DOI 10.1515/pterid-2015-0007 Pteridines 2015; 26(3): 113–133

Abstracts*) 34th International Winter Workshop

Clinical, Chemical and Biochemical Aspects of Pteridines and Related Topics

Society for Exploitation of Education and Research in Immunology and Infectious Diseases, Innsbruck, Austria in collaboration with The International Society of Pteridinology and The Austrian Society of Laboratory Medicine and Clinical Chemistry

Held in Innsbruck, Tyrol, Austria, February 24th–27th, 2015 Scientific committee: Dietmar Fuchs (Innsbruck), Andrea Griesmacher (Innsbruck), Bohuslav Melichar (Olomouc), Gilbert Reibnegger (Graz), Barbara Strasser (Hall), Guenter Weiss (Innsbruck) and Ernst R. Werner (Innsbruck) Organization: Dietmar Fuchs, Sektion für Biologische Chemie, Biozentrum, Medizinische Universität Innsbruck, Innrain 80, 6020 Innsbruck, Austria, e-mail: [email protected]

*)These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors’ responsibility. 114 34th International Winter Workshop

Circulating neopterin and citrulline concentrations Influence of carbon nanotubes, ZnO and in patients with germ-cell tumors during gold-doped TiO2 nanoparticles on human PBMC chemotherapy in vitro

Bartoušková M, Študentová H, Pejpková I, Zezulová M, Adam T, Becker K, Herlin N, Bouhadoun S, Gostner JM, Ueberall F, Schennach Melichar B H, Fuchs D Palacký University Medical School and Teaching Hospital, Olomouc, Divisions of Biological Chemistry and of Medical Biochemistry, Czech Republic Biocenter, Medical University, and Central Institute of Blood ([email protected]) Transfusion and Immunology, University Hospital, Innsbruck, Austria; Au Service des Photons, Atomes et Molécules - Laboratoire Francis Germ-cell tumors are relatively rare neoplasms that affect mostly Perrin, Gif-sur Yvette, France young adults. The poor prognosis of these tumors has significantly ([email protected]) improved after the advent of platinum compound, and most patients with metastatic germ cell tumors can be currently cured with com- Nanomaterials are increasingly produced and used throughout bined modality treatment that comprises surgery, chemotherapy or recent years. Consequently the probability of exposure of humans radiotherapy. With the improved long-term prognosis, acute and to nanoparticles has drastically risen. Because of their small chronic side effects of treatment represent an important issue. In the 1-100 nm size, the physicochemical properties of nanomaterials present study, we evaluated serum neopterin and plasma citrulline, may differ from standard bulk materials and may pose a threat to a biomarker of gut damage in germ-cell tumor patients before and human health. Only little is known about the effects of nanopar- during 3 to 4 cycles of standard platinum-based chemotherapy. We ticles on the human immune system [1]. In this study, we inves- report here the pilot results in the first 15 patients. Baseline serum tigated the effects of carbon nanotubes (CNT), zinc oxide (ZnO) neopterin concentrations were increased in a substantial proportion and gold-doped TiO2 nanoparticles employing the in vitro model of of patients, while plasma citrulline was mostly within the normal human peripheral blood mononuclear cells (PBMC) [2], cytokine- range. A significant negative correlation was observed between base- induced neopterin formation and tryptophan breakdown were line serum neopterin and hemoglobin concentrations while positive monitored (n = 4). PBMC were stimulated with mitogen phyto- concentration was noted between peripheral blood leukocyte count haemagglutinin (PHA). Activated T-cells release pro-inflammatory and neopterin. Changes in the investigated parameters were noted cytokines, leading to an enhanced formation of neopterin and during the course of therapy. The study is ongoing. tryptophan breakdown. Furthermore myelomonocytic THP-1-blue cells, a reporter-cell line, can detect the activation of the redox- sensitive transcription factor nuclear factor-κB (NF-κB) [3]. Stimu- lation of THP-1-blue cells with lipopolysaccharide (LPS) resulted Growing impact of psychoimmunological in the activation of NF-κB, which could be measured by assessing findings to understand pathogenesis of severe the SEAP activity in a colorimetric assay (n = 4). Treatment with mental illness CNT resulted in differential outcome in unstimulated and stimu- lated cells. Neopterin levels were significantly increased after CNT Bechter K, Maxeiner H, Kühne L, Reiber H, Fuchs D, Schneider M exposure (18.75-150 μg/ml) in unstimulated PBMC, while in PHA- Clinic for Psychiatry and Psychotherapy II, Ulm University, Germany; stimulated cells additional CNT (37.5 – 150 μg/ml) resulted in a Division of Biological Chemistry, Biocenter, Medical University, decrease of neopterin levels. The same observations were found Innsbruck, Austria for kynurenine to tryptophan ratios (Kyn/Trp) representing trypto- ([email protected]) phan breakdown rates. In THP-1-blue cells CNT were able to raise

NF-kB activation significantly in LPS-stimulated cells. TiO2 and

Etiology and pathogenesis of severe mental illness, core is the affec- gold doped TiO2 (9.37 – 150 μg/ml) were able to increase neopterin tive and schizophrenic spectrum, remains widely unknown, though production in both stimulated and unstimulated PBMC. However, many researchers assume some biological basis. CSF analysis is a Kyn/Trp levels were only influenced in stimulated cells, where key bodily fluid to assess rather directly the pathogenetic processes inhibition of tryptophan breakdown could be observed. Also ZnO involved in brain diseases. We demonstrated in several research pro- treatment resulted in distinct effects. Lower concentrations (2.34- jects, focused on therapy resistant cases of severe mental illness, 18.75 μg/ml) acted predominantly anti-inflammatory by inhibiting in about 40 % of patients some CSF abnormalities by conventional neopterin production in both stimulated and unstimulated PBMC. sophisticated CSF analysis, in addition increased CSF neopterin in Higher concentrations already influenced cell viability signifi- about 30 %, together indicating immune pathological responses cantly and were able to induce NF-κB activation significantly. Fur- within the intrathecal space in more than 50 % of cases. Also blood thermore results obtained with ZnO (50nm and 100nm and bulk CSF barrier dysfunction was found summing up to 70 % of cases with material) were not size dependent. CSF pathology. However in 100 % of these cases we demonstrated Our results show that not all types of nanoparticles had similar CSF cytokine abnormalities, often paralleled by corresponding effects on PBMC and THP-1-blue cells in vitro. Every type showed par- though not identical cytokine abnormalities in blood. Together our ticular results and have to be explored in more detail on other cell results reveal immune pathology in a large subgroup of patients of types. Additionally exposure to nanoparticle during inflammatory the affective and schizophrenic spectrum, focused within the intrath- conditions may induce different effects as compared with a “healthy” ecal spaces. and unstimulated milieu. 34th International Winter Workshop 115

Support by the Austrian Research Funds (Project 25150-B13) is Interferon-γ-mediated immune activation in patients gratefully acknowledged. with severe symptomatic aortic stenosis and implica- tions of baseline inflammatory markers for risk strati- [1] Becker K, et al. Food Chem Toxicol 2014;65:63-9. [2] Jenny M, et al. Inflamm Res 2011;60:127-35. fication in patients undergoing transcatheter aortic [3] Schroecksnadel S, et al. J Biomed Nanotechnol 2011;7:209-10. valve implantation

Csordas A, Fuchs D, Reibnegger G, Nietlispach F, Maier W, Stähli BE, Binder RK, Cahenzli M, Lüscher TF IDO and related pathways as targets for HIV Department of Cardiology, University Heart Center Zürich, Zürich, immunotherapy Switzerland; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; and Institute of Physiological Chemistry, Center of Physiological Chemistry, Medical University, Graz, Austria Boasso A ([email protected]) Centre for Immunology and Vaccinology, Chelsea & Westminster Hosp., Imperial College London, UK Transcatheter aortic valve implantation (TAVI) has emerged as a ([email protected]) viable treatment option for patients with severe symptomatic aortic stenosis and a high or prohibitive surgical risk. Neopterin, a pteridine Increased Tryptophan (Trp) catabolism via the Kynurenine (Kyn) mainly synthesized by activated macrophages, is considered a stable pathway is observed in several chronic inflammatory and infec- integral of TH1 immune activation, and elevated neopterin levels have tious diseases, and is caused by increased indoleamine 2,3-dioxy- a solid track record as risk estimates for mortality in atherosclerosis- genase (IDO) activity. In the particular case of HIV-1 infection, IDO related diseases. We prospectively included 128 patients undergoing dysregulation is considered to contribute to different aspects of TAVI. Neopterin, kynurenine, tryptophane, tyrosine and phenylala- immunopathogenesis, including immune deficiency, neurological nine levels were measured at baseline as well as at postoperative day impairment and tissue fibrosis. Increased IDO activity may result 1-3. Neopterin and the Euroscore II were dichotomized at the upper from direct activation of certain immune cell subsets by HIV (eg. percentile, and paramteric Royston-Parmar proportional odds models plasmacytoid dendritic cell; pDC), or may be driven by the general- were employed relating biomarkers and clinical risk scores to non- ized immune activation observed during chronic HIV infection via procedural mortality. Mortality was higher for patients with elevated inflammatory cytokines (such as type I and type II interferons) and neopterin ( > 21 nmol/L, upper percentile) (18.18% vs. 5.05%, P = ligand-receptor interactions (eg. binding of cytotoxic T lymphocyte 0.018) and Euroscore II ( > 7.5, upper percentile) (18.75% vs. 6.1%, P = antigen (CTLA)-4 to its ligands B7.1 and B7.2). Immunotherapeutic 0.031). In bivariate analysis adjusted for Euroscore II, elevated neop- approaches which target IDO directly or indirectly are being con- terin ( > 21 nmol/L) persisted as an independent predictor of mortality sidered for the treatment of HIV infection and have been tested (HR 2.41 95% CI [0.99-13.6], P = 0.050). A similar finding was obtained mainly at preclinical level. The competitive IDO inhibitor 1-methyl- in an external confirmation cohort encompassing 207 patients. In D-tryptophan (D-1mT) has been tested in both humanized mice conclusion, systemic markers of increased immune activation, as and SIV/macaques with encouraging results, indicating that IDO evidenced by enhanced tryptophan degradation and neopterin pro- inhibition may favour immune-mediated control of HIV replica- duction, provide an incremental risk estimate for mortality beyond tion [1,2]. Strategies interfering with the binding of CTLA-4 to its canonical surgical risk scores. Our data support a role for a biomarker- ligands B7.1 and B7.2 have shown ambiguous results, but the effect guided strategy for sharpening discrimination of high-risk patients in of CTLA-4 blockade may only be partially dependent on IDO sup- advance of TAVI and underscore the role of baseline inflammation for pression [3,4]. In addition, simultaneous administration of D-1mT prognosis in patients with severe aortic stenosis. and anti-CTLA-4 to Rhesus macaques caused fatal pancreatitis, calling for caution when considering strategies which may reverse immune tolerance [5]. Other therapeutic options aim at dampen- Specific in-vivo requirements for tetrahydrobiopterin ing the generalised immune activation by using compounds with broad anti-inflammatory properties, particularly those interfering revealed by cell-specific deletion of GTP cyclohydrolase I with pDC activation, a notable example is that of the antimalaric drug chloroquine and its derivative hydroxychloroquine (CQ and Channon KM hCQ). However, the direct effect of these compounds on IDO activ- Cardiovascular Medicine, R&D and NIHR Biomedical Research Centre, ity in vivo is yet to be determined. Thus, inhibition of IDO or IDO- John Radcliffe Hospital University of Oxford, Oxford, Great Britain related pathways may represent a valid therapeutic option in the ([email protected]) setting of HIV infection, but its application is still limited by the Abstract not received in time. paucity of specifically designed comparative preclinical and clini- cal studies. HFE deficiency critically affects cholesterol homeosta- [1] Potula R, et al. Blood 2005; 106(7): 2382-90. sis in mice [2] Boasso A, et al. J Immunol 2009; 182: 4313-20. [3] Hryniewicz A, et al. Blood 2006; 108: 3834-42. Demetz E, Haschka D, Heim C, Auer K, Aßhoff M, Schroll A, Dichtl S, [4] Cecchinato V, et al. J Immunol 2008; 180: 5439-47. Hilbe R, Lener D, Seifert M, Tymoszuk P, Pechlaner R, Willeit J, Kiechl [5] Vaccari M, et al. J Virol 2012; 86: 108-13. S, Theurl I, Tancevski I, Weiss G 116 34th International Winter Workshop

Medical University of Innsbruck, Department of Internal Medicine VI, Leptin has initially been described as hormone that is released by Infectious Diseases, Immunology, Innsbruck, Austria adipose tissue and controls nutritional intake by binding and activat- ([email protected]) ing POMC neurons in the hypothalamus. However, leptin has pleio- tropic functions and has been shown to have pro-inflammatory and Iron metabolism plays a crucial role in diseases characterized by immunomodulatory effects. In combination with interferon gamma chronic inflammation including atherosclerosis. Specifically, iron over- (IFNγ), leptin stimulates TH1 T-cells, which cause inflammation pro- load has been linked to an increased risk for atherosclerosis develop- moting insulin resistance. There is a clear established connection ment, and with its sequels stroke and myocardial infarction. However, between serum leptin levels and IFNγ levels, as ectopic leptin stim- the underlying mechanism has not been elucidated thus far. To study ulates IFNγ release. Santucci et al. (2011) found that plasma leptin this we used apoE-/- mice as a model of dyslipidemia which develop levels are positively correlated to the basal amount of IFNγ present spontaneous atherosclerosis over time and crossed these with Hfe-/- at 24 h in culture supernatants from tuberculosis patients [1]. Hence, mice, a model of genetic iron overload. When modulating dietary iron activation of IFNγ might lead to altered leptin levels in serum. supplementation, we did not observe any differences in cholesterol IFNγ is a potent inflammatory mediator that carries a polymor- metabolism in both wildtype and apoE-/- mice. However, mice lacking phism (+847T/A; rs62559044) in the first intron which is located Hfe, different iron loading markedly affected plasma cholesterol levels. within a canonical binding site of the activating transcription factor In both studied models, i.e. wildtype and apoE-/- mice, high iron feeding NF-κB and its functional importance was shown as the T-allele gives resulted in a strong induction of hepatic LDL receptor which was asso- rise to increased levels of IFNγ. We developed a simple genotyping ciated with decreased plasma LDL cholesterol levels. Furthermore, method for this polymorphism and genotyped the IFNγ +874 A/T pol- high iron fed animals displayed enhanced serum cholesterol efflux ymorphism in 508 patients (60% females) of the STYJOBS/EDECTA capacity, which may additionally inhibit cholesterol deposition in the cohort, which has been set up to investigate the preclinical phase arterial wall. Both, enhanced LDL cholesterol clearance together with of obesity by a well defined cohort of young and middle aged over- enhanced macrophage cholesterol efflux may be responsible for the weight/obese and normal weight subjects [2]. significant reduction in atherosclerotic lesion area observed in high- In a general linear model, rs 62559044 associated significantly iron fed Hfe-/- apoE-/- mice. In summary, we show that disruption of the with leptin levels (p = 0.032). The effect is sex specific, as the associa- gene coding for Hfe affects cholesterol metabolism and atherogenesis, tion between rs 62559044 and serum leptin levels in women is highly presumably through regulation of hepatic LDL receptor and by modu- significant (p = 0.003) while we detected no significant association in lating cholesterol efflux capacity, both mechanisms shown to be signifi- males. The effect is allele specific: using the Mann-Whitney Test, the cantly influenced by dietary iron supplementation. presence of the A allele versus the TT genotype did not return signifi- cant differences of serum leptin levels depending on IFNγ allelic status, while comparing the presence of the T allele versus the AA genotype Development of an ELISA for neopterin measurement resulted in a significant association (p = 0.004). Therefore, we conclude that the T-allele of rs62559044 might increase serum leptin levels.

