Reiichiro Kuwahara

Epithelial cell adhesion molecule (EpCAM) marks hepatocytes newly derived from stem/progenitor cells in humans

Epithelial cell adhesion molecule (EpCAM) is a surface marker on human hepatic stem/progenitor cells that is reported as absent on mature hepatocytes. However, it has also been noted that in cirrhotic livers of diverse causes, many hepatocytes have EpCAM surface expression; this may represent aberrant EpCAM expression in injured hepatocytes or, as we now hypothesize, persistence of EpCAM in hepatocytes that have recently derived from hepatobiliary progenitors. To evaluate this concept, we investigated patterns of EpCAM expression in hepatobiliary cell compartments of liver biopsy specimens from patients with all stages of chronic hepatitis B and C, studying proliferation, senescence and telomere lengths. We found that EpCAM(+) hepatocytes were rare in early stages of disease, became increasingly prominent in later stages in parallel with the emergence of ductular reactions, and were consistently arrayed around the periphery of cords of keratin 19(+) hepatobiliary cells of the ductular reaction, with which they shared EpCAM expression. Proliferating cell nuclear antigen (proliferation marker) and p21 (senescence marker) were both higher in hepatocytes in cirrhosis than in normal livers, but ductular reaction hepatobiliary cells had the highest proliferation rate, in keeping with being stem/progenitor cell–derived transit amplifying cells. Telomere lengths in EpCAM(+) hepatocytes in cirrhosis were higher than EpCAM(−) hepatocytes (P < 0.046), and relatively shorter than those in the corresponding ductular reaction hepatobiliary cells (P = 0.057). Conclusion: These morphologic, topographic, immunophenotypic, and molecular data support the concept that EpCAM(+) hepatocytes in chronic viral hepatitis are recent progeny of the hepatobiliary stem/progenitor cell compartment through intermediates of the transit amplifying, ductular reaction hepatobiliary cells. (HEPATOLOGY 2011)

A Randomized Study of Extended Treatment With Peginterferon α-2b Plus Ribavirin Based on Time to HCV RNA Negative–Status in Patients With Genotype 1b Chronic Hepatitis C

OBJECTIVES:The treatment of patients with hepatitis C virus (HCV) genotype 1 with peginterferon plus ribavirin treatment for more than 48 weeks demonstrated high sustained virological response (SVR) rates. Although many studies extended the duration of therapy from 48 weeks to 72 weeks, the optimal duration has not yet been determined.METHODS:A total of 113 genotype 1b patients with high viral load were randomized at baseline to the standard (n=56) or extended (n=57) treatment group. The standard group patients received 48 weeks of peginterferon plus ribavirin treatment. In the extended group, the treatment was performed for 44 weeks after patients became negative for HCV RNA (total duration 48–68 weeks).RESULTS:The SVR rate of the standard and extended group was 36% (20 of 56) and 53% (30 of 57; P=0.07). However, the extended group patients who became negative for HCV RNA between weeks 16 and 24 had a significantly higher SVR rate (78%; 7 of 9) than that of standard group (9%, 1 of 11; P=0.005). The predictive factors for the SVR were the treatment regimen (the standard vs. extended treatment) and the time to HCV RNA negative–status.CONCLUSIONS:The extended treatment significantly increased the SVR rate in patients who were HCV RNA negative at 16–24 weeks.

A real-time quantitative polymerase chain reaction method for hepatitis B virus in patients with chronic hepatitis B treated with lamivudine

OBJECTIVES:During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants.METHODS:Twenty-four patients who had received lamivudine therapy for >1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with >3.7 log genome equivalents/ml, the Roche Monitor kit for sera with ≥2.6 log copies/ml, and real-time PCR for sera with < 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the <1.7 log copies/ml group (eight patients), the 1.7–2.5 log copies/ml group (five patients), and the ≥2.6 log copies/ml group (11 patients).RESULTS:Pretreatment HBV DNA levels were significantly lower in the <1.7 copies/ml group than in the other two groups (p < 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the <1.7 copies/ml group. In contrast, in the 1.7–2.5 copies/ml and ≥2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p < 0.001). Virological breakthroughs were observed at a mean of 49.6 ± 18.4 wk in 11 the patients in the ≥2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7–2.5 copies/ml group.CONCLUSION:The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.

Low hepatocyte repopulation from stem cells: a matter of hepatobiliary linkage not massive production.

