OBJECTIVES:The accurate assessment of liver fibrosis is clinically important in patients with chronic hepatitis B (CHB). Blood tests and elastography are now widely used for the noninvasive diagnosis of liver fibrosis in CHB patients. The aim of this study was to develop a new and more accurate predictive model, which combines elastography data, serum biomarkers, and individual characteristics, to discriminate between CHB patients with and without significant liver fibrosis. METHODS:Two noninvasive methods, specifically, an ultrasound elastography technique termed acoustic radiation force impulse imaging (ARFI) and a blood test, were used to assess a cohort of 345 patients (estimation group, 218 patients; validation group, 127 patients) with CHB. Multivariate logistic regression analysis revealed that ARFI, the aspartate aminotransferase (AST) to platelet ratio, and age were significantly associated with fibrosis. Based on these results, we constructed and validated a model for the diagnosis of significant hepatic fibrosis. RESULTS:The area under the receiver operating characteristic (ROC) curve was 0.921 for the estimation group and 0.929 for the validation group, significantly higher than those for ARFI (0.887, 0.893) and for the AST-to-platelet ratio index (APRI; 0.811, 0.859). Using an optimal cutoff of 3.05 in the validation group, all the indices of the proposed model, including accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic odds ratio, were better than those for ARFI or APRI. CONCLUSIONS:We developed a simple noninvasive model that used ultrasound elastography, routine serum biomarkers, and individual characteristics to accurately differentiate significant fibrosis in patients with CHB. Compared with elastography or the biomarker index alone, this model was significantly more accurate and robust.

Assessing liver fibrosis with chronic hepatitis B (CHB) in patients is quite important. Some non-invasive approaches for evaluating liver fibrosis include blood tests and ultrasound elastography. How to effectively combine multiple methods to improve the diagnostic performance remains a challenging problem. The main goal of this paper is to assess and stage liver fibrosis in CHB using feature selection and machine learning methods based on multimodal data. Popular machine learning approaches (e.g., support vector machine (SVM)) and feature selection (FS) were explored to stage the CHB. 16 volunteers and 92 patients with CHB were investigated for liver fibrosis staging based on transient elastography (TE) and acoustical radiation force impulse imaging (ARFI) data. The accuracy of the staging result using FS and a SVM classifier was an accuracy of 90.68% for significant fibrosis (≥F2) and an accuracy of 93.52% for cirrhosis (F4), respectively. The proposed method also increased the sensitivity, specificity, and area under curve (AUC) values for both significant fibrosis and cirrhosis diagnosis, which is very promising for staging liver fibrosis from multimodal information. It outperforms any single method and their linear combination and also achieves a state-of-the-art performance.

AimWe aimed to investigate the efficacy and safety of telbivudine (LdT) on the intervention of mother-to-child transmission (MTCT) in different trimesters of pregnant women with high viral loads.MethodsIn this prospective cohort study, 160 cases of mothers with high viral loads were included. Eighty-two subjects received 600mg/day LdT therapy. Fifty of them started LdT therapy before the third trimester of gestation, including 17 cases before pregnancy, nine and 24 cases in the first and second trimesters of pregnancy, respectively. The other 32 cases started LdT in the third trimester of gestation. Control pregnant women (78 cases) did not take LdT therapy. MTCT rate was determined by hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA data of infants at the 51st week post-partum. Adverse events were also evaluated throughout the study.ResultsOne hundred and sixty infants were born from 160 pregnant women. Both LdT-treated groups displayed a marked decline in HBV DNA levels from the beginning to delivery. Positive rate of serum HBsAg in infants born from the above two groups of mothers were 0% and 3.1%, respectively, which was significantly lower than that in the untreated controls (24.4%). The incidence of detectable HBV DNA levels was significantly lower in infants born to LdT-treated mothers than in the controls (16.7%) at the 51st week post-partum. No infant had birth defects. No severe adverse event or complication were observed in LdT-treated mothers or infants followed until the 51st week post-partum.ConclusionThe earlier application of LdT during pregnancy, the better preventive effects it offered on MTCT.

