Streptococcal Extracts Research Articles

Streptococcus Induces Circulating CLA+ Memory T-Cell-Dependent Epidermal Cell Activation in Psoriasis

Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

IgG class antibodies to heat shock-induced streptococcal antigens in psoriatic patients.

Psoriasis is a chronic inflammatory skin disease. Probably autoimmune in nature, and associated with streptococcal throat infections as a triggering factor. Although many groups have associated the disease with other pathogens, Streptococcus pyogenes seems to be the most important microorganism related to this disease. Therefore, it is necessary to identify the streptococcal antigens involved in the process. In this work IgG class antibodies to soluble antigens obtained from Staphyloccus aureus, Candica albicans or S. pyogenes before and after heat shock induction, were analyzed by ELISA in 28 psoriatic patients and 30 healthy donors. In all cases, the patients and the controls had IgG class antibodies to the four antigens. Nevertheless, the IgG levels to the heat shock-induced S. pyognes were statistically different between the patients and the controls (P < 0.001). There was no difference between the groups when the IgG antibodies to the other antigens, including the noninduced streptococcal extract, were analyzed. Additionally, anti-streptolysin O titers and throat cultures were carried out in all patients and controls. No differences between ASO titers were found but the patients were more frequently colonized by pyogenes. Results obtained in this study suggest that heat shock-induced proteins from S. pyogenes are associated with psoriasis.

Non-M protein(s) on the cell wall and membrane of group A streptococci induce(s) IFN-gamma production by dermal CD4+ T cells in psoriasis.

Recently we have demonstrated that a disease-specific subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis produces interferon-gamma in response to group A streptococcal (GAS) antigens. To determine if these T cells recognize M or non-M protein, extracts from cell wall of type M6 GAS (M6W) and its isogenic M gene deletion mutant (M-W), M6 membrane extract (M6M) and recombinant M6 protein (rM6) were used to stimulate GAS-reactive T-cell lines from nine patients with chronic plaque psoriasis. T-cell lines were incubated with or without streptococcal extracts for 18 h in the presence of a transport inhibitor, stained for surface CD4 and intracellular cytokine expression, and analysed by flow cytometry. Variable numbers (0.2-34%) of CD4+ T cells produced interferon-gamma, in all but one of the T-cell lines tested, in response to M6W, M-W and M6M extracts. No significant difference between the response to M6W and M-W extracts was detected. In addition, rM6 protein failed to increase CD4+/interferon-gamma+ T-cell numbers in seven of nine T-cell lines compared to medium alone. For the group, there was a highly significant correlation between the responses to the three extracts (M6W vs M-W, P = 0.0005; M6W vs M6M, P = 0.0003; M-W vs M6M, P = 0.0001). Low or minimal numbers of interleukin-4- and interleukin-10-producing CD4+ T cells were occasionally induced. These findings suggest that a subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis patients produces interferon-gamma in response to non-M protein(s) present on the cell wall and membrane of GAS.

Autoantibodies to autologous skin in guttate and plaque forms of psoriasis and cross-reaction of skin antigens with streptococcal antigens.

Psoriasis is a chronic disease of the skin that appears to be of autoimmune nature. It has a strong association with throat streptococcal infections, as well as with stressful events. Although many groups consider psoriasis to be a T-cell-mediated autoimmune disease, autoantibodies could also play a role in the development of this process. In this work, we looked for autoantibodies to psoriatic skin in 21 psoriatic patients and four healthy donors (controls). The immunoperoxidase technique was used to look for autoantibodies in autologous sera in skin sections obtained from lesions or from healthy areas of the same patient, before and after immunoadsorption with a Streptococcus pyogenes extract. The skin biopsies were also analyzed with a pool of sera from mice immunized with the streptococcal extract. We found that all psoriatic patients had autoantibodies to antigens present in keratinocytes, whereas healthy subjects did not. These antibodies did not recognize epitopes on healthy skin from the same psoriatic patients or controls. Immunoadsorption of autologous sera removed the reactivity to antigens in skin lesions in all cases. Mouse anti-streptococcal sera recognized epidermal antigens present in lesional psoriatic skin, but not in healthy skin from psoriatic patients or controls. Deposits of immunoglobulin G (IgG) were not detected in the lesions. It seems that autoantibodies, although they do not appear to participate in the pathogenesis of psoriasis, are an important feature, and that skin antigens, which appear in lesional immature keratinocytes, cross-react with S. pyogenes and contribute to the autoimmune process in psoriasis.