Dressler D [1] Santucci N, et al., PLOS One 2011;6(10):e26363. F&E Gastroenterology and Lateral Flow Development, R-Biopharm AG, [2] Mangge H et al., Obesity (Silver Spring) 2013;21:E71-7. Darmstadt, Germany ([email protected])

An enzyme linked immunoassay for the detection of neopterin from Volatile breath markers of lung infection in mechani- human serum was developed. Results of 48 human serum samples were compared with a commercial ELISA from BRAHMS, Hennigs- cally ventilated patients dorf, Germany. The new test revealed very good sensitivity and there existed a significant correlation between the obtained results with Filipiak W, Sponring A, Filipiak A, Ager C, Nagl M, Beer R, Troppmair both assays, offering the possibility to distinguish serum samples J, Amann A† from sick or healthy subjects. Breath Research Institute of the Austrian Academy of Sciences, Dornbirn, Austria; Department of Anaesthesiology and Critical Care Medicine, Department of Hygiene, Microbiology and Social Medicine, Neurological Intensive Care Unit, Department of Neurology, and Serum leptin levels are associated with the interferon Daniel-Swarovski Research Laboratory, Department of Visceral-, gamma +847T/A polymorphism (rs62559044) Transplant- and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria Egger C, Wallner V, Weghuber D, Zelzer S, Paulweber B, Fuchs D, ([email protected]) Mangge H, Baumgartner GB †dedicated to the memory of Prof Anton Amann who passed away Clinical Institute of Internal Medicine, and Department of Pediatrics, January 6, 2015 Paracelsus Medical University, Salzburg, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University, Graz, The routinely used microbiological diagnosis of Ventilator-Asso- Austria, Division of Biological Chemistry, Biocenter, Medical University, ciated Pneumonia (VAP) is time consuming and often requires Innsbruck, Austria invasive methods for collection of human specimens (e.g. bronchos- STYJOBS/EDECTA (details: http://clinicaltrials.gov/ct2/show/ copy). Thus, the most desirable improvement related to diagnosis NCT00482924) of pneumonia is an earlier and non-invasive detection of the out- ([email protected]) break of infection and faster identification of the pathogens. This 34th International Winter Workshop 117 might be achieved by monitoring the presence of pathogen-derived role in the development of frailty including immobility (decreased volatile metabolites in the alveolar air of mechanically ventilated physical activity), balance impairment, depression and cognitive patients. For this purpose, in vitro experiments with microorgan- impairment. Phenotypic criteria that indicate compromised ener- isms frequently found in VAP patients, i.e. Staphylococcus aureus, getics include low grip strength, low energy, slowed waking speed, Pseudomonas aeruginosa, Streptococcus pneumonia, Haemophilus low physical activity, and/or unintentional weight loss [1]. Chronic influenzae and Candida albicans, were performed to identify patho- immune system activation and inflammation seem to contribute to gen-specific volatile organic compounds (VOCs). the development of frailty, e.g., enhanced neopterin concentrations Numerous VOCs were released by investigated bacteria and have been described recently in frail individuals [2]. fungi species in a concentration range varying from pptv to ppmv Smoking status and dietary habits are considered to impact level. The clear differences in the bacteria-specific profiles of VOCs on the performance. In the in vitro system of human peripheral production were found, particularly hydrocarbons released by Strep- blood mononuclear cells (PBMC), immunosuppressant properties tococcus pneumonia and Haemophilus influenzae and for aldehydes of several antioxidant vitamins and other compounds were shown, which were taken up by P. aeruginosa but released by S. aureus and reducing neopterin production and tryptophan breakdown rates [3]. for. Importantly, the in vitro experiments were performed under Moreover, significant inverse correlations between higher neopterin culture conditions and at bacterial densities which relate to the in blood levels and lower concentrations of several antioxidant com- vivo situation in the lungs of VAP patients. Moreover, the significant pounds and vitamins were observed in patients at cardiovascular VOCs release was found already 1.5 to 3 hours after inoculation, and risk [4]. Likewise, smokers were found to present lower neopterin their concentration profiles were very well correlated to the prolifera- and Kyn/Trp levels than non-smokers [5-7]. tion rate of cultured microorganisms. In this study, in 239 individuals (35 non-frail, 118 pre-frail and In a next step a clinical study at the Neurological Intensive 86 frail) aged > 63 (mean ± SD: 79.4+8.86) years serum neopterin Care Unit was performed to clarify whether (i) the volatile metabo- concentrations were determined by ELISA (BRAHMS, Germany) and lites produced by pathogens in vitro can be detected in alveolar air kynurenine to tryptophan ratios (Kyn/Trp) by HPLC, as biomarkers of of mechanically ventilated patients, and (ii) changes in bacterial immune activation. Results were compared to dietary habits includ- metabolites reflect disease progression and therapeutic interference. ing intakes of coffee, tea and alcohol as well as to smoking status. The results of breath analysis were referred to the patient’s health Intake of coffee, tea and alcohol was associated with lower neopterin status, in particular to the level of C-reactive protein (CRP). Pneumo- and Kyn/Trp concentrations, whereas tryptophan was higher. This nia was diagnosed on the basis of clinical (e.g. onset of fever, CRP was especially true in those who drank alcoholic beverages. Neop- level), microbiological (isolates) and imaging (CT or X-Ray) data. The terin and Kyn/Trp concentrations were lower and tryptophan levels developed methodological platform was proven to be suitable for higher in smokers vs. non-smokers. Tobacco consumers presented application in the ICU whereby end-tidal air was collected directly significantly higher blood tryptophan levels. No significant associa- from the respiratory circuit of MV patients. The relationship between tions existed between the number of daily cigarettes and the number the concentration profiles of selected breath-VOCs and the course of of years smoked and the concentrations of biomarkers. the microbial lung infection was found. A change in the breath-VOC The association between immune system activation and the patterns was observed in relation to the patients’ recovery. disturbed metabolism of tryptophan and neopterin in frail individu- In summary, we observed characteristic VOC patterns for each als appears to be related to smoking habits and nutritional aspects. microorganism that, though limitation to a small patient cohort, was Intake of nutrients rich in antioxidants like tea and coffee as well as suitable to detect the presence of pathogens in mechanically venti- smoking may influence immunoregulatory pathways. In accordance lated patients. The here presented results set fundament for further with in vitro data, antioxidant contents of beverages could be of impor- clinical study, preferably with real-time mass spectrometric tech- tance to underlie this effect, e.g., vitamins, resveratrol and humulones niques in ICU settings. in beverages, but also carbon monoxide in smokers could explain our findings. In a similar way, polyphenols from beer were found to reduce inflammatory biomarkers related to atherosclerosis [8]. Data of our study may point to a role of such behavior in suppressing the activated Neopterin concentrations and tryptophan breakdown immune system in older age. Thereby, the food compounds could also rates in the elderly are influenced by life style and influence nutrition behavior when intake of tryptophan-rescuing food dietary habits contributes to mood enhancement. The metabolic alterations espe- cially of tryptophan could be important for the amelioration of specific Fuchs D, Gostner JM, Sánchez-Flores M, Millán-Calenti JC, Marcos- symptoms like depression and cognitive deficits in the frail elderly. Pérez D, Maseda A, Pásaro E, Strasser B, Lorenzo-López L, Laffon B, Moreover, higher serum tryptophan levels were observed to relate to Valdiglesias V lower mortality in cardiovascular disease [9]. Biocenter, Medical University, Innsbruck, and Institute for Nutritional Sciences and Physiology, UMIT, Hall, Austria; DICOMOSA Group, [1] Fried LP, et al. J Gerontol A Biol Sci Med Sci 2001;56:M146–56. Department of Psychology, Area of Psychobiology, and Gerontology [2] Leng XS, et al. Age Ageing 2011;40:475–81. Research Group, Department of Medicine, Universidade da Coruña, [3] Gostner J, et al. Curr Pharm Des 2014;20:840-9. Spain. [4]Murr C, et al. Atherosclerosis 2009;202:543-9. ([email protected]) [5] Diamondstone LS, et al. J Clin Immunol 1994;14:368-74. [6] Walter RB, et al. Clin Chem Lab Med 2003;41:1314-9. Frailty is placed in an intermediate position between being functional [7] Pedersen ER, et al. Arterioscler Thromb Vasc Biol 2011;31:698-704. and the development of functional impairment and/or comorbidity [8] Chiva-Blanch G, et al. Nutr Metab Cardiovasc Dis 2015;25:36-45. directly associated with a disease process. Numerous factors play a [9] Murr C, et al. Eur J Clin Invest 2015 (in press). 118 34th International Winter Workshop

