Comparison of the frequency of detection of autoantibodies to epidermal antigens with the level of antibodies to polysaccharide of group A Streptococcus and the number of T-suppressors in glomerulonephritis]

By the acute glomerulonephritis (GN) of streptococcal etiology, autoantibodies (AA) reacting with the basal layer of skin epithelium (BLSE) are discovered. The presence of this AA's correlate with the high level of antibodies to the streptococcal group A polysaccharide (A-PS). In the control sera such AA's and the high level antibodies to A-PS are discovered very rarely. By the GN of non-streptococcal etiology, AA's to the BLSE apparently of other specificity are obtained in some cases, in spite of the absence of antibodies to A-PS. AA's reacting with the differentiated layers of skin epithelium are discovered in the high percent of cases by GN. The presence of these AA's do not correlate with the levels of antibodies to A-PS. The reduction of the number of T-lymphocyte suppressors is established in the blood by the presence of AA's to the BLSE by GN. This question is a subject of later investigations by the different autoimmune processes. Such data can apparently corroborate the previously expressed hypothesis, that AA's to BLSE, which as a rule react with endocrine thymus epithelium, are the cause of the beginning of immunoregulatory disorders, characteristic of autoimmune processes.     

Cytotoxic antibodies to thymocyte antigens in rheumatism and other diseases

Cytotoxic antibodies reacting with mouse and human thymocytes were detected in rheumatic patients' sera. The level of cytotoxic antibodies was considerably higher in active than in inactive process. A correlation was found between the antibody level and the clinical course of rheumatic fever. The cytotoxic index was the highest in sera of patients with acute rheumatic fever. Thymocytotoxic antibodies were also found in other autoimmune diseases. In sera of normal individuals, antibodies to thymocytes were revealed rarely and in small quantities. A possible role of thymocytotoxic antibodies as a cause of deficit of T suppressors in autoimmune diseases is discussed.     

Suppression of cytotoxic cellular reactions by the production of antibodies to rhamnose determinants of streptococcal group A polysaccharide cross-reacting with antigens of the skin epithelium]

By the BALB/c mice after different periods of immunization with the streptococci group A, treated with pepsin, antibodies belonging to autoantibodies to the determinants (DT) of polysaccharide (A-PS), cross-reactive (CR) with the epithelial skin cells, were investigated. In one of the mice groups, in the autologous system, on the target cells--macrophages of lymph nodes, the suppression of cytotoxic (CT) reactions was obtained. The CR are bound with the delayed type hypersensitivity appearing after the sensibilization with BCG. The suppression effect correlate (z-0.95) with the presence in the sera antibodies to the rhamnose DT'S of A-PS, which cross-react with the cells of basal and superbasal layers of skin epithelium. Antibodies to the group specific of the A-PS, cross-react only with the basal skin layer and not produce the suppression of CT reactions. It is possible that they also prevent the suppression of CT reactions, bound with the CR antibodies to the rhamnose DT-S of A-PS. The obtained data corroborate the earlier supposition that the autoantibodies to the CR DT'S of A-PS reacting with the skin epithelial cells as a rule common the thymus epithelial cells. It is possible that different IRD'S can prevent or stimulate the development of autoimmune processes by the infections with the streptococci group A.     

Antibodies to the rhamnose determinants of the polysaccharide of streptococci group A in the sera of rheumatism patients and donors]

In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.     

Autoantibodies to the common antigenic determinant of group A streptococcal polysaccharide and epithelial cells of mammalian thymus and skin]

A cross-reactive (CR) antigenic determinant in the polysaccharide of group A streptococus and the mammalian thymus and skin was studied. The CR antigen was found in adult humans and human embryos irrespective of the blood group, as well as in the tissues of all the animal species studied, including rabbits immunized with streptococcus and producing antibodies to A polysaccharide. Evidence was obtained that the antibodies elaborated to the CR determinant of polysaccharide A and of the tissue epithelium should be classed as autoantibodies. It is suggested that reaction of these antobodies with the CR antigen in the thymus could serve as one of the causes of autoimmune thymitis during rheumatic fever.     

Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates

Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.     

Determination of antibodies to Streptococcus group A polysaccharide by an immunodiffusion method in human sera in various pathological processes of streptococcal etiology

A method for the assay of antibodies to the specific antigenic determinant of group A streptococcal polysaccharide (A-polysaccharide) in human sera was developed. The sera were tested in the precipitation test in agar gel with different doses of A-polysaccharide. The presence of a high level of the above-mentioned antibodies is indicative of infection caused by group A streptococcus, but not streptococci of other groups or by the L-forms of streptococci. In 87.5% of patients with primary rheumatism a high level of antibodies to the specific antigenic determinant of A-polysaccharide was detected during the first day of the disease, which confirms most convincingly the etiological role of group A streptococcus in rheumatism. Considerable differences in the level of antibodies to A-polysaccharide in the active and non-active phases of rheumatism have been established, which makes it possible to use the presence of a high level of these antibodies as an indicator of the rheumatic process activity. A considerable percentage of sera with a high level of antibodies to A-polysaccharide was also detected in erysipelas and acute glomerulonephritis patients.     

