Objective To compare the structural genome variations of lamivudine resistant HBV strain from a patient with acute exacerbation after lamivudine therapy with pretretment HBV strain. Methods Full length genomes of two HBV strains before lamivudine treatment and after occurring severe acute exacerbation during lamivudine treatment were cloned and sequenced in a patient with chronic hepatitis B. Nucleotide sequence and predicted amino acid sequences of 4 open reading frames in two HBV strains were analyzed. Results Compared with pretreatment full length HBV genome, there were 30 amino acid substitutions in four ORFs of lamivudine resistant HBV strain, including substitution of valine for methionine at residue 550(M550V)accompanied by substitution of methionine for leucine at residue 526(L526M). The numbers of amino acid substitutions in ORF P, X, S, Precore/Core were 10(1.19%), 4(2.60%), 8( 2.00%) and 8 (3.77%) respectively. The total variation rate along the whole HBV genome was 1.86%. A G→A change at nucleotide position 1898 resulted in a precore stop codon and led to lack of HBeAg expression; At the preS2 initiation codon ATG was changed to ATA which caused the loss of preS2 protein expression. Some amino acid changes occurred within the known CTL, B or T cells epitopes.Conclusions In a patient with severe acute exacerbation during lamivudine therapy, together with 29 additional amino acid substitutions were detected outside the YMDD motif in the lamivudine resistant HBV strain.

Objective To detect anti-HCV in serum of hepatic disease patients by performing the confirmatory test, and further to confirm HCV infection. Methods Two recombinant immunoblot assays (CWT and CHIRON RIBA HCV 3.0 Strip Immunoblot Assay) were used respectively to detect anti-HCV in 477 human serum samples, which comprised 350 HCV-infected patients' specimens, 7 none-A none-E hepatitis specimens, 30 HBV-infected patients' specimens, 30 hepatitis E virus infected patients'specimens, and 60 specimens drawn from blood donors. The latter three groups served as controls. Results A total of 120 control non-HCV-infected patients' specimens were negative when tested by both assays. Among 350 HCV-infected patients, 341 were positive and 9 were indeterminated by CWT assay; 343 were positive and 7 were indeterminated by CHIRON RIBA HCV 3. 0 SIA. Seven none-A none-E hepatitis specimens tested by both assays turned out to be 2 positive, 4 negative and 1 indeterminate. The consistency rate of these two assays was 99. 16% (Kappa=0.98). Conclusion CWT assay is highly coherent with CHIRON RIBA HCV 3.0 SIA assay in the methodology of anti-HCV antibody detection, which can be applied in the determination of HCV infection among none-A none-E hepatitis patients.

Objective To investigate the effects of adefovir resistance related mutations in hepatitis B virus (HBV) reverse transcription (RT) region on the viral replication and hepatitis B surface antigen (HBsAg) secretion. Methods Twelve adefovir treated chronic hepatitis B (CHB) patients who experienced a viral breakthrough were enrolled in this study. The RT region was amplified by polymerase chain reaction (PCR) using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed to find out mutation patterns. Full-length HBV genome was amplified from 4 representative serum samples followed by direct sequencing. The dominant strain was cloned into vector PHY106 to construct a recombinant plasmid containing the 1.1 unit of HBV genome Which was transfected into Huh7 cells. HBsAg and hepatitis B e antigen (HBeAg) expression were determined by enzyme-linked immunosorbent assay (ELISA), meanwhile intracellular HBV DNA level was determined by quantitative real-time PCR. Furthermore strain harboring rtA181T/sW172 · mutation and strain without rtA181 mutation were cotransfected into Huh7 cells. HBsAg and intracellular HBV DNA were also determined after transfection. Results Ten out the 12 patients enrolled in this study exhibited mutations conferring resistance to adefovir. The rtA181T mutation was detected in 5 cases, and the rtA181T/S+rtN236T mutation was observed in 4 cases. Different mutants showed variable HBsAg secretion competency in vitro. Despite the defect of HBsAg secretion of the rtA181T/sW172 · mutant, the replication efficiency was almost the same in different mutants. When the strains with and without rtA181 mutation were cotranfected into cells, the HBsAg level increased in accordance with the amount of stains without rtA181 mutation. However, the intracellular HBV DNA level was not changed significantly. Conclusions The rtA181 mutation is common in patients with adefovir resistance, of which the rtA181T mutation is the major pattern. In vitro analysis reveals that the rtA181T/sW172 · mutant is defective in HBsAg secretion which could be rescued by coexistence of wild-type strains. The replication efficiency in various mutants shows no obvious differences.

