B and T lymphocyte attenuator is highly expressed on intrahepatic T cells during chronic HBV infection and regulates their function

Background

T cell antiviral function is impaired during chronic hepatitis B (CHB). Programmed death-1 (PD-1) impairs antiviral T cell responses, but dysfunction is not always reversed by blockade of PD-1 pathway. Whether distinct T cell populations expressing different sets of inhibitory molecules exist has not been determined.

Methods

We studied the expression of the B and T lymphocyte attenuator (BTLA) on both peripheral blood mononuclear cells (PBMC) and intrahepatic lymphocytes, and the effects of blocking BTLA on circulating and intrahepatic T cells in CHB patients. Sixty-three CHB patients who underwent liver biopsy were enrolled. The expression of BTLA and PD-1 on PBMC and intrahepatic T cells was assessed by flow cytometry with antibodies to T cell differentiation molecules. Functional recovery was evaluated by analyzing production of interferon (IFN)-γ and interleukin (IL)-2 after incubation of T cells with anti-CD3 and irradiated mature dendritic cells in the presence of anti-BTLA, anti-PD-1, or both.

Results

Intrahepatic T cells expressed higher levels of BTLA than their peripheral counterparts. A significant fraction of intrahepatic T cells coexpressed BTLA and PD-1 and showed deep exhaustion of T cell responses. Blockade of the BTLA pathway enhanced both intrahepatic and PBMC T cell proliferation and cytokine secretion, and exhibited an additive effect upon blockage of PD-1.

Conclusions

Upregulation of inhibitory receptor BTLA restricts T cell responses in CHB. T cell exhaustion by high antigen concentrations exacerbates dysfunction of peripheral and intrahepatic T cells. Blockage of BTLA is a potential therapeutic approach for chronic HBV infection that may act by restoring antiviral T cell responses.     

Regulatory Role of CD4+CD25+Foxp3+ Regulatory T Cells on IL-17-Secreting T Cells in Chronic Hepatitis B Patients

Background

Both interleukin (IL)-17-secreting CD4⁺ T (Th17) and CD4⁺CD25⁺Foxp3⁺ T regulatory (Treg) cells have been shown to be associated with disease progression or liver damage in chronic hepatitis B (CHB) patients. However, the relationship between Treg cells and IL-17-secreting T cells in hepatitis B virus (HBV) infections is unclear.

Methods

The frequencies of Treg cells and IL-17-secreting T cells in hepatitis B e antigen (HBeAg)-positive CHB patients and healthy subjects were measured by flow cytometric analysis. The role of Treg cells on the differentiation of Ag-specific IL-17-secreting T cells was determined by removing the Treg cells from peripheral blood mononuclear cells (PBMCs) in HBeAg-positive CHB patients.

Results

The frequencies of both Th17 (1.71 ± 0.58 vs. 1.08 ± 0.36 %; P < 0.01) and Treg cells (8.92 ± 4.11 vs. 6.45 ± 1.56 %; P < 0.01) were increased in the peripheral blood of HBeAg-positive CHB patients compared with healthy controls, but in not the IL-17-secreting CD8⁺ T (Tc17) cells. The frequency of Treg cells was significantly associated with that of Th17 cells (r = 0.625, P = 0.001) in CHB patients. Spearman analysis showed a positive correlation between the frequency of Treg cells and HBV DNA load (r = 0.508, P = 0.008), as well as between the frequency of Th17 cells and serum alanine aminotransferase level (r = 0.516, P = 0.007). The deletion of Treg cells significantly enhanced both Th17 and Tc17 cell development in PBMCs following recombinant HBV core antigen stimulation.

Conclusions

Our data indicate a clear inverse relationship between Th17 cells and Treg cells and that Treg cells can inhibit Th17 cell development in CHB patients.     

Significant association of different preS mutations with hepatitis B-related cirrhosis or hepatocellular carcinoma

Background

The associations of nucleotide substitution mutations in the preS region of hepatitis B virus (HBV) with hepatocellular carcinoma (HCC) and cirrhosis remain unknown. We aimed to determine the associations of preS mutations with HCC or cirrhosis.

