Objective To explore the correlation between the efficacy of lamivudine (LAM)therapy and changes of T lymphocyte subsets,CD4+ CD25+ regulatory T lymphocytes (Treg),and levels of interleukin-10 (IL-10)and interferon-gamma (IFN-γ)in the peripheral blood of patients with chronic hepatitis B (CHB).Methods Ninety CHB patients were enrolled in this study.T lymphocyte subsets in the peripheral blood were detected by flow cytometry at baseline and week 52 of LAM therapy.The frequencies of CD4+ CD25+ Treg in the peripheral blood were detected by flow cytometry and levels of IL-10 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA)at baseline,week 12,24,36 and 52.The comparisons of overall means between groups and within groups were done by analysis of variance or Dunnett's test.The comparison of means before and after LAM therapy was done by paired t test.Results In 32 complete-responders of 90 CHB patients,the proportions of CD4+ T lymphocytes,CD8+ T lymphocytes and CD4+/CD8+ ratio were increased significantly after LAM therapy (t=4.055、3.267、2.328,all P＜0.05); the frequencies of CD4+CD25+ Treg at baseline,week 12,24,36 and 52 were (5.40±0.60)%,(4.99±0.59)%,(4.54± 0.72)%,(3.86±0.95)% and (3.44±0.76)%,respectively; the levels of IFN-γ,IFN-γ/IL-10 ratio were increased,while the IL-10 level was decreased after LAM therapy.In 43 partial-responders,the proportion of CD4+T lymphocytes and ratio of CD4+/CD8+ were increased after LAM therapy (t= 3.484,2.018,both P＜0.05); the proportion of CD8+ T lymphocytes was not changed significantly after therapy; the frequencies of CD4+ CD25+ Treg at baseline,week 12,24,36 and 52 were (5.65±0.60)%,(5.23±0.63)%,(4.65±0.98)%,(4.42±0.97)% and (4.32±0.82)%,respectively;IFN-γ level,IFN-γ/IL-10 ratio were increased,while IL-10 level was decreased.In 15 non-responders,the proportion of T lymphocyte subsets,the frequency of CD4+ CD25+ Treg,the levels of IFN-γ and IL-10 were not changed significantly after LAM treatment.Conclusions In CHB patients who have achieved response after LAM therapy,the frequency of CD4+ CD25+ Treg in the peripheral blood is decreased,while ratios of CD4+/CD8+ and IFN-γ/IL-10 in the peripheral blood are increased.

Objective To investigate the relationship between dendritic cell (DC)and intestinal endotoxemia in patients with chronic hepatitis B (CHB).Methods Peripheral blood were collected from CHB patients (n = 80)and healthy controls (n = 21 ).Plasma endotoxin (ET)levels,liver function (alanine transaminase,total bilirubin)were detected.According to plasma ET concentration,all CHB patients were divided into two groups:ET positive and ET negative.The peripheral blood mononuclear cells (PBMCs)were isolated and then cultured with recombinant human granulocyte-macrophage colony-stimulating factor ( rhGM-CSF),recombinant human interleukin-4 ( rhIL-4 ),FMS-related tyrosine kinase 3 ligand (Flt3L)and tumor necrosis factor-alpha (TNF-α)to derive DC.The phenotypic patterns were characterized by flow cytometry.The proliferation of T lymphocytes was evaluated with mixed leukocytes reaction (MLR)and the levels of IL-12 and interferon-γ (IFN-γ)produced by DC were analyzed with enzyme-linked immunosorbent assay (ELISA).Comparisons among the two groups and healthy control group were done by single factor analysis of variance.Results Compared to healthy controls,the expressions of CD83,CD80,CD86,human leucocyte antigen (HLA)-DR and the proliferation of allogeneic T lymphocytes by DC were all significantly reduced in CHB patient groups.The expressions of CD83,CD80,CD86,HLA-DR and the activation of proliferation in ET positive subjects were lower than those in ET negative subjects [CD83 (8.25±3.63)% vs(11.39±4.35)% ,CD80 (10.63±4.52)% vs (13.56±5.13)%,CD86 (36.61±16.16)% vs (45.90±15.35)%,HLA-DR (61.65±14.33)% vs (70.35±18.89)%,the activation of proliferation0.812±0.311 vs 1.153±0.324; F=5.123,4.213,3.714,3.323 and 3.125,respectively; all P＜0.05].After cultured for 9 days,the secretions of IL-12 and IFN-γ by DC were significantly lower in CHB patients than in healthy controls [IL-12 (16.99± 6.74)pg/mL vs (44.51±14.56)pg/mL,IFN-γ (10.52±4.19)pg/mL vs (17.94±5.86)pg/mL].The level of IL-12 in the ET positive group was significantly lower than that ET negative group [( 13.14 ±5.71)pg/mL vs (20.98 ± 9.03)pg/mL; F= 3.225,P = 0.016].The level of IFN-γ was not different between two groups [(9.46 ± 3.24)pg/mL vs (11.54 ± 5.20)pg/mL; F = 2.003,P =0.076].Conclusion The intestinal endotoxemia may play a role in DC dysfunction in CHB patients.

