Association of human leukocyte antigen (HLA) footprints with the comorbidity of latent autoimmune diabetes in adults (LADA) and hepatitis C virus (HCV) infection: A multicenter cross-sectional study.

AIMS: This study aims to investigate the interplay between hepatitis C virus (HCV) infection and major forms of diabetes: type 1 diabetes (T1D), type 2 diabetes (T2D), and latent autoimmune diabetes in adults (LADA). METHODS: This multicenter study analyzed a cohort of 2699 diabetic and 7344 non-diabetic subjects who visited medical centers in China from 2014 to 2021. T1D, T2D, LADA, and HCV were diagnosed using standard procedures. High-throughput sequencing was conducted to identify genetic footprints of human leukocyte antigen (HLA) alleles and haplotypes at the DRB1, DQA1, and DQB1 loci. RESULTS: HCV infection was detected in 3 % (23/766) of LADA patients, followed by 1.5 % (15/977) of T2D patients, 1.4 % (13/926) of T1D patients, and 0.5 % (38/7344) of non-diabetic individuals. HCV prevalence was significantly higher in people with diabetes than in non-diabetic individuals (p < 0.01). HLA alleles (DQB1*060101, DQB1*040101) and haplotypes (DRB1*080302-DQA1*010301-DQB1*060101) in LADA patients with HCV revealed higher frequencies than in LADA patients without HCV (adjusted p < 0.03). Furthermore, a higher risk of diabetes complications was found among LADA patients with HCV infection (p < 0.001). CONCLUSIONS: LADA patients are susceptible to HCV infection, potentially associated with certain HLA alleles/haplotypes. Early diagnosis and treatment of HCV infection among people with diabetes are important for the management of severe complications.

miR-3 Encoded by Hepatitis B Virus Downregulates PTEN Protein Expression and Promotes Cell Proliferation.

PURPOSE: Chronic hepatitis B virus (HBV) infection is a key determinant of hepatocellular carcinoma (HCC). However, the mechanism by which HBV contributes to the development of HCC remains to be further explored. HBV-encoded miR-3 (HBV-miR-3) is a newly discovered microRNA that can affect the replication of HBV, but its influence on host genes is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is expressed at low levels in most cancer cells. How HBV-miR-3 acts on PTEN to induce tumorigenesis has not been clarified. MATERIALS AND METHODS: PTEN protein expression was evaluated in HBV-miR-3-transfected cells and HBV-related liver cancer and paracancerous tissues. A luciferase reporter assay was employed to identify the HBV-miR-3 binding site on the 3'-untranslated region (3'-UTR) of PTEN. Cell apoptosis was assessed by flow cytometry. Cell proliferation was evaluated by colony formation assays. Transwell assays were used to detect cancer cell invasion. RESULTS: HBV-miR-3 was identified only in HBV-replicating HCC cells and HBV-infected patients. HBV-miR-3 expression in liver cancer tissues was higher than that in paracancerous tissues, and the corresponding PTEN expression was significantly decreased. Wild-type HBV-miR-3 bound to the 3'-UTR of PTEN and downregulated its protein expression in a dose-dependent manner. Moreover, the inhibition of HBV-miR-3 rescued PTEN protein expression. Furthermore, HBV-miR-3 reduced liver cancer cell apoptosis, enhanced cell invasion, and promoted cell proliferation. CONCLUSION: HBV-miR-3 binds to the 3'-UTR of PTEN mRNA and downregulates PTEN protein expression, thereby reducing cell apoptosis and enhancing cell invasion and proliferation. These results indicate that HBV-miR-3 contributes to the development of HBV-related HCC and may be a therapeutic target for this cancer.

SARS-CoV-2 clearance in COVID-19 patients with Novaferon treatment: A randomized, open-label, parallel-group trial.

