Monoclonal antibodies cross-reacting with fibroblasts of interstitial connective tissue of the myocardium and cell wall protein antigens of group A Streptococcus

Monoclonal antibodies (MCA) B6/5 and C5/3 were obtained after immunization of BALB/c mice with the protein non-type-specific antigens (NTSA) of streptococcal group A cell wall. MCA B6/5 in the indirect immunofluorescence react with human and animal interstitial connective tissue (ICT) of the myocardium and human fibroblast culture cells. MCA C5/3 react with the bands of muscle fibers of the myocardium. MCA B6/5 and C5/3 are autoantibodies. It was revealed that these MCA are directed to two streptococcal cross-reacting antigens (CRA). Production of B6/5 and C5/3, apparently, does not depend on the possibility of some streptococcal antigens to bind fibrinogen. Bound immunoglobulins were not revealed in the ICT and in the muscle fibres by the cultivation of the C5/3 monoclone. Firstly it was stated that, MCA B6/5, reacting with fibroblasts and with streptococcal CRA, are capable to fix in the ICT of myocardium, what is typical for the phenomenon described in rheumatic fever.     

Heterogenetic antigens of gram-positive bacteria

Chorpenning, Frank W. (The Ohio State University, Columbus), and Matthew C. Dodd. Heterogenetic antigens of gram-positive bacteria. J. Bacteriol. 91:1440-1445. 1966.-Soluble antigens obtained by various methods from gram-positive bacteria were used to modify erythrocytes whose hemagglutinating reactions with immune rabbit sera and normal human sera were then studied. Antigens from all gram-positive organisms studied except corynbacteria altered red cells, causing them to react with specific bacterial antisera and with normal human sera; however, cross-absorption and inhibition tests indicated that at least three different specificites were involved. One of these antigens seemed to be similar to Rantz's streptococcal NSS, which is shared with Staphylococcus aureus and Bacillus spp., and is therefore heterogenetic. Another was found in streptococci but was apparently not present in S. aureus and Bacillus spp. A third antigen, also heterogenetic, appeared to be shared by several species of Bacillus and by S. aureus, but not by streptococci or any gram-negative bacteria. The third antigen was heat-stable at pH 8.0, and appeared to be essentially polysaccharide in nature. Normal human sera varied in their content of antibodies which reacted with erythrocytes modified by extracts from gram-positive bacteria. Whereas some sera reacted very broadly with red cells modified by extracts of practically any gram-positive organism, other sera agglutinated only cells which had been modified by streptococcal antigen.     

Monoclonal autoantibodies to different epithelial structures of the thymus gland obtained by the immunization with streptococcal group A antigens

By the indirect immunofluorescence method it was shown to which epithelial thymus structures monoclonal antibodies (mAT) reacting with the different epidermal structures are directed. These mAT related to the autoantibodies were obtained earlier, as a result of lymphoid cells polyclonal activation, by the immunization of BALB/c mice with streptococcal group A nonspecific protein antigens of the cell wall. It was shown that mAT A6/1, reacting with the basal layer of the skin epithelium are directed to the epithelium of the cortical and medullar thymus zones, which is regarded as the so called endocrinal epithelium. These mAT, by the study with immunoblotting method, react with the protein of mV SOkD, B5/1 mAT to the skin epithelium, on the thymus sections react with the single cells around the Hassel bodies.     

Isolation of non-type-specific protein antigens of Streptococcus group B

Hydrochloride extracts obtained from group B streptococcal strains of different serotypes have proved to be the source of type-nonspecific protein antigens, precipitated with ethanol and studied by gel chromatography and spectrophotometric scanning in ultraviolet rays. Thus, 2 or 3 antigens, one of them found to be common for streptococci of groups A, B and G, as well as the admixture of group-specific polysaccharide, have been detected. In extracts obtained from group B streptococcal strains of different serotypes a common protein antigen, specific only for group B, has been detected. The suitability of gel chromatography with the use Toyopearl gel HW-55F for the preparative isolation of the specific fraction of protein type-nonspecific antigen with a view to the subsequent study of immune response to group B streptococci has been shown.     

Immunocytochemical identification of bovine Langerhans cells by use of a monoclonal antibody directed against class II MHC antigens

We undertook a study to develop a reliable light microscopic technique for identifying Langerhans cells (LC) in bovine epidermis. Monoclonal antibodies (MCA) detecting bovine class II MHC antigens were used in conjunction with an avidin-biotin-peroxidase complex (ABC) immunocytochemical staining method. The specificity of the MCA for LC was confirmed ultrastructurally by use of gold-labeled second antibody. Epidermal sheets and epidermal single-cell suspensions examined by light microscopy confirm that bovine epidermal LC express class II antigens. Anti-bovine class II MCA is a dependable reagent for identification of LC in normal bovine epidermis.     

