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The differences between toxic or septic shocks in humans during infections by streptococci and gramnegative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokineinducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-la (IL-la), IL-1p, IL-6, IL-8, tumor necrosis factor alpha (TNF-a), and TNF-, induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-la, IL-1j0, IL-6, and TNF-a was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PBMC elicited large amounts of these cytokines, as well as IL-8 and TNF-j, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-la, IL-1,, IL-6, and TNF-a, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-0 in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-a was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.

The effect of triterpenes and flavonoids on the activity of several hyaluronic acid-splitting enzymes was investigated. Studies showed that the inhibitory effect of the triterpenes glycyrrhizin and glycyrrhetinic acid is dependent on the source of hyaluronate lyase. Hyaluronate lyase from Streptococcus agalactiae (Hyal B) and recombinant hyaluronate lyase from Streptococcus agalactiae (rHyal B) demonstrated strongest inhibition. In contrast, hyaluronate lyases from Streptomyces hyalurolyticus (Hyal S), Streptococcus equisimilis (Hyal C) and hyaluronidase from bovine testis (Dase) showed only reduced inhibition action. A non-competitive dead end inhibition with Ki=3.1+/-1.8x10(-6) mol/mL and Kii=6.7+/-2.4x10(-6) mol/mL was found for glycyrrhizin on recombinant hyaluronate lyase from Streptococcus agalactiae. The inhibitory effect of flavonoids on Hyal B, rHyal B and Dase was determined depending on the number of hydroxyl groups and side chain substituents in the molecule. Flavonoids with many hydroxyl groups inhibited hyaluronate lyase stronger than those with only a few. Native hyaluronate lyase (Hyal B) showed a more extensive inhibition than the recombinant protein (rHyal B). Accordingly, the inhibition by triterpenes and flavonoids is presumably specific for each hyaluronic acid (HA)-splitting enzyme.

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V␤2, V␤4, and V␤8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cellstimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V␤2 and V␤8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.

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