BACKGROUND: The antiviral effects of Novaferon, a potent antiviral protein drug, on COVID-19 was evaluated in the laboratory, and in a randomized, open-label, parallel-group trial. METHODS: In the laboratory, Novaferon's inhibition of viral replication in cells infected with SARS-CoV-2, and prevention of SARS-CoV-2 entry into healthy cells was determined. Antiviral effects of Novaferon in COVID-19 patients with treatment of Novaferon, Novaferon plus Lopinavir/Ritonavir, or Lopinavir/Ritonavir were evaluated. The primary endpoint was the SARS-CoV-2 clearance rates on day six of treatment, and the secondary endpoint was the time to SARS-CoV-2 clearance. RESULTS: Novaferon inhibited viral replication (EC50=1.02ng/ml), and prevented viral infection (EC50=0.10ng/ml). Results from the 89 enrolled COVID-19 patients showed that both Novaferon and Novaferon plus Lopinavir/Ritonavir groups had significantly higher viral clearance rates on day six than Lopinavir/Ritonavir group (50.0% vs. 24.1%, p=0.0400, and 60.0% vs. 24.1%, p=0.0053). The median time to viral clearance was six days, six days, and nine days for three groups, respectively, a 3-day reduction in both the Novaferon and Novaferon plus Lopinavir/Ritonavir groups compared with the Lopinavir/Ritonavir group. CONCLUSIONS: Novaferon exhibited anti-SARS-CoV-2 effects in vitro and in COVID-19 patients. These data justify further evaluation of Novaferon. TRIAL REGISTRATION NUMBER: Number ChiCTR2000029496 at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/).

Clinical characteristics of older and younger patients infected with SARS-CoV-2.

BACKGROUND: SARS-CoV-2 causes high mortality risk in older patients. This study aims to characterize the clinical features of older and younger SARS-CoV-2 infected patients. RESULTS: A total of 239 patients were divided into the younger group (<60 years; n=181) and the older group (≥60 years; n=58). In both groups, fever and cough were common symptoms. However, dyspnea was more frequent in older patients than younger patients (20.7% versus 9.9%, p=0.032). Compared with younger patients, older patients harbored more severe cases (37.9% versus 17.1%, p=0.001) and comorbidities (58.6% versus 21.0%, p<0.001) such as hypertension and diabetes. The baseline values of eosinophils and C-reactive protein were abnormal in older and younger groups. From baseline to day 14, significant decreases of three biomarkers (C-reactive protein, hemoglobin, albumin) and dramatic increases of three biomarkers (lymphocytes, platelets, blood urea nitrogen) were observed in older patients. CONCLUSION: Older and younger patients exhibited differences in dyspnea, comorbidities, and proportions of severe cases. Moreover, the disease progression of SARS-CoV-2 in older patients is observed with the dynamics of laboratory biomarkers, supporting their potential use in disease monitoring. METHODS: We retrieved clinical symptoms, laboratory findings, comorbidities, and hospitalization information of SARS-CoV-2 cases in Changsha.

Rapidly decreased HBV RNA predicts responses of pegylated interferons in HBeAg-positive patients: a longitudinal cohort study.

BACKGROUND: As an important anti-HBV drug, pegylated interferon α (PegIFNα) offers promising clinical efficacy, but biomarkers that accurately forecast treatment responses are yet to be elucidated. Here, we evaluated whether HBV RNA could act as an early monitor of pegylated interferon responses. METHODS: We analyzed a phase 3, multicenter, randomized cohort of 727 HBeAg-positive non-cirrhotic patients receiving a 48-week treatment of PegIFNα-2a or PegIFNα-2b and a 24-week treatment-free follow-up. Serum levels of HBV RNA, HBV DNA, HBeAg, and HBsAg were measured at weeks 0, 12, 24, 48, and 72. RESULTS: HBeAg seroconversion and HBsAg loss at week 72 were observed in 217 (29.8%) and 21 (2.9%) patients, respectively. During the 48-week treatment, HBV RNA decreased more rapidly than HBV DNA and HBsAg, but HBV RNA and HBeAg shared similar dynamics with positive correlations. Multivariate regression analyses consistently revealed the significance of HBV RNA at weeks 0, 12, 24, and 48 to monitor HBeAg seroconversion but not HBsAg loss. Although baseline HBV RNA only showed a modest AUC performance, HBV RNA with a significant increase of AUC at week 12 outperformed other HBV biomarkers to forecast HBeAg seroconversion (p value < 0.05). HBV RNA ≤ 1000 copies/mL was an optimized cutoff at week 12 that offered better prediction than other HBV biomarkers. This optimized cutoff plus patient age, HBV genotype B, and HBeAg offered a strong estimation of HBeAg seroconversion (accuracy 95.2%, true negative rate 99.8%). CONCLUSION: HBV RNA at week 12 is an effective monitor of HBeAg seroconversion in HBeAg-positive patients treated with pegylated interferons.

