Streptococcal Pyrogenic Exotoxin A Research Articles

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vβ regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3− lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1β. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3− T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.

Intravenous immune globulin affects cytokine production in T lymphocytes and monocytesjmacrophages

Immune globulin for intravenous use (IVIG) has been used in many inflammatory conditions due to its immunomodulatory potential. The effector mechanisms are incompletely understood. This study dealt with the effects of IVIG on cytokine production in vitro. Cytokine synthesis was identified at the single-cell level using cytokine-specific MAb and indirect immunocytochemical techniques. Peripheral blood mononuclear cells (PBMC) were stimulated for 96 h by immobilized anti-CD3 MAb or by a combination of a protein kinase C activator (PMA) and a calcium ionophore (ionomycin). The addition of IVIG (6 mg/ml) caused a marked inhibition of proliferation and blast transformation despite unaffected cell survival. Anti-CD3-stimulated cultures containing IVIG exhibited a significant inhibition of production of T-cell derived lymphokines IL-2, IL-10, TNF-beta, IFN-gamma and TNF-alpha (made by both monocytes and T cells), while synthesis of the monokine IL-8 was significantly increased. The expression of IL-2 receptors was significantly suppressed. Similar but transient inhibition of most T-cell products (IL-2, IL-3, IL-4, IL-5, IL-10, TNF-beta and GM-CSF) was noted in the PMA/ionomycin-containing cultures. In contrast, no effects were found on IFN-gamma or TNF-alpha production. The superantigen streptococcal pyrogenic exotoxin-A (SPE-A) induced vigorous cell activation and extensive cytokine synthesis. IVIG was added either at the beginning or 24 h after the initiation of cultures in order to elucidate the importance of direct toxin-neutralization. Addition of IVIG from the beginning of cultures induced a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular of IFN-gamma and TNF-beta. Supplementation with IVIG 24 h after initiation of cultures also led to a significant decrease in lymphokine synthesis. Monokine production (IL-1 alpha, IL-1 beta, IL-1ra, IL-6 and IL-8) was either unaffected or even increased. These two facts argue against direct antigen-neutralization as being the only mechanism at work. However, in IVIG-exposed PBMC stimulated with LPS, IL-6 production was significantly reduced. A significant upregulation of IL-1ra was noticed in unstimulated PBMC cultured with IVIG. The results in all the experiments did not indicate a cytotoxic effect by IVIG on cell survival and the production of certain cytokines were unaffected. Instead, the authors believe that the results suggest a previously little examined functional link where the humoral immune response may have direct immunoregulatory effects on the cellular immune system.

Superantigen‐presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Detection of Multiple Superantigen Genes in Stools of Patients with Kawasaki Disease

To investigate whether superantigens (SAgs) are involved in the development of Kawasaki disease (KD) by examining SAg genes in the stool of patients with KD. Stool specimens were obtained from 60 patients with KD and 62 age-matched children (36 children with acute illness and 26 healthy children). Total DNA was extracted from these stool samples. Using polymerase chain reaction, we examined genes of 5 SAgs: streptococcal pyrogenic exotoxin-A (SPE-A), SPE-C, SPE-G, SPE-J, and toxic shock syndrome toxin-1. At least 1 of the 5 SAg genes was detected in 42 (70%) specimens from patients with KD, 14 (38.9%) from the febrile group, and 7 (26.9%) from the healthy group. The detection rate between subjects with and without KD was of at least 1 of the 5 SAg genes (P < .001), and more than 2 SAg genes were significantly different (P = .002). SAg may be involved in the development of KD; data suggest that multiple SAgs may trigger KD.

Superantigens SPEA and SMEZ do not affect secretome expression in Streptococcus pyogenes

The superantigens, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB), were recently reported to repress global exoprotein synthesis in Staphylococcus aureus. To investigate if this phenomenon could be observed in a different Gram-positive pathogen, the effects of two major Streptococcus pyogenes superantigens on streptococcal secretome expression were examined. Using mutagenesis and genetic complementation, we demonstrated that neither streptococcal pyrogenic exotoxin A (SPEA) nor streptococcal mitogenic exotoxin Z (SMEZ) had any consistent effect on global protein expression or on transcription of genes encoding the secreted exoproteins, DNase B, SPEB and SPEG. In S. pyogenes, superantigen production does not appear to have a major regulatory role.