Objective To understand the clinical features of patients with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) complicated by cytomegalovirus (CMV)viremia.Methods The clinical data of 249 cases of HIV/AIDS patients hospitalized in Beijing Ditan Hospital from Oct 2008 to Nov 2009 were analyzed retrospectively,in which 43 HIV/AIDS patients were diagnosed with CMV viremia.The symptoms and signs,cerebrospinal fluid (CSF)tests,and pathological detections by bronchoscope,gastroscope and fibercoloscope were collected.The database was set up using Excel software.The association between cellular immunity and CMV DNA level was determined by SPSS12.0 software.Results Forty-three patients (17.3%)were diagnosed with CMV viremia by positive results of CMV pp65 antigen and CMV DNA tests; 14 patients manifested retinal bleeding or infiltration and 4 patients displayed retinal fibrosis; 1 patient was diagnosed with CMV pneumonitis by pathological results of bronchoalveolar lavage fluid.Low level of CD4+ T lymphocytes and CMV DNA levels were positively correlated.Conclusions CMV pp65 antigen and CMV DNA should be detected in HIV/AIDS patients with CD4+ T lymphocytes less than 100 × 106/L and anti-CMV treatment should be given according to the results.Ophthalmologic examination and bronchoalveolar lavage fluid pathological detection are effective methods in diagnose of CMV retinitis and pneumonitis.

Objective To evaluate the survivability of hand foot mouth disease(HFMD)virus,in tap water for daily use.Methods HFMD viruses were isolated from cases of HFMD in Shanghai and Zhejiang from in 2008.Six isolated strains (five subtype of enterovirus 71 and one coxackie virus)were selected in this study.These viruses were mixed with chloride 1.0 mg/L tap-water and then inoculated into Vero cells.The cytopathic effect (CPE)was checked everyday in order to survey the survivability of each virus strain.The decline of virus survivability was analyzed by scatter diagram.Results These six strains of HMFD virus could survive longer than one month in tap water with initial chloride concentration of 1.0 mg/L and still had celluar infectivity.The survivabilities were varied between viruses isolated from different HFMD cases.Conclusions The survivabilities of enterovirus 71 and coxackie virus stains are quite strong in water.Therefore,the transmission route of water-borne pathogens should be monitored in regions using tap water during HFMD epidemic period.

Objective To investigate the changes and clinical significance of the frequency of circulating CD4+ CD25+ regulatory T cells (Treg) in nonalcoholic fatty liver (NAFL) patients.Methods CD4+ CD25+ Treg in the peripheral blood from 50 NAFL patients and 50 healthy subjects were quantitatively analyzed using flow cytometry. Group t test or Mann-Whitney U test and Spearman's rank correlation test were used for statistical analysis.Results The proportion of circulating CD4+CD25+ Treg in NAFL patients was (5.39 ± 1.94)％,which was significantly higher than that in healthy controls [(4.21±1.52)％](t=3.385,P＜0.01).Further analysis revealed that the frequency of Treg was positively correlated with triglyceride (TG) level and body mass index (r=0.307 and 0.251,respectively; P=0.002 and 0.012,respectively),and negatively correlated with high density lipoproteincholesterol (HDL-C) (r=-0.306,P=0.002).Meanwhile,Treg in patients with high body mass index,high TG,low HDL-C,hypertension and metabolic syndrome (MS) were all higher than those in controls (t=2.294,2.533,3.154,2.010 and 4.454,respectively; all P＜0.05).But there was no significant difference between patients with high fasting blood glucose and controls (U=1143.500,P=0.471).Conclusion The increased frequency of peripheral Treg in NAFL patients may have some relations with the imbalance of proinflammation and anti-inflammation in NAFL patients who coexisting with MS.