Freezing conformational dynamics in phenylalanine higher than 0.99. The accuracy was in the range of 95.6-108%. The hydroxylase with pharmacological chaperones inter-day precision (n = 5 days), expressed as % RSD, was in the range 5-12%. The benefit of using UPLC is a short analysis period and thus, a very good sample throughput. Using this method, we ana- Fuchs JE, Santos-Sierra S, Lagler FB, Kirchmair J lysed plasma samples from transgenic rat model for tauopathies. Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, United Kingdom; Molecular and Clinical Pharmacology, [1] US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Innsbruck Medical University, Austria; Institute for Inborn Errors of Method Validation, 2001. http://www/fda.gov/cder/guidance/index.htm. Metabolism, Paracelsus Medical University, Salzburg, Austria; Center for Bioinformatics, University of Hamburg, Germany ([email protected]) Effect of occupational mercury exposure on neopterin Inherited mutations may lead to protein misfolding and thus loss of profile and tryptophan degradation in dental technicians function. Small molecule pharmacological chaperones aim to rescue proteins from misfolding by stabilization of the fold [1]. Dysfunction Girgin G, Tutkun E, Ünüvar S, Palabiyik SS, Yilmaz OH, Baydar T of phenylalanine hydroxylase causes the well-studied genetic disease Department of Toxicology, Faculty of Pharmacy, Hacettepe University, phenylketonuria, thus turning pharmacological chaperones stabiliz- Ankara; Ankara Occupational Diseases Hospital, Ankara; Department ing this enzyme against degradation into promising drug candidates of Toxicology, Faculty of Pharmacy, Inönü University, Malatya; [2]. In an attempt to rationalize chaperone activity on atomistic level, Department of Toxicology, Faculty of Pharmacy, Atatürk University, we perform molecular dynamics simulations to characterize intrinsic Erzurum; Turkey flexibility of phenylalanine hydroxylase, an enzyme with a complex ([email protected]) energy hypersurface and several regulatory pathways [3,4,5]. We show that conformational dynamics of the protein are frozen con- Amalgam has been widely used as a restorative dental material for siderably in presence of its co-factor tetrahydrobiopterin as well as over 150 years. Most standard dental amalgam formulations contain pharmacological chaperone candidates. The observed reduction of approximately 50% elemental mercury in a mixture of copper, tin, the enzyme’s conformational entropy [6] correlates to the experi- silver, and zinc. Mercury is a highly volatile metal, can easily vaporize to mentally observed stabilization versus proteolytic degradation, thus a colorless and odorless gas. It has been demonstrated that mercury is showing the potential of the simulation approach to guide develop- released from dental amalgam which is increased by chewing, eating, ment of innovative fold stabilizing agents. brushing and drinking hot liquids. Beside this, mercury is the main occupational exposure source for dental workers during amalgam [1] Santos-Sierra S, et al. Human Molecular Genetics 2012;21:1877-87. preparation. It is known that mercury exposure causes immune modu- [2] Underhaug J, et al. Curr Top Med Chem 2012, 12, 2534-5. lation in humans. In this study, it was aimed to evaluate the changes [3] Fuchs JE, et al. PLoS One 2012;7:e53005. in neopterin levels and tryptophan degradation in dental technicians. [4] Ronau JA, et al. Eur Biophys J 2013;42:691-708. 135 dental technicians who applied for routine check-up to the Occupa- [5] Fuchs JE, et al. Pteridines 2014;25:33-39. tional Diseases Hospital were recruited the study. Spot urinary mercury [6] Huber RG, et al. Protein Sci 2015;24:174-81. concentrations were detected by ICP-MS. Serum neopterin levels were determined by ELISA while tryptophan and kynurenine levels were analyzed by HPLC. The results were compared to 165 healthy controls. Determination of phenylalanine, tyrosine, tryptophan It was observed that occupational mercury exposure had an appar- and kynurenine in plasma of rat model for tauopathies ent effect on neopterin levels. The significant decrease in neopterin by HPLC with fluorescent and mass spectrometry levels and kynurenine/tryptophan in dental technicians compared to healthy controls (both, p < 0.01) indicates a possible immune sup- detection pression with occupational mercury exposure. Moreover, urinary mercury concentrations and serum neopterin levels had a significant Galba J, Michalicova A, Parrak V, Novak M, Kovac A positive correlation (p < 0.05), while serum kynurenine-to-tryptophan Axon Neuroscience SE and Faculty of Pharmacy Commenius University, ratios and mercury levels had a negative, non-significant relationship Bratislava, Slovakia (p > 0.05). The relationship between urinary mercury levels as an indi- ([email protected]) cator of occupational mercury exposure and neopterin reminded an effect on T-cell mediated immune response; however, this effect was We developed and validated a simple and sensitive ultra-performance not observed in tryptophan degradation. liquid chromatography (UPLC) method for the analysis of phenylala- nine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm × 50 mm, 1.7 μm particle size) using a 4 minutes ammonium Black tea consumption increases blood kynurenine acetate (pH 5) gradient and detected by Fluorescence and positive concentrations - a randomized controlled trial ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. Gostner JM, Becker K, Croft KD, Woodman RJ, Puddey IB, Fuchs D, The procedure was validated in compliance with the FDA guideline Hodgson JM [1]. The limits of quantification (LOQ) were 0.125 μmol/L for Kyn and Divisions of Medical Biochemistry and of Biological Chemistry, Medical 1.25 μmol/L for Phe, Tyr, Trp. (calculated from calibration curves). University of Innsbruck, Austria; School of Medicine and Pharmacology, The method showed excellent linearity with regression coefficients University of Western Australia, Perth, Western Australia, Australia 34th International Winter Workshop 119 and Flinders Centre for Epidemiology and Biostatistics, School of Transfusion-related immunomodulation (TRIM) is controversially Medicine, Flinders University, Adelaide, South Australia, Australia discussed as potential risk factor that favors the acquisition of post- ([email protected]) surgical infections, the reoccurrence of malignancies and the activa- tion of endogenous viral infections [1]. Otherwise, allogenic blood Circulating neopterin and the ratio of kynurenine to tryptophan transfusion was reported to enhance the survival or renal allografts (Kyn/Trp) concentrations are biomarkers of immune activation that and reduce the recurrence rate of Crohn disease [2]. However, there have been linked to cardiovascular and total mortality [1,2]. In vitro are only a small number of studies available that discuss such immu- data show that antioxidant compounds including tea flavonoids nomodulatory phenomena and results are often contradictory, and reduce immune stimulation and down-stream biochemical pathways still the blood product constituents responsible for TRIM have not such as neopterin formation and tryptophan breakdown in human been identified. peripheral blood mononuclear cells [3,4]. We aimed to assess the In this study we investigated the effect of the individual com- effects of regular black tea consumption on tryptophan and neop- ponents of blood storage solutions on indoleamine 2,3-dioxyge- terin metabolism in vivo [5]. nase (IDO) activity in unstimulated and mitogen-stimulated human Participants were healthy individuals with no major illnesses, peripheral blood mononuclear cells (PBMC), which has turned out having normal to mildly elevated systolic blood pressure. They were to be a reliable test system to assess potential immunomodulatory randomly assigned to consume 3 cups per day of either powdered properties [3]. The interferon gamma (IFN-γ)-induced enzyme IDO black tea solids (tea; n = 45) or a flavonoid-free caffeine-matched catalyzes the breakdown of the essential amino acid tryptophan to beverage (control; n = 49). Serum concentrations of tryptophan, kynurenine, which represents a metabolic checkpoint crucial for kynurenine and neopterin were assessed at baseline and again at 3 pathogen defense but also for immunosuppression [4,5]. and 6 months after daily ingestion of the respective beverage. Freshly isolated human PBMC obtained from healthy donors Compared to control, regular consumption of tea over 6 months, were preincubated with different concentrations of blood stabili- did not significantly alter neopterin or tryptophan concentrations, sation solution (CPD: citrate phosphate dextrose) or red cell pres- but did result in significantly higher kynurenine (p = 0.016) and ervation solultion components (SAGM: saline, adenine, glucose, Kyn/Trp (p = 0.012). Relative to the control group, in the tea group mannitol) and were stimulated or not with mitogen phytohemag- kynurenine and Kyn/Trp increased during the treatment period by glutinin (PHA). PHA stimulation activates T cells to produce IFN-γ, 0.28 μmol/L (95%CI: -0.04, 0.60) and 3.2 μmol/mmol (95%CI: -1.6, which induces IDO activity in monocytes/macrophages. After 48 8.0), respectively at 3 months and by 0.48 μmol/L (95%CI: 0.16, 0.80) hours, tryptophan (Trp) and kynurenine (Kyn) levels were deter- and 7.5 μmol/mmol (95%CI: 2.5, 12.5), respectively at 6 months. mined by HPLC in culture supernatants. IDO activity was indicated Increased circulation of kynurenine and Kyn/Trp following by the Kyn/Trp ratio [6]. Cell viability was assessed by analyzing the regular black tea consumption may indicate enhanced tryptophan metabolic activity of cells by measuring the conversion of the sub- breakdown, possibly due to increased activity of immune activation- strate resazurin into resorufin. induced tryptophan degrading enzyme indoleamine 2,3-dioxygenase Glucose treatment of cells did not influence IDO activity in the (IDO). This assumption is supported by the fact that Kyn/Trp corre- tested concentration range [11.1- 66.6 mM], but cell viability decreased lated with neopterin concentrations throughout the study. slightly. Mannitol treatment [0.625-20.0 mM] resulted in a ∼20% The influence of black tea consumption on biomarkers of decrease of Kyn/Trp ratio and cell viability in PHA-stimulated cells immune system activation could relate to its general health benefits with the highest concentrations. A significant and dose-dependent and also to its blood pressure lowering property [6] as kynurenine decrease of Kyn/Trp was measured upon treatment of PBMC with was identified as an endothelium-derived relaxing factor produced citric acid [6.25-100 mM] in stimulated but not unstimulated cells, during inflammation [7]. Data suggests that net effect strongly e.g., 50.0 mM citric acid addition reduced IDO to 13% of remaining depend on the individual immune state, being stimulatory in healthy activity, whereas cells viability was 80% of control cells. The higher individuals, while acting more immune dampening in situations concentration of 100 mM was already toxic, only ∼15% of both stimu- with an inflammatory background. lated and unstimulated cells remained viable, pH of the media was lowered to 5.5. Adenine [0.032-100 μM] upregulated IDO activity at [1] Fuchs D, et al. Curr Med Chem. 2009;16:4644-53. lower concentrations slightly but significantly, while the enzyme was [2] Pedersen ER, et al. Arterioscler Thromb Vasc Biol 2011;31:698-704. suppressed with the highest treatment concentration in stimulated [3] Zvetkova E, et al. Intern Immunopharmacol 2001;1:2143-50. but not in unstimulated cells. Cell viability was also affected, but to [4] Gostner J, et al. Curr Pharm Des 2014;20:840-9. a minor extent. [5] Gostner JM, et al. Biochim Biophys Acta Clinical 2015;3:31–5. Data indicate the potential of blood storage solutions compo- [6] Hodgson JM &Croft KD. Mol. AspectsMed 2010;31:495–502. nents to mediate immunomodulatory effects on PBMC in vitro. These [7] Wang H, et al., Nat Med 2010;16:279–85. findings are in line with our previous report on the effect of erythro- cyte concentrates on IDO activity in the same model system, where the SAGM storage solution was identified as a critical contributor [7]. In vitro effects of stabilizer compounds in blood Suppression of IDO activity by several types of antioxidants is dis- donations cussed as a relevant mechanism to inhibit T helper type 1 reactions, thereby promoting T helper type 2 responses [8].

Gostner JM, Becker K, Schennach H, Fuchs D Biocenter, Medical University, Innsbruck, and Central Institute of [1] Cata JP, et al. Br J Anaesth. 2013;110(5):690-701. Immunology and Blood Transfusion, University Hospital, Innsbruck, [2] Vamvakas EC, Am J Clin Pathol 2006;126(Suppl 1): S71-85. Austria [3] Becker K, et al. Phytomedicine. 2014;21(2):164-71. ([email protected]) [4]Brandacher G, et al. Curr Opin Organ Transplant. 2008;13(1):10-5. 120 34th International Winter Workshop

[5] Munn DH and Mellor AL, Trends Immunol. 34, 137–143 (2013). [1] Fried LP, et al. J Gerontol A Biol Sci Med Sci 2001;56:M146–56. [6]Widner B, Adv Exp Med Biol. 1999;467:827-32. [2] SX Leng, et al. Age Ageing 2011;40:475–81. [7]Schennach H, et al. Transfus Med Hemother 2014;41(suppl 1):2–108. [3] WidnerB, et al. Brain Behav Immun 2002;16:590-5. [8] Gostner J, et al. Curr Pharm Des 2014;20:840-9. [4] Gostner J, et al. Curr Pharm Des 2014;20:840-9.

Increased neopterin production and tryptophan Impact of resveratrol on tryptophan metabolism breakdown in the frail elderly, potential pathophysi- in vivo ologic consequences Gualdoni GA, Mayer KA, Fuchs D, Zlabinger GJ, Gostner JM Institute of Immunology; Center of Pathophysiology, Infectiology Gostner JM, Marcos-Pérez D, Millán-Calenti JC, Sánchez-Flores M, and Immunology, Medical University, Vienna; Division of Biological Maseda A, Valdiglesias V, Lorenzo-López L, Strasser B, Pásaro E, Chemistry, and Division of Medical Biochemistry, Medical University, Fuchs D, Laffon B Innsbruck, Austria Biocenter, Medical University, Innsbruck, and Institute for Nutritional ([email protected]) Sciences and Physiology, UMIT, Hall, Austria; DICOMOSA Group, Department of Psychology, Area of Psychobiology, and Gerontology Resveratrol is a polyphenol compound found in various nutrients that Research Group, Department of Medicine, Universidade da Coruña, was shown to have immunomodulatory, anti-cancerogenic, and car- Spain. dioprotective effects. The regulation of indoleamine-2,3-dioxygenase ([email protected]) (IDO), the rate-limiting enzyme in inflammatory tryptophan metab- olism, has been proposed to be involved in resveratrol’s biological Frailty is a geriatric syndrome describing the “weak” physical and effects. These observations, however, rely mainly on in vitro findings. mental state of an individual at increased risk for adverse health We assessed the impact of resveratrol on tryptophan metabolism in a outcomes. Numerous factors play a role in the development of frailty murine sepsis model and in humans after oral resveratrol intake. In including immobility (decreased physical activity), loss of balance, the murine sepsis model, balb/c mice were treated with 5 mg/kg bw, depression and cognitive impairment. Phenotypic criteria that indi- 20 mg/kg bw or placebo before inducing a sepsis by LPS adminis- cate compromised energetics include low grip strength, low energy, tration. We found that resveratrol lowered the kynurenine to trypto- slowed walking speed, low physical activity, and/or unintentional phan ratio which is indicative for a decreased IDO activity. This effect weight loss [1]. Inflammation could play a major role in the precipi- was accompanied by an overall decreased inflammatory response tation of characteristic symptoms. An association with increased as mirrored by a dampened pro-inflammatory cytokine induction. concentrations of immune system biomarker neopterin has been Furthermore, we have analyzed the impact of resveratrol on trypto- already described in frail elder individuals [2]. Disturbances in the phan metabolism in humans. Healthy volunteers were orally admin- biosynthesis and reduced availability of specific neurotransmitters istrated 5 g resveratrol (n = 8) or placebo (n = 2) in a pilot study. IDO like serotonin and adrenalin/noradrenalin are suggested to underlie activity was determined by analyzing plasma levels of tryptophan the high risk of depression and cognitive impairment [3]. With this and kynurenine. Determination of the immune activation marker regard, inflammation-induced breakdown of tryptophan can be of neopterin was included in the analysis. Resveratrol administration particular relevance. Also, loss of muscle mass and body weight may resulted in a 1.33- and 1.30-fold increase of the kynurenine to trypto- be a consequence from the developing limitations of this essential phan ratio at 2.5 h (p < 0.01) and 5 h (p < 0.01), respectively. Neopterin amino acid. levels were not affected by resveratrol administration. Concluding, In 239 individuals (35 non-frail, 118 pre-frail and 86 frail) resveratrol differentially affects tryptophan metabolism in situations aged > 63 (mean ± SD: 79.4 ± 8.86) years, measurements of serum of induced inflammation and in unstimulated conditions in vivo. neopterin concentrations by ELISA (BRAHMS, Germany) and of Considering the distinct oxidative milieus in these settings, reported tryptophan metabolism by HPLC were performed. The kynurenine findings point towards a mechanism of differential modulation of to tryptophan ratio (Kyn/Trp) was calculated. Higher neopterin con- IDO by resveratrol based on the cellular environment. centrations were associated with higher tryptophan breakdown rates (lower tryptophan and higher Kyn/Trp levels), with frailty status and with older age. The correlations remained significant also after age-adjustment and there was no influence of gender. Higher neop- Complex connections between marital satisfaction terin and Kyn/Trp as well as lower tryptophan levels were associated with significantly lower grip strength and lower physical activity (all and cellular immune activity in a woman with breast p < 0.001). cancer To summarize, higher age and specific symptoms of frailty are associated with disturbed metabolism of tryptophan. As results of Hannemann J, Singer M, Primetshofer M, Neises M, Schmid-Ott G, neopterin determinations show, enhanced tryptophan breakdown in Fuchs D, Ocaña-Peinado FM, Geser W, Schubert C frail elderly is associated with immune system activation and implies Psychoneuroimmunology Lab, Clinic for Medical Psychology, Medical an influence of cytokine-inducible tryptophan degrading enzyme University, Innsbruck, Austria indoleamine 2,3-dioxygenase (IDO) on the metabolic changes. Meta- ([email protected]) bolic alterations, especially of tryptophan, could be important for the development of specific symptoms like depression and cognitive While studies have shown that troubled marriages can lead to deficits in the frail elderly [4]. negative immune changes in spouses, a deeper understanding of 34th International Winter Workshop 121 the complex connections between marital satisfaction and immune Systematic investigation of the effects of dietary iron system activity is still lacking. This study on a patient with breast supplementation on the course of infection in mice cancer sought to investigate the dynamic relation between marital satisfaction and inflammation under as ecologically valid as pos- Haschka D, Demetz E, Tymoszuk P, Asshoff M, Seifert M, Nairz M, sible research conditions. The 60-year-old patient (ductal mamma Theurl I and Weiss G carcinoma diagnosis [pT1c, N0, M0, G2, Her2-/neu receptor] 5 years Medical University of Innsbruck, Department of Internal Medicine earlier) collected her entire urine for 31 days in 12-h intervals (from VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, 8 p.m. to 8 a.m. and from 8 a.m. to 8 p.m.; total: 63 measurements) Innsbruck, Austria for the determination of urinary neopterin (immune activation ([email protected]) marker) and creatinine levels using HPLC. Furthermore, she com- pleted a short questionnaire on marital satisfaction twice each Iron deficiency in children and its sequels growth and mental retarda- day (at approx. 8 a.m. and 8 p.m.). Cross-correlational analyses tion are an important health issue. Dietary iron supplementation has revealed that increases in marital satisfaction were followed first been considered as a beneficial strategy to overcome iron deficiency, by a urinary neopterin concentration increase after 72–84 h (lag 6: however, in several randomized trials in Africa and Asia iron supple- 0.263; p < 0.05) and then by decreases in urinary neopterin levels mentation was associated with an increased risk of severe infection after 132–144 h (lag 11: -0.426; p < 0.05). We suggest that the long and infection associated mortality with the mechanisms underlying temporal delay between increases in marital satisfaction and this devastating observation remaining elusive. We hypothesize that decreases in inflammation, as well as the biphasic psychoimmu- the increased risk of infection in iron supplemented individuals may nological response pattern seen in this patient actually point to be related to function of iron as a microbial nutrient and a modulator the dynamic impact of superordinate emotionally positive marital of anti-microbial immune effector function. Moreover, it is completely incidents on neopterin production. Such findings once again chal- obscure up to now which specific group of subjects is at the highest risk lenge the usual practice of generalization based on simple data for infection upon dietary iron supplementation and whether or not aggregation. dietary iron intake may be beneficial in another subgroup. To address these important questions we used different mouse models. To study the impact of baseline iron status (iron adequate Mycosporine like amino acids, their biological versus iron deficiency) on the course of infection we fed mice with a standard rodent diet or an iron deficient diet for four weeks. Subse- relevance in algae, isolation and pharmacological quently mice were split in two groups, one received dietary/interven- activities tional iron supplementation and one group remained on its previous diet. For an acute infection model we used Salmonella typhimurium, Hartmann A, Ganzera M an iron dependent bacterium causing severe infection. Institute of Pharmacy, Pharmacognosy, Center for Chemistry and We found that after infection with Salmonella for three days, Biomedicine, University of Innsbruck, Innsbruck, Austria bacterial loads in spleen and liver were highest in previously iron ([email protected]) deficient mice which received iron supplementation prior to infec- tion. This phenotype was strictly iron dependent, as infection with Mycosporine like amino acids, deriving from a broad range of a Salmonella mutant strain, which is lacking the three main iron mainly marine algae, cyanobacteria, terrestrial microalgae and even acquiring siderophores resulted in non-significant differences in lichens, are small water-soluble molecules with multi-target activi- bacterial loads in organs between the four groups. When analyzing ties. Recent research revealed MAAs as antioxidants in a variety gene expression levels in the spleen of Salmonella infected mice we of different assays such as AAPH and DPPH. The degree of radical found a down-regulation of ferroportin expression in spleens of mice scavenging was found to depend on their chemical structure. Hence with the worst outcome along with a significant increase of IL-10. Cor- mycosporine-glycin, an oxo-carbonyl MAA showed much higher respondingly hepcidin expression levels in the infected livers were antioxidative potential compared to MAAs with cyclohexenimine proportionally high in worst outcome group. structure [1-3]. Due to their already known photo-protective proper- Our results demonstrate that iron supplementation of iron defi- ties MAAs might also be potential target molecules for the inhibition cient mice results in an adverse clinical course of infection with the of collagenases, whose activity is triggered by UV-light. Therefore, intracellular bacterium S. typhimurium. This is due to low expression an optimized fluorescent collagenase assay was established and val- of ferroportin which is an important defense strategy of macrophages idated to study their biological activity. For this purpose four MAAs to restrict these pathogens of iron along with increased expression were isolated from marine red algae and one lichen by applying dif- of the macrophage deactivating cytokine IL-10. Our study will be of ferent techniques such as ion-exchange chromatography and pre- helpful in identifying these subjects in areas with a high burden of parative HPLC. Then they were assayed in-vitro for their inhibition infection who may or may not benefit from iron supplementation. of Chlostridium histolyticum collagenase (Chc). A dose-dependent response was observed for all tested compounds, with IC50 values sometimes lower than the positive control 1,10-phenanthroline Adherence to a Mediterranean dietary pattern and (42.86 μg/ml), e.g. that of the MAA porphyra-334 was found to be risk of cancer: systematic evidence from observa- 3 7.7 3 μg/ml. tional studies.