Methods

HBV from 603 asymptomatic hepatitis B surface antigen carriers (ASCs), 219 chronic hepatitis B (CHB) patients, 119 cirrhosis patients, and 231 HCC patients were genotyped and sequenced in the preS region. Nucleotides with the highest frequencies in HBV from the hepatitis B e antigen (HBeAg)-positive ASCs were treated as “wild-type” nucleotides. Twenty-one preS1 mutations and 14 preS2 mutations were evaluated. Multivariate regression analyses were applied to determine factors independently associated with cirrhosis or HCC.

Results

Most (85.7%) preS2 mutations were associated with CHB compared with ASCs, whereas most preS1 mutations were associated with HCC compared with the cirrhosis patients or CHB patients. Compared with the CHB patients, 81.0% preS1 mutations in genotype C were inversely associated with cirrhosis. Multivariate regression analyses showed that C2964A, C3116T, and C7A were novel factors associated with HCC compared with those without HCC, whereas A2964C and T3116C were independently associated with cirrhosis compared with ASCs and the CHB patients. Combined preS1 mutations had specificities greater than 95%, while C3116T and C7A had moderate sensitivities and specificities, for HCC.

Conclusions

C2964A, C3116T, and C7A are novel markers independently associated with an increased risk of HCC, while A2964C and T3116C are novel markers independently associated with an increased risk of cirrhosis. Combined preS1 mutations are specific for HCC.     

The Expression of Programmed Death-1 in Circulating CD4+ and CD8+ T Cells during Hepatitis B Virus Infection Progression and Its Correlation with Clinical Baseline Characteristics

Background/Aims

Programmed death-1 (PD-1) expression was investigated in CD4⁺ and CD8⁺ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages.

Methods

PD-1 expression in circulating CD4⁺ and CD8⁺ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0.

Results

PD-1 expression in CD4⁺ and CD8⁺ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4⁺ and CD8⁺ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent.

Conclusions

PD-1 expression in peripheral CD4⁺ and CD8⁺ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.     

Combination and evolution of HBV mutant strains in the HBeAg-positive status predict clinical outcomes after HBeAg seroconversion

Purpose

We investigated whether the combined presence and evolution of hepatitis B virus (HBV) mutant strains in the hepatitis B e antigen (HBeAg)-positive status can predict clinical outcomes after HBeAg seroconversion.

Methods

One hundred and eighty-six patients with spontaneous HBeAg seroconversion were enrolled into this longitudinal study. The sequences of pre-S, core promoter, and precore regions were determined at study entry and at the visit immediately before HBeAg seroconversion.

Results

Age ≥40 years at HBeAg seroconversion, male sex, and higher HBV DNA levels at entry were independent predictors for HBeAg-negative chronic hepatitis B (CHB). Patients with combined mutations of pre-S deletions and T1762/A1764 had a significantly increased risk of cirrhosis and hepatocellular carcinoma (HCC) compared to patients with the wild type at both genomic regions. Combinations of pre-S deletions and T1762/A1764 were found on the same HBV genome by cloning analysis of full-length HBV genomes. Patients with a persistent presence of pre-S deletions and T1762/A1764 mutations, and new development of pre-S deletions in the HBeAg-positive status were significantly at an increased risk of HBeAg-negative CHB, cirrhosis, and HCC after HBeAg seroconversion than those with a persistent presence of the wild type at both genomic regions. After adjusting the other risk factors, the evolution of pre-S deletions was an independent predictor for cirrhosis [hazard ratio (HR): 1.52, 95 % confidence interval (CI) 1.02–2.25] and HCC (HR: 4.0, 95 % CI 1.6–10.1).

Conclusions

The combined presence and evolution of pre-S deletions and T1762/A1764 in the HBeAg-positive status was a useful factor significantly predictive of clinical outcomes in patients with spontaneous HBeAg seroconversion.     

