Abstracts S32

124 125 HLA ALLELES IN ISOLATED POPULATIONS FROM NORTH HLA-DPB1* POLYMORPHISMS IN GREEK POPULATION SPAIN: ORIGIN OF THE BASQUES AND THE ANCIENT IBERIANS. Katerina E. Tarassi, Diamanto I. Kouniaki, Theophilos I. Athanassiades, Chryssa A. Papasteriades. Pablo Sánchez-Velasco2, Eduardo Gómez-Casado1, Jorge Martínez-Laso1, - Histocompatibility Dept, Evangelismos Hospital, Juan Moscoso1, Jorge Zamora1, Ernesto Lowy1, Carlos Silvera-Redondo1, Athens, Greece. Alberto Cemborain2, Francisco Leyva-Cobián2, and Antonio Arnaiz-Villena1. 1Department of Immunology and Molecular , Hosp. 12 de Octubre, Since the number of HLA-DPB1 identified alleles is continuously increasing Universidad Complutense, Madrid, Spain. E-mail: [email protected]. and their clinical relevance, particularly in Bone Marrow Transplantation, is 2Department of Immunology. Hospital Universitario Marqués de established, the aim of this work is to present the HLA-DPB1 polymorphism in Valdecilla. 39008 Santander, Spain. Greeks. The population studied consisted of 140 unrelated, unselected healthy individuals of Greek origin. Sixty-eight (68) of them were typed for HLA-A, -B, -DRB1, -DQA1 and -DQB1 alleles have been studied in three DPB1*0101-1901 using oligonucleotide typing, whereas 72 were typed for all relatively isolated populations of northern Spain from Cantabria (Pas Valleys known DPB1 alleles by PCR-SSOP and/or PCR-SSP. inhabitants or Pasiegos and Cabuernigos) and from the Basque Country (Arratia The DPB1* alleles detected are presented in the following table: Valley inhabitants). These populations have been compared to other neighbouring ones and other Mediterraneans by using neighbor-joining DPB1 Freq % DPB1 Freq % DPB1 Freq % dendrograms, plane genetic distances, correspondence and HLA haplotype (N=140) (N=140) (N=72) analyses. These populations are more related each other than to Spaniards and 0101 7.1 1101 5.7 2001 1.4 also are genetically close to North Africans. Pasiegos show a slight degree of 0201 40.0 1301 5.0 3101 1.4 admixture with central and western Europeans. The most feasible origin of these 0301 7.1 1401 1.4 3301 1.4 populations is regarded as being a remnant of the primitive Spanish population 0401 58.6 1601 2.1 3501 2.8 (ancient Iberians) or otherwise of North African refugees that could have 0402 33.6 1701 3.6 3701 1.4 occupied the area before their expulsion by the Catholic Kings after 1492 AD, 0501 1.4 3901 1.4 when North Africans definitively were expelled. The first theory is more favoured since there are no records of a permanent Muslim invasion in these 0801 0.7 5001 1.4 areas during Middle Ages. 0901 0.7 5101 1.4 1001 4.3 6001 1.4 Conclusions: In comparison to the data from the 11th and 12th IHW 1) DPB1*0201, 0401, 0402 are the most frequent alleles in Greeks, as in other Caucasians, but DPB1*0201 shows the highest frequency among all populations studied, 2) DPB1*1301, characterizing Blacks and Orientals, is presented in Greeks with the highest frequency among Caucasians (except Gypsy Spanish). So, it seems that DPB1* alleles distribution in Greeks show certain peculiarities, as we have also found in other HLA loci studied.

Poster Group 9 - Immunogenetics of Disease 126 127 THE ORIGIN OF SOUTHERN SIBERIA TUVINIANS: A COORDINATED DOWNREGULATION OF APM COMPONENTS POPULATION WITH BOTH ORIENTAL AND CAUCASOID IN MHC CLASS I-DEFICIENT METASTATIC TUMOUR CHARACTERISTICS. VARIANTS IMMUNOSELECTED BY T LYMPHOCYTES

Jorge Martínez-Laso2, Marina Sartakova1, Luis Allende2, Marisol Martinez, Angel M. Garcia-Lora, Ignacio Algarra*, Eva Jimenez and Vladimir Konenkov1, Juan Moscoso2, Carlos Silvera-Redondo2, Federico Garrido Serv. Analisis Clinicos, Hospital Univ. Virgen de las Nieves, Arancha Pacho2, Jorge Zamora2, Ernesto Lowy2, Eduardo Granada, Spain. *Dep. Ciencias de la Salud, Univ. de Jaén, Spain Gómez-Casado2, and Antonio Arnaiz-Villena2 1 Laboratory of Immunogenetics, Institute of Clinical Immunology, 630091 Our previous studies with the GR9 tumour model had shown that the MHC class Novosibirsk, Russia I phenotype of the metastatic tumour variants differs depending on the immune 2 Department of Immunology and Molecular Biology, H. 12 de status of the host: MHC class I-negative in immunocompetent BALB/c mice Octubre, Universidad Complutense, Madrid, Spain. E-mail: and MHC class I-positive in immunodeficient (nu/nu) mice. The results showed that in this tumour model the major factor contributing to the appearance of [email protected]. MHC class I-negative tumour variants is T cell immunoselection. HLA class I and class II alleles have been studied for the first time in the To explore the molecular mechanisms involved in the origin of these MHC Turkish-speaking Tuvinian population, which lives in Russia, North of class I-deficient tumour variants in our fibrosarcoma model, we analyzed the Mongolia and close to the Altai mountains. Comparisons have been done with expression of different components of the MHC class I -processing about 11000 from other worldwide populations, and extended machinery (APM). haplotypes, genetic distances, neighbour joining dendrograms and The MHC class I stability assays at 26ºC in the presence of exogenous 2- correspondence analyses have been calculated. Tuvinians show an admixture of microglobulin showed a partially recovered the cell surface expression of H-2 Mongoloid and Caucasoid characters, the latter probably coming from the class I molecules. Semiquantitative RT-PCR of different APM components ancient Kyrgyz background or, less feasibly, more recent Russian Caucasoid showed the constitutive expression of TAP-1, TAP-2, LMP-2, LMP-7, LMP-10, admixture. However, Siberian population traits are not found and thus Tuvinians tapasin and calnexin was downregulated in the B9 tumour clone as well as in all are closer to Central Asian populations. Siberians are more related to Na-Dene metastatic nodes obtained from immunocompetent BALB/c mice. Interestingly, and Eskimo American Indians; Amerindians (from nowadays Iberian-America) this was not the case for the nu/nu BALB/c-derived metastases. In conclusion, a are not related to any other group, including Pacific Islanders, Siberians or other coordinate transcriptional downregulation of multiple components of the APM American Indians. The `more than one wave' model for the peopling of the is responsible for the absence of H-2 class I antigen surface expression in Americas is supported. metastatic nodes generated in immunocompetent BALB/c mice.

Genes and Abstracts S33

128 129 DIFFERENT IMMUNOGENICITY AND PATTERN OF LOCAL MOLECULAR MECHANISMS INVOLVED IN THE TOTAL GROWTH IN METASTATIC TUMOUR VARIANTS THAT LOSS OF HLA CLASS I IN COLORECTAL TUMORS: 2M ARE/ARE NOT IMMUNOSELECTED BY T LYMPHOCYTES INACTIVATION AND LMP7 DOWNREGULATION.

Marisol Martinez, Angel M. Garcia-Lora, Ignacio Algarra*, Eva Jimenez and Carmen M. Cabrera, Pilar Jiménez, Teresa Cabrera, Francisco Ruiz- Federico Garrido Servicio de Analisis Clinicos, Hospital Universitario Virgen de las Cabello, Federico Garrido. Servicio de Análisis Clínicos. Hospital Nieves, Granada, Spain. *Departamento de Ciencias de la Salud, Universitario Virgen de las Nieves. Granada. Spain. Universidad de Jaén, Jaén, Spain The mechanisms to produce loss of MHC class I expression, in different types of tumors are partially known. Accordingly, we have studied colorectal tumors We previously reported that metastatic cell lines obtained in immunocompetent to elucidate the specific mechanisms evading T-cell immunoresponse. BALB/c mice have a H-2 class I-negative phenotype similar to that of the We have selected tumors with total loss of MHC class I expression by inducing fibrosarcoma tumour cell clone from which they derived. In contrast studying 124 colorectal tumors with immunohistochemical staining with anti when immunodeficient (nu/nu) BALB/c mice were used to produce metastases, HLA monoclonal (mAb). 14 tumors out of 124 (11%) exibit a 100% of the metastasis-derived cell lines were H-2 class I-positive for Kd, Dd phenotype with HLA class I total loss. Microsatellite instability (MSI) analysis and Ld molecules. The results showed that in this tumour model the major was also carry out in the same tumor samples. The expression of β2- factor contributing to the appearance of MHC class I-negative tumour variants is microglobulin, HLA-A, B, and C, TAP1, TAP2, LMP2, and LMP7 were T cell immunoselection. analysed using RT-PCR in microdissected tumor samples. 4 out of 14 MSI To determine the biological behaviour of these metastatic tumour variants we positive (MSI+) and W6/32 mAb negative tumors, showed biallellic inactivation performed local growth and immunogenicity studies. Different metastatic cell of β2m and accumulation of HLA heavy chain in the cytoplasm. MSI negative lines were injected subcutaneously into the footpad in syngeneic (MSI-)/W6/32 mAb negative tumors presented alterations in the expression of immunocompetent BALB/c mice. components of the antigen processing machinery (APM). 9 out of 10 tumor The results showed a different pattern of local growth: H-2 positive metastatic samples showed downregulation of LMP7 gene, and 4 out 10 presented TAP2 cell lines derived from nu/nu mice grew slowly, reaching a maximum of 5 mm disregulation. This group apparently expressed normal levels of heavy chain and of diameter after 15 days, then regressing and been rejected after a short period 2m proteins. of time (25 days). The H-2 negative metastatic cells (derived from Two major mechanisms in colorectal cancer appear to be responsible for total immunocompetent mice) grew faster reaching 14 mm of diameter in 25 days, loss of MHC class I surface expression (β2-microglobuline and the local tumours of these mice did not regress until they were sacrified. LMP7 downregulation). In conclusion, the results indicate that the H-2 class I-positive metastatic tumour cell variants, produced in nude mice, are more immunogenic that their H-2 class I-negative counterpart derived from immunocompetent mice.

130 131 ADDITIONAL GENETIC SUSCEPTIBILITY OF TYPE 1 TOTAL LOSS OF HLA CLASS I ANTIGEN EXPRESSION ON DIABETES LIES IN COMBINATIONS OF LOCI RATHER THAN MELANOMA CELLS: A RESULT OF SUCCESSIVE IN SINGLE LOCI MUTATIONAL EVENTS.

