Prenatal Exposure of Rats to Staphylococcal Enterotoxin B Alters the Expression of Th1 and Th2 Cytokines via Decreased Methylation in the Spleen of Adult but not Neonatal Offspring.

BACKGROUND: The methylation of IFN-γ and IL-4 genes is regarded as an epigenetic regulation that maintains the Th1 or Th2 phenotype. OBJECTIVE: To explore the influence of prenatal administration of the staphylococcal enterotoxin B (SEB) in pregnant rats, on the IFN-γ or IL-4 expression in the offspring spleen. METHODS: The SEB or PBS was administered intravenously to pregnant rats on the embryo-day 16. After normal delivery, the spleens from the fifth-day neonates and adult offspring were isolated under anesthesia. Quantitative PCR, western blot, ELISA and MeDIP-qPCR were applied to determine the levels of the splenic IFN-γ or IL-4 mRNAs, their protein levels, and methylation status, respectively. RESULTS: Prenatal administration of the SEB in pregnant rats decreased the levels of the splenic IFN-γ and IL-4 proteins in neonates, but increased their mRNA levels. However, prenatal administration of the SEB significantly augmented both mRNA and protein levels of the IFN-γ and IL-4 in the adult spleen. In addition, the prenatal SEB administration decreased the methylation of the splenic IFN-γ and IL-4 in adult but not neonatal offspring. CONCLUSION: The prenatal administration of SEB in pregnant rats can cause a mixed Th1 and Th2 cytokines response in the offspring spleen, and alter the cytokine expression of the Th1 and Th2 via decreasing the methylation in adult but, not neonatal offspring spleen.

[Immune Modulatory Effect of Outer Membrane Vesicles Derived from Salmonella on Mouse Bone Marrow-Derived Dendritic Cells].

OBJECTIVE: To study the effect of outer membrane vesicles (OMVs) derived from Salmonella typhimurium (ST) on the ultrastructural features and immune function of dendritic cells (DC). METHODS: Mice bone marrow cells were collected aseptically, and myeloid DC were generated by the combined induction and amplification with recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Cell morphology was observed under inverted phase contrast microscope and the phenotype was identified with flow cytometry. ST-OMVs were isolated through ultracentrifugation. The survival rate of DC was assessed with CCK-8 assay, and the stimulus concentration of OMVs was henceforth determined. The ultrastructural characteristics of DC loaded with OMVs were observed with transmission electron microscopy. The cytokine secretion, surface molecule expression and phagocytic capacity of DC were examined with flow cytometry. RESULTS: The DC induced and amplified in vitro displayed typical DC phenotype in morphological analysis and the purity of DC exceeded 85%. Transmission electron microscopy showed that there were large numbers of protrusions on the cell surface. After stimulation with ST-OMVs, it was observed that the dendritic structures on the surface of DC were reduced and a large number of phagolysosomes were found in the cytoplasm. In addition, increased numbers of mitochondria, swelling and typical apoptosis were observed. After treatment with ST-OMVs at 5 µg/mL and 10 µg/mL, the secretion of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) of DC increased significantly ( P<0.05). Furthermore, the immature DC could differentiate into mature DCs after stimulation with ST-OMVs, which were characterized by a decrease in phagocytic capacity ( P<0.05) and an upregulation of phenotypic markers ( P<0.05). CONCLUSION: ST-OMVs can stimulate DC to produce TNF-α and IL-1ß and promote DC maturation and antigen presentation.

Exposure of pregnant rats to staphylococcal enterotoxin B increases offspring splenic Treg number and function via decreasing FoxP3 methylation.