Antibodies reacting with thymus and skin epithelium and antibodies to cell nuclei in immunization with streptococcal group A polysaccharide conjugated with synthetic polyelectrolytes

The antibodies to streptococcal group A polysaccharide (A-PS) have been obtained upon immunization of BALB/c mice with A-PS conjugated with synthetic polyelectrolytes (PEL). Prolonged immunization in the majority of cases revealed antibodies to cross-reactive determinant of A-PS reacting with human and mouse epithelium of the thymus and basal skin layer. These antibodies belong to autoantibodies. Later on, after the beginning of immunization some animals produced antibodies reacting with cellular nuclei. The formation of autoantibodies to nuclei is not related to crossreactions with A-PS, because A-PS do not inhibit these reactions. No antibodies reacting with the epithelial cells or with cellular nuclei have been observed upon immunization with A-PS in Freund adjuvant or with PEL alone. The production of autoantibodies to cellular nuclei is probably a result of immunoregulatory disorders associated with the damage of thymus epithelium by autoantibodies during immunization with A-PS conjugated with PEL.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

Anti-human cardiac myosin autoantibodies in Kawasaki syndrome.

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.     

Autoantibodies against aggrecan in systemic rheumatic diseases

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.     

Epitope mapping of human aromatic L-amino acid decarboxylase

Autoimmune polyendocrine syndrome type I (APS I) is a rare hereditary condition considered a model disease for organ specific autoimmunity. A wide range of autoantibodies targeting antigens present in the affected organs have been identified. Autoantibodies against aromatic L-amino acid decarboxylase (AADC) are present in about 50% of APS I patients. In order to increase our understanding of autoantibody specificity in APS I, the aim of the present study was to localize target regions on AADC recognized by sera from APS I patients. Using several complementing strategies, we have shown that autoantibodies against AADC mainly recognize conformational epitopes. The major antigenic determinants were detected N-terminally to amino acid residue 237. Replacement of amino acids 227-230 (ERDK) with alanine residues reduced the reactivity towards AADC by >80% in all patient sera tested, suggesting that amino acids 227-230 are an important part of an immunodominant epitope.     

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.     

The incidence of the pituitary autoantibodies in Graves' disease

INTRODUCTION: It is known that in the sera of patients with Graves, Addison and other autoimmune endocrine diseases we can detect autoantibodies against pituitary antigens. The aim of the study was evaluation of pituitary autoantibodies in Graves' disease patients using immunoblotting methods. MATERIAL AND METHODS: Studies were performed in 32 Graves' disease patients, 25 women (age range: 31-67 yrs, median: 49.9 +/- 9.4) and 7 men (age range: 41-58 yrs, median: 51.0 +/- 7.1). All patients presented signs and symptoms typical of thyrotoxicosis. The diagnosis was confirmed by laboratory tests (TSH, fT(3), fT(4), TSH-R antibodies). Sera of control subjects were obtained from 10 healthy blood donors, 7 women, 3 men (age range 21-45 yrs, median: 30.6 +/- 7.1). Incidence of pituitary autoantibodies was assessed by polyacrylamide electrophoresis gel and western-blotting. Pituitary microsomes were obtained from human pituitary tissues by ultracentrifugation and solubilisation in 1% desoxycholic acid. RESULTS: In 23 sera from 32 we detected autoantibodies against pituitary microsomal antigens. 16 sera were reacting with 55 kDa antigen, 10 sera with 67 kDa, 6 sera with 60 kDa, 5 sera with 52 kDa and 4 sera with 105 kDa. It is important to note that 6 sera were reacting with 57 and 55 kDa, and 5 sera with 55, 60 and 67 kDa. CONCLUSIONS: In sera of Graves' disease patients autoantibodies against pituitary microsomal antigens can be frequently detected. The most frequent are antibodies against 55, 60 and 67 kDa antigens.     

Qualitative and quantitative analysis of autoantibodies to the membrane antigens of erythrokaryocytes

The study of the specificity, composition and concentration of autoantibodies (AB) active against erythrokaryocytes (EK) has been made by means of the enzyme immunoassay with sera obtained from patients. The specificity of AB embraces interspecific determinants of human, rabbit and mouse EK; determinants expressed on the EK of human and rabbit marrow; determinants expressed on human embryo EK. AB are mainly represented either by IgG or by IgM. Their quantitative evaluation permits the subdivision of sera into groups according to AB concentration. This phenomenon is due to the existence of the threshold value which, in most cases, sets the upper limit to the AB level detectable in the assay.     

