Soluble programmed death-1 is predictive of hepatitis B surface antigen loss in chronic hepatitis B patients after antiviral treatment.

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss, a functional cure in patients with chronic hepatitis B (CHB) undergoing antiviral therapy, might be an ideal endpoint of antiviral treatment in clinical practice. The factors that contribute to the functional cure remain unclear, and the predictors of functional cure are worth exploring. The concentration and kinetics of soluble programmed death-1 (sPD-1) in patients with CHB may play an important role in elucidating the immune response associated with functional cure after nucleos(t)ide analogs therapy. AIM: To investigate the factors associated with HBsAg loss and explore the influence of sPD-1 Levels. METHODS: This study analyzed the data and samples from patients with CHB who underwent antiviral treatment in a non-interventional observational study conducted at Peking University First Hospital in Beijing (between 2007 and 2019). All patients were followed up: Serum samples were collected every 3 mo during the first year of antiviral treatment and every 6 mo thereafter. Patients with positive hepatitis B e antigen levels at baseline and with available sequential samples who achieved HBsAg loss during antiviral treatment served as the case group. This case group (n = 11) was further matched to 44 positive hepatitis B e anti patients without HBsAg loss as controls. The Spearman's rank correlation test and receiver operating characteristic curves analysis were performed. RESULTS: The sPD-1 Levels were higher in patients with HBsAg loss than in those without HBsAg loss from baseline to month 96, and the differences were significant between the groups at baseline (P = 0.0136), months 6 (P = 0.0003), 12 (P < 0.0001), 24 (P = 0.0007), 48 (P < 0.0001), and 96 (P = 0.0142). After 6 mo of antiviral treatment, the sPD-1 levels were positively correlated with alanine transaminase (ALT) levels (r = 0.5103, P = 0.0017), and the sPD-1 levels showed apparent correlation with ALT (r = 0.6883, P = 0.0192) and HBV DNA (r = 0.5601, P = 0.0703) levels in patients with HBsAg loss. After 12 mo of antiviral treatment, the sPD-1 levels also showed apparent correlation with ALT (r = 0.8134, P = 0.0042) and HBV DNA (r = 0.6832, P = 0.0205) levels in patients with HBsAg loss. The sPD-1 levels were negatively correlated with HBsAg levels in all patients after 12 mo of antiviral treatment, especially at 24 (r = -0.356, P = 0.0497) and 48 (r = -0.4783, P = 0.0037) mo. After 6 mo of antiviral treatment, the AUC of sPD-1 for HBsAg loss was 0.898 (P = 0.000), whereas that of HBsAg was 0.617 (P = 0.419). The cut-off value of sPD-1 was set at 2.34 log pg/mL; the sensitivity and specificity were 100% and 66.7%, respectively. CONCLUSION: The sPD-1 levels at 6 mo can predict HBsAg loss after 144 mo of antiviral treatment.

Tumor-suppressive miR-3650 inhibits tumor metastasis by directly targeting NFASC in hepatocellular carcinoma.

In recent years, a growing body of evidence has provided support for the important role of microRNAs (miRNAs) in the progression of human cancers. A recent study showed that a novel miRNA miR-3650 expression was significantly decreased in hepatocellular carcinoma (HCC). However, the precise role of miR-3650 in HCC have remained poorly understood. In this study, we found that miR-3650 expression was frequently decreased in HCC tissues. Low expression of miR-3650 is positively associated with tumor metastasis and poor survival of HCC patients. Forced expression of miR-3650 significantly inhibited the migration and epithelial-mesenchymal transition (EMT) of HCC cells. Through bioinformatic analysis and luciferase assays, we confirmed that neurofascin (NFASC) is a directly target mRNA of miR-3650. Rescue experiment demonstrated that NAFSC overexpression could partially counteracted the inhibitory effect of miR-3650 in HCC metastasis and EMT. In conclusion, our findings are the first time to demonstrate that reduced expression of miR-3650 in HCC was correlated with tumor metastasis and poor survival. MiR-3650 repressed HCC migration and EMT by directly targeting NFASC. Our findings suggested that miR-3650 may serve as a potential prognostic marker and promising application in HCC therapy.

Serum hepatitis B virus RNA is a predictor of HBeAg seroconversion and virological response with entecavir treatment in chronic hepatitis B patients.