Development of serum IgM antibodies against superantigens ofStaphylococcus aureusandStreptococcus pyogenesin Kawasaki disease

To serologically determine the association of microbial superantigens and the pathogenesis of Kawasaki disease (KD), we conducted a case-control study. Serum IgG and IgM antibodies against staphylococcal enterotoxin A (SEA), SEB, SEC, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxin A (SPEA) were measured by an enzyme-linked immunosorbent assay in 293 serum samples from 65 KD patients on clinical days 1-28 and 120 control samples. The administration of immunoglobulin products, which contain high concentrations of IgG antibodies against all the superantigens, directly elevated antitoxin IgG antibodies in KD patients. In contrast, antitoxin IgM antibodies were not detected in immunoglobulin products. Actually, we found a significant elevation of IgM antibodies against SEA in KD patients in the first (median titre: 0.020, P < 0.01 versus control), second (0.024, P < 0.001), third (0.030, P < 0.001) and fourth (0.038, P < 0.001) weeks, compared to the controls (0.015). Significant differences of IgM antibodies were also true for SEB, TSST-1, and SPEA throughout the first to fourth weeks, and for SEC throughout the second to fourth weeks. The prevalence of KD patients having high IgM titres (> mean + 2SD of control values) to the 5 superantigens was increased with the clinical weeks, and reached 29-43% of KD subjects at the fourth week. This is the first study that describes kinetics of IgM antibodies against superantigens and clarifies the serological significance throughout the clinical course of KD. Our results suggest that multiple superantigens involve in the pathogenesis of KD.

Intranasal Exposure to Bacterial Superantigens Induces Airway Inflammation in HLA Class II Transgenic Mice

Staphylococcus aureus is widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections. S. aureus can elaborate a variety of superantigen exotoxins in "carrier" or "pathogenic" states. Streptococcus pyogenes can also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.

Detection of circulating superantigens in an intensive care unit population

Objective: Plasma concentrations of superantigens were measured in an intensive care unit (ICU) population and the relationship of superantigen positive rates with the presence of sepsis was investigated. Methods: Plasma samples were collected at least twice a week from 78 patients whose primary diagnoses were abdominal disorders ( n = 27), respiratory disorders ( n = 11), trauma ( n = 10), burns ( n = 10), cardiovascular disorders ( n = 4), neurological disorders ( n = 2), and others ( n = 14). Five different species of superantigens, i.e., staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC), toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxin A (SPEA), were measured using an enzyme-linked immunosorbent assay. Results: Significant levels of plasma superantigens were detected in 16 patients. SEA was found in seven patients, SEB in four patients, SEC in two patients, TSST-1 in six patients, and SPEA in five patients. Superantigen detection rates were 6% (1/17) in patients without systemic inflammatory response syndrome (SIRS), 0% (0/21) in SIRS patients without infection, 31% (5/16) in septic patients without shock, and 42% (10/24) in septic shock patients. Conclusions: The presence of superantigens was confirmed in part of the ICU population. The role of superantigens in the pathogenesis of sepsis remains to be determined.

Mutational effects on protein folding stability and antigenicity: the case of streptococcal pyrogenic exotoxin A

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.

Complementation of a speA negative Streptococcus pyogenes with speA: effects on virulence and production of streptococcal pyrogenic exotoxin A

We have shown previously that an isogenic SPEA-negative Streptococcus pyogenes strain did not attenuate virulence in a murine model of necrotizing fasciitis. The aim of this study was to confirm that streptococcal pyrogenic exotoxin A (SPEA) is not crucial for streptococcal invasiveness in murine invasive infection. The SPEA-negative S. pyogenes (H326) was complemented with speA extra-chromosomally to create strain H361 which produced 2.2-fold more SPEA compared with the parental speA+wild-type (H305). The growth phase-regulated expression of SPEAin vitro was unaffected in this strain. Complementation with speA resulted in reduced virulence and bacterial counts in invasive murine infection. SPEA production was quantitated from muscle tissue of infected mice. However, H361 did not produce more SPEA than H305 in vivo. We conclude that SPEA does not play a key role in invasive murine streptococcal infection.

