Sparse logistic regression revealed the associations between HBV PreS quasispecies and hepatocellular carcinoma.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC). AIMS: The purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies. METHODS: A total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves. RESULTS: Entropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data. CONCLUSIONS: Using NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.

Analysis and Follow up of Endoscopy Results in 1099 Patients with Terminal Ileum Lesions.

Objective: We aim to analyze the diagnostic yield, diagnostic accuracy, and delayed diagnosis of patients with terminal ileum lesions, providing follow-up suggestions for suspected patients. Methods: We carried out an analysis of 1099 patients who had terminal ileum lesions in our hospital from 2009 to 2019. The endoscopy reports and histopathology reports of terminal ileal biopsies were recorded. Clinical diagnosis and management were reviewed to determine whether there was a need to correct after a follow-up endoscopy result. Results: A total of 1099 patients were found to have terminal ileum lesions, among which 959 in 1099 patients (87.26%) were diagnosed as benign, 17 in 1099 patients (1.55%) were diagnosed as malignant, and 123 in 1099 patients (11.19%) were diagnosed as suspected. The diagnostic accuracies of terminal ileal polyp, cyst, cancer, eosinophilic enteritis, parasite, lymphofollicular hyperplasia, and amyloidosis were 100%. The diagnosis was delayed in 9.93% of Crohn's disease (CD) and 12.5% of lymphoma. Among the definite cases, the diagnosis was corrected during the follow-up in 12.5% of the patients, while the clinical treatment was corrected during the follow-up in 17.86% of the patients. Among the suspected cases, the diagnosis and treatment was corrected in 61.11% of the patients during the follow-up. Conclusion: Coincident diagnosis of ileitis and ileum ulcer is low. Delayed diagnosis of Crohn's disease and lymphoma were observed in a certain proportion of patients with terminal ileum lesions. A follow-up endoscopy was strongly recommended for these suspected patients with terminal ileum lesions.

Integrated Network Pharmacology and Metabonomics to Reveal the Myocardial Protection Effect of Huang-Lian-Jie-Du-Tang on Myocardial Ischemia.

Myocardial ischemia (MI) is one of the most common cardiovascular diseases with high incidence and mortality. Huang-Lian-Jie-Du-Tang (HLJDT) is a classic traditional Chinese prescription to clear "heat" and "poison". In this study, we used a deliberate strategy integrating the methods of network pharmacology, pharmacodynamics, and metabonomics to investigate the molecular mechanism and potential targets of HLJDT in the treatment of MI. Firstly, by a network pharmacology approach, a global view of the potential compound-target-pathway network based on network pharmacology was constructed to provide a preliminary understanding of bioactive compounds and related targets of HLJDT for elucidating its molecular mechanisms in MI. Subsequently, in vivo efficacy of HLJDT was validated in a rat model. Meanwhile, the corresponding metabonomic profiles were used to explore differentially induced metabolic markers thus providing the metabolic mechanism of HLJDT in treating MI. The results demonstrated the myocardial protection effect of HLJDT on ischemia by a multicomponent-multitarget mode. This study highlights the reliability and effectiveness of a network pharmacology-based approach that identifies and validates the complex of natural compounds in HLJDT for illustrating the mechanism for the treatment of MI.

Deep sequencing analysis of quasispecies in the HBV pre-S region and its association with hepatocellular carcinoma.

BACKGROUND: The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown. METHODS: We used deep sequencing to examine the dynamics of HBV quasispecies and their relationship to HCC development. Thirty-two chronic hepatitis B (CHB) patients with HCC (HCC group) and 32 matched CHB patients without HCC (controls) were recruited. Fourteen patients from each group had serial sera available up to 9 years before the time of the present study. Deep sequencing of the HBV pre-S regions was performed. HBV quasispecies complexity, diversity, and intrapatient prevalence of pre-S deletions/mutations were analyzed. RESULTS: Compared with control patients, HCC patients had a significant greater quasispecies complexity (p = 0.04 at the nucleotide level), greater diversity (p = 0.004 and 0.009 at the nucleotide level and the amino acid level respectively), and a trend of greater complexity at the amino acid level (p = 0.065). HCC patients had a higher intrapatient prevalence of pre-S deletions and point mutations (at codons 4, 27, and 167) compared with the control patients (all p < 0.05). Longitudinal observation in the sera of 14 HCC patients showed that quasispecies complexity (p = 0.027 and 0.024 at the nucleotide level and the amino acid level respectively) and diversity (p = 0.035 and 0.031 at the nucleotide level and the amino acid level respectively) increased as the disease progressed to HCC. CONCLUSIONS: Increased HBV quasispecies complexity and diversity in the pre-S region, probably reflecting enhanced virus-host interplay, was associated with disease progression from CHB to HCC.

