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Synovial tissue B-lymphocytes as source for the generation of human monoclonal antibodies
480
25 June 1997 - Poster presentations
Autoantibodies
idiotope is decreased only in the sera of ALS patients. (This work was supported by the Ministry of Science, Belgrade, Serbia.)
P.5.21.26
Synovlal tissue B-lymphocytes as source for the generatlon of human monoclonal antibodles
P. von Landenberg, I. Melchers. Clinical Research Unit on Rheumato/w University Medical Center, F&burg, Germany
Introduction: B-cell reactivities in the affected joints of patients with rheumatoid arthritis probably are an important factor in the chronic inflammation process. Our aim is to characterize the antibodies, being directly associated with the synovial tissue damage, in terms of specificity and V gene usage. For immortalisation of synovial B-lymphocytes from patients with rheumatoid arthritis we use the method of electrofusion. This technique allows to produce B-cell hybridomas with high fusion efficiency even from mixed cell populations containing only small numbers of &lymphocytes. Materials and Methods: Synovial tissue B-lymphocytes from patients with rheumatoid arthritis (defined according to the criteria of the American Rheumatism Association) were prepared by collagenase treatment and mechanical dissection. The viable leucocyte population in neatly all used samples contained only about 3% CD19 positive B-lymphocytes. The total number of mononuclear cells obtained after this treatment varied from 1 x IO5 to 5 x 105. This population was then immortalized by electrofusion without any in vitro stimulation or prior B-cell-selection. From this cell population we obtained B-cell hybrtdomas in a frequency of 1 in 10.000 total cells, i.e. 1 clone from 300 B-lymphocytes. Reeuh Up to now we established 10 antibody secreting hybrfdomas from synovial fluid and tissue. B-lymphocytes isolated from synovial fluid (1 patient) and from synovial tissue (2 patients) were used for electmfusion. 4 hybridomas secreted IgA and 6 hybrfdomas secreted IgG. Conclusion: Electrofusion apperantty allows to fuse B cells in a broader range of differentiation stages than conventional techniques. Synovial B-cells differ from peripheral B-cells in several aspects. Therefore the production of synovial hybtidomas is up to now difficult and rarely reported. With this technique we are now able to investigate the local B-cetl response in the synovial tissue of RA-patients. And we can expect, that antibody specificities detected with these monoclonal antibodies might reflect relevant autoantigens involved in the local inflammation process. Supported by a DFG fellowship (La 1051/l) and a DFG grant (Me 604./4).
( P.5.21.27 1 Prevalence of antlneutrophll cytoplasmlc antlbody (ANCA) In patients with autolmmune hepatltls 6. Malenica l, A. KrstuloviCl, R.OstojiC*, B. VuceliC*, D. BatlniCl. 1Div Immunol, Dept Lab Diagnosis, Zagreb Univ School A&d, Zagreb, Zagreb, Croatia, 2Div Gastroenterolog~ Dept lntemal Med., C/in Hosp Center Zagreb, Zagreb, Croatia
Introduction:ANCA autoantibodies have been described as a sensitive immunoserologic marker of certain types of systemic vasculiis, especially Wegener’s granulomatosis, microscopic arterftis and idiopathic crescentic glomerulonephritis. A few studies have also shown that patients with some inflammatory bowel and hepatobiliary diseases have autoantibodies directed against neutrophils, which am a different specificity than those associated with vasculitis. The prevalence, titer and clinical correlation of these antibodies we investigated in patients with various hepatobiliary diseases. PatIentaand Methodr:The ANCA was analyzed in 141 serum samples. The presence of ANCA was detected on ethanol-fixed cytospin preparations of peripheral blood neutmphils by indirect immunofluorescence. A perfnuclear (pANCA) and atypical (aANCA) staining pattern was considered positive. Westem blot was used for detection of autoantibodies which characterized different subtypes od autoimmune hepatitis. Reeut& Both pANCA and aANCA was detected in 20% (7/34) of patients with autoimmune hepatitis, in 7% (l/15) of patients with primary biliary cinhosis and in 4% (l/26) of patients with biliary cirrhosis. ANCA did not find in patients with chronic hepatitis (o/23) and in a group of patients with valious hepatobiliary disorders (O/43).The ANCA autoantibodies was selectively observed in patients with type 1 autoimmune hepatitis @/IO; 60%). Conclusion: These results indicate that ANCA can be used as useful clinical tool to distinguish different subtypes of autoimmune hepatitis.
