Objective:A mouse model of pancreatitis induced by coxsackievirus B3 (CVB3) was established. The pathological change of pancreas and the infiltration of Th17/Treg cells were observed.Methods:The BALB/c mice were inoculated intraperitoneally with CVB3 to induce acute viral pancreatitis model. Then the pathological changes of pancreas were observed by HE staining; the viral RNA load and relative expression of cytokines (IFN-γ, IL-6 and IL-17) mRNA were detected by q-PCR; the proportion of infiltrated CD45 + CD3 + T cells, CD4 + and CD8 + T cells, Th17 and Treg cells in the pancreas was determined by flow cytometry. Results:Three days after CVB3 infection, the viral RNA load in pancreas was the highest (0.96±0.18) and gradually decreased with prolongation of infection. Compared with the 3 dpi group, the viral RNA load in pancreas was decreased (0.96±0.18 vs. 0.62±0.14) at 7 dpi, but there was no statistically significant difference. In addition, the infiltration of immune cell in pancreas increased significantly after 7dpi and the pathological score >2. The percent of infiltrated Th17 cells (1.05±0.21 vs. 22.13±5.79) and Treg cells (3.11±0.78 vs. 8.25±1.30) among CD4 + T cells significantly increased after infection (P<0.05), and the Th17/Treg also increased (P<0.01). Compared with the control group, the relative mRNA expression of IFN-γ (1.05±0.23 vs. 672.6±47.67), IL-6 (1.00±0.38 vs. 68.28±4.57), and IL-17 (1.01±0.11 vs. 54.15±7.94) in pancreas increased at 7 days after CVB3 infection ( P<0.01). Conclusions:The infiltration of Th17/Treg cells and the expression of related cytokines related cytokines IL-6 and IL-17 mRNA were upregulated in pancreas, which promoted the process of CVB3-induced pancreatitis.

Objective:To establish a real-time fluorescent quantitative PCR for the detection of torque teno virus types 7 (TTV7), 8 (TTV8) and 10 (TTV10) and analyze its performance in clinical sample detection.Methods:Specific primers were designed based on the gene sequences of TTV7, TTV8 and TTV10 in GenBank. Recombinant plasmids of pMD19-T-TTV7, pMD19-T-TTV8 and pMD19-T-TTV10 were constructed and used as positive standard control to establish a real-time fluorescent quantitative PCR based on FAM-Eclipse probe method. The specificity and sensitivity of the established method were evaluated. Moreover, it was validated in terms of clinical sample detection.Results:The standard curve equations of the real-time fluorescent quantitative PCR for detecting TTV7, TTV8 and TTV10 were y=-0.340 2 x+ 114.780 0 ( R2=0.998 8), y=-0.351 1 x+ 114.940 0 ( R2=0.995 3) and y=-0.348 9 x+ 115.020 0 ( R2=0.991 7), respectively, and there was no cross-reaction with other viruses. The detection sensitivity of the established method for TTV7, TTV8 and TTV10 were 108 copies/μl, 84 copies/μl and 98 copies/μl, and the positive detection rates in clinical pediatric serum samples were 10.9%, 2.1% and 4.3%, respectively. Conclusions:The established real-time fluorescent quantitative PCR for detection of TTV7, TTV8 and TTV10 was featured by strong specificity and high sensitivity, which could be used for rapid TTV detection in clinical serum samples.

Objective:To construct the dual-luciferase reporter vector for identification of internal ribosome entry site (IRES).Methods:The hairpin structure was inserted between Renilla luciferase (R-Luc) and Firefly luciferase (F-Luc) genes based on psiCHECK-2 to form plasmid psiCHECK-IRES. IRES of Encephalomyocarditis virus (EMCV) was inserted between the hairpin structure and F-Luc genes of psiCHECK-IRES to form vector psiCHECK-IRES-EMCV. After psiCHECK-IRES-EMCV or psiCHECK-IRES was transfected into BHK-21 cells respectively, expressions of F-Luc and R-Luc were detected by RT-qPCR. Then Luciferase activity of transfected cells was detected with the dual-luciferase reporter assay system at 24 h post-transfection.Results:The hairpin structure was successfully inserted into psiCHECK-2 to form psiCHECK-IRES by sequencing. RT-qPCR result showed that there were the approximate expressing levels of mRNA between F-Luc and R-Luc. The result indicated that no aberrant monocistronic transcripts, which caused false positive F-Luc readings, were produced. Then IRES of EMCV was introduced into psiCHECK-IRES to form psiCHECK-IRES-EMCV. The F-Luc/R-Luc ratio in psiCHECK-IRES-EMCV-transfected cells was 53.35 times that of psiCHECK-IRES-transfected cells. The result confirmed that IRES of EMCV initiated effectively the translation of F-Luc.Conclusions:Dual-luciferase reporter vector psiCHECK-IRES was successfully constructed, which could be used to validate viruses and eukaryotic genes, the translation thereof was IRES-dependent.

