- Email: [email protected]
Molecular analysis of Class II sequences involved in autoimmune hepatitis in Mexicans
23 June 1997 - Poster presentations
P.5.04.03
promoter analysis of the matrix metalloprotelnase RASI-1, a novel autoantlgen In rheumatold arthritis
M. Mueller. S. Mauch, R. Sedlacek, U. Krawinkel. Departmentof /mmuno/og~! University of Konstanz, Konstanz, Germany introduction: The matrix metalloproteinase (MMP) RASI-1 was detected as a novel autoantigen in Rheumatoid Arthritis (RA) (1). Many MMPs seem to play a critical role in the pathology of systemic autoimmune diseases such as FiA (2, 3). In order to study the transcriptional regulation of RASI-1 we have isolated the complete rasi-7 gene and analyzed its 5’8anking sequence. This study allows first insights into possible regulatory mechanisms controlling the expression of RASI-1. Materials and Methods: We have isolated the complete rasi-I gene from a human genomic DNA library. The promoter/enhancer region has been sequenced and the transcription start site was identified by primer extension analysis. Potential DNA-binding sites for transcription factors were detected by database analysis. Fteaulta: We have found many potential DNA-binding sites for transcription factors, several of which are typical for matrtx metalloproteinases. The resi-I promoter contains a TATA-box at nucleotkte -29 relative to the transcription start site. We have detected a consensus sequence for the transcription factor AP-1 at position -73 and a putative PEA-3 element at position -155. This arrangement is typical for many MMPs (4). Furthermore potential binding sites for the transctiption factors NF-kappa& AP-2, SP-1 and elements which show similarity to the TGF-beta inhibitory element (TIE) and a retinoic acid response element (RARE) have been detected. In addition we recognized some other binding sites that can not be found in other MMPs. Conclusion: Taken together these data suggest that msi-I is a tightly controlled gene under physiological conditions resembling many of the regulatory characteristics of most other MMPs. Further experiments such as DNA-footprinting and promoter/reporter-gene assays have to proof the regulatory effect of the above mentioned transcription factors. Because MMPs play a crucial role in the pathogenesis of systemic autoimmune diseases, obtaining insights into their transcriptional regulation is of particular interest and may lead to the development of new therapy concepts to treat such disorders. [l] Sedlacek, R., Mauch, S., Eibel, H., Peter, H.-H. and Krawinkel, U. (1995) Immunobiol., 194,153 121 _ _ Bid&al-Hansen, H., Moore, W., Bodden, M.. Windsor, L., Birkedal-Hansen, B., DeCado, A. and Angler, J.(1993) Crit. Rev. Oral Biol. Med., 4 (2), 197-250 [3] Murphy, G. and Hembry, R. (1992) J. Rheumatol. Suppl., 32,61-84 [4] Mattisian, L. (1994) Ann. N. Y. Acad. Sci., 732, 42-50
P.5.04.04
Frequencies of HLA-ORB1 and HLA-DQBl alleles In patients buffering from vltlllgo
M. But’, H. FazekaSovB’, E. Hegyi 2, K. Kolib&Bov62, S. FerenEik3. ’ Department of Immunology, Faculty of Medicine, Comanius Univatsi~, Bmtislava, Slovakia, ‘Depamant of Darma~ology,Hospital Ru?inoy Bratislava, Slovakia, 31nstkuteof Immunoiog~ University Hospital, Essan, Germany Introduction: Our previous studies on the associations between vitiligo and HLA an&ens disclosed the increased frequency of HLA-Dw7 antigen (But et. al., Foliabiol. 42: 23-25, 1998). To confirmthe &lular typing results we retyped the studied group of patients for the frequencies of HIA-DRBl and HlA-DQBl alleles, resp&ti;ely. Matertal and Methods: 21 patients were investigated. 13 alleles of the HLA-DRBl locus and 17 alleles of the HLA-DQBl locus were determined by PCR-SSP method according to Qleriup et al. mssue Antigens, 39: 225-235, 1992). Results: The statistically significant deviations in frequencies of the following alleles were found: DRB1’0701 (28.5% compared to 15.3% in the healthy population, p .C 0.001) and WB1’0201 (38.0% compared to 21.1% in the control group, p < 0.0001). No other statistically significant differences were obsenred. Conctuslon: The allele DRB1’0701 encodes the antigen HLA-DR7 and the allele DQBl”O201 encodes HlA-Dw7, an epttope present on B-chain of the antigen HLA-DQ2. So DNA typing results confirm the previously recorded associations of HLA-DR7 and HLA-Dw7 antigens with vitiligo and indicate that HLA antigens do play a role in the immunopathogenesis of vitiligo.