Objective To investigate the central neurotoxicity induced by nucleoside analog reverse transcriptase inhibitors (NRTIs)-stavudine (D4T).Methods Mouse primary cortical neurons were cultured and treated with different concentrations of stavudine.Neuron apoptosis was analyzed by calcein/acetomethoxy/propidium iodide (AM/PI) staining. Morphological change of neuron was confirmed by immunofluorescence.Mitochondrial DNA copies which were usually evaluated through Cycloxygenase 2 (COX-2) and thymidine kinase2 (TK2) mRNA were determined by real-time quantitative polymerase chain reaction.Chi-square test,student t test and Wilcoxon nonparameter test were used to analyze the data.Results Neuronal apoptosis observed in 50 μmol/L D4T treatment group was more significant than that in 0μmol/L D4T treatment group and 25 μmol/L D4T treatment group (51.3％±12.4％ vs 24.9％±8.2％ and 26.5％±10.6％,respectively; x2 =7.25 and 6.93,respectively; both P＜0.01).The average neurite numbers of each neuron were 11.2±3.6 in 0μmol/L D4T treatment group,8.6±2.8 in 25 μmol/L D4T treatment group and 4.3±2.4 in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.06,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t =4.35,P＜ 0.01). Furthermore,the average lengths of neuritis were (319.9±100.2) μm in 0 μmol/L D4T treatment group,(298.3±83.9) μm in 25 μmol/L D4T treatment group and (258.4±82.2) μm in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.58,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t=4.65,P＜0.01).TK2 mRNA expression dramatically decreased along with the increasing D4T concentration.The fold changes were 0.34 in 25 μmol/L D4T treatment group and 0.08 in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0μmol/L D4T treatment group (Z=- 3.28,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (Z=-4.25,P＜0.01).Compared with 0μmol/L D4T treatment group,the relative fold changes of COX-2 copies were 1.01 in 25 μmol/L D4T treatment group and 1.12 in 50 μmol/L D4T treatment group.The differences were not significant among the three groups (Z=0.98 and 1.24,respectively; both P＞0.05).Conclsion It suggests that short-term exposure to D4T may result in neuron apoptosis,neurite shrink and down-regulated expression of TK2,but the level of mitochondrial DNA copies keeps stable.

Objective To understand the genetic and antigenic variations of influenza A (H1N1) isolates in Shanghai area in winter of 2010.Methods A total of 137 throat swabs were collected from patients with influenza-like illness in the sentinel hospital in Shanghai area from December 2010 to January 2011,then inoculated into Madin-Darby canine kidney (MDCK) cells.The types of influenza were identified by direct immunofluorescence assay (DIF) and influenza A (H1N1) subtype was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).The mutations of gene and amino acid locus were analyzed through the whole genome sequencing of hemagglutinin (HA),neuraminidase (NA) and polymerase (PB2) segments from some influenza A (H1N1) isolates.Results Total of 53 human influenza virus strains were isolated including 48 influenza A (H1N1) virus strains.Nineteen strains were selected for sequencing by simple random sampling.The phylogenetic tree of HA gene revealed that the latest isolates and most of influenza A (H1N1) viruses isolated before June 2010 were not in the same stem.Analysis of amino acid residues in HA protein showed that mutations were found in antigenic determinant region in some strains.Residues at the enzyme active sites of NA protein were strictly conservative,no change was observed in amino acid residues which were related to drug resistance against oseltamivir and zanamivir.The 627 and 701 residues in PB2 protein were glutamic acid and aspartic acid,respectively,which was still the feature of avian influenza virus,but E677G mutation was detected.Conclusion Compared to influenza A (H1N1) strains isolated in spring and summer,some variations have been detected in the strains isolated in Shanghai area in winter of 2010,some antigen drift and adaptive evolution in mammalian hosts have appeared.