Efficacy of Fluidized Bed Bioartificial Liver in Treating Fulminant Hepatic Failure in Pigs: A Metabolomics Study.

Bioartificial livers may act as a promising therapy for fulminant hepatic failure (FHF) with better accessibility and less injury compared to orthotopic liver transplantation. This study aims to evaluate the efficacy and safety of a fluidized bed bioartificial liver (FBBAL) and to explore its therapeutic mechanisms based on metabolomics. FHF was induced by D-galactosamine. Eighteen hours later, pigs were treated with an FBBAL containing encapsulated primary porcine hepatocytes (B group), with a sham FBBAL (containing cell-free capsules, S group) or with only intensive care (C group) for 6 h. Serum samples were assayed using ultra-performance liquid chromatography-mass spectrometry. The difference in survival time (51.6 ± 7.9 h vs. 49.3 ± 6.6 h) and serum metabolome was negligible between the S and C groups, whereas FBBAL treatment significantly prolonged survival time (70.4 ± 11.5h, P < 0.01) and perturbed the serum metabolome, resulting in a marked decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelinase, and fatty acids and an increase in conjugated bile acids. The FBBAL exhibits some liver functions and may exert its therapeutic effect by altering the serum metabolome of FHF pigs. Moreover, alginate-chitosan capsules have less influence on serum metabolites. Nevertheless, the alterations were not universally beneficial, revealing that much should be done to improve the FBBAL.

Hepatitis B virus full-length genomic mutations and quasispecies in hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatitis B virus (HBV) full-length genomic mutations and quasispecies characteristics in hepatocellular carcinoma (HCC) were investigated. METHODS: Hepatitis B virus DNA was extracted from the tumor and non-tumor tissues of 16 HCC patients. Overlapping DNA fragments covering the entire HBV genome were amplified and sequenced. To study HBV sequence at the quasispecies level, the preS region was amplified and clonally sequenced. HBV mutation profiles, quasispecies complexity and diversity, and phylogenetic characteristics were assessed. RESULTS: Fourteen patients had full-length HBV amplification. Hot-spot mutations at HBx aa130-131 and pre-S deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C regions were more frequently detected in the tumor than in the non-tumor tissues (P = 0.031). Compared with the non-tumor tissues, the tumor tissues had a lower quasispecies complexity (P = 0.014 and 0.043, at the nucleotide and amino acid levels, respectively) and diversity (P = 0.048 and 0.022, at the nucleotide and amino acid levels, respectively). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by the correlation coefficient testing on both the number and length of branches of viral populations (all P < 0.02). CONCLUSIONS: Hepatitis B virus mutation patterns in HCC tumor tissues and non-tumor tissues were different. HBV quasispecies within the preS region were compartmentalized, and tumor tissues had a lower genome complexity and diversity. Our study suggests HBV evolution is conditioned by the differential host cellular environment in HCC tumors.

Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Deletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development. METHODS: We studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1-3 years, 4-6 years, 7-9 years and ≥10 years prior to the recruitment of the study. RESULTS: Compared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005). CONCLUSIONS: High prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.

Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection.

BACKGROUND: The underlying mechanism of HBsAg-negative hepatitis B virus (HBV) infection is notoriously difficult to elucidate because of the extremely low DNA levels which define the condition. We used a highly efficient amplification method to overcome this obstacle and achieved our aim which was to identify specific mutations or sequence variations associated with this entity. METHODS: A total of 185 sera and 60 liver biopsies from HBsAg-negative, HBV DNA-positive subjects or known chronic hepatitis B (CHB) subjects with HBsAg seroclearance were amplified by rolling circle amplification followed by full-length HBV genome sequencing. Eleven HBsAg-positive CHB subjects were included as controls. The effects of pivotal mutations identified on regulatory regions on promoter activities were analyzed. RESULTS: 22 and 11 full-length HBV genomes were amplified from HBsAg-negative and control subjects respectively. HBV genotype C was the dominant strain. A higher mutation frequency was observed in HBsAg-negative subjects than controls, irrespective of genotype. The nucleotide diversity over the entire HBV genome was significantly higher in HBsAg-negative subjects compared with controls (p = 0.008) and compared with 49 reference sequences from CHB patients (p = 0.025). In addition, HBsAg-negative subjects had significantly higher amino acid substitutions in the four viral genes than controls (all p<0.001). Many mutations were uniquely found in HBsAg-negative subjects, including deletions in promoter regions (13.6%), abolishment of pre-S2/S start codon (18.2%), disruption of pre-S2/S mRNA splicing site (4.5%), nucleotide duplications (9.1%), and missense mutations in "α" determinant region, contributing to defects in HBsAg production. CONCLUSIONS: These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.

Effect of hepatitis B virus reverse transcriptase variations on entecavir treatment response.

