Effect of curcumin on superantigen TSST-1 induced inflammatory cytokines by splenocytes / 中国人兽共患病学报

In the present study ,we aimed to observe the effect of curcumin on TSST-1-induced inflammatory cytokines in splenocytes of mouse and provide evidence for the further study on the effect of curcumin on inflammatory shock .Lactate de-hydrogenase (LDH) release assay was used to determine cytotoxicity of different doses of TSST-1 and curcumin .Inflammatory cytokines were determined by ELISA and flow cytometry .The doses of TSST-1 and curcumin we used in the present study did not cause significant cytotoxicity .TSST-1 induced higher level of IFN-γand IL-2 production but relatively lower level of TNF-α.The production of IL-1β,IL-6 and IL-12 was undetectable .TSST-1 induced Th1 cytokines (IFN-γand IL-2) were not IL-12-dependent which was different from LPS-induced IFN-γ.Curcumin significantly reduced IFN-γand TNF-αproduction at the concentration of 15 umol/L (P0 .05) .It’s suggested that curcumin could significantly inhibit the production of IFN-γand TNF-αby splenocytes induced by TSST-1 ,but could not affect the prolifera-tion of T cells .

The use of mice as animal model for testing acute toxicity (LD-50) of toxic shock syndrome toxin / Utilização de camundongos como modelo animal para a verificação da toxicidade aguda da toxina-1 da síndrome do choque tóxico

Acute toxicity test (LD-50) using toxic shock syndrome toxin (TSST-1) was tested in BALB/c, C57BL/6 and Swiss mice. Animals (n = 10) were intraperitoneally injected with TSST-1 (0.01-10.0µg/mouse) followed 4h later by potentiating dose of lipopolysaccharide (75.0µg of LPS - E. coli O111:B4) and cumulative mortality was recorded over 72h. Control animals received either TSST-1 or LPS alone. The data were submitted to qui-Square test and acute toxicity test was calculated by probit analysis (confidence limits expressed as µg toxin/kg). BALB/c mice was the most sensitive (20.0µg/kg, 95 percent confidence limits: 9.0-92.0) followed by C57BL/6 (38.5µg/kg, 95 percent confidence limits: 9.11- 401.6). Data from Swiss mice was not conclusive, indicating only low sensitivity. Selection of the animal model and standardization of the experiment are fundamental for the development of serum neutralization tests used for final quality control of vaccine production.

A toxicidade aguda (DL-50) da toxina da síndrome do choque tóxico (TSST-1) foi testada em linhagens de camundongos BALB/c, C57BL/6 e Suíça. Os animais (n=10) inoculados intraperitoneal com doses crescentes de toxina (0,01 - 10,0µg/animal) receberam 4h após 75µg de LPS (E. coli O111: B4). A toxicidade aguda (DL50) foi observada por um período de 72h e os dados submetidos ao teste de qui- quadrado. Os resultados e os limites de confiança foram expressos em µg de toxina/kg. A linhagem BALB/c apresentou maior sensibilidade (20µg/kg - limite de confiança a 95 por cento entre 9,0- 92,0), seguida da C57BL/6 (38,5µg/kg - limite de confiança a 95 por cento entre 9,11 - 401,6). A amplitude dos limites de confiança deve-se à natureza da toxina, ao mecanismo de ação, a via de inoculação e ao animal utilizado. A seleção do modelo animal e a padronização do experimento são fundamentais para o desenvolvimento de testes de soro neutralização para fins de controle de qualidade do processo de produção de vacinas.

Produção de enterotoxinas e da toxina da síndrome do choque tóxico por cepas de Staphylococcus aureus isoladas na mastite bovina / Production of enterotoxins and toxic shock syndrome toxin by Staphylococcus aureus strains isolated from bovine mastitis

A total of 72 strains of Staphylococcus aureus were examined for the production of staphylococcal enterotoxins (SE) A, B, C, D and toxic shock syndrome toxin (TSST-1). The strains were isolated from milk samples from cows with mastitis in dairy herds of São Paulo State, Brazil. Off 72 isolates, 38 (52.8 percent) produced SEA, 38 (52.8 percent) SEB, 32 (44.4 percent) SED, 28 (38.9 percent) SEC and 27 (37.5 percent) TSST-1. From the 72 strains, 66 (91.7 percent) produced, at least, one or more toxin, including TSST-1

Inhibitory Effects of Dexamethasone on Production IL-1alpha and TNF-alpha in Peripheral Blood Mononuclear Cells

PURPOSE: IL-1alpha and TNF-alpha have a seminal role in the intial events of inflammation. An area of intense interest is the assessment of new therapeutic modalities that regulate inflammatory response in acute bacterial infection. We conducted this study to compare the inhibitory effects of dexamethasone on the secretion of proinflammatory cytokines, such as IL-lalpha and TNF-alpha from cord blood mononuclear cells(MC) to those of adult blood MC during stimulation with S. aureus TSST-1 and E. coli LPS. METHODS: MC were isolated by differential centrifugation on Ficoll-Hypaque gradients. Each MC were incubated with TSST-1(2ug/ml) or LPS(0.2ug/ml), with various concentrations of dexamethasone for 72hr. And the other MC were incubated with TSST-1 or LPS, using the same concentration of dexamethasone, which was added 4hr before or simultaneously or 4hr, 24hr, 48hr after the stimulation. Concentration of IL-1alpha and TNF-alpha was measured by ELISA kit. RESULTS: Dexamethasone showed significant inhibitory effects on secretion of IL-1alpha and TNF-alpha. In comparison with both cytokines, secretion of IL-1alpha was suppressed more severely than TNF-alpha. In comparison with each stimulators, inhibition of TSST-1 induced cytokines production was greater than LPS. There was no difference between adult and cord blood MC. When dexamethasone was added to MC 4hr before the stimulation, it had the best inhibitory effects on the production of proinflammatory cytokines. CONCLUSION: Proinflammatory cytokine production in cord and adult blood MC were inhibited by dexamethasone in a dose-dependent manner. Early treatment of dexamethasone is more effective and can be used for modulating or suppressing excessive proinflammatory cytokine production in acute bacterial infection.