Vaccination with whole-cell vaccine and bacterial protein extract protects tilapia against Streptococcus difficile meningoencephalitis

Formalin-killed Streptococcus difficile strains used as vaccines delivered intraperitoneally were able to protect tilapia against a challenge of 100 LD 50. The protection obtained was not strain specific. A vaccine based on an S. difficile extract containing 50% protein conjugated to alum also protected tilapia challenged with a virulent S. difficile strain. Protection in tilapia was correlated with the development of specific agglutinins. Western blot analysis supported the hypothesis that only a few proteins act as protective antigens in both the whole-cell vaccine and the streptococcal extract. The high efficacy of these vaccines make them good candidates for the control of streptococcal fish meningoencephalitis.

The protective M proteins of the equine group C streptococci

The group C streptococci are the most commonly isolated bacteria from disease states in the horse. Important virulence factors of S. equi and S. zooepidemicus are the hyaluronic acid capsule and the antiphagocytic fibrillar M protein located on the surface of the cell wall and extending into and through the capsule. The hyaluronic acid capsule is non-antigenic and so is not involved in protective immunity. The M protein, a superantigen, elicits very strong B and T cells responses that may result in protective immunity mediated by opsonic antibodies in plasma and by locally synthesized IgG and IgA on the pharyngeal mucosa. However, vaccines based on acid or mutanolysin extracted M protein do not confer a high level of protection against field exposure. Protective antibodies to S. equi or S. zooepidemicus can in part be assayed by the bactericidal test that measures opsonization for equine neutrophils. A mouse-challenge model has also been used to test immunizing potency of streptococcal extracts and in a passive protection test for protective antibody. There is as yet no means of distinguishing protective opsonic or mucosal antibodies from other antibodies produced against the many epitopes on the M molecule.

Evidence for actinlike proteins in an M protein-negative strain of Streptococcus pyogenes.

Antigens shared between Streptococcus pyogenes and heart tissue may play an important role in autoimmune cardiac injury associated with acute rheumatic fever. Antiheart/antistreptococcal antibodies found in the disease react with antigens of S. pyogenes, including M protein and a 60-kDa antigen distinct from M protein. Heart antigens recognized by these cross-reactive antistreptococcal antibodies include myosin and actin. To investigate the presence of a streptococcal actin, established protocols for the polymerization and isolation of eukaryotic actin were used to extract and concentrate actinlike proteins from M- streptococcal cells. The polymerized bacterial actin from the streptococcal extract was probed in immunoblots with an antiactin monoclonal antibody. Two proteins of about 60 kDa in the polymerized bacterial actin reacted with the antiactin antibody. Proteins in the polymerized bacterial actin extract of about 43 and 60 kDa behaved like eukaryotic actin by binding to myosin and DNase I affinity columns. Filaments were demonstrated by electron microscopy in the polymerized bacterial actinlike extract, which also enhanced the ATPase activity of eukaryotic myosin. The data suggest that proteins resembling actin are present in S. pyogenes.

The nature of the substance in acidic streptococcal extracts reacting with fibrinogen

Acid extracts from streptococci were shown to react with fibrinogen and synovial fluid. The reactive material was a teichoic acid and not M‐protein as previously suggested and is of high molecular weight and akin to polyglycerophosphate.

A group B streptococcal extract reduces neutrophil counts and induces neutrophil aggregation.