Dynamic Changes of Treg and Th17 Cells and Related Cytokines Closely Correlate With the Virological and Biochemical Response in Chronic Hepatitis B Patients Undergoing Nucleos(t)ide Analogues Treatment

Background:

The restoration of HBV-specific T-cell response during antiviral therapy is associated with CD4+T-cell activity. Treg cells and Th17 cells are subtypes of CD4+T cell. However, it has remained unknown how the Treg and Th17 cells and their associated cytokines affect nucleos(t)ide analogues (NA) antiviral efficacy.

Objectives:

The aim of the present study was to provide a new insight to evaluate the NA antiviral therapy for patients with chronic hepatitis B (CHB).

Patients and Methods:

Forty-four CHB patients hospitalized between July 2010 and August 2011 were enrolled in this study. They were received NA (entecavir, lamivudine and adefovir) treatment for 14.42 ± 13.08 weeks, and the peripheral blood was collected. The frequencies of Treg and Th17 cells were detected by flow cytometric analysis, and the levels of IL-10, TGF-β1, IL-17 and IL-23 were measured by enzyme-linked immunosorbent assay (ELISA).

Results:

In complete and partial-responders, Treg cells frequencies and IL-10, TGF-β1, IL-23 levels were all decreased significantly after NA therapy, while Th17 cells and the IL-17 levels were increased slightly. Treg/Th17 ratio was only dramatically declined in complete-responders. But there was no significant difference in non-responders. Either HBV DNA decreased by at least 2 log copies /mL or ALT turned to normal level, Treg cells frequencies and IL-10, TGF-β1, IL-23 levels were significantly reduced. Meanwhile, Treg cells were positively correlated with HBV DNA and ALT.

Conclusions:

The changes of Treg and Th17 cells and their associated cytokines were related to virological and biochemical responses.     

辅助性T淋巴细胞17/调节性T淋巴细胞比值失衡在HBV相关肝脏疾病进展中的作用

慢性乙型肝炎患者肝脏损害多发生在免疫介导作用下,考虑病毒感染和免疫反应的相互作用是疾病病情进展的重要原因。慢性乙型肝炎可发展至肝纤维化、肝硬化甚至肝细胞癌。回顾了辅助性T淋巴细胞17(Th17)与调节性T淋巴细胞(Treg)的发现起源,叙述了二者自身特性,同时阐述了在维持免疫系统稳定过程中二者所起的作用及作用机制;详细分析了细胞失衡在慢性乙型肝炎、肝纤维化、肝硬化、肝细胞癌等各个阶段中的作用,指出Th17/Treg失衡会促使HBV相关肝脏疾病病情加重。     

Kinetics of peripheral hepatitis B virus-specific CD8+ T cells in patients with onset of viral reactivation

Background

Patients with resolved hepatitis B virus (HBV) infection undergoing chemotherapy or immunosuppressive therapy are potentially at risk of HBV reactivation. However, it remains unclear how liver disease develops after HBV reactivation. To compare the host immune response against HBV, we performed immunological analyses of six HBV reactivation patients.

Methods

The numbers of peripheral HBV-specific CD8⁺ T cells were investigated longitudinally in six HLA-A2- and/or A24-positive patients with HBV reactivation. In addition, 34 patients with resolved HBV, 17 patients with inactive chronic hepatitis B (ICHB), 17 patients with chronic hepatitis B (CHB) and 12 healthy controls were analyzed. The number and function of HBV-specific CD8⁺ T cells were assessed by flow cytometry using tetramer staining and intracellular IFN-γ production. Furthermore, the numbers of CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells and serum inflammatory cytokine levels were analyzed.

Results

The frequency of HBV-specific CD8⁺ T cells was significantly increased in HBV reactivation patients compared with ICHB and CHB patients. In addition, the number of HBV-specific CD8⁺ T cells was increased in resolved HBV patients compared with ICHB patients. PD-1 expression was decreased in HBV reactivation patients compared with ICHB and CHB patients. The numbers of HBV-specific CD8⁺ T cells and CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells were negatively correlated following onset of HBV reactivation.