Objective:To investigate the influencing factors of significant liver fibrosis in patients with chronic hepatitis B (CHB) concurrent with non-alcoholic fatty liver disease (NAFLD).Methods:Those who underwent liver pathological examination and confirmed diagnosis of CHB and NAFLD in Tianjin Second People′s Hospital from August 2014 to September 2017 were enrolled. Data regarding their demographic information, laboratory tests results, and liver pathology results were analyzed. The latter results were used to categorize the patients either in non-significant liver fibrosis group (Metavir stage

Objective:To investigate the application value of reactive oxygen species (ROS) and adiponectin (ADPN) in the judgment of liver inflammation in chronic hepatitis B virus infection combined with nonalcoholic fatty liver disease (NAFLD).Methods:A total of 159 cases with NAFLD (21 cases), chronic hepatitis B virus infection (57 cases), and chronic hepatitis B virus infection combined with NAFLD (81 cases) were collected between June 2016 to December 2018, and the visited patients diagnosis were confirmed by histopathological examination of the liver. ROS and ADPN level retained in serum was determined by enzyme-linked immunosorbent assay. Histopathological examination of liver tissue was used as the gold standard to discuss the diagnostic value of the serum in patients with chronic hepatitis B virus infection combined with NAFLD for the occurrence of nonalcoholic steatohepatitis. One-way analysis of variance was used for the comparison among multiple groups, and LSD-t test was used for pairwise comparison between groups. Measurement data for non-normal distributions were expressed as M (P25, P75). Comparisons between groups were performed using the Mann-Whitney U or Kruskal-Wallis H test. Chi-square test was used to compare the count data between groups. Correlation analysis was performed using Spearman correlation analysis. Histopathological grouping of liver tissue was used as the gold standard, and the area under the receiver operating characteristic curve was used to evaluate the diagnostic efficacy of the regression formula.Results:(1) In patients with chronic hepatitis B virus infection combined with NAFLD, the levels of ROS in the non-hepatic steatosis group and the mild hepatic steatosis group were significantly lower than those in the moderate and severe hepatic steatosis group, while the ADPN level in the non-hepatic steatosis group was significantly higher than liver steatosis group, P < 0.05. (2) The results of correlation analysis showed that ROS was significantly correlated with NAS score, change in the degree of fatty liver and lobular inflammation (all P < 0.05).There was a significant negative correlation between ADPN and the change in the degree of fatty liver ( P < 0.05). (3) Logistic regression analysis results showed that the diagnostic formula for chronic hepatitis B virus infection combined with nonalcoholic steatohepatitis was 0.02 × controlled attenuation index + 0.584 × white blood cells/10 9 + 0.587 × ROS-10.982. The area under receiver operating characteristic curve of the subject was = 0.896. The sensitivity, specificity, positive and negative predictive value were 97.1%, 71.2%, 64.2%, and 97.9%. Conclusion:ADPN and ROS have certain reference value in differentiating the change in the degree of fatty liver and inflammation in chronic hepatitis B virus infection combined with NAFLD and the diagnostic formula has higher application value in the diagnosis and exclusion of chronic hepatitis B virus infection combined with nonalcoholic steatohepatitis.

Objective To investigate the expression level of fatty acid synthase (FASN) in different grades ofgliomas and its relationship with glioma malignancy.Methods Glioma U87 and U373 cell lines were routinely cultured in vitro;immunofluorescence assay was performed to detect the cellular localization of FASN in glioma U87 and U373 cell lines.Sixty-seven human glioma samples,collected in our hospital from March 2012 to March 2013,were used in our study;grade Ⅰ was noted in three samples by WHO grading,grade Ⅱ in 29,graded Ⅲ in 22,and grade Ⅳ in 13;immunohistochemistry was performed to detect the expression levels of FASN,CD34 and Ki-67 and microvascular density (MVD) in these human glioma samples.Spearman's correlation was used to analyze the relationship between FASN expression and both Ki-67 expression and MVD in human gliomas.Results (1) FASN could express in the cytoplasm of glioma U87 and U373 cell lines.(2) immumohistochemical staining indicated that FASN mainly located in the cytoplasm,Ki-67 in the cell nucleus,and CD34 in the cytomembrane.FASN expression in samples of grade Ⅰ (3.3683±0.6549) was significantly lower than that in samples of grade Ⅱ (4.0512±0.4859,P＜0.05),and FASN expression in samples of grade Ⅲ (4.1881±0.5755) was significantly lower than that in samples of grade Ⅳ (4.6996±0.5164,P＜0.05);MVD in samples of grade Ⅱ was significantly smaller than that in samples of grade Ⅲ(P＜0.05),and MVD in samples of grade Ⅲ was significantly lower than that in samples of grade Ⅳ(P＜0.05);Ki-67 expression in samples of grade Ⅱ was significantly lower than that in samples of grade Ⅲ (P＜0.05).(3) There was a positive and significant correlation between FASN expression and MVD (r=0.606,P=0.030);a positive and significant correlation between FASN expression and Ki-67 was also noted (r=0.636,P=0.014).Conclusion High expression level of FASN indicates high proliferation capability and high blood supply in haman gliomas.