Cytotoxic effect of lymphocytes on autologous blood monocytes in the presence of streptococcal group A antigens in patients with primary erysipelas

The present work deals with a modification of the cytotoxic test for the determination of the cytotoxic activity of lymphocytes in infectious diseases. This modification is based on the use of the suspension of mononuclear blood cells, simultaneously containing effector cells (sensitized lymphocytes) and target cells (autologous monocytes). The cytotoxic effect on monocytes is observed after the preliminary incubation of nonadhering cells (lymphocytes) with the antigen of microorganisms causing the infectious process. A statistically significant increase in the cytotoxic activity of lymphocytes was recorded in patients with primary erysipelas at the acute period of the disease. The cytotoxic effect has been found to persist at a high level for two weeks. By the end of the disease this effect drops to the level characteristic of clinically normal persons. An elevated level of the cytotoxic activity of lymphocytes in the presence of streptococcal antigens of one type has been detected in 72% of patients with primary erysipelas. This indicates that type-nonspecific streptococcal antigens take part in the formation of delayed hypersensitivity, which is also confirmed by the data obtained in animal experiments.     

The suppression of delayed hypersensitivity to BCG antigens in BALB/c mice during the production of antibodies to the rhamnose determinants of Streptococcus group A polysaccharide]

The data obtained for the first time in our studies indicate that the production of antibodies to group A streptococcal polysaccharide (A-PS), one of the cross-reacting streptococcal antigens, may suppress delayed hypersensitivity (DH) to microbial antigens. The existence of sharply pronounced correlation between the suppression of DH and the presence of antibodies to the rhamnose area of A-PS in the blood of BALB/c mice immunized with the pepsin-treated culture of group A streptococci has been shown. The suppression of DH is absent in the immunized animals of the same group whose blood contains antibodies to the determinant, specific for A-PS. As revealed in this study, the effect of the suppression of antigen-specific cytotoxicity linked with DH to BCG antigens can be reproduced by mixing lymph node cells taken from these two groups of the animals. The data thus obtained are possibly linked with the activation of nonspecific T suppressors in the production of antibodies to the rhamnose determinants of A-PS in the animals immunized with streptococci. The mechanism of the newly discovered phenomenon is discussed.     

Extraction of nontype-specific antigens of group A Streptococcus by potassium thiocyanate

A method for extraction of nontype-specific antigens of the group A streptococcus cellular wall from whole microbial cells is described. Potassium thiocyanate was used as an extracting agent. Nontype-specific antigens of the thiocyanate extract purified by ammonium sulfate precipitation were examined by immunodiffusion in agar gel. The thiocyanate extracts were found to contain several nontype-specific protein antigens part of which were absent from the HCl-extracts. No group A streptococcal polysaccharide was found in the thiocyanate extracts.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Streptococcal-related antigens stimulate production of IL6 and interferon-gamma by T cells from patients with Behcet's disease.

Greater attention has been recently paid to the role of certain strains of streptococcus as an etiologic agent of Beh?et's disease, in which T cell abnormalities are considered to be involved. We therefore examined whether T cells from patients with Beh?et's disease might to be stimulated by Streptococcus sanguis-related antigen (RRE KTH-1 antigens). T cells from 17 patients with Behcet's disease, but not those from 13 healthy individuals or from 13 patients with other rheumatic diseases, were stimulated to produce greater amounts of interleukin 6 (IL6) by addition of RRE KTH-1 antigens [stimulation index: 3.96 +/- 0.56 and 1.35 +/- 0.28 or 1.83 +/- 0.43 (mean +/- SEM), respectively]. The IL6 production by T cells required the presence of either fresh or paraformaldehyde-fixed monocytes. The enhancement of T cell IL6 production was not related to the presence of HLA-B51, which has been shown to be frequently associated with Beh?et's disease. These results indicate that T cells from patients with Beh?et's disease are stimulated by streptococcal antigens to produce IL6 through T cell-monocyte interactions in which binding of the antigens to monocytes, but not necessarily processing of the antigens by monocytes, is involved. Moreover, RRE KTH-1 antigens as well as Escherichia coli-derived antigens also enhanced the production of interferon-gamma by T cells from patients with Beh?et's disease. The data thus suggest that T cell hypersensitivity to several bacterial antigens may play a central role in the pathogenesis of Beh?et's disease.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Pathogenesis of passive Heymann glomerulonephritis: chlorpromazine inhibits antibody-mediated redistribution of cell surface antigens and prevents development of the disease

