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Liver-intestine cadherin (LI-cadherin) represents a novel type of cadherin within the cadherin superfamily that comprises seven cadherin repeats and a short cytoplasmic domain. In this study, we first examined LI-cadherin expression immunohistochemically in 34 specimens of human intrahepatic cholangiocarcinoma (ICC). LI-cadherin expression was positive (defined as positivity in > or = 10% of cells) in 18 of the ICCs (52.9%). LI-cadherin negativity was significantly correlated with tumor dedifferentiation (P=0.026) and vascular invasion (P=0.015). The cumulative survival rate of patients with LI-cadherin-negative ICC was significantly shorter than that of patients with LI-cadherin-positive ICC (P=0.021). Multivariate analysis identified the extent of LI-cadherin staining as an independent prognostic factor for ICC survival (P=0.027). Next, to elucidate the mechanism of loss of LI-cadherin-mediated aggressiveness in ICC, we knocked down LI-cadherin expression in an ICC cell line using small interfering RNA (siRNA) technology, and screened for genes that were expressed differentially between these cells and ICC cells transfected with scrambled siRNA using microarray analysis with real-time polymerase chain reaction confirmation. Among 21 identified genes, we focused on metal-responsive transcription factor-1 (MTF-1), whose target genes might contribute to tumor aggressiveness. Expression of placental growth factor (PlGF), one of the MTF-1 target genes, was up-regulated in the ICC cells transfected with LI-cadherin siRNA. Likewise, PlGF expression was up-regulated in LI-cadherin-negative ICC specimens. There was a significant inverse relationship between these expressions (P=0.033). Furthermore, the microvessel density of LI-cadherin-negative ICC specimens was higher than that of LI-cadherin-positive specimens. These findings suggest that loss of LI-cadherin in ICC is associated with tumor dedifferentiation and vascular invasion, and thus poor prognosis. Loss of LI-cadherin results in up-regulation of MTF-1 and PlGF, thereby regulating angiogenesis in ICC.

Aim: Hepatic stem cells are capable of dramatically changing and differentiating to form mature hepatocytes in acute and chronically damaged livers; however, the clinicopathological characteristics of these heterogeneous cell populations have not been sufficiently analyzed. Methods: In this study, cells in tissue sections from 12 cases of acute damaged livers and 31 cases of hepatocellular carcinomas (HCC), and the surrounding chronically damaged liver tissues, were analyzed by immunohistochemistry using the previously reported hepatic stem/progenitor cell marker CD133 (AC133) and the neural cell adhesion molecule (NCAM) marker. Results: In both the acute and chronically damaged livers, CD133+ cells and NCAM+ cells were present in ductular reactions (DR), which include hepatic stem/progenitor cells, and became more apparent in proportion to the degree of fibrosis or histological damage. Analysis of their distribution and morphological similarities revealed that the NCAM+ cell population included cells that were closer to, and morphologically more similar to, hepatocytes than were CD133+ cells. Analysis of HCC using these markers revealed that 9.7% of HCC expressed NCAM (two cases had abundant NCAM+ cells), while CD133+ HCC were not detected. Conclusion: These results suggest that CD133 and NCAM can be employed to enrich for hepatic stem/progenitor cells and that DR can be distinguished in greater detail using these markers. NCAM+ HCC were detected, but their function remains unresolved. Expression of CD133, a potent stem cell marker, may be extremely rare in the common human HCC examined.

The liver is a distinctive immune organ with predominant innate immunity, being rich in innate immune cells such as natural killer (NK) cells. In humans, NK cells comprise about 30%-50% of intrahepatic lymphocytes, whereas peripheral blood lymphocytes contain about 5%-20% NK cells. Accumulating evidence suggests that NK cells play an important role not only in host defense against invading microorganisms and tumor transformation in the liver but also in liver injury and repair. In recent years, significant progress has been made in terms of understanding how NK cells recognize their target cells and carry out their effector functions. It is now clear that NK cells are strictly regulated by numerous activating and inhibitory NK cell receptors that recognize various classes of cell surface ligands, some of which are expressed by normal healthy cells. Therefore, to further elucidate the involvement of NK cells in the pathogenesis of liver diseases, an understanding of recent advances in NK cell biology is crucial. This review provides an overview of recent advances in our knowledge of human NK cell receptors and their ligands in the context of liver diseases.