Objective To investigate the clinical significance of detection of hepatitis E virus (HEV) RNA in sera from patients with acute hepatitis E using real-time reverse transcription (RT)-PCR to detect hepatitis E virus RNA in sera from patients with acute hepatitis E.Methods A real-time RT-PCR assay, which can amplifies and detect the conserved region on ORF3, was used in this study. 434 outpatients and hospitalized patients with acute HEV infection was enrolled into this study.Simultaneously,the serum samples from 40 patients with HAV infection, 100 patients with HBV infection and 110 healthy blood donors were collected as the control The real-time RT-PCR was performed to detect HEV RNA in all these sera.Results 232 sera (53.5%) were positive for HEV RNA by real-time RT-PCR and all of the control were negative.The results of real-time RT-PCR and anti-HEV IgM (ELISA) were concordant in 67.1% samples.There was significant difference between the two methods ( Kappa = 0.308, P = 0.000 ).The first serum sample from five serum samples of the patients was positive for HEV RNA and negative for anti-HEV IgM.Follow-up studies showed all the five sera samples were positive for anti-HEV IgM.HEV RNA in serum could be detected between 2 and 10 days.Conclusions The real-time fluorescent RT-PCR method has high specificity, and can be applied to the qualitative detection of the serum with genotypes Ⅰ and Ⅳ of hepatitis E virus.Its clinical use can improve the early diagnosis of HEV.

s E in Beijing belong to HEV genotype Ⅳ.

significantly save the time of diagnosis and facilitate the clinical application of large samples.

OBJECTIVETo study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF.

METHODSThe ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver tissue. Liver function was assessed by measuring levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis. qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay.

RESULTSThe ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALF. Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the proinflammatory cytokines TNFa, IL-6 and IL-1 β, and reduced the extent of hepatocyte apoptosis.

CONCLUSIONERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.

Animals ; Apoptosis ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; Galactosamine ; adverse effects ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL

Objective Based on the theoretical connotation of"Dredge Du meridian remodeling god"intervention in PSD,two rounds of expert questionnaire were conducted in combination with Delphi survey method to optimize the diagnosis and treatment scheme of massage intervention in PSD,and to develop an optimal operation mode of Tuina for PSD.Methods Experts with rich experience in Tuina intervention in different provinces and cities in China were selected to participate in two rounds of questionnaires.Items were screened after statistical analysis of experts′ basic information,positive degree,coordination coefficient and authority coefficient of the two rounds of questionnaires.Results In the two rounds of Delphi survey,34 and 37 experts were selected for questionnaire survey,and the positive coefficients of experts were 94.44%and 94.47%,respectively.The average expert authority coefficients of the two rounds were 0.65 and 0.74.The expert coordination degree of the two rounds of questionnaires was medium,and Kendall coefficient W>0.4.Conclusion After two rounds of expert research in Delphi,the specific techniques,techniques and treatment frequency were screened and demarcated,and the optimized PSD diagnosis and treatment plan of"Dredge Du meridian remodeling god"Tuina intervention with high expert authority and good consensus was formed,which can be promoted and applied to PSD patients clinically.