Figure 1. A, Label (BrdrU) retaining peribiliary hepatocytes (black nuclei) in murine acute acetaminophen toxicity represent the majority of label retaining cells in this injury model and probably derive from the smallest cholangiocytes of the canals of Hering (CoH; DAB stained pan-keratin, brown) to which they are connected (stain: hematoxylin counterstain; original magnification: 40). B, Laminin staining in human liver is continuous around all ducts and ductules, but is interupted at the CoH: hepatocyte interface (arrow), a distinctive discontinuity that may relate to a specialized repopulating function of the CoH as a stem cell niche and be responsible for the unique ductular reactions that are seen in liver, but no other organ (DAB;stain: hematoxylin counterstain; original magnification: 40). C, Laminin staining of human pancreas shows continuous basement membrane around all duct, acinar, and islet structures (*; DAB; stain: hematoxylin counterstain; original magnification: 20). D, Laminin staining of human colon shows continuous basement membrane around all intestinal crypts (DAB; stain: hematoxylin counterstain; original magnification: 10). (Laminin staining images provided by Christine Sempoux, Université Catholique de Louvain, Belgium.) Low Hepatocyte Repopulation From Stem Cells: A Matter of Hepatobiliary Linkage Not Massive Production

Lamivudine treatment‐related morphological changes of esophageal varices in patients with liver cirrhosis

Aim:  Many studies have reported the therapeutic effects of lamivudine on cirrhotic patients with hepatitis B; however, no study has investigated the morphological changes of esophageal varices after lamivudine treatment.

Adefovir Dipivoxil as a Treatment for Hepatic Failure Caused by Lamivudine-Resistant HBV Strains

Hepatitis B virus (HBV) infection is a serious worldwide problem because it is one of the major causes of cirrhosis and hepatocellular carcinoma in endemic areas. The number of people with chronic HBV infection is 350 million globally (1). Until recently, the therapeutic option for chronic HBV infection has been limited to interferon. Seroconversion from hepatitis B e antigen (HBeAg) to anti-hepatitis B e antibody (anti-HBe) occurs in up to about 40% of patients treated with interferon monotherapy (2–4). Recently, lamivudine, a nucleoside analogue, has become the main therapeutic option for chronic HBV infection. Some clinical trials showed that lamivudine suppressed HBV replication effectively and induced histological improvement (5–7). However, the prolonged therapy with lamivudine has been associated with the emergence of drug-resistant viruses with the mutations in the polymerase gene coding for the YMDD (tyrosine, methionine, aspartate, aspartate) motif (8, 9). The lamivudine-resistant HBV strains have been observed in 14 to 32% of patients undergoing a 1-year treatment regimen of 100 mg daily (6, 7). Several new nucleoside analogues are now under development. Adefovir dipivoxil, a prodrug of the acyclic deoxyadenosine monophosphate analogue adefovir, displays potent antiviral activity against HBV (10, 11) and an in vitro study demonstrated the antiviral activity to be against both wild-type and lamivudine-resistant strains of

Subclones of Drug-Resistant Hepatitis B Virus Mutants and the Outcome of Breakthrough Hepatitis in Patients Treated with Lamivudine

Breakthroughs during lamivudine therapy were assessed according to hepatitis flares and mutational polymorphism of hepatitis B virus (HBV) infecting patients. Of 42 patients with chronic hepatitis B and positive for hepatitis B e antigen in serum, 13 (30%) harbored HBV mutants with lamivudine resistance after a mean duration of 29 months on lamivudine. The virological breakthrough occurred 14.5 months after the start of lamivudine treatment, and all the patients with it developed breakthrough hepatitis 3 months later. The clinical course of breakthrough hepatitis was self-limited except in one patient who had already developed cirrhosis at the baseline. One year after breakthrough hepatitis, serum ALT, albumin, prothrombin time and platelet counts were maintained well on conventional treatments without resorting to interferon. Major HBV mutants during breakthrough hepatitis were those with M552I in the YMDD motif of viral DNA polymerase/reverse transcriptase in 7 patients (54%), M552I/L528M in 4 patients (31%) and M552V/L528M in 2 patients (15%). There were no patients in whom mutations at nucleotide 529 occurred including the 2 who later developed hepatocellular carcinoma. There was no clear relationship between distinct mutational patterns and clinical courses. Further studies are needed for making out the effects of lamivudine-resistant mutants on clinical outcomes, taking into considerations genotypes of HBV.

