Objective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro and assess the influence of Naloxone on Morphine2s effect.Methods MT2 cells were randomly assigned into 4 groups: (1) Morphine treatment for MT2 group, (2) Morphine+Naloxone co-treatment for MT2 group, (3) Naloxone treatment for MT2 group and (4) MT2 Control;Macrophages were also randomly assigned into 4 groups: (5) Morphine treatment for Macrophage group, (6) Morphine+Naloxone co-treatment for Macrophage group, (7) Naloxone treatment for Macrophage group and (8) Macrophage Control. Group (2), (3), (6) and (7) were pre-treated with 10-8 mol/L Naloxone for 0.5 h, and then group (1) and (2) were treated with 10-12, 10-10 and 10-8 mol/L Morphine for 24 h;group (5) and (6) were disposed of 10-10 mol/L Morphine for 24 h.All 8 groups were added in HIV-1 viral strain with 50% tissue culture infective dose(TCID50).P24 antigen in MT2 cells culture supernatant at day 3, 4, 5 and 6, and in Macrophages culture supernatant at day 4, 6, 8, 10 and 12 after infection were determined with ELISA.Student2s t-test and ANOVA were used to compare the differential expression in different groups, and repeated measures ANOVA was used to compare the increasing or decreasing expression of p24 antigen in morphine treatment groups than that in the control group at different time points.Results On the 3rd day of infection with HIV-1 in MT2 cells, the expression of p24 antigen in 10-12, 10-10 and 10-8mol/L dose of group (1) were (4.44?.30), (5.59?.25) and (4.60?.24) ng/ml respectively, compared to control[(1.93?.05) ng/ml, t= 14.15, 24.74 and 19.14, all P<0.01].On the 4th day, 10-12, 10-10 and 10-8mol/L dose of group (1) resulted in a significant increase of p24 antigen expression [(24.30?.66), (31.73?.17) and (26.02?.37) ng/ml]in culture supernatants compared to control[(8.03?.09) ng/ml, t=10.59, 34.92 and 81.2, all P<0.01].On the 5th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (56.30?.26), (81.77?.49) and (63.66?.57) ng/ml respectively, compared to control [(15.30?.91) ng/ml, t= 45.83, 43.51 and 30.07, all P<0.01].On the 6th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (150.70?.97), (243.09?.93) and (173.72?.73) ng/ml respectively, compared to control [(41.01?.84) ng/ml, t= 21.09, 39.02 and 29.55, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of morphine treatment group compared to control increased with HIV-1 infected MT2 cells time, trend analysis of repeated measurements showed statistically significant time effect (F=842.18, P<0.01). On the 4th day of infection with HIV-1 in Macrophage cells, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (0.68?.15), (0.87?.41) and (0.75?.09) ng/ml respectively, compared to control [(0.60?.01) ng/ml, t= 7.27, 11.06 and 3.02, all P<0.05]. On the 6th day, 10-12, 10-10 and 10-8 mol/L dose of group (5) resulted in a significant increase of p24 antigen expression[(1.64?.57) , (2.07?.12 ) and (1.75?.17) ng/ml]in culture supernatants compared to control [(1.16?.07) ng/ml, t=8.93, 11.3 and 5.45, all P<0.01].On the 8th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (6.31?.17), (8.81?.34) and (7.19?.11) ng/ml respectively, compared to control [(3.84?.45) ng/ml, t=8.83, 15.11 and 12.42, all P<0.01]. On the 10th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of Morphine treated group were (32.30?7.55), (50.74?7.55) and (39.74?.56) ng/ml respectively, compared to control [(17.55?.86) ng/ml, t= 13.65, 17.84 and 36.69, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of group (5) compared to control increased with HIV-1 infected Macrophage cells time, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01).Conclusions Morphine has the ability to enhance HIV-1 replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression can be blocked by Naloxone.

