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 evaluate the sensitivity and specificity of a nested polymersase chain reaction (nested PCR) for detection of the Mycobacterium tuberculosis IS6110 in paraffin embedded tissues. Methods 31 samples from tuberculosis patients and 5 biopsy specimens from patients with hepatitis were subjected to detection of Mycobacterium tuberculosis IS6110 by nested PCR. PCR products of two randomly selected samples were cloned and sequenced. Results Mycobacterium tuberculosis IS6110 was positive in 28 of 31 samples of tuberculosis. The sensitivity and specificity of nested PCR for detection of IS6110 were 90.3% and 100%, respectively. The predictive value of nested PCR was 100%. The sequences of two samples were compared with known sequence of H37Rv isolate (reported by Thierry D) and with nucleotide homology of 97% and 95.3%, respectively. Conclusions Nested PCR is sensitive and specific in the diagnosis of Mycobacterium tuberculosis infection in tissues of routinely paraffin embedded. We propose the diagnostic application of nested PCR for the identification of Mycobacterium tuberculosis infection, especially in the cases which can not be distinguished with certainty from other diseases by histopathology and Ziehl Neelsen's staining.

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 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 identify subtypes of human immunodeficiency virus 1(HIV-1)strains from the AIDS patients in Shenzhen and determine whether the HIV-1 subtypes differ in disease progression.Methods HIV-1 env gene was amplified by the nest-RT-PCR from plasma obtained from 26 patients with AIDS in Shenzhen. The C2-V3 regions were sequenced to identify subtypes The plasma viral loads and CD4T lymphocyte were measured as the same time.Results Phylogenetic trees showed that the 12 AIDS patients had subtype B in which, one was close with the U.S reference strain and 11 with the Chinese Yunnan reference strain;13 AIDS patients had subtype CRF01-AE from Thailand;There were no differences in the CD4 cell count and plasma HIV-RNA levels between individuals infected with subtypes B and CRF01-AE.Conclusion Our study indicated that HIV-1 subtype B and CRF01-AE strains were present in AIDS patients in Shenzhen. There was no evidence that the subtypes of virus could determine disease progression.

Objective To evaluate the homing molecules expression of CD49d, CCRg, CD62L on T lymphocytes in AIDS patients before and after treatment with HAART. Methods The study was per-formed in 42 cases of AIDS patients and 18 cases of healthy controls. The expression of CD49d, CCR9 and CD62L on T lymphocytes in AIDS patients and healthy controls were analyzed by flow cytometry. Software BD FACSDiva was used to calculate the percentage of expression. Results The number of peripheral CD4~+ T lymphocytes in group on-HAART was significantly increased compared with group pre-HAART (P<0.01) ; the frequency of CD3~+ CD49d~+, CD3~+CCR9~+, CD3~+CD62L~+, CD3~+CD4~+, CD4~+CD49d~+,CD4~+CCR9~+, CD4~+CD62L~+, CD8~+CD49d~+, CD8~+CD62L~+T lymphocyte in group pre-HAART were statistically decreased compared with group on-HAART and controls(P<0.05) ; The frequency of CD3~+ CD8~+ T lymphocytes was significantly increased compared with group on-HAART(P<0.05) ; the frequency of CD3~+ CCR9~+, CD8~+CCR9~+, CD8~+CD62L~+ T lymphocytes in group on-HAART were significantly de-creased than controls (P<0.001). Conclusion Not only the number of T lymphocytes sub-group, but the expression rate of gut homing molecules CD49d and CCR9, lymph node homing molecule CD62L on T lym-phocytes was changed in AIDS patients : the lower expression frequency of gut homing molecules CD49d and CCR9, lymph node homing molecule CD62L. Anti-virus therapy could partially reverse the immunologic pathological phenomena. CD49d, CCR9 and CD62L may be suggested to indicate the progression of AIDS and immunologic reeonstitution after HAART.