BACKGROUND: Characteristics of alterations of serum hepatitis B virus (HBV) RNA in different chronic hepatitis B (CHB) patients still cannot be fully explained. Whether HBV RNA can predict HBeAg seroconversion is still controversial. AIM: To investigate whether HBV RNA can predict virological response or HBeAg seroconversion during entecavir (ETV) treatment when HBV DNA is undetectable. METHODS: The present study evaluated 61 individuals who were diagnosed and treated with long-term ETV monotherapy at the Department of Infectious Diseases of Peking University First Hospital (China) from September 2006 to December 2007. Finally, 30 treatment-naive individuals were included. Serum HBV RNA were extracted from 140 µL serum samples at two time points. Then they were reverse transcribed to cDNA with the HBV-specific primer. The product was quantified by real-time quantitative PCR (RT-PCR) using TAMARA probes. Statistical analyses were performed with IBM SPSS 20.0. RESULTS: Level of serum HBV RNA at baseline was 4.15 ± 0.90 log10 copies/mL. HBV RNA levels showed no significant difference between the virological response (VR) and partial VR (PVR) groups at baseline (P = 0.940). Serum HBV RNA significantly decreased among patients who achieved a VR during ETV therapy (P < 0.001). The levels of HBV RNA in both HBeAg-positive patients with seroconversion group and those with no seroconversion increased after 24 wk of treatment. Overall, HBV RNA significantly but mildly correlated to HBsAg (r = 0.265, P = 0.041), and HBV RNA was not correlated to HBV DNA (r = 0.242, P = 0.062). Furthermore, serum HBV RNA was an independent indicator for predicting HBeAg seroconversion and virological response. HBeAg seroconversion was more likely in CHB patients with HBV RNA levels below 4.12 log10 copies/mL before treatment. CONLUSION: The level of serum HBV RNA could predict HBeAg seroconversion and PVR during treatment. In the PVR group, the level of serum HBV RNA tends to be increasing.

Efficacy and safety of combined directly acting antivirals for treatment of Chinese chronic hepatitis C patients in a real-world setting.

AIM: To assess the efficacy and safety of combined directly acting antivirals (DAAs) for the treatment of Chinese chronic hepatitis C (CHC) patients in a real-world setting. METHODS: Hospitalized CHC patients who were treated with DAAs at Peking University First Hospital between January 2015 and December 2016 were enrolled. Samples and clinical data were collected at 0 wk, 2 wk, 4 wk, 8 wk, 12 wk, or 24 wk during DAAs treatment and at 4 wk, 12 wk, and 24 wk after the end of treatment. RESULTS: Fifty-four patients who underwent DAAs treatment were included in our study, of whom 83.3% (45/54) achieved rapid virological response at 2 wk after treatment initiation (RVR 2) and 94.4% (51/54) achieved sustained virological response at 24 wk after the end of treatment (SVR 24). Serum creatinine and uric acid levels at the end of treatment were significantly increased compared with baseline levels (83.6 ± 17.9 vs 88.8 ± 19.4, P01 < 0.001; 320.8 ± 76.3 vs 354.5 ± 87.6, P01 < 0.001), and no significant improvements were observed at 24w after the end of treatment (83.6 ± 17.9 vs 86.8 ± 19.1, P02 = 0.039; 320.8 ± 76.3 vs 345.9 ± 89.4, P02 = 0.001). The total frequency of adverse events (AEs) during treatment was 33.3% (18/54), with major AEs being fatigue (16.7%), headache (7.4%), anorexia (7.4%), and insomnia (5.6%). CONCLUSION: Though based in a small cohort of patients, the abnormal changes in renal function indices and relative high frequency of AEs during combined DAAs treatment should be taken as a note of caution.

Dynamic Characteristics of Serum Hepatitis B Surface Antigen in Chinese Chronic Hepatitis B Patients Receiving 7 Years of Entecavir Therapy.

BACKGROUND: The ultimate goal of hepatitis B treatment is hepatitis B surface antigen (HBsAg) seroclearance. Several factors have been suggested to be associated with the rate of HBsAg reduction in antiviral-naive or lamivudine therapy cohorts. However, there are few studies evaluating the factors during long-term entecavir (ETV) therapy. In the present study, we aimed to evaluate the factors to predict the outcome of ETV therapy for 7 years. METHODS: A total of 47 chronic hepatitis B (CHB) patients treated with ETV monotherapy were included in this study. Liver biochemistry, hepatitis B virus (HBV) serological markers, serum HBV DNA, and HBsAg titers were tested at baseline, 3 months, 6 months, and yearly from 1 to 7. The associations between factors and HBsAg reduction were assessed using multivariate tests with repeated measure analysis of variance. RESULTS: At baseline, serum HBsAg levels showed a positive correlation with baseline HBV DNA levels (r = 0.625, P < 0.001). The mean HBsAg titers after ETV treatment were significantly lower than the baseline titers (P ranges from 0.025 to 0.000,000,6). The HBsAg reduction rate during the 1st year was greater compared to after 1 year of treatment (P < 0.05). Multivariate test showed that hepatitis B e antigen (HBeAg) seroclearance and/or HBsAg reduction ≥0.5 log10 IU/ml at 6 months had a high negative predictive value (96.77%) for HBsAg seroclearance (P = 0.002, P = 0.012, respectively). CONCLUSIONS: The HBsAg reduction rate during the 1st year was greater than that after 1 year of treatment. Further, HBeAg status and HBsAg levels at month 6 are the optimal factors for the early prediction of HBsAg seroclearance after long-term ETV therapy in CHB patients.