Staphylococcus aureus is an infectious pathogen that is relatively common, but that can cause severe disease in pregnant women and their fetus. We previously demonstrated that exposing pregnant rats to staphylococcal enterotoxin B (SEB) altered splenic CD4/CD8 T cell frequencies in their offspring. Whether prenatal SEB exposure impacts Tregs in these offspring, however, remains to be determined. As such, in this study, we intravenously injected pregnant rats with 15 µg of SEB on gestational day 16. Splenic tissue was then harvested from 1-, 3-, and 5-day-old neonatal rats and analyzed via flow cytometry to assess Treg numbers. In addition, FoxP3 expression levels were assessed via qPCR and western blotting, while FoxP3 methylation status was evaluated via methyl-DNA immunoprecipitation qPCR. Immunosuppression assays were additionally used to gauge Treg suppressive functionality. We found that exposing pregnant rats to SEB resulted in a significant increase in Treg numbers, FoxP3 expression, and Treg suppressive capacity in the spleens of both neonatal and adult offspring. In addition, total T cell, CD4+T cell, and non-Treg CD4+ T cell numbers were elevated in the spleens of offspring following prenatal SEB exposure. We additionally determined that SEB exposure resulted in a significant reduction in FoxP3 DNA methylation. Together, our results indicate that prenatal SEB exposure can markedly enhance offspring splenic Treg numbers and functionality at least in part by decreasing FoxP3 methylation.

Phenotypic and genetic characteristics of Streptococcus mutans isolates from site-specific dental plaque in China.

Introduction. Streptococcus mutans is an important cariogenic microbe.Hypothesis/Gap Statement. The potential characteristics of S. mutans isolates from site-specific dental plaque are still not clear.Aim. This study aimed to investigate the phenotypic and genetic characteristics of S. mutans isolates from site-specific dental plaque in China.Methodology. We used S. mutans isolated from children with early-childhood caries (ECC) and caries-free children to compare the phenotypic and genetic characteristics of S. mutans from site-specific dental plaque samples. The ECC subjects presented two sites: a cavitated lesion and a sound surface. The caries-free subjects presented one sound surface. Growth pattern, biofilm, decrease in pH, extracellular polysaccharide, expression levels of virulence-related genes, multilocus sequence typing (MLST) and phylogenetic trees were evaluated among these three sites.Results. The phenotypes detected between the cavitated and sound surfaces of ECC children were similar. However, the capacity for biofilm formation, pH drop and expression levels of genes (gtfB and spaP) of S. mutans in the caries-free group were lower compared with those of the ECC group. We identified 44 new alleles and 77 new sequence types. More than 90 % of the children with ECC shared an identical sequence type. The distribution of sequence types among different subjects showed diversity, and child-to-child transmission was detected.Conclusions. This is the first report of MLST on site-specific dental plaques in a single subject, and indicates that S. mutans isolated from site-specific dental plaque of a single subject showed similar phenotypes as a result of the isolates were closely related.

Exposure of pregnant rats to staphylococcal enterotoxin B attenuates the response of increased Tregs to re-exposure to SEB in the thymus of adult offspring.

Regulatory T cells (Tregs) play an essential role during homeostasis and tolerance of the immune system. Based on our previous study that exposure of pregnant rats to staphylococcal enterotoxin B (SEB) can alter the percentage of CD4/CD8 subsets in the thymus of the offspring, in this study, we focus on the influence of exposure of pregnant rats to SEB on number, function and response of Tregs in the thymus of the offspring. Pregnant rats at gestational day of 16 were intravenously injected with 15 µg SEB and the thymuses of the neonatal and adult offspring were harvested for this study. We found that exposure of pregnant rats to SEB could significantly increase the absolute number of Tregs and the FoxP3 expression level in the thymus of not only neonatal but also adult offspring. Re-exposure of adult offspring to SEB remarkably reduced the suppressive capacity of Tregs to CD4+ T cells and the expression levels of TGF-ß and IL-10 in the thymus, but had no effect on production of IL-4 and IFN-γ. Furthermore, it also notedly decreased the absolute number of Tregs and the FoxP3 expression level. These data suggest that prenatal exposure of pregnant rats to SEB attenuates the response of increased Tregs to re-exposure to SEB in the thymus of adult offspring.

Prenatal exposure of staphylococcal enterotoxin B attenuates the development and function of blood regulatory T cells to repeated staphylococcal enterotoxin B exposure in adult offspring rats.