Objective To evaluate the diagnostic value of two tuberculosis-specific IFN-γ release assays in latent tuberculosis infection among HIV-infected individuals. Methods The levels of tuberculosis antigen-specific IFN-γin 102 HIV patients from AIDS Outpatient Clinic of Shenzhen Third People's Hospital were detected by in-house tuberculosis-specific IFN-γ ELISpot assay and commercial T-SPOT TB kit, and tuberculin skin test (TST) were done at the same time. There were 66 males and 36 females,and the average age was 35. Results Seventeen HIV infected patients were positive in both IFN-γ ELISpot and T-SPOT TB methods, the sensitivity, specificity positive predictive value(PPV), negative predictive value(NPV) and compliance rates of ELISpot were 94. 4% ,94. 0% ,77. 3% ,98. 8% and 94. 1% ,respectively. Three patients were positive in both IFN-γELISpot and T-SPOT TB methods, the sensitivity, specificity, PPV, NPV and compliance rates of TST were 16. 7%, 98. 8%, 75.0%, 84. 7% and 84. 3%, respectively. The average number of spots using three kinds of antigen ESAT-6, Pool A,Pool B obtained were 26. 89 ±5. 77,18. 96 ±4. 75 and 14. 51 ± 3.77, respectively. Only ESAT-6 and Pool B have a statistically significant difference (H=7.557,P = 0.022 9), no significant difference was shown between other groups. There was no significant difference between the positive rate and the CD4+ T cellls number(x2 =0. 860 8 ,P =0. 650 2) ,as the same as the T-SPOT TB (x2 = 1. 396 4, P = 0. 497 5 ). Conclusions The performance of this in-house tuberculosis-specific IFN-γ ELISPot assay was comparable to T-SPOT assay in diagnosis of latent tuberculosis infection, and the sensitivity and specificity of both these two assays were all much higher than TST. They canbe recommended in diagnosing latent tuberculosis infection in HIV infected patients.

Objective To investigate the role of CD4 ~+ CD25~+ regulatory T lymphocytes (Treg)in modulating the cellular immune response and pathogenesis of murine pulmonary tuberculosis.Methods Inactivation of Treg was achieved by intraperitoneal injection anti-CD25 (clone PC61,50 μ/mouse) in PC61 group, and rat-IgG (50 μ/mouse) was injected intraperitoneally in control group. All the mice were inoculated intravenously with H37Rv 0. 1 mL (1 × 10~6 CFU) 3 days after Treg inactivation. The effects of Treg inactivation in different tissues were analyzed by flow cytometry. The cellular immune response, pulmonary histopathology and bacterial load were determined in vitro at different time points. The data were compared using homogeneity of variance F test and non-paired t test. Results In spleen, the percentages of Treg/CD4 T lymphocytes in PC61 group and control group were (21. 13± 3. 58)% and (30. 42± 4. 20)%, respectively at day 10 of inoculation (t = 2. 38, P < 0. 05), and those were (16. 12 ± 1. 26)% and ( 17. 34± 1. 62)%,respectively at day 30 of inoculation (t = 0. 84,P>0. 05). The percentages of Foxp3~+/CD4~+ T lymphocytes in PC61 group and control group were (32. 07 ± 3. 95)% and (60. 55 ± 5. 48)%,respectively at day 10 of inoculation (t = 5. 96, P<0. 05). Similar results were achieved in the peripheral blood. Bacillus calmette-guerin (BCG)-specific 1L-17 (ng/L) secreted by murine spleen cells in PC61 group and control group at day 10, 30 and 60 of inoculation were 5. 1± 0.9 vs 0, 43. 1± 10.0 vs5. 9± 2. 8 and 124.8 ± 5.8 vs 102. 5±8. 1, respectively (t = 7. 90, t=5. 10,t = 3. 19; all P<0.05); those of BCG-specific IFN-γ (ng/L) were 28. 4 ± 8. 2 vs 4. 0±1. 3, 685. 9± 128. 6 vs418. 7±20.4 and 310.9±119. 7 vs 32. 8±7. 5, respectively(tO = 4. 21,t = 8. 43, t = 3. 27; all P<0.05);those of TNF-a (ng/L) were 38. 6±5.0 vs 16. 3±4. 0, 112. 9 ±12. 3 vs 71. 5±12. 6 and 86. 2±8. 2vs0, respectively(t = 4. 95, t=3. 33,t/=14.8; all P<0. 05). The lung bacterial load at day 10 of inoculation in PC61 group was lower than that in control group (t = 4. 63, P < 0. 01), but the differences were not significant thereafter. The changes of lung histopathology at late stage of infection (day 120) in PC61 group were less severe than those in control group. Conclusions Murine Tregs increase dramatically after Mycobacterium tuberculosis infection. Treg could inhibit the specific cellular immunity against Mycobacterium tuberculosis, and therefore, may facilitate the persistent infection of Mycobacterium tuberculosis and development of tuberculosis.

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.