Objective To analyze the difference of the delivery date reckoned by traditional and modified formula for calculating the expected date of confinement (EDC).Methods The data of 2055 women (37-41+6 week) were collected who gave monotocousa term spontaneous birth in the Chongqing Health Center for Women and Children from Jan.2014 to Feb.2015.Of which 1300 were primipara,and 755 were multipara;and the data of 1224 women (39-41week) were collected,of which 832 were primipara,and 392 were multipara.The expected date was calculated with traditional and modified calculating formula,and then the actual delivery date was used for comparison and statistical analysis.Results The coincidence of actual delivery date with the estimated due date reckoned by traditional formula (39-41week) was 8.4％ in primipara and 9.7％ in multipara,and the coincidence reckoned by modified formula was 11.9％ in primipara and 14.8％ in multipara.The EDC estimated by modified formula was more precise than that calculated by traditional formula (P＜0.05).Conclusion The EDC calculated with modified formula is more accurate than that calculated with traditional formula.

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.

AIM: Two models of experimental myocardial ischemia were set up to observe the protection of Danshen glucose injection against ischemia in rats. METHODS: Models of experimental acute myocardial ischemia were made by ligature or medication (pituitrin) to check the indices of ECG, hemodynamics, and morphology. RESULTS: A dosage of Danshen glucose injection 4.0 g/kg, 8.0 g/kg was given to rats by intravenous injection; the rats had undergone a thirty-minute ligatute of coronary left anterior descending branch, and used as a model of ischemic reperfusion. The observations showed that Danshen glucose injection exerted a recovery effect on heart rate, blood pressure, internal pressure of left ventricule and its peak/trough rate (?dp/dt max ), and ST segment (electrocardiogram). The injection markedly reduced the myocardial infarct size of the coronary-ligatured rats. The injection benefited the rats with pituitrin (iv) induced acute myocardial ischemia to reverse and T-wave fall. CONCLUSION: Danshen glucose injection has a protective and therapeutic action on the experimental myocardial ischemia of rats.

AIM: To investigate the effect of peripheral administration of arginine vasopressin (AVP) on lipopolysaccharide (LPS)-induced fever and hyperalgesia in rats and its relationship with interleukine-1β (IL-1β) and prostaglandin E2 (PGE2).METHODS: The core temperature (Tc), brown adipose tissue (BAT) temperature and activity were measured by telemetry in adult male Sprague-Dawley rats at an ambient temperature of 23 ℃ during a 12 h light/12 h dark photoperiod (lights on at 06:00 and lights off at 18:00).The rats were intraperitoneally injected with LPS (50 μg/kg), AVP (10 μg/kg) or V1a vasopressin receptor antagonist (V1a antagonist, 30 μg/kg) at 10:00 or 11:30.Hyperalgesia was assessed by measuring the latency to withdraw a hindpaw from radiant heat (Hargreaves test).The concentrations of IL-1β and PGE2 in the serum were tested by ELISA.RESULTS: Intraperitoneal administration of LPS induced periods of biphasic fever accompanied by hyperalgesia.AVP reversed LPS-induced fever, and decreased the hyperalgesia and BAT thermogenesis.Peripheral administration of V1a antagonist enhanced the fever produced by LPS, but did not affect the hyperalgesia.AVP significantly attenuated LPS-induced IL-1β and PGE2 production.CONCLUSION: Peripheral administration of AVP reverses LPS-induced fever and decreases hyperalgesia by reduction of BAT thermogenesis and inhibition of IL-1β and PGE2.Endogenous AVP attenuates the fever induced by LPS, but does not affect the nociceptive thresholds.

Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.

Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.

Objective To investigate the role of CD4+CD25+Foxp3 regulatory T cells in chronicity of hepatitis B and viral clearance of hepatitis B virus(HBV).Methods Nineteen patients with chronic active hepatitis B(CAH).21 HBV carriers(AsC)and 12 patients with resolved HBV infection and 1 5 healthy controls were enrolled.The frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells were detected by flow cytometry.CD4+CD25+T cells were sorted by magnetic-activated cell sorting(MACS)assay.Level of Foxp3 mRNA in CD4+CD25+T cells was examined by real time polymerase chain reaction(PCR)assay.The data were analyzed by one-way ANoVA or nonparametric statistics.Results Both frequencies of CD4+CD25+Foxp3+T cells and levels of Foxp3 mRNA in CD4+CD25+T ceils in patients with CAH or AsC were significantly higher than those in healthy controls Or resolved HBV infection(F=6.8,F=3.72,respectively;both P<0.05).Accumulation of Foxp3+T cells in liver tissue of CAH patients was higher than that of healthy controls,while that in AsC was lower than CAH.The frequency of CD4+CD25+Foxp3+T cells of hepatitis B e antigen(HBeAg)positive patients(including CAH and AsC)was significantly higher than that of HBeAg negative patients(t=2.3,P<0.05),and that of antFHBe negative patients were significantly higher than anti-HBe positive patients(t=2.4,P<0.05).Furthermore,the frequency of CD4+CD25+Foxp3 regulatory T cells was positively correlated with serum HBV DNA level of patients with chronic hepatitis B(r=0.56,P<0.01).Conclusion The findings have important implication in the understanding of the role of CD4'CD25'regulatory T cells in chronicity and viral clearance in HBV infection.

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 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 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.