Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.

Objective To investigate the effects of exogenous prostaglandin E 1 (PGE 1) on expression of platelet derived growth factor B (PDGF-B) mRNA and its receptor ? protein in rabbit with schistosomiasis. Methods In this study, 14 rabbits were infected with cercaria of S. japonicum percutaneously. PGE 1 ( 2.5 ?g/kg?d -1 ) was given intravenously to 7 rabbits from the 60th day to day 120. The expressions of PDGF-B mRNA, PDGFR ? protein and ?-SMA were detected by RT-PCR, Western blotting and immunohistochemistry, respectively. Endogenous IFN-? was measured by in situ hybridization. Results Up-regulated expressions of PDGF-B mRNA, receptor ? protein as well as ?-SMA were observed in rabbits with Schistosome hepatic fibrosis. The increased expressions of PDGF mRNA and receptor ? were suppressed in rabbits treated with exogenous PGE 1 (29.42?5.05 vs 41.37?7.23, P

0.05 ). The rate of hypertensive gastropathy, compared with PCDV (66.74%), was significantly attenuated in patiens who underwent PCDV+ORP (22.78%, P

Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization .Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group:sham (n=6), unilateral ureteral obstruction(UUO)3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group:bilateral ureteral obstruction(RBUO)0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6).The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining . Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein -1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68.Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF.TGF-β1 levels were determined by ELISA .Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats , and aggravated as time prolonged, but alleviated in rats with recanalization .TGF-β1 levels were increased obviously in the UUO group , but decreased in rats with recanalization compared with those in BUO rats .In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased .MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats .The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD 68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte -macrophages , which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction .Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation .

Objective To investigate the expression of a dismtegnn and metalloproteinase-12 (ADAM12) and proliferating cell nuclear antigen (PCNA) in human bladder carcinoma,and to explore their correlation with different grades and stages of bladder cancer.Methods Biopsies of 12 normal bladder and 43 bladder tumors were performed.And immunohistochemistry was conducted to detect the expression of ADAM12 and PCNA in the biopsies.Results Positive expression signals of ADAM12 were detected significantly higher inthe bladder cancer biopsies than that in the normal ones (Z =4.879,P ＜ 0.05),and the expression level of ADAM-12 in lower histological grade was significantly higher than that in the moderate and higher histological grades (x2 =22.3685,P ＜ 0.01).Positive expression signals of PCNA were detected significantly higher in the bladder cancer biopsies than that in the normal ones (Z =4.879,P ＜ 0.05)).Those with lower histological grade had a higher expression level of PCNA when compared with the moderate and higher histological grades (x2 =10.665,P =0.0137).The expression of ADAM-12 was positively correlated with PCNA in bladder cancer (r =1.000,P ＜ 0.0001).Conclusion The over expression of ADAM12 and PCNA maybe play an important role in development of the bladder tumors.And ADAM12 may be a promising biomarker of bladder cancer in the clinical behavior.

Objective To investigate the phenotype, frequency of Th17 cells and the association between Th17 cells and viral clearance in patients with H1N1 influenza A. Methods Three groups including 70 confirmed patients with H1N1 influenza A, 30 patients with seasonal influenza as well as 68 healthy subjects as controls were enrolled in this study. The percentages of Th1, Th2, Treg and Th17 lymphocytes in the peripheral blood were determined by intracellular staining and flow cytometry. The levels of interferon-γ (IFN-γ), transforming growth factor-beta (TGF-β),interleukin-6 (IL-6) in plasma and supernatant of the peripheral blood mononuclear cell (PBMC)culture were quantified by enzyme-linked immunosorbent assay (ELISA). Viral load in nasopharyngeal swabs was detected by real time quantitative reverse transcription-polymerase chain reaction (RTPCR). Data were analyzed by one way ANOVA and liner correlation analysis. Results The percentage of Th17 cells in H1N1 influenza A patients was (2. 740±0. 210)%, which the percentage of was significantly decreased compared to healthy subjects (3. 443 ±0. 154)% and seasonal influenza patients (3. 443±0. 277) % (F=4. 242, P＜0. 05); while the percentage of Thl, Th2 and Treg cells were not significantly different among these groups. Moreover, the TGF-β level in plasma of H1N1 influenza A patients was (10±8) ng/mL, which was significantly lower than healthy subjects (43 ±32 ) ng/mL and seasonal influenza patient ( 18 ± 10) ng/mL ( F= 17.72, P＜0.01 ). The TGF-β level in the supernatant of PBMC culture of H1N1 influenza A patients was (782 ± 736) pg/mL, which was significantly lower than healthy subjects (1462±315) pg/mL and seasonal influenza patients (1481 ±348) pg/mL (F=5. 730, P＜0.01). Additionally, the viral clearance period was inversely correlated with the percentage of Th17 cells (r=-0.38, P=0.02). Conclusions The proportion of Th17 cells in patients with H1N1 influenza A is significantly decreased, which is closely correlated with the level of TGF-β. This decrease may results in the delayed viral clearance.