Diagnosis, Differential ; Humans ; Liver Diseases, Alcoholic ; diagnosis ; Non-alcoholic Fatty Liver Disease ; diagnosis

OBJECTIVETo investigate the value of controlled attenuation parameter (CAP) in the diagnosis of fatty liver using FibroScan in patients with chronic liver disease (CLD).

METHODSA prospective cohort study was performed for the patients with chronic hepatitis B (CHB), chronic hepatitis C (CHC), and non-alcoholic fatty liver disease (NAFLD) who underwent liver pathological examination followed by CAP measurement within 1 week in The Second People's Hospital of Tianjin from February 2013 to May 2014. According to related guidelines, hepatocyte steatosis was classified as S0: <5%, S1: 5%-33%, S2: 34%-66%, or S3: ≥67%. The receiver operating characteristic (ROC) curves were plotted with positive results as the diagnostic criteria, and the optimal cut-off values were determined at the maximum Youden index. Single linear regression and multiple stepwise regression were applied to analyze the influencing factors for CAP.

RESULTSA total of 427 patients were enrolled, consisting of 19 patients (4.4%) with NAFLD, 383 (89.7%) with CHB, and 25 (5.9%) with CHC. The optimal cut-off values for CAP in the diagnosis of steatosis ≥5%, ≥34%, and ≥67% were 230 dB/m, 252 dB/m, and 283 dB/m, respectively, and the areas under the ROC curve were 0.803, 0.942, and 0.938, respectively (Z = 14.194, 28.385, and 16.486, respectively, all P < 0.01). CAP differentiated S0 from S1, S1 from S2, S0 from S2, S0 from S3, and S1 from S3 (Z = 10.109, 10.224, 47.81, 29.917, and 10.999, all P < 0.01), but was not able to differentiate S2 from S3 (Z = 0.656, P = 0.5116). The single linear regression and multiple stepwise regression analyses showed that only body mass index (BMI; B = 4.001, P < 0.01) and hepatic steatosis (B = 33.015, P = 0.000) were correlated with CAP. The coincidence rates between CAP and liver pathological diagnosis were 77.4%, 81.0%, and 96.2% for S0, S3, and ≥S2, respectively.

CONCLUSIONCAP has a good value in the diagnosis of fatty liver in CLD patients, and can well differentiate between all stages of fatty liver except S2 and S3. CAP is influenced by BMI, but is not found to be associated with liver fibrosis, inflammation, liver stiffness measurement, and etiology.

Area Under Curve ; Biopsy ; Body Mass Index ; Cell Differentiation ; Elasticity Imaging Techniques ; Hepatitis B, Chronic ; complications ; Hepatitis C, Chronic ; complications ; Humans ; Inflammation ; complications ; Linear Models ; Liver Cirrhosis ; complications ; Multivariate Analysis ; Non-alcoholic Fatty Liver Disease ; complications ; diagnosis ; Prospective Studies ; ROC Curve