Is HBV viral load at admission associated with development of acute-on-chronic liver failure in patients with acute decompensation of chronic hepatitis B related cirrhosis?

BACKGROUND: Hepatitis B virus (HBV) reactivation is one of the most common precipitating events associated with acute decompensation (AD) or acute-on-chronic liver failure (ACLF) in chronic hepatitis B (CHB)-related cirrhotic patients. However, whether their serum HBV deoxyribonucleic acid (DNA) levels are associated with ACLF incidence and short-term mortality rate is still ambiguous. METHODS: The ACLF incidences, 28-day and 90-day liver transplantation (LT)-free mortality rates, previous nucleoside/nucleotide analogues (NUCs) treatments and serum HBV DNA levels at admission (ad-levels) of 111 hospitalized patients with AD of CHB-related cirrhosis were analyzed. RESULTS: 43 (38.7%) patients developed ACLF. The 28-day and 90-day LT-free mortality rates of the ACLF cases were 15.4 and 40.9%, respectively. Though NUCs inhibited HBV replication effectively, there were no differences in the ACLF incidence between antiviral treatment-naïve patients and NUCs treatment-experienced patients with or without interruptions (37.5, 41.7 and 45.5%, respectively, P>0.05). The serum HBV DNA ad-level was similar between the patients with and without ACLF development (logarithms: 4.50 ± 1.96 vs 4.32 ± 1.99; ≥2000 IU/ml: 67.4% vs 67.6%; both P>0.05), so was between the ACLF patients died or survived in 28 or 90 days (logarithms: 4.31 ± 1.91 vs 5.54 ± 2.53, 4.81 ± 1.76 vs 4.84 ± 2.40, respectively, both P>0.05). CONCLUSION: Serum HBV DNA ad-level and previous NUCs treatment are not associated with incidence of ACLF and short-term mortality rate in the hospitalized patients with AD of CHB-related cirrhosis.

Genotype distribution of hepatitis C virus in 952 cases from 2014 to 2016 in Hunan Province, China.

INTRODUCTION: Few large-scale investigations on genotype (GT) distribution of hepatitis C virus (HCV) in Hunan Province, China, are reported. MATERIAL AND METHODS: We recruited all of the 952 patients in the census register of Hunan Province who were first diagnosed with HCV infection in the Second Xiangya Hospital, Central South University in 2014-2016. HCV genotypes were surveyed. The genotype distribution pattern was compared with those of the neighboring regions in China. RESULTS: Among the 952 patients, genotype 1 (GT1) (69.9%) was the most common HCV genotype, followed by GT6 (19.0%), GT3 (8.4%), and GT2 (2.6%). GT4 and GT5 were not found. One case had mixed infection of GT3 and GT6. Predominance of GT1 HCV was more evident in the patients aged ≥ 40 years than in those aged < 40 years (79.5% vs. 47.9%, χ2 = 95.993, p < 0.001). HCV genotype distribution had gender difference (χ2 = 44.695, p < 0.001), with GT3 and GT6 more prevalent in males than in females (36.2% vs. 18.2%, χ2 = 39.088, p < 0.001) while GT1 more prevalent in females than in males (80.1% vs. 60.3%, χ2 = 44.276, p < 0.001). Though Hunan Province is located in central China, its HCV genotype priority was similar with the change trend in south and southwest China, while distinguished from those of other regions, in particular from the neighboring central province, Hubei Province. CONCLUSIONS: HCV GT1 was the most predominant HCV genotype in Hunan Province, and GT6 and GT3 accounted for a significant percentage, especially in young patients. The HCV distribution pattern was more similar to those of the regions in south China.

Analysis of Transmission Routes of Hepatitis C Virus Based on Virus Genotyping in 341 Cases with Different Suspected Initial Infection Time Points in Hunan Province, China.

