[Monocytes of human blood as a target of Streptococcus pyogenes components].

AIM: Study the effect of components of destroyed streptococci on human blood monocyte functions related to processes of trans-endothelial migration in vitro. MATERIALS AND METHODS: Mononuclear leukocytes, isolated from blood of healthy donors, endothelial cells of EA.hy 926 line and supernatant of ultrasound disintegrated Streptococcus pyogenes (DSS) were the objects of the study. Evaluation of adhesion and monocyte migration, level of expression of adhesion molecules and phosphokinases on monocytes was carried out by flow cytometry using monoclonal antibodies. Cytokine concentration was determined by using standard commercial test systems in enzyme immunoassay. RESULTS: Under the effect of DSS, expression of adhesion molecules CD162 and CD11b, as well as phospho-p38 MAPK changed, IL-6 and IL-8 secretion induction took place. DSS caused enhancement of migration and adhesive activity of monocytes, however, inhibited intensity of trans-endothelial migration. CONCLUSION: Products of destroyed streptococci have a multi-directional effect on human blood monocytes, that could be explained by the presence of components with varying biological activity in DSS.

[Fc-receptor proteins of Streptococcus pyogenes and pathogenesis of post-infection complications].

Phenomenon and mechanism of non-immune binding of immunoglobulins G and A by various emm-genotypes of group A streptococcus and in particular M-family proteins--main factors of pathogenicity of this causative agent of widespread human diseases are examined. The role of these receptor proteins in pathogenesis of post-streptococcal damage of kidneys (glomerules) and heart (myocarditis) are proved. Results of long-term studies that confirm hypothesis of initiating function of Fc-receptor M proteins in genesis of immune inflammation in organ tissues that precede development of glomerulonephritis and myocarditis are provided. According to the basic position, Fc-binding of an immunoglobulin by M proteins initiates production of anti-IgG, immune complexes of various composition and complement activation, deposition of those in tissues results in lymphocyte infiltration and production of pro-inflammatory cytokines. Literature data on the role of Fc-binding proteins in genesis of IgA-nephropathies and rheumatoid factor is also examined. An important role of other factors of the microbe is discussed such as cross-reacting antigens, erythrogenic toxin B, system of streptokinase-plasmin receptor or endostreptosin in post-streptococcal processes in kidneys. Their participation in the process must be mediated by an inflammation reaction in the tissue that is initiated by interaction of immunoglobulins with Fc-binding proteins of the microbe. A novel approach to understanding the nature of this pathology allowed to establish the ability of Fc-fragments of immunoglobulin G to suppress the development of the process.

[Regulation of endothelial cells functions by ultrasonic supernatant of Streptococcus pyogenes].

Angiogenesis and vascular remodeling are vital components of inflammation. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. This study demonstrates that ultrasonic supernatant of Streptoccocus pyogenes has anti-angiogenic properties: inhibit EA.hy 926 human endothelial cells metabolism, adhesion, migration, proliferation. At the same time Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. These effects are not associated with necrosis or apoptosis in cell culture. Taking together, our results suggest that impairing angiogenic function of endothelial cells might contribute to the reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis caused by Streptococci group A.

[Influence of products of bacterial origin on the expression of surface molecules in monocyte-derived and endothelial cells].

AIM: To study the influence of lypopolysaccharide (LPS) of Gram-negative bacterium (Escherichia coli O55:B5) and lysate of Gram-positive bacteria (Streptococcus pyogenes - group A, type M1, strain 40/58) on the level of expression of important surface molecules of monocyte-derived cells from continuous cell line THP-1 and endothelial cells from continuous cell line EA.hy 926. MATERIALS AND METHODS: Expression of surface molecules HLA-DR, CD11b, CD14, CD16, CD32, and CD54 was assessed using FITC- or PE-labeled monoclonal antibodies (Beckman Coulter, USA). Intensity of fluorescence was measured by flow cytometer Epics Altra manufactured by Beckman Coulter (USA). RESULTS: Studied components of Gram-positive and Gram-negative bacteria stimulated expression of CD14, CD16, CD32, and CD54 molecules on cells from THP-1 line; incubation of cells from EA.hy 926 line in the presence of the same bacterial components increased expression levels of CD54 and HLA-DR molecules. CONCLUSION: Endothelial cells of EA.hy 926 line was less sensitive to LPS of E. coli and lysate of S. pyogenes compared to monocyte-derived cells of THP-1 line. Usage of THP-1 cells allowed to reveal differences between effects of components of Gram-positive and Gram-negative bacteria. The stimulating effect of LPS was more pronounced compared to effect of S. pyogenes lysate in relation to expression of HLA-DR, CD11b, and CD54 molecules, whereas lysate of S. pyogenes better stimulated expression of CD14, CD16, and CD32 molecules.