Peter Eerligh, Bobby P.C. Koeleman, Bart O. Roep and Marius J. Giphart Méndez Rosa1 Paschen Annette2, Jiménez Pilar1, Sucker Antje2, Department of Immunohaematology and Blood Transfusion, Leiden Ruiz-Cabello Francisco1, Song M.2, Garrido Federico1 and University Medical Centre, Leiden, The Netherlands Schadendorf Dirk2. 1Servicio de 1Análisis Clínicos, Hospital After more than 25 years of research, significant association with Type 1 Universitario Virgen de las Nieves, Universidad de Granada, Diabetes (T1D) has been found for only two chromosomal regions (HLA Granada, Spain. 2Skin Cancer Unit (German Cancer Research DR/DQ and INS VNTR); this could be due to the fact that T1D is usually Center); University Hospital Mannheim; Theodor Kutzer Ufer; 68135 analysed as a single gene rather than a multi-gene disorder. Polymorphisms in Mannheim, Germany. the regulatory regions of several cytokines and metabolic important factors have been described extensively. Those with functional consequences for expression Alterations in the surface expression of HLA class I molecules have been provide a pool of candidate genes for a variety of different diseases. described as a strategy of tumors to evade recognition by cytotoxic T cells. We In this study we have selected (functional) polymorphisms of the IL-2, IL-12, detected complete loss of HLA class I antigen presentation for two tumor cell IFN-γ (T-helper-1 group), IL-4, IL-6, IL-10 (T-helper-2 group), IGF-I, VDR lines derived from one melanoma patient, the first originated from a regional and INS (metabolic/growth group) genes. These polymorphisms were typed in lymph node lesion diagnosed simultaneously with the primary tumor and the 206 Dutch simplex families with juvenile onset T1D and the results were second established 8 months later from a metastatic pleural effusion sample. analysed using the transmission disequilibrium test (TDT). Polymorphisms were Antigen presentation was not inducible with IFN-gamma but could be restored β β analysed both separate and combined in the predefined groups. after transfection of tumor cells with 2m cDNA, indicating a defect in 2m We found no single locus association for T1D except for INS and VDR. expression. Analysis of the nature of this defect revealed that it originated from However, when analysed together, the combination of T-helper-2 and at least 2 mutational events affecting both copies of the β2m gene: a metabolic/growth alleles Il-10*196, Il-4*C, VDR*C, IGF-I*192 was found to microdeletion of 498 bp in one β2m gene, including its entire exon 1, and a be transmitted more frequent than expected (67%, Pcorrected=0.0021). macrodeletion involving the entire copy of the second β2m gene. Microsatellite Remarkably, none of the markers from the Th-1 group seemed to contribute to analysis pointed to the macrodeletion by demonstrating LOH for several the disease, neither alone nor in combination with Th2 markers. specific markers on the long arm (q) of 15. FISH studies also We conclude that additional genetic predisposition to T1D will be found in indicated the coexistence of a structurally abnormal variant of chromosome 15q combinations of markers rather than in a single marker. with two apparently entire chromosomes 15q harboring the homozygous β2m microdeletion. Block of β2m expression in tumor cells builds a barrier to immunotherapy of cancer patients,. and its result should be of major considerations in the development and design of immunotherapeutic strategies.

Genes and Immunity Abstracts S34

132 133 MHC-EXTENDED HAPLOTYPES DEFINED BY SEGREGATION HLA HAPLOTYPE LOSS IN COLORECTAL CARCINOMAS ANALYSIS IN FAMILIES - PORTUGAL (ECRAF) Isabel Maleno, Carmen Cabrera, Antonia Collado*, Teresa Cabrera, Laura Paco, Antonio Ferrón*, Miguel López-Nevot, Federico Garrido. Helena Alves1,3, M. Fernandes2, C. Maia2, B. Lima1, J.M. Bernardes2, R. Serv. Análisis Clínicos & Unidad de Cirugía Experimental*. Hospital Gonçalves1, S. Tafulo1, F. Mendes1, M. Dias1, A . Pires1, A . Mendes1, A . Universitario Virgen de las Nieves. Granada. Spain.. Lopes Vaz2 , D. Charron4 for ECRAF (European Consortium for Rheumatoid Arthritis Families). 1North Histocompatibility Centre - Porto; 2 Rheumatology The importance of alterations in the expression of HLA class I in the Service - Hospital S. João - Porto; 3 Medical Genetic Service, Faculty of escape of tumors from immune surveillance is well stablished. One of the Medicine - Porto; 4 Hôpital Saint Louis, Paris, France. molecular mechanisms of these losses is HLA haplotype deletion. To stablish the frequency of HLA haplotype loss in colorectal carcinomas, 8 Introduction: The association of HLA- DR with Rheumatoid Arthritis (RA) STR markers from 6q and 6p21-3 were used with DNA obtained from 94 is well established. HLA-DQB has been also investigated. However, HLA- colorectal carcinomas. were obtained by microdissection of cryostatic DRB1*04, *01 or *10 do not account for the full MHC susceptibility nor sections previously analyzed with immunohistological techniques. In addition, HLA-DQB does explain it. HLA Class I genes have not been neither HLA genomic typing by sequence specific oligonucleotide analysis was done to adequately nor exhaustively looked at. RA association studies using allele define the HLA haplotype lost. frequencies did not show any significant differences between patients and 42 (44%) colorectal carcinomas had HLA haplotype loss (Phenotype II), of controls and the known haplotypes reports are computing deduced based. In these tumors in 27 samples the HLA haplotype could be defined. They showed this study we analysed extended defined (not deduced) haplotypes, using complete loss of one allele from at least 3 informative STRs markers, and also HLA-A,-C,-B,-DRB1 and –DQB1, as well as microsatellites polymorphisms lost of one HLA-ABC haplotype. In the other 15 cases a decrease of 25-50% in the short arm of chromosome 6, from groups of RA and control families. was detected on one allele of three informative STRs but by SSO the band pattern of A and B locus was normal. 8 cases (8%) showed microsatellite A group of 100 unrelated RA patients is also studied. instability (MSI). Material and methods: 142 RA patients. 42 from 27 RA families (94 In summary, out of 79% colorectal tumors with HLA class I alterations individuals, from Portugal – ECRAF) and a group of 100 unrelated patients. (showed by immunohistology or by microsatellite analysis) 44% cases showed 172 allele and haplotype RA matched control families from the North of LOH with HLA haplotype loss. Portugal. The HLA typing was assigned by DNA typing using PCR-SSP and PCR-Reverse hybridization methods. Microsatellites genotyping was performed using the 13th IHWG protocol. Chi square test and Linkage Disequilibrium were calculated. Results: We could define by segregation analysis 104 HLA- A, -C, -B, - DRB1, -DQB haplotypes in RA families and 109 HLA- A, -C, -B, -DRB1, - DQB haplotypes in control families. Comparing allele and haplotype frequencies between RA and control families we could find a different linkage disequilibrium (LD) between the most frequent DRB1 alleles found in RA and other loci. DRB1*04 / B*40 and DRB1*04 /B*49 association is related with RA susceptibility. DRB1*04 /B* 57, DRB1*04 /C*06 and DRB1*04 /C*15 are “protective”. Our findings point to a second susceptibility region in chromosome 6.

134 135 REDUCTION IN MRNA LEVELS OF 2-M AND HEAVY CHAIN A SHORT TANDEM REPEAT POLYMORPHISM IN THE AS THE MAJOR MECHANISM FOR HLA CLASS I TOTAL NEONATAL FC RECEPTOR -CHAIN GENE LOSS IN BLADDER TUMORS. Sachs U.J.H., Braeunlich C.G., Santoso S. José M. Romero¹, Pilar Jiménez¹, Teresa Cabrera¹, José M. Cozar², Institute for Clinical Immunology and Transfusion Medicine, Justus- Miguel Tallada², Rosa Méndez¹, Rosario Marin¹, Francisco Ruiz- Liebig-University, Giessen, Germany Cabello¹, Federico Garrido¹. ¹Serv. Análisis Clínicos, ²Serv. de Antibodies are transported from the maternal blood across the placenta to fetal Urología. H.U. Virgen de las Nieves, Granada, Spain. circulation, protecting the fetus and neonate against infectious diseases. On the other hand, this transport subjects the infant to the risk of diseases caused by Downregulation of HLA class I molecules is a frequent event observed in maternal alloantibodies, such as hemolytic disease of the newborn (HDN) and human tumors and it is believed to be related with tumor immune escape. In this neonatal alloimmune thrombocytopenia (NAIT). Until now, several studies context, we examined the mechanism responsible for the HLA class I total loss failed to show an association between severity of disease and titer or specificity in 11 bladder HLA negative tumors selected by immunohistochemistry. of the involved platelet- or red cell-specific alloantibodies. Thus, one might Mutations in 2-microglobulin ( 2-m) have been involved in HLA class I total speculate whether a variable capability of transport loss. In fact, we have previously described this alteration in colorectal across the placenta plays a role in this respect. Recently, the neonatal Fc carcinomas with microsatellite instability. However, in bladder tumors manual receptor (FcRn) has been isolated from human placenta and it`s ability to bind microdissection of tumor cells and subsequent cDNA sequence analysis did not and transport IgG through cells has been demonstrated. FcRn is a heterodimer reveal any mutations. In adittion, these tumors did not show microsatellite composed of heavy chain and 2 microglobulin. instability. Then, we analysed expression levels of 2-m and heavy chain genes To determine whether polymorphisms of this receptor could contribute to by quantitative real time PCR performed in a Light Cycler . To determine inter-individual variability, the promoter and entire coding regions of the FcRn downregulation of these genes we compared RNA levels in this negative tumors α-chain was sequenced. Analysis of 16 unrelated individuals did not differ from with 11 HLA positive tumors. We used GPDH as the reference gene. The gene the wildtype sequence. In contrast, analysis of the 5` untranslated gene revealed expression profiles showed reduced RNA levels of 2-m and heavy chain in the a region of short tandem repeats (STR) consisting of a 37 bp long motif which is negative tumors in comparison to the positive tumors. Statistical analysis repeated up to five times (37-185 bp). This region is located 306 bp upstream performed by Smirnoff-Kolmogorov test identified statistically significant from ATG. Genotyping of 427 healthy Caucasian blood donors showed five differences in 2-m (p=0.0001) and heavy chain (p=0.002) gene expression different alleles (STR A-E) with frequencies of 0.1, 7.5, 92, 0.2 and 0.2%, between HLA positive and negative tumors. respectively. STR A represents the allele with one, STR E with five repeats. The These data support a possible regulatory transcriptional mechanism underlying biological relevance of this polymorphism regarding the quantity of FcRn HLA class I total loss in bladder tumors. mRNA in monocytes from genotyped individuals is currently under investigation.