Conclusions

During HBV reactivation, the frequency of HBV-specific CD8⁺ T cells increased even though the administration of immunosuppressive drugs and interactions with CD4⁺ regulatory T cells may be important for the onset of liver disease.     

Expression and significance of peripheral myeloid-derived suppressor cells in chronic hepatitis B patients

Background

Myeloid-derived suppressor cells (MDSCs) exert their suppressive effects on multiple immune response and contribute to the development of many diseases. However, limited data is available on the involvement of MDSCs in human chronic HBV infection.

Long-term effect of lamivudine treatment on the incidence of hepatocellular carcinoma in patients with hepatitis B virus infection

Background

Nucleotide analogues have recently been approved for the treatment of patients with hepatitis B virus (HBV) infection. However, it is still controversial whether the decrease of HBV-DNA amount induced by treatment with nucleotide analogues can reduce the risk of hepatocellular carcinoma (HCC) development in HBV patients.

Methods

A total of 293 HBV patients without HCC who were treated with lamivudine (LAM) were enrolled in a multicenter trial. The incidence of HCC was examined after the start of LAM therapy, and the risk factors for liver carcinogenesis were analyzed. The mean follow-up period was 67.6?±?27.4?months.

Results

On multivariate analysis for HCC development in all patients, age ≥50?years, platelet count <14.0?×?10⁴/mm³, cirrhosis, and median HBV-DNA levels of ≥4.0?log copies/ml during LAM treatment were significant risk factors. The cumulative carcinogenesis rate at 5?years was 3% in patients with chronic hepatitis and 30% in those with cirrhosis. For the chronic hepatitis patients, the log-rank test showed the significant risk factors related to HCC development to be age ≥50?years, platelet count <14.0?×?10⁴/mm³, and hepatitis B e antigen negativity, but median HBV-DNA levels of <4.0?log copies/ml (maintained viral response, MVR) did not significantly suppress the development of HCC. In cirrhosis patients, however, the attainment of MVR during LAM treatment was revealed to reduce the risk of HCC development.

Conclusions

These results suggest that the incidence of HCC in HBV patients with cirrhosis can be reduced in those with an MVR induced by consecutive LAM treatment.     

Characteristics and Management of Patients with Chronic Hepatitis B in an Integrated Care Setting

Background

Few population-based studies have described characteristics and management of patients with chronic hepatitis B (CHB) in the USA.

Methods

We retrospectively studied adults with CHB in the Northern California Kaiser Permanente Medical Care Program (KPNC) from July 2009 to December 2010 (n = 12,016). Laboratory tests, treatment patterns, and hepatocellular carcinoma (HCC) surveillance were ascertained during a “recent” 18-month study window (July 2009–December 2010), or as “ever” based on records dating to 1995.

Results

The mean age was 49 years; 51 % were men, 83 % Asian, and 87 % KPNC members >5 years. Overall, 51 % had ≥1 liver-related visit, 14 % with gastroenterology or infectious disease specialists, and 37 % with primary care providers (PCP) only. Less than 40 % of patients had both hepatitis B virus (HBV) DNA and ALT testing conducted recently, while 56 % of eligible patients had received HCC surveillance. Recent laboratory testing and HCC surveillance were more frequent in patients seen by a specialist versus PCP only (90 vs. 47 % and 92 vs. 73 %, respectively, p values <0.001). During the study period, 1,649 (14 %) received HBV treatment, while 5 % of untreated patients had evidence of treatment eligibility. Among 599 patients newly initiated on HBV therapy, 76 % had guideline-based indications for treatment.

Conclusions

Most patients initiated on HBV treatment met eligibility, and very few patients with evidence of needing treatment were left untreated. However, monitoring of ALT and HBV DNA levels, as well as HCC surveillance, were not frequent, underestimating the proportion of patients that warranted HBV therapy. Viral monitoring and cancer surveillance are therefore important targets for improving the scope of CHB care in the community setting.     