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)diagnostic kit for early rapid detection of sarum anti-EV71 antibody and evaluate its clinical application value.Methods Recombinant protein VP1 of EV71 were prepared and purified as an immobilized antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG.Compared with RT-PCR.isolation of EV71 and micro-neutralizing assay.the clinical application value of anti-EV71 IgM and anti-EV71 ISG in the diagnosis of EV71 disease was evaluated.Results In comparison with RT-PCR.the sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81%and 87%,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68%and 77%.respectively.Compared with viral isolation assay.the sensitivity and specificity of anti-EV71 IgM antibody were 85%and 97%,respectively.The sensitivity and specificity of anti-EV71 IsG antibody were 75%and 77%,respectively.In addition.the titers of anti-EV71 IgG antibody were significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysis(r=0.72,P＜0.05).Finally,the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage(P＜0.01),but the titers of anti-IgM had no significant difference(P＞0.05).Conclusions With VP1 recombinant protein used as an immobilized antigen,an indirect ELISA diagnostic kit was successfully develooed for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies.

Objective To Screen for safe and effective vaccine candidates for EV71,provide a theoretical basis for development of EV71 vaccines in the future.Methods VP1 gene of enterovirus was used to design a target for development of EV71 vaccines.Different vaccine candidates,including inactivated EV71 vaccines,VP1 protein vaccine,DNA vaccines of different doses,were used to challenge female BALB/c mice by intramuscular injection at baseline (0),2 weeks,4 weeks,and caudal vein blood was collected at 0,2,4,6,8,10,and 16 weeks,and BALB/c mice were sacrificed and the spleen cells were collected for detection of both humoral immunity and cellular immunity to evaluate the efficacy and safety of the vaccine candidates.Results IgG antibody titers were increased at 2 weeks,remarkably increased at 4 weeks,reached a peak at 8 weeks,at least sustained for 16 weeks during the whole observation period,subtypes of IgG1 and IgG2a were the major component.The three vaccines could induce cellular immunity characterized by EV71 specific γ-IFN and IL-4 production.Our results indicated that inactivated EV71 vaccine was superior to the other vaccine candidates.Conclusions Inactivated EV71 vaccines,VP1 protein vaccine,DNA vaccines can induce both strong and sustainable humoral and cellular immunities in challenged mice,and the inactivated EV71 vaccine is superior to the other vaccine candidates,which needs to be proved their immunity by challenge assay in the future.

Objective To detect mutations of the ABCA12 gene in 2 Chinese families with autosomal recessive congenital ichthyosis (ARCI).Methods According to the typical clinical manifestations,two probands were diagnosed with ARCI.DNA was extracted from the peripheral blood samples collected from the patients and their parents.High-throughput sequencing was conducted by using multi-gene array for genetic skin disorders to determine mutation sites in the probands,and then DNA isolated from the probands and their parents were bidirectionally verified by Sanger sequencing.Results Two compound heterozygous mutations (c.2759A＞G and c.7004A＞G) in the ABCA12 gene were found in the proband 1,and another two compound heterozygous mutations (c.6163_6164insT and c.7406G＞A) were identified in the proband 2.The parents of the two probands were heterozygous carriers of one of the two mutations in the ABCA12 gene.Function prediction for the 4 mutations showed that all of the 3 missense mutations (c.2759A＞G,c.7004A＞G and c.7406G＞A) may exert pathogenic effect,and fragnin encoded by the frameshift mutation c.6163_6164insT may also affect protein function,c.2759A＞G and c.6163_6164insT were newly identified mutation sites.Conclusion The compound heterozygous mutations in the ABCA 12 gene are the causative mutations responsible for ARCI in the two probands of the two pedigrees.

Objective To evaluate the reliability and validity of SF-36 in patients with advanced schistosomiasis,so as to proride scientific basis for the selection of suitable tools for health measure.Methods A Chinese version of SF-36 scale was applied to evaluate the health of patients with advanced schistosomiasis by a household survey in Hanshou County of Hunan Province and Jiangling County of Hubei Province,then the reliability and validity of the scale were tested.Results Atotal of 326 patients were investigated in the two counties.The split-half reliability(with a split-half coefficient of 0.95) and the internal consistency (Cronbach'α coefficients of the eight dimensions ranged from 0.86 to 0.88)were satisfying;the convergent and discriminative validity were high with the test successful rates of 97.14%and 87.86%,respectively;the criterion validity was acceptable with a correlation coefficient between the total score of SF-36 and EQ-5D+C VAS score of 0.70.However,the construct validity seemed to be not so reasonable as only 2 dimensions out of 8 were completely in accordance with the theoretical model on factor loading.The percentages of floor effect and ceiling effect in most dimensions were not significant except RP and RE(with the percentages of floor effect of 50.31%and 48.16%,respectively).Conclusions SF-36 is appropriate to be used in patients with advanced schistosomiasis.but some items need to be improved according to the local settings of endemic areas.