AIM To investigate the combined diagnostic accuracy of acoustic radiation force impulse (ARFI), aspartate aminotransferase to platelet ratio index (APRI) and Forns index for a non-invasive assessment of liver fibrosis in patients with chronic hepatitis B (CHB). METHODS In this prospective study, 206 patients had CHB with liver fibrosis stages F0-F4 classified by METAVIR and 40 were healthy volunteers were measured by ARFI, APRI and Forns index separately or combined as indicated. RESULTS ARFI, APRI or Forns index demonstrated a significant correlation with the histological stage (all P < 0.001). According to the AUROC of ARFI and APRI for evaluating fibrotic stages more than F2, ARFI showed an enhanced diagnostic accuracy than APRI (P < 0.05). The combined measurement of ARFI and APRI exhibited better accuracy than ARFI alone when evaluating ≥ F2 fibrotic stage (Z = 2.77, P = 0.006). Combination of ARFI, APRI and Forns index did not obviously improve the diagnostic accuracy compared to the combination of ARFI and APRI (Z = 0.958, P = 0.338). CONCLUSION ARFI + APRI showed enhanced diagnostic accuracy than ARFI or APRI alone for significant liver fibrosis and ARFI + APRI + Forns index shows the same effect with ARFI + APRI.

目的 探讨采用声触诊组织量化(VTQ)技术联合谷草转氨酶/血小板比值(APRI)对慢性乙型病毒性肝炎患者肝纤维化分期的价值.方法 将117例慢性乙型病毒性肝炎患者按照穿刺活检病理结果,分为F1～F4组,均接受VTQ检查,测量并计算肝脏右叶sS～s8段的剪切波速度作为VTQ值,同时计算每例患者的APRI.随机选择34名健康志愿者作为对照组(F0组),同法测量、记录所有指标.分析VTQ、APRI与病理学分期的相关性,采用ROC曲线评价VTQ、APRI及二者线性结合(VTQ+ APRI)分期F2以上级别肝纤维化的效能.结果 各组VTQ值比较,除F1与F2组间差异无统计学意义(P＞0.05)外,其余各组两两比较差异有统计学意义(P＜0.05);APRI除F0与F1组间差异无统计学意义(P＞0.05),其余各组间比较均差异有统计学意义(P＜0.05).患者VTQ和APRI与肝纤维化病理学分期均呈正相关(r-0.814,0.677,P＜0.001).评价≥F2级肝纤维化时,VTQ比APRI显示出更好的特异度(89.83％ vs71.19％),阳性预测值(92.11％ vs 82.47％),阳性似然比(7.48 vs 3.02),ROC曲线下面积(AUC)亦较大(0.898 vs0.845);而VTQ+ APRI的AUC、敏感度、阴性预测值分别为0.913、82.61％、76.47％,均高于单独应用VTQ(0.898、76.09％、70.67％).结论 VTQ与APRI均与慢性乙型病毒性肝炎患者肝纤维化程度相关.对于F2以上级别肝纤维化,VTQ的诊断准确性高于APRI,而二者结合的诊断效能更高.

目的 分析肺结核患者(TB)外周血单核细胞中miR-34a-5p和miR-708-5p的表达水平,并探讨其临床诊断价值.方法 选取TB患者和健康对照(HD)各25例,应用TaqMan qPCR技术测定两组人群外周血单核细胞中miR-34a-5p和miR-708-5p表达水平,以U6 snRNA作为内参.应用ROC曲线评价miR-34a-5p和miR-708-5p诊断TB的临床意义.结果 miR-34a-5p在TB患者中的表达水平为(10.08±0.68),显著高于HD的(7.67±0.36),差异有统计学意义(P＜0.01);miR-708-5p在TB患者中的表达水平显著高于HD的(13.52±0.98 Vs7.71±0.57),差异有统计学意义(P＜0.01).ROC曲线分析表明在肺结核诊断中,miR-34a-5p的敏感性、特异性分别为72％、76％;miR-708-5p的敏感性、特异性为76％、76％.结论 miR-34a-5p和miR-708-5p分子在TB患者外周血单核细胞中异常表达,对TB辅助诊断具有一定参考价值.