Aim: Intra-arterial cisplatin appears to have high therapeutic efficacy, but this has not been studied in detail. Accordingly, we developed a fine-powder cisplatin formulation and tested it in advanced hepatocellular carcinoma (HCC) patients in an open-label, uncontrolled study in which 27 institutions participated. Methods: The patients in this study had inoperable advanced HCC without extrahepatic metastases. All received two infusions of high-concentration cisplatin (1.43 mg/mL) via the hepatic artery at a dose of 65 mg/m2, with an intervening 4–6 week interval. Results: Cisplatin efficacy and safety were assessed in 80 patients. We found partial responses in 27 cases, no change in 37, and progressive disease in 11 (five were not evaluated). The overall response rate was 33.8%. The 1-year survival rate was 67.5% and the 2-year survival rate was 50.8%. Severe adverse effects (≥grade 3) included anorexia in 22.5%, vomiting in 6.3%, abdominal pain in 1.3%, thrombocytopenia in 25%, neutropenia in 13%, leukopenia in 1.3%, hypochromia in 1.3%, elevated serum aspartate aminotransferase in 32.5%, elevated serum alanine aminotransferase in 11.3%, elevated serum bilirubin in 3.8%, decreased serum albumin in 1.3%, elevated serum alkaline phosphatase in 1.3%, elevated γ-glutamyltranspeptidase in 3.8%, and elevated serum creatinine in 2.5%. Death from myocardial infarction occurred as an incidental event in one case, and no other life-threatening, adverse events were observed. Conclusion: Although intra-arterial cisplatin has substantial local and systemic toxicity, high therapeutic efficacy suggests the potential usefulness of this agent in the treatment of advanced HCC.

Aim: Previous studies have revealed that functional impairment of innate immune cells, including natural killer (NK) and natural killer T (NKT) cells, might be associated with the persistence of hepatitis C virus (HCV) infection. However, the involvement of innate immune cells, which predominate in the liver, in therapeutic HCV clearance is still unclear. Methods: To clarify the role of intrahepatic innate immune cells in the clinical outcome of patients with chronic hepatitis C (CHC) treated with interferon-α plus ribavirin (IFN/RBV), we prospectively investigated the status of NK and NKT cells in paired liver biopsy and peripheral blood (PB) samples obtained from 21 CHC patients before and immediately after IFN/RBV treatment by flow cytometry. Normal liver and PB samples were obtained from 10 healthy donors for living donor liver transplantation. Results: Before treatment, intrahepatic NK and NKT cells constituted a significantly lower proportion in CHC patients than in healthy individuals (P

The uricosuric drug benzbromarone (3,5-dibromo-4-hydroxyphenyl)-1-(2-ethyl-3-benzofuranyl)methanone, a benzofurane derivative, was studied for its effects on parameters related to hepatic peroxisome proliferation. Groups of male F-344 rats were fed either basal diet, the peroxisome proliferator clofibrate at 5000 ppm as a comparison compound, or benzbromarone at two doses, 1000 and 2000 ppm. Benzbromarone and clofibrate produced hepatomegaly and increases in the activities of catalase, acyl CoA oxidase, malate dehydrogenase, and glycerol-3-phosphate dehydrogenase. Benzbromarone and clofibrate also both induced similar histologic and ultrastructural changes in hepatocytes, including induction of peroxisomes. Therefore, benzbromarone acted as a peroxisome-proliferating agent in rats under these conditions. Benzbromarone differs from other peroxisome proliferators in its chemical structure, uricosuric action, and the morphology of liver peroxisomes that were induced by exposure.

The liver has a high potential to regenerate however, the relation between oval cells and endothelial cells in the portal area during liver regeneration has not been adequately described. We have focused on sca-1+ endothelial cells (SPEC: sca-1+CD31+CD45- cells) and analyzed their localization, growth potential, and the role of these cells in damaged liver. SPECs are localized in the portal area and comprise approximately 20-30% of CD31+CD45- cells. These cells have higher growth potential than sca-1- endothelial cells and grow aggressively when the liver is severely damaged on the lateral side of the oval cells. In an in vivo study we show that when the liver is severely damaged in the presence of a VEGF (vascular endothelial growth factor)-inhibitor, the frequency of SPECs decreased and the recovery of liver volume was also delayed. These results strongly suggest that SPECs play important roles in the recovery of severely damaged liver.