Objective To detect the distribution of H.pylori ureA, vacA s1 gene and cagA subtype(ABC, ABD, ABAB, AAD, et al) in patients with digestive diseases in Guangzhou and investigate the relationship with the pathological findings of gastric mucosa.Methods A total of 227 randomly selected gastric mucosa from patients with digestive diseases were enrolled in the research, including 46 without pathological changes, 130 with chronic gastritis, 29 with peptic ulcer, 15 with atrophic gastritis and 7 with gastric cancer.Real-time PCR assay were used to detect Helicobacter pylori ureA gene and vacA s1 gene.EPIYA motifs in the 3′ region of cagA were amplified by conventional PCR followed by subtype sequencing. The conserved gene ureA was used to detect H.pylori infection.Results Among the 227 patients with digestive diseases, 50.7% (115/227) patients were H.pylori positive, in which 91.3%(105/115) carried vacA s1 and 78.3% (90/115) carried cagA. Four types of cagA-EPIYA subtype were detected, including ABC 17.8%(16/90), ABD 78.9%(71/90), AAD 2.2%(2/90) and ABAB 1.1%(1/90).In the non-pathological change group, 32.6% (15/46) were H.pylori positive, in which 28.3% (13/46) carried vacA s1 and 26.1% (12/46) carried cagA;in chronic gastritis group, it was 48.5% (63/130), 43.8% (57/130) and 36.2% (47/130), respectively;in ulcer group, it was 72.4% (21/29), 65.5% (19/29) and 55.2% (16/29), respectively;in atrophic gastritis group, it was 66.7% (10/15), 66.7% (10/15) and 66.7% (10/15), respectively;in gastric cancer group, it was 85.7% (6/7), 85.7% (6/7) and 71.4% (5/7), respectively.The distribution of H.pylori among the 4 groups had statistical significance (χ2=16.72;P<0.01). H.pylori was more prevalent in ulcer, atrophic gastritis and cancer group than in inflammation group and non-pathological change group (χ2=16.02;P<0.01).In patients infected by H.pylori, there was no significant difference in the distribution of vacA s1 gene as high virulence factors among non-pathological change, inflammation, ulcer, atrophic gastritis and cancer group (χ2=2.00;P=0.74), as well as cagA (χ2=3.44;P=0.49) and EPIYA subtypes (χ2=3.66;P=0.45).Conclusions H.pylori infection is significantly associated with the pathological change of gastric mucosa for patients with digestive diseases in Guangzhou, while the relationship with the pathogenicity of H.pylori with high virulence genotype is not significant.More samples and diseases reclassification are needed to make an advanced analysis of the effect of H.pylori with high virulence in gastrointestinal diseases development.

Objective To evaluate the potential value of IgG antibodies against recombinant PPE65 protein (rPPE65) of Mycobacterium tuberculosis in serodiagnosis of tuberculosis.Methods The gene encoding PPE65 protein of M.tuberculosis was cloned into the PET-28a vector and then expressed in Escherichia coli.The rPPE65 was purified with Ni-NTA affinity and ion exchange chromatography.After dialysis renaturation, the concentration of rPPE65 was determined using Lowry assay.ELISA was used to detect the levels of specific IgG against rPPE65 and recombinant PstS1 protein (rPstS1) in sera from 144 patients with pulmonary tuberculosis (PTB patients), 144 health controls, and 56 patients with non-tuberculosis pulmonary diseases.ROC curves were used to determine cut-off values with the results of IgG antibodies against rPPE65 and rPstS1 for 144 PTB patients and 97 controls with negative PPD skin test.The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of rPPE65 and the combination of rPPE65 and rPstS1 were counted.Results The PPE65 protein of M.tuberculosis was successfully expressed in E.coli. The purity and concentration of rPPE65 were 95% and 0.5 mg/ml, respectively.ROC analysis showed that the cut-off of ELISA using rPPE65 was 0.64.The sensitivity, specificity, PPV, NPV, and accuracy of rPPE65 were 34.7%(50/144), 93.5%(187/200), 79.4%(50/63), 66.5%(187/287), and 68.9%(237/344), respectively.The sensitivity, specificity, PPV, NPV, and accuracy of the combination of rPPE65 and rPstS1 were 59.0%, 91.0%, 82.5%, 75.5%, 77.6%, respectively.Conclusions The rPPE65 of M.tuberculosis appears to be a candidate antigen for serodiagnosis of tuberculosis.Detection of IgG antibodies against the combination of rPPE65 and rPstS1 can increase the sensitivity of serological test for tuberculosis.