Evaluation of Resistance-Associated Substitutions in NS5A Using Direct Sequence and Cycleave Method and Treatment Outcome with Daclatasvir and Asunaprevir for Chronic Hepatitis C Genotype 1.

Background The aim of this study was to evaluate the efficacy of daclatasvir plus asunaprevir therapy in patients infected with hepatitis C virus and determine its relevance to resistant variants. Methods A total of 629 consecutive patients infected with hepatitis C virus genotype 1 were assessed. Daclatasvir (60 mg/day) plus asunaprevir (200 mg/day) was given for 24 weeks. The virological responses and resistance-associated substitutions of hepatitis C virus mutants were examined by the direct sequence and cycleave methods were evaluated. Results Overall, 89.4% (555/621) of patients exhibited a sustained virological response (SVR). The SVR rates in the patients with wild type, mixed, and mutant type Y93 by direct sequencing were 92.5% (520/562), 70.3% (26/37), and 42.9% (9/21), respectively. The SVR rates in the patients with 100%, 90%, 80%-30%, and 20%-0% Y93 wild by the cycleave method were 93.4% (456/488), 88.2%(30/34), 56.0%(14/25), and 36.8%(7/19), respectively. In contrast, the SVR rates for the wild type and mixed/mutant type L31 by direct sequencing were 90.2% (534/592) and 72.4% (21/29), respectively. In the multivariate analyses, the wild type Y93, no history of simeprevir therapy, the wild type L31, and low HCV RNA level were independent factors of SVR. Conclusion NS5A resistance-associated substitutions, especially Y93H, were major factors predicting the SVR. Although direct sequencing can predict the SVR rate, the cycleave method is considered to be more useful for predicting the SVR when used in combination.

Clinical course of patients with chronic hepatitis B with viral breakthrough during long-term lamivudine treatment

BackgroundWe evaluated the clinical course of patients with chronic hepatitis B who showed viral breakthrough during long-term lamivudine therapy.MethodsWe initially studied 141 patients treated with lamivudine for 1 year or more, and 49 patients who showed viral breakthrough were the subjects of this study. Their mean lamivudine administration period was 2.3 ± 0.9 years.ResultsAfter viral breakthrough, breakthrough hepatitis occurred in 47 patients (95.9%), but did not occur in the other 2 (4.1%). Four of the 47 patients with breakthrough hepatitis were observed without further treatment, and the alanine transferase (ALT) level was normalized in 2 of them but fluctuated in the other 2. Breakthrough hepatitis was treated by injection of glycyrrhizin or ursodeoxycholic acid administration in 36 of the remaining 43 patients, and by antiviral drug administration in the other 7 (entecavir in 2 patients, adefovir in 2, and interferon in 3). The ALT level was normalized in 5 of the 36 patients treated with glycyrrhizin or ursodeoxycholic acid, but persistently fluctuated in the other 31. In those with normalized ALT after the occurrence of breakthrough hepatitis, the peak ALT level at that point was significantly lower (86 ± 47 IU/l) than that in the patients without normalization (206 ± 167 IU/l).ConclusionsThese results showed that there were a few patients who did not develop breakthrough hepatitis after showing viral breakthrough, and some who showed normalization of the ALT level after the occurrence of breakthrough hepatitis, but in many patients, ALT continuously fluctuated.

Drug-induced liver injury associated with Agaricus blazei Murill which is very similar to autoimmune hepatitis

Agaricus blazei Murill (ABM) is one of the most popular complementary alternative medicines (CAM). We experienced a case of a 60-year-old woman with severe hepatitis associated with extract of ABM and extract of Ganoderma lucidum, and a case of a 75-year-old man with drug-induced liver injury (DILI) associated with extract of ABM and fucoidan. Their clinical courses from the start of CAM until the onset of DILI were observed unexpectedly, because they were under observation for stable malignant neoplasms: stage III malignant thymoma and stage IV lung cancer, respectively. However, they did not talk about taking CAM with their physicians. There were two common points between these two cases. First, they were diagnosed as compatible with DILI by using an international diagnostic scale, the Roussel Uclaf Causality Assessment Method. The second point was that histological findings of the liver were very similar to autoimmune hepatitis (AIH). In addition, serum immunoglobulin G and zinc sulfate turbidity tests gradually increased from the start of CAM to the onset of DILI. Their clinical course and liver histology suggested that the immunostimulating action of ABM caused liver injury which was very similar to that seen in AIH.