Metabolomic Analysis Uncovers Energy Supply Disturbance as an Underlying Mechanism of the Development of Alcohol-Associated Liver Cirrhosis.

Alcohol-associated liver disease (ALD) is caused by alcohol metabolism's effects on the liver. The underlying mechanisms from a metabolic view in the development of alcohol-associated liver cirrhosis (ALC) are still elusive. We performed an untargeted serum metabolomic analysis in 14 controls, 16 patients with ALD without cirrhosis (NC), 27 patients with compensated cirrhosis, and 79 patients with decompensated ALC. We identified two metabolic fingerprints associated with ALC development (38 metabolites) and those associated with hepatic decompensation (64 metabolites) in ALC. The cirrhosis-associated fingerprint (eigenmetabolite) showed a better capability to differentiate ALC from NC than the aspartate aminotransferase-to-platelet ratio index score. The eigenmetabolite associated with hepatic decompensation showed an increasing trend during the disease progression and was positively correlated with the Model for End-Stage Liver Disease score. These metabolic fingerprints belong to the metabolites in lipid metabolism, amino acid pathway, and intermediary metabolites in the tricarboxylic acid cycle. Conclusion: The metabolomic fingerprints suggest the disturbance of the metabolites associated with cellular energy supply as an underlying mechanism in the development and progression of alcoholic cirrhosis.

Metabolomic Signatures of Autoimmune Hepatitis in the Development of Cirrhosis.

Objectives: Autoimmune hepatitis (AIH) can progress into severe outcomes, i.e., decompensated cirrhosis, from remarkable and persistent inflammation in the liver. Considering the energy-expending nature of inflammation, we tried to define the metabolomics signatures of AIH to uncover the underlying mechanisms of cirrhosis development and its metabolic biomarkers. Methods: Untargeted metabolomics analysis was performed on sera samples from 79 AIH patients at the stages (phenotypes) of non-cirrhosis (n = 27), compensated cirrhosis (n = 22), and decompensated cirrhosis (n = 30). Pattern recognition was used to find unique metabolite fingerprints of cirrhosis with or without decompensation. Results: Out of the 294 annotated metabolites identified, 2 metabolic fingerprints were found associated with the development of cirrhosis (independent of the decompensated state, 42 metabolites) and the evolution of decompensated cirrhosis (out of 47 metabolites), respectively. The cirrhosis-associated fingerprints (eigenmetabolite) showed better capability to differentiate cirrhosis from non-cirrhosis patients than the aminotransferase-to-platelet ratio index. From the metabolic fingerprints, we found two pairs of metabolites (Mesobilirubinogen/6-Hydroxynicotinic acid and LysoPA(8:0/0:0)/7alpha-Hydroxycholesterol) calculated as ratio of intensities, which revealed robust abilities to identify cirrhosis or predict decompensated patients, respectively. These phenotype-related fingerprint metabolites featured fundamental energy supply disturbance along with the development of AIH cirrhosis and progression to decompensation, which was characterized as increased lipolysis, enhanced proteolysis, and increased glycolysis. Conclusions: Remodeling of metabolism to meet the liver inflammation-related energy supply is one of the key signatures of AIH in the development of cirrhosis and decompensation. Therefore, drug regulation metabolism has great potential in the treatment of AIH.

Optimization of hepatobiliary phase delay time of Gd-EOB-DTPA-enhanced magnetic resonance imaging for identification of hepatocellular carcinoma in patients with cirrhosis of different degrees of severity.

AIM: To optimize the hepatobiliary phase delay time (HBP-DT) of Gd-EOB-DTPA-enhanced magnetic resonance imaging (GED-MRI) for more efficient identification of hepatocellular carcinoma (HCC) occurring in different degrees of cirrhosis assessed by Child-Pugh (CP) score. METHODS: The liver parenchyma signal intensity (LPSI), the liver parenchyma (LP)/HCC signal ratios, and the visibility of HCC at HBP-DT of 5, 10, 15, 20, and 25 min (i.e., DT-5, DT-10, DT-15, DT-20, and DT-25 ) after injection of Gd-EOB-DTPA were collected and analyzed in 73 patients with cirrhosis of different degrees of severity (including 42 patients suffering from HCC) and 18 healthy adult controls. RESULTS: The LPSI increased with HBP-DT more significantly in the healthy group than in the cirrhosis group (F = 17.361, P < 0.001). The LP/HCC signal ratios had a significant difference (F = 12.453, P < 0.001) among various HBP-DT points, as well as between CP-A and CP-B/C subgroups (F = 9.761, P < 0.001). The constituent ratios of HCC foci identified as obvious hypointensity (+++), moderate hypointensity (++), and mild hypointensity or isointensity (+/-) kept stable from DT-10 to DT-25: 90.6%, 9.4%, and 0.0% in the CP-A subgroup; 50.0%, 50.0%, and 0.0% in the CP-B subgroup; and 0.0%, 0.0%, and 100.0% in the CP-C subgroup, respectively. CONCLUSION: The severity of liver cirrhosis has significant negative influence on the HCC visualization by GED-MRI. DT-10 is more efficient and practical than other HBP-DT points to identify most of HCC foci emerging in CP-A cirrhosis, as well as in CP-B cirrhosis; but an HBP-DT of 15 min or longer seems more appropriate than DT-10 for visualization of HCC in patients with CP-C cirrhosis.