Objective To verify the value of routine laboratory markers for assessment of liver fibrosis status in chronic hepatitis B virus (HBV) carriers.Methods A total of 196 patients who were clinically diagnosed with chronic HBV carriers with liver biopsy and routine laboratory test were included in this study. The data of complete blood count, aspartate aminofransferase/alanine aminotransferase (AST/ALT),aspartate aminotransferase to platelet ratio index (APRI) and ageplatelet index (API) were collected and calculated.Patients were divided into group S0 (n=112) and group S1- S3 (n =84) based on liver fibrosis stages.Measurement data were analyzed by Wilcoxon rank sum test and enumeration data were analyzed by chi square test.Results All 196 enrolled HBV carriers were HBV DNA positive,with 156 (79.6％) HBeAg-positive.Age,ALT,AST,AST/ALT,APRI and API were all significantly higher in group S1- S3 than those in group S0 (statistic value=7.705,6.33,7.095,4.977,11.059,8.936,respectively; all P＜0.05).However,PLT level was lower in the former group compared to that in the latter group (statistic value=10.196,P＜0.05 ).The area under receiver operating characteristic curve (AUROC) of APRI and API were 0.827and 0.829,respectively.The highest sensitivity and negative prediction value (NPV) were 70.46 ％and 71.43 ％ respectively when using API.The best specificity and positive prediction value (PPV)was 92.94％ and 92.86％,respectively when using APRI.When APRI≥0.30 was used as the cut-off of liver fibrosis,97.62 ％ of 119 patients were diagnosed with liver fibrosis; when API≥4.0 was used as the cut-off of liver fibrosis, 96.43％ of 112 patients were diagnosed with liver fibrosis.Conclusion APRI and API are two simple and feasible non-invasive biochemical markers that can be used to determine liver fibrosis status in chronic HBV carriers.

Objective To establish real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) for human coronaviruses(HCoV)-NL63,HCoV-HKU1,HCoV-OC43 and HCoV-229E,and to investigate the prevalence of the four coronaviruses in children with acute respiratory tract infection(ARTI) in Fuzhou area.Methods Totally of 538 nasopharyngeal swabs were collected from pediatric patients with ARTI,including 289 specimens from children with acute upper respiratory tract infection (AURTI) and 249 from acute lower respiratory tract infection (AURTI) during three consecutive winter-spring seasons from December to April of 2006 - 2009 in Fuzhou area.All the specimens were subjected to FQ-PCR specific for HCoV-NL63,HCoV-HKU1,HCoV-OC43 and HCoV-229E,respectively.The enumeration data were analyzed by chi square test.Results The FQ-PCR methods were established for detecting HCoV-NL63,HCoV-HKU1,HCoV-OCA3 and HCoV-229E.The intraassay coefficient of variation (CV) and interassay CV were both ≤ 1.6％.The coronaviruses were detected in 41 (7.6％) children with ARTI,including HCoV-NL63 in 8 (1.5％)children (1 with AURTI,7 with ALRTI),HCoV-229E in 5 (0.9％; 1 with AURTI,4 with ALRTI),HCoV-HKU1 in 6 (1.1％; 1 with AURTI,5 with ALRTI),and HCoV-OC43 in 22(4.1％; 13 with AURTI,9 with ALRTI).The four coronaviruses were detected during each of the three winter-spring seasons and the positive rates of different periods were not significantly different (P＞0.05).The HCoV-OC43 positive rate was significantly higher than HCoV-NL63,HCoV-229E and HCoV-HKU1 (x2 =6.721,10.979,9.387; respectively; all P＜0.01).ConclusionsIt is suggested that the four coronaviruses might be important virus pathogens in children with ARTI in Fuzhou,China.And detection of them is needed for etiology and epidemiology evaluations for children with ARTI.

Objective To determine the prevalence,trends and risk factors of drug-resistant tuberculosis (TB) in Ningbo during 2007-2010,and to explore the efficient control strategy of drugresistant TB.Methods A cross-sectional study of regional anti-TB drug resistance was conducted in Ningbo.The registered and culture-positive TB patients were enrolled and drug sensitivity test was performed.The demographic and clinical information were collected from the national TB report system.Logistic regression model was used to determine the risk factors of drug resistance.Results Of 1613 enrolled TB patients,39.3％-48.3％ were resistant to any first-line anti-TB drug and 14.0％-19.9％ were multidrug resistant (MDR)-TB.The proportion of new cases resistant to any first-line anti-TB drug was 35.4 ％-42.1％ and MDR TB was 9.8％- 12.2 ％,which were both significantly lower than those of retreated patients (69.5％-72.7％ and 33.9％ - 54.5％,respectively).The multivariate Logistic regression model showed that anti-TB treatment history and migration were significantly associated with any drug resistance (OR=3.298,95 ％ CI 2.391 4.550and OR=0.771,95 ％CI 0.608 - 0.978,respectively) ; while age,treatment history and migration were also significantly associated with MDR-TB.Conclusions Drug-resistant TB prevalence showed a decrease trend in Ningbo,while the resistant rates in both new cases and retreated cases still remain at high levels. Improved case management,including directly observed treatment short-course and appropriate treatment regimens specifically for drug-resistant TB,should be developed to prevent further transmission and development of drug-resistant TB in this setting.