Dynamics of Genotypic Mutations of the Hepatitis B Virus Associated With Long-Term Entecavir Treatment Determined With Ultradeep Pyrosequencing: A Retrospective Observational Study.

The aim of the study is to explore the evolution of genotypic mutations within the reverse transcriptase region in partial virological responders (PVRs) receiving long-term entecavir (ETV) treatment. A total of 32 patients were classified as completely virological responders (CVRs) (n = 12) or PVRs (n = 20). Five partial responders were hepatitis B virus (HBV)-DNA positive after long-term therapy, which lasted for >3 years. A total of 71 serum samples from these 32 patients were assayed by ultra-deep pyrosequencing (UDPS): 32 samples were from all patients at baseline, and 39 were from PVRs with sequential inter-treatment. Approximately 84,708 sequences were generated per sample. At baseline, the quasispecies heterogeneity did not significantly differ between the 2 groups. The frequencies of substitutions indicating pre-existence of nucleos(t)ide analog resistant (NAr) mutants ranged from 0.10% to 6.70%, which did not statistically differ between groups either. However, the substitutions associated with the NAr mutants were significantly different from those associated with the non-NAr mutants in 13 patients; 6 of these patients were PVRs and the others were CVRs. Five patients were HBV DNA positive after regular ETV monotherapy for >3 years, and 4 of these patients underwent mild NAr substitution fluctuations (<20%). One patient developed virological breakthrough while bearing single, double, and triple (rtL180 M, rtM204 V, rtS202G) substitutions. In addition to the common substitutions, unknown amino acid substitutions, such as rtL145 M/S, rtF151Y/L, rtR153Q, rtI224 V, rtN248H, rtS223A, rtS256C, need to be further verified. NAr substitutions are observed at frequencies of 0.10% to 6.7% before therapy. Long-term ETV therapy generally results in virological responses, as long as the proportion of resistance mutations remains at a relatively low level. Genotypic resistance to ETV is detected in all PVRs receiving long-term ETV therapy.

Thyroid dysfunction in Chinese hepatitis C patients: Prevalence and correlation with TPOAb and CXCL10.

AIM: To investigate the relationship among pretreatment serum CXC chemokine ligand 10 (CXCL10), thyroid peroxidase antibody (TPOAb) levels and thyroid dysfunction (TD) in Chinese hepatitis C patients. METHODS: One hundred and thirty-nine treatment-naive genotype 1 chronic hepatitis C patients with no history of TD or treatment with thyroid hormones were enrolled in this study. Patients underwent peginterferon alfa-2a/ribavirin (PegIFNα-2a/RBV) treatment for 48 wk, followed by detection of clinical factors at each follow-up point. Hepatitis C virus (HCV) antibodies were analyzed using microsomal chemiluminescence, and serum HCV RNA was measured by real-time PCR assay at 0, 4, 12, 24 and 48 wk after the initiation of therapy and 24 wk after the end of therapy. To assess thyroid function, serum thyroid stimulating hormone (TSH), free thyroxine (FT4), free triodothyronine (FT3) and TPOAb/thyroglobulin antibody (TGAb) levels were determined using chemiluminescent immunoassays every 3 mo. Serum CXCL10 levels were determined at baseline. RESULTS: The prevalence of TD was 18.0%. Twenty-one (84.0%) out of twenty-five patients exhibited normal thyroid function at week 24 after therapy. The rate of sustained virological response to PegIFNα-2a/RBV in our study was 59.0% (82/139), independent of thyroid function. Pretreatment serum CXCL10 levels were significantly increased in patients with euthyroid status compared with patients with TD (495.2 ± 244.2 pg/mL vs 310.0 ± 163.4 pg/mL, P = 0.012). Patients with TD were more frequently TPOAb-positive than non-TD (NTD) patients (24.2% vs 12.3%, P = 0.047) at baseline. Three of the one hundred and fifteen patients without TPOAb at baseline developed TD at the end of treatment (37.5% vs 2.6%, P = 0.000). Female patients exhibited an increased risk for developing TD compared with male patients (P = 0.014). CONCLUSION: Lower pretreatment serum CXCL10 levels are associated with TD, and TD prevalence increases in female patients and patients who are positive for TPOAb at baseline.