We describe a 64-year-old man with decompensated hepatitis B virus (HBV)-related cirrhosis who became resistant to lamivudine. He was started on adefovir at 10 mg daily while continuing lamivudine therapy. Several months later, his liver function improved and subsequently his ascites disappeared. The serum HBV-DNA level became undetectable 11 months later. Twenty months after the start of additional treatment with adefovir, one hepatocellular carcinoma (HCC) was detected, and the patient underwent a successful hepatectomy. Our findings suggest that the addition of adefovir to ongoing lamivudine therapy is useful for improving liver function in patients with decompensated lamivudine-resistant HBV-related cirrhosis, allowing surgery for HCC.

The aim of this study was to determine to what extent hypermethylation of the p16 INK4A (p16) gene promoter is increased in nontumorous liver tissues compared with in normal liver, using two quantitative methylation-specific polymerase chain reaction (MS-PCR) methods and a bisulfite sequencing method. Methylation of the p16 gene was detected more frequently in nontumorous liver than in normal liver using the TaqMan PCR method. Methylation indices also were significantly higher in nontumorous than in normal liver. However, the bisulfite sequencing method did not detect significantly more methylation of the p16 gene in nontumorous than normal liver, nor was there a significant difference in the level of p16 mRNA. There may be a greater proportion of cells which contain methylated p16 in nontumorous than in normal liver. However, the difference was so small that the functional relevance to hepatocarcinogenesis remains elusive.

Backgrounds/Aims Toll-like receptors (TLRs) have emerged as a key component of the innate immune system that triggers antimicrobial responses. Altered monocyte responses to ligands for TLRs have been reported in patients with primary biliary cirrhosis (PBC), yet the precise mechanism remains unknown. Methods We investigated in vitro responses to a TLR4 ligand, lipopolysaccharide (LPS), using peripheral blood mononuclear cells and monocytes from 25 patients with PBC, 10 patients with chronic viral hepatitis (CVH), and 20 healthy individuals. Results After stimulation with LPS, we found significantly higher amounts of IL-1β, IL-6, and IL-8 production in PBC patients. Through the TLR4 signaling pathway, activation of NF-κB and expression of MyD88 mRNA were significantly increased in PBC patients, and the level of TLR4 expression was significantly increased on PBC monocytes as compared with CVH patients and controls. Of significance, the surface expression of RP105, which has recently been shown to be involved in negative regulation of TLR4 signaling, on PBC monocytes was significantly decreased in comparison with CVH patients ( P =0.016) and controls ( P Conclusions These results suggest that expression of RP105 and TLR4 is altered on PBC monocytes, which appear to be hypersensitive to LPS, resulting in increased secretion of pro-inflammatory cytokines.

Fulminant hepatic failure (FHF) is a dramatic clinical syndrome characterized by massive hepatocyte apoptosis and very high mortality. The c-Jun-N-terminal kinase (JNK) pathway is an important stress-responsive kinase activated by several forms of liver injury. The aim of this study is to assess the role of JNK during D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury, an experimental model of FHF, using SP600125, a small molecule JNK-specific inhibitor. Mice were given an intraperitoneal dose of GalN (800 microg/g body weight)/LPS (100 ng/g body weight) with and without subcutaneous SP600125 (50 mg/kg body weight) treatment (at 6 and 2 h before and 2 h after GalN/LPS administration). GalN/LPS treatment induced sustained JNK activation. Administration of SP600125 diminished JNK activity, suppressed lethality and the elevation of both serum alanine aminotransferase and aspartate aminotransferase, but had no effect on serum tumor necrosis factor-alpha, and reduced hepatocyte apoptosis after GalN/LPS administration. In support of the role of JNK in promoting the mitochondria-mediated apoptosis pathway, SP600125 prevented cytochrome c release, caspase-9 and caspase-3 activity. Moreover, SP600125 downregulated the mRNA and protein expression of Bad in the early periods following GalN/LPS injection and prevented Bid cleavage in the late periods. These results confirm the role of JNK as a critical apoptotic mediator in GalN/LPS-induced FHF. SP600125 has the potential to protect FHF by downregulating Bad and inhibiting Bid cleavage.