Crucial Role of the CB3-Region of Collagen IV in PARF-Induced Acute Rheumatic Fever

Acute rheumatic fever (ARF) and rheumatic heart disease are serious autoimmune sequelae to infections with Streptococcus pyogenes. Streptococcal M-proteins have been implicated in ARF pathogenesis. Their interaction with collagen type IV (CIV) is a triggering step that induces generation of collagen-specific auto-antibodies. Electron microscopy of the protein complex between M-protein type 3 (M3-protein) and CIV identified two prominent binding sites of which one is situated in the CB3-region of CIV. In a radioactive binding assay, M3-protein expressing S. pyogenes and S. gordonii bound the CB3-fragment. Detailed analysis of the interactions by surface plasmon resonance measurements and site directed mutagenesis revealed high affinity interactions with dissociation constants in the nanomolar range that depend on the recently described collagen binding motif of streptococcal M-proteins. Because of its role in the induction of disease-related collagen autoimmunity the motif is referred to as “peptide associated with rheumatic fever” (PARF). Both, sera of mice immunized with M3-protein as well as sera from patients with ARF contained anti-CB3 auto-antibodies, indicating their contribution to ARF pathogenesis. The identification of the CB3-region as a binding partner for PARF directs the further approaches to understand the unusual autoimmune pathogenesis of PARF-dependent ARF and forms a molecular basis for a diagnostic test that detects rheumatogenic streptococci.     

Autoimmunity induced by HgCl2 in Brown-Norway rats. II. Monoclonal antibodies sharing specificities and idiotypes with mouse natural monoclonal antibodies

Spleen cells derived from BN rats receiving HgCl2 were fused with the nonsecreting rat myeloma cell line IR983F. We screened 59 supernatants from immunoglobulin-secreting hybrids for antibody activity against actin, tubulin, autologous and heterologous myosin, myoglobin, dsDNA, peroxidase, and the haptens TNP, NIP, NNP, and NBrP. Six monoclonal antibodies (mAb) were found to react with antigen(s) of the panel. At least three groups of antibody specificities were identified: clones reacting with TNP (1 IgM, 1 IgE); clones reacting with horseradish peroxidase (1 IgM); and clones possessing widespread reactivity for several antigens as found for mouse natural autoantibodies (2 IgM, 1 IgE). We also analyzed the idiotypic (Id) determinants of the 59 mAb by using anti-Id antibodies described elsewhere prepared in rabbits against the BALB/c D23 natural monoclonal autoantibody and recognizing a BALB/c recurrent Id (Id D23) of natural polyspecific autoantibodies. We found that all rat mAb that possessed widespread reactivities bore this Id. We performed similar studies in sera from normal and mercury-stimulated rats. The results indicate a role for HgCl2 in the stimulation of natural antibodies producing cells and the existence of interspecies cross-reactive Id among mouse and rat natural antibodies.     

Prevalence of various antiphospholipid antibodies in pregnant women

Antiphospholipid antibodies (APAs) are characterized as a heterogeneous population of autoantibodies directed against different target antigens, predominantly anionic phospholipids or phospholipid-containing structures. The presence of APAs has been strongly associated with a variety of clinical disorders including adverse pregnancy complications such as spontaneous abortions, pregnancy-induced hypertension, preeclampsia and intrauterine growth retardation. The purpose of this study was to compare the prevalence of anticardiolipin antibodies (ACAs), which are routinely examined, with APAs directed against phosphatidylserine (APS), phosphatidylinositol (API), phosphatidylethanolamine (APE) and phosphatidylcholine (APC) in the sera of pregnant women. We examined 410 serum samples of pregnant women hospitalized in the department for pathological pregnancies. They underwent prenatal biochemical screening of fetal congenital abnormalities in the first and the second trimester of gravidity. Anticardiolipin IgG and IgM were measured using commercial ELISA kits (ImmuLisa Anti-Cardiolipin Antibody), whereas APS, APE, API and APC were determined by our modified ELISA kit. Among 410 pregnant women we found 21 patients (5.1%) positive for ACA IgG (>20 GPL) and 30 patients (7.3%) positive for ACA IgM (>10 MPL). It was found that 7.8% of pregnant women had at least one high-titer APA IgG and 9.8% high-titer APA IgM. One third of ACA IgG or IgM positive sera contained polyspecific autoantibodies reactive to at least two various phospholipids. In the group of IgG ACA positive women, 28.6% patients were positive for APS, 28.6% were positive or moderately positive for API, 23.8% for APC and 19% for APE. In the group of IgM ACA positive women, 33.3% were also positive for APS, 26.7% for APE, 26.7% for API and 23.3% for APC were present. IgG and IgM ACA negative patients exhibited a significantly lower incidence of other APA than the group of ACA positive pregnant women. It still remains to clarify if the routine examination of APA reacting with other anionic and zwitterionic antigens other than cardiolipin would improve the probability of identifying women liable to adverse pregnancy complications.     

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sj?gren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.