Toshihiko Kirishima

Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment

BACKGROUND/AIMS The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant. METHODS We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients). RESULTS We could detect as little as 0.01-0.001% of mutant viruses coexisting in 10(5)-10(9) copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients. CONCLUSIONS We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.

Clinical significance of elevated serum interferon- inducible protein-10 levels in hepatitis C virus carriers with persistently normal serum transaminase levels.

The aim of this study was to assess the immuno‐ logical profile in hepatitis C virus carriers with persistently normal serum transaminase levels. Forty‐two serum HCV RNA positive patients with persistently normal serum transaminase levels (22 natural ‘asymptomatic HCV carriers’ and 20 biochemical responders to IFN therapy) and 23 complete responders to IFN therapy were enrolled. The HCV genotypes and serum HCV RNA levels were determined before IFN therapy in treatment responders, and at entry in the others. The serum levels of IFN‐inducible protein‐10 (IP‐10) (a protein mainly induced by IFN‐γ), interleukin (IL)‐10, and IL‐4 were measured in all patients while the serum transaminase levels were normal. The serum transaminase levels and platelet counts were then monitored for the next 4 years and the changes in liver fibrosis were assessed. The serum levels of IP‐10 in infected and biochemically normal patients were significantly higher than the levels in complete responders to therapy, whereas the serum levels of IL‐10 and IL‐4 did not vary significantly among the different groups. During the 4‐year follow‐up period, 10/20 (50%) biochemical responders and 12/22 (55%) asymptomatic carriers had an elevation of the serum transaminase levels. A significant (P=0.0370) increase in platelet count after 4 years and improvement in liver fibrosis were noted in treatment responders but not in infected patients. The weak but significant residual immune response as reflected by the increased serum IP‐10 level may underlie the outcome of HCV carriers with persistently normal serum transaminase levels.

Transient biochemical response in interferon therapy decreases the development of hepatocellular carcinoma for five years and improves the long-term survival of chronic hepatitis C patients

The development of hepatocellular carcinoma (HCC) was significantly reduced in both sustained responders (SR) and transient biochemical responders (TR) in chronic hepatitis C (CH-C) patients who received interferon (IFN) therapy. However, the long-term clinical outcome of TR remains unclear. One thousand three hundred and seventy CH-C Japanese patients who received IFN therapy and 54 control cirrhotic patients were enrolled. TR were defined as those patients who showed a normal serum alanine aminotransferase level (<==30 IU/l) at the end of therapy and then relapsed. Mean follow-up period was 5.6 years (6.1 years in 48 cirrhotic patients) in the IFN group and 8.3 years in the 54 control cirrhotic patients. HCC was detected in 114 patients in the IFN group among whom 4 were in the 425 SR, 21 were in the 359 TR and 89 were in the 586 non-responders (NR). The cumulative incidence of HCC was significantly (P=0.0001) inhibited in both SR and TR compared with NR. Its inhibitory effect in TR was within 5 years. Platelet count did not significantly decrease for 2-4 years after IFN therapy in TR, but it significantly decreased in NR 2 years after IFN therapy. The cumulative survival in both SR and TR was significantly higher than NR (SR vs NR; P=0.0001, TR vs NR; P=0.0305). These results indicate that IFN therapy lowers the rate of the progression of HCC and improves the long-term survival even in CH-C patients who transiently respond to IFN therapy.

Activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A-induced hepatic injury in mice

BACKGROUND/AIMS: To examine whether or not activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A (Con A)-induced hepatic injury in mice. METHODS: Con A was administered to Balb/c mice pretreated with or without gadolinium chloride (GdCl(3)). Kupffer cell activation was evaluated by their ability to produce superoxide anions in situ under liver perfusion with nitro blue tetrazolium (NBT). Hepatic concentration of cytokines was measured by ELISA and the mRNA expression of CXC chemokine receptor 3 (CXCR3) was evaluated by RT-PCR. Immunohistochemical detection of CD4 positive lymphocytes in the liver was also performed. RESULTS: GdCl(3)-pretreatment significantly (P<0.01) reduced the serum levels of alanine aminotransferase (ALT) in Con A-treated mice. Formazan deposition in Kupffer cells, the hepatic concentration of tumor necrosis factor-alpha and interferon-gamma, the mRNA expression of CXCR3 and the CD4 positive lymphocytes in the liver were decreased in GdCl(3)-pretreated mice as compared with those without GdCl(3)-pretreatment (P<0.05, respectively). CONCLUSIONS: Activated Kupffer cells, which produce superoxide anions, are involved in Con A-induced hepatic necro-inflammation in mice possibly through the activation of Th1-associated immune response mediated by CD4 and/or CXCR3 positive cells recruited into the liver.

Direct cholangioscopy combined with double- balloon enteroscope-assisted endoscopic retrograde cholangiopancreatography

Double-balloon enteroscope (DBE)-assisted endoscopic retrograde cholangiopancreatography (ERCP) is an effective endoscopic approach for pancreatobiliary disorders in patients with altered gastrointestinal anatomy. Endoscopic interventions via DBE in these postoperative settings remain difficult because of the lack of an elevator and the use of extra-long ERCP accessories. Here, we report the usefulness of direct cholangioscopy with an ultra-slim gastroscope during DBE-assisted ERCP. Three patients with choledocholithiasis in postoperative settings (two patients after Billroth II gastrojejunostomy and one patient after Roux-en-Y gastrojejunostomy) were treated. DBE was used to gain access to the papilla under carbon dioxide insufflation, and endoscopic sphincterotomy was performed with a conventional sphincterotome. For direct cholangioscopy, the enteroscope was exchanged for an ultra-slim gastroscope through an incision in the overtube, which was inserted directly into the bile duct. Direct cholangioscopy was used to extract retained bile duct stones in two cases and to confirm the complete clearance of stones in one case. Bile duct stones were eliminated with a 5-Fr basket catheter under direct visual control. No adverse events were noted in any of the three cases. Direct cholangioscopy with an ultra-slim gastroscope facilitates subsequent treatment within the bile duct. This procedure represents another potential option during DBE-assisted ERCP.