Antibody to hepatitis B core antigen levels in the natural history of chronic hepatitis B: a prospective observational study.

Previous studies have revealed antibody to hepatitis B core antigen (anti-HBc) levels as a predictor of treatment response in hepatitis B early antigen (HBeAg)-positive chronic hepatitis B (CHB) patients in both interferon and nucleos(t)ide analog therapy cohorts. However, there is no information about anti-HBc levels in the natural history of CHB. This study aimed to define anti-HBc levels of different phases in the natural history of CHB. Two hundred eleven treatment-naive CHB patients were included in the study. They were classified into 4 phases: immune tolerance (IT) phase (n = 39), immune clearance (IC) phase (n = 48), low or no-replicative (LR) phase (n = 55), and HBeAg-negative hepatitis (ENH, n = 69). Fifty patients who were HBsAg negative and anti-HBc positive were also recruited as past HBV infection (PBI) control group. Anti-HBc levels were measured by a newly developed double-sandwich immunoassay. Correlation of anti-HBc levels with alanine aminotransferase (ALT) and other HBV-related markers within each phase was performed. Serum anti-HBc levels were statistically significant between patients in different phases of CHB (P < 0.001). The median anti-HBc levels were: IT (3.17 log 10 IU/mL), IC (4.39 log 10 IU/mL), LR (3.29 log 10 IU/mL), ENH (4.12 log 10 IU/mL), and PBI (0.61 log 10 IU/mL). There existed a strong correlation in IC (r = 0.489, P < 0.001), a poor correlation in ENH (r = 0.275, P = 0.042), and no correlation in patients with ALT reached 5 times upper limit of normal (r = 0.120, P = 0.616). Anti-HBc levels show significant differences during the natural course of CHB. These results may provide some potentially useful insights into hepatitis B pathogenesis and immune activation against hepatitis B virus.

Kinetics and prediction of HBsAg loss during long-term therapy with nucleos(t)ide analogues of different potency in patients with chronic hepatitis B.

BACKGROUND & AIMS: About 350-400 million people are infected with hepatitis B virus (HBV) chronically and 1 million people die of hepatitis B virus (HBV)-related liver diseases. Nucleos(t)ide analogues (NAs) have been used for the treatment against HBV. However, few studies have investigated the long-term effects of different nucleos(t)ide analogues on levels of hepatitis B surface antigen (HBsAg) in patients with chronic hepatitis B (CHB). The aims of this study were to measure the magnitude of HBsAg reduction by long-term monotherapy with adefovir dipivoxil (ADV) and entecavir (ETV), to compare HBsAg reduction between the two drugs of different potency and to predict the expected time needed to achieve HBsAg loss. METHODS: We retrospectively evaluated the kinetics of HBsAg in 67 patients with CHB who all exhibited persistent viral suppression. These patients were treated with ADV or ETV for at least 6 years. HBV genotype was determined at baseline. Liver biochemistry, HBV serological markers, serum HBV DNA and HBsAg titers were determined at baseline, half year and yearly from year 1 to 6. RESULTS: Serum HBsAg titers after treatment with ADV or ETV were significantly lower than the baseline titers (P<0.05). HBsAg reduction rate of patients treated with ETV (0.11 log10 IU/mL/ year) was higher than that treated with ADV (0.10 log10 IU/mL/year), and the calculated expected time to HBsAg loss for patients treated with ETV (approximate 24.99 years) was shorter than that with ADV (approximate 30.33 years), but there was no statistically significant difference between two groups (P>0.05). CONCLUSION: Serum HBsAg titers gradually decreased during long-term treatment with either ADV or ETV. It appears that the potency of ADV on HBsAg reduction is close to that of ETV, as long as patients have achieved persistent viral suppression.

Characteristics of HIV-1 natural drug resistance-associated mutations in former paid blood donors in Henan Province, China.