Soluble FGL2, a novel effector molecule of activated hepatic stellate cells,regulates T-cell function in cirrhotic patients with hepatocellular carcinoma

Purpose

To investigate the effects of soluble FGL2 (sFGL2) secreted by hepatic stellate cells (HSCs) on immune suppression in cirrhotic patients with hepatocellular carcinoma (HCC).

Methods

Serum sFGL2 levels were examined by ELISA in 40 patients with HCC, liver cirrhosis (LC) or chronic HBV (CHB) infection. A double staining of the immunofluorescence analysis of α-SMA and FGL2 was performed in two cirrhotic liver specimens. The expression of FGL2 in the LX2 cell line was analyzed by immunofluorescence, Western blot and flow cytometry. T-cells purified from HCC patients using magnetic beads were cultured with LX2 cells at different ratios with anti-CD3-stimulating or FGL2-blocking antibodies. The proliferation index (PI) of CD8 + T cells was assessed by flow cytometry, and the secretion of IFN-γ was measured by ELISA.

Results

sFGL2 levels are significantly higher in patients with HCC or LC compared with those with CHB (p = 0.0039/p = 0.0020). Among HCC patients, those with cirrhosis exhibited significantly higher levels of sFGL2 compared with non-cirrhotic individuals (p = 0.0108). The expressions of FGL2 and α-SMA overlapped in HSCs in liver specimens. FGL2 protein secreted by LX2 cells inhibited T-cell proliferation of HCC patients in a dose-dependent manner in vitro. The PI of CD8 + T cells was significantly enhanced following addition of FGL2 antibody to the culture system (LX2/T-cell ratio of 1:10, p = 0.002). The level of IFN-γ in mixed cultures was inversely correlated with the number of HSCs and was reversed by incubation with FGL2 blocking antibody.

Conclusion

sFGL2 protein is a novel effector molecule of activated HSCs, which suppresses CD8 + T cell proliferation and interferon-γ production, and it subsequently might contribute to immune suppression during fibrosis and tumorigenesis in the liver.     

Cholecystectomy in Sweden 2000 – 2003: a nationwide study on procedures, patient characteristics, and mortality

Background

The role of leptin in the course of liver disease due to chronic viral hepatitis (CVH) remains controversial. Our aims were to investigate the relationship between serum leptin concentrations and the severity of liver disease in a cohort of subjects with HBeAg negative chronic hepatitis B (CHB) and C (CHC) and to analyze the effect of body composition, the leptin system and insulin resistance together with viral factors on virologic response to antiviral treatment.

Methods

We studied 50 (36 men) consecutive patients suffering from biopsy-proven CVH due to HBV (n = 25) or HCV (n = 25) infection. Thirty-two (17 men) healthy volunteers served as controls. Levels of serum leptin and insulin were determined by immunoassays at baseline and at the end of the treatment.

Results

A significant association between serum leptin levels and the stage of hepatic fibrosis was noted; patients with cirrhosis presented higher serum leptin levels compared to those with lower fibrosis stage [CHB patients (17436 pg/ml vs 6028.5 pg/ml, p = 0.03), CHC patients (18014 pg/ml vs 4385 pg/ml, p = 0.05]. An inverse correlation between lower leptin levels and response to lamivudine monotherapy was noted in patients with CHB; those with a virologic response presented lower serum leptin levels (5334 vs 13111.5 pg/ml; p-value = 0.003) than non-responders. In genotype 1 CHC patients, insulin resistance played a significant role in the response to antiviral therapy.

Conclusion

Our data clearly suggest that cirrhosis due to CHB or CHC is associated with higher leptin levels. Increased serum leptin levels represent a negative prognostic factor for response to lamivudine monotherapy in patients with CHB. In CHC patients insulin resistance strongly influences the response to antiviral treatment in patients infected with genotype 1.     

Association of Hepatitis B Virus Mutations of A1846T and C1913A/G With Acute-on-Chronic Liver Failure Development From Different Underlying Chronic Liver Diseases

Background

As most HBV-related acute-on-chronic liver failure (ACLF) have concurrent cirrhosis, it is important to clarify the association of viral factors with ACLF with or without cirrhosis.