1P.5.21.28 1 Antlbodles agalnst cardlollpln and oxLDL In lilkyear-old men predict myocardlal lnfarctlon a prospective 20 years’ study Ruihua Wu’ , Soniya Nityanand l, Larsberglund 2, Hans Lithell *, G&an Hdm ‘, Ann Karl Lefverl ‘. ‘Medicineof Dept. Karvlinska Hospital, Stc&ho/m, Sweden, *Department of Geriatrics, University of Uppsala, Uppsala, Sweden Abstract: Autoantibodies against oxidatively modified low density lipoproteins (oxLDL) and cardiolipin occur in patients with vascular diseases, including atherosclerosis. The ability of such antibodies to predict myocardial infarctton (Ml) was investigated in a prospective nested case control study in samples collected from 2322 males aged 50 years in Uppsata, Sweden during 197&1972. This cohort made up 82% of all 50 year old men living in Uppsala at that time. The cohort was then followed for 20 years. Autoantibodies against oxLDL and cardiolipin were measured with an enzyme-linked immunosorbent assay method. Raised levels of antibodies against oxLDL and cardidipin at 50 years of age correlated positively with the incidence of Ml and mortality related to Ml 10 to 20 years later. IgG and IgA antibodies against cardidipin were associated with Ml between 50-60 of age and IgG and IgA antibodies against oxLDL with Ml at 60-70 yean of age. Moreover, higher antibody levels were noted in those who died from acute Ml in comparison to those who survived. The predictive power of IgA and IgG antfbodies was strong and independent of that of other strong risk factors. In conclusion, raised levels of antibodies against oxLDL and cardiolipin may predict Ml and Ml-related death.
P.5.21.29 LI_l
The spectrum of autoantlbodles In HIV seroposltlve subjects in varlous stages of HIV infection
A. Tsimyianni, M. Economou, K. Terzoglou. J. Economidou. Deperhnent of lmmunolosv-Hist~m~ocompatibiliiy, Evangelismos Hospital, Athens, Greece
Intoduction:Autoimmune reactions and different types of autoantibodies (AA) have been mcognised in HIV infection. This study analyses serological aspects of organ-specific AA (PCA, AMA, SMA, ATA), non organ-specific AA (ANA, antidsDNA, anti-CL, anti-histones) and anti-neutrophil antibodies (ANCA), in individuals with different stages of HIV infection according to CDC classification (1993). MaterialandMethods:Sera from two groups of HIV-l seropositive subjects were investigated. Group 1 consisted of 24 asymptomatic carders (stage Al and A2) and group 2, of 23 patients with AIDS (stages A3,83, C). For the detection of AA, indirect immunofluorescence (IIF) and ELISA techniques were used. Results:The percentage of the various AA detected is shown in the table. Autoantibodies
Al(N=lO)%
AZ (N = 14)%
A3,S3,C(N=32)%
WA
0
14.3
ANA
0
7.1
anti-DNA anti-histones anti-cardiolipin IgG anti-cardiolipin IgM ANCA -
0
0
0
50
64.3
58.4
20 0
14.3 0
30
14.3
Q-4 8.2
6.3 6.3 53.1
Concluelon:In general, fewer specificities and a lower frequency of AA was found in stage Al than in stage A2 or in AIDS, with the exception of anti-histones AA which occured with equal frequency in all stages. ANCA were found to be especially increased in AIDS, they were exclusively C-ANCA and with an atypical staining pattern in IIF. These results indicate that a variety of mechanisms including apoptosis may be implicated for the immunological disregulation resulting in autoimmune reactions against a wide spectrum of autoantigens. 1P.5.21.30 ] Frequency and speclflcltles anti-neutrophll antlbodles (ANCA) In Inflammatory bowel disease M. Economou, A. Tsimyianni, K. Tetzoglou, J. Economidou. Department of Immunolcgy-Hi&compatibility, Evangelismos Hospital, Athens, Greece Introduction: Anti-neutmphil antibodies producing a p-ANCA pattern by indirect immunofluorescense (IIF) have been detected recently in sera of patients with inflammatory bowel disease (IBD). To evaluate their clinical significance, sera from patients with ulceratiie colitis (UC) and Crohn’s disease (CD) in phases with active and inactive disease have been investigated. Yaterlal and Methods: Sera from Q4 patients with IBD (69 with UC and 25 with CD) were tested for the detection of ANCA using IIF and ELISA techniques forthespecificitiesanti-PRS, anti-MPO, anti-elastase (E), anti-cathepsin G (C-G) and anti-lactoferrin (1). Reeulk The frequency of ANCA is shown in the table. No statistically significant difference was found between males and females with UC and CD
25 June 1997 - Poster presentations
Autoantibodies
idiotope is decreased only in the sera of ALS patients. (This work was supported by the Ministry of Science, Belgrade, Serbia.)