BACKGROUND: Natural drug resistance is a major cause of antiviral treatment failure. The characteristics of HIV-1 natural drug resistance-associated mutations in former paid blood donors in Henan Province remain unclear. METHODS: One hundred and fifty HIV-1-positive plasma samples were collected. Plasma viral RNA was extracted for pol gene amplification and sequencing. The sequencing results were submitted to the HIV-1 drug resistance database for drug-resistance analysis. RESULTS: The rates of natural drug resistance and resistance-associated mutations were 17.7% (19/107) and 40.2% (43/107), respectively. The rates of PI major, PI minor, NRTI, and NNRTI mutations were: 0, 30.8% (33/107), 10.3% (11/107), and 18.7% (20/107), respectively. Nine cases (8.4%) had both NRTI and NNRTI resistance-associated mutations. Seven cases (6.5%) had PI minor, NRTI and NNRTI resistance-associated mutations. NNRTI resistance was the most serious, followed by NRTI resistance and PI resistance. Polymorphism mutation sites with mutation rates in the protease region higher than 60.0% were: L63A/P/S/T 89.7%, V77I 82.2%, I72E/M/K/T/V 80.4%, I93L 75.7%, and E35D 72.9%. Polymorphism mutation sites with mutation rates in the RT region higher than 60.0% were: I135A/L/M/R/T/V 93.5%, T200A/E/I/P/V 89.7%, Q278E/K/N/T 88.8%, S162C/Y 82.2%, and K277R/S 66.4%. The distribution of 107 gene sequences was scattered, with some drug-resistant strains grouped in the same cluster. CONCLUSION: The natural drug resistance mutation rate of HIV-1 in former paid blood donors in Henan Province was 17.7%, with NNRTI resistance the most serious. The distribution of drug-resistant strains was scattered, with some correlations found in certain resistance loci.

[Antiviral nucleotide-induced dynamic change of HBV DNA and HBsAg and significance of quarterly and annual quantitative measurements over 5-year follow-up of chronic hepatitis B patients].

OBJECTIVE: To analyze the dynamic changes in hepatitis B virus (HBV) DNA and hepatitis B surface antigen (HBsAg) levels in chronic hepatitis B (CHB) patients following treatment by antiviral nucleotide drugs over a 5-year follow-up period and to assess the clinical significance of quarterly and annual quantitative measurements. METHODS: One-hundred-and-ten patients with CHB were enrolled in the study and administered on-going standard mono-therapy with various antiviral nucleotide drugs. Over a 5-year period, the HBV DNA level was measured by quantitative PCR every three months and the HBsAg levels were measured by chemiluminescence once a year. The dynamic changes in HBV DNA and HBsAg levels were assessed by Chi-squared test and ANOVA. RESULTS: Only 90 of the CHB patients completed the 5-year follow-up and were included in the analysis. The patients who showed HBeAg-positivity at baseline (study start) had higher levels of HBV DNA and HBsAg than the patients showing HBeAg-negativity. In general, the antiviral nucleotide drug therapy induced downward trends in HBsAg and HBV DNA level over time (F = 17.1, 151.53, all P less than 0.05). However, the most robust reduction in HBV DNA occurred during the first year. The HBsAg level followed an opposite trend, with the most robust reductions occurring in the 3rd, 4th and 5th years of treatment. CONCLUSION: Long-term antiviral nucleotide mono-therapies induced decreases in HBV DNA and HBsAg levels in CHB patients, with the former being most reduced in the short-term and the latter in the long-term.

Effect of nuclear transcription factor kappaB pathway on proliferation in rat pulmonary artery smooth muscle cells induced by platelet-derived growth factor / 中华实用儿科临床杂志

Objective To explore the effect of nuclear transcription factor kappaB(NF-κB) pathway on proliferation of rat pulmonary artery smooth muscle cells (PASMC) induced by platelet-derived growth factor (PDGF).Methods PASMC isolated from rats and cultured in vitro were divided into 3 groups according to the randomization principle:control group(cultured by M199),PDGF treatment group(cultured by M199 and stimulated by PDGF),PDGF + parthenolide treatment group (cultured by M199 and stimulated by PDGF,and intervented by the NF-κB inhibitors parthenolide).MTT colorimetric assay and flow cytometry were performed to detect cell proliferation and cell cycle distribution.Immunohistochemistry was performed to detect the expressions of NF-κB and COX-2 protein.Fluorescence quantitative RT-PCR was performed to detect NF-κB and COX-2 mRNA expressions.One-way ANOVA was used for statistical analysis,multiple comparisons were analyzed by LSD.Results Compared with the control group,MTT value of PASMC was increased significantly when induced by PDGF at each time points(all P ＜ 0.05).MTT value decreased dramatically after the intervention of NF-κB inhibitor parthenolide(P ＜0.05).Data from flow cytometry detection showed that cell proportion of S phase and G2 + M phase increased significantly in PDGF treatment group,which had statistical difference compared with control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,cell proportion of S phase and G2 + M phase ratio decreased dramatically (P ＜ 0.05).The expressions of NF-κB and COX-2 protein and mRNA were promoted in the PDGF induced group compared with the control group (all P ＜ 0.05).Compared with PDGF induced group,after the intervention of parthenolide,the expressions of NF-κB and COX-2 protein and mRNA decreased dramatically(all P ＜ 0.05).Conclusions PDGF can induce proliferation of PASMC,promote cell cycle process and enhance the expressions of NF-κB and COX-2 protein and mRNA.NF-κB pathway involves in the proliferation of PASMC induced by PDGF.