Objectives

The aim of this study was to analyze the association of HBV genotypes and mutations with ACLF development underlying different chronic liver diseases.

Patients and Methods

Eighty-seven ACLF patients including 29 patients with chronic hepatitis (ACLF-CHB) and 58 patients with liver cirrhosis (ACLF-LC) were enrolled. Age and sex matched patients with chronic hepatitis (CHB) and liver cirrhosis (LC) were enrolled as controls. The genotypes and mutations at HBV basic core promoter (BCP), precore (PC), and partial C regions were determined by nested PCR and direct sequencing.

Results

Our results revealed significantly higher incidences (P < 0.05) of genotype B with C1913A/G or A1846T in patients with ACLF-CHB than those with CHB; genotype C with C1913A/G or A1846T in patients with ACLF-CHB and ACLF-LC than those with CHB and LC, respectively. Multivariable analysis indicated that A1846T and C1913A/G mutations were independent factors for ACLF (OR = 2.86 and 5.93, respectively), suggesting an association between the mutations and development of ACLF. In addition, there were no significant differences in mutations at T1753V, A1762T, G1764A, G1896A, and G1899A which were found between either CHB and ACLF-CHB or LC and ACLF-LC patients， suggesting no associations of these mutations with ACLF development.

Conclusions

Our findings suggest that CHB or LC patients infected with HBV A1846T and C1913A/G mutants are more susceptible to develop ACLF.     

A proinflammatory CD4<Superscript>+</Superscript> T cell phenotype in gestational diabetes mellitus

Aims/hypothesis

Numerous adaptations of the maternal immune system are necessary during pregnancy to maintain immunological tolerance to the semi-allogeneic fetus. Several complications of pregnancy have been associated with dysregulation of these adaptive mechanisms. While gestational diabetes mellitus (GDM) has been associated with upregulation of circulating inflammatory factors linked to innate immunity, polarisation of the adaptive immune system has not been extensively characterised in this condition. We aimed to characterise pro- and anti-inflammatory CD4⁺ (T helper [Th]) T cell subsets in women with GDM vs women without GDM (of similar BMI), during and after pregnancy, and examine the relationship between CD4⁺ subsets and severity of GDM.

Methods

This is a prospective longitudinal case–control study of 55 women with GDM (cases) and 65 women without GDM (controls) at a tertiary maternity hospital. Quantification of proinflammatory (Th17, Th17.1, Th1) and anti-inflammatory (regulatory T cell [Treg]) CD4⁺ T cell subsets was performed on peripheral blood at 37 weeks gestation and 7 weeks postpartum, and correlated with clinical characteristics and measures of blood glucose.

Results

Women with GDM had a significantly greater percentage of Th17 (median 2.49% [interquartile range 1.62–4.60] vs 1.85% [1.13–2.98], p?=?0.012) and Th17.1 (3.06% [1.30–4.33] vs 1.55% [0.65–3.13], p?=?0.006) cells compared with the control group of women without GDM. Women with GDM also had higher proinflammatory cell ratios (Th17:Treg, Th17.1:Treg and Th1:Treg) in pregnancy compared with the control group of women without GDM. In the control group, there was a statistically significant independent association between 1 h glucose levels in the GTT and Th17 cell percentages, and also between 2 h glucose levels and percentage of Th17 cells. The percentage of Th17 cells and the Th17:Treg ratio declined significantly after delivery in women with GDM, whereas this was not the case with the control group of women. Nevertheless, a milder inflammatory phenotype persisted after delivery (higher Th17:Treg ratio) in women with GDM vs women without.

Conclusions/interpretation

Dysregulation of adaptive immunity supports a novel paradigm of GDM that extends beyond hyperglycaemia and altered innate immunity.