BACKGROUND: Entecavir therapy often reduces hepatitis B virus (HBV) DNA to an undetectable level, but HBV DNA remain detectable in some patients. We investigated whether baseline HBV reverse transcriptase (rt) polymorphism and quasispecies complexity and diversity were associated with treatment response. METHODS: Pretreatment HBV DNA levels, HBV rt sequence, serology, and quasispecies complexity and diversity from 305 entecavir-treated patients were determined. These data were tested for their association with year 1 virological outcome, defined by optimal response (undetectable HBV DNA; lower limit of detection, ≤12 IU/mL) or partial response (detectable HBV DNA). RESULTS: Four rt variants were more frequently detected in the 64 partial responders than in the 241 optimal responders (all P < .05). Multivariate analysis revealed that high baseline HBV DNA level (P < .0001; odds ratio [OR], 2.32), HBV e antigen (HBeAg) positivity (P < .001; OR, 3.70), and rt124N (P = .002; OR, 3.06) were associated with a partial entecavir response. Compared with the optimal responders, the partial responders had a lower quasispecies complexity and diversity. CONCLUSIONS: Apart from the known factors (high baseline HBV DNA level and HBeAg positivity), a novel single nucleotide polymorphism (rt124N) and lower quasispecies complexity and diversity were associated with partial entecavir response at year 1.

Metabolomic analyses of faeces reveals malabsorption in cirrhotic patients.

BACKGROUND: The study of faeces offers a unique opportunity to observe cooperation between the microbiome and the metabolism of mammalian hosts, an essential element in the study of the human metabolome. In the present study, a global metabolomics approach was used to identify metabolites differentially excreted in the faeces of cirrhotic patients compared to controls. METHODS: Seventeen cirrhotic patients and 24 healthy individuals were recruited. Faecal metabolites were detected through non-targeted reversed-phase ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. RESULTS: A total of 9215 peaks were detected. Using unequal variance t-tests, 2393 peaks were observed with P≤0.05, approximately 74.0% of which were due to decreased faecal metabolite concentrations in liver cirrhosis vs. healthy controls. Integrating multivariate data analyses, we identified six major groups of metabolites. Relative levels of identified metabolites were as follows: strong increase in lysophosphatidylcholines, aromatic amino acids, fatty acids, and acylcarnitines, and a dramatic decrease in bile acids and bile pigments. CONCLUSION: With severe hepatic injury in patients with liver cirrhosis, malabsorption occurs along with disorders of fatty acid metabolism, potentially due to changes in gut microflora.

Bioartificial liver system based on choanoid fluidized bed bioreactor improve the survival time of fulminant hepatic failure pigs.

Bioartificial liver (BAL) support system has been proposed as potential treatment method for end-stage liver diseases. We described an improved BAL system based on a choanoid fluidized bed bioreactor containing alginate-chitosan encapsulated primary porcine hepatocytes. The feasibility, safety, and efficiency of this device were estimated using an allogeneic fulminant hepatic failure (FHF) model. FHF was induced with intravenous administration of D-galactosamine. Thirty FHF pigs were divided into three groups: (1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation, and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, and properties of the encapsulations and hepatocytes before and after perfusion were also evaluated. Compared to the two control groups, the BAL-treated group had prolonged the survival time and decreased the blood lactate levels, blood glucose, and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed. This new fluidized bed BAL system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.

Fluidized-bed bioartificial liver assist devices (BLADs) based on microencapsulated primary porcine hepatocytes have risk of porcine endogenous retroviruses transmission.

PURPOSE: Bioartificial liver assist devices (BLADs) are expected to bridge liver failure patients to liver transplantation, but porcine endogenous retroviruses (PERVs) still pose a potential risk in pig-to-human xenotransplantation and thereby limit the use of bioartificial liver therapy. In our lab, fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have been successfully used to treat liver failure pigs. We detected the risk of PERVs transmission of microencapsulated primary porcine hepatocytes-the key component of fluidized-bed BLADs, to evaluate the biosafety of this device for further clinical applications. METHODS: Microencapsulated primary porcine hepatocytes (cell diameter = 300 µm) were cultured in Dulbecco's modified Eagles medium (DMEM). Microencapsulated cell culture supernatants were collected at 6, 12, 24 and 72 h. HEK-293 were cocultured with these supernatants, and the cocultured cells were harvested every 7 days. RT-PCR was used to detect PERVs transmission. RT-qPCR was used to get the number of virus copies. PK-15 was used as the positive control whereas HepG2 was used as the negative control. RESULTS: PERV was detected in all supernatants, and the viral load of the supernatants increased with time. Moreover, cocultured 293 cells were positive for PERV-specific sequences. CONCLUSION: The kind of fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have risk of PERVs transmission. Further extensive pre-clinical study focused on biosafety is warranted.