Objective To investigate the clinical application of multiplex allele-specific PCR assays for simultaneous detection of the mitochondrial 12S rRNA A1555G and C1494T mutations associated with aminoglycoside-induced hearing impairment.Methods Three standard plasmids of different genotypes (wild-type, A1555G mutant and C1494T mutant) were constructed for templates and allele-specific primers aiming directly at wild-type and mutant of mitochondrial DNA nt1555 and nt1494 were designed for developing a multiplex allele-specific PCR technique to detect the A1555G and C1494T mutations.Then the method was applied to clinical screening of 138 non-syndromic hearing loss subjects and confirmed by DNA sequencing.Results Multiplex allele-specific PCR was successfully applied to the detection of A1555G and C1494T mutations in a cohort of 138 Han Chinese genetically unrelated hearing-loss subjects.Finally, 11(7.97%) unrelated affected subjects harbored the A1555G and C1494T mutations in the 12S rRNA gene(10 cases for A1555G and 1 cases for C1494T), which was well consistent with results of DNA sequencing [7.97%(11/138), Kappa=1.000, P＜0.01].Conclusion This study indicates that the multiplex allele-specific PCR assay is useful, convenient and reliable in the detection of the A1555G and C1494T mutations, which could identify the subjects at risk and effectively prevent of aminoglycoside-induced hearing loss.

Objective To investigate the level of B7-H1 and its counter-receptor PD-1 expression in mDC and different subsets of T lymphocytes in HIV infected individuals in China and to analyze the correlation between the level of B7-H1/PD-1 and disease progression, and to demonstrate that PD-1/PD-L1-dependent inhibition is operating in HIV infected patients.Methods Percentage of B7-H1 and PD-1 expression in mDC, CD+4 T cells and CD+8 T cells from thirty-six untreated HIV infected patients and 20 health controls were selected and detected by flow-cytometry, its correlations with CD+4 T cell absolute counts and plasma viral loads were analyzed.Results The percentage of B7-H1 expression in mDC, CD+4 T cells and CD+8 T cells (mean 15.21, mean 20.63, mean 13.5)were higher than that of health controls (all P<0.05).The percentage of PD-1 expression in CD+4 T cells and CD+8 T cells (mean 17.91, mean 19.21)were higher than that of health controls (P<0.05, P<0.05). The level of B7-H1 and PD-1 expression were inversely correlated with CD+4 T-cell counts(mDC+B7-H1+:r=-0.647, P<0.01;CD+4B7-H1+:r=-0.489, P=0.002;CD+8B7-H1+:r=-0.372, P=0.026;CD+4PD-1+:r=-0.374, P=0.025;CD+8PD-1+:r=-0.455, P=0.005) and positively correlated with HIV viral load(mDC+B7-H1+:r=0.662, P<0.01;CD+4B7-H1+: r=0.426, P=0.01;CD+8B7-H1+:r=0.531, P=0.001;CD+4PD-1+:r=0.362, P=0.03;CD+8PD-1+:r=0.380, P=0.022).Conclusion The level of B7-H1 and PD-1 expression was associated with HIV disease progression, which provides a useful marker to define disease progression of HIV infection.

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method.

