Peter A. Berg

Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis

Anti‐M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha‐ketoacid dehydrogenase complex of the inner mitochondria! membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC). 70 kD; protein X of the PDC, 56 kD; alpha‐ketoglutarate dehydrogenase complex, 52 kD; branched‐chain alpha‐kctoacid dehydrogenase, 52 kD; Elalpha subunit of PDC, 45 kD; and Elbeta‐subunit of PDC, 36 kD). These epilopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria. The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera. However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC‐E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the rccombinant PDC‐E2. In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC‐specific epilope at 70 kD. Naturally occurring mitochondrial antibodies (NOM A) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders. They recognized cither a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta).None of the sera from 100 blood donors had anti‐M2 but 14 had NOM A. Testing anti‐M2 and NOMA‐positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E. coli it could be shown that‐like mammalian mitochondria—they contain both the PBC‐specific M2 antigen as well as the non‐PBC‐specific naturally occurring mitochondrial antigen system (NOM Ag). The observation that PBC‐specific antibodies were preferentially induced in patients suffering from a mycobactcrial infection may provide some new clues to the still unknown etiology of PBC.

Clinical relevance of antibodies against serotonin and gangliosides in patients with primary fibromyalgia syndrome

The fibromyalgia syndrome (FMS) is a non-articular rheumatic disorder associated with disturbances in serotonin metabolism. In order to evaluate whether patients with FMS suffer from an autoimmune disorder, we tested sera from 50 clinically well-defined FMS patients for non-organ-specific and organ-specific antibodies by enzyme-linked immunosorbent assay and immunofluorescence test. Common antibodies against nuclei, mitochondria, and microsomes were not increased in these patients compared to healthy controls. However, 74% had antibodies against serotonin and gangliosides. The clinical and diagnostic relevance of these antibodies is supported by the absence of anti-serotonin antibodies in other rheumatic disorders such as rheumatoid arthritis, polymyalgia rheumatica, and collagen diseases. These antibodies may belong to the group of antireceptor antibodies, considering the fact that gangliosides are an important component of the serotonin receptor. It remains to be determined whether these antibodies are of pathogenetic relevance, interfering with serotonin binding and thereby inducing symptoms associated with FMS.

Effects of Clozapine on In Vitro Immune Parameters: A Longitudinal Study in Clozapine-Treated Schizophrenic Patients

Clozapine is an atypical antipsychotic agent with immunomodulatory properties. We hypothesized that in vitro immune parameters of peripheral blood mononuclear cells (PBMC) are affected in the course of clozapine treatment and that clozapine per se, added in vitro to PBMC cultures of clozapine-treated patients, exerts differential effects in the timecourse of treatment in vivo. We measured proliferation and cytokine secretion of PBMC, serum autoantibodies, and immunoglobulin levels in 17 patients before and during the first 6 weeks of clozapine treatment. Independent of clozapine dosage and rectal temperature, clozapine treatment in vivo suppressed proliferation and shedding of sIL-2r by PBMC, and the addition of clozapine in vitro induced, relative to unstimulated conditions, PBMC proliferation and secretion of IL-6 and sIL-2r. Serum IgG levels were increased; whereas, autoantibody pattern was unaffected. Thus, clozapine treatment and the addition of clozapine in vitro exert differential effects on various in vitro immune parameters independent of clozapine dosage and rectal temperature in the course of treatment.

The lymphocyte transformation test--a debated method for the evaluation of drug allergic hepatic injury.

In this study, 42 hepatitis C virus (HCV)-infected patients included in different treatment protocols were assessed by semiquantitative Amplicor assay to evaluate the changes in HCV-RNA titer during antiviral therapy. Amplicor detected change to negative HCV-RNA at the end of treatment in four biochemically responding patients; a decrease in HCV-RNA titer from base line was observed in 2/6 complete responder patients, in 7/11 responders with relapse, and in 6/21 nonresponder patients. Furthermore, Amplicor also detected an increase in HCV-RNA titer from the end of treatment to the follow-up sample in 3/6 complete responder patients, in 7/11 responders with relapse, and in 3/6 nonresponder patients in whom HCV-RNA titer at the end of treatment was lower than in the basal sample. None of the nonresponder patients showed a change to negative HCV-RNA either at the end of treatment or at follow-up. When comparing the detection of HCV-RNA by this semiquantitative method and by nested-PCR, we found a concordance in 113/118 polymerase chain reaction (PCR) results (96%), including three negative results by both methods. With respect to the nonconelated results, negative PCR samples were found to be positive by Amplicor assay in three cases (correspondent to final samples of treatment); by contrast, two positive PCR samples were found to be negative by Amplicor assay (correspondent to final and follow-up samples of different patients)

Serology of Primary Biliary Cirrhosis

The aetiology of primary biliary cirrhosis (PBC) still remains unknown. Many features point towards an autoimmune disorder [25]: the female preponderance, the familial aggregation, the association with other autoimmune conditions, the protracted course, the presence of various types of antibodies (smooth muscle (SMA), nuclear (ANA), and especially mitochondrial antibodies (AMA)), and the histological lesions (showing granuloma formation and infiltration of bile ducts by lymphocytes). In addition in a high percentage of patients circulating cryoglobulins have been detected [91] and there is a striking abnormality of the complement system [45] and impairment of cell mediated immunity [62]. Also cellular immunity against biliary antigens [64] and mitochondria [18] has been described, and patients’ lymphocytes also seem to interfere in vitro with some mitochondrial functions [16, 24]. AMA apparently play no role in the pathogenesis of PBC but their incidence in almost 90% of all PBC patients led to the speculation that these antibodies could be related to the aetiology of the disease.