Enhanced Susceptibility to Superantigen‐Associated Streptococcal Sepsis in Human Leukocyte Antigen–DQ Transgenic Mice

Bacterial superantigens are believed to cause septic shock, although, because of the lack of superantigen-sensitive infection models, proof that superantigenicity underlies shock pathogenesis is lacking. This work demonstrates a clear superantigen effect in septic shock resulting from bacterial infection. Transgenic expression of human leukocyte antigen (HLA)-DQ, but not HLA-DR, specifically augments lymphocyte responses to streptococcal pyrogenic exotoxin A (SPEA). HLA-DQ transgenic mice had increased mortality after administration of SPEA or infection with Streptococcus pyogenes. Immune activation during infection was HLA-DQ transgene-dependent and was manifested by Vbeta-specific T cell repertoire changes and widespread lymphoblastic tissue infiltration. Unlike earlier models, which used toxin-induced shock, these T cell superantigen responses and lymphoblastoid changes were observed during invasive streptococcal sepsis. Lymphoid activation was undetectable in HLA-DQ mice infected with an isogenic SPEA(-) strain, which proves that a single superantigen can play a role in sepsis pathogenesis.

Dexamethasone and Cyclosporin A Affect the Maturation of Monocyte-Derived Dendritic Cells Differently

In contrast to the confirmed effects of glucocorticoids (GCs) and cyclosporin A (CyA) on T cells, the effects of both agents on antigen-presenting cells (APCs), especially on dendritic cells (DCs), are still poorly understood. In this study, we cultured monocyte-derived DCs (MoDCs) under a variety of stimulations in the presence or absence of these immunosuppressants and compared their effects on the activation of MoDCs by these stimulations. The stimulations used were the following: three bacterial toxins, including lipopolysaccharide (LPS), staphylococcal enterotoxin A (SEA) and streptococcal pyrogenic exotoxin A (SPEA), the combination of IL-1β and TNF-α, and an agonistic anti-CD40 antibody. All of these stimulations increased the expression of CD54, CD83, CD86, and HLA-DR antigen, and the production of TNF-α in MoDCs. When MoDCs were treated with dexamethasone (Dex) during the stimulation, Dex significantly suppressed the augmentation of CD86 expression and TNF-α production induced by all of these stimulations. In contrast, when MoDCs were treated with CyA, it inhibited only the effects induced by the superantigens, SEA and SPEA, but not that induced by LPS, the combination of cytokines, or anti-CD40 antibody. The augmentation of CD54 or HLA-DR antigen expression was not significantly suppressed by either Dex or by CyA. When we used MoDCs pretreated with each of these stimulations + Dex or + CyA as APCs, however, significant suppression of T cell proliferation was observed only in the case of the pretreatment with IL-1β/TNF-α + Dex. The allogeneic T cell stimulation by MoDCs pretreated with the other combinations did not significantly differ from that treated with the stimulation alone. Our present study succeeded in demonstrating a clear difference between Dex and CyA in the activation of MoDCs. These differences may induce a significant difference in their final immunological responses.

Invasive and noninvasive group A streptococcal isolates with different speA alleles in The Netherlands: genetic relatedness and production of pyrogenic exotoxins A and B.

Streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B have been implicated in the pathogenesis of severe group A streptococcal (GAS) disease. We studied 31 invasive GAS strains including 18 isolates from patients with toxic shock syndrome and 22 noninvasive strains isolated in The Netherlands between 1994 and 1998. These strains were associated with the different allelic variants of the gene encoding SPE-A. We selected endemic strains with speA-positive M and T serotypes: speA2-associated M1T1 and M22-60T12 strains, speA3-associated M3T3 strains, and speA4-associated M6T6 strains. Since speA1-positive isolates were not frequently encountered, we included speA1 strains of different serotypes. The GAS strains were compared genotypically by pulsed-field gel electrophoresis and phenotypically by the in vitro production of SPE-A and SPE-B. All strains within one M and T type appeared to be of clonal origin. Most strains produced SPE-A and SPE-B, but only a minority of the speA4-positive isolates did so. Among our isolates, speA1- and speA3-positive strains produced significantly more SPE-A than speA2- and speA4-carrying strains, while SPE-B production was most pronounced among speA1- and speA2-containing strains. There was a marked degree of variability in the amounts of exotoxins produced in vitro by strains that shared the same genetic profile. We conclude that the differences in the in vitro production of SPE-A and SPE-B between our selected strains with identical M and T types were not related to either genetic heterogeneity or the clinical course of GAS disease in the patient from whom they were isolated.

Relative avidities of human immunoglobulin G antibodies for streptococcal pyrogenic exotoxins A and B.