Introduction. Staphylococcal enterotoxin B (SEB) is an extensively studied super-antigen. A previous study by us suggested that SEB exposure during pregnancy could alter the percentage of CD4+ and CD8+ T cells in the peripheral blood of neonatal offspring rats.Aim. It is unknown whether SEB exposure during pregnancy can influence the development of regulatory T cells (Tregs) in the peripheral blood of neonatal offspring rats.Methodology. Pregnant rats at gestational day 16 were intravenously injected with 15 µg SEB. Peripheral blood was acquired from neonatal offspring rats on days 1, 3 and 5 after delivery and from adult offspring rats for determination of Treg number by cytometry, cytokines by ELISA, and FoxP3 expression by real-time PCR and western blot.Results. SEB given to pregnant rats significantly increased the absolute number of Tregs and the expression levels of FoxP3, IL-10 and TGF-ß (P<0.05, P<0.01) in the peripheral blood of not only neonatal but also adult offspring rats. Furthermore, repeated SEB exposure in adult offspring rats significantly decreased the absolute number of Tregs (P<0.01), and the expression levels of FoxP3, IL-10 and TGF-ß (P<0.05, P<0.01) in their peripheral blood.Conclusion. Prenatal SEB exposure attenuates the development and function of Tregs to repeated SEB exposure in the peripheral blood of adult offspring rats.

[Effect of cordycepin on apoptosis and autophagy of tongue cancer cells in vitro and the molecular mechanism].

OBJECTIVE: To study the effect of cordycepin on cell cycle, apoptosis and autophagy of human tongue cancer TCA-8113 cells and explore the mechanism of cordycepin for inhibiting the occurrence of tongue cancer. METHODS: CCK-8 method was used to assess the inhibitory effect of cordycepin on TCA-8113 cell proliferation in vitro. The cell cycle and cell apoptosis of TCA-8113 cells treated with different concentrations of cordycepin were analyzed using flow cytometry. The expressions of apoptosis-related genes caspase-3, caspase-9, Bcl-2, and Bax were examined using quantitative real-time PCR and Western blotting, and immunohistochemistry was used to detect the expressions of autophagy-related proteins LC-3ß, P62, p-mTOR, and AMPK. RESULTS: CCK-8 assay showed that cordycepin significantly inhibited the proliferation of TCA-8113 cells in a concentration-dependent manner with an IC50 of 3.548 mg/mL at 24 h and an IC50 of 1.185 mg/mL at 48 h. Flow cytometric analysis showed that cordycepin caused cell cycle arrest at S phase and dose-dependently increased the apoptotic rate of TCA-8113 cells. Treatment of the cells with cordycepin enhanced the expressions of Bax, caspase-3 and caspase-9 at both the mRNA and protein levels and inhibited the expression of the antiapoptotic gene Bcl-2. Immunohistochemistry demonstrated that cordycepin promoted the expression of LC-3ß and AMPK and inhibited the expression of P62 and p-mTOR. CONCLUSION: Cordycepin inhibits the proliferation and induces apoptosis of HCT-116 cells through the mitochondrial pathway and induces autophagy via the AMPK/mTOR pathway.

[Influence of Ureaplasma Urealyticum Infection on Testicular Tissue Structure and Secretion Function in Rats].