Genetics of autoimmuni~
P.5.04.05
167
Study of the HLA-BZ;Nankyloslng spondylltls association about 34 cases
H. Makni 1.4,N. Lahlani-Mahfoudh ‘, S. Baktouti 2, 2. Bahloul 3, H. Ayadi 4.
’ Laboratoire d’hishxwmpatibili~6, EfS H. Chakac S&x, Tunisia,2San&a da Rhumatolwia, EPS H. Chakar, S&x, Tunisia,3Sawim da Medacina lnfeme. EPS H. Chakac Sfax, Tunisia,4La~tatoira d’lmmunobgia at da Biokqia Mol&u/aire, Faculte da hMdecine, Sfax, Tunisia Thirty four patients from south Tunisia fulfilled the new York criteria of ankylosing spondylitis (AS) and were phenotyped for HLA-A and -B loci. A complement dependent micrccytotoxictty assay was perfom?ed using a battery of local sera screened from multiparous women: A sample of 105 healthy and unrelated individuals was used as controls. Nineteen out of 34 patients (55.85%) were HLA-B27 positive whereas the HLA-B27 frequency was of 1.9% in controls. The relative Risk value (RR) was of 85.23: The most frequent haplotype associated to AS was A2 827. As for most populations worldwide, we found a significant increased frequency of HLA-B27 in patients with AS. Nevertheless, HLA-B27 was associated to AS at a lower frequency than in Caucasians. Moreover, the RR value found was lower than those reported in Caucasians. Our results showed an equal distribution of HLA-827 in the population of south Tunisia, in Kabyles and in Saudi Arabs. In addition, 15 AS patients of 34 (44.2%) lacked the HlA-B27 antigen. Altogether, these data suggest that besides the HLA-B27 antigen, additional genes might also be involved in the susceptibility to develop AS.
P-5.04.06
Polymorphism of the human lmmunoglobulln heavy chain locus In rheumatoid arthritis
F. Fakhfakh ‘, A. Maalej I, 2. Bahloul ’ , A. Jarraya ‘, H. Ayadi ‘, M.Zouali 2. ’ LaborMoire d’lmmunokgia at da Biologic Mol&ulaira, FaculM da M6decina. St&x, Tunisia,2LMpartement d’lmmunolcgia, lnsritot Pastaq 28, rue du Dr Roux, Paris, France The genetic origin of Rheumatoid Arthritis (RA) is largely unknown. The purpose of this investigation was to assess the potential genetically determined involvement of the immunoglobulin (lg) heavy chain variable region (VH) locus in the pathogenesis of RA. We tested the hypothesis of whether there is a genetic linkage between a structural abnormality of the VH gene complex and autoantibody hyperproduction in RA. We used restfiction endonuclease generated polymorphism in human VH gene-family specific probes to examine genomic DNA from a RA family and from unrelated RA patients from both the Tunisian and the European populations. The use of DNA samples from these ethnic origins permitted a further evaluation of the polymorphism of the humanVH locus. While we found that the polymorphism of the VH locus was lower in the Tunisian population, we could not detect a restriction site polymorphism pattern restricted to RA. Our results argue against a genetic linkage between RA and polymorphism in the VH locus. However it is possible that the method of restriction fragment length polymorphism was not able to detect subtle variations in DNAs of RA patients and, hence, reveal minor abnormalities in the VH locus of the RA patients. Likewise, genomic changes or somatic variation in a B-cell subset, both undetectable by our restriction fragment analysis, would need to be further investigated.
1P.5.04.07 ] Molecular analysis of Class II sequences Involved In autolmmune hepatitis In Mexicans M.N. Vtiquez-Garcia 1.3,C. Alaez ‘, A. Olivo ‘, H. Debaz I, E. P&z-Luque ‘, G. De La Rosa ‘, S. Cano ‘, A. Burguete ‘, J. Bandera 2, D. Kershenobich 2, C. Gorodezky ’ ’ Department of Immunogenetics, INDRE, SSA, Mexico Ci& Mexico, 2Gastroentarol~y Sarvica, lnsikuto National da la Nutriciidn,SSA, Mexico Ciw Mexiw, 3Prvgrama da Biomedicina Molecular CINVESTAV-IPN, Mexico Ci& Maxkw Introduction: Autoimmune hepatitis (AH) is a progressive condition of the liver characterized by piecemeal necrosis, the presence of diierent autoantibodies and by its prevalence in females. DRBl’O301 and DRBl l0401 are associated in Caucasians and DRB1.0405 is a susceptibility gene in Japanese. This is the first study done looking for HLA class I antigens and class II DNA alleles in Mexicans with the disease. Materials and Methods: We analyzed 30 Mexican Mestizo patients with type I AH compared to 175 healthy controls. The following autoantibodies were determined: (r-DNA, smooth muscle, ANA, mitocondrtal, thyroglobulin and LKMl (microsomal). Serotogical class I typing was done using the microcytotoxicity technique with magnetic beads and double fluorescence staining. DNA typing for DRBI, DQAl, and DQBl loci was performed with the protocols, primers and probes developed for the 12th International Wokshop. PCR-SSP was done to confirm certain DRBl, DQAl or WBI alleles. Frequencies were calculated and the most probable combinations were deduced.