Genes and Immunity Abstracts S35

136 137 HLA-DRB1 ALLELES DEFINE PROTECTIVE, NEUTRAL OR INFLUENCE OF CYTOKINE GENE POLYMORPHISMS ON PREDISPOSING EFFECTS IN RHUMATOID ARTHRITIS. SUSCEPTIBILITY TO AND PROGNOSIS IN BREAST CANCER

Morgane Gibert 1,3, Nathalie Balandraud-Pieri 2,4, Pierre Mercier 1, Jean W. Martin Howell, Katherine C. Smith, Helen M. Fussell, Adrian C. Roudier 2,4, Denis Reviron 1,2 Bateman. H & I Laboratory and Departments of Cellular Pathology 1 E.F.S. “Alpes-Méditerranée”, Marseille, France 2 Immuno- and University Human , Southampton General Hospital, rheumatology, INSERM EMI 9940, Faculté de Médecine ; Marseille, Southampton, SO16 6YD, UK. France 3 UMR 6578 CNRS / Faculté de Médecine ; Marseille, France 4 Service de Rhumatologie, hôpital La Conception, Marseille, France. Single polymorphisms (SNPs) in the promoter regions of a number of cytokine genes are associated with differential levels of cytokine expression Aim. Because of recombination, DRB1 alleles may come to resemble at each in vitro. In accordance with our findings in other malignancies, we hypothesised other in area coding for peptide binding regions (PBR). PBR of HLA molecules that these SNPs might influence breast tumour development and progression by could be involved in predisposition. Using such functional affecting the efficiency of the anti-tumour immune response and/or pathways of categorisation based on PBR motifs, we examined RA association with 7 groups angiogenesis. of HLA-DRB1 alleles in a Southern French population. 144 female breast cancer patients and 263 cancer-free controls were Methods. DRB1 alleles were classified in 7 Restrictive Supertype Patterns genotyped for selected pro- and anti-inflammatory and angiogenic cytokine (RSPs) within pocket 4 of peptide binding groove. DRB1* RSPs frequencies SNPs, including IL-1 –511 (T/C), IL-6 –174 (G/C), TNF –308 (A/G), IL-10 – were then studied in 200 patients versus 200 controls. 1082 (A/G), IL-8 –251 (A/T) and VEGF –1154 (A/G), using ARMS-PCR and Results. RSP “DE” (“D” in 70 and “E” in 71) including TaqMan® 5’ nuclease assays for allelic discrimination. Patient-control DRB1*0103,*0402,*1102,*1103,*1301,*1302 and RSP “Q” (“Q” in 74) comparisons revealed that the TNF –308 GG genotype was increased in including DRB1*0701,*0703 had a significantly protective effect: OR 0.33 (pc patients v controls (79.7% v 68.2%, P=0.03, OR=1.83, 95% CI=1.08-3.09). < 0.01), and OR 0.40 (pc < 0.01) respectively. Stratification of the patient group according to the Nottingham Prognostic Index RSPs "E" (in 74), (DRB1*0403,*0407,*0901,*1401), "R" (in 74) revealed a number of significant associations, including IL-8 –251 AA and poor (DRB1*0301,*0302) and "a" (“A” in 71) (DRB1*1501,*1502) had a neutral prognosis disease (35.3% v 12.7%; P=0.02) and IL-6 -174 GC and Grade 2/3 v effect OR: 0.71, OR: 0.76, OR: 0.94 (p NS). RSP “A” containing the shared Grade 1 disease (51.3% v 31.4%; P=0.04). epitope alleles had a predisposing effect (OR 4.35, pc < 0.01). These results provide preliminary evidence that polymorphisms in the Conclusion. Beside the classical predisposing effect of DRB1 shared epitope promoter regions of cytokine genes may affect both susceptibility to and alleles, functional categorisation discriminate among DRB1* alleles, those that prognosis in breast cancer, although causality cannot be directly inferred. confer a protective effect or those that confer a neutral effect in RA. Confirmation of these findings is currently being sought in an expanded patient and control group, with full SNP exclusion for selected genes.

138 139 IL-10 POLYMORPHISMS IN PATIENTS AFFECTED BY ACUTE HLA–DR AND DQ ASSOCIATIONS WITH AUTOIMMUNE MYOCARDIAL INFARCTION. ADDISON’S DISEASE IN ITALIAN PATIENTS.

Domenico Lio, Letizia Scola, Antonio Crivello, Giuseppina Candore, M. Paola Albergoni,* M. Vittoria Gazzola*, Elisa Slanzi*, Carlo Giuseppina Colonna-Romano, Martina Chiappelli, Cecilia Tampieri, Federico Carcassi˚, Chiara Dal Prà^, Moscon Alessandro^, Corrado Betterle^. Licastro, Angelo Branzi, Marco Caruso, Enrico Hoffmann, Calogero Caruso. Gruppo di Studio sull’immunosenescenza, Dipartimento di Biopatologia e *Dept of Pediatrics University of Padova, ^Medical and Surgical Metodologie Biomediche dell’Università di Palermo, Corso Tukory 211, 90134 Sciences, University of Padova, ˚, University of Palermo. Cagliari, Italy For long time, hypercholesterolaemia has been claimed as be the most important Autoimmune Addison’s disease (AAD) is a rare (prevalence 100 per million in risk factor for atherogenesis. However, recently it has been appreciated that Europe) and chronic disorder of the adrenal gland. AAD may occur in isolation inflammatory mechanisms couple dyslipidaemia to atheroma formation and or as a manifestation of autoimmune polyendocrine syndrome (APS) type 1 or inflammatory processes play an important role for the progression of 2. APS1 is characterized by AAD, chronic candidiasis, hypoparathyroidism and atherosclerosis. Thus, atherosclerotic patients display a pro-inflammatory is caused by mutations of AIRE gene, APS2 is characterized by the association phenotype and the control of might play a protective role in of AAD with autoimmune tyroid disease and/or . atherosclerosis complications, including acute myocardial infarction (AMI). We studied 85 patients: 14 with APS1, 49 with APS2 and 22 with isolated Interleukin (IL)-10 is a multifunctional anti-inflammatory cytokine that has an AAD. HLA DRB1 and DQB1 typing was performed by PCR-SSP method. The essential role in regulation of local and systemic inflammatory processes. The frequencies were compared with those obtained from a group of healthy BM quantitative production of IL-10 is subject to genetic influence based on donors using the Fisher exact test. polymorphisms in the promoter of its gene. We tested the hypothesis that the IL- Results. No HLA correlation was found in the group of patients with APS1. In 10 genotype, by influencing the capacity to control systemic inflammation, may the group with APS2 the haplotypes DRB1*03-DQB1*02 and DRB1*04- play a role in AMI. One hundred sixty subjects (mean age 67) affected by AMI DQB1*03 resulted positively associated to AAD, independently of the presence and 213 controls, both from northern Italy, were genotyped for the polymorphic of diabetes. In contrast DRB1*01 and DRB1*13 correlated in a negative way. base at position -1082 of the IL-10 promoter by sequence specific probes. The - Moreover, we observed a significant high relative risk in the heterozygous 1082A allele, which is associated with low production of the cytokine IL-10, patients DRB1*03, *04 (RR=9.8). In the isolated form only the haplotype was significantly increased in AMI patients (57.5% vs. 31.9%, p = 1 x 10-7). To DRB1*03-DQB1*02 appeared positively correlated. validate these results we examined this IL-10 SNP in another cohort of patients Conclusions. This study, the first and the largest in the Italian population, (N = 57), younger (under 45 years) and from Sicily. Also in this cohort of confirms that AAD is associated with the haplotypes DRB1*03-DQB1*02 and patients the -1082A allele was significantly increased in AMI patients respect DRB1*04-DQB1*03, except when associated to APS1. to 212 controls (60.5% vs. 48.1%, p = 0.019). So, we may conclude that presence of –1082A allele, associated to a low production of anti-inflammatory cytokine IL-10, may be considered an independent genetic risk factor for AMI. It is noteworthy that the converse –1082G allele, associated to an increased production of IL-10, has been shown to significantly increase the possibility to reach the extreme limit of human lifespan in men. However, these data necessitate of further confirmations looking for the interaction with the well established AMI risk factors as genes regulating cholesterol levels.

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140 141 THE CD14 –260C-T PROMOTER POLYMORPHISM IN KIR GENES IN TYPE 1 DIABETES (T1D) AUSTRIAN PATIENTS Arno R. van der Slik, Bobby P.C. Koeleman, Willem Verduijn, Bart O. Roep Andreas Lutterotti1, Christoph Gassner2, Rainer Ehling1, Thomas and Marius J. Giphart. Berger1 and Markus Reindl1 Department of Immunohaematology and Blood Transfusion, Leiden 1 Department of Neurology, University of Innsbruck, Innsbruck, University Medical Center, Leiden, The Netherlands. Austria. 2 Central Institute of Blood Transfusion and Immunological Killer Cell Immunoglobulin-like Receptors (KIR) are membrane glycoproteins Department, General Hospital and University Clinics, Innsbruck, of the immunoglobulin (Ig) superfamily that modulates natural killer (NK) cell Austria. function upon recognition of HLA class I. Since KIRs have opposing effects on signal transduction pathways and effector function, it is conceivable that KIR Introduction: Multiple sclerosis (MS) is a chronic inflammatory demyelinating genes may play a role in immune regulation including the development of disease of the central nervous system of assumed autoimmune etiology. The autoimmune diseases, such as Type 1 Diabetes Mellitus (T1D). inflammatory infiltrates within the demyelinating lesions consist mainly of To determine a putative role of KIR genes in susceptibility to develop T1D, lymphocytes and mononuclear cells of the monocyte/macrophage system. we performed PCR-SSP genotyping on patients with juvenile onset diabetes Susceptibility to MS and the course of the disease are influenced by genetic and (n=149) and a random panel of the Dutch population (n=215). We typed for environmental factors. CD14 is a LPS binding receptor expressed on KIR genes responsible for inhibitory functions (2DL1, 2DL2, 2DL3, 2DL4, monocytes/macrophages and activated granulocytes. A promoter polymorphism 2DL5, 3DL1, 3DL2, 1D) and activating signals (2DS1, 2DS2, 2DS3, 2DS4, in the CD14 gene has been shown to influence the transcriptional activity of the 2DS5, 3DS1). All individuals were typed for HLA class I and HLA class II protein. genes. Materials and Methods: We examined the CD14 genotypes in 113 RR-MS and The results show no significant increase or decrease of overall KIR gene 54 SP-MS patients and 95 organ donors. The CD14 –260C-T promoter frequencies in the diabetic population as compared to the healthy controls. polymorphism was analyzed by PCR with sequence specific primers. Serum However, we observed differences in the distribution of KIR and their levels of soluble CD14 were measured with ELISA (IBL-Hamburg, Germany) corresponding HLA Class I ligand clusters between patients and controls. in 67 patients. Furthermore, the HLA associated high-risk group showed a small increase in Results: The CD14 –260C-T was not associated with MS when compared to activating KIR gene frequencies in patients as compared to controls. 95 organ donors. We found significant higher degree of clinical disability measured by the EDSS score in MS patients carrying the C allele. There was no significant difference in serum levels of soluble CD14 between patients carrying different genotypes. Conclusion: Our data indicate a negative influence of the C allele on the clinical disability in the course of MS. This polymorphism was not associated with higher serum levels of soluble CD14 in our patients. In conclusion we have evidence that a variation in a gene of the innate immune systems influences the clinical course of MS.