BACKGROUND Few investigations have been reported on the changing trends in transmission routes of hepatitis C virus (HCV) and the corresponding HCV genotype (GT) distribution in Hunan province, China. MATERIAL AND METHODS HCV GTs, suspected viral transmission routes, and time of initial infections were investigated in 341 HCV-infected patients in 2016. RESULTS Genotype 1 (GT1) (72.1%) was the most prevalent HCV GT, followed by GT6 (17.6%), GT3 (7.6%), and GT2 (2.6%). GT4 and GT5 were not found. The predominant HCV transmission routes were blood-related routes (57.5%) and intravenous drug use (IDU) (15.0%); 52.2% of the patients got HCV infection before 1994, 25.6% from 1994 to 1998, and 22.2% after 1998; 93.5% of the infections via blood-related transmission routes were with HCV GT1, 61.5% via IDU or feculent sexual contact were with HCV GT6, and 50.0% via non-healthcare invasive procedures were with HCV GT6. HCV infections via IDU or feculent sexual behavior were more prevalent in young males, while infections via invasive cosmetic procedures occurred more in young females, and both had a shorter time interval from suspected infection to confirmed clinical diagnosis. Multinomial logistic regression confirmed the time points of the initial HCV infections and suspected viral transmission routes were correlated with HCV GT distribution. CONCLUSIONS HCV GT1 infections via blood-related transmission routes in Hunan province have continually decreased since 1994. However, younger patients infected with HCV, especially with HCV GT6 via IDU, feculent sexual behavior, and non-healthcare invasive procedures, have significantly increased.

Hepatitis B virus X protein-induced upregulation of CAT-1 stimulates proliferation and inhibits apoptosis in hepatocellular carcinoma cells.

The HBx protein of hepatitis B virus (HBV) is widely recognized to be a critical oncoprotein contributing to the development of HBV-related hepatocellular carcinoma (HCC). In addition, cationic amino acid transporter 1 (CAT-1) gene is a target of miR-122. In this study, we found that CAT-1 protein levels were higher in HBV-related HCC carcinomatous tissues than in para-cancerous tumor tissues, and that CAT-1 promoted HCC cell growth, proliferation, and metastasis. Moreover, HBx-induced decreases in Gld2 and miR-122 levels that contributed to the upregulation of CAT-1 in HCC. These results indicate that a Gld2/miR-122/CAT-1 pathway regulated by HBx likely participates in HBV-related hepatocellular carcinogenesis.

A preliminary investigation on single nucleotide polymorphism rs2287622 of bile salt export pump gene in patients with chronic hepatitis C virus infection in Hunan, China.

BACKGROUND: European researchers have underscored associations between single nucleotide polymorphism (SNP) rs2287622 of the hepatobiliary bile salt export pump (BSEP) gene and the risk of hepatitis C virus (HCV) infection. The distributions of SNP rs2287622 are racially specific. This study was aimed to preliminarily investigate the distribution of BSEP gene SNP rs2287622 in the Han patients with chronic HCV-infection (CHC) in Hunan, China. METHODS: BSEP gene SNP rs2287622 of 165 CHC patients, 99 patients with chronic hepatitis B virus infection (CHB) and 99 healthy individuals were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis and nucleotide sequencing. RESULTS: The overall frequencies of the C allele of BESP gene SNP rs2287622 in the CHC patients, CHB patients and healthy individuals were 74.2, 72.7 and 74.2%, respectively (P > 0.05). The overall odds ratios (ORs) aiming at predicting CHC risk by comparing the ratios of the frequency distribution of alleles or genotypes in the CHC group with those in the non-CHC group had no statistical significance (P > 0.05). However, the CHC ORs of CC vs TT, TC vs TT and CC + CT vs TT among the individuals aged over 40 years were 2.680, 3.122 and 2.824 respectively (P < 0.05), and the higher risk did not relate to gender, HCV genotypes and presence of HCV-related liver cirrhosis. CONCLUSIONS: Among the Han individuals aged over 40 years in Hunan, China, genotype CC or CT of BSEP gene SNP rs2287622 may correlate with higher risk of CHC in comparison with genotype TT. Further study with a larger cohort is essential.

Differences in IP­10, TLR4 and IRF5/3 between SVR and non­SVR HCV­1 patients treated with PEG­IFN and ribavirin.