Study of enterovirus 71-induced autophagy in human malignant glioma cell line U251 / 中华实用儿科临床杂志

Objective To show evidences that autophagy is induced in human malignant glioma cell line U251 by enterovirus(EV71) and its effect on the expression of microtubule-associated protein 1 lightchain 3 (LC3) in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously.After 12 hours post infection of U251 cells with EV71,autophagic vacuoles of U251 cells were marked by monodansylcadaverine staining and observed under fluorescence microscope.After 24 hours post infection,expression and intracellular distribution of LC3 protein in U251 cells were observed under fluorescence microscope by immunofluorescence.Expressions of LC3-I and LC3-Ⅱ protein were measured by Western blot and analysis of LC3-Ⅱ protein expression was performed with semi-quantitative calculation at 2,4,8,12,24 and 48 hours post infection of U251 cells with EV71,respectively.Results Autophagic vacuoles stained by MDC in U251 cells appeared as distinct dot-like structures distributed under fluorescence microscope.The number of autophagic vacuoles were increased significantly at 12 hours post infection of U251 cells with EV71 when compared with control group.The morphological features of these cells became significantly shrunken,smaller and irregular shape at 24 hours post infection of U251 cells,and the expressions of LC3 protein were significantly higher in experimental group than those in control group.Under a fluorescence microscope,LC3 protein distributed within the cytoplasm or localizing in the perinuclear regions.At 4 hours post infection of U251 cells with EV71,the expression of LC3-Ⅱ protein started to increase,and was significantly higher than that in control group (P ＜ 0.01).Conclusion These results indicate that EV71 can effectively induce autophagy of human malignant glioma cell line U251,and play its oncolytic effect.

Anti-tumor effects of enterovirus 71 on human malignant glioma cell line U251 in vitro / 中华实用儿科临床杂志

Objective To investigate the effects of enterovirus 71 (EV71) on growth,apoptosis and human telomerase reverse transcriptase (hTERT) expression of human malignant glioma cell line U251 in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In the experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously for 72 hours with U251 cells,but in the control group,EV71 were not put.The morphological features and growth of cells were observed under inverted microscope at 0 hour,24 hours,48 hours and 72 hours of EV71 treatment,respectively.At the same time,cell apoptosis was measured by flow cytometry using Annexin V/PI double staining method.hTERT expression was detected with semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR).Results The growth of U251 cells was inhibited significantly at 24 h of EV71 treatment under inverted microscope in experimental group when compared with control group,and at 48 h and 72 h,the morphological features of these cells became significantly shrunken,smaller and irregular shape in experimental group,but which were uniform size in control group.At 24 h,48 h,72 h of EV71 treatment,flow cytometry showed the apoptosis rates were significantly higher in experimental group than that in control group [24 h:(12.55 ± 2.38) ％ vs (1.42 ± 0.21) ％,48 h:(65.60 ± 8.48)％ vs (1.42 ±0.17)％,72 h:(87.52 ±3.05)％ vs (1.41 ±0.16)％,all P =0.000],and there was upward trend day by day,but no change in control group.At the same time,hTERT mRNA expression levels of U251 cells were significantly lower in experimental group than control group [24 h:(0.58 ± 0.05) vs (0.89 ± 0.05),48 h:(0.23 ± 0.04) vs (0.89 ± 0.03),72 h:(0.10 ± 0.03) vs (0.90 ± 0.06),all P =0.000],and the downward trend was observed day by day,but there was no this trend in control group.Conclusions EV71 can effectively inhibit the growth of U251 cells,induce the cell apoptosis,and has the potential anti-tumor effect.Its mechanisms may be partly related to down regulation the hTERT expression of U251 cells.

G6PD genotype and its associated enzymatic activity in a Chinese population.