[1] del la Coba F, et al. J Appl Phycol 2009;21:191-6. Hoffmann, G., Schwingshackl, L. [2] Tao C, et al. Fisheries Sci 2008;74:1166-72. Department of Nutritional Sciences, University of Vienna, Althanstreet [3] Rastogi RP & Incharoensakdi A. J Photochem Photob B 14 UZA II, A-1090 Vienna, Austria 2014;130:287-92. ([email protected]) 122 34th International Winter Workshop

The aim of this systematic review was to meta-analyze the effects of were calculated (NCI was defined as a GDS ≥ 0.5). Non-parametric adhering to a Mediterranean Diet (MD) on overall cancer risk and risk statistical analyses included Kruskal-Wallis and Mann-Whitney U of different types of cancer. Literature search was performed using tests, and multivariate logistic regression. the electronic databases MEDLINE, SCOPUS, and EMBASE until Groups were comparable for gender and ethnicity. HIV+ partici- 10th January 2015. Only cohort as well as case-control studies were pants were older (median age 40 vs. 35; P = 0.027) and had higher rates included. Study specific risk ratios (RR) were pooled using a random of NCI (39% vs. 14%; P = 0.002) and MDD (46% vs. 15%; P < 0.001). effect model by the Cochrane software package Review Manager IA marker concentrations were increased and TRP concentrations 5.2 (©). 21 cohort studies (1,368,736 subjects), and 12 case-control decreased in the CSF and plasma of HIV+ participants, including studies (62,725 subjects) met the objectives and were enclosed for a sub-group of aviraemic HIV+ participants receiving ART (n = 44), meta-analyses. compared with controls. There were no differences in CSF and plasma The highest adherence to MD category resulted in a significant KYN between the HIV+ groups and controls. The KYN/TRP ratio in risk reduction for overall cancer mortality+incidence [cohort; RR: plasma was increased in the total HIV+ group only (P = 0.027). The 0.90, 95% CI 0.86 to 0.95, p < 0.0001; I2 = 55%], as well as a significant PHE/TYR ratios in CSF, and nitrite concentrations in plasma, were risk reduction for incidence of either colorectal [cohort/case control; significantly increased in the HIV+ groups. In multivariate analysis, RR: 0.86, 95% CI 0.80 to 0.93, p < 0.0001; I2 = 62%], prostate [cohort; detectable viral loads in CSF and plasma, use of ART and reduced RR: 0.95, 95% CI 0.92 to 0.99, p = 0.03; I2 = 0%], or aerodigestive cancer current CD4+ cell counts were predictors of increased concentrations [case-control studies; RR: 0.34, 95% CI 0.18 to 0.65, p = 0.001; I2 = 76%]. of TNF-α (R2 = 0.354; P = 0.03) and NEO (R2 = 0.364; P < 0.001) in CSF. In Non-significant changes were observed for breast cancer, gastric a multiple regression model, TNF-α and NEO correlated with KYN/ cancer, and pancreatic cancer. TRP ratios in both CSF (R2 = 0.277; P = 0.008) and plasma (R2 = 0.674; According to these results, adherence to a Mediterranean diet is P < 0.001). In a logistic regression model, KYN/TRP ratios in plasma associated with a significant reduction in the risk of overall cancer negatively correlated with NCI (χ2[1,n = 82] = 4.312; B = -1.926; P = 0.038), mortality (by 10%), colorectal cancer (by 14%), prostate cancer (by but not with MDD status (χ2[1,n = 84] = 3.458; B = -1.643; P = 0.063) in 4%), and aerodigestive cancer (by 56%), respectively. This is of clini- HIV+ participants. The KYN/TRP ratios in aviraemic participants cal as well as public health significance, since dietary habits have were negatively correlated with MDD (χ2[1,n = 32] = 4.874; B = -4.054; changed over the past 40 years in favor of a dietary pattern typical P = 0.066), but not with NCI (χ2[1,n = 32] = 3.388; B = -3.078; P = 0.066). for northern European countries. Recommendations to reverse this No associations were observed in viraemic patients or controls. process, i.e. promoting a Mediterranean dietary pattern typical for Significant correlations were not observed for PHE/TYR ratios with southern European regions might prove to be beneficial. either NCI or MDD in any of the groups. In this heterogeneous HIV+ population, we observed increased concentrations of IA biomarkers, decreased TRP, and increases in the KYN/TRP ratio in plasma and the PHE/TYR ratio in CSF. NCI Tryptophan metabolism and its relationship with and MDD were associated with a lower KYN/TRP ratio in plasma in immune activation, depression, and neurocognitive both the overall HIV+ group and the aviraemic sub-group. This asso- impairment among HIV-infected individuals ciation was not observed in HIV+ participants with detectable viral loads. The causes of NCI and MDD in HIV+ individuals are likely to be multifactorial and these findings warrant further exploration. Keegan MR, Chittiprol S Winston A Letendre S, Fuchs D, Boasso A, Iudicello J, Ellis RJ Imperial College London, London; ViiV Healthcare Ltd., United Kingdom; University of California, San Diego, San Diego, CA, Biocenter, Decreased HDL-cholesterol in still healthy Medical University, Innsbruck, Austria oberweight/obese offsprings indicates parental ([email protected]) myocardial infarction and stroke whereas leptin is Neurocognitive impairment (NCI) and major depressive disorder increased only in male offsprings with positive family (MDD) are highly prevalent during HIV-1 disease. Two possible mech- anamnesis anisms that could contribute to both NCI and MDD are the accelerated degradation of tryptophan (TRP) via the kynurenine (KYN) pathway, Kofler P, Stelzer I, Zelzer S, Reininghaus E, Mangge H resulting in increased production of neurotoxic catabolites, and the Clinical Institute for Medical and Chemical Laboratory Diagnosis, decreased production of serotonin and dopamine, both of which Austria can result from increases in immune activation (IA). We explored ([email protected]) whether IA is associated with these two biochemical pathways and their relationship with NCI and MDD in HIV+ individuals. Atherosclerosis causes fatal clinical endpoints such as myocar- We performed a cross-sectional analysis of archived CSF and dial infarction and stroke. To prevent this, we must improve the plasma from HIV+ individuals (n = 91; receiving antiretroviral therapy understanding of cardiovascular risk factors and early upcoming [ART], n = 65; not receiving ART, n = 26) and HIV- controls (n = 66), pathophysiologic mechanisms which can induce clinical useful measuring concentrations of IA biomarkers (neopterin [NEO], and biomarkers. tumour necrosis factor [TNF]-α), TRP and KYN, with KYN/TRP ratios Herein we investigated a possible connection between positive as an index of indoleamine 2,3-dioxygenase (IDO) activity, phenyla- family history of cardiovascular events and associated biomarker lanine (PHE) and tyrosine (TYR), with PHE/TYR ratios as an index of patterns of offspring. phenylalanine hydroxylase (PAH) activity, and nitrite, as well as neu- Study participants are from the prospective, observational study rocognitive function and mood status. Global Deficit Scores (GDS) STYJOBS/EDECTA (STYrian Juvenile Obesity Study/Early DEteCTion 34th International Winter Workshop 123 of Atherosclerosis; ClinicalTrials.gov Identifier NCT00482924) inves- Upon treatment, disease activity improved significantly tigating metabolic/cardiovascular parameters in obese young and (p < 0.001), and also CRP and OPG declined significantly (p = 0.023 adults free of chronic health conditions. for CRP, p < 0.01 for OPG), while ESR tended to be lower (p = 0.086). We analyzed 15 biomarkers with a possible relation to cardio- However, there were no significant changes of neopterin, sRANKL and vascular events in male normal weight, male overweight/obese, the sRANKL/OPG ratio. After 12 weeks of adalimumab therapy, the female normal weight and female overweight/obese STYJOBS/ correlation between OPG and neopterin was not detectable anymore. EDECTA participants. Differences (p < 0.05) between participants We conclude that therapy with adalimumab was very effective with a positive family history of myocardial infarction > 55years, to modify disease activity (as reflected by the decrease in the number myocardial infarction < 55years, and stroke were compared to a of tender and swollen joints) and also slow-down inflammatory and control group with negative family history of both cardiovascu- bone remodelling cascades. lar events. Depending on the distribution of the data, all investi- gations were performed by using the T-Test or the Mann-Whitney Pre-, pro- and psychobiotics in old age U Test. Male ow/ob participants: In the groups with parental MI, MI < 55y and stroke, triglycerides were significantly higher, and Leblhuber F, Schuetz B, Fuchs D HDL-Cholesterol significantly lower compared to the control group Department of Neurological and Psychiatric Gerontology, Linz; Division without cardiovascular events. Leptin was found significantly higher of Biological Chemistry, Biocenter, Medical University, Innsbruck, in MI < 55 and in the stroke group. Austria; Biovis Diagnostik MVZ GmbH, Limburg, Germany Male normal weight participants: Significantly higher levels of ([email protected]) triglycerides and leptin were seen only in the stroke group. Female ow/ob participants had significantly lower levels of HDL-Cholesterol Gastrointestinal health is of increasing interest in preventive medi- in the MI and MI < 55 group. Female normal weight persons showed cine. It includes normal intestinal microbiota and effective immune no significant differences of any biomarker. status. Gastrointestinal barrier function and gut microbiom seem to These data underline the importance of decreased HDL choles- be the key to good health. terol as an “overall generation” cardiovascular risk factor. All other Prebiotics are nondigestible food ingredients that beneficially markers (i.e. CRP, IL-6, LDL, oxLDL, TG, HOMA etc.) failed in this effect the host by selectively stimulating the growth and activity of context. Leptin may be of additional interest. bacteria in the colon and thus improve host health that is especially This work was partially supported by funding under the European important in the elderly, as there is an age-related decrease in the FP7 program “NanoAthero”-NMP4-LA-2012-3099820. diversity of gut microbiota. Probiotics are living organisms, which when administered in adequate amounts confer health benefits on the host: probiotics sup- Osteoprotegerin and RANKL in patients with rheuma- press pathogens, stimulate epithelial cell proliferation, differentiate toid arthritis and fortify the intestinal barrier and mediate immmune modulation. However, there is still confusion in the minds of the authorities over whether a probiotic is a drug, a food, or a dietary supplement. Kurz K, Herold M, Klotz W, Mur E, Weiss G, Fuchs D Gut microbes can produce hormones and neurotransmitters Department of Internal Medicine VI and Division of Biological like short chain fatty acids, g-amino-butyric acid (GABA), serotonin, Chemistry, Biocenter, Medical University, Innsbruck, Austria tryptophan and other biogenic amins like adrenalin and noradrena- ([email protected]) lin that are identical to those produced by the host. Therefore such probiotics are also named psychobiotics, which act on the brain-gut Rheumatoid arthritis is an autoimmune disease which is charac- axis. The effects are mediated via the vagus nerve, spinal cord and terized by joint erosions, progressive focal bone loss and chronic the hypothalamic-pituitary-adrenal axis. inflammation. Pro-inflammatory cytokines like TNF-alpha and inter- Dysbiosis (perturbation in microbiom composition) results in feron-gamma are involved in enhanced bone resorption. dysbalance of neurotransmitters and consecutively in depression, 22 patients (20 women, 2 men) with long-standing, moderate to anxiety, altered pain perception and other symptoms like cognitive severe rheumatoid arthritis were included in our study and treated decline and dementia [1]. with a monoclonal TNF-antibody (Adalimumab 40 mg subcutane- Administration of prebiotics and probiotics like Omnibiotic ously every other week) in addition to their concomitant but inad- (Allergosan, Graz, Austria) may alter the gut microbiom and improve equate anti-rheumatic therapies. Patients were assessed for overall the disturbed resorption of neurotransmitters important for the neu- disease activity using the DAS28- score and blood samples were ropsychiatric status in the elderly. collected to determine neopterin, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) concentrations as well as oste- [1] Leblhuber F, et al. J Neural Transm 2015, Feb 14, DOI 10.1007/ oprotegerin (OPG) and soluble Receptor Activator of NF-κB Ligand s00702-015-1381-9. (sRANKL) concentrations before adalimumab therapy and at week 12. Neopterin as well as OPG and sRANKL were determined by ELISA (neopterin: Brahms, Henningsdorf, Germany, and OPG/sRANKL: Routine serum and urine neopterin measurement in a Biomedica, Vienna, Austria). clinical central laboratory Before adalimumab therapy, neopterin concentrations corre- lated with OPG (rs = 0.536, p = 0.01) and ESR (rs = 0.552, p < 0.01), Loacker L, Weigel G, Griesmacher A and tendentially with CRP (rs = 0.404, p = 0.062), but not with Central Institute for Medical and Chemical Laboratory Diagnostic, DAS28. sRANKL levels were neither associated with OPG concentra- University Hospital, Innsbruck, Anichstraße 35, Austria tions nor with those of other inflammatory markers. ([email protected]) 124 34th International Winter Workshop