Two hypotheses were tested: first, that in LEW rats the interaction of sheep (or rabbit) anti-brush border antibodies with antigens (Heymann antigens) expressed on the plasma membrane of glomerular visceral epithelial cells is characterized by initial redistribution of immune complexes on the cell surface and by subsequent shedding of immune complexes in the subepithelial part of the capillary wall; and secondly, that this interaction is inhibited by chlorpromazine, a drug that displaces calcium ions from binding sites linking the plasma membrane to the cytoskeleton, and which blocks the redistribution of IgG on the surface of B lymphocytes exposed to anti-IgG antibodies. The studies were performed in vitro on cultured LEW glomerular epithelial cells and in vivo in LEW rats. In cultured glomerular epithelial cells exposed at 37 degrees C to anti-brush border IgG, chlorpromazine prevented, in a dose-dependent manner, the redistribution ("capping") of Heymann antigens and the fixation of complement. The renal glomeruli of chlorpromazine-treated LEW rats examined 6 and 48 hr after transfer of anti-brush border antibodies had punctate and, later, punctate and diffuse deposits of sheep (or rabbit) IgG on glomerular epithelial cells, but not similar deposits of rat C3. Moreover, granular subepithelial deposits of sheep (or rabbit) IgG and rat C3, characteristic of passive Heymann glomerulonephritis, did not develop, although deposits of sheep IgG were detected by immunoelectron microscopy on the microvilli of glomerular epithelial cells. Comparative studies on rats with similar reductions in glomerular filtration rates, produced by high doses of chlorpromazine or with renal artery stenosis, showed that the findings were not the consequence of insufficient delivery of antibody to glomerular epithelial cells. The results are consistent with the interpretation that Heymann glomerulonephritis is induced by mechanisms of redistribution of cell surface antigens comparable to those that govern the interaction of surface antigens (or receptors) with appropriate ligands in B lymphocytes and other classical in vitro systems.     

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium

Summary The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le^x were demonstrated to be uninfluenced by the genetic background. A and Ale^y antigens were exclusively demonstrated in endometria from blood group A individuals, while Le^y was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe^y antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le^x, sialosyl-Le^x, Le^y) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.     

An immunoferritin labeling study of H-2 antigens on dissociated epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from stomach, duodenum-jejunum, ileum, trachea, diestrus uterus, gall bladder, and vas deferens. Before cell dissociation, most of the organs were prefixed in periodate-lysine-paraformaldehyde to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. Five kinds of epithelial cells expressed H-2 antigens on lateral and basal membranes but not on apical membranes. These were the lining cells of the upper intestine, ileum, gall gladder, uterus, and the tracheal brush cell. The antigens were continuously distributed on the lateral and basal membranes of these cells and appeared to be absent from the apical membranes, rather than masked by the fuzzy coat. On four other epithelial cell types H-2 antigens could not be detected. These were the lining cells of the vas deferens, parietal and chief cells from the stomach, and ciliated tracheal cells. It does not seem to be uncommon for normal nucleated cells to lack H-2 antigens. On fixed and labeled epithelial cells from the upper intestine the zonula occludens membranes were unlabeled, while the zonula adherens and desmosome membranes were labeled as densely as the remainder of the lateral membranes. The zonula occludens membrane thus constituted the boundary betewen the unlabeled apical membrane and the labeled lateral membrane of these cells. Intestinal epithelial cells dissociated without prefixation showed a patchy distribution of H-2 antigens on their lateral membranes after indirect labeling, indicating antigen mobility in this membrane. On the same unfixed dissociated cells the antigens were able to migrate from lateral to apical membranes, a movement which appears to be prevented in the intact epithelial layer by the occluding junction. The absence of H-2 antigens from apical membranes and their inability to migrate through an intact zonula occludens suggest that these molecules must reach the lateral membranes of epithelial cells by a pathway which is distinct from that followed by apical membrane components.     

Role of T- and B-lymphocytes in the heterogeneity of human cell-mediated reactions to bacterial antigens]

Leucocytes from 30 patients with allergy to tuberculin and bacterial antigens were treated with antithymus (ATS) and anti-immune globulin (AIGS) sera. The leucocyte migration inhibition test (LMIT) was performed with these antigens. ATS abolished the LMIT induced by tuberculin and sometimes by bacterial antigens (staphylococcal, streptococcal etc.). AIGS frequently abolished the LMIT induced by bacterial antigens, but not by tuberculin. In some cases the treatment with any serum abolished the LMIT induced by the antigens, or, on the contrary, it was abolished only by a successive treatment with both sera. The lymphocyte types (T or B) determining the secondary immune response to the same antigen are different in various patients, as well as they differ in the same patients in relation to diverse antigens. Five types of lymphocyte - antigen interrelation in the LMIT have been distinguished.     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

Serum antibodies to group A streptococcal extracellular and cell-associated antigens in Egyptians with post-streptococcal diseases

We investigated serum antibodies to a comprehensive array of group A streptococcal antigens and superantigens in Egyptian subjects. Antibodies to Streptococcus pyogenes cell-associated proteins and to proteins released by rapidly dividing S. pyogenes were compared in four patient groups with different post-streptococcal diseases and in healthy controls. Enzyme-linked immunosorbent assays showed that total Ig and IgG to extracellular antigens were significantly higher in patients with acute rheumatic fever (ARF) compared to healthy controls, but no differences were found in either total Ig or IgG titres to cell-associated proteins between any of the groups. Western blotting showed that multiple extracellular and cell-associated antigens, covering a wide range of molecular masses, were recognised by all sera, including healthy controls. No evidence was obtained for putative dominant antigens associated with any disease group, although a low molecular mass cell-associated protein (approximately 4 kDa) was clearly recognised by two-thirds of subjects irrespective of disease status. These findings demonstrate that raised serum Ig and IgG titres to extracellular, but not cell-associated, S. pyogenes antigens are a feature of ARF in this population, and suggest that multiple S. pyogenes antigens contribute to this response.