CSH guidelines for the diagnosis and treatment of drug-induced liver injury.

Drug-induced liver injury (DILI) is an important clinical problem, which has received more attention in recent decades. It can be induced by small chemical molecules, biological agents, traditional Chinese medicines (TCM), natural medicines (NM), health products (HP), and dietary supplements (DS). Idiosyncratic DILI is far more common than intrinsic DILI clinically and can be classified into hepatocellular injury, cholestatic injury, hepatocellular-cholestatic mixed injury, and vascular injury based on the types of injured target cells. The CSH guidelines summarized the epidemiology, pathogenesis, pathology, and clinical manifestation and gives 16 evidence-based recommendations on diagnosis, differential diagnosis, treatment, and prevention of DILI.

Management of hepatitis C virus infection in hemodialysis patients.

The prevalence of hepatitis C virus (HCV) infection in patients on maintenance hemodialysis (MHD) is relatively higher than those without MHD. Chronic HCV infection detrimentally affects the life quality and expectancy, leads to renal transplant rejection, and increases the mortality of MHD patients. With the application of erythropoietin to improve uremic anemia and avoid blood transfusion, the new HCV infections during MHD in recent years are mainly caused by the lack of stringent universal precautions. Strict implementation of universal precautions for HCV transmission has led to markedly decreased HCV infections in many hemodialysis units, but physicians still should be alert for the anti-HCV negative HCV infection and occult HCV infection in MHD patients. Standard interferon alpha and pegylated interferon alpha monotherapies at a reduced dose are currently the main treatment strategies for MHD patients with active HCV replication, but how to increase the sustained virological response and decrease the side effects is the key problem. IFNα-free treatments with two or three direct-acting antivirals without ribavirin in MHD patients are waiting for future investigations.

[Meta-analysis of magnesium isoglycyrrhizinate combined with nucleoside analogues in patients with chronic hepatitis B].

OBJECTIVE: To evaluate the efficacy and safety of the magnesium isoglycyrrhizinate plus nucleoside analogues (MGL + NA) combination therapy in patients with chronic hepatitis B using a meta-analysis approach. METHODS: The Chinese Biochemical literature on Disc (CBMDisc) and the Chinese Scientific Journal database, CNKI, were searched for randomized controlled trials (RCTs) of MGL+NA in patients with chronic hepatitis B published between 1995 and 2013. Data related to treatment type (combination therapy vs. mono-therapy) and outcome (markers of efficacy and safety, including levels of hepatitis B virus (HBV) DNA, hepatitis B e antigen (HBeAg), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). Weighted mean differences (WMD) were calculated and the Peto method was used to determine the relative risk (RR), both with 95% confidence intervals (CIs). RESULTS: Meta-analysis of the six included RCTs of MGL + NA, representing a 704 patients with chronic hepatitis B, showed WMDs for ALT of -12.98 (95% CI: -18.24 to -7.71, P less than 0.01) and for AST of -9.49 (95% CI: -14.53 to -4.45, P = 0.0002) and RRs for HBeAg of 1.79 (95% CI: 1.17 to 2.76, P = 0.008) and for HBV DNA of 1.35 (95% CI: 1.05 to 1.74, P = 0.02). The therapeutic efficacy of MGL+NA combination therapy was better than that of NA mono-therapy (P less than 0.01). CONCLUSION: For patients with chronic hepatitis B, MGL combination therapy may enhance the antiviral efficacy of NA treatment and help to improve liver function during treatment.

