Yu-Bao Zheng

Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: Short-term and long-term outcomes†‡

Our study aimed to investigate the short‐term efficacy and long‐term prognosis of liver failure patients caused by hepatitis B after a single transplantation with autologous marrow mesenchymal stem cells (MMSCs). A total of 527 inpatients with liver failure caused by hepatitis B were recruited and received the same medical treatments, among whom 53 patients underwent a single transplantation with autologous MMSCs. A total of 105 patients matched for age, sex, and biochemical indexes, including alanine aminotransferase (ALT), albumin, total bilirubin (TBIL), prothrombin time (PT), and Model for End‐Stage Liver Disease (MELD), comprised the control group. A total of 120 mL of bone marrow was obtained from each patient and then diluted and separated. Then, the MMSC suspension was slowly transfused into the liver through the proper hepatic artery. The success rate of transplantation was 100%, without serious side effects or complications. Levels of ALB, TBIL, and PT and MELD score of patients in the transplantation group were markedly improved from 2‐3 weeks after transplantation, compared with those in the control group. At 192 weeks of follow‐up, there were no dramatic differences in incidence of hepatocellular carcinoma (HCC) or mortality between the two groups. Additionally, there were no significant differences in the incidence of HCC or mortality between patients with and without cirrhosis in the transplantation group. Conclusion: Autologous MMSC transplantation is safe for liver failure patients caused by chronic hepatitis B. Short‐term efficacy was favorable, but long‐term outcomes were not markedly improved. In respect to several parameters, this method is preferable for patients with liver cirrhosis and may have potential for reducing their incidence of HCC and mortality. (HEPATOLOGY 2011;)

CHARACTERIZATION AND HEPATOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM HUMAN AMNIOTIC FLUID AND HUMAN BONE MARROW: A COMPARATIVE STUDY

Since stem cells can differentiate into hepatocyte, stem cell‐based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid‐derived mesenchymal stem cells (AF‐hMSCs) and human bone marrow‐derived mesenchymal stem cells (BM‐hMSCs). Our results show that AF‐hMSCs possess higher proliferation and self‐renewal capacity than BM‐hMSCs. Cytogenetic studies indicate that AF‐hMSCs are as genetically stabile as BM‐hMSCs. Following incubation with specific hepatogenic agents, AF‐hMSCs showed a higher hepatic differentiation potential than BM‐hMSCs. Expression of several liver‐specific markers was significantly greater in AF‐hMSCs than in BM‐hMSCs, as shown by real time RT‐PCR and immunofluorescence (IF). In conclusion, AF‐hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.

Human bone marrow mesenchymal stem cells are resistant to HBV infection during differentiation into hepatocytes in vivo and in vitro

Hepatocyte‐like cells induced from bone marrow mesenchymal stem cells (BMSCs) recover liver function in animal models with liver failure. Our initial findings revealed that human BMSCs improved liver function in hepatitis B patients with end stage liver disease. However, the susceptibility of BMSCs to HBV infection during induction toward hepatocytes remains unknown. We have assessed whether BMSCs‐derived hepatocyte‐like cells can function like liver cells and be infected by HBV. A new and efficient way to direct the differentiation of BMSCs into functional hepatocytes was developed. BMSCs obtained from hepatitis B patients were induced to differentiate into hepatocytes through exposure to HGF, FGF‐4, and EGF. After 6 days of exposure, BMSCs‐derived hepatocyte‐like cells that expressed a subset of hepatic genes and showed hepatic functions were obtained. HBV was used to infect the differentiated cells, and subsequently these cells were assayed for the presence of HBeAg, HBsAg, and HBV DNA. BMSCs proved resistant to HBV infection, both in vitro and during differentiation into hepatocytes in vitro. This demonstrates that BMSCs are resistant to HBV infection. BMSCs are viable for transplantation and should facilitate further research exploring the in vivo HBV‐resistance of the hepatocytes derived from BMSCs after transplantation, a characteristic that could form the basis for hepatocyte transplantation.

Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure.

Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid–derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1–receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra–expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine–induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.