142 143 MOLECULAR BASIS OF THE GRANULOCYTE GLYCO- HLA CLASS I ALLELE ASSOCIATIONS WITH SECONDARY PROTEIN NB1 (CD177) INVOLVED IN THE PATHOGENESIS DENGUE INFECTIONS IN ETHNIC THAIS OF IMMUNE NEUTROPENIAS AND TRANSFUSION REACTIONS Klaythong R., Bangkok, Sirikong M., Bangkok, Vaughn D., Bangkok, Green S., Worcester; Endy T, Bangkok, Libraty D., Karin Kissel, Sentot Santoso, Christine Hofmann, Steffi Scheffler, Worcester, Innis B., Bangkok, Rothman A., Worcester; Ennis F., David Stroncek, Mohammed Kerowgan and Juergen Bux. Worcester, Chandanayingyong D., Bangkok, Stephens Henry, Institute for Clinical Immunology and Transfusion Medicine, London Giessen, Germany. The role of classical HLA-A and -B class I alleles in determining resistance, The human granulocyte glycoprotein NB1 is heterogeneously expressed on the susceptibility and severity of acute viral infections, are relatively unknown. neutropil plasma membrane of healthy individuals. Immunization to NB1 can Appropriate paradigms of acute viral infections, are dengue fever (DF) and cause alloimmune neonatale neutropenia, transfusion-related lung injury, dengue hemorrhagic fever (DHF). Both primary and secondary infections with immune neutropenia after bone marrow transplantation, drug induced immune dengue virus (DEN) serotypes 1 –2, -3 or -4, can result in either clinically less neutropenia and autoimmune neutropenia. However, the molecular nature of the severe DF or the more severe DHF. In secondary exposures, a memory response glycoprotein was still unknown. is induced in immunologically primed individuals, that can both clear the Therefore, we isolated NB1 glycoprotein from granulocyte lysate by infecting dengue virus and contribute to pathology. In a case control study of immunoaffinity chromatography. Next, we determined its NH2-terminal amino 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected acid sequence and designed NB1-specific primers. Performing rapid HLA class I associations with secondary infections, but not in immunologically amplification of cDNA ends (RACE) PCR and molecular cloning, a naive patients with primary infections (see Tissue Antigens 60:309-318, 2002). complementary DNA for NB1 was identified, which was confirmed by the HLA-A*0203 associated with the less severe DF, regardless of the secondary expression of recombinant NB1 glycoprotein in mammalian cells. A 1311 bp infecting virus serotype (Pc=0.012). By contrast, HLA-A*0207 associated with sequence was thought to represent the entire coding region for NB1which codes susceptibility to the more severe DHF, in patients with secondary DEN-1 and for 437 amino acids. Database searches revealed homology to Ly-6 (uPAR) DEN-2 infections only (P=0.0084). Conversely, HLA-B*51 associated with the domain suggesting that NB1 glycoprotein belongs to the uPAR/CD59/Ly-6 development of DHF in patients with secondary infections (Pc=0.021), and snake toxin superfamily. Analysis of the RNA from NB1 deficient individuals HLA-B*52 associated with DF in patients with secondary DEN-1 (P=0.046) suggest that the NB1-negative phenotype is the result of different off-frame and DEN-2 (Pc=0.0000096) infections. Moreover, HLA-B44, B62, B76 and insertions on RNA level, resulting in NB1 deficiency on granulocyte cell B77 also appeared to be protective against developing clinical disease after surface. secondary dengue virus infection (P=0.005-0.02). These results confirm that These results provide the basis for understanding the biological role of NB1. classical HLA class I alleles associate with the clinical outcome of exposure to Additionally, it may allow typing of NB1 antigen on molecular level in future. dengue virus, in previously exposed and immunologically primed individuals.

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144 145 RAGE GENE POLYMORPHISM AND JUVENILE IDDM INSULIN-LIKE GROWTH FACTOR 1 IS NOT ASSOCIATED WITH TYPE 1 DIABETES IN NORWAY Ilaria Campo, Michele Zorzetto*, Valeria Bozzi, Nicoletta Carlo- Stella, Miryam Martinetti*, Laura Salvaneschi*, Mariaclara Cuccia. Benedicte A. Lie, Hanne E. Akselsen, Erik Thorsby, Dag E. Undlien Dip. Genetica e Microbiologia, Università di Pavia, Italy; *IRCCS Institute of Immunology, Rikshospitalet University Hospital, Policlinico S. Matteo, Pavia, Italy University of Oslo, Oslo, Norway. The IDDM (Insulin Dependent Diabetes Mellitus) Component of 13th Type 1 diabetes (T1D) is a complex disease. Several factors, both genetic and International Histocompatibility Workshop found evidence that polymorphic environmental, determine the disease susceptibility. The major genetic loci in the HLA class III (TNFd, D6S273) contribute to IDDM susceptibility determinant resides in the HLA complex, however, numerous other unidentified independently of HLA-DR and -DQ. For this reason we investigated the genes contribute to the disease risk. polymorphism of RAGE gene (Receptor of Advanced Glycation End products) Insulin-like Growth Factor-1 (IGF-1) is involved in glucose homeostasis. The located close to this putative region. In particular it is known that in type 1 effects of IGF-1 and insulin are complementary. Insulin stimulates secretion of diabetes, the accumulation of AGE results in an increase in the severity of the IGF-1, and IGF-1 suppresses insulin secretion. IGF-1 occurs at reduced complications associated with the disease. The RAGE gene sequence is rather circulating levels in individuals with T1D. This is probably a consequence of polymorphic; in particular it has been shown that RAGE Gly82Ser dimorphism insulin deficiency. In addition, IGF-1 has been shown to stimulate proliferation (G/A 557) is associated to peculiar HLA class II haplotypes in type 1 diabetes. of T lymphocytes. Finally, a microsatellite upstream in the promoter of IGF-1 Moreover it is known that RAGE expression increases when has been found by others to be associated with circulating levels of IGF-1, type diabetes intervenes, and the upregulation of the receptor could be 2 diabetes and body weight. In this study, we evaluated the IGF-1 gene as a candidate gene for T1D, by sustained over the years. With that in mind we have considered the genotyping the promoter microsatellite in 527 Norwegian simplex families. The RAGE promoter that contains two NF-kB sites, along with an transmission of microsatellite alleles to children with T1D was tested using interferon- response element and nuclear factor interleukin 6 transmission disequilibrium tests (ETDT and TDT). The overall transmission DNA binding motif. These regulatory elements could have an was not significantly distorted (ETDT, p=0.5). Likewise, none of the individual important role in ligand-associated upregulation of the gene. alleles showed a significant distortion from the expected 50% transmission (pnc>0.2). Taken together, there is no indication that the microsatellite upstream We have investigated if a transition T/A upstream of the start codon could be a of IGF-1 is associated with T1D in Norway. genetic factor implicated in juvenile IDDM. To perform our research we have analyzed and HLA typed 213 healthy blood donors, and 70 children affected by IDDM and HLA typed. Our results show the following genotype frequencies: 23% AA, 46% AT, 31% TT in the control group and 15% AA, 33% AT, 52% TT in IDDM patients, with TT genotype being significantly more frequent in this group (P=0,008).

146 147 RAGE ASSOCIATION WITH CORONARY ARTERY DISEASE CYTOKINE GENE POLYMORPHISMS ON A PORTUGUESE RHEUMATOID ARTHRITIS POPULATION Ilaria Campo, Colomba Falcone˚, Michele Zorzetto*, Ilaria Sbarsi, Nicoletta Carlo-Stella, Maria Paola Buzzi˚, Laura Salvaneschi*, Helena Alves1, C. Maia2, R. Gonçalves1, M. Fernandes2, C.Vaz2, F. Mendes1,Aroso Dias2, S. Tafulo1, M. Dias1, A. Pires1, B. Lima1, A . Mendes1, Mariaclara Cuccia. A. Lopes Vaz2, D. Charron3, for ECRAF. Dip. Genetica e Microbiologia, ˚Dip. Cardiologia, Università di 1North Histocompatibility Centre, Porto, Portugal , 2 Rheumatology Pavia;*IRCCS Policlinico S. Matteo, Pavia, Italy. Department - Hospital S. João, Porto, Portugal, 3 Hôpital Saint RAGE (Receptor for Advanced Glycation End products) is a 35 kDa Louis, Paris, France. polypeptide of the Ig superfamily, coded for by a gene located on the short arm Introduction: Rheumatoid Arthritis (RA) is a chronic autoimmune disease which of chromosome 6, in the HLA class III region near the junction with class II. In complex inflammatory processes within the joints contribute to erosion and our study we have investigated if a T/A transition in the promoter region of the articular destruction. Extra articular features such as rheumatoid nodules, RAGE gene could be implicated in the development of coronary artery disease. vasculitis and scleritis are common. Both pro-inflammatory and anti- The accumulation of advanced glycation end products, a heterogenous group inflammatory cytokines can be found in synovial fluid and tissue of RA patients. of compounds, leads to a chronic inflammation present in atherosclerosis and in There is chronic activation and high levels of inflammatory cytokines such as microvascular lesions. These can be found in numerous organic diseases such as TNF-alfa, IL-6 and IFN-gama causing persistent inflammatory changes. Anti- ischaemic heart disease, diabetes mellitus and paraphysiological complications inflammatory cytokines, such as IL-10 are inhibitors and regulators. Higher or of aging (cerebral ischemic disease). In particular, it is now established that lower production of cytokines are associated with allelic variants of cytokine coronary artery disease (CAD) is a result of localized atherosclerosis and its genes. Polymorphisms on the regulatory regions of the genes for the TNF-alfa, inflammatory consequences. AGEs modified molecules, in the blood vessel TGF-beta, IL-6, IL-10 and IFN-gama have functional significance and could be wall, exert their cellular effects via engagement of the surface receptor RAGE. involved in the susceptibility to or the severity of autoimmune diseases and Our approach was to analyse 102 patients affected by CAD and 213 healthy infectious diseases. These Single Nucleotide Polymorphisms (SNPs), are related blood donors, also HLA typed. Our results show the following genotype with high and low cytokine levels production and with control production in frequencies: 12.8% AA, 50% AT, 38.3% TT in CAD patients and 23% AA, specific cells (lymphocytes, monocytes, macrophages). Onset age, disease 46% AT, 31% TT in the control group. The A allele frequency is 37% and 46% severity, family extended haplotypes and linkage disequilibrium (LD) with respectively. These data demonstrate that the AA genotype (P=0.032) and the A HLA alleles must be evaluated with these new markers. allele (P=0.043) are significantly less frequent in CAD patients than in the Material and Methods: cytokine genotyping was performed in 80 unrelated control group. We can hypothesized a change in gene expression, pivotal for the RA patients using PCR-SSP method (One Lambda, Inc). HLA-A, B, C, DRB1 functionality of this receptor. and DQB1 typing was done by PCR-SSP(Pel-Freeze) and Dot Blot Hybridization (Abbott). Cytokine gene frequencies were compared with Caucasian control populations previously reported. Results: The frequencies are: TNF-alfa: G/G(low)=76%, G/A(high) =24% (p=ns). TGF-beta1: a) codon 10: CC=12.5% (p=ns), TC=42.9% (p=0.05, OR= 0.54, CI: 0.27-1.08), TT=44.6% (p=0.01, OR= 2.22, CI: 1.09-4.54). b) codon 25: GG=96.4% % (p=0.02, OR= 4.89, CI: 1.04-31.56), GC=3.6% (p=0.02, OR= 0.2, CI: 0.03-0.96), and CC=0% (p=ns). IL-6: G/G (high)= 32% (p=0.04, OR= 0.54, CI: 0.28-1.02), G/C(high)= 54.7% (p=0.06, OR= 1.73, CI: 0.93-3.21), C/C

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148 (low)=13.3% (p=ns). IFN-gama: high= 16.1%, int=58.1%, low=25.8% (all MOLECULAR MODEL FOR HLA CLASS II ASSOCIATIONS IN p=ns). IL-10: high=19%, int=53%, low=28%, GCC/ATA (int.) = 30.8% COMMON AUTOIMMUNE DISEASES. (p=0.05 OR=1.81, CI: 0.95 – 3.45). There was neither association with onset age nor with DR. To TNF-alfa (high )/DQB*0201:LD=5.6. Milena Ivanova, Snezina Michailova, Anastasia Michailova, Discussion: In RA patients there is not higher frequencies of high producers of TNF-alfa. The IL-6 and IL-10 genotypes differences are borderline and need Antoaneta Nedialkova, Elissaveta Naumova. Central Laboratory of confirmation. TGF-Beta1 high producer genotypes in both codons present Clinical Immunology, University Hospital Alexandrovska, Sofia, significant differences and clearly are risk values to develop and/or maintain Bulgaria. the disease. The found TNF- alfa HLA LD seems to be ancestral haplotype related. Disease severity and answer to anti-TNF-alfa antibody needs further Our study on IDDM, MS, JCA, pemphigus vulgaris and SLE showed that investigation. common haplotypes confer predisposition or protection in the Bulgarian population. In order to explain these associations we compared amino acid sequences in the hipervariable regions of molecules encoded by protective and predisposing DRB1 and DQB1 alleles. The -chains coded by predisposing DRB1*0301, 0401, 0402, 0404 alleles have similarities in HVRIII: a motif 57D58A in pocket 9 and an electric charge of pocket 4, generated by residues 70-74. Similar characteristics are observed for molecules coded by other predisposing in our population DRB1 alleles: 1401 and 1404 (pemphigus vulgaris); 1501 and 1001 (MS); 0701 (SLE). The protective DRB1*1104 codes a molecule with a neutral charge of pocket 4 and absence of 57D58A in pocket 9. Predisposing DQB1*0302 and 02 alleles encode a common motif 13G,26L,57A. Part of this motif (13G,26L) is shared by other predisposing for our population alleles: DQB1*0602 (MS), 0604 and 0303 (SLE). The -chain, encoded by the protective DQB1*0301 is characterized by different, hydrophobic amino acids –13A,26Y, and similarly to DQB1*0503, other protective for IDDM allele in Bulgarians, has D at position 57. In conclusion, based on these results we suggest that more than one amino acid regions in - chains of DR and DQ molecules, predicted to be involved in processed antigen binding and helical regions, are important for disease predisposition and protection. The approach could be useful to evaluate further the role of HLA molecules in the development of autoimmune diseases.