Objective To explore the immune response of T helper cells 17 in patients with hepatitis B virus (HBV) and the relation with Th1,Th2 and Treg cells.Methods The percentages of Th17 cells were detected by intracellular cytokine staining with flow cytometry.Using Realtime PCR method,we assayed the mRNA expression of IL-17,T-bet,GATA-3 and FoxP3 in 58 HBV patients and 40 healthy controls in our research.Results Compared with control group (3.56％ ± 1.26％),the percentage of Th17 cells in the peripheral blood of HBV group (4.72％ ± 1.80％) was higher (P ＜0.001).The mRNA of IL-17 expression of the HBV group (4 ±0.49) was higher than in healthy control group (1.19 ±0.19,P ＜ 0.0001).The mRNA of T-bet expression of the HBV group (2.27 ± 0.24) was higher than in healthy control group (1.10 ± 0.13,P ＜ 0.05).The mRNA of GATA-3 expression of the HBV group (2.29 ± 0.16) was higher than in healthy control group (1.10 ± 0.10,P ＜ 0.0001).The mRNA of FoxP3 expression of the HBV group (2.03 ±0.15) was higher than in healthy control group ((1.05 ±0.10,P ＜ 0.0001).Conclusions The Th17 cells participated in immune response of the hepatitis B virus infection patients,and there has certain adjustment relations among Th1,Th2 and Treg cells.

Objective To produce influenza virus-like particles(VLPs) with drosophila S2 cell line and identify its bioactivity.Methods The persistent expression vector sof pAc-V5-HA,NA and M1 suitable for drosophila S2 cell line were constructed at the earlier stage,which were co-transfected into S2 cell with pCoblast vector.Then we screened monoclonal S2 cell lines which integrated HA,NA and M1 genes through blasticindin combined with limited dilution methods.We used PCR assay to identify whether HA,NA and M 1 genes were successfully integrated into genome,and used Western-Blot assay to identify whether there was VLPs in the supernatant.The HA activity was evaluated with hemagglutination test,and NA activity was reflected with the ability to hydrolyze sialic acid.Results The HA,NA and M1 genes were successfully amplified from the monoclonal S2 cell using PCR assay.The VLPs integrated HA,NA and M1 protein was identified with Western-Blot assay.The hemagglutination test confirmed influenza VLPs could agglutinate chicken red blood cells with titer of 1 ∶ 160.The NA could dose-dependent hydrolyze sialic acid,and no significance was observed at the high dose(250 ng,500 ng)between wild type virus and VLPs.Conclusion We successfully established the monoclonal S2 cell line which integrated influenza HA,NA and M 1 genes,and it could persistently produce influenza VLPs with perfect HA and NA activity.VLPs could be considered as effective influenza vaccine candidate in the future.

In this prospective study, we aimed to evaluate the efficacy and safety of tenofovir disoproxil fumarate (TDF) in Chinese chronic hepatitis B (CHB) patients after multiple nucleoside/nucleotide analog (NA) treatment failures.

Background & AimsAccurate assessment of liver fibrosis in patients with chronic hepatitis B (CHB) is necessary not only to predict the long-term clinical course but also to determine an appropriate antiviral therapy scheme. Several noninvasive approaches - serum markers and elastography - have been proposed as alternatives for the histopathological analysis of liver biopsies. The aim of this study was to evaluate two ultrasound elastography methods (ARFI and TE) and one biochemical test (APRI), as well as their optimal linear combination, in the assessment of liver fibrosis in CHB.MethodsNinety five patients with CHB and 16 volunteers underwent ARFI, TE and APRI; and liver fibrosis was staged in the patients by a liver biopsy. An optimal linear combination of the three methods was developed, and its diagnostic performance was evaluated by a 10-fold cross-validation.ResultsThe accuracy of the linear combination was 83.86% and 91.88% for significant fibrosis (F2) and cirrhosis (F4), respectively, higher than those obtained for ARFI (83.50%, 88.76%), TE (75.27%, 87.61%) and APRI (73.29% and 81.67%). The combination also increased the sensitivity and the negative predictive values for the diagnosis of significant fibrosis and cirrhosis.ConclusionsThe optimal linear combination algorithm is effective for noninvasive staging of liver fibrosis in CHB. However, linear combination has its own limitations; nonlinear methods may eventually reveal even clearer diagnostic results.