Dynamic Changes of Lipopolysaccharide Levels in Different Phases of Acute on Chronic Hepatitis B Liver Failure

Background High serum levels of lipopolysaccharide (LPS) with LPS-MD-2/TLR4 complex activated NF-kb and cytokine cause hepatic necrosis in animal models. We investigated the dynamic changes of LPS levels in patients with acute on chronic hepatitis B liver failure (ACHBLF). Methods We enrolled ACHBLF patients for a 12-week study. Patients’ LPS levels were measured along with 10 healthy controls. Patients on supportive care and recovered without intervention(s) were analyzed. Patients’ LPS levels during the disease progression phase, peak phase, and remission phase were compared with healthy controls. Results Among 30 patients enrolled, 25 who received interventions or expired during the study period were excluded from the analysis, five patients on supportive care who completed the study were analyzed. Significant abnormal distributions of LPS levels were observed in patients in different phases (0.0168±0.0101 in progression phase; 0.0960±0.0680 in peak phase; 0.0249±0.0365 in remission phase; and 0.0201±0.0146 in controls; respectively, p<0.05). The highest level of LPS was in the peak phase and significantly elevated when compared to controls (0.0201±0.0146 vs. 0.0960±0.0680, p = 0.007). There were no statistically significant differences in LPS levels between healthy controls and subjects in the progression phase or remission phase. Dynamic changes of LPS were correlated with MELD-Na in the progression phase (p = 0.01, R = 0.876) and in the peak phase (p = 0.000, R = −1.00). Conclusions Significant abnormal distributions of LPS levels were observed in ACHBLF with the highest level in the peak phase. The dynamic changes of LPS were correlated with disease severity and suggested LPS causing secondary hepatic injury.

Short-term entecavir versus lamivudine therapy for HBeAg-negative patients with acute-on-chronic hepatitis B liver failure.

BACKGROUND Selection of drugs for antiviral therapy of patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) remains difficult. This study was undertaken to evaluate the short-term efficacy of entecavir versus lamivudine on hepatitis B e antigen (HBeAg)-negative patients with ACLF. METHODS The data of 182 HBeAg-negative patients with ACLF were retrospectively collected from patient profiles of the hospital. In these patients, 93 HBeAg-negative patients with ACLF were treated orally with 0.5 mg of entecavir and 89 were treated orally with 100 mg of lamivudine every day. The gender and age were matched between the two groups. Biochemical items, the model for end-stage liver disease (MELD) score, and HBV DNA level were matched at baseline between the two groups and monitored during treatment. The 3-month mortalities of the two groups were compared. RESULTS No significant differences were found in biochemical items, MELD score, and HBV DNA level at baseline (P>0.05). HBV DNA level decreased within 3 months in both groups (P<0.05), regardless of the pretreatment MELD score. In patients with the same range of pretreatment MELD scores, treatment duration, posttreatment HBV DNA levels, percentage of HBV DNA level <2.7 lg copies/mL, biochemical items, MELD scores and 3-month mortality were similar in the two groups (all P>0.05). Pretreatment MELD score was not related to posttreatment HBV DNA levels (P>0.05), but related to a 3-month mortality in both groups (both P<0.001). CONCLUSIONS In HBeAg-negative patients with ACLF, the short-term efficacy of entecavir versus lamivudine was similar. The degree of pretreatment liver failure significantly affected the outcome of treatment.

High level of IL-27 positively correlated with Th17 cells may indicate liver injury in patients infected with HBV

Interleukin‐6/IL‐12 family cytokines play a key role in inflammatory diseases via their effects on the differentiation or regulation of T helper cells.

Plasma Interleukin-10: A Likely Predictive Marker for Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

Background: The pathogenesis of HBV-related acute-on-chronic liver failure (HBV-ACLF) is mainly based on a heightened immune-inflammatory reaction; however, the intimate underlying mechanism remains unclear. Objectives: The aim of the study was to explore potential key immune molecular targets that could serve as early predictive markers for HBV-ACLF. Patients and Methods: Twenty-seven patients with acute exacerbation of chronic hepatitis B (CHB) (defined by: alanine transaminase ≥ 20 ULN, total bilirubin ≥ 5 ULN, 40% < prothrombin time activity ≤ 60%) and without cirrhosis were divided into 18 cases which did not progress to HBV-ACLF (defined by: prothrombin time activity < 40% and development within four weeks of hepatic encephalopathy and/or ascites) and nine cases that developed HBV-ACLF. Nine healthy people defined the normal control group (NC). Interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-α and IFN-γ protein levels were assayed by Cytometric Bead Array (CBA) in blood plasma. The ELISA method was applied to confirm IL-10 detection using the CBA method. Results: IL-4, IL-12p70 and IFN-γ were undetectable; IL-1β, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher than in NC. Moreover, cytokines reached the highest levels in acute exacerbation of CHB, with the exception of IL-2 and IL-8. When comparing the HBV-ACLF patients prior to and at the time of ACLF diagnosis, IL-10 was the only cytokine that exhibited a significant decrease (P = 0.008). IL-10 concentrations were positively correlated to ALT levels (r = 0.711, P < 0.001). Conclusions: The assessment of plasma IL-10 levels in chronic hepatitis B acute exacerbation may provide an early predictive marker for progression to HBV-ACLF.