     

HBsAg, HBeAg and HBV DNA level changes and precore/basal core promoter mutations in the natural history of chronic hepatitis B in Indonesian patients

Introduction

Chronic hepatitis B (CHB) is a state of complex interactions between the hepatitis B virus (HBV) and host. We studied the changes in hepatitis B surface antigen (HBsAg), hepatitis B ‘e’ antigen (HBeAg) and HBV DNA levels, considering the implications of HBV genotype, basal core promoter (BCP) A1762T/G1764A and precore G1896A mutations in CHB.

Methods

One hundred fifty-two treatment-naïve CHB patients were classified into immune-tolerant (IT), immune-clearance (IC), low/non-replicative (LR) and ‘e’-negative hepatitis B (ENH) phases, based on HBeAg status, HBV DNA and ALT levels. HBV DNA was detected and quantified by polymerase chain reaction, then analyzed by sequencing. HBsAg and HBeAg levels were measured serologically.

Results

HBsAg and HBV DNA levels varied between CHB phases, with HBsAg highest in IT and lowest in LR, and HBV DNA high in IT and IC, and lowest in LR. Both markers increased in ENH. Correlation between HBsAg and HBV DNA was significant in IT and IC, modest in ENH, but missing in LR. HBeAg and HBV DNA levels were dissociated in HBeAg-positive patients. Genotypes B and C were similarly distributed, with precore mutations higher in HBeAg-negative patients and BCP mutations comparable in all phases. Temporal association between HBeAg seroconversion and an increase of BCP/precore mutations was observed.

Conclusion

HBsAg and HBV DNA levels were high and correlated in early CHB phases and dissociated after HBeAg seroconversion, indicating different controls affecting HBV replication and HBsAg production. Selection of BCP/precore mutants may affect disease course and explain the HBeAg–HBV DNA dissociation, a precaution for clinical application of quantitative HBeAg.     

Bortezomib induces tumor-specific cell death and growth inhibition in hepatocellular carcinoma and improves liver fibrosis

Background

Human hepatocellular carcinoma (HCC) is highly ubiquitinated. The ubiquitination is important to the generatation of HCC. The antitumor and antifibrosis effects of an ubiquitin–proteasome system inhibitor, bortezomib, on HCC with liver cirrhosis (LC) were analyzed in vitro and in vivo.

Methods

The effect of bortezomib was analyzed in the rat hepatocarcinogenesis model using a DEN and CDAA diet (DEN/CDAA model), which shows severe LC and generation of HCC. The decrease of GST-P-positive foci and HCC were analyzed in vivo. Cell death was analyzed by cell death detection kit. Liver fibrosis was checked by sirius-red staining and α-smooth muscle actin staining. The in vitro study involved 3 HCC cell lines (HepG2, HuH7, and HLF) and primary rat and human hepatocytes. The proliferation rate of the HCC cell line was analyzed using the MTT assay and FACS analysis. The toxicity of bortezomib was checked using the LDH release assay for primary human and rat hepatocytes.

Results

In the rat hepatocarcinogenesis model, bortezomib prevented the development of preneoplastic lesions during the early stages of hepatocarcinogenesis and specifically induced cell death in HCC. Furthermore, bortezomib inhibited cell proliferation and induced tumor-specific cell death in HCC cell lines with decrease of cyclin D1 and phospho-Rb expression. Further, bortezomib showed no hepatotoxicity of primary rat and human hepatocytes, suggesting that it might be an HCC-specific drug. Bortezomib also prevented the activation of hepatic stellate cells and inhibited the liver fibrosis of the DEN/CDAA model.

Conclusions

Bortezomib appears to be an ideal target drug for HCC with LC.     

Regulatory T cells are associated with post-cryoablation prognosis in patients with hepatitis B virus-related hepatocellular carcinoma

Background

We carried out this study to evaluate the association between regulatory T cells (Treg) and prognosis and progression after cryoablation in patients with hepatitis-B virus-related hepatocellular carcinoma.

Methods

Peripheral Treg frequency in 111 patients with hepatocellular carcinoma (HCC) was detected by flow cytometry. Treg frequency and function were re-examined during patient follow up. A possible association between Treg and α-fetoprotein (AFP) was also analyzed, and the distribution of resident CD4⁺ and CD8⁺ T cells and FoxP3⁺ T cells in the liver tissue of patients with HCC was examined by immunohistochemistry.