OBJECTIVE: To study the influences of ureaplasma urealitycum (UU) infection on testicular tissue structure and secretion function in rats. METHODS: Forty clean grade male SD rats were randomly divided into the experiment group A (at 7 d after surgery), experiment group B (at 14 d after surgery), control group C (at 7 d after surgery) and control group D (at 14 d after surgery). There were 10 rats in each group. The experimental groups were injected with 0.6 mL UU4 through bladder. In the same way, the control groups were injected with the same volume of UU liquid medium. At day 7 and 14 after injection, the structures of testis of all rats were observed by light microscopy and spermatogenic cells by transmission electron microscopy. The content of testosterone in plasma and testicular fluid were detected by chemiluminescence method. RESULTS: The changes of inflammatory pathology (including the layer and amount of spermatogenic cell decreasing, inflammatory cell infiltrating and mature sperms decreasing) in the testis of group A and group B were found by light microscopy, and the inflammatory changes in group B were lighter than those in group A. The structures of testicular tissue in group C and group D were normal. The apoptosis performances of germ cell (including the cell membrane corrugated, nuclear chromatin concentration and nuclear rupture) in the testis of group A and group B were found by transmission electron microscopy, and the changes in group B were lighter than those in group A. The structures of germ cell in group C and group D were normal. The levels of plasma testosterone in group A and group B were significantly lower than that in group C and group D (P<0.01), the difference between group A and group B was not statistically significant. The testosterone level in testis interstitial fluid in group A was significantly lower than that in other groups (P<0.01), the differences between other groups were not statistical significant. CONCLUSIONS: The testicular tissue of UU infected rats can have various pathological damage and functional changes, further confirming that UU infection can cause male infertility. The pathological damage and functional changes of the testicular tissue of rats after UU infection can be gradually restored with the extension of the duration of the disease.

Staphylococcal enterotoxin B administration in pregnant rats alters the splenic lymphocyte response in adult offspring rats.

BACKGROUND: Our previous study suggested that SEB exposure in pregnant rats could lead to the change of T cells subpopulation in both peripheral blood and thymus of the offspring rats. However, rarely is known about the influence of SEB exposure in pregnant rats on T cell subpopulation in the spleens of offspring rats. RESULTS: SEB was intravenously administered to the pregnant rats at gestational day 16 in this study. The percentages, in vivo and in vitro responses of CD4 and CD8 T cells were investigated with flow cytometry. The prenatal SEB exposure obviously increased splenic CD4 T cell percentages of both neonates and adult offspring rats, and obviously reduced splenic CD8 T cell percentages of both the fifth day neonates and adult offspring rats. After spleens in the adult offspring rats were re-stimulated with SEB in vivo or in vitro, in vivo SEB stimulation could lead to the marked decrease of splenic CD4 T cell percentage and the marked increase of splenic CD8 T cell percentage. While in vitro SEB stimulation to the cultured splenocytes markedly decreased the proliferation of the splenic lymphocytes and the CD4 T cell percentage, and had no influence on CD8 T cell percentage. CONCLUSION: The prenatal SEB exposure could alter the percentages of CD4/CD8 T cell subpopulation and the response of CD4 and CD8 T cells to the in vivo and in vitro secondary SEB stimulation in the splenocytes of adult offspring rats.

Staphylococcal enterotoxin B administration during pregnancy imprints the increased CD4:CD8 T-cell ratio in the peripheral blood from neonatal to adult offspring rats.

Our previous study demonstrated that Staphylococcal enterotoxin B (SEB) administration during pregnancy could alter the percentage of T cells subpopulation in the thymus of the neonatal rats; however, little is known about the effect of maternal SEB administration during pregnancy on T cells subpopulation in the peripheral blood of the offspring rats. In the present study, pregnant rats at gestational day 16 were intravenously injected with 15 µg SEB. The present study found that prenatal exposure to SEB significantly decreased the percentages of CD8 T cells in the peripheral blood of both neonatal rats on the fifth day after delivery and the adult offspring rats. Furthermore, it significantly increased the percentage of CD4 T cells as well as the ratios of CD4 to CD8 T cells in both neonatal and adult offspring rats. Prenatal exposure to SEB significantly decreased the expression levels of IL-4 and IFN-γ in the plasma of neonatal and adult offspring rats. Furthermore, SEB restimulation significantly increased the percentage of CD8 T cells and significantly decreased the percentage of CD4 T cells. These data suggest the prenatal exposure to SEB can imprint the increased CD4:CD8 T cell ratio in the peripheral blood from the neonate to adulthood through the decreased CD8 T cells and the increased CD4 T cells, and altered the response characteristics of CD4 and CD8 T cells to secondary SEB administration in the peripheral blood of the adult offspring rats.

