TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88.

TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.

[Validity of the Inpatient Satisfaction Questionnaire].

OBJECTIVE: To assess the validity of the self-designed inpatient satisfaction questionnaire using Structural Equation Model (SEM). METHODS: The questionnaire survey was conducted in a tertiary hospital in Sichuan in April 2016,with participants selected through a systematical sampling approach. The structural validity of the inpatient satisfaction questionnaire was assessed using SEM. The statistical analyses were performed using Lisrel8.70. RESULTS: About 98.5% of returned questionnaires were valid for data analyses,which resulted in a total sample of 2562. A good model fit was achieved: df=8.36,root mean square error of appoximation (RMSEA)=0.054,root mean square residual (RMR)=0.021,goodness-of-fit index (GFI)=0.93,adjusted GFI (AGFI)=0.91,com-parative fit index (CFI)=0.98,non-normed fit index (NNFI)=0.98,parsimony GFI(PGFI) =0.74. Factor loadings on exogenous latent variables ranged from 0.59 to 0.94,with 0.52-0.87 AVE. CONCLUSION: The questionnaire has a good construct validity,which can be used for evaluating inpatient satisfaction in tertiary hospitals.

Effect of ginkgolide B on the platelet-activating factor induced changes of chemotaxis and cytoskeleton of macrophages.

AIM: To study the inhibitory effect of ginkgolide B (BN52021) on the PAF induced changes of chemotaxis of murine peritoneal macrophages and the related polymerization of F-actin. METHODS: Chemotaxis assays were performed using a modified 48-well Boyden chamber. Actin polymerization of murine peritoneal macrophages was analyzed by flow cytometry using a specific fluorescent stain. RESULTS: Peritoneal macrophages significantly migrated toward platelet-activating factor (PAF) through a micropore filter; however, in the presence of PAF receptor antagonist BN52021 (0.01 nmol x L(-1) -0.1 micromol x L(-1)), the migration was significantly inhibited. Moreover, BN52021 inhibited the actin polymerization of murine peritoneal macrophages induced by PAF in the presence of Ca2+, but not in Ca2+ -free medium. CONCLUSION: The results suggested that preventing polymerization of F-actin may be a pathway by BN52021 to inhibit the chemotaxis of macrophages, and this effect seems to be Ca2+ dependent. The data further indicated that inhibition of PAF induced macrophage chemotaxis is an important mechanism underlying the anti-inflammatory action of BN52021.

[Inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation].

AIM: To investigate the inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation and the possible mechanisms. METHODS: The murine chronic granulomatous air pouch model was used to observe the anti-angiogenesis effect of ginkgolide B. The vascular index was determined by colorimetry of carminic acid, and angiogenesis was observed by histology method. The interleukin-1beta (IL-1beta) levels in mice serum and in supernatants of U937 cell culture stimulated by phorbol 12-myristate 13-acetate (PMA) were detected by radioimmunoassay (RIA). The tumor necrosis factor-alpha (TNF-alpha) levels in mice serum and in supernatant of U937 cell culture were measured by cytotoxicity bioassay. The mRNA expression of IL-1beta and TNF-alpha of U937 cell culture was investigated by RT-PCR. RESULTS: Oral administration of ginkgolide B 25 and 100 mg x kg(-1) was shown to significantly inhibit the vascular index of murine chronic granulomatous air pouch model with the inhibitory rate of 22.52% and 25.29%, respectively. This result was supported by histological observation. Concomitantly, the IL-1beta levels in mice serums were also significantly decreased with the inhibitory rate of 50.61% and 58.66%; so were the TNF-alpha levels with the inhibitory rate of 28.91% and 52.41%. Ginkgolide B at concentration of 1 x 10(-5) to 1 x 10(-8) mol x L(-1) could also reduce both the IL-1beta and TNF-alpha contents in the supernatants of U937 cell culture stimulated by PMA, but the scopes of changes were much different. For IL-1beta the IC50 was 1.93 x 10(-8) mol x L(-1), while ginkgolide B at concentration of 1 x 10(-5) mol x L(-1) only decreased the release of TNF-alpha by 25.99%. Furthermore, ginkgolide B at concentrations of 1 x 10(-5) to 1 x 10(-7) mol x L(-1) was shown to significantly inhibit TNF-alpha mRNA expression of U937 cells; and at concentrations of 1 x 10(-5) and 1 x 10(-6) mol x L(-1) could inhibit IL-1beta mRNA expression. CONCLUSION: Ginkgolide B was shown to significantly inhibit angiogenesis of the murine chronic granulomatous air pouch model, reduce the IL-1beta and TNF-alpha levels in mice serums, and significantly inhibit IL-1beta and TNF-alpha mRNA expression and protein secretion in supernatants of U937 cell culture. It was suggested that reduction of proangiogenic cytokines IL-1beta and TNF-alpha secretion may contribute to the anti-angiogenesis effect of ginkgolide B in the murine chronic granulomatous air pouch model.