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.

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.

Objective To investigate the antimicrobial and antitumor activities of extracts from the root of mangrove plant,Kandelia candel.Methods Micro-dilution and MTT method were used to test the antimicrobial and antitumor activities of extracts from the root of K.candel.Results The petroleum ether extracts of root could inhibit the growth of Staphylococcus aureus,S.epidermidis,Bacillus subtilis and Sarcina lutea with the minimal inhibitory concentration(MIC) of 3.125 mg?mL-1,1.5625 mg?mL-1,0.78125 mg?mL-1 and 3.125 mg?mL-1 respectively.The ethyl acetate extracts and ethyl alcohol extracts could inhibit the growth of S.epidermidis with MIC of 3.13 mg?mL-1 and 6.25 mg?mL-1 respectively.At the same time,the petroleum ether extracts could inhibit the proliferation of tumor cells,such as SMMC-7721,B16,BGC803 and HepG2 with IC50 of 355.2?g?mL-1,378.6?g?mL-1,275.6?g?mL-1 and 411.6?g?mL-1 respectively,while the other extracts could stimulate the proliferation of tumor cells apparently.Conclusion The antimicrobial and antitumor activities of extracts from the root of K.candel indicate the potential pharmaceutical usage of this plant.

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 investigate the clinical characteristics and imaging manifestations of AIDS complicated with disseminated Penicillium marneffei (PM) infection. Methods A total of 12 patients with AIDS complicated with disseminated PM infection were collected and the symptoms, signs, laboratory examination results and image manifestations of these patients were analyzed retrospectively. Results (1) The diagnosis of PM infection in all the 12 cases were confirmed by peripheral blood culture.All the 12 cases (100％) had irregular fever (38-41 ℃) and enlarged lymph nodes, 8 cases (66％) had skin rashes; 8 cases (66％) had hepatomegaly; 9 cases (75％) had splenomegaly while 8 cases (66％) had anemia. (2) Imaging manifestation: Five cases manifested bilateral pulmonary disseminated miliary nodular shadows or lattice signs; 1 case showed enlarged hilar lymph node and 2 zases showed patchy shadow with pleuritis. One case presented sub-pleural curve line shadow at the posterior part of the right lower lung,and adhesion between the intestinal wall and intestinal mesentery in mass form in the abdomen by CT examination. Conclusion Patients suffering from AIDS (CD4T lymphocytes ＜50/μ L) with impaired immunity might be susceptible to complication of disseminated PM infection, which presents mainly damage of multiple organs and symptoms such as fever; enlargement of liver,spleen and lymph nodes, as well as specific skin maculopapular rashes. Imaging manifestations in the lungs were revealed as miliary nodular shadows and lattice-like shadows. Intensified abdominal CT might reveal presence of several enlarged postperitoneal lymph nodes and intestinal adhesion in shape of cakes.

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 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 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 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 explore CD59 expression on CD4+T cells in HIV infected patients and its relationship with apoptosis.Methods 12 HIV infected patients and 10 healthy donors were performed in this study.The PBMC(peripheral blood monocyte)were collected and cell surface cytokine were stained,and then were evaluated with the BD FACSCanto flow cytometry.The expression of CD59 on T lymphocyte subsets were analyzed by FACSDiva software,and the apoptosis rate of CD59+CD4+T cells and CD59-CD4+T cells in every group was analyzed respectively,then the results were compared between groups.Results Compared with healthy donor,the expression of CD59 on T cells in HIV infected patients was significantly hisher(t=5.198,P＜0.01),and the apoptosis rate of CD59+CD4+T cells had significantly higher(t=5.968,P＜0.01).The apoptosis rate of CD59-CD4+T cells was no difference between two groups (t=0.1353,P=0.8577).Condnsion HIV infection increase CD59 expression on CD4+T cells,and CD59+CD4+T cells were prone to apoptosis.