Accurate on-line ν-support vector learning.

The ν-Support Vector Machine (ν-SVM) for classification proposed by Schölkopf et al. has the advantage of using a parameter ν on controlling the number of support vectors and margin errors. However, comparing to standard C-Support Vector Machine (C-SVM), its formulation is more complicated, up until now there are no effective methods on solving accurate on-line learning for it. In this paper, we propose a new effective accurate on-line algorithm which is designed based on a modified formulation of the original ν-SVM. The accurate on-line algorithm includes two special steps: the first one is relaxed adiabatic incremental adjustments; the second one is strict restoration adjustments. The experiments on several benchmark datasets demonstrate that using these two steps the accurate on-line algorithm can avoid the infeasible updating path as far as possible, and successfully converge to the optimal solution. It achieves the fast convergence especially on the Gaussian kernel and is faster than the batch algorithm.

Regularization path for ν-support vector classification.

The v-support vector classification (v-SVC) proposed by Schölkopf has the advantage of using a regularization parameter v for controlling the number of support vectors and margin errors. However, compared to C-SVC, its formulation is more complicated, and to date there are no effective methods for computing its regularization path. In this paper, we propose a new regularization path algorithm, which is designed on the basis of a modified formulation of v-SVC and traces the solution path with respect to the parameter v. Through theoretical analysis and confirmatory experiments, we show that our algorithm can avoid the infeasible updating path under several assumptions (i.e., Assumptions 1 and 2), and fit the entire solution path in a finite number of steps. When the regularization path of v-SVC is available, a novel approach proposed by Yang and Ong can be applied to obtain the global optimal solution of common validation functions for v-SVC, and the computation for the whole process is minimal. Numerical experiments show that it is more efficient than various kinds of grid search methods for selecting the optimal regularization parameter v.

[Construction and expression of vectors carrying chemokine receptors CXCR4/ CCR5 in SKOV3 cells].

AIM: To construct the eukaryotic expression vector of human CXCR4 and CCR5, transfect SKOV3 cells with them. METHODS: The cDNA of CXCR4 and CCR5 was amplified by RT-PCR. After purification, the gene was cloned into a vector pEGFP. The sequence of inserted CXCR4 and CCR5 gene fragment was identified by enzyme digestion of EcoR I/Sal I and sequencing, and then the recombinant plasmid was transfected into SKOV3 cells which did not express CXCR4 and CCR5 by lipofectamine-mediated gene transfection method. The SKOV3 cells transfected with CXCR4 and CCR5 were examined by FCM. RESULTS: Vectors pEGFP-CXCR4 and pEGFP-CCR5 were obtained. Cell clones which were screened by G418 were obtained. The results of FCM indicatedthat transfected SKOV3 cells expressed CXCR4 and CCR5. CONCLUSION: SKOV3 cells that can express CXCR4 and CCR5 protein stably have been established successfully, which facilitates the researches of epithelial ovarian.

GnRH antagonist cetrorelix inhibits mitochondria-dependent apoptosis triggered by chemotherapy in granulosa cells of rats.

OBJECTIVE: Exposure to chemotherapy causes various adverse effects on the ovaries including premature ovarian failure and infertility. GnRH antagonist cetrorelix could reverse the ovarian damage during chemotherapy, but the mechanism remains unclear. The objectives of this study were to examine the role of the cetrorelix for prevention of mitochondria-dependent apoptosis in granulosa cells of rats during the treatment with cyclophosphamide(Cy), if the mitochondria-dependent apoptotic process was involved. METHODS: Female SD rats were injected with cetrorelix before and after administration of saline, or Cy. Main outcome measures were the apoptotic indexes, serum hormones, ultrastructure of granulosa cells, mitochondrial membrane potential, the kinetics of cytochrome c (Cyt-c) processing in cells, and apoptotic markers. RESULTS: The ovarian apoptotic indexes as shown by TUNEL assay were reduced by cetrorelix pretreatment and the rats regained normal hormonal profile. The ultrastructure and JC-1 fluorescence intensity assessments showed cetrorelix pretreatment inhibited mitochondrial dysfunction in granulosa cells induced by chemotherapy. Western blot analysis showed that cetrorelix suppressed the release of Cyt-c from mitochondria to cytoplasm. Meanwhile, cetrorelix pretreatment expressed less Bax, caspase-3 and Cyt-c in granulosa cells compared with chemotherapy alone. CONCLUSION: Cetrorelix could reduce the apoptosis in granulosa cells through inhibiting mitochondria-dependent apoptosis triggered by chemotherapy.