In this pilot study, we investigated the relative avidities for streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B of antibodies in sera from patients with fatal streptococcal toxic shock-like syndrome and from healthy individuals and in intravenous immunoglobulin (IVIG) preparations. We observed a great variation in the relative avidities of patient, control, and IVIG immunoglobulin G (IgG) (values estimated to be between 10(-7) and 10(-11) M), with mean values for patient IgG about 10-fold lower than those of control IgG.

Molecular analysis of the role of streptococcal pyrogenic Exotoxin A (SPEA) in invasive soft-tissue infection resulting from Streptococcus pyogenes.

Epidemiological studies strongly implicate the bacterial superantigen, streptococcal pyrogenic exotoxin A (SPEA), in the pathogenesis of necrotizing soft-tissue infection and toxic shock syndrome resulting from Streptococcus pyogenes. SPEA can act as a superantigen and cellular toxin ex vivo, but its role during invasive streptococcal infection is unclear. We have disrupted the wild-type spea gene in an M1 streptococcal isolate. Supernatants from toxin-negative mutant bacteria demonstrated a 50% reduction in pro-mitogenic activity in HLA DQ-positive murine splenocyte culture, and up to 20% reduction in activity in human PBMC culture. Mutant and wild-type bacteria were then compared in mouse models of bacteraemia and streptococcal muscle infection. Disruption of spea was not associated with attenuation of virulence in either model. Indeed, a paradoxical increase in mutant strain-induced mortality was seen after intravenous infection. Intramuscular infection with the SPEA-negative mutant led to increased bacteraemia at 24 h and a reduction in neutrophils at the site of primary muscle infection. Purified SPEA led to a dose-dependent increase in peritoneal neutrophils 6 h after administration. SPEA is not a critical virulence factor in invasive soft-tissue infection or bacteraemia caused by S. pyogenes, and it could have a protective role in murine immunity to pyogenic infection. The role of this toxin may be different in hosts with augmented superantigen responsiveness.

Activation of mouse liver natural killer cells and NK1.1(+) T cells by bacterial superantigen-primed Kupffer cells.

Although bacterial superantigens have been well characterized as potent stimulators of T cells, their role in natural killer (NK)-type cells remains largely unknown. In the present study, we examined the effect of bacterial superantigens on mouse liver NK cells and NK1.1 Ag(+) (NK1(+)) T cells. C57BL/6 mice were intravenously injected with staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A), and mononuclear cells (MNC) of various organs were obtained from mice 4 hours after being injected with superantigen. MNC were cultured for 48 hours, and interferon gamma (IFN-gamma) levels of supernatants were measured. The antitumor cytotoxicities of the liver and spleen MNC were also evaluated 24 hours after the mice were injected with superantigen. Liver MNC produced more IFN-gamma than did splenocytes, and peripheral blood and lung MNC did not produce any detectable IFN-gamma. In addition, liver MNC acquired a potent antitumor cytotoxicity by the SEB injection, and both NK cells and NK1(+)T cells but not cluster of differentiation (CD)8(+) T cells were responsible for the cytotoxicity as demonstrated by either in vivo or in vitro cell depletion experiments, and the NK-type cells were partly responsible for the increased serum IFN-gamma. Activation of liver NK-type cells was also supported by the fact that liver NK cells proportionally increased and NK1(+) T cells augmented their CD11a expressions after SEB injection. The pretreatment of mice with anti-IFN-gamma Ab and/or with anti-interleukin-12 (IL-12) Ab diminished the SEB-induced cytotoxicity of liver MNC. Furthermore, the in vivo depletion of Kupffer cells decreased the SEB-induced cytotoxicity of liver MNC. Consistent with these results, liver MNC stimulated with superantigens in the presence of Kupffer cells in vitro produced a greater amount of IFN-gamma than did the liver MNC without Kupffer cells or splenocytes. Our results suggest that bacterial superantigen-primed Kupffer cells produce IL-12 and other monokines, while also nonspecifically activating both NK cells and NK1(+) T cells to produce IFN-gamma.