Neopterin is a biologically stable and helpful marker reflecting the (ApoE mice, rabbits) by means of NMRI. Under the scope of the EU cell-mediated immunity and is therefore able to enfold the state of project NanoAthero this approach is examined for a development to activation of the entire immunological system. Moreover it provides the human pipeline. an opportunity to monitor and estimate the prognosis of multiple Nevertheless, the main challenge remains: which asympto- diseases such as infections, autoimmune diseases, malignancies, matic individual should be screened? At which time? Furthermore, cardiac and renal failure or allograft rejection. In routine laboratories it is essential to act specific, effective, without side effects because neopterin can be determined by ELISA, RIA or HPLC. the “patient” may yet feel healthy at the time of the successful In a retrospective examination we investigated the quantity and prediagnosis. numeric development of all neopterin analyses in the period of 2008 This work was partially supported by funding under the European to 2014 at the Innsbruck University Hospital, the frequency of differ- FP7 program “NanoAthero”-NMP4-LA-2012-3099820. ent underlying diagnoses and the distribution of requesting medical departments. neopterin was determined in human serum and urine samples using an ELISA or RIA assay in a routine laboratory setting. There is a distinct increase of 34% of urine and 198% of serum IDO1 controls anti FVIII antibody response in neopterin measurements comparing the analysis frequency in 2008 hemophilia A and 2014. Main areas of clinical interests for determination actually are infectious diseases, HIV monitoring and autoimmune diseases. It Matino D, Gargaro M, Santagostino E, Di Minno D, Landolfi R, Morfini is therefore most frequently requested by the infectiology, gastroen- M, Rocino A, Mancuso ME, Di Minno MND, Coppola A, Volpi C, Vacca terology and dermatology departments. C, Iannitti R, azzucconi MG, Santoro C, Tosti A, Tagariello G, Belvini Neopterin measurement is widely established at the Innsbruck D, Radossi P, Castaman G, Fuchs D, Boon E, Grohmann U, Puccetti P, University Hospital. Beside its known value in the assessment of Iorio A, Fallarino F severity, course, activity and treatment control of immunological Department of Experimental Medicine, University of Perugia, diseases neopterin is also getting relevant in recent clinical fields of Perugia, Italy; Angelo Bianchi Bonomi Hemophilia and Thrombosis application such as cardiovascular or metabolic diseases. Centre, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico Milan, Italy; Regional Reference Centre for Coagulation Disorders, Improved diagnosis and stabilization of vulnerable Department of Clinical and Experimental, Medicine, Federico II University, Naples, Italy; Institute of Internal Medicine and Geriatrics, atherosclerotic plaques – a main challenge to fight Haemostasis Research Center, Catholic University, Rome, Italy;Centro myocardial infarction and stroke Emofilia, Azienda Ospedaliera Careggi, Florence, Italy; Haemophilia and Thrombosis Centre, San Giovanni Bosco Hospital, Naples, Italy: Mangge H, Almer G, Opriessnig P, Reininghaus E, Prassl R Department of Cellular Biotechnology and Haematology, La Sapienza Clinical Institute for Medical and Chemical Laboratory Diagnosis, University, Rome, Italy. Department of Medicine University of Perugia, Medical University, Graz, Austria Perugia, Italy; Transfusion Service, Hemophilia and Regional Blood ([email protected]) Disease Centre, Castelfranco Veneto, Italy; Dept. of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy; Division of Biological Vulnerable atherosclerotic (AS) plaques cause fatal clinical end- Chemistry, Biocenter, Medical University, Innsbruck, Austria; Bioceros, points such as myocardial infarction and stroke. To prevent this, we Utrecht, The Netherlands; Departments of Clinical Epidemiology and must improve the early diagnosis and treatment of these fatal vas- Biostatistics and Medicine, McMaster University, Canada cular lesions. Because vulnerable AS plaques are frequently non- stenotic, they are preclinical undetectable by conventional imaging. Inhibitory antibodies to FVIII are a major obstacle to hemophilia Blood lipids, C-reactive protein, and interleukin-6 are insufficient to A treatment. Congenital absence of FVIII prevents onset of central indicate the process of critical perpetuation prior to endpoints. More tolerance to FVIII, thus foisting effective control of FVIII-reactive specific biomarkers (e.g., troponin, copeptin, natriuretic peptides, lymphocytes on peripheral tolerance mechanisms. Indoleamine 2,3 growth differentiation factor-15, soluble ST2) indicate acute coronary dioxygenase-1 (IDO1) is a key regulatory enzyme of peripheral toler- syndrome or cardiac insufficiency, but not critically destabilized AS ance in adult life. We investigated the association between IDO1 com- lesions. Thus, valuable time (months to years) that could be used to petence and inhibitor status in hemophilia A patients, enrolled in six treat the patient is wasted. An improved management of this dilemma Italian centers, with F8 null mutations and either an inhibitor-nega- may involve better detection of variations in degrees of immune tive (N = 50) or positive (N = 50) status. By analyzing IDO1 induction, inflammation in plaques by using new biomarkers in blood and/or expression, and function for any relationship with inhibitor occur- within the lesion by nanotechnological aided molecular imaging. rence by multivariable logistic regression, we found that defective Macrophage and T-cell polarization, innate- and adaptive IDO1 induction by Toll-like receptor (TLR)9 activation was associ- immune responses (e.g., The Toll-like receptors 2, 4, 7), are involved in ated with an inhibitor-positive status. In an experimental model of the process of plaque “vulnerabilisation”. New biomarkers involved the disease using hemophilic mice with or without functional IDO1, in these mechanisms include adiponectin, interleukins, pentraxin 3, we found that tryptophan metabolites resulting from IDO1 activity calprotectins S100A8/A9, myeloperoxidase, and chemokines. These prevented the generation of anti-FVIII antibodies and that a TLR9 proteins may be candidates for molecular imaging by using NMRI/ agonist caused suppression of FVIII-specific B cells by a mechanism nuclear imaging tools. involving IDO1-dependent induction of regulatory T cells. Thus IDO1 We found out recently that globular adiponectin or interleu- competence may represent a novel patient risk stratification tool in kin-10 targeted pegylated “stealth” liposomes are promising nano- hemophilia A, thus implementing novel strategies for preventing or constructs for molecular imaging of AS lesions in animal models eradicating inhibitors to therapeutically administered FVIII protein. 34th International Winter Workshop 125

Kynurenine metabolites in acute alcohol withdrawal patients (n = 72 and n = 474). Urinary neopterin was determined by high-performance liquid chromatography. LMR calculated based Mechtcheriakov S, v. Gleissenthall G, Arnhard K, Oberacher H on automated and manual counts was significantly different. LMR Department of Psychiatry and Psychotherapy, and Institute of Legal and PLR were higher in patients compared to controls. A signifi- Medicine and Core Facility Metabolomics, Medical University, cant correlation was observed between peripheral blood cell counts Innsbruck, Austria (PBC)-derived ratios, but no correlation was noted between urinary ([email protected]) neopterin and PBC-derived ratios. In a cohort of 72 patients NLR ≥ 3, but not urinary neopterin was significant predictor of survival. In Recent research has suggested that alcohol-associated immune contrast, in an unselected cohort of 474 patients urinary neopterin activation may contribute to the pathophysiology of alcohol with- and NLR ≥ 3.5 were significant predictors of survival. In multivari- drawal and dependence. We studied the relationships between an able analysis, age, hemoglobin, urinary neopterin and NLR were immune activation marker neopterin and parameters of tryptophan independent predictors of survival. In conclusion, PBC-derived and kynurenine metabolism during the first 10 days of alcohol with- ratios did not correlate with urinary neopterin. In different cohorts, drawal in alcohol dependent patients. both NLR and urinary neopterin were significant predictors of Our results show a significant correlation between serum survival. neopterin values and kynurenine/tryptophan ratio as indicator of IDO-activation (r = 0.695, p < 0.001). Serum levels of quinolinic acid (QA) were also strongly related to neopterin concentrations as Investigating the NAD metabolome in Ewing Sarcoma measured by linear mixed model (p < 0.001) while the kynurenic acid (KYNA) concentrations showed no relationship with neopterin Mutz Cornelia N, Schwentner Raphaela, Kauer Maximilian O, Ban (p = 0.929). Jozef, Aryee Dave NT, Erhardt Sophie, Fuchs Dietmar, Heitger Andreas,* These findings demonstrate (1) an immune-triggered activa- Kovar Heinrich tion of IDO in acute alcohol withdrawal causing the increase of Children’s Cancer Research Institute and Department of Pediatrics, kynurenine production and suggest (2) that immune activation also Medical University, Vienna, Austria; Department of Physiology and stimulates the production of quinurenic acid that is known to exert Pharmacology, Karolinksa Institutet, Sweden; Division of Biological glutamatergic (potentially excitotoxic) effects. These data emphasize Chemistry, Biocenter, Medical University, Innsbruck, Austria; the specific role of the immune activation in regulation of tryptophan *deceased and kynurenine metabolism in alcohol disease. Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor Prognostic significance of urinary neopterin development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in generation of the chimeric transcription compared with neutrophil-to-lymphocyte, lympho- factor EWS-FLI1. cyte-to-monocyte and platelet-to-lymphocyte ratios Recently, ES has been reported to be exquisitely sensitive to in patients with breast cancer inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). This enzyme uses NAD as substrate and was demonstrated to regulate EWS-FLI1 Melichar B, Zezulová M, Bartoušková M, Kalábová H, Študentová H, in a feed-back mechanism. NAD is a key metabolite essential for sus- Vitásková D, Kujovská Krčmová L, Solichová D taining cellular energy metabolism. It plays a central role in cellular Palacký University Medical School and Teaching Hospital, Olomouc; redox reactions, DNA repair, and in the maintenance of genomic sta- Charles University Medical School and Teaching Hospital, Hradec bility serving as a donor of ADP-ribose. The other major mammalian Králové, Czech Republic NAD consumer is the deacetylase SIRT1 which we observed to be ([email protected]) specifically highly expressed in ES metastases. PARP1 and SIRT1 are crucial for coupling cellular metabolism to transcriptional gene regu- Predictive and prognostic biomarkers play and increasingly impor- lation as well as to stress response. Usually, NAD is regenerated from tant role in the management of cancer patients. The significance of nicotinamide in the NAMPT-dependent salvage pathway or from the inflammatory reaction that reflects the host response to neoplastic reduction of pyruvate via LDHA (Warburg effect), but can also be syn- growth is being increasingly recognized. A number of biomarkers thesized de novo from tryptophan. Interestingly, the knockdown of of the inflammatory response have been introduced into the man- EWS-FLI1 in ES cells comes along with alterations in the expression agement of cancer patients over the last decades, including, for of multiple enzymes involved in NAD biosynthesis and regeneration, example, C-reactive protein (CRP) and neopterin. In the last few including TDO2, KMO, NMNAT1, NAPRT, NAMPT, and LDHA. We are years considerable research has been focused on simple biomark- interrogating the role of EWS-FLI1 mediated modulation of these ers obtained by calculating the ratios of lymphocytes to other cellu- enzymes and of specific small molecule inhibitors on cellular tryp- lar components of peripheral blood. The neutrophil-to-lymphocyte tophan consumption, kynurenine production and intracellular NAD ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to- levels of Ewing sarcoma cells. Additionally, we are investigating the lymphocyte ratio (PLR) have been demonstrated to represent inde- functional consequences thereof on constitutive and stress-induced pendent prognostic biomarkers across a spectrum of solid tumors. protein poly(ADP)-ribosylation and acetylation. We performed a retrospective analysis of previously published Preliminary results suggest that NAMPT inhibition diminishes cohorts of patients with history of breast cancer. NLR, LMR and PARP1 activity due to low NAD, implying a pivotal role for the regen- PLR were calculated from peripheral blood cell counts obtained erative salvage pathway in ES cells. TDO2-dependent tryptophan as part of routine care. Survival data were updated for 2 cohorts of consumption increases in the absence of the oncogene. These studies 126 34th International Winter Workshop aim at a better understanding of factors influencing Ewing sarcoma Plasma levels of tyrosine, phenylalanine, tryptophan sensitivity to therapies targeting PARP1 and the NAD metabolome. kynurenine and neopterin in chronic lymphocytic Supported by the Austrian Science fund, grant I1225-B19. leukemia (CLL) patients