149 150 PATIENT AND DONOR HSP70-HOM GENE POLYMORPHISM THE ROLE OF D6S265, D6S273, AND MIB POLYMORPHISMS INFLUENCES THE OUTCOME OF ALLOGENEIC HSCT IN DEVELOPEMENT OF IDDM

Katarzyna Bogunia-Kubik and Andrzej Lange. L. Hirszfeld Institute Katarina Štingl1, Zorana Grubi 1, Renata unec1, Esma e uk-Jeli i 1, Ana of Immunology and Experimental Therapy, Polish Academy of Radica2, Miroslav Dumi 2, Vesna Kerhin-Brklja i 1 Sciences, Wroclaw, Poland 1National Referral Organ Transplantation and Tissue Typing Centre, 2Clinic of Paediatric, University Hospital Zagreb, Zagreb, CROATIA In the present study HSP70-hom polymorphism was analysed in 105 recipients Recent investigations have indicated the existence of non-HLA loci associated of HLA matched sibling and alternative donor transplants and in 50 alternative with IDDM in addition to HLA class II genes. donors in relation to DRB1 alleles and transplantation outcome. Dimorphism One hundred patients selected on the basis of their HLA class II alleles, where within the HSP70-hom gene +2763 (G/A) was analysed with the use of ARMS classified in 4 groups (DR3/DR4, DR3, DR4, non DR3/DR4). Control group technique (Jenkins et al. 2000). Distribution of HSP alleles were similar in consisted of 150 healthy individuals. PCR-SSP method was performed for HLA patients and donors. DRB1*03 was more frequently detected in GG-positive class II typing, while 3 microsatellites (D6S265, D6S273, and MIB) were individuals than those having A allele (15/47 vs 10/58, p=0.06 and 11/20 vs analysed using automated ALF express sequencer. 6/30, p=0.012 for patients and donors, respectively). Conversely, DRB1*11/12 Frequency of D6S273 126bp (p=0.02565), 138bp (p=0.01967) and 140bp was rarer in HSP-GG+ donors (1/20 vs 10/30, p=0.018) and patients carrying G (p=0.00035) were significantly higher among patients, while the frequency of allele (5/75 vs 6/30, p=0.05). Toxic complications were significantly affected D6S273 130bp was significantly lower. Analysis of MIB alleles in IDDM with the HSP genotype of the recipient. Patients having HSP-AA more revealed an increased frequency of alleles 348bp (p=0.01302), 350bp frequently developed grade II-IV toxic complications than patients with HSP-G (p=0.00677) and 352bp (p=0.02289), while allele 334bp (p=0.00105) was allele (0.91 vs 0.56, p=0.02). In variance, the donor genotype influenced more decreased. There was no difference between patients and controls at D6S265. significantly aGvHD than toxicity. Patients transplanted from DRB1*03 Comparison of IDDM patients positive for DRB1*0301 and/or *04 with non positive donors with HSP-GG more frequently developed aGvHD and toxic DRB1*03/*04 patients revealed a significantly higher frequency of D6S273 complications than those grafted from donors lacking this DRB1*03/HSP-GG 132bp allele (p=0.00283) among DRB1*0301/*04 negative patients. This genotype (1.00 vs 0.44, p=0.035 and 0.75 vs 0.22, p=0.05 for grade > I aGvHD suggests that all other D6S273 and MIB alleles showed association with IDDM and grade III-IV toxic complications, respectively). HSP70-hom polymorphic because of linkage disequilibrium with DRB1 alleles. When non DRB1*03/*04 features of patients and donors of allogeneic HSCT preferentially influence the group was compared with matched controls, difference in frequency of 132bp development of toxic and aGvHD complications, respectively. allele was also significant (p=0.03986). Our results lead to conclusion that allele 132bp has an independent role in development of IDDM.

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151 152 PECULIAR HLA POLYMORPHISM IN ITALIAN PATIENTS HLA–DR,DQ HAPLOTYPES ASSOCIATED WITH DIFFERENT WITH PRIMARY BILIARY CIRRHOSIS MANIFESTATIONS OF PRIMARY SJÖGREN’S SYNDROME F. MARTINOVA*, V. POPOVA*, M. PANCHOVSKA** Pietro Invernizzi1, Ilaria Bianchi1, Francesca Poli2, Alejandro *Dept. Blood Transfusion and Immunogenetics, Hospital “Pirogov”, Sofia, Espadas De Arias2, Sara Frison2, Elena Longhi2. 1Department of Bulgaria; ** Clinic of Rheumatology, Medical University, Plovdiv, Bulgaria Medicine, San Paolo Hospital Medical School, University of Milan, Milan, Italy; 2Transplantation Immunology and Blood Transfusion Primary Sjögren’s Syndrome (pSS) is a chronic autoimmune exocrinopathy characterized by lymphocytic infiltration into lacrimal and salivary glands. Service, I.R.C.C.S., Ospedale Maggiore Policlinico, Milan, Italy. The extraglandular manifestations include nonerosive arthritis, Raynaud’s Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown phenomenon, vasculitis, neuropathy, lymphadenopathy, lymphoma. Primary etiology that has heterogeneous clinical features and outcome. Data concerning Sjögren’s syndrome is frequently associated with other autoimmune diseases HLA polymorphisms in PBC come from a limited number of geographical (thyroid diseases and systemic lupus erythematosus) with autoantibodies areas. The aim of the present study was to investigate whether HLA polymorphisms contribute toward disease susceptibility. With this purpose, we production: anti-SS-A(anti-Ro), anti-SS-B(anti-La), antinuclear antibodies. compared 186 well defined Italian PBC patients with 58 healthy subjects The aim of our study was to investigate the role of HLA-DR, DQ matched by age, gender and geographical area. Patients and controls were HLA haplotypes for genetic predisposition and association with different typed at low resolution by PCR-sequence specific oligonucleotides for the loci manifestation of primary Sjögren’s syndrome in Bulgarian patients. Fifty A and B; DRB1 alleles were typed by reverse line blot assay. patients with pSS and 100 healthy Bulgarian unrelated individuals were We found that HLA-DRB1*11 was associated with a markedly reduced risk included in the study. Several types of linkage analysis between pSS and of developing PBC (OR: 0.3; 95%CI:0.2-0.5). Differently from what observed HLA-DR, DQ haplotypes were performed. in a series of published studies, no association was found with HLA-DRB1*08. In comparison to our healthy control we found: 1) increased frequency of B*15 (2.5; 1.3-4.6), B*41 (12.0; 2.7-72.1), B*55 (2.9; 1.1-7.5) and B*58 (6.8; the predicted haplotype HLA-DR3, DR52, DQ2 (p<0.001) in total group of 1.1-46.3) were more frequent in PBC. The frequency of HLA polymorphisms patients with pSS, in patients with autoantibodies (anti-Ro and anti-ssDNA), was similar in PBC patients with progressive or non-progressive disease, and in in patients with thyroid dysfunction and/or Raynaud’s phenomenon and in those with or without anti-mitochondrial antibodies. Our data on a large series patients with severe systemic course of pSS; 2) increased frequency of the of Italian patients suggest that PBC may have a peculiar genetic background in haplotype HLA-DR2, DR51, DQ1 in total group of patients with the mediterranean area. pSS(p<0.01) and in patients with anti-dsDNA autoantibodies (p<0.001); 3) increased frequency of the haplotype HLA-DR1, DR51, DQ1 (p<0.01); 4) decreased frequency of the haplotype HLA-DR5, DR53, DQ3 (p<0.001); 5) increased frequency of the HLA-DR2-DR3 (p<0.01) and HLA-DQ1-DQ2 (p<0.001) heterozygote haplotypes. Our data suggest a role of the HLA-DR3, DR52, DQ2 haplotype and HLA- DR2-DR3 and HLA-DQ1-DQ2 heterozygote haplotypes in genetic predisposition to the disease pSS in Bulgarian population. Moreover, the frequency of haplotype HLA-DR3, DR52, DQ2 is higher in comparison with Caucasoid patients with pSS. The HLA-DR3, DR52, DQ2 was also found to be associated with autoantibody production and severe systemic manifestation of disease. The haplotype HLA-DR5, DR53, DQ3 probably is protective.

153 154 THE COMMON C-159T POLYMORPHISM OF THE VARIABILITY OF HLA-B27 ALLELES IN GERMAN PATIENTS LIPOPOLYSACCHARIDE RECEPTOR CD14 GEN IS NOT SUSPETED ANKYLOSING SPONDYLITIS ASSOCIATED WITH IGE LEVELS IN HEALTHY BLOOD DONORS Woelpl Alois,Kastl F.,Simon A.,Stauber D.,Lenggeler I.,Klein H. Labor für Medizinische Genetik, Martinsried Kathrin Hasemann, Anette Bohnert, Gregor Bein. Institute of Clinical The distribution of B*27 alleles (B*2701-2725) was characterized on 1719 Immunology and Transfusion Medicine, University of Giessen, serologically typed patients living in Germany suspected ankylosis spondylitis Germany or related spondyloarthropathies. The allelic typing was done by PCR-SSP high resolution. Testing with DNA methods 127 (7,3%) samples were B*27 negative. Atopy is a complex immune disorder characterized by heightened IgE levels, As documented in the following table 10 different B*27 alleles were identified. and it leads to asthma, eczema and rhinitis. The chromosomal region 5q31 consistently shows linkage to asthma and related phenotypes. This region HLA-B27 NumberGenotype- contains the cytokine gene cluster and other candidate genes like CD14. An Subtype of Pts frequencies association of the C-159T polymorphism in the 5’ flanking region of the CD14 B*2702 233 0.0760 gene with circulating soluble CD14 (sCD14) levels and with total serum IgE in B*2703 1 0.0003 atopic children was reported by Baldini et al. (1999, Am J Respir Cell Mol Biol, B*2704 4 0.0013 20, 976-983). B*2705 1330 0.5937 We genotyped the C-159T polymorphism in 888 healthy blood donors after B*2707 7 0.0022 elucidation of the haplotype structure of the CD14 gene. All individuals were B*2709 5 0.0016 analyzed for serum IgE levels and in a subgroup serum sCD14 levels were B*2710 3 0.0010 determined. B*2712 1 0.0003 We confirmed the association of C-159T polymorphism with the height of B*2713 1 0.0003 serum sCD14 levels in a gene dose dependent manner. No significant B*2714 8 0.0025 association of genotype and total serum IgE levels was observed in the whole group or after stratification for age, sex and season of blood sampling. The genotype frequencies of the most common alleles in Germany B*2702 The CD14 C-159T polymorphism is associated with the height of serum and B*2705 were comparable with the data published. 19 patients (1%) with sCD14 levels which may also control LPS sensitivity of immune cells. low frequency alleles were German origin, 11 coming from Turky or oriental However, this does not influence total serum IgE levels in normal donors. countries. To confirm the results the rare subtypes are being sequenced.

