Yersinia enterocolitica Exploits Signal Crosstalk between Complement 5a Receptor and Toll-like Receptor 1/2 and 4 to Avoid the Bacterial Clearance in M cells

In the intestinal mucosal surface, microfold cells (M cells) are the representative gateway for the uptake of luminal antigens. At the same time, M cells are the primary infection site for pathogens invading mucosal surface for their infection. Although it is well recognized that many mucosal pathogens exploit the M cells for their infection, the mechanism to infect M cells utilized by pathogens is not clearly understood yet. In this study, we found that M cells expressing complement 5a (C5a) receptor (C5aR) also express Toll-like receptor (TLR) 1/2 and TLR4. Infection of Yersinia enterocolitica, an M cell-invading pathogen, synergistically regulated cyclic adenosine monophosphate-dependent protein kinase A (cAMP-PKA) signaling which are involved in signal crosstalk between C5aR and TLRs. In addition, Y. enterocolitica infection into M cells was enhanced by C5a treatment and this enhancement was abrogated by C5a antagonist treatment. Finally, Y. enterocolitica infection into M cells was unsuccessful in C5aR knock-out mice. Collectively, we suggest that exploit the crosstalk between C5aR and TLR signaling is one of infection mechanisms utilized by mucosal pathogens to infect M cells.

Effect of AngⅡ on Insulin Gene Expression in RIN-m Cells and Its Molecular Mechanism / 中国药师

Objective:To discuss the effect of AngⅡ on insulin gene expression IN RIN-m cells and its molecular mechanism. Methods:RIN-m cells were cultured and divided into three groups, including the control group, 100 nmol·L-1 AngⅡ group and losartan pretreatment group. After 24-hour incubation, insulin gene expression in RIN-m cells was detected by RT-PCR, the mean flu-orescent intensity of 2', 7'-dichlorofluorescein ( DCF) was detected by flow cytometry, PDX-1 and MafA mRNA expression were detec-ted by RT-PCR and the protein expression was detected by Western-blot. Results: Insulin expression in RIN-m cells, cellular ROS level, PDX-1 expression and MafA expression in 100 nmol · L-1 AngⅡ group were significantly different from those in the control group and losartan pretreatment group (P0. 05). Conclusion:AngⅡ can down-regulate PDX-1 and MafA expression inβ-cells through oxidative stress pathway, and then inhibit insu-lin gene expression. Pretreatment with losartan can antagonize the effect of AngⅡ, and has protective effect onβ-cells in the aspect of insulin gene expression.

Viriditoxin Induces G2/M Cell Cycle Arrest and Apoptosis in A549 Human Lung Cancer Cells

Viriditoxin is a fungal metabolite isolated from Paecilomyces variotii, which was derived from the giant jellyfish Nemopilema nomurai. Viriditoxin was reported to inhibit polymerization of FtsZ, which is a key protein for bacterial cell division and a structural homologue of eukaryotic tubulin. Both tubulin and FtsZ contain a GTP-binding domain, have GTPase activity, assemble into protofilaments, two-dimensional sheets, and protofilament rings, and share substantial structural identities. Accordingly, we hypothesized that viriditoxin may inhibit eukaryotic cell division by inhibiting tubulin polymerization as in the case of bacterial FtsZ inhibition. Docking simulation of viriditoxin to beta-tubulin indicated that it binds to the paclitaxel-binding domain and makes hydrogen bonds with Thr276 and Gly370 in the same manner as paclitaxel. Viriditoxin suppressed growth of A549 human lung cancer cells, and inhibited cell division with G2/M cell cycle arrest, leading to apoptotic cell death.

The inhibition effects of continuous low dose rate radiation of 125I radioactive seeds on KYSE150 esophageal cancer cells / 中华放射医学与防护杂志