The possibility that group B streptococci (GBS) may induce neonatal neutropenia by promoting neutrophil aggregation and the entrapment of aggregates in the lung was studied in vivo and in vitro utilizing a cell free GBS extract [(GBS)-trichloroacetic acid (TCA)]. The intravenous infusion of the extract into neonatal lambs induced reductions of circulating white blood cells (0 time, 3.1 X 10(3)/mm3 +/- 0.5 versus 2.2 X 10(3)/mm3 +/- 0.7) 5 min after infusion (p less than 0.01). At necropsy these lambs had prominent accumulation of polymorphonuclear leukocytes in their pulmonary interstitium. Subsequently, neutrophil aggregation was studied by incubating GBS-TCA in human serum or phosphate-buffered saline with subsequent addition to human polymorphonuclear leukocytes in an aggregometer. GBS-TCA incubated in human serum induced prompt polymorphonuclear leukocyte aggregation (mean delta T 12.3% +/- 2.8 in human serum versus delta T 2.5% +/- 2.1 in phosphate-buffered saline, p less than 0.001). Preincubation of GBS-TCA followed by incubation in human serum with human GBS hyperimmune IgG significantly reduced aggregation (GBS-TCA in serum mean delta T 14.9 +/- 2.44 versus 5.42 +/- 1.80, p = 0.002). Cell-free GBS products may induce polymorphonuclear leukocyte aggregation in the presence of whole serum. This phenomenon might contribute to the pulmonary injury experienced by infants with GBS pneumonia and sepsis.

Interaction of group A type 1 streptococcal M protein with fibrinogen.

Adsorption chromatography of streptococcal extracts on immobilized fibrinogen allows isolation of components that are linked to the corresponding receptors. In this study it is shown by an indirect bactericidal test that fibrinogen binds the M proteins of the streptococcal strains used. Phage-associated lysin extracts of group A type 1 streptococci precipitated with fibrinogen in a double-diffusion test. Fibrinogen reactive components of other streptococcal types inhibited this precipitation reaction. This suggests that the fibrinogen receptors in different types of group A streptococci have identical activity. The interaction between M protein and fibrinogen does not interfere with the interaction between M protein and the corresponding type specific antibodies. The streptococcal antigen components isolated by immobilized fibrinogen showed mitogenic activity in a lymphocyte transformation test.

Clinical evaluation of the improved streptex method for grouping streptococci

The improved Streptex method for serogrouping streptococci incorporates a new extraction enzyme and a simplified procedure requiring no centrifugation. A total of 114 clinical isolates of beta-hemolytic streptococci were serogrouped from primary plates, isolation plates, and Todd-Hewitt broth cultures using this system. Results were compared to those of the heat extraction Lancefield precipitin method. An additional 33 stock culture isolates of related streptococcal species and 5 strains of Listeria were serogrouped to assess the specificity of the test. Agreement between the two methods was 82.5% with primary plates and 96.5% with both isolation plates and broth cultures. Four isolates from three different serogroups were nongroupable by the Lancefield method, but did agglutinate in specific Streptex antisera; therefore, the enzyme extraction procedure appeared more sensitive than the heat extraction method. Streptex accurately grouped five isolates of gamma-hemolytic group B streptococci, but failed to detect antigen in 33% of the group D streptococcal extracts tested. In addition, cross-reactions were observed with strains of alpha-hemolytic streptococci. Streptex produced fewer ambiguous results and required fewer repeat tests. When used with isolation plates or broth cultures, Streptex is both sensitive and specific for the grouping of beta-hemolytic streptococci of groups A, B, C, F, and G.

Studies of short-term pulmonary and peripheral vascular responses induced in oophorectomized sheep by the infusion of a group B streptococcal extract.

Short-term (0-30 min) pulmonary and systemic vascular responses of oophorectomized ewes infused intravenously with a trichloroacetic acid (TCA) extract of a type III (strain 878) group B streptococcus (GBS) were studied. TCA-878 extract induced significant pulmonary hypertension, reduction in femoral artery pressure and reduced femoral artery PO2. These responses were similar to those observed after the infusion of sublethal doses of E. coli endotoxin and could be prevented by priming the animal with ibuprofen or indomethacin. Ewes rechallenged 48 h after their initial dose of TCA-878 extract experienced pulmonary arterial pressure significantly higher than those induced by the first infusion. Similar augmented responses were not seen after rechallenge with endotoxin. Larger doses of TCA-878 extract resulted in increasingly higher pulmonary arterial pressure. We conclude that the venous infusion of TCA extracts of GBS-878 induces significant pulmonary hypertension in sheep and that this response may be mediated by prostaglandins. The vasoactive substance extracted from these streptococci could play a role in promoting the vascular instability experienced by the human neonate with early onset GBS disease.

The occurrence of R-protein among isolates of group B streptococci from human sources.