THE ROLE OF LIPOPOLYSACCHARIDE, PRO-INFLAMMATORY CYTOKINES AND BACTERIAL SUPERANTIGENS IN THE TRANSCRIPTIONAL REGULATION OF LYMPHOTOXIN α AND β IN MOUSE SPLENOCYTES

Lymphotoxin α (LT-α) and lymphotoxin β (LT-β) are members of the tumour necrosis factor (TNF) ligand family. Because of the importance of TNF in the pathogenesis of septic shock, the expression of LT-α and LT-β mRNA in murine splenocytes stimulated with different pro-inflammatory cytokines, sepsis-associated mediators such as lipopolysaccharide (LPS) and bacterial superantigens was investigated. The authors show that the bacterial superantigens, toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin B (SEB) upregulate LT-α mRNA expression in vitro in murine cells. Basal expression of LT-β mRNA was found in unstimulated murine splenocytes, and could be increased by the addition of the mitogen concanavalin A (Con A). Despite this suggested inducibility of the murine LT-β transcript, sepsis-associated mediators did not affect its regulation. Neither the pro-inflammatory cytokines interleukin 2 (IL-2), TNF-α nor LPS alone or in combination with interferon γ (IFN-γ) had any effect on LT-β mRNA expression. The bacterial superantigens TSST-1, SEB and streptococcal pyrogenic exotoxin A (SPEA) were also unable to upregulate LT-β mRNA transcript, in contrast to the observation with LT-α.

Comparative effects of clindamycin and ampicillin on superantigenic activity of Streptococcus pyogenes.

We have tested the ability of Streptococcus pyogenes to produce streptococcal pyrogenic exotoxin A (SPEA) and superantigenic activity in the presence of sub-inhibitory concentrations of ampicillin and clindamycin. After 6 h of culture, SPEA production by S. pyogenes was higher in broths containing ampicillin (715.7 +/- 296.4 pg/10(6) cfu) than in broths containing clindamycin (167.1 +/- 31.9 pg/10(6) cfu), a difference that was not significant (P = 0.25). Promitogenic activity of bacterial supernatants was also greater in ampicillin-treated cultures (467.5 +/- 17.2 ccpm/10(6) cfu) than in clindamycin-treated cultures (169.2 +/- 8.9 ccpm/10(6) cfu), a difference that was highly significant (P = 0.0001). The data support the use of clindamycin in the treatment of streptococcal toxic shock-like syndrome, in order to inhibit superantigen synthesis.

Streptococcal Pyrogenic Exotoxin A Release, Distribution, and Role in a Murine Model of Fasciitis and Multiorgan Failure Due to Streptococcus pyogenes

The role of streptococcal pyrogenic exotoxin A (SPEA) was evaluated in a murine model of fasciitis and multiorgan failure due to a toxigenic strain of Streptococcus pyogenes. Increased serum levels of SPEA at 15 and 21 h were associated with a survival time of <24 h. Levels of SPEA correlated with interleukin-6 levels. Immunostaining showed SPEA localized to renal and hepatic cells. Neutralizing rabbit antibody to SPEA was administered to mice challenged with S. pyogenes, but no effect on survival was observed. Vaccination of mice with recombinant SPEA enhanced mortality due to streptococcal infection, despite the development of neutralizing immunity to the toxin prior to infection. Hence, SPEA is produced systemically during S. pyogenes soft-tissue infection, and increased levels are associated with reduced survival. In this model, however, SPEA did not appear to play a dominant role in pathogenesis; passive immunization against SPEA was not protective, and active immunization enhanced mortality.

Intravenous Immune Globulin Has Effects on Superantigen-Induced Cytokine Synthesis

Increased incidences of invasive group A streptococcal infections occurred in the Western Society in the 1980s, and the number of streptococcal toxic shock syndrome (STSS) cases continue to rise. Among the hypotheses entertained to explain the pathogenesis of STSS has been the concept that streptococcal superantigens (Sags), including streptococcal pyrogenic exotoxins (SPEs), can trigger potent inflammatory responses and release of cytokines of TH 1 type (TNF-oc, IL-1 IFN-γ and TNF-β). These may mediate tissue damage, organ failure, and shock.Pooled human immune globulin G (IgG) preparations for intravenous administration (ivIgG) have been used in many inflammatory conditions due to their immunomodulatory potential. The effector mechanisms are incompletely understood. This study dealt with the effects of ivIgG on cytokine production in vitro. Cytokine synthesis was identified at the single-cell level using cytokine-specific monoclonal antibodies (mAb) and indirect immunocytochemical techniques. The super-antigen streptococcal Pyrogenic exotoxin-A (SPE-A) induced vigorous cell activation and extensive cytokine synthesis. ivIgG was added either at the beginning or 24 h after the initiation of cultures of peripheral blood mononuclear cells (PBMNC) in order to elucidate the importance of direct toxin neutralization. Addition of IVlg (6 mg/ml) from the beginning of cultures induced a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular of IFN-γ and TNF-β. Supplementation of ivIgG 24 h after initiation of cultures also led to a significant decrease