Knowledge of the G6PD genotype and its associated enzyme activity is significant for population genetics, diagnosis of disease, and management of patients. We tested 2,872 unrelated subjects from a Hakka population in China for G6PD activity by the WHO standard method and for genotype by DHPLC and DNA sequencing. Among female heterozygotes, 78.5% had relatively normal enzyme activity. The phenotype frequency of G6PD deficiency is 0.028, and the causal allele frequency is 0.060 in females. The accuracy, sensitivity, and specificity of DHPLC are more than 98% for detecting G6PD-deficient hemizygotes, heterozygotes, and homozygotes. Measuring enzyme activity alone is not sufficient for the diagnosis of heterozygotes. A combination of enzyme activity and DNA analysis should be used.

Risk factors of heart and lung failure in children with severe hand, foot and mouth disease and treatment experience / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study risk factors for severe hand, foot and mouth disease (HFMD) complicated by heart and lung failure and treatment experience.</p><p><b>METHODS</b>A total of 198 children with severe HFMD between March and August in 2011 were enrolled. Univariate analysis and logistic regression model were used to analyze the risk factors severe HFMD complicated by heart and lung failure. The effects of combination therapy with immunoglobulin+dexamethasone+ribavirin were observed.</p><p><b>RESULTS</b>Univariate analysis indicated that HFMD patients with heart and lung failure had higher proportions of consciousness, tachypnoea, abnormal hemodynamics, increased troponin and EV71 infection than HFMD patients without heart and lung failure (P<0.05).Multivariate logistic regression analysis indicated that tachypnoea, abnormal hemodynamics and EV71 infection were the main risk factors for heart and lung failure. Compared with combination therapy with dexamethasone+ribavirin, combination therapy with immunoglobulin+dexamethasone+ribavirin was more effective for preventing hemodynamic changes in children with severe HFMD (P<0.01). Compared with HFMD patients with heart and lung failure, the effect of the combination therapy with immunoglobulin+dexamethasone+ribavirin was better in HFMD patients without heart and lung failure (P<0.01).</p><p><b>CONCLUSIONS</b>The main risk factors for heart and lung failure in children with severe HFMD include tachypnoea, abnormal hemodynamics and EV71 infection. Early combination therapy with immunoglobulin+dexamethasone+ribavirin can reduce the incidence of heart and lung failure in children with severe HFMD.</p>

[Comparison of lamivudine or interferon monotherapy and sequential therapy in chronic hepatitis B: a random controlled trial].

OBJECTIVE: To compare the efficacy and safety of lamivudine or interferon monotherapy and sequential therapy in HBeAg positive chronic hepatitis B patients. METHODS: A total of 225 patients with HBeAg positive chronic hepatitis B were randomized into 3 groups: sequential group (group A, 83 patients), lamivudine group (group B, 89 patients) and interferon group (group C, 53 patients). Group A was administrated with lamivudine 100 mg/d for 32 week, and 5 million units of interferon alpha 2b injected subcutaneously every other day lasting for 24 week were added since week 25. Group B was administrated with lamivudine 100 mg/d for 48 week. Group B was injected with 5 million units of interferon alpha 2b subcutaneously every other day for 24 week. All subjects were followed up for 24 week. Serum HBV DNAs were measured quantitatively by PCR. HBV mutations were analyzed by PCR-RFLP. RESULTS: For groups A, B and C, baseline HBV DNAs were 7.8±1.0, 7.9±1.1 and 8.0±0.9 log10 copies/mL, respectively, P>0.05. Baseline ALTs were 167.5 (99.0, 267.8), 134.0 (101.0,275.0) and 131.0 (99.0, 192.8)U/L, respectively, P>0.05. At the end of the treatment, HBV DNA decrease rates for groups A, B and C were 78.2%, 87.8% and 78.4% (P>0.05), respectively. At the end of the follow-up, HBV DNA decrease rates for groups A, B and C were 54.4%, 63.6% and 66.7% (P>0.05), respectively. At the end of the treatment, group B (83.5%, P<0.05) achieved the highest response rate and group C achieved the lowest (39.6%, P<0.05). At the end of the follow-up, the response rates for groups A, B and C were 36.2%, 54.4% and 42.1% (P>0.05), respectively. YMDD motif mutation rate in group A was lower than that of group B (10.5% vs 26.9%, P<0.05) at the end of the treatment. CONCLUSION: Sequential therapy decreased hepatitis B virus mutation. But no efficacy advantages were found in sequential therapy than in lamivudine or interferon monotherapy.