Objective To analyze the antibiotic susceptibility, ESBL genotype of clinical Klebsiella pneumoniae strains isolated from People′s hospital and facilitate the control of resistance spread. Methods Identification and antibiotic susceptibility tests of 1 205 strains from 2001 to 2007 were done by VITEK-2 system.The antibiotic susceptibility results were analyzed by whonet5.3.The ESBL gene was detected by PCR and the Chi-square test was used for statistical analysis.Results The rate of ESBL-producing strains in klebsiella pneumoniae has increased from 2001 to 2007[18.8% (40/213) in 2001, 20.9% (53/253) in 2002, 32.8% (42/128) in 2003, 33.6% (45/137) in 2004, 36.6% (60/164) in 2005, 45.3% (68/150) in 2006 and 45.6% (73/160) in 2007].The SHV gene was the most dominant in ESBL genotypes.There were 83.3% (50/60) ESBL strains in 2005 with SHV gene, 82.3%(56/68) in 2006 and 83.6%(61/73) in 2007.The rated of strains with CTX-M gene were increasing.There were 26.7%(16/60) ESBL strains with CTX-M gene in 2005, 36.7%(25/68) in 2006 and 54.8%(40/73) in 2007.The isolates with more than one type of ESBL gene were increasing.There were 45%(27/60) ESBL strains in 2005 with two types of ESBL gene, and no one had more than two types of ESBL gene in that year.There were 47.9%(35/73) ESBL strains in 2007 with two types of ESBL gene.In 2007 there were 9.6%(7/73) and 2.7%(2/73) ESBL strains with three types and four types of ESBL gene respectively.There was a statistical difference between the antibiotic resistance rates of cefotaxime, ceftriaxone and ceftazidime in SHV-gene-phore strains (χ2=13.22, P＜0.01).The strains with SHV gene were more resistant to cefotaxime than ceftriaxone and ceftazidime.There also was a statistical difference of the antibiotic resistance rate of cefotaxime, ceftriaxone and ceftazidime between strains with TEM gene (χ2=9.91, P＜0.01) and CTX-M gene (χ2=34.84, P＜0.01) respectively.None of the strains with CTX-M gene was sensitive to cefotaxime, and they were more resistant to ceftriaxone than ceftazidime.The strains with TEM gene were more resistant to cefotaxime than ceftriaxone and ceftazidime.There were statistical differences of the antibiotic resistance rate to cefotaxime (χ2=29.65, P＜0.01), ceftriaxone (χ2=20.26, P＜0.01) and ceftazidime (χ2=20.26, P＜0.01) between the strains with SHV gene only and strains with SHV and CTX-M gene concurrently.There were also statistical differences of the antibiotic resistance rates to cefotaxime (χ2=11.01, P＜0.01), ceftriaxone (χ2=9.93, P＜0.01) and ceftazidime (χ2=7.01, P＜0.01) between the strains with SHV gene only and strains with SHV and TEM gene concurrently.The antibiotic resistance rates to cefotaxime (χ2=11.54, P＜0.01), ceftriaxone (χ2=17.58, P＜0.01) and ceftazidime (χ2=14.11, P＜0.01) were statistically different between the strains with SHV gene only and strains with SHV and OXA gene concurrently.The antibiotic resistance rates to ceftazidime (χ2=23.61, P＜0.01) were statistically different between the strains with CTX-M gene only and strains with SHV and CTX-M gene concurrently. There was no statistical difference in antibiotic resistance rates to cefotaxime (χ2=3.55, P＜0.01) and ceftriaxone (χ2=3.35, P＜0.01) between the strains with CTX-M gene only and strains with SHV and CTX-M gene concurrently. The antibiotic resistance rates to ceftazidime (P=0.01) were statistically different between the strains with only TEM gene and strains with SHV and TEM gene concurrently, and there was no statistical difference of the antibiotic resistance rates to cefotaxime (P=0.29) and ceftriaxone (P=0.26) between the strains with TEM gene only and strains with SHV and TEM gene concurrently. ConclusionsThe producing rate of ESBL is increasing year after year and the SHV type of ESBL is the dominant one.Strains with more than one type of ESBL gene are increasing.The antibiotic resistance rates to cefotaxime, ceftriaxone and ceftazidime are statistically different between strains with same ESBL genotype.

Objectives To explore the associations between drug efflux pump gene expression and phenotypic drug resistance as well as gene mutation patterns related to drug resistance of Mycobacterium tuberculosis.Methods Forty-five Mycobacterium tuberculosis isolates resistant to one or more of drugs including isoniazid, rifampicin, streptomycin and ethambutol, and 26 isolates all sensitive to the above four drugs from Tianjin Tuberculosis Control Institute in 2007 were involved in this study. Direct sequencing was applied to detect the mutations in the corresponding resistance genes(isoniazid:katG, inhA, oxyR-ahpC, ndh, rifampicin:rpoB, streptomycin:rpsL, rrs, and ethambutol:embB, embC and embA). After RNA extration and reverse transcription, real-time PCR was conducted to assess the expressions of putative drug efflux pump genes Rv1410c, Rv2136c, Rv0783c and Rv2136c, and Students' t test and ANOVA analysis were used to analyze the expression differences in Mycobacterium tuberculosis with different phenotypic drug resistance and drug resistance related gene mutation patterns.Results Compared to pan-sensitive isolates[(5.67±3.29)×10-5], Rv1410c showed higher expression in streptomycin[(8.48±6.33)×10-5, t'=2.18, P<0.05], isoniazid[(8.43±6.38)×10-5, t'=2.20, P<0.05], rifampicin[(9.59±7.27)×10-5, t'=2.29, P<0.05], multi-drug[(10.37±7.86)×10-5, t'=2.34, P<0.05] resistant isolates, and in isoniazid + streptomycin resistant isolates[(9.39±6.81)×10-5, t'=2.43, P<0.05];Rv2136c showed higher expression in isoniazid resistant[(3.51±2.43)×10-5, t'=2.03, P<0.05], multidrug-resistant isolates[(4.21±2.94)×10-5, t'=2.22, P<0.05] and resistant to isoniazid+streptomycin[(3.81±2.46)×10-5, t'=2.28, P<0.05] isolates . The expression of Rv0783c in rifampicin resistant isolates with rpoB 531 mutations [(5.41±3.03)×10-6] was higher than those with wild type of rpoB 531[(2.29±1.62)×10-6, t=2.81, P<0.05].Conclusions The expression of Rv1410c and Rv2136c are associated with mutiple-drug resistance of Mycobacterium tuberculosis.The expression of Rv0783c in rifampicin resistant isolates is associated with mutation in rpoB 531.