Objective To determine the biological effectiveness of 125I radioactive seeds with continuous low dose rate radiation on the human esophageal cancer cell line KYSE150 in vitro and explore the underlying cellular mechanisms.Methods The cells were divided into three cell groups:control group,single dose radiation group (SDR) and 125I radioactive seeds with continuous low dose rate radiation group (125 I-CLDR).The KYSE150 cells were exposed to radiation of X-ray at a high dose rate of 1.052 Gy/min or 125I radioactive seeds at a low dose rate of 2.77 cGy/h.The responses of KYSE150 cells to two modes of irradiation were evaluated by the colony-forming assay,cell apoptosis as well as cell cycle analysis.Furthermore,the expression levels of γ-H2AX and Bax were detected by Western blot.Results KYSE150 cells were more radiosensitive to 125I-CLDR than SDR.The relative biological effectiveness (RBE) for 125I-CLDR related to SDR was 1.56.Compared with SDR,125I-CLDR yielded more proportions of the early and late apoptosis rate (t =4.07,11.08,P ＜0.05) as well as cells at G2/M phase (t =11.25,P ＜0.05).Moreover,γ-H2AX and Bax expression levels in 125I-CLDR significantly increased compared with SDR.Conclusions Compared with the high dose rate X-ray radiation,the continuous low dose rate radiation of 125I radioactive seeds had stronger inhibition effect on KYSE150 esophageal cancer cells by impairing clonogenic capacity,inducing apoptosis and G2/M cell cycle arrest,and increasing radiosensitivity.

Antigen targeting to M cells for enhancing the efficacy of mucosal vaccines

Vaccination is one of the most successful applications of immunology and for a long time has depended on parenteral administration protocols. However, recent studies have pointed to the promise of mucosal vaccination because of its ease, economy and efficiency in inducing an immune response not only systemically, but also in the mucosal compartment where many pathogenic infections are initiated. However, successful mucosal vaccination requires the help of an adjuvant for the efficient delivery of vaccine material into the mucosa and the breaking of the tolerogenic environment, especially in oral mucosal immunization. Given that M cells are the main gateway to take up luminal antigens and initiate antigen-specific immune responses, understanding the role and characteristics of M cells is crucial for the development of successful mucosal vaccines. Especially, particular interest has been focused on the regulation of the tolerogenic mucosal microenvironment and the introduction of the luminal antigen into the lymphoid organ by exploiting the molecules of M cells. Here, we review the characteristics of M cells and the immune regulatory factors in mucosa that can be exploited for mucosal vaccine delivery and mucosal immune regulation.

Targeted Delivery of VP1 Antigen of Foot-and-mouth Disease Virus to M Cells Enhances the Antigen-specific Systemic and Mucosal Immune Response

Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and-mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infection has been initiated, which is hardly achievable through parenteral immunization. Given that effective delivery of vaccine materials into immune inductive sites is prerequisite for effective oral mucosal vaccination, M cell-targeting strategy is crucial in successful vaccination since M cells are main gateway for luminal antigen influx into mucosal lymphoid tissue. Here, we applied previously identified M cell-targeting ligand Co1 to VP1 of FMDV in order to test the possible oral mucosal vaccination against FMDV infection. M cell-targeting ligand Co1-conjugated VP1 interacted efficiently with M cells of Peyer's patch. In addition, oral administration of ligand-conjugated VP1 enhanced the induction of VP1-specific IgG and IgA responses in systemic and mucosal compartments, respectively, in comparison with those from oral administration of VP1 alone. In addition, the enhanced VP1-specific immune response was found to be due to antigen-specific Th2-type cytokine production. Collectively, it is suggested that the M cell-targeting strategy could be applied to develop efficient oral mucosal vaccine against FMDV infection.

The effects of interleukin-18 on M cells of intestinal mucosa in murine acute pancreatitis / 中华普通外科杂志

Objective To investigate the effects of interleukin-18 ( IL-18 ) on M cells of intestinal mucosa in acute pancreatitis (AP) in rats. Methods Seventy SD rats were divided into seven groups: AP 6 hour group, 12 hour group, 24 hour group, IL-18 6 hour group, 12 hour group, 24 hour group, and control group at random. The models of AP were established. Serum, pancreatic tissue, and ileal mucosa were harvested. Serum amylase, glutamate pyruvate transaminse, total bilirubin, endotoxin, IL-27, and TNF-α were detected. Pathologic changes of pancreatic tissue and ileal mucosa were observed. The protein with reverse transcription-polymease chain reaction (RT-PCR). Results The level of serum TNF-α increased obviously in IL-18 6 hour group, 12 hour group, and AP group. The level of serum IL-27 increased in IL-18 6 hour group and AP group and back to normal in IL-18 24 hour group. The in IL-18 group than AP group (P < 0. 05 ). Conclusions IL-18 may affect M cells function and play important roles in intestinal barrier in acute pancreatitis.