A total of 241 clinical isolates of group B streptococci were tested for presence of R-protein by applying the double diffusion-in-gel technique on streptococcal extracts against monospecific anti-R protein serum, radio-labelled protein A and anti-R protein serum or S. aureus, Cowan I coated with antibodies against group A streptococci, T-type 28. The first two methods proved more sensitive than the latter. R-protein was detected in 51% of 43 type II strains and in 87% of 102 type III strains, whereas none of 79 type Ia and none of 17 type Ib strains contained R-protein. Among 30 isolates from blood and cerebrospinal fluid of infants with neonatal infections, R-protein was detected in 43%, one of two type II strains and 12/14 type III strains. Tests of 37 paired strains from infant/mother pairs showed concordant results with respect to presence or absence of R-protein in the isolates.

Immunochemical detection of a common antigen among Streptococcus uberis isolates

Fifty-three isolates of Streptococcus uberis from various sources were examined for the presence of a common antigen. Initially, a serum was produced in rabbits which, by using rocket line immunoelectrophoresis, proved to react with identity to all of the S. uberis crude extracts as well as group B and E streptococcal extracts. The antigen(s) responsible for this cross-reactivity was partially purified by Sephacryl S-200 gel chromatography and analyzed by fused rocket immunoelectrophoresis. Further analysis by immunodiffusion showed that probably two antigens in the gel chromatography-consolidated fractions were common to the S. uberis and group B and E isolates, but that one of the antigens present was unique to S. uberis. Trypsin destroyed the immunoreactivity of this antigen. Isolation of this common antigen could possibly alleviate some of the tedium associated with the identification of this organism.

Isolation of heart- and kidney-binding protein from group A streptococci.

Tritium-labeled, water-soluble components of Streptococcus pyogenes type M6 absorbed to cardiac tissue in vitro. Tissue binding was time dependent, saturable, and reversible. Chromatography of the crude bacterial extract on Bio-Gel P-300 indicated a molecular weight greater than 300,000 for the heart-binding component. Sodium dodecyl sulfate (SDS) dissociated this aggregate into a protein of 18,000 to 20,000 daltons as determined by Sephacryl S-200 chromatography and SDS-polyacrylamide disc gel electrophoresis. The tissue-binding protein was also purified from streptococcal extracts by absorption to immobilized heart components. SDS-polyacrylamide gel electrophoresis of the protein desorbed from tissue revealed a radioactive band of 19,000 daltons. Indirect immunofluorescence tests on cardiac tissue treated with streptococcal extract showed an accumulation of a bacterial antigen on the sarcolemmal sheaths. Streptococcal components also adsorbed to basement membranes of kidney. Antisera prepared to isolated cytoplasmic membranes and water-soluble extracts of S. pyogenes type M6 were the most sensitive reagents for the detection of bacterial components bound to tissue. Antisera prepared to isolated cell walls and to intact bacteria were weakly reactive in these assays.

正常胎児の細胞性免疫機能の発達

Human fetal lymphoid cells from the thymus, spleen and peripheral blood of 25 fetuses (14-25 weeks of fetal age) were studied for their ability to respond to phytohemagglutinin(PHA), pokeweed mitogen(PWM), and streptolysin-0(SLO).Lymphocyte transformation by PHA and PWM was observed in the thymus, spleen and peripheral blood at the 14-16th week of fetal age. The percentage of transformed thymic lymphocytes, observed 3 days after inoculation, decreased as the age of fetuses increased. On the other hand, splenic lymphocyte transformation increased according to age. The percentage of thymic lymphocytes transformed by PHA, observed 3 days after inoculation, was comparable to that of transformed splenic and peripheral blood lymphocytes. However, a lower percentage of transformed lymphocytes in the thymus was observed 6 days after inoculation compared to that of transformed splenic lymphocytes. In the thymus, the response to PHA observed 3 days after inoculation was higher in ratio than that observed 6 days after. These results suggest that thymic lymphocytes consist of two subpopulations which exhibit “maximum response” 3 or 6 days after inoculation. The percentages of thymic lymphocytes and splenic lymphocytes transformed by PWM were the same. In PWM-stimulated transformation, no difference was detectable between these two organs. There was no difference in transformation whether observed 3 or 6 days after inoculation with PWM. Lymphocyte transformation to SLO was observed in the thymus at the 17th week and in the spleen at the 19th week. Although cord blood lymphocytes were not exposed to streptococcal extracts, they were transformed by SLO. These results suggested that SLO was a nonspecific mitogen.Human fetal lymphoid cells from the thymus and the spleen of 9 fetuses (14-25 weeks) were examined for the presence of spontaneous rosette-forming cells (RFC) with sheep red blood cells. RFC were found in the thymus and spleen at the 14th week. The average percentage of RFC was 78.1% in the thymus and 12.1% in the spleen.