The human hepatocellular carcinoma (HCC)-derived cell line KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastasis of human HCC, because it often shows cell scattering in vitro and intrahepatic metastasis in vivo. We previously found that integrin-mediated extracellular signals inactivated E-cadherin in KYN-2, and caused loss of cell-cell contact with gain of cell motility, which is considered to be a critical step in the process of cancer cell invasion and metastasis. To further understand molecular mechanisms involved in biological aggressiveness of HCC, we investigated intracellular signaling involved in integrin-mediated scattering of KYN-2 cells. Cultured KYN-2 cells formed trabecular aggregates in suspension, but when adhering to integrin-stimulating substrata, they scattered according to phosphorylation of extracellular signal-regulated kinase (ERK). Upon treatment with ERK kinase (MEK) inhibitor PD98059, adhered KYN-2 cell scattering was inhibited, tight cell-to-cell contact was recovered, and both E-cadherin and actin filaments accumulated in the area of intercellular contact zone. In contrast, constitutively active MEK1-transfected KYN-2 cells showed reduced E-cadherin and actin filaments in the intercellular contact zone, showing a flattened phenotype with broad lamellipodia. Enforced signaling of MEK-ERK pathway in KYN-2 cells suppressed cadherin-mediated homotypic adhesion and increased the potential of cell motility. An antibody-based protein microarray analysis revealed that the cytoplasmic protein c-Cbl was significantly downregulated in MEK1-transfected KYN-2 cells, suggesting that c-Cbl might be a candidate downstream mediator of integrin/MEK/ERK-mediated cell scattering. In conclusion, cell scattering of the highly metastatic cell line KYN-2 is regulated through the integrin-MEK-ERK signaling cascade, suggesting that this molecular pathway may be critical in intrahepatic metastasis of human HCC.

The incidence of hepatocellular carcinoma among patients who have seroconverted to anti-hepatitis B surface antigen (anti-HBs) remains controversial.We report four patients with chronic hepatitis B virus (HBV) infection who had cleared HBsAg and had developed anti-HBs at a later time, but who developed hepatocellular carcinoma (HCC) eventually.The common clinicopathological characteristics of the four patients were: An established diagnosis of precirrhosis or liver cirrhosis more than a decade previously, a long-standing normalization or stabilization at a low level of ALT values due to undetectable HBV DNA by the Amplicore Monitor assay, and a marked reduction of the fibrosis level in the non-tumorous liver obtained at HCC surgery or autopsy compared to the previous histology more than a decade previously. There was no fibrosis in the needle biopsy specimen from one patient.Our findings suggest that HCC due to HBV can occur in the serologically-cured stage if progression to pre-cirrhosis or cirrhosis already has occurred, where the fibrosis level has improved considerably because of the long-term absence of active HBV viremia and inflammation. Active medical intervention to prevent liver cirrhosis for chronic hepatitis B may have an important role in the inhibition of HCC in patients with chronic hepatitis B.

Prognosis of recurred HCV recipient after both deceased and live liver transplantation is quite worse in foreign countries, recently. The reason of this poor prognosis is believed as immune-suppressant, steroid bolus, aged donor and liver donor in USA and Europe. However, in Japan, survival ration of HCV recipients is 5% reduction than mean survival of live donor liver transplantation. In addition, post transplant treatment of recurred HCV recipient is not good result in out of Japan, because of high rate of withdraw or drop out with peg interferon and ribavirin. On the contrary, some of Japanese investigator reported with 40-50% SVR with same drug management. It is still controversy whether HCV recipients are poor prognosis after transplantation and treatment resistance.

For patients with hepatocellular carcinoma (HCC), the selection of effective therapeutic options depends on a reliable prognostic assessment of both the tumor characteristics and liver impairment. This study sought to provide a modified Japan Integrated Stage (m-JIS) score to predict more accurately the survival of patients with HCC.We analyzed the records of 42,269 patients diagnosed with HCC that were registered between 1992 and 1999 in a nationwide Japanese database. The m-JIS score was calculated from the tumor-node-metastasis stage and the grade of liver damage as defined by the Liver Cancer Study Group of Japan. The predictive accuracy for patient survival based on the m-JIS score was compared with that determined by the modified Cancer of the Liver Italian Program (m-CLIP) score using the cross-validation method.Patients were divided randomly into two groups, a training sample for construction of prediction models (n = 21,127 patients with 8,458 deaths) and a validation sample for assessment of those prediction models (n = 21,142 patients with 8,434 deaths). Both the m-JIS score and the m-CLIP score showed good discriminatory ability in the training sample. In the validation sample, the residuals of prediction based on the m-JIS score were smaller than those of the m-CLIP score (P0.0001).The m-JIS scoring system had better predictive accuracy than the m-CLIP score system for survival of Japanese patients with HCC.

