 Vlahos, Ross 1017 1018 1031 5103 5106 6053 Yan, Weixing 7112 Tedla, Nicodemus 5048 7066 Yanchina, E 5102 Teh, Cheng Lay 5040 Vohra, Harpreet 5034 Yang, Yuan 6029 Teixeira, Catarina F 7041 Wågsäter, Dick 5057 Yang, Yuan H 7002 Teixeira, Catarina Fátima 7040 Wade, A L 5080 Yang, Yuanhang 1036 Teteris, Simon A 7124 Wagner, Andreas 7024 Yang, Zheng 5048 Thaworn, Athiwat 6045 Waldman, Alla 1026 Yao, Jun 5084 Thiele, Michael 6084 Wall, Mark J 5039 Yeoh, George C 6050 Thim, Kerstin 5101 Wallace, Eli 7027 Yeung, Yee-Guide 3004 Thomas, Ranjeny 5028 Wallace, Graham R 7062 7063 Yip, Kwok Ho 7029 Thompson, Alexander 7064 Wan, Jennifer Man Fan 5050 Yokomakura, Aya 6063 Thompson, Michelle 5103 5106 Wang, Shugui 7057 Yona, Simon 2045 Thompson, Philip J 6037 Wang, Wansheng 7008 Yoshikawa, Hideki 1037 5031 Thomson, Napier M 7124 Wang, Xian-song 5072 Young, Paula 5049 Thong, Bernard YH 5040 Wang, Xiao J 3039 7008 Young, Tim 3020 Thumboo, Julian 5013 5014 Wannberg-Anderson, Sharon L 7039 Younos, Ibrahim H 6088 Thurmond, Linda M 4006 Warton, Kristina 1013 5024 Yu, Liming 6032 Thwin, Maung-Maung 5019 Watson, Jessica 5097 Yu, Violeta 6010 Tin, SK 5013 5014 Watt, Paul M 7033 Zabihian, Shahla 7018 Tipping, Peter G 2041 7122 Way, Kerrie J 6021 Zackrisson, Catharina 5101 Togonu-Bickersteth, Babajide 7058 Weber, Christian 3034 Zafarullah, Muhammad 3011 5008 S 232 Infl amm. res. Supplement 2 (2005)

Zamani, Alireza 6011 6012 6044 Zamunér, Silvia Fernanda 7040 Zamuner, Stella R 7041 Zargarizadeh, Ahad 6060 Zee, OkPyo 6063 Zeman, Krzysztof 7108 7109 Zhang, Guishui 1026 Zhang, Neng 5091 Zhang, Xuxia 7051 Zhao, Jing 5082 Zheng, Timothy S 2021 5015 Zhu, H 5081 Zhu, Hua 5091 Zhu, Yan 7105 Zhuo, Jia L 7076 7123 Zingali, Russolina 2036 Zuliani, Juliana P 7041 Zuliani, Juliana Pavan 7040