[Construction of RNA recombinant vector expressing STAT3 short hairpin and its effects on proliferation and apoptosis of SiHa cells].

AIM: To investigate the effects of eukaryotic vector expressing short hairpin RNA (shRNA) of signal transducers and activators of transcription 3 (STAT3) on the proliferation and apoptosis of SiHa cells. METHODS: shRNA templates were designed based on STAT3 gene sequence and cloned into pSilencer2.1-U6-neo vector. The resultant plasmid was transfected into SiHa cells with Lipofectamine 2000. The STAT3 protein and mRNA were detected by Western blot and RT-PCR, respectively. The cellular growth activity was assayed by MTT and the apoptosis was tested by flow cytometry. RESULTS: The plasmid pSilencer2.1-U6-neo-STAT3 was successfully constructed and transfected into SiHa cells. The expression of STAT3 in SiHa cells decreased, the cellular growth activity decreased, and the cell apoptosis increased. CONCLUSION: The siRNA expressing plasmid pSilencer2.1-U6-neo-STAT3 can inhibit cellular proliferation and induce the apoptosis of cervical cancer cells by suppressing the expression of STAT3.

[Characteristics of HBcAg(18-27) CTL epitopes of the main epidemic HBV strains in China].

OBJECTIVE: To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China. METHOD: 30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites. RESULTS: We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24). CONCLUSION: The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.

Exvivo experiments of human ovarian cancer ascites-derived exosomes presented by dendritic cells derived from umbilical cord blood for immunotherapy treatment.

OBJECTIVES: Exosomes, a type of membrane vesicles, released from tumor cells have been shown to be capable of transferring tumor antigens to dendritic cells and activating specific cytotoxic T-lymphocytes. Recent work has demonstrated the presence of high numbers of exosomes in malignant effusions. Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and from which a significant number of dendritic cells can be produced. We hypothesized that the exosomes released from metastatic ovarian carcinoma were able to present tumor specific antigen to dendritic cells derived from unrelated umbilical cord blood, then could stimulate resting T cells to differentiate and induce effective cytotoxicity. STUDY DESIGN: Exosomes were isolated by ultracentrifugation of malignant ascites from ovarian cancer patients (n = 10). Purified exosomes were further characterized by Western blot analyses and immunoelectronic microscopy. Dendritic cells were collected from unrelated umbilical cord blood and cultured in the presence of GM-CSF, IL-4 and TNF-α. Resting T cells were mixed with dentritic cells previously primed with exosomes and the cytotoxicity were measured by MTT method. T cells were activated by DCs presented with exosomes. RESULTS: 1) the exosomes isolated from the ascites were membrane vesicles of about 30-90nm in diameter; 2) the exosomes expressed MHC class I molecules, HSP70, HSP90, Her2/Neu, and Mart1; and 3)umbilical cord blood-derived DCs previously exosome-primed stimulated resting T cells to differentiate and produce effective cytotoxicity. CONCLUSIONS: These results suggested that tumor-specific antigens present on exosomes can be presented by DCs derived from unrelated umbilical cord blood to induce tumor specific cytotoxicity and this may represent as a novel immunotherapy for ovarian cancer.

[Expression of TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcriptase gene core promoter in ovarian cancer cell line SKOV3].

AIM: To construct the TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcription gene core promoter and to evaluate the expression of the TRAIL gene in ovarian cancer cell line SKOV3 in vitro. METHODS: The amplified TRAIL gene fragment was subsequently cloned into hTERT promoter-pIRES2-EGFP vector and CMVpromoter-pIRES2-EGFP vector. The hTERT promoter-pIRES2-EGFP-TRAIL and CMVpromoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were obtained, respectively. The plasmids were transfected into human ovarian carcinoma SKOV3 cell line by lipofeclin mediation and the positive clones were screened by G418. The expression of TRAIL gene was examined by RT-PCR, Western blot, immunocytochemistry and flow cytometry. RESULTS: All the constructed vectors were verified by enzyme digestion. The TRAIL gene of transfected cells was increased significantly (P<0.01). CONCLUSION: An eukaryotic expression vector of the TRAIL gene modulated by the human telomerase reverse transcription gene core promoter has been constructed successfully and its steady expression in human ovarian cancer cell line SKOV3, which will be beneficial to further research into its role in regulating the biological behavior of ovarian cancer cells.