In this study we evaluated the potential role of preoperative h-TERT mRNA expression in peripheral blood as a tool for predicting prognosis and tumor recurrence after living-related liver donor transplantation (LRLDT).The study included patients with unresectable HCC who underwent LRLDT from July 1999 to May 2003.There was no significant difference between the survival curves of those patients who met the Milan criteria and those who did not. However, there was a statistically significant difference (p=0.032) between the survival curves of those patients with positive preoperative h-TERT mRNA expression, and those who either had an initially negative preoperative h-TERT mRNA or who converted from positive to negative after neoadjuvant immunochemotherapy.In conclusion, the presence or absence of h-TERT mRNA in the peripheral blood may be a useful criterion in evaluating HCC patients for transplantation, as well as a valuable method of assessing anti-tumor therapy and tumor relapse.

Background & Aims: To elucidate the survival of the patients with unresectable hepatocellular carcinoma (HCC) who underwent transcatheter arterial lipiodol chemoembolization (TACE) and to analyze the factors affecting the survivals. Methods: During the last 8 years, a nationwide prospective cohort study was performed in 8510 patients with unresectable HCC who underwent TACE using emulsion of lipiodol and anticancer agents followed by gelatin sponge particles as an initial treatment. Exclusion criteria were extrahepatic metastases and/or any previous treatment prior to the present TACE. The primary end point was survival. The survival rates were calculated by the Kaplan–Meier method. The multivariate analyses for the factors affecting survival were evaluated by the Cox proportional hazard model. The mean follow-up period was 1.77 years. Results: For overall survival rates by TACE, median and 1-, 3-, 5-, and 7-year survivals were 34 months, 82%, 47%, 26%, and 16%, respectively. Both the degree of liver damage and the tumor-node-metastasis (TNM) system proposed by the Liver Cancer Study Group of Japan demonstrated good stratification of survivals (P = .0001). The multivariate analyses showed significant difference in degree of liver damage (P = .0001), α-fetoprotein value (P = .0001), maximum tumor size (P = .0001), number of lesions (P = .0001), and portal vein invasion (P = .0001). The last 3 factors could be replaced by TNM stage. The TACE-related mortality rate after the initial therapy was .5%. Conclusions: TACE showed safe therapeutic modality with a 5-year survival of 26% for unresectable HCC patients. The degrees of liver damage, TNM stage, and α-fetoprotein values were independent risk factors for patient survival.

The human liver contains significant numbers of innate immune cells, such as natural killer (NK) cells and natural killer T (NKT) cells, which express both T-cell receptors and NK-cell receptors simultaneously. It has been suggested that the innate immune system plays a crucial role in the liver. In this report, the distribution of NK and NKT cells in the liver and peripheral blood of two patients with drug-induced fulminant hepatic failure (FHF) who had undergone living donor liver transplantation was examined. In both the liver and peripheral blood, the proportions of NK and NKT cells markedly decreased compared with those in healthy donors. It was also revealed that, unlike murine NKT cells, human CD56(+) T cells and CD57(+) T cells did not constitutively express CD28, which is one of the important costimulatory molecules on T cells. Additionally, the residual CD56(+) T cells and CD57(+) T cells in the patients expressed more CD28 than in controls. This result suggests that NKT cells might be more activated in FHF. Although the accumulation of further cases is required, it is suggested that both NK and NKT cells might be involved in hepatic injury in FHF.

Acute renal failure after liver transplantation can occur in some and is an important postoperative complication. Our goal is to clarify the risk factors of acute renal failure after living-related donor liver transplantation (LDLT).From March 1999 to August 2000, ten consecutive patients were investigated the changes of the systemic hemodynamics and the renal function. They were classified into Group A (Creatinine (Cre) was over 2.0 mg/dL) and B (Cre was below 2.0 mg/dL). Retrospective variables were examined with two groups A and B being compared.In both groups, Cardiac Index (CI) was above standard levels. However, the CI levels in Group B were significantly higher than those in Group A (p=0.031). The early postoperative transaminase levels were significantly higher in Group A than in Group B (p=0.049) and graft liver volume/recipient body weight ratio was significantly smaller in Group A than in Group B (p=0.016).Our study suggests that small-for-size graft or hypovolemia, resulting in the delay of the recovery of graft liver function, may be an important cause of acute renal failure during the early postoperative period in adult LDLT.

