Reinhild Klein

Clinical significance of antibodies against neutrophils in patients with inflammatory bowel disease and primary sclerosing cholangitis.

The presence of perinuclear antibodies against neutrophils (pANCA) has been detected recently in sera of patients with inflammatory bowel disease and primary sclerosing cholangitis. In order to evaluate their clinical significance, sera from 126 patients with inflammatory bowel disease (80 Crohns disease and 46 ulcerative colitis and 22 patients with primary sclerosing cholangitis were examined for pANCA by indirect immunofluorescence on liver sections and cytocentrifuge slides of neutrophils and by immunoblot. Perinuclear antibodies against neutrophils were found in 83% of patients with ulcerative colitis in 88% of patients with primary sclerosing cholangitis and inflammatory bowel disease, in 40% of patients with primary sclerosing cholangitis but without inflammatory bowel disease, and in 25% of patients with Crohns disease using the immunofluorescence test. Titres of pANCA ranged from 1:10 to 1:1000 in ulcerative colitis and primary sclerosing cholangitis (median 1:100), whereas in Crohns disease only four patients had titres of more than 1:10. The occurrence of pANCA did not correlate with clinical activity of Crohns disease and primary sclerosing cholangitis whereas in ulcerative colitis high titres of pANCA were found mainly in active disease. Using an immunoblot system with sonified neutrophils as antigen, 82% of sera from patients with primary sclerosing cholangitis reacted with up to five different determinants, whereas only 12% of sera from patients with Crohns disease and 11% of sera with ulcerative colitis detected one of the determinants, suggesting different antigens involved in pANCA reaction.

Antiphosphatidylserine antibodies are elevated in normal tension glaucoma

The two main entities of open‐angle glaucoma are primary open‐angle glaucoma (POAG) and normal tension glaucoma (NTG). Both diseases may be associated with autoimmune processes. Therefore, IgG and IgM antibodies to phospholipids (APL) and their subspecies cardiolipin (ACL), phosphatidylserine (APS) and β2‐glycoprotein (β2GP) were determined in 43 NTG patients, 40 POAG patients and 40 healthy controls in a prospective study. The most prominent observation was the increase in APS concentrations in NTG patients (IgG 20·6 ± 2·7 U/ml, IgM 24·4 ± 3·4 U/ml) compared with POAG patients (IgG 8·8 ± 1·2 U/ml, IgM 11·0 ± 1·7), and controls (IgG 7·7 ± 1·3 U/ml, IgM 12·8 ± 1·5 U/ml). APS may be important due to their binding specificity to phosphatidylserine molecules which become accessible during apoptosis; this in turn may lead to local thrombosis.

ATPASE-ASSOCIATED ANTIGEN (M2): MARKER ANTIGEN FOR SEROLOGICAL DIAGNOSIS OF PRIMARY BILIARY CIRRHOSIS

Serum samples from 94 patients with primary biliary cirrhosis (PBC) and 17 patients with chronic cholestatic hepatitis (CCH) were tested in the fluorometric immunoassay (FIAX) against the nonorgan-specific ATPase-associated antigen (M2) and against submitochondrial from beef heart and rat liver, to evaluate the specificity and sensitivity of the M2 antigen for the diagnosis of PBC. As controls serum samples from 42 patients with other antimitochondrial antibody (AMA) specificity (against M1, M3, M5, and M6) as well as samples from 417 patients with various other hepatic and non-hepatic disorders were used. Serum samples from 91 of the 94 PBC patients (97%) and all 17 with CCH reacted with the M2 antigen. However, when SMP from rat liver and beef heart were tested in parallel in the FIAX, AMA could be detected in all PBC serum samples. None of the 42 patients with different types of AMA had reactions with the M2 antigen but all had reactions with SMP from rat-liver or beef-heart mitochondria or both. Among the other 417 patients with hepatic and non-hepatic disorders only 4(1%), all with collagen diseases, had anti-M2 antibodies.

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.

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.

Autoantibody reactivity in serum of patients with major depression, schizophrenia and healthy controls

The present study assessed 25 patients with unipolar major depression and 34 patients with schizophrenia along with 50 healthy, non-psychiatric controls for the presence of serum antinuclear (ANA), smooth muscle (SMA), anti-endothelial (AEA), anti-sarcolemma (ASA), thyroid gland (TGA) and parietal cell (PCA) antibodies. In the group of patients with major depression, the frequency of elevated ANA, TGA and PCA was significantly higher than in the control group. In addition, the group of patients with schizophrenia significantly more often showed increased levels of ANA and SMA than the control group of healthy volunteers. When the two psychiatric groups were compared, PCA serum titers in major depression and SMA values in schizophrenia were significantly more frequently elevated, whereas values of AEA and ASA showed no difference. These results point towards the existence of an unspecific (auto) immune disposition or reaction in at least a subgroup of patients with major depression and schizophrenia.

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.

Promiscuous survivin peptide induces robust CD4+ T‐cell responses in the majority of vaccinated cancer patients

CD4+ T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide‐based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin‐derived CD4+ T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA‐DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4+ T cell responses, as monitored by IFN‐γ ELISPOT and intracellular cytokine staining. Thus, this HLA‐DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors.

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)