Objective To Screen for safe and effective vaccine candidates for EV71,provide a theoretical basis for development of EV71 vaccines in the future.Methods VP1 gene of enterovirus was used to design a target for development of EV71 vaccines.Different vaccine candidates,including inactivated EV71 vaccines,VP1 protein vaccine,DNA vaccines of different doses,were used to challenge female BALB/c mice by intramuscular injection at baseline (0),2 weeks,4 weeks,and caudal vein blood was collected at 0,2,4,6,8,10,and 16 weeks,and BALB/c mice were sacrificed and the spleen cells were collected for detection of both humoral immunity and cellular immunity to evaluate the efficacy and safety of the vaccine candidates.Results IgG antibody titers were increased at 2 weeks,remarkably increased at 4 weeks,reached a peak at 8 weeks,at least sustained for 16 weeks during the whole observation period,subtypes of IgG1 and IgG2a were the major component.The three vaccines could induce cellular immunity characterized by EV71 specific γ-IFN and IL-4 production.Our results indicated that inactivated EV71 vaccine was superior to the other vaccine candidates.Conclusions Inactivated EV71 vaccines,VP1 protein vaccine,DNA vaccines can induce both strong and sustainable humoral and cellular immunities in challenged mice,and the inactivated EV71 vaccine is superior to the other vaccine candidates,which needs to be proved their immunity by challenge assay in the future.

Objective To investigate significance of Methyl-prednisone(MP) and intravenous immunoglobulin(IVIG) treatment for patients with severe hand,foot and mouth disease(SHFMD).Methods 178 children of three groups with SHFMD were retrospectively studied from the Third People's Hospital of Shenzhen City from March 2010 to October 2012,including MP treatment group with 71 patients(group A),IVIG treatment group with 45 patients(group B) and control group with 62 patients(group C).The illness progress,fever maintenance time,rash maintenance time and changes of the blood,white blood cell(WBC),neutrophil(N),lymphocyte(L),monocyte(MONO) of three groups before and after treatment were compared.Results There were respectively three and four cases of limb muscle strength obstacles appeared in group A and C,and one case of neurogenic pulmonary edema appeared in group C,no case of limb muscle strength obstacles and neurogenic pulmonary edema appeared in group B.There were shorter fever time in group A and B [(3.31±1.64),(3.20±1.84) d] than group C [(4.35±1.89) d],and the difference was statistically significant(P < 0.05).There were shorter rash time in group B [(5.33±1.21) d] than group C [(6.18±1.73) d],and the difference was statistically significant(P < 0.05).There were much more WBC,N,L in group A [(9.06±2.30) ×109/L,(3.13± 1.24) ×109/L,(4.99±2.08) ×109/L] than group C [(7.20±2.04) ×109/L,(2.59±0.98) ×109/L,(3.82±1.78) ×109/L] after treatment,and the differences among them were significant(P < 0.05).However,there were no significant difference about MONO between group A and group C(P > 0.05),WBC,N,L,MONO between group B and group C had no significant difference(P > 0.05).Conclusion Early use of IVIG can improve clinical symptoms,shorten the duration of fever and rash for patients with SHFMD.It is more effective,more secure than MP.

OBJECTIVE:To express soluble HA of A/H1N1 influenza virus in drosophila S2 cell line and identify its bio-activity.METHODS:HA gene was amplified from A/Shenzhen/71/09 virus strain using RT-PCR, then we constructed pAC5.1-HA expression vector, which was co-transfected into S2 cell with pCoblast vector. After transfection, stable S2 cell was selected through Blasticindin. HA in the supernatant was identified with Western Blot assay and purified with Ni-column. Recombinant HA was immunized into BALB/c mice 3 times, and the Abs titers were evaluated with ELISA.RESULTS:We successfully cloned HA gene with 1.7 x 10(3) bp of A/Shenzhen/71/09 virus strain and got recombinant pAC5. 1-HA expression vector. Stable S2 cell line was established after transfection and selection, which continuously expressed HA with molecular weight 75 x 10(3) D. After immunization with HA, the Abs titers were 1:1280 and 1: 5120 respectively on 10 d, 30 d.CONCLUSION:We expressed soluble HA with good bio-activity, which contributed to research on immune diagnosis, subunit vaccine, and monoclonal Abs for influenza.