BACKGROUND Advances in the diagnosis and surgical treatment of hepatocellular carcinoma (HCC) have improved the prognosis for patients with HCC who undergo liver resection. The objective of this study was to evaluate prognostic predictors for patients with HCC who underwent liver resection in a Japanese nationwide data base. METHODS In this study, the authors analyzed 12,118 patients with HCC in a Japanese nationwide data base who underwent liver resection between 1990 and 1999 and compared them with a previous analysis of patients between 1982 and 1989. All patients were evaluated for prognostic factors. RESULTS During the last decade, the increases in patients who were without hepatitis B virus surface antigen, who had small tumors, and who had portal vein invasion were noted. The 5-year overall survival rates for patients with HCC improved to 50.5%, compared with < 40% in the previous analysis. A multivariate analysis using a stratified Cox proportional hazards model according to associated liver disease indicated that age, degree of liver damage, α-fetoprotein level, maximal tumor dimension, number of tumors, intrahepatic extent of tumor, extrahepatic metastasis, portal vein invasion, hepatic vein invasion, surgical curability, and free surgical margins were independent prognostic predictors for patients with HCC. Operative mortality decreased from 2.3% in 1990–1991 to 0.6% in 1998–1999. CONCLUSIONS Outcomes and operative mortality rates in patients with HCC improved during the last decade. Age, degree of liver damage, α-fetoprotein level, maximal tumor dimension, number of tumors, intrahepatic extent of tumor, extrahepatic metastasis, portal vein invasion, hepatic vein invasion, surgical curability, and free surgical margins were prognostic factors for patients with HCC who underwent liver resection. Cancer 2004. © 2004 American Cancer Society.

We investigated the influence of HTK solution against natural killer T cells and thymic T cells in liver graft before and after perfusion in adult living related donor liver transplantation.Graft samples were obtained before liver resection, after perfusion, and one hour after liver transplantation. Flowcytometry analysis was conducted using several human natural killer markers; CD16, CD56, CD57, and CD161.Natural killer T cells existed prominently in the liver leukocytes compared with their presence in peripheral blood lymphocytes, and the difference was significant. CD56+ T and CD161+ T cells, in comparison with CD16+ T cells and CD57+ T cells, were especially numerous in the liver. The proportion of CD56+ T and CD161+ T cells increased in the graft immediately after perfusion with HTK solution. However, CD16+ T cells and CD57+ T cells decreased in the graft immediately after perfusion and reperfusion of portal blood flow. Thymus-derived cells also decreased significantly after perfusion. The proportion of CD56+ T cells among CD3+ cells showed a significant increase immediately after perfusion. All types of natural killer cells in the graft immediately increased after perfusion by HTK solution and reperfusion of portal blood flow. Compared with CD57+ NKT cells, CD56+ NKT cells showed a significant tendency to stay in the liver graft against the perfusion. CD57+ NKT cells tended to wash out from the liver into the systemic circulation. Moreover, thymus-derived T cells showed the strongest tendency to wash out from the liver graft.CD56+ NKT cells and natural killer cells are more involved in local immunity, whereas thymus-derived cells and CD57+ NKT cells are involved in regulation of systemic immunity. Alloimmunity between local and systemic systems may be affected by the dynamic changes in hepatic circulation associated with living related donor liver transplantation.