P.5.21.26
Synovlal tissue B-lymphocytes as source for the generatlon of human monoclonal antibodles
P. von Landenberg, I. Melchers. Clinical Research Unit on Rheumato/w University Medical Center, F&burg, Germany
Introduction: B-cell reactivities in the affected joints of patients with rheumatoid arthritis probably are an important factor in the chronic inflammation process. Our aim is to characterize the antibodies, being directly associated with the synovial tissue damage, in terms of specificity and V gene usage. For immortalisation of synovial B-lymphocytes from patients with rheumatoid arthritis we use the method of electrofusion. This technique allows to produce B-cell hybridomas with high fusion efficiency even from mixed cell populations containing only small numbers of &lymphocytes. Materials and Methods: Synovial tissue B-lymphocytes from patients with rheumatoid arthritis (defined according to the criteria of the American Rheumatism Association) were prepared by collagenase treatment and mechanical dissection. The viable leucocyte population in neatly all used samples contained only about 3% CD19 positive B-lymphocytes. The total number of mononuclear cells obtained after this treatment varied from 1 x IO5 to 5 x 105. This population was then immortalized by electrofusion without any in vitro stimulation or prior B-cell-selection. From this cell population we obtained B-cell hybrtdomas in a frequency of 1 in 10.000 total cells, i.e. 1 clone from 300 B-lymphocytes. Reeuh Up to now we established 10 antibody secreting hybrfdomas from synovial fluid and tissue. B-lymphocytes isolated from synovial fluid (1 patient) and from synovial tissue (2 patients) were used for electmfusion. 4 hybridomas secreted IgA and 6 hybrfdomas secreted IgG. Conclusion: Electrofusion apperantty allows to fuse B cells in a broader range of differentiation stages than conventional techniques. Synovial B-cells differ from peripheral B-cells in several aspects. Therefore the production of synovial hybtidomas is up to now difficult and rarely reported. With this technique we are now able to investigate the local B-cetl response in the synovial tissue of RA-patients. And we can expect, that antibody specificities detected with these monoclonal antibodies might reflect relevant autoantigens involved in the local inflammation process. Supported by a DFG fellowship (La 1051/l) and a DFG grant (Me 604./4).
( P.5.21.27 1 Prevalence of antlneutrophll cytoplasmlc antlbody (ANCA) In patients with autolmmune hepatltls 6. Malenica l, A. KrstuloviCl, R.OstojiC*, B. VuceliC*, D. BatlniCl. 1Div Immunol, Dept Lab Diagnosis, Zagreb Univ School A&d, Zagreb, Zagreb, Croatia, 2Div Gastroenterolog~ Dept lntemal Med., C/in Hosp Center Zagreb, Zagreb, Croatia
Introduction:ANCA autoantibodies have been described as a sensitive immunoserologic marker of certain types of systemic vasculiis, especially Wegener’s granulomatosis, microscopic arterftis and idiopathic crescentic glomerulonephritis. A few studies have also shown that patients with some inflammatory bowel and hepatobiliary diseases have autoantibodies directed against neutrophils, which am a different specificity than those associated with vasculitis. The prevalence, titer and clinical correlation of these antibodies we investigated in patients with various hepatobiliary diseases. PatIentaand Methodr:The ANCA was analyzed in 141 serum samples. The presence of ANCA was detected on ethanol-fixed cytospin preparations of peripheral blood neutmphils by indirect immunofluorescence. A perfnuclear (pANCA) and atypical (aANCA) staining pattern was considered positive. Westem blot was used for detection of autoantibodies which characterized different subtypes od autoimmune hepatitis. Reeut& Both pANCA and aANCA was detected in 20% (7/34) of patients with autoimmune hepatitis, in 7% (l/15) of patients with primary biliary cinhosis and in 4% (l/26) of patients with biliary cirrhosis. ANCA did not find in patients with chronic hepatitis (o/23) and in a group of patients with valious hepatobiliary disorders (O/43).The ANCA autoantibodies was selectively observed in patients with type 1 autoimmune hepatitis @/IO; 60%). Conclusion: These results indicate that ANCA can be used as useful clinical tool to distinguish different subtypes of autoimmune hepatitis.