<正>患者男,48岁,超市管理员,曾居住于"牛羊屠宰场"附近共2年。1981-1986年曾患"右耳化脓性中耳炎",于1986年行切开引脓术后,同时出现右侧面瘫及右耳听力丧失。临床表现:主因"反复头晕、呕吐1年,视力、听力下降7个月,乏力1个

OBJECTIVEThis study aimed at evaluating the efficacy and safety of a combination treatment of entecavir and Peginterferon alpha-2a for HBeAg positive chronic hepatitis B patients with high serum hepatitis B viral loads.METHODS60 treatment-naive HBeAg-positive CHB patients with high serum hepatitis B viral loads were enrolled and randomly divided into three groups: group A received Peginterferon alpha-2a monotherapy for 48 weeks (n = 20); group B received entecavir monotherapy for more than 48 weeks (n = 20); group C received Peginterferona alpha-2a combined with entecavir for 12 weeks, then Peginterferon alpha-2a monotherapy for 36 weeks (n = 20). Virological response, ALT normalization, HBeAg and HBsAg seroclearance rate were analysed at the end of 4, 12 and 24 weeks after the treatment.RESULTSThe ratio of undetectable hepatitis B virus (HBV) DNA were 50% and 10%, 95% and 25% and 100% and 30% in group C and group A respectively, 50% and 20%, 95% and 75% and 100% and 90% in group C and group B respectively at the end of 4, 12 and 24 weeks of treatment. The differences were significant between group C and A (Z = -4.6, P < 0.001), group C and B (Z = -2.53, P = 0.0114). ALT normalization rate was significantly lower in group A than that of group C (Z = -2.63, P = 0.0086). HBeAg levels declined more in group C than the other two groups after 24 weeks of treatment.CONCLUSIONSFor HBeAg positive chronic hepatitis B patients with high serum hepatitis B viral loads, combination treament of Peginterferon alpha-2a with entecavir is more effective than Peginterferon alpha-2a monotherapy in virologic response and ALT normalization after 24 weeks of treatment.

Objective To express the Neuramidinase(NA) protein of A/H1N1 influenza virus in prokaryotic expression system E.coli and prepare the specific monoclonal antibodies against NA.Methods NA gene was amplified using RT-PCR from A/Shenzhen/71/09 viral strain,and recombination protein NA with 6×His tag was expressed in prokaryotic expression system E.coli,then the recombinant protein was purified with Nickel ion affinity column(Ni-column).BALB/C mice were immunized with purified NA protein,and hybridoma cells which secreted anti-NA monoclonal antibodies were screened after 3 times immunization.Finally the monoclonal antibody(mAb) was identified with western blot method.Results NA gene fragment was cloned successfully with about 1400bp and inserted into pET-21b(+) vector.Recombination protein NA was detected in the inclusion bodies after IPTG induction,whose molecular weight was about 50kDa.NA protein was purified with Ni-column after denaturation with Urea.Mice were immunized with purified NA protein,then,the spleen cells were fused with myeloma SP2/0 cells.A hybridoma cell line called 7C11 which secreted anti-NA mAb continuously was screened by detecting anti-NA mAb level in cell supernatant with ELISA.It was proved that mAb could bind with NA protein with Westernblot assay.Conclusion We expressed recombinant NA protein of A/H1N1 influenza virus successfully and got one hybridoma cell line which could secret anti-NA mAb.This result would benefit the diagnosis and vaccine research of A/H1N1 influenza in the future.

Abstract Objective Various immune cells in patients with CHB have been demonstrated to play critical roles in HBV infection. The goal of this study is to observe changes in Th17, Treg, Th1 and B lymphocytes from peripheral blood and to evaluate immune status of CHB patients undergoing antiviral treatment. Methods Total of 49 CHB patients, 19 asymptomatic carriers and 29 healthy donors were included in our present study. The frequencies of peripheral Th17 cells (CD3+CD4+IL-17+Tcells), Treg cells (CD3+CD4+CD25+CD127- T cells), Th1 cells (CD3+CD4+IFN-γ T cells) and B lymphocytes in chronic hepatitis B (CHB) were analyzed by flow cytometry. Results The frequency of Th17 cells increased after treatment for 6 months, but there was no statistically significant difference of IL-17 expression between baseline and 6 months after treatment. The frequencies of Treg cells, momory B cells and total CD19+ B cells decreased after antiviral treatment. The frequencies of Th1 cells and plasma cells increased after antiviral treatment. Conclusions This study highlights that the reestablishment of immune function during antiviral treatment in CHB patients, which caused by the antiviral drugs or the patients themselves. CHB patients may exhibit varied responses to these antiviral drugs. It is essential to supplement immune therapy during the antiviral treatment, but Th17 may play a limited role in inflammation during antiviral treatment, targeting Th17 therapy may not be useful for CHB treatment. More time and more experiments are critical to explain it.