Objective To investigate the effects of different stabilizers, time and temperature before centrifugation on the stability of homocysteine (Hcy) and other related thiols levels involving EDTA-containing whole blood.Methods Blood was drawn from 17 healthy adults and collected into tubes containing EDTA, EDTA plus NaF and EDTA plus 3-deazaadenosine(3DA),then stored on crush ice(0-4 ℃) immediately or at room temperature(25 ℃).Plasma was separated at 0, 3, 6, 24 and 48 hours, respectively.The levels of plasma total Hcy (tHcy), total cysteine (tCys), tatal cysteinylglycine (tCysGly) and tatal glutathione (tGSH) were measured by HPLC.The plasma immediately separated was used as baseline sample. Results In EDTA tubes stored at room temperature, tHcy levels increased by 38.5%, 64.2%, 141.9%, 225.4% for 3, 6, 24, and 48 h, respectively.The levels of tCysGly and tGSH increased by 20.0% and 37.9% within 3 h, however, tCys decreased by 3.5%.The levels of the thiols increase by less than 5% up to 6 h in EDTA tubes stored on crush ice.In EDTA-3DA and EDTA-NaF tubes, no statistical differences were observed in the plasma levels of tHcy, tCys,tCysGly and tGSH compared with their respective baseline values at room temperature for 3 h(EDTA-3DA tubes:F=0.01,0.94,0.09,0.01,all P>0.05;EDTA-NaF tubes:F=0.85,0.04,0.03,0.02,all P>0.05).Conclusions The EDTA-plasma levels of tHcy and other related thiols are time and temperature-dependent. There is a strong need for standardization of blood sample collection and processing in tHcy and other thiols assays. The plasma concentrations of tHcy, tCys, tCysGly and tGSH were stable for 3 h at least in the EDTA-3DA and EDTA-NaF tubes kept at room temperature.

Objective To investigate the molecular characterization of a Chinese pedigree with rare β thalassemia genotype.Methods Phenotypic analysis was performed using standard hematological tests to measure red blood cell parameters, including RBC,Hb,MCV,MCH,MCHC and RDW.SPIFE automatic Hb agarose gel electrophoresis instrument was used to measure hemoglobin fraction Hb A,Hb A2 and Hb F.The alleles of β thalassemia mutation were determined by RDB assay, and then cloning and sequencing were performed to define the mutation sites.Results The proband and his father had typical microcytic hypochromic anemia with low MCV and MCH(79.8, 63.1 fl and 19.9, 20.9 pg, respectively) and high level of Hb A2 (5.66% and 5.60%, respectively).The proband′s mother had normal MCV and MCH. β thalassemia mutation analysis with RDB assay showed that the proband had thalassemia minor resulting from double mutations on one globin gene.One showed codons 41/42 (-TTCT) mutation and the other was CAP mutation from positions +40 to +43 in the promoter region.These two mutations were inherited from his father.The genotype of the proband and his father was β41/42、CAP/βA ,and the genotype of his mother was βA/βA.Conclusions It′s rare that double mutations occur on single β globin gene, with one mutation on CD41/42(-TTCT) and the other mutation from positions +40 to +43 relative to the mRNA cap site in the promoter region.The findings enrich knowledge of the mutation spectrum of β thalassemia.