Immunodiffusion studies on synovial fluid from patients with rheumatoid arthritis: the reaction of fibrinogen with a streptococcal extract.

Synovial fluids (83.3%) from patients with rheumatoid arthritis gave a positive reaction when examined by immunodiffusion against a Lancefield extract of Group A Type 12 streptococci. None of the patients with osteoarthrosis reacted positively. An antiserum was prepared to the material in synovial fluid reacting with the streptococcal extract. This antiserum reacted positively with all synovial fluids tested including the osteoarthrosis patients. It also reacted with all plasma samples tested but only with a few (18.2%) of serum samples. Immunodiffusion and absorption experiments demonstrated that the material reacting with the streptococcal extract was fibrinogen. Of 12 other streptococci examined only one, a Group C organism, gave a similar positive reaction with synovial fluids.

Capacity of group A, B, C, D, and G streptococci to agglutinate sensitized sheep red cells.

The reactivity of streptococci with sheep red cells, sensitized with a subagglutinating dose of rabbit anti‐sheep red cell antibodies was studied; agglutination of sensitized and not non‐sensitized sheep red cells by streptococci was demonstrated in isolates belonging to groups A, B, C, D and G. The frequencies of isolates which had the capacity to agglutinate were 97, 88 and 100 per cent in group A, C and G, but only 47 and 33 in group B and D. The agglutination of sensitized sheep red cells by streptococci diminished after treatment of the streptococci with protease from Streptomyces griseus, but was not affected by pepsin or trypsin treatment. Heating the streptococci at 60°C for 30 minutes and repeated subculturing did not suppress their agglutinating activity. Besides, various streptococcal extracts agglutinated sensitized sheep red cells. The presence of an immunoglobulin G reactivity in streptococci might cause certain difficulties in the serological grouping and typing of streptococci.

Lymphocyte Surface Effects of Soluble Extracts of Streptococcal Membranes (TGCM)

Abstract A water-soluble, dialyzable glycoprotein extract of group A type 12 nephritogenic streptococcal membranes, which has previously shown transplantation antigen-like activity in mice and induced nephritis in rhesus monkeys and in dogs, has the capacity to protect human lymphocytes from the cytotoxic effects of isospecific HL-A typing antisera. This effect is not a consequence of a serologic reaction between HL-A antibodies and streptococcal extract (TGCM). Rather, it is due to the firm binding of TGCM to the lymphocyte surface, in a manner which resists dissociation by vigorous repeated washings. The protective effects of TGCM against HL-A antisera appear thus far to be limited to lymphocytes, as TGCM failed to protect human fibroblasts obtained from donors of known HL-A genotypes from the cytotoxic effects of such antisera. Immunofluorescence studies indicate that TGCM binding sites occur in diffuse fashion on the lymphocyte surface. In addition, the results of absorption-inhibition tests utilizing lymphocytes from donors of known HL-A phenotypes, speciesspecific anti-human heteroantisera and isospecific HL-A antisera point to a close spatial relationship between HL-A, species-specific, and TGCM binding sites on the surface of the human lymphocyte.

Lymphocyte stimulation by a glycopeptide isolated from Streptococcus pyogenes C203S : I. Isolation and partial purification

Lymphocyte transformation is induced by a streptococcal extract rich in streptolysin S (SLS). The lymphocyte-stimulating principle is distinct from SLS as shown by separation on DEAE-cellulose, by absorption spectroscopy, by amino acid analysis, and by its ability to retain full activity in the absence of stabilizers such as RNA digest required for the activation of SLS. The lymphocyte-transforming factor has been designated LTF. LTF is heat stable (100 ° for 5 min) and is resistant to proteolytic enzymes. It retains activity after exposure to acid or alkali in the pH range 1–12. Two peaks were eluted from DEAE-cellulose with a step-wise gradient. With two exceptions the amino acid composition of the two peaks was similar, suggesting polymerization. The molecular weight is estimated at less than 51,000.