Efficacy and safety of sorafenib in very elderly patients aged 80 years and older with advanced hepatocellular carcinoma

Sorafenib is the standard systemic therapy for patients with advanced hepatocellular carcinoma (HCC). We aimed to assess the efficacy and safety of sorafenib therapy in very elderly patients aged 80 years and older with advanced HCC.

Prediction of breakthrough hepatitis due to lamivudine-resistant hepatitis B virus by a sensitive semiquantitative assay using peptide nucleic acids.

Objective: The aim of this study was to predict breakthrough hepatitis and analyze the dynamics of lamivudine-resistant hepatitis B virus in patients treated with lamivudine. Methods: Fifty-five chronic hepatitis B patients treated with lamivudine were included. The emergence of YMDD motif mutants was detected by peptide nucleic acid (PNA) mediated PCR clamping with a detection limit of 101 YMDD mutants. We then performed a semiquantitative PCR assay of subjects in whom YMDD mutants were detected. This assay detects 102.7–107.7 copies of mutant virus per 1 ml of serum. Results: YMDD mutants were detected in 28 (51%) of the 55 patients. Eight patients stopped medication before viral breakthrough. YMDD mutants appeared transiently despite the continuance of lamivudine therapy in 12 patients. In all 8 patients with breakthrough hepatitis, the quantities of YMDD mutants ranged from 102.7–104.7 copies/ml in the two to three months before clinical breakthrough. In contrast, in 12 patients without viral breakthrough, there were always less than 102.7 copies/ml YMDD mutants. Conclusions: Lamivudine-resistant viruses sometimes disappear even during lamivudine administration. Our sensitive quantitative assay proved useful for early detection of YMDD mutants and a threshold of 102.7 copies/ml is suggested for predicting viral breakthrough.

Novel single nucleotide polymorphisms of the cytokeratin 19 pseudogene are associated with primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by chronic inflammation and destruction of intra-hepatic bile ducts. However, the pathogenesis of PBC has not been fully delineated. We examined whether patients with PBC harbor genomic mutations of the cytokeratin 19 (CK19) gene since that gene is specifically expressed in biliary epithelial cells. Thirty-six patients with PBC, 26 patients with other liver diseases, and 36 healthy volunteers were enrolled in this study, but there were no significant differences in the genomic sequence of the CK19 gene between those groups. On the other hand, novel single nucleotide polymorphisms (SNPs) of the CK19 pseudogene, C341T, T524G and A754G, were frequently detected in PBC patients. These results suggest that those novel SNPs of the CK19 pseudogene may be associated with PBC and may prove useful for predicting susceptibility to PBC.

Lamivudine Therapy for Japanese Patients with Cirrhosis B

Objective: The aim of this study was to investigate the emergence of YMDD mutants in patients with chronic hepatitis B during lamivudine therapy and to compare the emergence patterns of YMDD mutants in cirrhotic and noncirrhotic patients. Methods: Eighteen cirrhotic and 37 noncirrhotic patients with chronic hepatitis B were studied. The emergence of YMDD mutants was determined before, as well as at 1, 3, 6, 9 and 12 months after treatment using a highly sensitive method based on polymerase chain reaction. Results: Although YMDD mutants were elicited early, the emergence of YMDD mutants was not always associated with breakthrough hepatitis. YMDD mutants appeared in cirrhotic and noncirrhotic patients: in 22 and 8% at 1 month, 13 and 21% at 3 months, 46 and 19% at 6 months, 30 and 19% at 9 months, and 83 and 27% at 12 months, respectively. Conclusion: YMDD mutants emerge more frequently in cirrhotic than noncirrhotic patients during the early period on lamivudine treatment. The highly sensitive method may be useful for monitoring the development of YMDD mutants in patients with chronic hepatitis B during lamivudine therapy.

Retreatment with interferon-α at dosages or period increased by 1.3 times is effective for treatment for transient responders and non-responders in chronic hepatitis C patients

Abstract The objectives of retreatment with interferon (IFN) in chronic hepatitis C (CH-C) patients are sustained response and a reduction in the risk of hepatocellular carcinoma (HCC). However, it is still unclear, as to which patients are candidates for retreatment with IFN. Eighteen transient responders (TRs) and 15 non-responders (NRs) to IFN therapy in CH-C received retreatment with IFNα. Of the 18 TRs, five showed sustained disappearance of hepatitis C virus, two showed sustained biochemical response, 10 continued as TR and one was a NR. Of the 15 NRs, six showed a TR while nine continued as NRs. Responsive cases, which included the virologically or biochemically sustained and transient responders, received either a dose of IFN 1.3 times greater or were treated for a period of 1.3 times longer in the retreatment than the original treatment. We submit that IFN treatment consisting of either a time period or a dosage 1.3 times those of the original IFN administration may be beneficial in the case of TR and NR in chronic hepatitis C patients.