168
23 June 1997 - Poster presentations
Cytokine-directed therapy
Results: The patients were mainly females (90%) with an X age at onset = 32.1 f 12.5 years. All of them had ANA and/or SMA antibodies but not LKMl ; and were negative for hepatitis virus B and C. Ten of them had another autoimmune disease (SLE, thyroiditis, Sj6gren and Fisher-Evans syndrome). No association was detected with dass I antigens. However, a significant increase was found for DRB1.0404 (pc = 0.0001; RR = 7.71). An important decrease of DQB1’0301 is informed for the first time (pc = 0.03) suggesting the presence of a strong DC&linkedresistance gene in Mexicans, which is DR independent. Conctualon: The 67-72 “motif” LLEQKR may be very important in the expression of AH, because the “hepatogenic peptide” leading to autoimmune recognition must have complementary sequencles for DRB1’0405 and DRB1.0404, which share the same residues at 67-72. On the other hand, DRB1*0301 and DRB1’0404 have only one substitution in position 71 (Rfor A), but both are basic aminoacids. Therefore, we propose that this “motif” is crucial for the hepatogenic peptide recognition and for enhancing the disease process. Moreover, DRB1’0301 is associated with early onset in Caucasians (~30 y) and in our patients the onset is early too (X = 32.1 y) with DRB1.0404 as the susceptibility gene. Both, DRB1’0301 and DRB1’0404 have a Vat position 66, contrasting with DRB1’0401 and DRB1’0405 that have a G and are associated with a late onset. Thus, these aminoacid substitutions in the antigen binding groove of the DR molecule, may determine disease susceptibility as well as severity. Understanding of the mechanisms of peptide binding are crucial for the development of novel prophylactic or treatment methods.
09:00-18:30/12:0&14:00
Forum lounges
P.5.05
Cytokine-directed therapy
P.5.05.01
Immunological pa&meters have predictive value on the clinical response to interferon-a therapy of hepatitis C patients
B. Moragues, A. Pdeto, J. Monserrat, A. Albillos’, R. Moreno *, G. Gacho 3, J. Calleja3, F. Esquivel, M. Barque*, M. Alvarez-Mon 4. ’ Hc@tat Ram& Y Cajai, hadrid, Skin, 3Hospital.’ PuerB de Hierro, Madrid. S&n, * Hospit& de la Princesa, Madrid, Spain, 4Hospital. Principe de Asturias, Universidad de Alcald, Madrid, Spain Introduction: Currently, there is a not valid marker to predict the heterogeneous clinical response chronic hepatitis C (CHC) patients to interferon IY(IFNa). The antiviral and the immunomodulator effects of IFNa appear to be involved in its mechanism of action. The aim of this study was to investigate the potential prognostic value of immunologic parameters to predict the clinical response to IFNaPb in CHC patients. Materials and Methods: We have evaluated the Natural Killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) and the expression of the CD2, 3, 4, 6, 16, 56 antigens and the proliferative response to PHA, antiCD3 monoclonal antibodies of T lymphocytes from 35 CHC patients before a standard course of IFNaPb (3 MU, t.i.w., 12 months). We have also studied their T lymphocyte subpopulations by flow cytometry. After 3 months, patients were divided in responders (normal ALT or >.60% ALT reduction) or nonresponders (~60% ALT. reduction). Results: The NK activity of PBMC in responders (37.6% specific lysis at a 50/l effector-to-target cell ratio) was significantly lower than that found in nonresponder oatients (50.3%) (II < 0.01). Furthermore. the percentaoe of specific lysis measured before’treatment correlates strong and‘inverseqwith the percentage of decrease of ALT levels at third month of treatment (p = 0.006, R = 0.464). The correlation between the specffic lysis measured before treatment and the percentage of decrease of ALT after twelve months of treatment was also significant (p = 0.016, R = 0.461). These data show that the patients with the lowest levels of NK activity before IFNa2b treatment had the strongest decrease in ALT levels in response to this treatment. The percentage of CD4+ T lymphocytes in PMNC from responders patients was lower than that found in non responder patients. In contrast the percentages of CD6+ and CD16+ cells were in responder patients significantly higher than in non responders. CD4 to CD6 ratio was lower in responder patients than in non responder patients. Concfuslon: The clinical response of CHC patients to IFNaPb treatment is related to their odor NK actfvttv of their PBMC and to their distribution of T cell subsets. Thus, ‘immunologic parameters have prognostic value to predict the clinical response to IFNc2b in CHC patients suggesting that not only antiviral effects of Interferon but inmunomodulatory effects are also relevant in the therapeutic effect of IFNaPb treatment on HCV patients.