Genes and Immunity Abstracts S40 155 156 TUMOUR NECROSIS FACTOR-ALPHA (TNF-α) GENE INFLUENCE OF PATIENT AND DONOR GENOTYPES ON POLYMORPHISMS AND NARCOLEPSY RENAL ALLOGRAFT IMMUNE RESPONSE.

Cheryl M. O'Rourke1, Craig J. Taylor 1, John M. Shneerson 2 and Reyna S. Angelica Canossi, Anna Aureli, Antonina Piazza, Daniela Piancatelli, Goodman.1 Marilena Di Rocco, Giuseppina Ozzella, Tiziana Del Beato, Elvira Addenbrooke's NHS Trust1, Papworth NHS Trust2, Cambridge,UK Poggi, Gabriella Liberatore, Carlo Umberto Casciani and Domenico Introduction: Narcolepsy is a disorder characterised by recurrent daytime sleep Adorno. Istituto C.N.R. Trapianti D’Organo e l’Immunocitologia, episodes and cataplexy, and exhibits a strong association with HLA- L'Aquila and Rome Sections, Italy. DRB1*1501 and DQB1*0602. In addition, elevated levels of tumour necrosis factor alpha (TNF-α) have been observed in patients with narcolepsy and TNF- This analysis sought to investigate the gene polymorphism of TNF-α, IL-6, α promoter gene polymorphisms encoded within the HLA region have been TGF-β, IL-10 and IFN-γ in 58 cadaveric renal transplant donor-recipient pairs, correlated with disease susceptibility. In particular, the TNF-α -857 ‘T’ allele is in order to evaluate the impact of some genotypes on the incidence and severity reported to be significantly increased in patients with narcolepsy compared to of acute rejection (AR) and the donor-specific antibody (DS-Ab) production healthy controls. after renal transplantation. Transplant recipients were divided into rejectors Methods: We have investigated three polymorphisms (-308, -857 and -863) (ARpos, n=15) and non rejectors (ARneg, n=43), anti-HLA DS-Ab positive within the promoter region of the TNF-α gene in 75 patients with narcolepsy, (n=15) and negative (n=43) groups. A PCR-SSP method was performed for the α 104 random controls and an additional 25 HLA-DRB1*15/DQB1*0602 positive analysis of the polymorphism in the TNF- (-308, G/A), IL-6 (-174, C/G), β controls. Control and patient TNF-α, HLA-DRB and -DQB genotypes were TGF- (codons 10, 25 in exon 1), IL-10 (-1082,-819, -592 in promoter) and IFN- compared. γ(+874 in intron1). DS-Abs monitoring was carried out using a flow cytometric Results: Of 75 patients with narcolepsy, 68 (91%) were HLA-DRB1*15 and technique. As for recipient cytokines, we evidence an increased percentage of 72 (96%) were HLA-DQB1*0602, compared to 21 (20%) and 22 (21%) of 104 IL-10 high-intermediate genotypes in Abpos (80.0%, p=0.064) and ARpos random controls respectively (P<0.0001). The TNF-α -857 ‘T’ allele was (73.0%) compared to ARneg/Abneg (50.0%) group. IL-10 high/int producers present in 12 of 75 (16%) patients with narcolepsy compared to 15 of 104 (14%) also showed a high incidence of chronic rejection (100%). As for donors, we random controls and 8 of 46 (17%) HLA-DRB1*15 controls. No correlation observed an increment of TNF-α high producers in ARpos (33.0%) compared to independent of linkage with HLA-DRB1*15 was observed between the three ARneg/Abneg group (17.0%). In addition, we noticed an unexpected result for TNF-α polymorphisms and susceptibility to narcolepsy. donor IFN-γ high genotype, which was significantly decreased in ARpos (0%, Conclusion: The results of this study do not support the findings of previous p=0.0058), Abpos (7.0%, p=0.032) and ARpos/Abpos (0%, p=0.031) compared studies indicating a link between TNF-α promoter gene polymorphism and to ARneg/Abneg group (36.0%). These data may be explained either from a narcolepsy. recipient suppressive effect on donor cytokines production or from mechanisms of cytokine network acting in allograft.

157 158 PML STAINING PATTERNS IN COLORECTAL TUMORS WITH THE ASSOCIATION OF SCE FREQUENCIES WITH HLA-B51 TOTAL LOSS OF HLA CLASS I. POSITIVITY IN BEHCET’S DISEASE

Carmen M. Cabrera, Teresa Cabrera, Pilar Jiménez, Federico Garrido, Mevlit IKBALa, Ibrahim PIRIMa, Saliha KARATAYb, M. Teoman ERDEMc, Francisco Ruiz-Cabello. Servicio de Análisis Clínicos, Hospital Tulay TOSa, Naci EZ RM Kd. Ataturk University, Medical Faculty, Departments of Medical Universitario Virgen de las Nieves. Granada. Spain. Geneticsa, Physical Medicine and Rehabilitationb, Dermatologyc, The RING-finger promyelocytic leukemia (PML) protein is the product of the and Orthopedicsd 25240 - Erzurum, Turkey. PML gene that fuses with the retinoic acid receptor- gene (RARα) in acute promyelocytic leukaemia (APL) produced disruption of PML bodies. Wild-type The analysis of sister chromatid exchange is a cytogenetic technique used to PML localizes in the nucleus with a typical speckled pattern. PML bodies show DNA damage due to an exchange of DNA fragments between sister accumulate several proteins involved in multiple cellular pathways as apoptosis, chromatids. We investigated if HLA-B51 positive patients with Behcet’s disease transcriptional regulation and proteasomal degradation of ubiquitined proteins. (BD) were associated with higher SCE frequencies than patient without HLA- The ubiquitin-proteasome pathway at PML bodies is dependent of proteasome B51. In this study, lymphocytes from 62 patients (35 females, 27 males) and components recruitment. lymphocytes were cultured in darkness for 72 hours in culture BrdU added. Components of antigen processing machinery as TAPs and LMPs are Metaphase chromosomes were stained with a fluorescence plus Giemsa frequently downregulated in different tumor tissues that present MHC class I technique after a standard harvest procedure. For HLA-B51 typing, DNA is expression impaired. We recently, have documented LMP7 downregulation in extracted from EDTA blood samples and HLA-B5 allele genotyping was colorectal tumors with total loss of MHC class I antigens. The performed by the PCR-SSP method. 24 out of 62 patients with BD were immunohistochemical study of PML protein with PG-M3 genotyped with HLA-B51 . Furthermore, the SCE frequencies in HLA-B51 in these tumors revealed a disrupted pattern of PML bodies in a nuclear diffuse positive patients were higher than HLA-B51 negative ones (p<0.001). The study form similar to observed in APL cells. results presented that there is a significant association between elevated SCE Therefore, the disruption of the PML bodies was clearly associated with MHC frequencies and HLA-B51 than that of HLA-B51 absent with BD. class I antigen loss and marked LMP7 downregulation.

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159 160

161 162 HLA I AND II CLASS POLYMORPHISM IN MYASTHENIA PATIENTS WITH THYMIC HYPERPLASIA OR THYMOMA

Bubnova Ludmila, Pavlova I., Berkos M.,Glazanova T,Baranov V. Research Institute of Hematology and Transfusiology lab. of immunohematology, St. Petersburg Myasthenia Gravis (MG) is an autoimmune disease of the neuromuscular junction having multigene control. It seemed of interest to study the HLA I and II class associations of this disease in patients of North-West region of Russia. We have marked out 2 groups of generalized form myasthenia Gravis (GMG) patients: 1st - with histologically verified thymic hyperplasia, who undervent transsternal thymectomia, (n=23, average age 33,5 years, 64,3% -female), in 21,4% of cases was observed a severe form of disease; 2nd group – with thymoma (n=7, average age 38,2 years, 100% - female), without any cases of severe form. HLA I class antigens were typed serologically (control group consisted of 1011 healthy citizens of North- West Russia), while HLA II class - using PCR-SSP method (control group, n=208). Chi-square values were calculated with Yates correction for small groups. We have revealed that in Noth-West Russian region in patients with MG with thymic hyperplasia (1st group), there was observed statistically significant associations with HLA-A1 (43,5% vs. 22,4% in control group, chi-sq=3,84), HLA-B8 (47,8% vs. 14,8%, chi-sq =18,6), HLA-B18 (39,1% vs. 14,2%, chi-sq =11,04), DRB1*03 (43,5% vs. 15,9%, chi-sq =7,24) and DRB1*16 (21,7% vs. 5,8%, chi-sq =7,74). In patients with thymoma (2nd group) we have not revealed any statistically significant difference between the HLA frequencies listed above neither when compared to 1st group, nor to control group. Nevertheless, some difference in HLA-distribution between the two groups of patients were revealed. Thus, in thymoma patients there was increased frequency of HLA-A3 as compared to 1st group (17,4% vs. 57,1% in the 1st and 2nd group, respectively, chi-sq=4,33 ) and HLA-B7 (8,7% vs. 42,9%, respectively, chi-sq.=4,51). The data suggest that different HLA genes can predispose to two different forms of MG, which thus are likely to have different pathogenetic mechanisms.

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163 164 TUMOUR NECROSIS FACTOR-ALPHA –308(G/A) PROMOTER GENETIC MARKERS ASSOCIATED WITH MULTIPLE POLYMORPHISM IN CZECH PATIENTS WITH PULMONARY SCLEROSIS IN BULGARIANS SARCOIDOSIS 1Snejina Mihailova,1Milena Ivanova,2Olia Mikova,1Elissaveta Frantisek Mrazek, Vitezslav Kolek, Zuzana Ambruzova, Beata Naumova.1University Hospital Alexandrovska, Central Laboratory Hutyrova, Roland M. du Bois, Ken I. Welsh & Martin Petrek Of Clinical Immunology, 2University Hospital St. Naum, Dept. Of Dept of Immunology, Medical Faculty of Palacky University, Neuroimmunology And Microbiology, Sofia, Bulgaria. I.P.Pavlova str. 6, CZ-775 20, Olomouc, Czech Republic Our aim was to investigate the role of HLA class II, mtDNA and cytokine gene Tumour necrosis factor-alpha (TNFα) is implicated in the pathogenesis of polymorphisms in development of MS in the Bulgarian population. 52 MS sarcoidosis, a granulomatous disease most frequently affecting the lung. Acute patients and 88 healthy controls from the Bulgarian population were studied for onset of sarcoidosis with erythema nodosum and bilateral hilar HLA class II, TNF-α, TGF-β, IL-10, IL-6 and IFN-γ gene polymorphism by lymphadenopathy (Löfgren' s syndrome) is often associated with spontaneous PSR-SSP. Screening of mtDNA polymorphism, including MS associated resolution of the disease. Promoter polymorphism TNFα-308(G→A) has been mtDNA 4216, 13708 and 14798 mutations, was performed by PCR-RFLP. Most previously shown to affect production of TNFα. of individuals were classified in previously described mtDNA haplogroups. We have, therefore, analyzed TNFα-308 genotype in 61 patients with Significant predominance of 4216 in the patients group (p<0.05, OR- sarcoidosis and 232 healthy control subjects. All individuals were unrelated 2.93) was observed. Our results revealed that in one of the mt DNA regions Caucasians of Czech origin. Genotyping for TNFα - 308 polymorphism was (11932-14007 bp, restricted with BstN I) one of the polymorphic variants performed by PCR-SSP. (morph1) was protective (p=0.002, OR=0.22), while another one (morph2) was There were no significant differences in distribution of TNF -308 G/A alleles predisposing (p-0.001, OR=5.73). Observation of HLA predisposing DRB1*15 between patients with sarcoidosis and control subjects (C). Subgroup analysis and protective DRB1*11 related haplotypes in Bulgarians was in agreement with established MS associations in Caucasians. In the controls 82% of those revealed that the frequency of uncommon TNFα-308*A allele is significantly who carried MS protective HLA DRB1*11 allele also had protective for our increased in sarcoidosis patients presenting with Löfgren' s syndrome (LS) in population mtDNA morph 1. We observed that all healthy individuals with comparison with sarcoidosis patients without LS (NLS) and with control predisposing DRB1*15 allele carried protective mtDNA morph1. That could subjects (allelic frequency: LS, 37.5%; NLS, 16.7%; C, 17.9%). The odds ratio suggest a probable combined protective effect of these two genetic markers. No for carriage of TNFα-308*A allele was 4.5 for Löfgren´s syndrome within the significant correlation was detected between cytokine gene polymorphism and group of patients with sarcoidosis (p=0.012). MS. In conclusion our date revealed an association between HLA and mtDNA α We report here an association of TNF -308*A allele with Löfgren' s polymorphisms and MS in Bulgarians and lack of genetic predisposition related syndrome among the Czech patients with pulmonary sarcoidosis. Observed to cytokine gene polymorphisms. genetic difference may be related to different clinical outcome of investigated sarcoid subgroups.