Myocardial tissue damage in rabbits injected with group A streptococci, types M1 and M22. Role of bacterial immunoglobulin G-binding surface proteins.

Acute rheumatic fever (ARF) and acute poststreptococcal glomerulonephritis (APSGN), two important sequelae of streptococcal throat or skin infections, according to current concepts may be elicited by autoimmune mechanisms due to molecular mimicry between group A streptococci (GAS) and human tissue. In the case of APSGN, however, our experimental data have indicated that GAS immunoglobulin-binding surface proteins (IgG BPs) might be of pathogenic significance by triggering anti-IgG production and immune complex formation leading to renal damage. Thus, rabbits injected with IgG-binding, as opposed to non-binding, GAS strains were found to develop renal deposition of IgG and complement factor C3 and inflammatory and degenerative glomerular changes resembling the picture seen in APSGN. In the present study, cardiac tissue material from rabbits injected with GAS was investigated. After 8 or more weeks of intravenous (i.v.) injections, minimal changes were seen in those animals receiving an IgG non-binding GAS strain, type T27, whereas those animals receiving either of two IgG-binding GAS strains, types M1 or M22, developed strong inflammatory and degenerative myocardial changes accompanied by deposition of IgG and C3. Furthermore, on injecting rabbits with defined mutants of a type M22 strain, the development of myocardial tissue damage proved to be dependent on the presence of streptococcal IgG-binding activity. Our results demonstrate that myocardial tissue changes may be induced in the rabbit by i.v. injection of whole heat-killed GAS of at least two M serotypes. Conceivably, induction of immune complexes by bacterial IgG BPs may lead to myocardial deposition of IgG, in turn triggering a series of events, involving the complement system and proinflammatory cytokines, with resulting tissue damage. Though many virulence factors may be involved in the development of ARF and APSGN, and a given GAS strain will never cause both, our results may suggest a new pathogenetic mechanism common to these two major non-suppurative complications.

Triggering of renal tissue damage in the rabbit by IgG Fc-receptor-positive group A streptococci.

Our previous studies have shown that streptococcal IgG Fc receptors (FcR) act to elicit circulating anti-IgG as well as renal glomerular deposition of IgG in rabbits immunized with group A streptococci (GAS). In order to study if other FcR-positive bacteria might have similar effects, rabbits were immunized with either group G streptococci (GGS; strain G148) or Staphylococcus aureus (strain Cowan I) for two periods of 8 and 6 weeks, respectively. At the end of immunization, circulating anti-IgG was found in 6 of 20 (30%) and 4 of 19 (21%) animals receiving G148 and Cowan I, respectively, compared to all 28 receiving FcR-positive GAS strains of types M1, M4, M15 or M22 (p < 0.05 for both comparisons); furthermore, anti-IgG appeared earlier and at higher levels in the GAS groups. Weak glomerular IgG deposits occurred in 5 out of 10 (50%) and 2 out of 8 (25%) animals immunized with G148 and Cowan I, respectively. In contrast, all 11 rabbits examined, given GAS of types M1 or M15, displayed heavy deposits. None of four control animals immunized with either of two FcR-negative strains, GAS type T27 or group B streptococci (GBS) type Ia, exhibited any renal IgG deposits or circulating anti-IgG. Renal tissue materials from rabbits immunized with any of the four FcR-positive GAS strains showed strong inflammatory and degenerative glomerular changes, compatible with the picture seen in acute poststreptococcal glomerulonephritis (APSGN). Only transient renal changes were found in those rabbits immunized with G148 or Cowan I, or the controls injected with the FcR-negative strains, GAS type T27 or GBS. Thus, only the FcR-positive GAS strains showed capacity to induce high levels of anti-IgG, pronounced tissue deposition of IgG as well as irreversible glomerular changes. Our experimental data suggest that streptococcal IgG FcR activity might play an important role in triggering APSGN.

Role of streptococcal IgG Fc receptor in tissue deposition of IgG in rabbits immunized with Streptococcus pyogenes.