I..P 5 05 .02 1Soluble CD62E as a measure of an attenuated inflammatory response in sepsis patients treated with G-CSF B. Harms I, W. GroBWeege *, K. Dumont *, R.E. Scharf ‘, EM. Schneider ‘. ’ Immunology Laboratory. Institute of Hem&as& and Tmnsfusionsmedicine, University of Diisseldmf, Mcotenstr. 5, DiksekJmf, Germany, 2Department of Surgew University of Dijsseldorf, Mocrenstr. 5, Dtisseldorf. Germany Introduction: Inflammatory cytokines (IL-l, TNFa) induce the expression of adhesion molecules such as CD62E (El-AM-l) on endothelial cells. This selectin mediates the primary adhesion of neutmphils to endothelium via CD15, slex, LAM-1 and thus enables transendothelial migration. Material8and Methods:To study endothellal functions in states of acute inflammation we examined patients at risk of sepsis with or without treatment of mu G-CSF (I &kg daily for ten days) with regard to CD62E expression. Increased expression of endothelial CD62E also increases soluble CD62E (s CD62E) in the plasma of these patients which was madsorpted on granulocytes and can be quantified by flow cytometry using CD62E antibodies. IL-6 and soluble IL-6 receptor levels were measured by ELISA techniques. Results:Sofar, results of 14 patients, 7 G-CSF treated and 7 non-treated, am available. On the granulocytes of all patients tested we could detect CD62E with 26.94 f 17.09% on day 0 (begin of the study). This relative amount increased in the patients of both groups during the first days of analysis. The maximal CD62E detection levels on the granulocytes were on day 2.43 f 0.49 for the control group followed by a decline to day 0 levels. In the treatment group the peak of CD62E determined on granulocytes was later, on day 3.7 f 1.75. In addition, the inflammatory cytokfne IL-6 dropped to non-detectable levels in the plasma much earlier in the treatment group (day 1.5 f 0.5) as compared to the control group (day 3.63 f 3.3). Declining of IL-6 correlated to increases of IL-6 receptor which follows an anti-inflammatory response pattern induced by G-CSF. Conclusion: According to these findings, G-CSF affects endothelial cells by delavina the peak of shedded and madsorbed CD62E levels. Because of the reduced LAM-1 expression on granulocytes after G-CSF treatment (Weiss et al., Cytokine 8: 260-266, 1996) a delayed CD62E expmssicn on endothelial cells appears to be favourable for less non specific-binding of pmactivated neutrophils to endothelial cells. A substitution of G-CSF appears to counterregulate hyperactivation of proinflammatory process of endothelial cells.
P.5.05.03 U
Cytokines in the pathogenesis of the hu/mu SCID arthritis
H. Schadlich, J. Ermann, F. Sperling, W. Falk’, W.B. van den Berg*, F. Emmdch, U. Sack. institute of Clinical lmmunolcgy and Tmnsfusion Medicine, Univetsiiy of Leipzig, Netherlands, ’ Department of Medicine, University of RegensLwg, Netherlands, *Department of Ftheumatoi~ University Hospital, Nijmegen, Netherlands Introduction: Rheumatoid synovial membrane (RA-SM) is implanted into the knee joint of mice wfth severe combined immunodeffciency (SCID). In this hu/mu SCID-anhrftis model human cells induce an arthdtis dominated by attracted mudne inflammatory cells. Both murine IL-1 and TNF-czcould be detected in inflamed joints using immunohistochemistry on cryostat sections. We aimed to modulate the hu/mu SCID-arthritis by injection of anti-cytokine-antibodies. MaMaIs and Methods:Beginning on day 1 after implantation of RA-SM one group of mice was given intrapedtoneal injections of 2 mg of a polyclonal rabbit-anti-murine-IL-la and b-antibody once a week or 120 fig of a monoclonal rat-anti-mudne-TNFn-antibody every 4 days, respectively. Two other groups of mice were used as control, one of this treated with inactive isotype antibody. Joint swelling was measured weekly and serum levels of of murine IL-6 and human IL-6 were determined regularly during the experiment. All mice were sacrificed after 6 weeks. Cryocut sections of the knee joints were prepared for histology and immunohistochemistry. Resutts: Untreated mice developed considerable joint swelling postoperatively which had decreased to an intermediate level at 4 weeks post implantation. Mice treated with anti-TNF-antibody showed a shortening of this acute phase with significantly less swelling at 2 weeks. Anti-IL-l-antibody did not effect the acute phase. However the isotype antibody rabbit-IgG lead to diminished joint swelling. Joint destruction was not influenced by anti-IL-1 antibody therapy either. Human IL-6 could only be detected in the untreated gmup in the anti-IL-1 experiment, but neither in the other groups of this experiment nor in the groups of the anti-TNF experiment. Murine IL-6 was only detected in the anti-TNF experiment. On the first day after transplantation IL-6 was increased in all groups. On day 3.7 and 10 IL-6 could only determined in the group with specific antibody. Conclurlon: The aoolied antibodies influenced the hu/mu SCID-arthritis differently. TNF-a p1ays.adominant role in the acute phase of the inflammatory process. Other cytokines, such as IL-6, am controled by TNF-u and IL-1 in different ways.