The present study aimed to investigate alterations in Toll­like receptor 4 (TLR4), interferon regulatory factor 5 (IRF5) and interferon­Î³­inducible protein­10 (IP­10), and evaluate whether these factors may be associated with a sustained virological response (SVR) among patients with hepatitis C virus genotype­1 (HCV­1) who were treated with peginterferon plus ribavirin (PEG­IFN­RBV). A total of 31 Chinese patients infected with HCV­1 were enrolled in the present study and 25 patients obtained SVR. The expression levels of IP­10 declined significantly during PEG­IFN­RBV therapy at the 24 and 48 week time­points, compared with the baseline (P<0.005, 0.001 and 0.001, respectively). In addition, it was observed that IRF5 mRNA expression and the number of TLR4+ peripheral blood mononuclear cells exhibited similar correlations with IP­10 concentration (R2=0.0726, P=0.001, R2=0.1634, P<0.0001, respectively) in the SVR group patients; however, these correlations were not observed to be present in the non­SVR group patients. In conclusion, the results of the present study suggest that marked alterations in IP­10, TLR4 and IRF5 expression may serve as indicators for the development of SVR in patients with HCV­1 treated with PEG­IFN­RBV.

HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN.

Hepatitis B virus X protein (HBx) is involved in the initiation and progression of hepatocellular carcinoma (HCC). However, the mechanism is still needed to be elucidated. In this study, we explored the relationship between HBx and microRNA and their roles in hepato-carcinogenesis. Firstly, by global microarray-based microRNA profiling and qRT-PCR, we found miR-181a was strongly up-regulated in HepG2.2.15 cells (HBV positive) and pHBV1.3-expressing HepG2 cells, and HBx played a major role in it. Secondly, reduced PTEN protein expression in the presence of HBx was aslo mediated by miR-181a, and in the Luciferase reporter system, miR-181a inhibited the PTEN translation by binding the PTEN 3'-untranslated-region (UTR), and PTEN protein was decreased when epigenetic expression of miR-181a and rescued by knocking down miR-181a. Finally, HBx interrupted the balance between apoptosis and proliferation, which contributed to the development of hepatocellular carcinoma, was also related to the interaction of miR-181a and PTEN. Taken together, we presented here a novel cross-talk between miR-181a and PTEN which was raised by HBx, and this shined a new line in HBV-related hepato-carcinogenesis.

Artesunate, an anti-malarial drug, has a potential to inhibit HCV replication.

Hepatitis C virus (HCV) infection is a major global health issue. Although the search for HCV treatments has resulted in great achievements, the current treatment methods have limitations, and new methods and drugs for hepatitis C treatment are still required. The aim of the present study was to investigate the effects of artesunate (ART) on HCV replication and compared these effects with those of ribavirin (RBV) and interferon-2b (IFN). The study was performed in HCV-infection cell models (JFH1-infected Huh7.5.1 and OR6 cell lines). Our results showed that the antimalarial drug ART inhibited HCV replicon replication in a dose- and time-dependent manner at a concentration that had no effect on the proliferation of exponentially growing host cells, and the inhibitory effect on HCV replication was stronger than RBV but weaker than IFN, as determined by qPCR, luciferase assays, and Western blot analysis. Furthermore, the combination of ART and IFN resulted in a greater inhibition of HCV replication. These findings demonstrated that ART had an inhibitive effect on HCV replication and may be a novel supplemental co-therapy with IFN and RBV for HCV and as an alternative strategy to combat resistance mechanisms that have emerged in the presence of DAA agents.

HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) co-infection is characterized by higher serum HCV RNA loads compared with HCV mono-infection. However, the relationship between HIV and HCV replication remains to be clarified. HIV Vpr has been shown to play an essential role in HIV replication. In this study, we aimed to explore the role of Vpr in HCV replication and pathogenesis. We therefore used the genotype 2a full-length HCV strain JFH1 infection system and the genotype 1b full-length HCV replicon OR6 cell line to analyse the effects of Vpr on HCV replication. We found that Vpr promoted HCV 5' UTR activity, HCV RNA replication and HCV protein expression in two HCV infection cell models. Additionally, lymphocyte-produced Vpr significantly induced HCV 5' UTR activity and HCV replication in hepatocytes. We also found that Vpr upregulated the expression of miR-122 by stimulating its promoter activity. Furthermore, an miR-122 inhibitor suppressed the Vpr-mediated enhancement of both HCV 5' UTR activity and HCV replication. In summary, our results revealed that the Vpr-upregulated expression of miR-122 is closely related to the stimulation of HCV 5' UTR activity and HCV replication by Vpr, providing new evidence for how HIV interacts with HCV during HIV/HCV co-infection.