165 166 HLA, TNFA, LTA AND TNFR2 POLYMORPHISMS IN SLE IN PROSPECTIVE ANALYSIS OF HLA CLASS I AND II THE NORTH OF PORTUGAL PHENOTYPE FREQUENCIES IN PATIENTS WITH PEYRONIE’S DISEASE Clara Pereira1, Claudia Carvalho3, Sara Casimiro1, Zulmira Quintas3, Carlos Vasconcelos2, Berta Martins da Silva1,3. Arne Hauptmann, Ekkehard W. Hauck, Wolfgang Weidner, Gregor Bein, 1Departamento de Patologia e Imunologia Molecular–ICBAS/UP. Holger Hackstein 2Departamento de Medicina - Hospital Santo Antonio, Porto. Institute of Clinical Immunology and Transfusion Medicine, 3Instituto Naciona Saúde Dr. Ricardo Jorge–Porto. Giessen Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease of The etiopathology of Peyronie's disease remains unclear. Previous unknown aetiology. Association with HLA antigens, namely HLA-DR2 and studies have described possible associations between various human HLA-DR3 has been reported in several populations. Tumor Necrosis Factor (TNF), localised in HLA class III region, is considered a candidate gene to SLE leukocyte antigens (HLA) and Peyronie's disease. However, only susceptibility given its role in the inflammation process. This study analyses a small patient series comprising 9 to 52 patients were analyzed in possible relationship between HLA-DR, TNFA and LTA and TNF Receptor 2 these studies and the results published were controversial. The aim of (TNFR2) gene exon 6 allelic polymorphisms and susceptibility to SLE in this study was to investigate prospectively a possible association of Portuguese patients. Seventy-one patients with SLE – 19 of those with nephritis, Peyronie’s disease with HLA class I and II in a large patient group. a severe complication of the disease - and 99 healthy control subjects were 154 consecutive patients with idiopathic Peyronie's disease were analysed. The frequency of HLA-DR*03 allele was significantly increased and HLA-DR*13 decreased in SLE patients (P=0.005, OR=3,11 and P=0.044, typed serologically for HLA class I antigens. Molecular typing of OR=0.443, respectively). The increased frequency of HLA-DR*03 was HLA class II was performed by the polymerase chain reaction sustained and higher in the subgroup with SLE nephritis (p=0.007, OR=4,34). sequence specific primer method (PCR-SSP). The results were The frequency of LTA3 allele was decreased but only significantly in SLE compared with healthy bone marrow donors for HLA class I (n = nephritis (p=0.040, OR=0.32). Interestingly, TNFA3 was absent in SLE 2,450) and for HLA class II (n = 316) as representative controls nephritis. Differences of TNFR2 196M/R genotype frequencies between SLE selected from the local population. Additionally, HLA distribution in and controls were observed but didn’t reach statistical significance (p=0.054, a German normal cohort (n=14,835) was used as a control. OR=0.36 for TNFR2). This study supports the known association of HLA-DR3 to SLE predisposition, particularly when kidney involvement is considered. A No significant difference in HLA class I and II phenotype protective role for HLA-DR*13 in susceptibility to SLE without nephritis is frequencies was evident in patients with Peyronie's disease compared suggested. The role of the other immunogenetic markers could not be ruled out. to healthy controls. The results of this study do not indicate any significant association between Peyronie's disease and the antigens of the HLA system.

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167 168 HFE MUTATIONS IN PATIENTS AFFECTED BY ACUTE MYOCARDIAL INFARCTION.

Giuseppina Candore, Carmela Rita Balistreri, Florinda Listì, Domenico Lio, Giuseppina Colonna-Romano, Martina Chiappelli, Cecilia Tampieri, Federico Licastro, Angelo Branzi, Marco Caruso, Enrico Hoffmann, Calogero Caruso. Gruppo di Studio sull’immunosenescenza, Dipartimento di Biopatologia e Metodologie Biomediche dell’Università di Palermo, Corso Tukory 211, 90134 Palermo. There is substantial interest in the role of iron in coronary heart disease (CHD) and other atherosclerotic diseases. In fact, studies of the relation between elevated iron levels and cardiovascular disease have demonstrated that the excessive tissue iron deposition could be harmful, because of the potential of Fe++ to react with H2O2 to form OH- radicals and Fe++ to react with O2 to forms ROS. These free radicals react with cell membranes and cell organelles and they are believed to play a role in the development of CHD. In fact, as regards CHD it has been reported that these radicals can initiate lipid peroxidation, causing LDL to undergo an oxidative modification that targets it for uptake by macrophages and may damage arterial endothelium directly and interfere with normal vasomotor regulation. Following the hypothesis that iron overload could be risk factor for CHD, the hereditary hemochromatosis provides an additional model to study the effects of iron in cardiovascular disease. In particular, three studies have been shown that individuals who carried HFE mutations may be at greater risk of developing CHD, than those without the mutations. In contrast, a larger number of studies have reported no associations between HFE gene mutations and atherosclerosis including coronary artery disease and acute myocardial infarction (AMI). We studied the relation between HFE mutations and AMI in 172 subjects (139 males and 33 females; mean age 67) affected by AMI and 207 controls (91 males and 116 females), both from northern Italy. We were not able to show significant differences in frequencies of the different HFE alleles (C282Y and H63D) between controls and AMI patients. To confirm these negative results, we examined HFE mutations in another cohort of patients (N = 57), younger (under 45 years) and from Sicily. Also in this cohort of patients, no significant difference in frequencies of the different HFE alleles between controls and AMI patients was observed. Thus, our study does not support the suggestion that HFE mutations may be a genetic risk factor for AMI. On the other hand, a recent study performed on 41038 subjects in the USA, including elderly subjects, screened for C282Y and H63D

169 mutations, suggests that the clinical penetrance of the mutation is very low. In CYTOKINE GENE POLYMORPHISMS AND SUSCEPTIBILITY fact, in this study, only one of the 152 homozygotes had signs and symptoms of TO ORAL LICHEN PLANUS. hemochromatosis and, concerning other conditions, there was only a significantly increased prevalence of a history of hepatitis. Besides, the authors Uboldi de Capei Mariafederica1, Dametto Ennia1, Fasano add that it would be quite extraordinary for common polymorphisms such as HFE mutations to have an adverse phenotypic effect. Thus, they believe that one M.Edvige1, Brancatello Francesca1, Carrozzo Marco2, Vezza must resist the temptation to conclude that the adverse associations, which have Denise2, Pentenero Monica2, Arduino Paolo2, Rendine Sabina1, been observed, are related by cause and effect. Accordingly, in Sicilian and Gandolfo Sergio2, Curtoni Emilio Sergio1. Sardinian populations, the HFE mutations are found to be over represented in 1 Transplantation Immunology Service, Dept. Genetics and 2 Dept. very old people. Oral Medicine, University of Torino, Italy. Lichen planus (LP) is a chronic inflammatory disease that affects skin and mucous membranes: the Oral form (OLP) seems more common and chronic that cutaneous type. LP probably represents a cell-mediated immunological response to an induced antigenic change in the skin or mucosa. The aim of the present study was to analyze whether the polymorphisms of several pro- and anti-inflammatory cytokines may influence the susceptibility to OLP in a Northern-Italian population. Cytokine typing was performed by a Sequence Specific Polymerase Chain Reaction (PCR-SSP) assay. The Cytokine typing tray kit was supplied by Heidelberg University: it permits to analyse 13 cytokine genes with 22 Single Nucleotide Polymorphisms (SNPs). Statistical analyses were performed using chi square test. Forty-four unrelated Caucasian patients with OLP took part in the study. As controls, 140 healthy unrelated donors serologically typed for HLA-A and B and without evidence or history of LP, were studied. Patients and controls were all Italian and their geographic origin had been carefully checked to compare subjects from the same areas. Considering all the comparisons between allele and genotype frequencies in OLP patients and controls we found that the following genotypes seem to contribute to the susceptibility to OLP: a) TT of UTR5644 IFN- , GG of –1098 IL4, GA of –308 TNF-α and allele may contribute to the susceptibility to OLP.

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170 171 IL-6, IL-10 AND IFN-GAMMA GENE POLYMORPHISMS IFN-GAMMA AND HLA POLYMORPHISM IN SARCOIDOSIS ACTING IN CONCERT IN PATIENTS UNDERGOING HSCT Barbara Wysoczanska, Katarzyna Bogunia-Kubik, Andrzej Lange Lidia Karabon and Andrzej Lange L. Hirszfeld Institute of Immunology and Experimental Therapy , Lower Silesian Center for Cellular Transplantation, Wroclaw, Poland Polish Academy of Sciences, Wroclaw, Poland Several polymorphic features of cytokine encoding genes were described to Our previous reports documented correlation between HLA-DRB1 and DRB3 influence the outcome of HSCT. Our own observations and those of Cavet et al. alleles and the clinical variants of sarcoidosis (Bogunia-Kubik et al. 2001) and (2001) and Socie et al. (2001) showed that IL-6, IFN-gamma and IL-10 association between expression of IFN-gamma gene and stages of the disease recipient genotypes associate with susceptibility to toxic or GvHD (Swider et al. 1995). In the present study STR polymorphism within the 1 intron complications. In the present analysis we found that patients suffered from of IFN-gamma gene in connection with HLA specificities was analysed in 43 aGvHD grade II-IV were more frequently having unfavourable pattern of sarcoidosis patients (F/M: 25/18, age 24-58, median 36, stage I/II-III: 21/22), 14 cytokine alleles IL-6(-174)G, INF(+874)A, IL-10(-1082)G than patients lacking of which presented with Löfgren’s syndrome symptoms. Four IFN-gamma aGvHD complications (9/11 vs 27/77, p=0.087). Therefore it appears that alleles, numbered 2, 3, 4 and 5, were detected (with 12, 13, 14, 15 CA repeats, polymorphic features within different chromosomes can act in concert, making respectively) with following frequencies: allel 2: 0.37; allel 3: 0.55, allel 4: the host more or less susceptible to the advert effect of transplant related 0.05, allel 5: 0.03. As was previously showed, DRB1*03 and DRB3*0101 complications. In the next analysis we looked whether the presence of cytokine associated with susceptibility to Löfgren’s syndrome (9/14 vs 7/29, p=0.014 and genotypes may be preferably linked with class II DR specificities in patients 11/12 vs 9/19, p=0.014 for DRB1*03 and DRB3*0101, respectively) while suffering from haematological disorders. It appeared that DR2, 5 and 4 DRB1*11 prevailed in stage II/III disease (7/22 vs 2/21, p=0.076). Patients prevailed in patients having IL-10 GCC+, ACC+, ATA+ alleles, respectively. carrying DRB1*03 were less frequently 2,2 homozygotes than patients having Therefore we can assume that IL-10 gene polymorphism and DR associated other IFN genotypes (2/8 vs 14/35, ns). A tendency was observed towards the features may contribute together in shaping some haematological diseases. lower incidence of Löfgren’s syndrome among patients having 2,2 (1/14 vs 7/29, ns). Patients lacking 2,2 homozygosity but having DRB1*03 more frequently developed Löfgren’s syndrome symptoms than those carrying the other IFN and DRB1 genotypes (9/14 vs 5/29, p=0.003). In addition, DRB1*11 positive patients lacking 2,2 alleles prevailed in stage II/III sarcoidosis as compared to those without DRB1*11 but carrying IFN 2,2 (0.83 vs 0.29, p=0.078). These results imply that IFN-gamma gene polymorphism in addition to HLA-DRB associates with the course of sarcoidosis.