Ultrastructural Changes of Epithelium Covering Peyer's Patch by Simple Obstruction of Gerbil Ileum / 체질인류학회지

The light and electron microscopic studies were carried out to find the morphological changes of epithelial cells covering Peyer's patch after simple observation of gerbil (Mongolian gerbil) ileum. Animals were classified as the control, 6 hour-ligation and 18 hour-ligation groups. Terminal ileum was ligated with white silk around Peyer's patch without the vascular injury. In control group, epithelia of the gerbil ileum was consisted of villous epithelium and follicle-associated epithelium (FAE) covering Peyer's patch. FAE represented typical dome structure, and was composed of the cuboid absorptive cells mainly and M cells. M cells were distributed at the periphery rather than central portion of dome-like FAE that are distinguishable from absorptive cells, owing to their typically short and thick microvilli on its free surface. In the light mictoscopy on 6 hour-ligation group, cells with vacuoles were appeared in FAE, and some lymphocytes in lymphoid follicle were condensed and then densely stained. There are many lymphocytes in FAE, infiltrated through the interrupted basement membrane. In the electron microscopic findings of 6-hour-ligated group, absorptive cells appeared to have many vesicle and vacuoles in various size, some lipid droplets and membranous structure contained inclusion bodies. Microvilli of M cell appeared to be destroyed at the central portion on its free surface. In the light microscopy of 18 hour-ligation group, FAE destructed partially and lymphoid follicle was hypertrophied and atrophied simultaneously. In the electron microscopic findings of 18 hour-ligation group, absorptive cells appeared to have the irregular and densely stained nucleus, and have many lipid droplets other than structures observed in 6 hour-ligation group. M cell appeared to have various-sized vacuoles, and have the bleb-like and irregular membrane-limited structures that protrude into the lumen and have less the cytoplasmic cell organs. These results suggested that the simple ligation of ileum gives rise to the inflammatory response on FAE of 6 hourligated group and then lead to the various response; degeneration, necrosis and atrophy of cells in FAE, and the hypertrophy and atrophy of lymphocytes in lymphoid follicle. M cell might have no special function and have the degenerative change with the adjacent absorptive cells during simple obstruction.

Genistein Induces G2/M Cell Cycle Arrest and Apoptosis in Rat Neuroblastoma B35 Cells; Involvement of p21(waf1/cip1), Bax and Bcl-2

BACKGROUND: The effect of genistein on different types of cells has been investigated. However, its effect on the nervous system is still unclear. The aim of the present work is to explore the effect of genistein on rat neuroblastoma B35 cells. METHODS: The effect of genistein on the proliferation of B35 cells, its cytotoxicity, the cell-cycle distribution, the ultra-structural changes and the induction of apoptosis were determined using MTT assay, LDH assay, Flow-cytometric analysis, transmission electron microscopy and Hoechst staining, respectively. Furthermore, Real-time quantitative RT-PCR and Western blotting were used to examine the transcriptional and post-translational alterations of the G2/M cell-cycle arrest marker cyclin-dependent kinase inhibitor p21(waf1/cip1) and the apoptosis-related genes after genistein treatment. RESULTS: Genistein significantly inhibits cell survival, slightly elevates the release of lactate dehydrogenase and induced apoptosis in B35 cells. Genistein increased the number of cells at S-phase and induced cells to accumulate at the G2/M phase. These G2/M arrested cells are associated with a marked up-regulation of p21(waf1/cip1) at both the mRNA and protein levels. We observed that genistein up-regulates pro-apoptotic Bax with concurrent down-regulation of the anti-apoptotic Bcl-2 protein. CONCLUSION: These observations suggest that the anticancer effect of genistein on B35 neuroblastoma cells is mediated through multiple cellular pathways including G2/M cell-cycle arrest and the induction of apoptosis.

Effects of chloride channel blockers on H_2O_2 induced apoptosis in pancreas RIN-m beta cells / 中国药理学通报

Aim To investigate the effects of chloride channel blockers on the apoptosis of RIN-m? cells of pancreatic islet induced by H2O2.Methods The apoptotic model was made by H2O2 exposed for six hours with a concentration of 500 ?mol?L-1.The chloride channel blockers:DIDS,NPPB and NFA were administered to pretreat the samples respectively.The cell viability,morphological changes,and apoptosis rate were observed.Results Chloride channel blockers alone have no marked effects on the cell viability of RIN-m? cell.However,they elevated the cell viability of RIN-m?cell disposed of by H2O2.Compared to H2O2 group,the groups of DIDS +H2O2,NPPB+ H2O2 and NFA+H2O2 have significant difference in cell viability and apoptosis rate(P