1P.5.21.28 1 Antlbodles agalnst cardlollpln and oxLDL In lilkyear-old men predict myocardlal lnfarctlon a prospective 20 years’ study Ruihua Wu’ , Soniya Nityanand l, Larsberglund 2, Hans Lithell *, G&an Hdm ‘, Ann Karl Lefverl ‘. ‘Medicineof Dept. Karvlinska Hospital, Stc&ho/m, Sweden, *Department of Geriatrics, University of Uppsala, Uppsala, Sweden Abstract: Autoantibodies against oxidatively modified low density lipoproteins (oxLDL) and cardiolipin occur in patients with vascular diseases, including atherosclerosis. The ability of such antibodies to predict myocardial infarctton (Ml) was investigated in a prospective nested case control study in samples collected from 2322 males aged 50 years in Uppsata, Sweden during 197&1972. This cohort made up 82% of all 50 year old men living in Uppsala at that time. The cohort was then followed for 20 years. Autoantibodies against oxLDL and cardiolipin were measured with an enzyme-linked immunosorbent assay method. Raised levels of antibodies against oxLDL and cardidipin at 50 years of age correlated positively with the incidence of Ml and mortality related to Ml 10 to 20 years later. IgG and IgA antibodies against cardidipin were associated with Ml between 50-60 of age and IgG and IgA antibodies against oxLDL with Ml at 60-70 yean of age. Moreover, higher antibody levels were noted in those who died from acute Ml in comparison to those who survived. The predictive power of IgA and IgG antfbodies was strong and independent of that of other strong risk factors. In conclusion, raised levels of antibodies against oxLDL and cardiolipin may predict Ml and Ml-related death.
P.5.21.29 LI_l
The spectrum of autoantlbodles In HIV seroposltlve subjects in varlous stages of HIV infection
A. Tsimyianni, M. Economou, K. Terzoglou. J. Economidou. Deperhnent of lmmunolosv-Hist~m~ocompatibiliiy, Evangelismos Hospital, Athens, Greece
Intoduction:Autoimmune reactions and different types of autoantibodies (AA) have been mcognised in HIV infection. This study analyses serological aspects of organ-specific AA (PCA, AMA, SMA, ATA), non organ-specific AA (ANA, antidsDNA, anti-CL, anti-histones) and anti-neutrophil antibodies (ANCA), in individuals with different stages of HIV infection according to CDC classification (1993). MaterialandMethods:Sera from two groups of HIV-l seropositive subjects were investigated. Group 1 consisted of 24 asymptomatic carders (stage Al and A2) and group 2, of 23 patients with AIDS (stages A3,83, C). For the detection of AA, indirect immunofluorescence (IIF) and ELISA techniques were used. Results:The percentage of the various AA detected is shown in the table. Autoantibodies
Al(N=lO)%
AZ (N = 14)%
A3,S3,C(N=32)%
WA
0
14.3
ANA
0
7.1
anti-DNA anti-histones anti-cardiolipin IgG anti-cardiolipin IgM ANCA -
0
0
0
50
64.3
58.4
20 0
14.3 0
30
14.3
Q-4 8.2
6.3 6.3 53.1
Concluelon:In general, fewer specificities and a lower frequency of AA was found in stage Al than in stage A2 or in AIDS, with the exception of anti-histones AA which occured with equal frequency in all stages. ANCA were found to be especially increased in AIDS, they were exclusively C-ANCA and with an atypical staining pattern in IIF. These results indicate that a variety of mechanisms including apoptosis may be implicated for the immunological disregulation resulting in autoimmune reactions against a wide spectrum of autoantigens. 1P.5.21.30 ] Frequency and speclflcltles anti-neutrophll antlbodles (ANCA) In Inflammatory bowel disease M. Economou, A. Tsimyianni, K. Tetzoglou, J. Economidou. Department of Immunolcgy-Hi&compatibility, Evangelismos Hospital, Athens, Greece Introduction: Anti-neutmphil antibodies producing a p-ANCA pattern by indirect immunofluorescense (IIF) have been detected recently in sera of patients with inflammatory bowel disease (IBD). To evaluate their clinical significance, sera from patients with ulceratiie colitis (UC) and Crohn’s disease (CD) in phases with active and inactive disease have been investigated. Yaterlal and Methods: Sera from Q4 patients with IBD (69 with UC and 25 with CD) were tested for the detection of ANCA using IIF and ELISA techniques forthespecificitiesanti-PRS, anti-MPO, anti-elastase (E), anti-cathepsin G (C-G) and anti-lactoferrin (1). Reeulk The frequency of ANCA is shown in the table. No statistically significant difference was found between males and females with UC and CD