Administration of low-dose heparin in total nutrient admixture prevents central venous catheter-related infections in neonates / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Peripherally inserted central catheter (PICC) is widely used to provide a long-term access for the administration of total parenteral nutrition and medications. Catheter-related infections (CRI) are common complications of PICC. The purpose of this retrospective study was to investigate the role of low-dose heparin added to the total nutrient admixture (CTNA) in the prevention of CRI.</p><p><b>METHODS</b>Eighty-three neonates who underwent PICC received TNA with (heparin group, n=43) or without heparin (0.5 U/mL) (control group, n=40). The incidence of CRI was compared between the two groups.</p><p><b>RESULTS</b>The incidences of catheter obstruction (5% vs 20%) and the catheter-tip colonization (2% vs 18%) in the heparin group were significantly lower than those in the control group (p<0.05). None of the neonates in the heparin group had clinical evidence of catheter-related bloodstream infection, but 5 cases in the control group (p<0.05).</p><p><b>CONCLUSIONS</b>The administration of low-dose heparin in TNA may decrease the incidences of catheter obstruction and CRI.</p>

hTERT gene expression in children with beta-thalassemia major / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Human telomerase reverse transcriptase (hTERT) is a rate-limiting enzyme which dictates the activity of human telomerase and thus decides the life span of cells. The aim of this study was to explore the expression of hTERT in bone marrow from children with beta-thalassemia major and the relationship between the expression of hTERT and hemoglobin levels.</p><p><b>METHODS</b>Multiple allele specific polymerase chain reaction (MASPCR) was used for targeted DNA amplification and gene mutation analysis of beta-thalassemia. hTERT mRNA expression in bone marrow was examined using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis in 29 children with beta-thalassemia major, in 10 children with agranulocytosis and in K562 cell line. The hemoglobin levels in peripheral blood were measured. The relationship between hTERT expression and hemoglobin levels was evaluated by the Spearman test in the beta-thalassemia major group.</p><p><b>RESULTS</b>hTERT mRNA expression significantly increased in bone marrow from children with beta-thalassemia major compared with that from children with agranulocytosis (0.2928+/- 0.0838 vs 0.0993+/- 0.0336; P<0.01), but was significantly lower than that in K562 cell line (0.8291+/- 0.0908) (P<0.01). A significantly inverse correlation was found between hTERT mRNA expression and hemoglobin levels (r=-0.841, P<0.01).</p><p><b>CONCLUSIONS</b>A low hemoglobin concentration might contribute to the up-regulation of marrow hTERT expression in children with beta-thalassemia major.</p>

Comparative study on clinical features between TEL-AML1 positive and negative childhood acute lymphoblastic leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the incidence of TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) and to compare the clinical features between TEL-AML1 positive and negative patients.</p><p><b>METHODS</b>Samples of bone marrow or peripheral blood were collected from 95 newly diagnosed ALL children and the TEL-AML1 fusion gene was detected using nested reverse transcription-polymerase chain reaction (RT-PCR). The ALL patients were stratified into TEL-AML1 positive and negative groups and the clinical features were compared.</p><p><b>RESULTS</b>Among 95 patients, 20 (21.05%) were TEL-AML1 positive. The median age of TEL-AML1 positive patients was 5.9 years old and M/F ratio was 1.22:1. There were significant differences between TEL-AML1 positive and negative patients in hepatomegaly (2.75 cm vs. 4 cm below costal arch, P=0.006), splenomegaly (0 cm vs. 3 cm below costal arch, P < 0.001), initial white blood cell count (median 7.40 x 10(9)/L vs.18.70 x 10(9)/L, P=0.011), initial peripheral blood blast (median 2.45 x 10(9)/L vs.11.66 x 10(9)/L, P=0.013), hemoglobin level [(61.45 +/- 13.46) g/L vs. (75.89 +/- 23.11) g/L, P=0.003] and serum lactate dehydrogenase [(621.47 +/- 335.85) U/L vs.(1566.64 +/- 1720.45) U/L, P=0.020], while no differences were found between two groups in age, gender ratio, initial platelet count, percentage of blast in bone marrow, immunophenotypes and the expression of myeloid antigen CD13, CD33 and CD34. The prednisone sensitivity test showed that all 12 TEL-AML1 positive patients were good responders, while there were 11 prednisone poor responders among 40 negative patients (27.50%, P < 0.05). Bone marrow examination on day 15 showed no difference in the rate of complete remission between TEL-AML1 positive and negative patients.</p><p><b>CONCLUSION</b>The incidence of TEL-AML1 fusion gene in cases of ALL is 21.05%. The load of leukemia cells in TEL-AML1 positive patients is significantly smaller than its counterparts, and the blast cells in TEL-AML1 positive patients are more sensitive to prednisone, indicating childhood ALL with TEL-AML1 fusion gene has a favorable prognosis.</p>