Hepatitis C virus NS5A drives a PTEN-PI3K/Akt feedback loop to support cell survival.

BACKGROUND & AIMS: Decreased levels of phosphatase and tensin homologue (PTEN) are associated with hepatocellular carcinoma (HCC) pathogenesis and poor prognosis in hepatitis C virus (HCV)-infected HCC patients. The molecular processes governing the reduction in PTEN and outcome of PTEN dysfunction in hepatocytes are poorly understood. METHODS: The levels of proteins and mRNA were assessed by real time PCR and immunoblot. PTEN promoter activity was measured by reporter assay. Signalling pathways were perturbed using siRNAs or pharmacological inhibitors. RESULTS: Here, we report that HCV down-regulates PTEN expression at the transcriptional level by decreasing its promoter activity, mRNA transcription, and protein levels. We further identify NS5A protein as a key determinant of PTEN reduction among HCV proteins. NS5A-mediated down-regulation of PTEN occurs through a cooperation of reactive oxygen species (ROS)-dependent Nuclear Factor- kappa B (NF-κB) and ROS-independent phosphoinositol-3-kinase (PI3K) pathways. Moreover, NS5A protects cells against apoptosis. In addition, we found that down-regulation of PTEN relieves its inhibitory effect on PI3K-Akt pathway and triggers cumulative activation of Akt. This PTEN-PI3K/Akt feedback network mediates the suppression of cell apoptosis caused by NS5A. CONCLUSIONS: These data demonstrate that HCV NS5A down-regulates PTEN expression through a cooperation of ROS-dependent and -independent pathways that subsequently drives a PTEN-PI3K/Akt feedback loop to support cell survival. Our findings provide new insights suggesting that NS5A contributes to HCV-related hepatocarcinogenesis.

HBx down-regulated Gld2 plays a critical role in HBV-related dysregulation of miR-122.

miR-122 is a liver-rich-specific microRNA that plays an important role in hepatic gene expression via post-transcription regulation, and it is potentially associated with the development of hepatocellular carcinoma. It has been confirmed that miR-122 is down-regulated during HBV infection; however, how HBV affects miR-122 is still debated. One research provided evidence that HBx could reduce the miR-122 transcription level, but the other insisted that HBV had no significant effect on miR-122 transcription level but reduce miR-122 level via binding and sequestering endogenous miR-122. It is determinate that Gld2 could increase the specific miRNA stabilization by monoadenylation which was a post-transcription regulation. In this study, we aimed to investigate the mechanism of HBV-induced reduction of miR-122 and examine whether Gld2 is involved in it. According to the results of a microRNA microarray, we found miR-122 was the most down-regulated microRNA in HepG2.2.15 compared to HepG2. As revealed by qRT-PCR and western blotting analyses, both miR-122 and Gld2 levels were reduced in hepatic cell lines with expression of HBV or HBx but not other proteins of HBV, and over-expression of Gld2 could abolish the effect of HBV and HBx on the miR-122 level. What's more, both HBV and HBx have no significant effect on pre-miR-122 levels. And the dual-luciferase assay implicated that HBx could reduce the Gld2 promoter activity but had no significant effect on miR-122 promoter activity. In conclusion, HBx is a critical protein derived from HBV, which regulates miR-122 via down-regulating Gld2.

[Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy].

OBJECTIVE: To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA. RESULTS: The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05). CONCLUSION: Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.

[Demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on peripheral blood mononuclear cells in chronic hepatitis B patients].

OBJECTIVE: To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients. METHODS: A total of 162 subjects, including 144 CHB patients and 18 healthy blood donors, were enrolled. The expression of PD-1 on PBMCs was detected by flow cytometry. And the serum HBV markers, HBV DNA load and liver function were also measured. DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli. Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined. RESULTS: With the PD-1 expression in normal controls (10.8% ± 4.4%) as a baseline level, the expression of PD-1 in CHB patients significantly increased. In CHB patients, the serum expression of PD-1 in PBMCs from patients with positive HBeAg (27.1% ± 18.4%) was much higher than that from those with negative HBeAg (19.6% ± 15.6%). And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase. The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601, -553, -538, -483, -463, -317 bp) were significantly correlated with PD-1 expression level (P < 0.05). CONCLUSION: The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.