Results

Treg frequency significantly increased with disease progression. Our longitudinal study showed that Treg frequency had significantly decreased in 17 patients with HCC regression following cryoablation, but the frequency had dramatically increased in 14 patients with HCC recurrence or progression. Furthermore, AFP levels varied in a way comparable with Treg frequency in patients with elevated AFP recorded before therapy. Significantly increased suppressive effects of Treg on proliferation and cytokine secretion of CD8⁺ and CD4⁺ T cells were observed during follow up in patients with tumor progression, but not in patients with tumor response. Moreover, the numbers of CD8⁺, CD4⁺, and FoxP3⁺ cells infiltrating the tumors around the cryotherapeutic zones were significantly decreased after argon–helium cryoablation, and this was associated with a reduction in the FoxP3/CD8 ratio. Importantly,increased quantities of circulating CD4⁺CD25⁺FoxP3⁺ Treg and tumor infiltrating FoxP3⁺ cells before cryoablation were associated with high recurrence or risk of progression in HCC patients after cryoablation.

Conclusions

Treg variation is associated with tumor regression or progression in HCC following cryoablation and may be used as a marker to estimate HCC progression.     

Influence of serum HBV DNA load on recurrence of hepatocellular carcinoma after treatment with percutaneous radiofrequency ablation

Background

High serum load of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) is a strong risk factor of hepatocellular carcinoma (HCC) development, independent of hepatitis B e antigen, serum alanine aminotransferase level, and liver cirrhosis. We evaluated whether serum HBV DNA load is associated with the risk of recurrence of HBV-related HCC treated with radiofrequency ablation (RFA).

Methods

The study population was 69 consecutive patients with HBV-related HCC treated locally completely with RFA between January 2000 and September 2007. The risk factors for HCC recurrence were analyzed based on laboratory data, including serum HBV DNA load, together with tumor size and number using univariate and multivariate proportional hazard regression analyses.

Results

HCC recurrence was observed in 42 of 69 patients during the median observation period of 1.5 years. Cumulative recurrence rates at 1, 3, and 5 years were 26.5, 57.8, and 74.3%, respectively. In univariate analysis, albumin (<3.5 g/dl), platelet count (<150 × 10³/mm³), prothrombin activity (PT) (<70%), Child-Pugh class B, serum HBV DNA load (>4.0 log10 copies/ml), and tumor number (>3) were associated with the recurrence at p ≤ 0.15. Multivariate Cox regression analysis with stepwise variable selection showed that the tumor number (risk ratio, 4.63; 95% CI, 1.50–14.25, P = 0.0076), low PT (3.39, 1.52–5.78, P = 0.0029), and high HBV DNA load (2.67, 1.16–6.14, P = 0.021) were independent risk factors for HCC recurrence.

Conclusion

Serum HBV DNA load is associated with the risk of recurrence of HBV-related HCC after RFA.     

Cancer-derived matrix metalloproteinase-9 contributes to tumor tolerance

Background and aims

Tumor-specific T regulatory cells (Treg) play a critical role in tumor cell survival. The development of tumor-specific Treg is not fully understood. This study aims to elucidate the role of matrix metalloproteinase (MMP)9 in tumor tolerance development.

Methods

We recruited 38 patients with laryngeal cancer (LC) in this study. MMP9 levels in the LC were measured by western blotting. Immune cells were isolated from LC tissue for indicated experiments. The cells?? activities were characterized by flow cytometry.

Results

High levels of MMP9 were detected in LC that plays a critical role in the development of tolerogenic dendritic cells and LC-specific Tregs. The isolated LC Tregs have the ability to suppress tumor-specific CD8 T cells in a tumor antigen-specific manner.

Conclusions

This study reveals a novel mechanism in tumor tolerance in which MMP9 plays a critical role in tumor survival. The data imply that MMP9 may be a potential target in the treatment of malignant tumors.