[Construction of TRAIL gene eukaryotic expression vector modulated by hTERT gene core promoter and its effect on apoptosis of ovarian cancer cells].

AIM: To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells. METHODS: Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing. The hTERTpromoter-pIRES2-EGFP-TRAIL and CMV promoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were constructed respectively. The recombinant plasmids were transfected into ovarian carcinoma cell line, SKOV3, and the levels of mRNA were determined by RT-PCR. The cell cycle and apoptosis rate of SKOV3 cells were determined by FCM. RESULTS: The constructed two recombinant vectors were verified by restriction enzyme digestion analysis and DNA sequencing. After being transfected with two recombinant vectors, the growth of SKOV3 cells was strongly inhibited and apoptotic features appeared. CONCLUSION: The recombinant eukaryotic expression vector has been constructed successfully. TRAIL gene driven by hTERTpromoter can be obviously expressed in ovarian carcinoma SKOV3 cells, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

[Construction of tumor cell-specific expression vector modulated by human telomerase reserse transcription gene core promoter].

AIM: To construct the tumor cell-specific expression vector modulated by human telomerase reserse transcription gene (hTERT) core promoter. METHODS: The hTERT gene core promoter fragment was amplified by PCR using the total genomic DNA from SKOV3 cells as template. The amplified gene fragment was subsequently cloned into pIRES2-EGFP vector. Then the expression vector modulated by hTERT core gene promoter was transfected into tumor cell lines, HO-8910, Hela and normal ECV304 cells through lipofectamine, respectively. The activity of reporter gene GFP was detected 48 h after transfection. RESULTS: There was significant transcriptional activity in 3 kinds of transfected tumor cells, while no transcriptional activity was detected in transfected normal cells. CONCLUSION: The hTERT core gene promoter has the tumor specificity, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

[Analysis of T cell receptor BV dominant usage and CDR3 sequences during acute exacerbation in patients with chronic hepatitis B].

OBJECTIVES: To understand the role cellular immunology plays in the pathogenesis of chronic hepatitis B (CHB) through analysis of T cell receptor (TCR) beta chain variable region gene (BV) family dominant usage and beta chain complementarity determining region3 (CDR3) sequences of peripheral blood mononuclear cells of the patients. METHODS: TCR BV families were amplified by inverse polymerase chain reaction (RT-PCR), and the dominant usage of BV families and CDR3 repertoire were analyzed by immunoscope technology for 8 CHB patients during their acute exacerbations and for 4 healthy blood donors who served as controls. The clonality of the T cells suspected by immunoscope was further confirmed by CDR3 sequencing. RESULTS: The TCR BV CDR3 repertoire of the 4 healthy blood donors showed a Gaussian distribution. In the 8 CHB patients, however, the clonal expansion of T cells showed different TCR BV families with each patient. The T cells of the clonal expansion shared different CDR3 sequences. CONCLUSION: The peripheral blood T cells of CHB patients during their acute exacerbation showed significantly a clonal expansion and their T cell clonal expansion may be stimulated by several HBV epitopes. These results indicate that cellular immunology is involved in the pathogenesis of the liver inflammation process of CHB.

[A comparison of clinical and virological characteristics of 1686 cases of HBeAg-negative and HBeAg-positive chronic hepatitis B].

OBJECTIVE: To investigate the clinical characteristics of HBeAg-negative and HBeAg-positive chronic hepatitis B (CHB). METHODS: A total of 1686 hospitalized CHB cases were analyzed retrospectively. The serum ALT values, HBV DNA levels and hepatic inflammation and fibrosis were analyzed by their serum HBeAg status. RESULTS: Among the 1686 cases, 628 (37.3%) were HBeAg-negative and 1058 (62.7%) were HBeAg-positive. Compared with HBeAg-positive group, HBeAg-negative group had a lower serum ALT and HBV DNA levels. However, hepatic necroinflammation grading and fibrosis staging in HBeAg-negative group were more advanced than that of HBeAg-positive group. Irrespective to serum HBeAg status, patients with serum HBV DNA less than 10(5)copies/ml, had a lower hepatic necroinflammation activity. CONCLUSIONS: HBeAg-positive CHB is still the predominant form of CHB in Chinese patients. Compared with patients with low HBV replication, patients with active HBV replication had a higher hepatic necroinflammation activity. The liver histological grading and staging in HBeAg-negative CHB patients were more advanced than that in HBeAg-positive patients.