Induction of anti-IgG during hyperimmunization of rabbit with Streptococcus pyogenes (group A streptococci; GAS) was previously shown to require the presence of IgG Fc receptors (FcR) in the vaccine strain. In the present work, we examined whether streptococcal FcR activity might also be of importance for heart and kidney deposition of IgG, known to occur in poststreptococcal sequelae as well as during experimental immunization of animals. Each of three IgG-binding (GAS types M1, M12 and M22) and two non-binding (GAS type T27 and S. agalactiae (GBS) type Ia) streptococcal strains were used for intravenous immunization of rabbits during two periods of eight and six weeks, respectively, separated by an interval of one month. Before use, vaccine strains were treated with KSCN and carefully washed in order to remove any surface-bound immunoglobulins. No deaths occurred among injected rabbits. No tissue deposition was elicited by the GAS type T27 or the GBS strain. In contrast, the strains of types M1, M12 and M22 all induced deposits of IgG in kidney and heart tissue, beginning during the first immunization period. In two tested animals, receiving GAS of types M1 or M22, circulating immune complexes containing anti-IgG antibodies were also detected. Finally, serum autoantibodies reacting with preparations of heart and kidney, but not lung or liver, were demonstrated in each of six animals receiving M1 or M22, reaching maximum levels during reimmunization; such antibodies were not evoked by the two strains not binding IgG. Our results suggest that, in GAS with capacity for non-immune binding of IgG, triggering of anti-IgG acted to enhance tissue deposition of IgG or immune complexes in immunized rabbits. Furthermore tissue-specific antibodies were elicited only by the IgG-binding strains and occurred comparatively late during immunization, suggesting that those antibodies might have been triggered due to the exposition of hidden kidney and heart determinants.

Antigenic diversity of IgA receptors in Streptococcus pyogenes.

In a previous study, group A and group B streptococcal IgA receptors were shown to differ serologically, in agreement with their known structural unrelatedness. The present study was undertaken to serologically compare the IgA binding epitopes of group A streptococcal strains representing various serotypes by the use of antisera to this species. It was found that blocking antibodies occurred in antisera to IgA binding but not to non-binding strains and that binding of IgA to a streptococcal strain was generally blocked by antiserum to the homologous type. However, cross-testing of a panel of 11 IgA binding strains, representing various M and T serotypes, with 10 different antisera to group A streptococci, demonstrated that IgA receptors were inhibited to a highly variable degree and that inhibition patterns were unique for each type. Comparing solubilized IgA receptors of various strains in immunoblot experiments, a variation in the molecular mass, between approximately 35 and 45 kDa, emerged. The IgA binding epitopes, analogous to protective sites of streptococcal M-protein, thus exhibited hypervariability which may suggest that IgA binding also plays a key role for evading host immune defence mechanisms.

Induction of myocarditis in rabbits injected with group A streptococci.

BACKGROUND & OBJECTIVES: We have earlier proposed that group A streptococcal (GAS) immunoglobulin binding surface proteins (IgGBPs) might trigger anti-IgG production and immune complex formation leading to glomerulonephritis. In the present study, cardiac tissue material from rabbits injected with heat-killed GAS was investigated. METHODS: Rabbits were injected intravenously with 10(9) colony forming units of streptococci three times weekly for 8 wk. Cardiac tissue samples were obtained at different times and deposition of IgG, C3, TNF-alpha and IL-6 was studied. RESULTS: After 8 or more weeks of intravenous (iv) injections, minimal changes were seen in animals receiving an IgG non-binding GAS strain, type T27, whereas in those animals receiving either of two IgG binding GAS strains, types M1 or M22, strong inflammatory and degenerative myocardial changes accompanied by deposition of IgG and C3 were noted. Furthermore, on injecting rabbits with defined mutants of a type M22 strain, the development of myocardial tissue damage proved to be dependent on the presence streptococcal IgGBPs. INTERPRETATION & CONCLUSION: The present data supported a role of streptococcal IgGBPs in the induction of myocardial tissue injury by GAS.

[New concepts of the mechanisms of immunopatholgies+ of Streptococcal etiology]. / Novye predstavleniia o mekhanizmakh immunopatologicheskikh sostoianii streptokokkovoi etiologii.

The paper summarizes the data on the role of immunoglobulin Fc-receptor proteins of group A streptococci as one of the leading agents of the pathogenicity that initiates severe poststreptococcal complications in the comprehensive immunological and morphological study of acute experimental glomerulonephritis. In addition to some pathogenic effects, evidence is presented for the capacity of IgG FcR-positive, rather than IgG FcR-negative, group A streptococci of inducing the synthesis of great quantities of anti-IgG which has been detected both in free circulation and as part of immune complexes. IgG FcR proteins induced the release of antiinflammatory cytokines (TNF-alpha in particular) and anti-IgG FcR proteins, concurrently with complement C, formed deposits in the structures of glomerules, resulting in destructive and degenerative changes. The morphological criteria for delayed hypersensitivity processes, which are typical of inflammatory reactions in immunopathologies are discussed in the paper. Immunomorphological and electron microscopic studies at early stages revealed signs of membrane-proliferative glomerulonephritis with a predominant response of podocytes and mesangial cells, which later progressed to developed fibrous, atrophic, glomerulonephritis. It should be stressed that IgG FcR proteins, such as A and G staphylococcal protein, group G streptococcal protein, unlike group A streptococcal protein with the similar function either failed to cause the described events or the latter occurred rarely or delayed. The findings provide evidence for the author's concept of the role of IgG Fc-receptors of group A streptococci in the development of complications of streptococcal etiology.