Parrak V, Secnik P, Mistrik M, Melichar B, Galba J, Michalicova A, Comparison of LC/MS and LC coupled with UV and Kovac A, Katina S, Fuchs D University Hospital, and Slovak Academy of Science, Bratislava and fluorescence detection for the analysis of tryptophan SK-Lab, Lucenec, Slovak Republic; University Hospital Olomouc metabolism and Masaryk University, Brno, Czech Republic; Biocenter, Medical University, Innsbruck, Austria Oberacher H, Arnhard K, Pitterl F, Sperner-Unterweger B, Fuchs D ([email protected]) Institute of Legal Medicine and Core Facility Metabolomics, Department of Psychiatry and Psychotherapy, and Division of Biological Chemistry, Chronic lymphatic leukemia (CLL) is the most common leukemia in Biocenter, Innsbruck Medical University, Austria adults in the Western world with highly variable clinical course, with ([email protected]) unclear prognosis and still uncurable. A lot of studies investigated predictive markers which can be helpful for predicting the individual The kynurine pathway is the most important pathway in tryptophan risk in different stage of the disease, or to find a diagnostic marker (TRP) catabolism which accounts for more than 90% of TRP breakdown or marker which can follow up the course of therapy. The immune in mammals. Important TRP metabolites include kynurenine (KYN), system plays an important role in CLL and immunological markers kynurenic acid (KYNA) and quinolinic acid (QUIN), and they are known shows changes in their levels in disease follow up. to be active in the nervous and/or immune systems. For studying the There are some early experiences with neopterin determina- physiological roles of TRP and its metabolites, the availability of reli- tions in urine (1). Hausen et al. and our own experiences which show able, sensitive and comprehensive quantitative methods is of utmost significant elevation of neopterin in plasma in patients with CLL in importance. Such analytical techniques are liquid chromatography more than 200 patients (about 600 plasma samples) within 3 years with UV and fluorescence detection (LC) as well as liquid chromatogra- of observation. phy coupled to high resolution mass spectrometry (LC/MS). We have collected EDTA plasma samples from blood sampling The LC method used for comparison in this study [1] enables the upon routine examinations (blood count) aliquotted samples are accurate and reproducible quantification of TRP and KYN: 100 μl of stored at -80o C. Neopterin was analysed by Enzyme-Linked Immu- plasma are prepared for reversed-phase LC analysis by protein precipi- nosorbent Assay tation with trichloroacetic acid, 3-nitro-L-tyrosine is added as internal (ELISA) BRAHMS, Henningsdorf, Germany with a reference standard. Aqueous solutions of TRP spiked with 70 g/l albumin are value of 5.3 ± 2.7 nmol/L; kynurenine, tryptophan, phenylalanine, used as surrogate matrix for preparing calibration standards. Ten μl and tyrosine concentrations were determined by UPLC/FLD/MS of produced supernatants is injected on a reversed-phase column. Run Waters. These parameters were measured in 75 EDTA plasma samples time is 7 min. Quantification is based on peak height comparison to (34 females, 31 males) with same diagnosis, similar age, collected in 1 an external standard. The LC method was validated. Reproducibility month from blood count, aliquotted and stored at -80o C. was found to be < 5%. Recoveries were 99% for TRP and 102% for KYN. Our first results of statistical analyses allow the following pre- To enable the quantification of TRP and its three metabolites, liminary conclusions however, from a small number of samples only: a LC/MS method was developed. 50 μL of plasma are prepared for On average, CLL patients show increased plasma neopterin concen- LC-MS analysis by protein precipitation with acetonitrile. Deuter- trations and compared to healthy controls (2) also the kynurenine to ated analogues of TRP, KYNA, and QUIN serve as internal standards. tryptophan ratios (Kyn/Trp) and the phenylalanine to tyrosine ratios TRP metabolite-free plasma obtained from treatment with activated (Phe/Tyr) were increased. Moreover, there existed significant correla- charcoal is used as surrogate matrix for preparing six calibration tions between neopterin and Kyn/Trp as well as Phe/Tyr. standards and two quality control samples. Analytes are separated We also found some gender differences in amino acids related. on a HILIC column using a gradient of 50-5% acetonitrile in aqueous New questions about the influence of age and of therapy will be ammonium formate (5 mM, pH 9.5), and detected on a quadrupole addressed during further statistical analyses. – quadrupole – time-of-flight instrument (TripleTOF5600+, ABSciex, Foster City, CA, USA) using electrospray ionization (ESI) in negative [1] Hausen A, et al. Clin Biochem 1982;15:34-7. ion mode. The acquisition strategy involves the collection of full scan [2] Geisler S, et al. Pteridines 2015;26:31–6. MS/MS spectra and post-acquisition extraction of analyte-specific fragment ion mass traces. Run time is 25 min. The developed method was validated. The parameters tested for each compound included calibration model, limit of quantification, accuracy, reproducibility, Laboratory diagnostic markers: is there a potential in sample stability, recovery, and matrix effects. The assay was well diagnosis of Alzheimer’s disease? within tolerances prescribed by regulatory guidance for validation of quantitative LC/MS assays. Parrak V, Secnik P, Kontsekova E, Kovacech B, Kovac A, Katina S, 89 samples were analysed with both methods available. TRP Novak M. and KYN levels showed good correlation. The Spearman’s rank cor- Axon-Neuroscience SE and Slovak Academy of Science, relation coefficients were found to be 0.87 and 0.67. Neuroimmunological Institute, Bratislava, and SK-Lab, Lucenec, Slovak Republic; and Masaryk University, Brno, Czech Republic [1] Laich A, et al. Clin Chem 2002;48:579-81. ([email protected]) 34th International Winter Workshop 127

Alzheimer’s disease (AD) is a chronic, irreversible neurodegenerative schedule were reviewed prospectively. Histology confirmed primary disease that causes progressive impairment of memory and cognitive carcinoma in situ (CIS) in 1 (12.5%), urothelial carcinoma pTa high function. AD is a leading cause of dementia worldwide. Epidemio- grade in 4 (50%), pT1 high grade in 2 (25%) and pTa+pT1 high grade logical data about dementia (1) reveals that currently 20 to 30 million in another 1 (12.5%) patient. A second resection (2-6 weeks after individuals suffer from dementia today, with 4.6 million new cases initial surgery) was routinely performed in 7 patients (except CIS) of dementia every year. Neurofibrillary degeneration represents one to exclude persistent tumor and understaging by initial resection. of the key hallmarks of AD. In spite of intensive research, there is no Urine and blood samples were collected before therapy (baseline), disease modifying drug for AD treatment available. The therapeutic during each instillation and 3 months after BCG induction therapy. intervention is considered to be most effective when administered Cystoscopy 3 months after completed BCG induction therapy con- early in the disease’s progression, before neurodegeneration is too firmed cancer recurrence (pTa low grade) in 2 (25%) of 8 patients. severe and widespread through the brain. Serum neopterin concentrations were lowest in patients with cancer The great effort is spent in a search for novel diagnostic tools recurrence (n = 2) before (median, range: 6.9, 5.4-8.4 nmol/L), during or laboratory markers that accurately identify AD in the very early (median after BCG1: 9.6 nmol/L, after BCG2: 7.95 nmol/L, after BCG3: phase of the disease progression. Such marker must be sensitive to 8.7 nmol/L, after BCG4: 8.85 nmol/L, after BCG5: 8.15 nmol/L; after earliest changes in AD and should differentiate among preclinical BCG6: 10.55 nmol/L) and 3 months after BCG therapy (median, range: AD, normal aging and other brain disorders that cause memory loss. 7.25, 7.1-7.4 nmol/L) compared to those patients without recurrence We have developed the first-in-man and first-in-class tau vaccine for (n = 6): before (median, range: 12.9, 6.1-14.4 nmol/L), during (median AD denominated as AADvac1. AADvac1 immunotherapy is currently after BCG1: 12.0 nmol/L, after BCG2: 11.3 nmol/L, after BCG3: 12.2 in Phase 1 clinical trial in four hospitals in Austria. According to our nmol/L, after BCG4: 13.8 nmol/l, after BCG5: 14.5 nmol/L, after BCG6: first results, AADvac1 has a favorable safety profile and acceptable 15.05 nmol/L) and 3 months after BCG induction (median, range: 16.6, antibody response in patients. Samples of human biological fluids 5.8-21.8 nmol/L). In addition, urinary neopterin levels were higher in were collected at every hospital visit and banked. In the future, this recurrence-free patients (median, range: 201, 124-305 μmol/mol cre- collection should represent the unique opportunity to search for atinine) compared to those patients with cancer recurrence (median, novel AD diagnostic tool or laboratory markers. range: 95.5, 51-140 μmol/mol creatinine) before, during and 3 months after (median, range: 344, 127-384 versus 149, 144-155 μmol/mol cre- [1] Ferri CP, et al. Lancet 2005;366:2112-7. atinine) BCG therapy. From this preliminary data obtained serum and urinary neop- terin could be a potentially promising and easily reproducible moni- toring marker in patients with high-risk NMIBC and intravesical BCG Neopterin as a monitoring marker in patients with therapy. However, these incidental findings must be further studied high-risk, non-muscle invasive bladder cancer and in a larger prospective trial prior to drawing any conclusion. intravesical bacillus Calmette-Guèrin (BCG) therapy: a pilot study [1] Babjuk M, et al. Eur Urol 2013;64:639-53. [2] Biot C, et al. Sci Transl Med 2012, 6;4(137):137ra72. [3] Werner-Felmayer G, et al. Cancer Res 1990;50:2863-7. Pichler R, Fuchs D, Fritz J, Brunner A, Horninger W, Culig Z [4] Maggi E, et al. J. Immunol 1992;148:2142-8. Department of Urology, Division of Biological Chemistry, Biocenter, [5] Mack D, et al. Eur J Cancer 1995;31A(6):1025-6. Department of Medical Statistics, Informatics and Health Economics, Department of Pathology, Division of General Pathology, Medical University Innsbruck, Austria ([email protected]) Determination of the neopterin concentration- is it a useful parameter in the pediatric department? Intravesical instillation of bacillus Calmette-Guérin (BCG) for the treatment of high-risk, non-muscle invasive bladder cancer (NMIBC) Plata- Nazar K, Luczak G, Kaminska B represents one of the most successful tumor immunotherapies Department of Pediatrics, Pediatric Gastroenterology, Hepatology and with 60-70% clinical response [1]. Nevertheless, 30-40% of patients Nutrition, Medical University of Gdansk, confirm recurrence and 15% have progression to muscle invasive Gdansk, Poland cancer with the necessity of cystectomy. As a delay in cystectomy ([email protected]) affects the oncological outcome, an early identification of patients suited for bladder preservation with BCG therapy or radical cystec- Evaluation of the usefulness of clinical parameters of inflamma- tomy is essential [1-2]. tion in clinical practice is still a topical issue. These parameters are Due to the fact that both biochemical pathways (neopterin routinely performed in the pediatric departments, in almost every production) as well as the therapeutic efficiency of intravesical patient. Commonly identified are: blood count and differential white BCG therapy are induced and triggered by Th1-type cytokine IFN-γ blood cell count, erythrocyte sedimentation rate (ESR), C- reactive [3-4], concentrations of serum and urinary neopterin could play a protein (CRP) and procalcitonin (PCT) level in serum. All of these crucial role in monitoring patients during BCG therapy. A significant parameters are non-specific. Erythrocyte sedimentation rate study increase of serum and urinary neopterin concentrations 48 hours is imprecise, lazily rising and slowly returns to normal. Highly after each BCG instillation has already been shown in 71% of 30 increased level of CRP- acute phase protein- is observed mainly in patients [5]. Since September 2014, 8 (2 women, 6 men) patients with the course of bacterial infections. PCT is an early marker of systemic a mean (range) age of 76 (65-89) years who were treated once a week bacterial infections. Therefore researchers are looking for more spe- with intravesically instilled BCG according to the empirical 6-weekly cific parameters of viral infections which could be applied for routine 128 34th International Winter Workshop clinical practice. One of these parameters seems to be neopterin association with suicide attempt history will be stronger in those (NPT) level. Elevated levels of neopterin are observed in diseases in with high kynurenine levels. 950 patients with schizophrenia were which cellular immune response is stimulated. Monitoring changes evaluated clinically, anti-T.gondii IgG antibodies were measured with of the immune system in the course of illness, by means of a rapid using solid-enzyme immunoassay as well as kynurenine levels using and sensitive method, helps to explain the disease etiology and gives with high performance liquid chromatography. In T. gondii sero- the ability to implement appropriate treatment. positive patients only, KYN levels in the upper 25th percentile were The study group consisted of 363 children with different inflam- associated with nonfatal suicidal self-directed violence. The results matory diseases. The control group consisted of 105 healthy children. argue against a linear mediation of T. gondii effect by kynurenine The serum neopterin concentration was analyzed by immune-enzy- and its metabolites. We propose that the immunosuppressive effect matic method (ELISA) using a commercial test set (Brahms, Hennigs- of high levels of kynurenine, resulting from a dysregulated kynure- dorf, Germany). Based on the results obtained in the control group, nine pathway, may contribute to T. gondii reactivation, and suicidal the reference level of NPT in serum was estimated as 11nmol/L, behavior. If confirmed in future longitudinal studies, this result may regardless of age and gender. In our studies determination of neop- have both theoretical and practical treatment implications. Addition- terin concentration was considered as a useful parameter in differen- ally, interactions with stress induced activation tial diagnosis of infections etiology. High levels of NPT were observed Supported by an American Foundation for Suicide Prevention Dis- in viral and intracellular bacterial infections while normal levels of tinguished Investigator Award (PI Postolache) with additional support NPT are characteristic for other typical bacterial infections. Determi- from the Rocky Mountain MIRECC, Denver, Co. Drs Postolache and nation of NPT was considered as a useful parameter in monitoring Rujescu equally contributed to this project. progress, course and treatment of autoimmune diseases (inflamma- tory bowel disease, rheumatoid disease). There was a significant positive correlation between serum levels of NPT and CRP only in the group of children with inflamma- In psychiatrically healthy individuals, overweight tory bowel disease. In other groups of children correlation between women but not men have lower tryptophan levels NPT and CRP levels was not observed. There was also no significant correlation between NPT and PCT. Postolache TT, Raheja UK, Giegling I, Mohyuddin I, Rovner SF, Determination of serum NPT level, as parameter independent Weghuber D, Mangge H, Rujescu D, Fuchs D of other markers of inflammation, is valuable for the diagnosis of Mood and Anxiety Program, Department of Psychiatry, University inflammatory diseases in children. of Maryland School of Medicine, Baltimore, MD, USA; Psychiatry Residency Training Program, Saint Elizabeths Hospital, Washington, DC, USA; Division of Biological Chemistry, Biocenter, Medical Toxoplasma gondii seropositivity and high kynure- University, Innsbruck, Austria; Department of Psychiatry, Martin- nine levels are associated with suicide attempts in Luther-Universität Halle-Wittenberg, Halle, Germany; Frontier Medical Group, El Paso, TX, USA; Department of Pediatrics, Paracelsus Medical patients with schizophrenia: Potential interaction School, Salzburg, Austria; Research Unit on Lifestyle and Inflammation- with stress induced inflammation associated Risk Biomarkers, Clinical Institute for Medical and Chemical Laboratory Diagnosis, Medical University of Graz, Graz, Austria Postolache TT, Okusaga O, Duncan E, Langenberg P, Brundin L, Groer Teodor T. Postolache, and Dan Rujescu equally contributed to this MW, Giegling I, Hartmann AM, Konte B, Friedl M, Rujescu D, Fuchs D project Department of Psychiatry and of Epidemiology and Public Health, University of Maryland-Baltimore School of Medicine, Baltimore, MD; Obesity has been shown to be associated with elevated kynurenine Emory University School of Medicine, Department of Psychiatry and (KYN) and kynurenine-tryptophan (KYN/TRP) ratio, an index estimat- Behavioral Sciences, Atlanta, GA; Division of Psychiatry and Behavioral ing tryptophan breakdown rate. Previous research showed that obese Medicine, College of Human Medicine, Michigan State University and women are more likely to be depressed than obese men. To exclude the Van Andel Research Institute, Grand Rapids, Michigan; Biocenter, effects of depression on tryptophan breakdown, we repeated the above Medical University, Innsbruck, Austria; University of South Florida, investigations in psychiatrically healthy individuals. In 1000 par- Tampa FL; Department of Psychiatry, Martin-Luther-University Halle- ticipants, plasma KYN, TRP, and their ratio were compared between Wittenberg, Halle Germany; Veterans Integrated Service Network overweight/obese and normal-weight individuals using ANCOVA with (VISN) 19, Mental Illness Research Education and Clinical Center adjustment for age and gender and bivariate post-hoc tests. (MIRECC), Denver, CO; Veterans Integrated Service Network (VISN) There was no significant relationship between KYN, TRP, and 5, Mental Illness Research Education and Clinical Center (MIRECC), KYN/TRP ratio and overweight/obese status. A significant gender by Baltimore, MD weight category interaction was identified for TRP only, with over- ([email protected]) weight/obese women having lower TRP than overweight/obese men (p = 0.02). The results suggest that the previously described associa- Both Toxoplasma gondii (T. gondii) chronic “latent” infection, and tion between obesity and KYN may have been mediated by depres- molecules of the kynurenine pathways (kynurenine and quinolinic sion or other psychopathology. Our results also affirm that lower acid), have been previously associated with suicidal self-directed TRP levels in obese women are not secondary to depression (e.g., via violence. As infection with T. gondii is known to elevate kynurenine indoleamine 2,3-dioxygenase activation), that multiple vulnerabili- levels, and because high kynurenine levels are in turn immunosup- ties are required for obesity related depression in women, and that pressive, thus potentially contributing to reduced immune pressure manipulations of TRP levels could be used to advance theoretical and induce reactivation of T. gondii, we hypothesized that T. gondii knowledge and therapeutic control of depression in obese women. 34th International Winter Workshop 129

