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Stich N.,Biomedizinische ForschungsgmbH | Model N.,Biomedizinische ForschungsgmbH | Samstag A.,Immunology Outpatient Clinic | Gruener C.S.,Biomedizinische ForschungsgmbH | And 2 more authors.
Toxins | Year: 2014

Toxic shock syndrome (TSS) results from the host's overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure. © 2014 by the authors; licensee MDPI, Basel, Switzerland. Source

Roetzer A.,Biomedizinische ForschungsgmbH | Haller G.,Biomedizinische ForschungsgmbH | Beyerly J.,Biomedizinische ForschungsgmbH | Geier C.B.,Immunology Outpatient Clinic | And 4 more authors.
BMC Microbiology | Year: 2016

Background: Nosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters. Results: Fifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7 % of all strains, in selw (92.2 %) and in selx (15.7 %). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates. Conclusion: Production rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins. © 2016 Roetzer et al. Source

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