Dynamic changes of clinical features that predict the prognosis of acute-on-chronic hepatitis B liver failure: a retrospective cohort study.

Objective: The natural history of acute-on-chronic hepatitis B liver failure (ACHBLF) is complex and highly variable. However, the global clinical characteristics of this entity remain ill-defined. We aimed to investigate the dynamic patterns of the natural progression as well as their impact on the outcomes of ACHBLF. Methods: The clinical features and disease states were retrospectively investigated in 54 patients with ACHBLF at the China South Hepatology Center. The clinical and laboratory profiles including hepatic encephalopathy (HE), hepatorenal syndrome (HRS), and spontaneous bacterial peritonitis (SBP) were evaluated. The disease state estimated by the model for end-stage liver disease (MELD) score and the dynamic patterns during the clinical course of ACHBLF were extrapolated. Results: Twenty-two patients died during the 3-month follow-up period (40.74%). The patients were predominantly male (88.89%). Baseline characteristics showed that there were significant differences in only hepatitis B virus (HBV) DNA levels and platelet count between the deceased and surviving patients (P=0.014 and P=0.012, respectively). Other baseline characteristics were similar in both groups. The dynamic state of the MELD score gradually increased from an initial hepatic flare until week 4 of ACHBLF progression. There were notable changes of the dynamic state of the MELD score at two time points (week 2 and week 4) during ACHBLF progression. The MELD scores were significantly greater in the death group (24.80±2.99) than in the survival group (19.49±1.96, P<0.05) during the clinical course of ACHBLF; the MELD scores of the survival group began to decrease from week 4, while they continued to rise and eventually decreased as more patients died. The gradients of the ascent and descent stages could predict exactly the severity and prognosis of ACHBLF. Conclusions: The natural progression of ACHBLF could be divided approximately into four stages including ascent, plateau, descent, and convalescence stages according to different trends of liver failure progression, respectively. Thus, the special patterns of the natural progression of ACHBLF may be regarded as a significant predictor of the 3-month mortality of ACHBLF.

Effect of GM-CSF in combination with hepatitis B vaccine on revacination of healthy adult non-responders.

OBJECTIVE To assess the immune effects and safety of using GM-CSF with the yeast-recombinant hepatitis B virus (HBV) vaccine for the re-vaccination of healthy adults who did not respond to a previous vaccination. METHODS Study participants included 1784 healthy adults and 100 individuals diagnosed as non-responders. These healthy non-responders were randomly assigned to one of the three treatment groups: Group A (34 individuals) was given 150 microg of granulocyte-macrophage colony stimulating factor (GM-CSF) the first day, then 20 microg of the vaccine; Group B (33 individuals) was given 40 microg of the vaccine only; and, group C (33 individuals) was injected with 20 microg of vaccine each time. All participants were injected three times, at time of study enrollment and one and six months later. Anti-HB surface antigen (HBs) antibody titers were tested before treatment and at one (T1), two (T2) and eight (T8) months post-first injection. RESULTS At T1, the rate of anti-HBs antibody(+) in groups A, B and C was 26.47%, 48.48% and 18.18%, respectively (p = .027). At T8, the seropositive rate of group A (64.71%) and group B (75.76%) was significantly higher than in group C (39.39%) (p = .011); the geometric mean of the antibody titer for groups A and B was higher than for group C (p = .0173). All three treatments were safe and well-tolerated. CONCLUSIONS Augmentation of the vaccine dose and co-administration of GM-CSF and the standard vaccine dose are effective for HBV vaccine non-responders. In fact, changing the vaccine dose had a better seropositive response than injecting the vaccine in combination with GM-CSF.