Objective: To investigate the effects and underlying mechanisms of the macrophage migration inhibitory factor(MIF) inhibitor(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester(ISO-1) on sepsis-induced acute kidney injury(AKI). Methods: Human renal tubular epithelial HK-2 cells were divided into Con group(without any treatment), ISO-1 group(10 μg/ml ISO-1 treatment for 24 h) and LPS group(10 μg/ml LPS treatment for 24 h), LPS+ISO-1 group(10 μg/ml LPS treatment for 24 h followed by 10 μg/ml ISO-1 treatment for 24 h). ELISA was used to measure the levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) in the cell supernatants. Reactive oxygen species(ROS) levels were assessed using the 6-carboxyl-2 ′,7′-dichlorodihydrofluorescein diacetate fluorescent indicator(DCFH-DA) method. Apoptosis levels were detected by TUNEL staining, and Western blot was employed to analyze the expression of proteins of Kelch like ECH associated protein 1(Keap1), NFE2 like bZIP transcription factor 2(Nrf2), heme oxygenase-1(HO-1), as well as apoptosis-related proteins Bcl-2, Bax, and cleaved Caspase-3(c-Caspase-3). A sepsis mouse model was established using the cecal ligation and puncture(CLP) method, and the mice were divided into four groups: sham-operated(Sham), ISO-1 control(ISO-1), CLP, and ISO-1 treatment(CLP+ISO-1). After the experiment, mouse kidney tissues were collected for HE staining to observe pathological changes. Blood urea nitrogen(BUN), serum creatinine(Scr), myeloperoxidase(MPO) levels in kidney tissues, glutathione(GSH) and superoxide dismutase(SOD) activities were measured. Western blot was also used to detect the expression of MIF and proteins in the Nrf2/Keap1 signaling pathway and apoptosis-related proteins in kidney tissues. Results: Compared to the Con group, the LPS and LPS+ISO-1 groups showed significantly increased levels of TNF-α, IL-1β, IL-6, TUNEL-positive rates, ROS levels, and protein expressions of Keap1, Bax, and c-Caspase-3 in HK-2 cells(P<0.05), while the expressions of Nrf2, HO-1, and Bcl-2 significantly decreased(P<0.05). The ISO-1 group showed no significant changes(P>0.05). Compared to the LPS group, the LPS+ISO-1 group exhibited significantly decreased levels of TNF-α, IL-1β, IL-6, TUNEL-positive rates, ROS levels, and protein expressions of Keap1, Bax, and c-Caspase-3, while the expressions of Nrf2, HO-1, and Bcl-2 significantly increased(P<0.05). In the mouse experiments, compared to the Sham group, the CLP and CLP+ISO-1 groups showed severe kidney tissue damage, increased levels of serum BUN, Scr, and kidney MIF, Keap1, Bax, and c-Caspase-3 protein expressions(P<0.05), while GSH, SOD activities, and protein expressions of Nrf2, HO-1, and Bcl-2 significantly decreased(P<0.05). The ISO-1 group showed no significant changes(P>0.05). Compared to the CLP group, the CLP+ISO-1 group showed significant improvements in the aforementioned indicators(P<0.05). Conclusion The specific MIF inhibitor ISO-1 can ameliorate sepsis-induced AKI by inhibiting oxidative stress, inflammatory response, and apoptosis bothin vitroandin vivo. The mechanism may be through Nrf2/Keap1 signaling pathway.

Objective: To recognize the efficacy and safety of paritaprevir/ritonavir-ombitasvir combined with dasabuvir (OBV/PTV/RTV+DSV) in the treatment of genotype 1b chronic hepatitis C. Methods: Patients with genotype 1b chronic hepatitis C who were admitted to the People's Hospital of Henan Province, Huashan Hospital of Shanghai and the Fifth Medical Center of the General Hospital of the People's Liberation Army of China between November 2017 to August 2018 were enlisted. All patients received OBV/PTV/RTV+DSV antiviral therapy. HCV RNA levels were measured at baseline, weeks 1, 2, 3, 4, 8, 12, and 24, then 12 weeks, and 24 weeks after completion of treatment; patients’ comorbidity, concomitant medications, and clinical adverse events were recorded. Results: 108 patients were enrolled in the study, with an average age of 49.1 years, 44 patients were male (40.8%), 96.3% (104/108) were newly diagnosed, and four patients had previous treatment history, of whom three were treated with IFN and one with IFN + DAA. Ninety-eight cases completed 12 weeks treatment and 89 cases were in follow up for 12 weeks, after discontinuation of the drug. Overall, 89 cases (100%) achieved SVR12.One patient treated with PR and DAA had HCV RNA level of 869175 IU/mL at 4 weeks of treatment, which was significantly higher than the baseline HCV RNA level (301776IU/ML), and was judged as failure of treatment; and follow-up was discontinued. Of all enrolled patients, 19 (17.6%) had underlying diseases and 15 (13.9%) had combined medications. During treatment, adverse events (AE) occurred in 11 patients (10.1%). The main adverse events were pruritus and elevated bilirubin. Conclusion Combined antiviral therapy (OBV/PTV/RTV+DSV) of 12 weeks are highly effective with good safety profile in the treatment of Chinese patients with genotype 1b chronic hepatitis C.