P.5.04.03
promoter analysis of the matrix metalloprotelnase RASI-1, a novel autoantlgen In rheumatold arthritis
M. Mueller. S. Mauch, R. Sedlacek, U. Krawinkel. Departmentof /mmuno/og~! University of Konstanz, Konstanz, Germany introduction: The matrix metalloproteinase (MMP) RASI-1 was detected as a novel autoantigen in Rheumatoid Arthritis (RA) (1). Many MMPs seem to play a critical role in the pathology of systemic autoimmune diseases such as FiA (2, 3). In order to study the transcriptional regulation of RASI-1 we have isolated the complete rasi-7 gene and analyzed its 5’8anking sequence. This study allows first insights into possible regulatory mechanisms controlling the expression of RASI-1. Materials and Methods: We have isolated the complete rasi-I gene from a human genomic DNA library. The promoter/enhancer region has been sequenced and the transcription start site was identified by primer extension analysis. Potential DNA-binding sites for transcription factors were detected by database analysis. Fteaulta: We have found many potential DNA-binding sites for transcription factors, several of which are typical for matrtx metalloproteinases. The resi-I promoter contains a TATA-box at nucleotkte -29 relative to the transcription start site. We have detected a consensus sequence for the transcription factor AP-1 at position -73 and a putative PEA-3 element at position -155. This arrangement is typical for many MMPs (4). Furthermore potential binding sites for the transctiption factors NF-kappa& AP-2, SP-1 and elements which show similarity to the TGF-beta inhibitory element (TIE) and a retinoic acid response element (RARE) have been detected. In addition we recognized some other binding sites that can not be found in other MMPs. Conclusion: Taken together these data suggest that msi-I is a tightly controlled gene under physiological conditions resembling many of the regulatory characteristics of most other MMPs. Further experiments such as DNA-footprinting and promoter/reporter-gene assays have to proof the regulatory effect of the above mentioned transcription factors. Because MMPs play a crucial role in the pathogenesis of systemic autoimmune diseases, obtaining insights into their transcriptional regulation is of particular interest and may lead to the development of new therapy concepts to treat such disorders. [l] Sedlacek, R., Mauch, S., Eibel, H., Peter, H.-H. and Krawinkel, U. (1995) Immunobiol., 194,153 121 _ _ Bid&al-Hansen, H., Moore, W., Bodden, M.. Windsor, L., Birkedal-Hansen, B., DeCado, A. and Angler, J.(1993) Crit. Rev. Oral Biol. Med., 4 (2), 197-250 [3] Murphy, G. and Hembry, R. (1992) J. Rheumatol. Suppl., 32,61-84 [4] Mattisian, L. (1994) Ann. N. Y. Acad. Sci., 732, 42-50
P.5.04.04
Frequencies of HLA-ORB1 and HLA-DQBl alleles In patients buffering from vltlllgo
M. But’, H. FazekaSovB’, E. Hegyi 2, K. Kolib&Bov62, S. FerenEik3. ’ Department of Immunology, Faculty of Medicine, Comanius Univatsi~, Bmtislava, Slovakia, ‘Depamant of Darma~ology,Hospital Ru?inoy Bratislava, Slovakia, 31nstkuteof Immunoiog~ University Hospital, Essan, Germany Introduction: Our previous studies on the associations between vitiligo and HLA an&ens disclosed the increased frequency of HLA-Dw7 antigen (But et. al., Foliabiol. 42: 23-25, 1998). To confirmthe &lular typing results we retyped the studied group of patients for the frequencies of HIA-DRBl and HlA-DQBl alleles, resp&ti;ely. Matertal and Methods: 21 patients were investigated. 13 alleles of the HLA-DRBl locus and 17 alleles of the HLA-DQBl locus were determined by PCR-SSP method according to Qleriup et al. mssue Antigens, 39: 225-235, 1992). Results: The statistically significant deviations in frequencies of the following alleles were found: DRB1’0701 (28.5% compared to 15.3% in the healthy population, p .C 0.001) and WB1’0201 (38.0% compared to 21.1% in the control group, p < 0.0001). No other statistically significant differences were obsenred. Conctuslon: The allele DRB1’0701 encodes the antigen HLA-DR7 and the allele DQBl”O201 encodes HlA-Dw7, an epttope present on B-chain of the antigen HLA-DQ2. So DNA typing results confirm the previously recorded associations of HLA-DR7 and HLA-Dw7 antigens with vitiligo and indicate that HLA antigens do play a role in the immunopathogenesis of vitiligo.