The expression of cathelicidin gene is reduced in the D vitamin deficiencies are risk factors for the development of age- prefrontal cortex of depressed suicides related diseases. The aim of this study was to evaluate the effects of B and D vitamin supplementation on telomere biology in healthy elderly people. Postolache TT, Zhang H, Lee E, Streeten E, Brenner LA, Dwivedi Y In a double-blind study 60 subjects ( > 54 years) were randomly University of Maryland, Baltimore, MD, Rocky Mountain MIRECC, assigned to receive a daily combination of vitamin D3 (1200 IU), folic Denver CO, University of Alabama School of Medicine, Birmingham, AL acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg) and calcium carbonate (456 mg) (Group A) or vitamin D3 and calcium carbon- Severe hypovitaminosis D leads to immune dysregulation, increased ate alone (Group B). Blood concentrations of 25-hydroxy-vitamin D, vulnerability to infections, and is associated with suicidal self- vitamin B12, vitamin B6, folate and several metabolites were meas- directed violence (SSDV). Vitamin D stimulates the production of ured. Furthermore, LINE-methylation and telomere length in periph- cathelicidin, an antimicrobial peptide identified in humans in the eral blood leucocytes were analyzed at baseline and after 1 year of skin. As brain cathelicidin-homologues were previously located supplementation. in rodents, we endeavored to uncover brain cathelicidin gene Baseline gender- and age-adjusted telomere length correlated (CAMP) expression in humans. We further hypothesized that among with 5-methyl-tetrahydrofolate (r = 0.35) and total folate (r = 0.33). depressed suicides, brain CAMP expression is decreased and of At the end of the study gender- and age-adjusted telomere length vitamin D receptor gene (VDR) is increased. Expression levels of showed the following correlations: Group A: methylmalonic acid CYP24A1 and CYP27B1, metabolic genes critical for the local produc- (r = -0.46) and choline (r = 0.39); Group B: 5,10-methenyl-tetrahydro- tion of activated Vitamin D were also measured. folate (r = -0.57), dimethyl-glycine (r = -0.39), and LINE-1 methylation Expression levels of CAMP, Vitamin D receptor (VDR) genes, (r = -0.43). The significant increase of choline and decrease of meth- CYP24A1and CYP27B1 in the dorsolateral prefrontal cortex (dlPFC) and ylmalonic acid after one-year treatment with B and D vitamins was anterior cingulate cortex (ACC) were compared between depressed significantly related to improved telomere length. suicides (n = 15), and matched (age, gender, and postmortem-interval) The present results suggest a functional relationship between non-psychiatric controls (n = 15). Gene expression was analyzed with vitamin B status and telomere length in elderly subjects. One-year qRT-PCR with TaqMan® primers and probes, with GAPDH and β-actin treatment with B and D vitamins significantly changed the pattern of genes as endogenous controls. Statistical analyses included ANOVAs correlations observed at baseline. The improved vitamin B12 status with post-hoc t-tests with Bonferroni corrections. after vitamin supplementation showed a significant relation to tel- CAMP expression was significantly down-regulated and VDR omere length. Moreover, a sub-optimal vitamin B status may result in expression significantly up-regulated (p < 0.05 corrected) in dlPFC telomere dysfunction through altered DNA methylation. and ACC In depressed suicides, with no significant differences in CYP24A1 or CYP27B1 expression. Future studies should include other brain regions, larger N, psychiatric controls, protein measures, and tissue localization with in-situ hybridization. Elevated VDR and Quantum chemical studies on pterin radicals lower CAMP gene expression in depressed suicides is consistent with associations of depression and SSDV with hypovitaminosis D, Reibnegger G inflammation and infection. Institute of Physiological Chemistry, Center of Physiological Medicine, The study was partly funded by R01MH082802 (Y. Dwivedi), Medical University Graz, Graz, Austria by the American Foundation for Suicide Prevention (T.T. Postolache) ([email protected]) and the Rocky Mountain MIRECC, Denver, CO (T.T.Postolache, L.A. Brenner). Drs. Postolache and Dwivedi equally contributed to this Pteridines are an important class of heterocyclic molecules which project. play important roles in many biological systems. In particular, pteri- dines interact with free radicals, and may trigger free radical forma- tion [1]. The occurrence of pteridine radicals has been hypothesized as intermediates in photobiological processes [2], and the electronic The role of telomeres in cellular aging: effect of nature of such systems appears of considerable interest. vitamins supplementation on LINE-methylation and Using reliable quantum chemical computations, I present a telomere length in elderly report on the electronic structure of neutral radicals formally derived from pterin by abstraction of one of the five hydrogen atoms. Pusceddu I, Herrmann W, Kirsch S, Obeid R, Hübner U, Geisel J, All calculations are done in gas phase. Starting with the molecu- Herrmann M lar geometry of neutral pterin in its most stable tautomeric form [3,4] Department of Clinical Chemistry, Saarland University Hospital, optimized using density functional theory (B3LYP/6-31G(d)), the five Homburg, Germany; Department of Clinical Pathology, District potential radicals are built by deleting one of the hydrogen atoms. Hospital Bolzano, Italy For each pterin radical, a new geometry optimization is performed, ([email protected]) followed by a vibrational analysis in order to ensure that a true minimum structure is found. Using this optimized structure, a more Telomeres are essential for the maintenance of genomic integrity. accurate single-point computation at the B3LYP/6-311+G(2d,p) level Telomere length declines with age and telomere shortening/dys- is done yielding the molecular wave function. The electron density, function has been proposed as biomarker for age-related diseases. the spin density and the electrostatic potential (ESP) functions B and D vitamins are essential cofactors for numerous cellular pro- are obtained in cubes surrounding the molecules and employing cesses including the synthesis of purines and nucleotides, DNA a 192x192x192 grid. These calculations are done using G09W soft- methylation, cell differentiation, proliferation and apoptosis. B and ware (Gaussian Inc., Pittsburgh, PA, USA). Analysis according to the 130 34th International Winter Workshop quantum theory of atoms in molecules (QT-AIM) is performed by the Linking body and mind: A psychoneuroimmunological AIMAll package (TK Gristmill Software, http://aim.tkgristmill.com/). study of pteridine metabolism and world-view This software is also employed for building molecular graphs and graphical representation of the charge density gradient fields and the Schnell T, Hefti R, Schubert C, Fuchs D atomic basins of attraction. Electron localization functions (ELF) are Department of Psychology, Leopold Franzens University, and Division computed using the free program DGRID (M Kohout, http://www2. of Biological Chemistry, Biocenter, Medical University, Innsbruck, cpfs.mpg.de/~kohout/dgrid.html). Visualization of electron as well Austria; and Research Institute for Spirituality and Health, Langenthal, as spin density function, ESP, ELF and the Laplacian of the electron Switzerland density function is done with AVS Express 5.3 software (Advanced ([email protected]) Visual Systems Inc., Waltham, MA, USA). The results demonstrate that the five possible radicals derived Associations between biological parameters and dimensions of from neutral pterin fall into two markedly different classes: abstrac- worldview, such as religion and meaning in life, have repeatedly tion of one of the three hydrogen atoms bonded to a nitrogen atom been established. Psychoneuroimmunological processes are not yet yields radicals exhibiting a strong delocalization of the unpaired clearly understood. To contribute to the clarification of links between electron over both the pyrimidine and the pyrazine part of the pterin worldview and biological functioning, a questionnaire-plus-exper- system, while the radicals lacking one of the two carbon bonded imental study has been carried out. A sample of N = 50 persons hydrogen atoms show strong localization of the spin density at the completed online questionnaires and participated in the Trier Social affected carbon atoms without any delocalization via the p-electron Stress Test for Groups (TSST-G) (1). Sixty-four percent of the sample system. Hence, the latter two radicals are somewhat less stable than were female; mean age was 23 years (SD = 4). The TSST is a proce- the resonance-stabilized variants. Further differences between the dure to induce stress by means of two socially evaluative situations: radicals in terms of planar versus non-planar geometries, strengths the first is a public speaking task framed as part of a job interview; of the chemical bonds, ESP and ELF are discussed. the second is a mental arithmetic task. Both take place in front of The presented calculations provide a basis for further investiga- a jury of two, as well as up to five other participants; performances tions, including also the biologically important dihydro and tetrahy- are also (mock) videotaped. Saliva samples were taken at baseline, dro pteridine derivatives as well as anionic and cationic radicals. after stressor 1 and 2 and four times during the following recovery period. Enzyme-linked immunosorbent assay (ELISA) was employed [1] Öttl K, et al. Helvetica Chimica Acta 2000;83:954-65. to determine the amount of neopterin in saliva. [2] Lorente C, et al. Pteridines 2011;22:111-9. The mean neopterin level during baseline was 5.26 nmol/L [3] Soniat M & Martin CB. Pteridines 2008;19:120-4. (SD = 3.10); values (and standard deviations) decreased over [4] Reibnegger G. Pteridines 2014;25:41-8. the course of the stress tasks and reached a stable low value during recovery (4.53/1.3, 4.13/1.22, 3.47/0.86, 3.52/0.72, 3.37/0.65, 3.48/0.87; F(6,44) = 11.42, p < 0.001, η2 = 0.61). Women had signifi- cantly higher levels of baseline neopterin than men (5.96/3.69 vs. Alkylglycerol monooxygenase in 3T3-L1 adipocyte 4.02/0.62). Controlling for sex, baseline neopterin was positively differentiation correlated with crisis of meaning (r = 0.27; p < 0.05). A nega- tive correlation was established between baseline neopterin and Sailer S, Geley S, Hermetter A, Golderer G, Werner-Felmayer G, Werner acceptance of existential self-responsibility (r = -0.28, p < 0.05). ER, Watschinger K Mean neopterin values were not significantly correlated with Department of Biological Chemistry, and Division of Molecular worldview dimensions. Pathophysiology, Biocenter, Medical University, Innsbruck, Austria; Results tie in with previous findings of positive correlations Institute of Biochemistry, Graz University of Technology, Graz, Austria between existential conflict and interleukin-6 [2], and negative corre- ([email protected]) lations between meaning in life and pro-inflammatory interleukin-6 and daily saliva cortisol [3]. Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin- dependent transmembrane protein and is the only enzyme capable [1] Von Dawans A, et al. Psychoneuroendocrinology 2011;36(4):514-22. of metabolizing alkylglycerols. However very little is known about its [2] Ai A, et al. Psych Rel Spir 2009;1(2):112128. physiological role. Our laboratory was able to identify the sequence [3] Ryff, C, et al. Phil Trans R Soc Lond B 2004;354(1449):1383-94. coding for alkylglycerol monooxygenase in 2010 [1]. Its expression and activity can be detected in a variety of tissues and cell lines including adipose tissue. 12 hour-to-12 hour cause-effect relations between IL-6 We could detect AGMO activity in the murine pre-adipocyte cell concentrations and specific and unspecific symptoms line 3T3-L1. Enzyme activity was determined by HPLC based on the conversion of 1-O-pyrenedecyl sn-glycerol to 1-pyrenedecanoic acid [2]. in a patient with mild SLE disease activity: A time- To investigate the role of AGMO in the differentiation process from pre- series analysis approach adipocytes to mature adipocytes we manipulated AGMO expression. We will show expression datat of adipocyte markers, AGMO enzyme Schubert C, Singer M, Ocaña-Peinado FM, König P, Sepp N, Schnapka- activity and Oil Red O staining of lipid droplets in mature adipocytes. Köpf M, Fuchs D Psychoneuroimmunology Lab, Clinic for Medical Psychology, Medical [1] Watschinger K, et al. Proc Natl Acad Sci (USA) 2010;107:13672-7. University, Innsbruck, Austria [2] Werner ER, et al. J Lipid Res. 2007;48:1422-7. ([email protected]) 34th International Winter Workshop 131