BACKGROUND/AIMS: This study aimed to verify the reliability of the alcoholic liver disease (ALD)/nonalcoholic fatty liver disease (NAFLD) index (ANI) for distinguishing ALD in patients with hepatic steatosis from NAFLD, and to investigate whether ANI combined with γ-glutamyl transferase (GGT) would enhance the accuracy of diagnosis in China. METHODS: A hundred thirty-nine cases of fatty liver disease (FLD) were divided into two groups of ALD and NAFLD. The ANI was calculated with an online calculator. All indicators and ANI values were analyzed using statistical methods. RESULTS: ANI was significantly higher in patients with ALD than in those with NAFLD (7.11 ± 5.77 vs. -3.09 ± 3.89, p < 0.001). With a cut-off value of -0.22, the sensitivity, specificity, and area under the receiver operating characteristic curve (AUROC) of diagnosed ALD cases was 87.1%, 92.5%, and 0.934 (95% confidence interval [CI], 0.879 to 0.969), respectively. The corresponding values for aspartate aminotransferase (AST)/alanine transaminase (ALT), mean corpuscular volume (MCV), and GGT were 75.29%, 72.94%, and 0.826 (95% CI, 0.752 to 0.885); 94.34%, 83.02%, and 0.814 (95% CI, 0.739 to 0.875) and 80.23%, 79.25%, and 0.815 (95% CI, 0.740 to 0.876), respectively. ANI AUROC was significantly higher than the AST/ALT, MCV, or GGT AUROCs (all p < 0.001), moreover, ANI showed better diagnostic performance. The combination of ANI and GGT showed a better AUROC than ANI alone (0.976 vs. 0.934, p = 0.016). The difference in AUROCs between AST/ALT, MCV, and GGT was not statistically significant (all p > 0.05). CONCLUSIONS: ANI can help distinguish ALD from NAFLD with high accuracy; when ANI was combined with GGT, its effectiveness improved further.
Alcoholics* ; Aspartate Aminotransferases ; China ; Diagnosis ; Diagnosis, Differential ; Erythrocyte Indices ; Fatty Liver* ; gamma-Glutamyltransferase ; Humans ; Liver Diseases, Alcoholic* ; ROC Curve ; Sensitivity and Specificity ; Transferases*

Objective To investigate the differences and similarities between drug-induced liver injury (DILI) and autoimmune hepatitis (AIH) in serum biochemical parameters and liver pathology,and to provide some thoughts for clinical diagnosis and differentiation of these two diseases.Methods A retrospective analysis was performed for the biochemical,immunological,autoantibody,and liver pathological data of 106 DILI patients and 63 AIH patients who were hospitalized,diagnosed,and treated in our hospital from January 2012 to October 2014.The patients' general data,biochemical parameters,immunological data,Ishak score,and qualitative changes in liver tissue were analyzed and compared.The Kruskal-Wallis test was used for comparison of nonparametric data between multiple groups,the Nemenyi test was used for comparison of nonparametric data between any two groups,the Wilcoxon rank sum test was used for comparison of Ishak scores,and the chi-square test was used for comparison of constituent ratio of categorical data.Results There were significant differences between AIH group and DILI hepatocyte injury group/mixed-type DILI group in the following serum biochemical parameters:alanine aminotransferase (187.2 U/L vs 1 326.5 U/L and 455.6,P ＜ 0.05),aspartate aminotransferase (172.2 U/L vs 759.5 U/L and 349.5 U/L,P ＜0.05),alkaline phosphatase (209.3 U/L vs 157.3 U/L and 169.4 U/L,P ＜ 0.05),gamma-glutamyl transferase (254.8 U/L vs 176.5 U/L and 170.5 U/L,P ＜ 0.05),total bilirubin (37.2 μmol/L vs 95.8 μmol/L and 52.6 μmol/L,P ＜ 0.05),serum iron (18.9 μmol/L vs 36.2 μmol/L and 23.9 μmol/L,P ＜ 0.05),serum ferritin (122.5 μmol/L vs 410.4 μmol/L and 186.5 μmol/L,P ＜ 0.05),immunoglobulin G (18.4 g/L vs 12.6 g/L and 12.3 g/L,P ＜ 0.05),and immunoglobulin M (1.8 g/L vs 1.3 g/L and 1.1 g/L,P ＜ 0.05).There were also significant differences between AIH group and DILI hepatocyte injury group/mixed-type DILI group in the Ishak score for interface inflammation (2.2±0.8 vs 1.3±0.7 and 1.3±0.6,P ＜ 0.05),Ishak score for portal inflammation (2.3±0.9 vs 1.5±0.7 and 1.4±0.8,P ＜ 0.05),and fibrosis score (2.8±1.1 vs 1.5±0.7 and 1.3±0.7,P ＜ 0.05).There were significant differences between AIH group and DILI hepatocyte injury group/mixed-type DILI group in the proportion of wax-like deposition (0 vs 29.2％ and 34.5％,P ＜0.05) and proportion of iron deposition (11.1％vs 52.1％ and 25.9％,P ＜ 0.05).Conclusion There are differences in biochemistry,immunology,and liver histology between DILI and AIH patients.AIH patients have more serious interface inflammation and portal inflammation and a higher fibrosis degree compared with DILI patients,while DILI patients have greater proportions of wax-like deposition and iron deposition compared with AIH patients.