Genetics of autoimmuni~
P.5.04.05
167
Study of the HLA-BZ;Nankyloslng spondylltls association about 34 cases
H. Makni 1.4,N. Lahlani-Mahfoudh ‘, S. Baktouti 2, 2. Bahloul 3, H. Ayadi 4.
’ Laboratoire d’hishxwmpatibili~6, EfS H. Chakac S&x, Tunisia,2San&a da Rhumatolwia, EPS H. Chakar, S&x, Tunisia,3Sawim da Medacina lnfeme. EPS H. Chakac Sfax, Tunisia,4La~tatoira d’lmmunobgia at da Biokqia Mol&u/aire, Faculte da hMdecine, Sfax, Tunisia Thirty four patients from south Tunisia fulfilled the new York criteria of ankylosing spondylitis (AS) and were phenotyped for HLA-A and -B loci. A complement dependent micrccytotoxictty assay was perfom?ed using a battery of local sera screened from multiparous women: A sample of 105 healthy and unrelated individuals was used as controls. Nineteen out of 34 patients (55.85%) were HLA-B27 positive whereas the HLA-B27 frequency was of 1.9% in controls. The relative Risk value (RR) was of 85.23: The most frequent haplotype associated to AS was A2 827. As for most populations worldwide, we found a significant increased frequency of HLA-B27 in patients with AS. Nevertheless, HLA-B27 was associated to AS at a lower frequency than in Caucasians. Moreover, the RR value found was lower than those reported in Caucasians. Our results showed an equal distribution of HLA-827 in the population of south Tunisia, in Kabyles and in Saudi Arabs. In addition, 15 AS patients of 34 (44.2%) lacked the HlA-B27 antigen. Altogether, these data suggest that besides the HLA-B27 antigen, additional genes might also be involved in the susceptibility to develop AS.
P-5.04.06
Polymorphism of the human lmmunoglobulln heavy chain locus In rheumatoid arthritis
F. Fakhfakh ‘, A. Maalej I, 2. Bahloul ’ , A. Jarraya ‘, H. Ayadi ‘, M.Zouali 2. ’ LaborMoire d’lmmunokgia at da Biologic Mol&ulaira, FaculM da M6decina. St&x, Tunisia,2LMpartement d’lmmunolcgia, lnsritot Pastaq 28, rue du Dr Roux, Paris, France The genetic origin of Rheumatoid Arthritis (RA) is largely unknown. The purpose of this investigation was to assess the potential genetically determined involvement of the immunoglobulin (lg) heavy chain variable region (VH) locus in the pathogenesis of RA. We tested the hypothesis of whether there is a genetic linkage between a structural abnormality of the VH gene complex and autoantibody hyperproduction in RA. We used restfiction endonuclease generated polymorphism in human VH gene-family specific probes to examine genomic DNA from a RA family and from unrelated RA patients from both the Tunisian and the European populations. The use of DNA samples from these ethnic origins permitted a further evaluation of the polymorphism of the humanVH locus. While we found that the polymorphism of the VH locus was lower in the Tunisian population, we could not detect a restriction site polymorphism pattern restricted to RA. Our results argue against a genetic linkage between RA and polymorphism in the VH locus. However it is possible that the method of restriction fragment length polymorphism was not able to detect subtle variations in DNAs of RA patients and, hence, reveal minor abnormalities in the VH locus of the RA patients. Likewise, genomic changes or somatic variation in a B-cell subset, both undetectable by our restriction fragment analysis, would need to be further investigated.
1P.5.04.07 ] Molecular analysis of Class II sequences Involved In autolmmune hepatitis In Mexicans M.N. Vtiquez-Garcia 1.3,C. Alaez ‘, A. Olivo ‘, H. Debaz I, E. P&z-Luque ‘, G. De La Rosa ‘, S. Cano ‘, A. Burguete ‘, J. Bandera 2, D. Kershenobich 2, C. Gorodezky ’ ’ Department of Immunogenetics, INDRE, SSA, Mexico Ci& Mexico, 2Gastroentarol~y Sarvica, lnsikuto National da la Nutriciidn,SSA, Mexico Ciw Mexiw, 3Prvgrama da Biomedicina Molecular CINVESTAV-IPN, Mexico Ci& Maxkw Introduction: Autoimmune hepatitis (AH) is a progressive condition of the liver characterized by piecemeal necrosis, the presence of diierent autoantibodies and by its prevalence in females. DRBl’O301 and DRBl l0401 are associated in Caucasians and DRB1.0405 is a susceptibility gene in Japanese. This is the first study done looking for HLA class I antigens and class II DNA alleles in Mexicans with the disease. Materials and Methods: We analyzed 30 Mexican Mestizo patients with type I AH compared to 175 healthy controls. The following autoantibodies were determined: (r-DNA, smooth muscle, ANA, mitocondrtal, thyroglobulin and LKMl (microsomal). Serotogical class I typing was done using the microcytotoxicity technique with magnetic beads and double fluorescence staining. DNA typing for DRBI, DQAl, and DQBl loci was performed with the protocols, primers and probes developed for the 12th International Wokshop. PCR-SSP was done to confirm certain DRBl, DQAl or WBI alleles. Frequencies were calculated and the most probable combinations were deduced.