Little is known about the real-time cause-effect relations between Schizophrenia and the immune system: possible IL-6 concentrations and SLE symptoms. A 52-year old woman with pathophysiological interactions – an overview mild SLE activity collected her entire urine for the determination of IL-6/creatinine and protein/creatinine levels (ELISA, HPLC) for a Sperner-Unterweger B, Fuchs D period of 56 days in 12h-intervals. Additionally, she answered ques- Department of Psychiatry and Psychotherapy, Division of tionnaires (VAS) on fatigue, weakness, joint pain, oral ulceration and Psychosomatic Medicine;and Division of Biological Chemistry, facial rash, and measured her oral temperature twice a day. Time Biocenter, Medical University, Innsbruck, Austria series analyses consisted of ARIMA modeling and cross-correlational ([email protected]) analyses (one lag = 12h, significance level = p < 0.05). Statistical analyses showed that increased urinary IL-6 con- Based on genetic vulnerability, exposure to different kinds of infec- centrations preceded increased urinary protein by 36–48h (lag3: r = tions and immune activation e.g. due to stress interaction leads to +0.225) and that, in opposite direction of effect, increased urinary dysbalance of the immune system. Increased inflammatory processes protein preceded urinary IL-6 decreases by 12–24h (lag1: r = –0.322). in the periphery as well as in CNS may cause neurobiological changes Moreover, urinary IL-6 increases co-occurred with increased oral including changed neurotransmitter metabolisms which result in ulceration (lag0: r = +,186) and tendentially preceded decreased neurodevelopmental/neurodegenerative alterations yielding to the oral ulceration by 48–60h (lag4: r = –0.170; n.s.). Also, urinary IL-6 heterogenous clinical symptoms of schizophrenia. increases co-occurred with decreased fatigue (lag0: r = –0.193) and Analyses of interleukin-6 (IL-6) and kynurenine (KYN) and facial rash preceded decreases in urinary IL-6 by 84–96h (lag7: r = kynurenic acid- both downstream metabolites of tryptophan – in cer- –0.215). Body temperature, weakness and joint pain did not signifi- ebral spinal fluid of schizophrenia patients indicated that IL-6 interferes cantly correlate with urinary IL-6 concentrations. with the KYN pathway [1]. These findings are in line with earlier studies Some of the results of this study refer to the existence of real- which point to a shift in the kynurenic pathway towards enhanced life feedback mechanisms in SLE. These findings are preliminary and KYNA formation playing a role in the pathophysiology of schizophre- require replication to draw firm conclusions about the real-time asso- nia. Recent results support earlier conclusions drawn from low or even ciation between IL-6 and SLE disease activity. decreased concentrations of macrophage product and pteridine deriva- tive neopterin, on a role of Th2-type immune activation rather than Th1- type immunity in the majority of schizophrenia patients [2, 3]. Also the Routine performance of an “in house” testosterone elevated phenylalanine concentrations and phenylalanine to tyrosine LC-MS/MS assay: data from the first 500+ days ratios in patients with schizophrenia [4] point towards a role of impaired phenylalanine hydroxylase (PAH) activity and probably tetrahydropteri- Seger C dine (BH4) cofactor availability in the pathogenesis of schizophrenia. Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), Immune alterations in schizophrenia may reflect underlying University Hospital, Innsbruck, Austria pathophysiological mechanisms at least for a subgroup of schizo- ([email protected]) phrenia patients which might be used to a better diagnostic charac- terization of this heterogenous disease and on the other side these Compared to IVD-CE certified automated ligand binding assays tes- abnormalities may offer further development options of immune tosterone routine quantification by LC-MS/MS can still be consid- related therapeutic strategies ered a highly experimental procedure which typically relies almost exclusively on laboratory developed measurement procedures. Only [1] Schwieler L, et al. J Psychiatry Neurosci 2014; 39:140126. doi: a limited number of IVD-CE certified assays are currently available; 10.1503/jpn.140126. none of the vendors provides a complete IVD-CE certified instru- [2] Sperner-Unterweger B, et al. Schizophr Res 1989; 2:417-21. ment / assay solution. Consequently outmost care must be taken [3] Bechter K, et al. J Psychiatr Res 2010; 44:321-30. by individual laboratories to ensure the analytical integrity of a lab [4] Okusaga O, et al. PLoS ONE 2014; 9:e85945. developed or an incompletely certified assay over prolonged times. Preferably calibration systems based on traceable higher order ref- Bright versus dim ambient light affects subjective erence materials are utilized, participation in international quality assessment platforms with strong peer groups (e.g. UK-NEQAS) to well-being but not serotonin-related biological factors review calibration preparation accuracy and long-time assay stabil- ity are highly recommended. Stemer B, Melmer A, Fuchs D, Ebenbichler C, Kemmler G, Deisenhammer With the lab developed ZIMCL assay, a online-SPE-HPLC- EA ESI-MS/MS based testosterone quantification platform approx. Department of General and Social Psychiatry, Center of Psychiatry 15000 samples have been quantified within the past 18 months. and Psychotherapy, Department of Internal Medicine I, and Division of Multilevel calibration is based on materials measured against Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria ERM DA345a and DA346a. Assay accuracy is tested against the UK- NEQAS steroid hormone proficiency testing scheme (72 samples / Light falling on the retina is converted into an electrical signal year). Here the assay was found to be accurate (mean deviation which stimulates serotonin synthesis. Previous studies described an in the LCMS group < 1.3%) and precise (standard deviation of this increase of plasma and CNS serotonin levels after bright light expo- mean 8.8%). The quantification level was set to 0.10 ng/ml which sure. Ghrelin and leptin are peptide hormones which are involved allowed to measure trough almost the complete female reference in the regulation of hunger/satiety and are related to serotonin. range – an impossible undertaking if ligand binding assays are ­Neopterin and kynurenine are immunological markers which are utilized. also linked to serotonin biosynthesis. In this study, 29 healthy male 132 34th International Winter Workshop volunteers were exposed to bright (5000 lux) and dim (50 lux) light Macrophages and breast cancer: an iron-hard conditions for 120 minutes in a cross-over manner. Subjective well- relationship being and hunger as well as various serotonin associated plasma factors were assessed before and after light exposure. Subjective well- Tymoszuk P, Aßhoff M, Doppler W, Weiss G, Theurl I being showed a small increase under bright light and a small decrease Department of Internal Medicine VI, Innsbruck Medical University, under dim light, resulting in a significant interaction between light Innsbruck, Austria condition and time. Ghrelin concentrations increased significantly ([email protected]) under both light conditions, but there was no interaction between light and time. Correspondingly, leptin decreased significantly under Tumor-associated macrophages (TAMs) are the major population both light conditions. Hunger increased significantly with no light- of tumor infiltrating leukocytes in a plethora of human and animal time interaction. We also found a significant decrease of neopterin, malignancies whose presence strongly correlates with faster tumor tryptophan and tyrosine levels, but no interaction between light progression and metastatic dissemination. The mechanisms which and time. In conclusion, ambient light was affecting subjective well- account for the carcinogenic properties of TAMs are plenty; pro- being rather than serotonin associated biological factors. motion of angiogenesis, suppression of anti-tumor immunity and growth factor supply are best described. Clinical and animal studies on breast cancer provide evidence The influence of bioactive lipid mediators on choles- for crucial role of iron in growth of malignant cells and orchestra- terol metabolism in sepsis tion of tumor-promoting inflammation. In non-malignant tissues as well as in the whole organism macrophages perform the recycling Tancevski I, Auer K, Asshoff M, Bischof D, Hilbe R, Demetz E, of aged red blood cells and hence regulate iron supply for metaboli- Tymoszuk P, Weiss G cally active and proliferating cells. The analogical function of TAMs Medical University of Innsbruck, Department of Internal Medicine VI, as iron providers for malignant cells attracted, however, only little Infectious Diseases, Immunology, Innsbruck, Austria attention so far. Here, we demonstrate that TAMs present in murine ([email protected]) mammary cancer express a broad spectrum of iron turnover proteins and are the sole tumor cell population donating iron to neoplastic Sepsis is a systemic inflammatory condition following severe bac- epithelium. Furthermore, TAMs, in a close analogy to professional terial infection resulting in high mortality. Sepsis is a global health erythrophagocytes of the liver and spleen, ingest aged or deteriorated problem with an increasing incidence over the past years, account- red blood cells and hence contribute to tumor-promoting effects of ing for > 700,000 cases/year in the United States. During the past blood transfusions. years, much has been discovered about the uncontrolled, over- Summarizing, our findings shed new light on TAM biology by whelming inflammatory condition taking place in an organism stressing their function in iron turnover. This previously unrecog- affected by sepsis. However, one observation in septicaemic patients nized aspect has potentially broad clinical implications for the safety still remains enigmatic: why plasma levels of high-density lipoprotein of treatment of tumor-accompanying anemia. cholesterol (HDL-C) decrease, followed by a decrease in low-density lipoprotein cholesterol (LDL-C). We recently identified a conserved interplay between bioactive lipid mediators including leukotrienes (LTs) and lipoxins (LXs), and HDL-C and LDL-C metabolism [1]. Alkylglycerol monooxygenase in murine macrophages These lipid mediators are typically released by highly specialized white blood cells (neutrophils and monocytes/macrophages) during Watschinger K, Keller MA, McNeill E, Alam MT, Laid S, Sailer S, Rauch infection, thereby contributing to inflammation and resolution of V, Patel J, Hermetter A, Golderer G, Geley S, Werner-Felmayer G, Plumb infection. During the acute response of innate immunity to bacte- RS, Astarita G, Ralser M, Channon KM, Werner ER rial infection mainly neutrophils are attracted to sites of infection Divisions of Biological Chemistry and Molecular Pathophysiology, with the aim of bacterial killing. These activated neutrophils in turn Biocenter, Medical University of Innsbruck, Innsbruck, Austria; release cytokines, interleukins, and LTs to attract further neutro- Department of Biochemistry and Cambridge Systems Biology Centre, phils. After 12-24 hours, neutrophils themselves begin to release so- University of Cambridge, Cambridge, UK; Division of Cardiovascular called stop signals including LXs to slow down the chemoattraction Medicine, University of Oxford, Oxford, UK; Waters Corporation, of leukocytes, and to initiate resolution of infection which includes Milford, USA; Institute of Biochemistry, Graz University of Technology, activation of monocytes/macrophages. Our recent studies suggest Graz, Austria that sequential generation of these lipid mediators by different leu- ([email protected]) kocytes may lead to critical alterations of HDL- and LDL-receptor expression in the liver, explaining the typical changes in these lipo- Tetrahydrobiopterin-dependent alkylglycerol monooxygenase is proteins during sepsis. Here we show a systematic analysis of spati- essential for irreversible ether lipid cleavage. To investigate the otemporal generation of lipid mediators combined with changes in impact of alkylglycerol monooxygenase expression and tetrahydro- cholesterol metabolism in an animal model of Gram-negative sepsis. biopterin biosynthesis we analysed the lipidome of RAW264.7 with In summary, our studies will help to further understand the physi- modulated enzymatic activities by liquid chromatography/mass ology underlying the cholesterol-lowering mechanisms observed in spectrometry. We could identify more than 1000 lipid species, clus- sepsis, placing the proposed thesis at the forefront of immunome- tered them according to LipidMaps categories/classes/subclasses tabolism research. and performed enrichment analyses. Profound changes in differ- ent lipid species beyond the class of ether lipids were revealed and [1] Demetz E, et al. Cell Metab 2014;20:787-98. thus, our results demonstrate a central role of tetrahydrobiopterin 34th International Winter Workshop 133 and alkylglycerol monooxygenase in the ether lipid metabolism of life expectancy of currently about 10 years. Several enzymes in the murine macrophages. ether lipid metabolic pathway have not been characterized by their Supported by the Austrian Science Funds (FWF) [P22406, J3264] sequence yet, such as the delta-1 desaturase introducing the vinyl and by the autonomous province Bozen-Südtirol, division educational ether bond to form the plasmalogens. support, university and research. A well characterized biologically active ether lipid mediator is platelet activating factor, a pleiotropic inflammatory messenger. Effects of exogenously added submicromolar concentrations of ether lipids described in the literature include stimulation of mac- Ether lipids, metabolism and functional roles rophages, alteration of the cytokine profiles of T-lymphocytes, inhibi- tion of tumour growth and impact on phospho-signalling pathways, Werner ER, Golderer G, Werner-Felmayer G, Watschinger K e.g. by inhibition of protein kinase C. Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Austria ([email protected]) Urinary neopterin and prognosis of patients with Our interest in ether lipids has been initiated by the tetrahydrobi- gastrointestinal stromal tumors opterin-dependent irreversible degradation of these compounds by alkylglycerol monooxygenase. Alkylglycerols are lipids character- Zezulová M, Bartoušková M, Študentová H, Kujovská Krčmová L, ized by the attachment of a fatty alcohol by an ether linkage to the Solichová D, Melichar B glycerol backbone which is the basis of their designation as ether Palacký University Medical School and Teaching Hospital, Olomouc; lipids. The ether bond distinguishes them from the more common Charles University Medical School and Teaching Hospital, Hradec glycerol based lipids which have the fatty acid side chains attached Králové, Czech Republic by an ester bond. Ether lipids are as abundant and as diverse as their ([email protected]) ester counterparts, but the ether bond makes them chemically and metabolically more stable. A subclass of these ether-bound lipids Increased concentrations of neopterin in serum or urine have been has a double bond adjacent to the ether bond. These vinyl ethers reported in patients with tumors across the spectrum of different occur mostly as 1-alkenyl-2-acyl-sn-glycero-phospholipids, the plas- primary locations, but there are no reports on neopterin in patients malogens, which are the most abundant lipid species in mammalian with gastrointestinal stromal tumors (GIST). We have studied urinary brain. The vinyl ether bond makes these lipids sensitive to oxidation neopterin in 51 patients with metastatic GIST. Urinary neopterin was and acid cleavage. determined by high performance liquid chromatography Increased The biosynthesis of ether lipids is initiated by two key enzymes urinary neopterin concentrations were observed in a significant pro- present in peroxisomes. If one of these two biosynthetic enzymes is portion of patients with metastatic GIST. No statistically significant inactivated in mice this leads to cataract, male sterility due to the changes of urinary neopterin were observed during the therapy with lack of seminolipid, and to alterations in brain structure develop- imatinib, but a marked decrease was observed occasionally during ment. In humans, lack of the first two biosynthetic enzymes in ether the first days of imatinib treatment. Increased urinary neopterin lipid biosynthesis leads to severe inherited disease called rhizomelic did not predict poor survival. In conclusion, urinary neopterin is chondrodysplasia punctata types 2 and 3, respectively. These dis- increased in patients with metastatic GIST. No significant changes eases are associated with cataract, developmental disorders of bone, were observed in association with imatinib therapy. Increased joints and brain. This results in severe mental retardation and a urinary neopterin was not associated with inferior survival.