[Interrelationships between the virulence and the presence of M-protein in hemolytic streptococcus group A]. / Vzaimootnosheniia mezhdu virulentnost'iu i nalichiem M-belka u gemoliticheskikh streptokokkov gruppy A.

Experiments on mice demonstrated that of hemolytic streptococci A M types 2, 3, 12, 22, 46, and 49 characteristic is the absence of parallelism between their virulence for mice and the presence of M+-colonies in the microbial population. There was also a greater virulence for mice of M-variants. The established changes in serological properties of the cultures passaged in vitro were brought to the acquisition of polyagglutinability by the passage cultures, and to the loss of type-specific protection by the immune sera against these cultures. A possibility of acquisition by passage cultures of a factor determining their high virulence for mice, nonidentical to M-protein, is discussed.

[Role of hyaluronic acid in the virulence of group A streptococci]. / Rol'gialuronovoi kisloty v virulentnosti streptokokkov gruppy A.

The effect of hyaluronic acid and hyaluronidase on the virulent properties of group A streptococci in mice were studied. The passage of streptococci belonging to different serotypes in mice was accompanied by a considerable increase of their virulence and the accumulation of hyaluronic acid. The electron microscopy of the initial and passaged strains allowed one to reveal the presence of considerable differences in their cell wall structure. The passaged strains had a pronounced filamentous and sharply thickened medium layer containing hyaluronic acid. The treatment of passaged streptococci of the infected mice with hyaluronidase did not induce decrease of virulence in the passaged cultures. The data obtained indicate that hyaluronic acid is not a virulence factor for mice. Such virulence may be determined by some component, not yet identified, of the filamentous layer of the cell wall in group A streptococci.

Transduction of M+- and SOR+-markers in group A streptococci. / Transduktsiia M+- i SOR+-priznakov u streptokokkov gruppy A.

Phagolysates obtained in passage of the virulent phage CA1 on M+ SOR+-cultures of streptococcus, serotype 22, offered a possibility of transduction of the M22+-sign to M-SOR--strains of serotype T12. Transductants were selected in direct bactericidal test with subsequent determination of their type in the indirect bactericidal test with homo- and heterologous immune sera. The UV-lysates of the lysogenic M+ SOR+-cultures of serotypes 4, 22, and 49 offered a possibility of transducing both signs conjointly. In this case transductants were selected by the SOR-phenotype in the dishes with serum agar. All the transductants retained the T-serotype of the recipient's strain and segregated M-- and SOR--clones with high frequency. There were for the first time obtained in streptococcus of group A proofs of transduction of M+- and SOR+-signs pointing to their determination by different, but closely conjoint genes. Conditions of the marker transduction, selection and study of transductant were chosen, they opened new possibilities for genetic analysis of the virulence of streptococcus, group A.

[The use of streptococcal immunoglobulin Fc receptors in the technology of producing a pseudotuberculosis diagnostic test system]. / Ispol'zovanie streptokokkovykh immunoglobulinovykh Fc-retseptorov v tekhnologii polucheniia diagnosticheskoi psevdotuberkuleznoi test-sistemy.

The conditions of obtaining the IgM from hyperimmune sera to Yersinia pseudotuberculosis have been established. The process includes the adsorption of these sera by streptococci belonging to serological groups G and A and having receptors which bind the Fc-fragment of IgG and IgA molecules. The data confirming the fact that specific antibodies to Y. pseudotuberculosis belong to IgM have been obtained. The use of isolated IgM fractions for the preparation of a pseudotuberculosis diagnostic assay system has made it possible to establish that the new method of obtaining sensitin is economical and ensures a 3.5-fold greater yield of the conjugate and simplifies the technological cycle of its preparation.

[Detection and determination of the biological activity of IgG Fc receptors in strains of streptococci isolated from carriers and patients]. / Vyiavlenie i otsenka biologicheskoi aktivnosti IgG Fc-retseptorov u shtammov streptokokkov, vydelennykh ot nositelei i bol'nykh.

Mechanism of anti-IgG induction during the immunization of animals by IgG Fc+ streptococcal strains has been studied. Streptococcal proteolytic activity did not influence microbial IgG Fc-receptor ability to induce synthesis of anti-IgG. The induction of anti-IgG by purified Fc-receptors and rabbit IgG, eluted from streptococcal IgG Fc-receptors has been examined. Biological activity of streptococcal IgG Fc+ strains, isolated from carriers and patients with acute streptococcal diseases has been evaluated according to their ability to induce synthesis of anti-IgG; data on the distribution of IgG Fc+ and IgG Fc- streptococcal strains among carriers and patients with streptococcal diseases are presented. IgG Fc+ strains were mostly isolated from patients with streptococcal diseases. Mechanisms of the transformation of rabbit IgG into autoantigen are discussed.