168
23 June 1997 - Poster presentations
Cytokine-directed therapy
Results: The patients were mainly females (90%) with an X age at onset = 32.1 f 12.5 years. All of them had ANA and/or SMA antibodies but not LKMl ; and were negative for hepatitis virus B and C. Ten of them had another autoimmune disease (SLE, thyroiditis, Sj6gren and Fisher-Evans syndrome). No association was detected with dass I antigens. However, a significant increase was found for DRB1.0404 (pc = 0.0001; RR = 7.71). An important decrease of DQB1’0301 is informed for the first time (pc = 0.03) suggesting the presence of a strong DC&linkedresistance gene in Mexicans, which is DR independent. Conctualon: The 67-72 “motif” LLEQKR may be very important in the expression of AH, because the “hepatogenic peptide” leading to autoimmune recognition must have complementary sequencles for DRB1’0405 and DRB1.0404, which share the same residues at 67-72. On the other hand, DRB1*0301 and DRB1’0404 have only one substitution in position 71 (Rfor A), but both are basic aminoacids. Therefore, we propose that this “motif” is crucial for the hepatogenic peptide recognition and for enhancing the disease process. Moreover, DRB1’0301 is associated with early onset in Caucasians (~30 y) and in our patients the onset is early too (X = 32.1 y) with DRB1.0404 as the susceptibility gene. Both, DRB1’0301 and DRB1’0404 have a Vat position 66, contrasting with DRB1’0401 and DRB1’0405 that have a G and are associated with a late onset. Thus, these aminoacid substitutions in the antigen binding groove of the DR molecule, may determine disease susceptibility as well as severity. Understanding of the mechanisms of peptide binding are crucial for the development of novel prophylactic or treatment methods.
09:00-18:30/12:0&14:00
Forum lounges
P.5.05
Cytokine-directed therapy
P.5.05.01
Immunological pa&meters have predictive value on the clinical response to interferon-a therapy of hepatitis C patients
B. Moragues, A. Pdeto, J. Monserrat, A. Albillos’, R. Moreno *, G. Gacho 3, J. Calleja3, F. Esquivel, M. Barque*, M. Alvarez-Mon 4. ’ Hc@tat Ram& Y Cajai, hadrid, Skin, 3Hospital.’ PuerB de Hierro, Madrid. S&n, * Hospit& de la Princesa, Madrid, Spain, 4Hospital. Principe de Asturias, Universidad de Alcald, Madrid, Spain Introduction: Currently, there is a not valid marker to predict the heterogeneous clinical response chronic hepatitis C (CHC) patients to interferon IY(IFNa). The antiviral and the immunomodulator effects of IFNa appear to be involved in its mechanism of action. The aim of this study was to investigate the potential prognostic value of immunologic parameters to predict the clinical response to IFNaPb in CHC patients. Materials and Methods: We have evaluated the Natural Killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) and the expression of the CD2, 3, 4, 6, 16, 56 antigens and the proliferative response to PHA, antiCD3 monoclonal antibodies of T lymphocytes from 35 CHC patients before a standard course of IFNaPb (3 MU, t.i.w., 12 months). We have also studied their T lymphocyte subpopulations by flow cytometry. After 3 months, patients were divided in responders (normal ALT or >.60% ALT reduction) or nonresponders (~60% ALT. reduction). Results: The NK activity of PBMC in responders (37.6% specific lysis at a 50/l effector-to-target cell ratio) was significantly lower than that found in nonresponder oatients (50.3%) (II < 0.01). Furthermore. the percentaoe of specific lysis measured before’treatment correlates strong and‘inverseqwith the percentage of decrease of ALT levels at third month of treatment (p = 0.006, R = 0.464). The correlation between the specffic lysis measured before treatment and the percentage of decrease of ALT after twelve months of treatment was also significant (p = 0.016, R = 0.461). These data show that the patients with the lowest levels of NK activity before IFNa2b treatment had the strongest decrease in ALT levels in response to this treatment. The percentage of CD4+ T lymphocytes in PMNC from responders patients was lower than that found in non responder patients. In contrast the percentages of CD6+ and CD16+ cells were in responder patients significantly higher than in non responders. CD4 to CD6 ratio was lower in responder patients than in non responder patients. Concfuslon: The clinical response of CHC patients to IFNaPb treatment is related to their odor NK actfvttv of their PBMC and to their distribution of T cell subsets. Thus, ‘immunologic parameters have prognostic value to predict the clinical response to IFNc2b in CHC patients suggesting that not only antiviral effects of Interferon but inmunomodulatory effects are also relevant in the therapeutic effect of IFNaPb treatment on HCV patients.