Recurrent first trimester spontaneous abortion associated with antiphospholipid antibodies: A pilot study of treatment with intravenous immunoglobulin

Background. Antiphospholipid antibodies, unassociated with an underlying connective tissue disease, have repeatedly been detected in women suffering from recurrent spontaneous abortions. Several therapeutic regimens have been advocated for pregnant women with recurrent fetal loss and antiphospholipid antibodies. However, most of these approaches were empirical, using several drugs simultaneously, and most reports describe single cases or limited series.

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes

Anti‐liver‐kidney microsome antibodies (anti‐LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti‐LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti‐LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti‐LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti‐LKM‐positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254–339 (254–273, 257–269, 270–294, 291–310, 307–324, 321–339, 373–389) as well as the novel peptide 196–218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6‐dependent bufuralol 1′‐hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257–269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196–218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254–273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti‐LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti‐LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257–269 a further immunodominant epitope exists on CYP2D6 which is recognized by sera from patients with AIH II but hardly by sera from patients with hepatitis C.

Antimitochondrial antibody profiles in patients with primary biliary cirrhosis before orthotopic liver transplantation and titres of antimitochondrial antibody-subtypes after transplantation

Four antimitochondrial antibody profiles (A-D) have been defined in primary biliary cirrhosis according to the presence of antibodies to M2, M4, M8, and M9 in ELISA and the complement fixation test: A: anti-M9 positive in ELISA and western blot, B: anti-M9 and/or anti-M2 positive in ELISA, C: anti-M2, -M4 and/or -M8 positive in ELISA, D: anti-M2, -M4, and/or -M8 positive in ELISA and complement fixation test. These profiles predict the outcome of primary biliary cirrhosis in the early stages and reflect differences in the natural course of the disease (benign versus progressive). In this study sera from 29 patients with advanced primary biliary cirrhosis who had received liver transplant were retested before and after orthotopic liver transplantation. Twenty-eight were antimitochondrial antibody/anti-M2 positive, and one patient had only antibodies to nuclear dots in the immunofluorescence test on cell cultures. When the antimitochondrial antibody-profiles in these 28 anti-M2 positive patients were analysed, it became evident that 26 of them belonged to subgroup C or D before orthotopic liver transplantation. Two patients had profile B; one had high titres of antinuclear and smooth muscle antibodies indicating an overlap syndrome between primary biliary cirrhosis and autoimmune chronic active hepatitis. The other patient had antibodies to nuclear dots in association with anti-M2. None of the patients had profile A. Antibody titres were studied after orthotopic liver transplantation in 23 of the 28 patients who survived for 1 to 13 years.(ABSTRACT TRUNCATED AT 250 WORDS)

CMV iNFECTION AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION IS ASSOCIATED WITH THE OCCURRENCE OF VARIOUS AUTOANTIBODIES AND MONOCLONAL GAMMOPATHIES

Recent findings indicate that the kinetics of B‐cell reconstitution after marrow transplantation mimic normal ontogeny. The early B‐cell repertoire during ontogeny is characterized by a high degree of autoreactivity and interconnectivity. Therefore, in a prospective analysis, 95 consecutive recipients of an allogeneic marrow transplant were screened for the occurrence of various autoantibodies and 47 of these 95 were also screened for monoclonal gammopathies. None of the patients developed antibodies specific for systemic autoimmune disorders. In contrast, a high prevalence of natural antibodies (79/95) was found early post‐transplant, with 58 of these 79 patients developing two or more autoantibodies. According to multiple regression, the mean number of natural antibodies (95% confidence limits in parentheses) depends significantly (P = 0.006) on the status of CMV infection: 0.9 (0.4; 1.6) CMV‐negative; 2.0 (1.0; 3.3) asymptomatic CMV infection; 3.1 (1.7; 5.0) CMV disease. Sex, age, underlying disease, conditioning therapy, acute graft‐versus‐host disease and CMV serology of donor and recipient pretransplant did not affect the number of natural autoantibodies. Monoclonal gammopathies were detected in 12/47 patients with a predominance of the IgG‐kappa subtype. All these 12 patients suffered from a viral infection (CMV, n = 11; influenza strain A, n = 1). The high degree of self‐reactivity post‐transplant further supports the hypothesis that B‐cell reconstitution mimics ontogeny. Moreover, these data indicate nonspecific polyclonal, CMV‐mediated, presumably T‐cell independent B‐cell stimulation and disturbed T‐cell regulatory function following allogeneic BMT.

11 Immunology of primary biliary cirrhosis

Abstract 1. (1) The serological diagnosis of PBC is possible in almost 100% of cases when appropriate methods and specific antigen preparations are used such as the purified ATPase fraction by ELISA for the detection of anti-M2, sonicated mitochondria by immunodiffusion for the demonstration of precipitating antibodies against M-A or M-B, and cell cultures by immunofluorescence for the detection of antibodies against nuclear dots. 2. (2) The establishment of AMA profiles obtained by ELISA and CFT seems to be a sensitive approach to a better definition of the natural course of PBC. A distinction between a rather benign and a more progressive course seems especially possible in the presence of the AMA profiles A and B (anti-M9 and/or anti-M2-positive only by ELISA) versus D (anti-M2-, anti-M4-, anti-M8-positive in the CFT). 3. (3) The analysis of cellular immune reactions in vitro and in vivo suggests an activation of cytotoxic T cells as well as a defect in the function of T suppressor cells. 4. (4) Although the aetiology of PBC is unknown, the detection of MHC Class II antigens on bile duct epithelial cells in liver biopsies of patients with PBC but not of normal individuals may imply that an infectious agent being exposed in association with these MHC structures may trigger the disease. The inability of the immune system in controlling this infectious process would then lead to an ongoing inflammatory reaction which is responsible for the continuous destruction of bile ducts within portal triads.