Decreased Vß8.2 T-cells in neonatal rats exposed prenatally to Staphylococcal enterotoxin B are further deleted by restimulation in an in vitro cultured thymus.

Staphylococcal enterotoxin B (SEB) administration during adulthood can cause the anergy or deletion of variable portion of the ? chain (V?)?expressing T cells. However, the effect of maternal SEB administration during pregnancy on the thymocytes of neonatal rats remains to be elucidated. In the present study, pregnant rats at gestational day 16 were intravenously injected with 15 µg SEB. The present study revealed that prenatal exposure of SEB significantly increased the proportion of cluster of differentiation (CD)4?single positive (SP) T cells and decreased the proportions of CD8?SP, CD4+ V?8.2+ and CD8+ V?8.2+ T cells in the thymus of neonatal rats between day 0 and 5 after delivery. In an in vitro cultured thymus, SEB restimulation significantly increased the proportion of double positive cells and decreased the proportions of CD4?SP, CD8?SP, CD4+ V?8.2+ and CD8+ V?8.2+ T cells. Furthermore, the decreased V?8.2+ T?cells in neonatal rats exposed prenatally to SEB were further deleted by SEB restimulation in an in vitro cultured thymus. These data suggested the special response pattern of the remaining SEB?specific T cells to SEB restimulation in neonatal rats exposed prenatally to SEB.

Staphylococcal enterotoxin B and α­toxin induce the apoptosis of ECV304 cells via similar mechanisms.

Staphylococcal enterotoxin B (SEB) and α­toxin produced by Staphylococcus aureus (S. aureus) are important in the pathogenesis of diseases. In the present study, we investigated the effects of SEB and α­toxin on ECV304 cells. It was identified that both SEB and α­toxin were capable of inducing the apoptosis of ECV304 cells in a dose­ and time­dependent manner. In addition, SEB and α­toxin were able to induce the expression of TNF­α and the activation of caspase­3 and ­8 in the ECV304 cells. The inhibition of TNF­α (with its neutralizing antibody) and caspase­3 and ­8 [with the corresponding inhibitory peptides; z-N-acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin (DEVD)-fluoromethyl ketone (FMK) for inhibition of caspase­3 and z-N-acetyl-Ile-Glu-Thr-Asp (IETD)-FMK) for inhibition of caspase­8] significantly decreased the rates of cell apoptosis induced by SEB and α­toxin, but was not able to completely block the induced cell apoptosis. These data suggest that SEB and α­toxin induce ECV304 cell apoptosis via a similar mechanism, which is partially mediated by the extrinsic death pathway involving TNF­α and caspase­8. These results provide insights into the synergistic pathogenicity of SEB and α­toxin during S. aureus infection.

Teratogenicity of Staphylococcus aureus L-forms using a mouse whole-embryo culture model.

Our previous studies have suggested that Staphylococcus aureus L-forms are able to pass through the placental barrier of mice from the maternal side to the fetal body and affect fetal growth and development, but little is known about the direct influence of S. aureus L-forms on embryos during the critical period of organogenesis. Mouse embryos at gestational day 8.5 were cultured in vitro for 48 h with 0, 50, 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1). At the end of the culture period, the mouse embryos were assessed morphologically for viability, growth and development. Bacteriological and immunohistochemical staining were used to determine the existence of S. aureus L-forms in embryonic tissues. We found that both crown-rump length and head length of mouse embryos exposed to S. aureus L-forms at a concentration of 50 c.f.u. ml(-1) were reduced. When the mouse embryos were exposed to 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1), the total morphological score, number of somites, dry embryo weight, yolk sac diameter, crown-rump length and head length were significantly lower than those of the control group. With the increased concentration of S. aureus L-forms in the culture medium, there were fewer normally developed embryos and more embryos with abnormalities or retardation in growth. S. aureus L-forms detected by Gram-staining and immunohistochemical detection of antigen were found in the tissues of embryos infected by S. aureus L-forms. These data suggest that S. aureus L-forms exert a direct teratogenic effect on cultured mouse embryos in vitro.