I..P 5 05 .02 1Soluble CD62E as a measure of an attenuated inflammatory response in sepsis patients treated with G-CSF B. Harms I, W. GroBWeege *, K. Dumont *, R.E. Scharf ‘, EM. Schneider ‘. ’ Immunology Laboratory. Institute of Hem&as& and Tmnsfusionsmedicine, University of Diisseldmf, Mcotenstr. 5, DiksekJmf, Germany, 2Department of Surgew University of Dijsseldorf, Mocrenstr. 5, Dtisseldorf. Germany Introduction: Inflammatory cytokines (IL-l, TNFa) induce the expression of adhesion molecules such as CD62E (El-AM-l) on endothelial cells. This selectin mediates the primary adhesion of neutmphils to endothelium via CD15, slex, LAM-1 and thus enables transendothelial migration. Material8and Methods:To study endothellal functions in states of acute inflammation we examined patients at risk of sepsis with or without treatment of mu G-CSF (I &kg daily for ten days) with regard to CD62E expression. Increased expression of endothelial CD62E also increases soluble CD62E (s CD62E) in the plasma of these patients which was madsorpted on granulocytes and can be quantified by flow cytometry using CD62E antibodies. IL-6 and soluble IL-6 receptor levels were measured by ELISA techniques. Results:Sofar, results of 14 patients, 7 G-CSF treated and 7 non-treated, am available. On the granulocytes of all patients tested we could detect CD62E with 26.94 f 17.09% on day 0 (begin of the study). This relative amount increased in the patients of both groups during the first days of analysis. The maximal CD62E detection levels on the granulocytes were on day 2.43 f 0.49 for the control group followed by a decline to day 0 levels. In the treatment group the peak of CD62E determined on granulocytes was later, on day 3.7 f 1.75. In addition, the inflammatory cytokfne IL-6 dropped to non-detectable levels in the plasma much earlier in the treatment group (day 1.5 f 0.5) as compared to the control group (day 3.63 f 3.3). Declining of IL-6 correlated to increases of IL-6 receptor which follows an anti-inflammatory response pattern induced by G-CSF. Conclusion: According to these findings, G-CSF affects endothelial cells by delavina the peak of shedded and madsorbed CD62E levels. Because of the reduced LAM-1 expression on granulocytes after G-CSF treatment (Weiss et al., Cytokine 8: 260-266, 1996) a delayed CD62E expmssicn on endothelial cells appears to be favourable for less non specific-binding of pmactivated neutrophils to endothelial cells. A substitution of G-CSF appears to counterregulate hyperactivation of proinflammatory process of endothelial cells.
P.5.05.03 U
Cytokines in the pathogenesis of the hu/mu SCID arthritis
H. Schadlich, J. Ermann, F. Sperling, W. Falk’, W.B. van den Berg*, F. Emmdch, U. Sack. institute of Clinical lmmunolcgy and Tmnsfusion Medicine, Univetsiiy of Leipzig, Netherlands, ’ Department of Medicine, University of RegensLwg, Netherlands, *Department of Ftheumatoi~ University Hospital, Nijmegen, Netherlands Introduction: Rheumatoid synovial membrane (RA-SM) is implanted into the knee joint of mice wfth severe combined immunodeffciency (SCID). In this hu/mu SCID-anhrftis model human cells induce an arthdtis dominated by attracted mudne inflammatory cells. Both murine IL-1 and TNF-czcould be detected in inflamed joints using immunohistochemistry on cryostat sections. We aimed to modulate the hu/mu SCID-arthritis by injection of anti-cytokine-antibodies. MaMaIs and Methods:Beginning on day 1 after implantation of RA-SM one group of mice was given intrapedtoneal injections of 2 mg of a polyclonal rabbit-anti-murine-IL-la and b-antibody once a week or 120 fig of a monoclonal rat-anti-mudne-TNFn-antibody every 4 days, respectively. Two other groups of mice were used as control, one of this treated with inactive isotype antibody. Joint swelling was measured weekly and serum levels of of murine IL-6 and human IL-6 were determined regularly during the experiment. All mice were sacrificed after 6 weeks. Cryocut sections of the knee joints were prepared for histology and immunohistochemistry. Resutts: Untreated mice developed considerable joint swelling postoperatively which had decreased to an intermediate level at 4 weeks post implantation. Mice treated with anti-TNF-antibody showed a shortening of this acute phase with significantly less swelling at 2 weeks. Anti-IL-l-antibody did not effect the acute phase. However the isotype antibody rabbit-IgG lead to diminished joint swelling. Joint destruction was not influenced by anti-IL-1 antibody therapy either. Human IL-6 could only be detected in the untreated gmup in the anti-IL-1 experiment, but neither in the other groups of this experiment nor in the groups of the anti-TNF experiment. Murine IL-6 was only detected in the anti-TNF experiment. On the first day after transplantation IL-6 was increased in all groups. On day 3.7 and 10 IL-6 could only determined in the group with specific antibody. Conclurlon: The aoolied antibodies influenced the hu/mu SCID-arthritis differently. TNF-a p1ays.adominant role in the acute phase of the inflammatory process. Other cytokines, such as IL-6, am controled by TNF-u and IL-1 in different ways.