172 173 HLA-DRB1 GENES IN ITALIAN PATIENTS WITH D6S273 AND MIB POLYMORPHISMS IN PATIENTS WITH MEDULLARY OR PAPILLARY THYROID CARCINOMA. PSORIATIC ARTHRITIS

Vincenza Luongo, Elena Cosentini, Luciano Pezzullo, Gelsomina Zorana Grubi 1, Katarina Štingl1, Porin Peri 2, Esma e uk-Jeli i 1, Renata Lupoli, Giuseppe Amato, Giuseppe Pirozzi and Maria Luisa une c1, Bo idar urkovi 2, Vesna Kerhin-Brklja i 1 1National Referral Organ Transplantation and Tissue Typing Centre, Lombardi., Istituto Nazionale Tumori and Facoltà di Medicina, University Hospital Zagreb, 2Clinic of Rheumatology, Zagreb, Università Federico II, Napoli, Italy. CROATIA Several studies have reported the association between a variety of malignancies Psoriatic arthritis (PsA), inflamatory arthritis followed by psoriasis, is related and HLA genes. Conflicting data, however, have been reported on HLA with B13, 27, 37, 38, 39, and 57 antigenes at HLA-B locus in Caucasians. association and thyroid carcinomas. No association indeed, was found between We investigated polymorphism of 2 microsatellites in proximity of HLA-B HLA genes and papillary, follicular or medullary thyroid carcinoma in German locus (D6S273 and MIB) to prove whether additional genetic markers also patients (Exp Clin Endocrinol, 99: 134; 1992), whereas, significant increase of DR2 in medullary carcinoma or DR7 and DR1 in differentiated thyroid increase disease susceptibility. carcinoma were found in patients of different ethnic groups (Cancer, 63: 1318; Fifthy-five unrelated PsA patients, fullfiling criteri a of disease and 165 1989). controls were studied. Molecular typing of HLA-A, -B, Cw, and DRB1 alleles We here report molecular analysis, by using PCR-SSP technique, of HLA was performed by PCR-SSP. Microsatellites were analysed by electrophoresis class II loci in 76 patients affected by thyroid carcinoma (TC), 37 with on polyacrilamide gel (ALF express). medullary thyroid carcinoma (MTC) and 39 with papillary thyroid carcinoma From this study we concluded: (1) the frequency of HLA-B*13, *27, *39, and (PTC). HLA frequencies were compared with those obtained in 128 ethnically *57 alleles was largely increased in PsA; (2) Cw*02 allele showed a slight matched healthy controls. Preliminary results showed a significant increase of association with the disease, while Cw*0602 did not show association with PsA; DRB1*15 (10.5% vs 5.1%; p= 0.032) in TC patients also present in both (3) at DRB1 locus we did not find significantly increased frequency of MTC and PTC patients. Moreover an increase of DRB1*04 (14.8% vs 8.9%; DRB1*04 and *0701 in PsA; (4) no significant differences of D6S273 and MIB p=0.108) and a decrease of DRB1*01 (1.4% vs 5.4%; p=0.112) were noted in alleles between PsA and controls were found; (5) reassessment of results after MTC patients whereas an increase of DRB1*13 (16.6% vs 8.8%; p=0.195) was the patients were divided into 2 groups based on presence or absence of one of present only in the PTC group. Because some frequencies are close to above mentioned HLA-B alleles, showed significantly lower frequency of allele significant values a larger number of subjects have to be analyzed in order to 130bp and 136bp at D6S273 locus in PsA, as well as for 346bp at MIB; (6) better define the possible association between HLA genes and thyroid higher frequency of D6S273 132bp and 134bp is due to linkage disequilibrium carcinoma in our patient group. with B locus.

Genes and Immunity Abstracts S45

174 175 DRB1*03 IN ALZHEIMER: A POSIBLE GENETIC MARKER . FLOW CYTOMETRY FOR HLA B27 DETECTION : V.J.León, J.L.Cacho1, C. González THRESHOLD FOR POSITIVITY USING 4 DIFFERENT Servicio de Bioquímica, 1 Servicio de Neurología. Hospital REAGENTS Universitario de Salamanca. Introduction : Our Spanish region, Castilla and León, presents a tight population X. Lafarge, A. Hrycaj, D. Fizet, G. Vezon with more than 2 million of inhabitants which has not received emigrants in the Histocompatibility Laboratory, Etablissement français du Sang last centuries, instead of it the population has emigrated and, moreover, it is a Aquitaine-Limousin, Bordeaux, France very aged population in comparison with other Spanish regions. Thus, More than 90% of patients suffering ankylosis or reactional spondylathritis, everything becomes suitable for populational studies of diseases that appear in inflammatory pathologies have HLA B27 gene. So HLA B27 detection is now a people of high ages and their markers. We have chosen in our study the alele routinely tool for clinical statement. DR3, a marker that has begun to be studied recently with contradictory results, Flow cytometry used by many laboratories is validated so there is no cross in populations of patients with Alzheimer (EA) in order to elucidate their reaction with other HLA antigens. On the other hand, one must be sure there are possible use as genetic marker in this group of patients. no false negative reaction depending HLA B27 subtype determined by Material and Methods: We have studied a series of 20 diagnosed patients of molecular biology. Probable EA according to approaches NINCDS-ADRDA, and 29 healthy 52 samples were typed by PCR SSO reverse for generic level and then PCR control fellows (SC). The DNA was extracted using the technique of Salting- SSP for allelic specificity : 47 been B*2705, 2 B*2702, 3 B*2707 and none Out. The HLA-DR3 was studied using the Kit HLA-DRB1* of Dynal, it was B*2708. also studied a well defined genetic marker of EA as it is the Apo E Lymphocytes were labelled with different anti HLA B27 antibodies FITC (Innogenetics). labelled (One Lambda, ComB27 Bioatlantic, Immunotech) . Mean fluorescence Results: We have found a significant increment of the frequency of intensity was compared with antibody irrelevant control. Becton Dickinson kit is appearance of the DR3 (p = 0,008) and of the allele E4 (p = 0,038) in the used not only with their calibrated beads but with an isotypic control. patients with EA in front of the SC. Concretely, it was found that 13 of the 20 One Lambda has an intensity 3 times lower than the others and B* 2702 were (55%) patient with EA presented allele DR3, while alone 3 of the 20 (15%) SC weaker than other allells but not B*2707.Homozygot cells B*2705 were always had a DR 3. In the group EA, didn't have relationship among presence of E4 and more fluorescent than heterozygots. Some subtypes have not been tested of DR3 (p = 0,193). In accordance with these results, the DR3 appears as a because rare in our country . Important labelling intensity was always obtained probable factor of risk to suffer EA and, also, it is independent of the presence with B*2705 and B*2707. However, BD kit gives false negative results because or absence of APOE E4. we estimate necessary other studies with more number threshold with fluorescent calibration beads is too high. of patients, to be able to confirm these preliminary results Flow cytometry is a simple and valid technology to detect HLA B27 as soon as one makes sure of no cross reactive reactions.

176 177 ASSOCIATION STUDY ON ABDOMINAL AORTIC ASSOCIATION OF HLA-B*5 IN HLA-B5-POSITIVE TURKISH ANEURYSM USING MICROSATELLITE MARKERS LOCATED PATIENTS WITH BEHÇET’S DISEASE IN THE HLA REGION. Orkunoglu Funda E.(*), Sengul A(*), Akar Ahmet(**), Tastan Bulent M. Ota1), T. Sugimoto2), M Sada2), Y. Katsuyama3), R Goto2), K (**), mirzalioglu Necat(***), Gata School Of Medicine, Dept Of Yamada2), T Miyamoto4), H. Inoko5): Dept. of Legal Med1)., and Dept Immunology (*),Dept Of Dermatology(**), and Dept Med. Genetics of Pharmacy3), Shinshu Univ. School of Med., Matsumoto, Dept of HLA B*5 allele genotyping was studied in Turkish Behcet’s disease (BD) Regenerative Med2)., National Cardio Vascular Center Research PAT ENTS (n=30) and healthy controls (n=90) in order to investigate the Institute, Dept of Thoracic Surgery, Hyogo Collage of Med4), Dept of subtyping of the B5 antigen. Among the HLA-B51-positive BD patients, Genetic Information, Division of Molecular Life Science, Tokai Univ. B*5101 was found to be the predominant allele, identified in 86 % of all BD School of Med5). patients. HLA-B*5101 was also the predominant allele in HLA-B51-positive healthy controls. HLA-B*5108 allele was identified in 14% of B51-positive BD Abodminal aneurysms (AAA) are common disease of maturity. patients, respectively non of other 25 sub-alleles were determined. The HLA- has been proposed to play a role in the pathogenetics of the AAA. In the B*5201 allele was not identified in the patient and control group. Our study spectrum of autoimmune disorders certain HLA alleles play a key role in the suggests that both HLA-B*5101 is the dominant alleles of HLA-B5 in Turkish presentation of self proteins as autoantigens in the different specific conditions. BD patients. We previously reported a linkage of the HLA-A2, -B61 and –DR15 antigens to Keywords: Behçet’s disease; HLA genotyping the susceptibility to the AAA in Japan. In the present study we performed association analysis to refine the genetic contribution of the HLA region in the development of AAA using 19 microsatellite markers (D6S439, TAP1, D6S2444, D6S2443, T16, CAT, DQ-CARII, D6S273, TNFd, TNFa, C1-2-A, MICA-TM, MIB, C1-4-1, C1-2-5, C1-3-1, C2-4-4, C3-2-11, D6S276). We analyzed microsatellite polymorphisms at each locus with genomic DNAs from 49 patients and 248 unrelated Japanese healthy controls. None of microsatellite markers localized in the class I and class III regions showed any statistically significant association with the disease by the chi-square method and Fishers’ exact probability test. However, only TAP marker located between HLA-DQ and –DP loci displayed statistically weak association (allele 200, P=0.003, Pc=0.02) with AAA. These results suggest that TAP gene seems to control the development of the AAA.

Genes and Immunity