Inhibitory Effects of Actinomycin-D and Pentoxifylline on the Production of IL-1alpha and TNF-alpha by Human Cord and Adult Blood Mononuclear Cells

PURPOSE: An area of intense intrest is assessment of new therapeutic modalities that regulate the inflammatory response in acute bacterial infection is proving to be an area of interest these days. So we conducted this study to compare the inhibitory effects of actinomycin-D and pentoxifylline on the production of IL-lalpha and TNF-alpha from cord blood mononuclear cells (MC) to those from adult blood MC stimulated with S. aureus toxic shock syndrome toxin (TSST)-1 and E. coli lipopolysaccharide (LPS). METHODS: Cord and adult blood MC were isolated by differential centrifugation on Ficoll-Hypaque gradients. Each mononuclear cells were incubated with TSST-1 (2 microgram/ml) or LPS (0.2 microgram/ml), simultaneously with various concentrations of actinomycin-D or pentoxifylline added for inhibition. Concentration of interleukin-1alpha (IL-lalpha) and tumor necrosis factor-alpha (TNF-alpha) were measured by means of ELISA. RESULTS: In comparison with each inhibitory drug, actinomycin-D showed more potent inhibitory effects on the production of IL-1alpha and TNF-alpha from adult and cord blood MC stimulated by TSST-1 and LPS, than pentoxifylline (P<0.05). There was no difference between adult and cord blood MC. In comparison with each stimulator, inhibition of TSST-1 induced cytokines production with actinomycin-D was greater than pentoxifylline, in contrast to inhibition of LPS induced cytokines production with pentoxifylline which was greater than actinomycin-D in adult blood MC (P<0.05). CONCLUSION: Proinflammatory cytokine production in cord and adult blood MC were inhibited by each drug in the same manner except, the inhibition of pentoxifylline for LPS in cord blood MC. So it is possible that these drugs can be used to modulate or suppress excessive proinflammatory cytokine production in acute bacterial infection.

Mechanism of Toxic Shock Syndrome Toxin-1 as a Superantigen / 감염

BACKGROUND: Staphylococcus aureus TSST-1 is a microbial superantigen which requires interaction between MHC class II molecules and T-cell receptors. To characterize the mechanism of TSST-1 as a superantigen, we investigated the influence of TSST-1 on the binding of T-cell receptor and MHC II. METHODS: For control, A20 cells and lymphocytes from healthy volunteers were measured by the IL-2 bioassay with or withtout TSST-1, which was then purified and confirmed using immunoblot. mRNA for TNF from PBMC was prepared and RT-PCR for mRNA expression was performed. RESULTS: When stimulated by TSST-1(100pg/ mL), lymphocytes produced 135microgram/ml of IL-2 compared to the control A20 cells which produced 5microgram/ ml of 1L-2. TSST-1 was confirmed to be the strong inducer for mRNA expression by RT-PCR. CONCLUSION: The binding to T-cell receptor was enhanced by TSST-1 as confirmed by the bioassay of IL-2. This data may contribute to the understanding of mechanism of cell-toxin interaction.

Production of Interleukin-1alpha and Tumor Necrosis Factor-alpha by Human Cord and Adult Blood Mononuclear Cells Stimulated by TSST-1 and LPS

PURPOSE: Immature immunological defens mechanism in the neonate may contribute to the high susceptibility to overwhelming sepsis. S. aureus TSST-1 and E. coli LPS known as one of the important pathogens of septic shock or toxic shock induce massive release of various cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha) produced from peripheral blood mononuclear cells (PBMC). In contrast, limited information has been provided so far concerning the capacity of cytokine production from neonatal immune cells. METHODS: This study was conducted to compare the secretion of proinflammatory cytokines, such as IL-l and TNF-alpha from cord blood PBMC to those from adult blood PBMC stimulated by S. aureus TSST-1 and E. coli LPS. RESULTS: 1) IL 1-alpha was secreted in a time-dependent manner from cord & adult blood PBMC stimulated with several cytokine inducers, and LPS stimulated adult & cord blood PBMC secreted IL 1-alpha in a dose-dependent manner. 2) TNF-alpha secretion from cord blood PBMC stimulated with LPS and IFN- significantly decreased in a time dependent manner, but not from adult PBMC. And secretion of TNF- from cord blood PBMC reached the highest level 24 hours after stimulated with LPS or IFN-gamma. The secretion of TNF-alpha from adult blood PBMC showed similar pattern to those from cord blood PBMC, but higher than cord blood PBMC. 3) IL-1alpha & TNF-alpha secretion from cord & adult blood PBMC stimulated with TSST-1 had no significant difference except in TNF- secretion by TSST-1 at 96 hours. 4) The secretion of IL-1alpha from adult PBMC stimulated with LPS showed higher and longer than that from cord blood PBMC. 5) IL-1alpha & TNF-alpha secretion from cord & adult blood PBMC stimulated with IFN-gamma had no significant difference. CONCLUSIONS: In summary, proinflammatory cytokines such as IL-1alpha, TNF-alpha in cord blood PBMC were secreted in a time dependent manner, but the amounts of IL-1alpha and TNF-alpha secretion were lesser than those of adult blood PBMC, especially stimulated by LPS. These results suggest that increased susceptibility to infection in neonatal period may be partially from a functional immaturity of cord blood mononuclear cells.