Antigens given orally or through the portal vein are known to be less immunogenic and to induce immunologic unresponsiveness. The mechanisms responsible for graft enhancement are still unclear. Moreover, in actuality, it is difficult to perform transfer of donor antigens via the portal vein in clinical transplantation. We investigated the effect of transfer of donor blood via the portal vein intra- and post-operatively in living related donor liver transplantation for recurrent multiple hepatocellular carcinoma. A 62-year-old female, who suffered from recurrent multiple hepatocellular carcinoma with hepatitis C virus, underwent living related donor liver transplantation with the right lobe of her daughter. Eleven hepatocellular carcinomas were recognized in the resected specimen. Donor blood was administered via the portal vein using a catheter inserted in the middle colic vein intra- and postoperatively. Mononuclear cells were obtained by operative liver biopsy or postoperative biopsy using fine needle aspiration biopsy, and from peripheral blood. They were analyzed by two or three color-flow cytometry using several antibodies. The differentiation between donor and recipient was estimated by means of anti-HLA antibodies of donor and recipient. The postoperative course was uneventful. She did not suffer from acute cellular rejection and was discharged on day 30 the after operation. CD56+ CD3+ T cells in the liver increased notably from 20% to 50% after transplantation. One half of the CD56+ CD3+ T cells in the liver graft were of the donor type (donor anti-HLA A2 antibody) on day 8 after surgery. Donor type CD56+ CD3+ T cells occupied 17.4% of the total CD56+ CD3+ T cells even on day 42 after the operation. Stimulation index by mixed lymphocyte reaction continued at a low level (2) from day 1 after the operation. Steroids were discontinued after 40 postoperative days. FK506 was also reduced to 0.5 mg/day 4 months after the operation. There was no recurrence of hepatocellular carcinoma and hepatitis C virus for two years after the operation. Macrochimerism of donor type CD56+ CD3+ T cells in a graft might be induced by the transfer of donor blood via the portal vein and may play an important role in transplantation tolerance. Inoculation of donor blood via the portal vein may also be very useful for rapid reduction of immunosuppression.

Adult living related liver transplantation seeks a balance between donor safety and the need to save the recipient's life. A small-for-size graft is a major obstacle for high-risk patients. We experienced a case of heterotopic auxiliary partial liver transplantation with extremely small-for-size graft for fulminant liver failure. The other reasons why we chose to perform heterotopic auxiliary partial liver transplantation were acute renal failure, subshock state, and a left lobe volume of 24% in the standard liver volume of the donor. Hepatic vein reconstruction was made using an inferior meserteric vein patch graft. Portal vein reconstruction was made using end-to-side anastomosis employing an interposed left external iliac vein. The left hepatic artery of the graft was connected to the distal gastroduodenal artery. The patient was discharged 3 months after transplantation. We would recommend heterotopic auxiliary partial liver transplantation as an optional procedure for patients with severe preoperative conditions or extremely small-for-size graft donors.

We report a 54-year-old Japanese woman who developed liver tumors 102 months after hepatic resection for hepatocellular carcinoma (HCC) and percutaneous transluminal angioplasty (PTA) for membranous obstruction of the inferior vena cava (MOVC), which is one form of Budd-Chiari syndrome. In the present admission workup showed no evidence of co-infection with hepatitis B and C viruses. Dynamic computed tomography (CT) and magnetic resonance imaging showed an enhanced lesion, 1.5 cm in diameter, in segment 3 of the liver, and no obstruction of the inferior vena cava after PTA. CT during both arterial portography and hepatic arteriography revealed another lesion, showing different hemodynamics, in segment 2. The patient therefore underwent hepatic resection, and the tumors were diagnosed histologically as HCC. The two tumors differed in their morphological features, one containing abundant fibrous stroma, whereas the other did not. The nontumorous liver tissue showed central zonal fibrosis, i.e., reversed lobulation, and partial expansion of nodule-like formations, indicating lack of progression since the situation seen at the initial hepatectomy. The presence of nontumorous liver tissue showing the above features suggests that, even after successful treatment for relief of congestion, patients who have had MOVC should be followed closely for as long as possible because of the risk of HCC recurrence. This is the first reported case of HCC recurrence after successful treatment of MOVC.

p27(Kip1) (p27) might act as an adverse prognostic marker for various types of cancers. However, its clinical usefulness remains uncertain, because it is sometimes overexpressed in aggressive types.To precisely evaluate the practical significance of p27 in hepatocellular carcinoma (HCC), we immunohistochemically compared the level of p27 expression with Ki-67 labeling in 74 HCCs and focused on tumors in which cell proliferation increased despite a high level of p27 expression. We then analyzed the status of p27 and related cell-cycle regulators using kinase and immunoprecipitation assays, Western blotting, and methylation-specific PCR to understand the rationale for the functional inactivation of p27 in HCC. We also evaluated relationships between the key biological characteristics of HCC and survival.Immunohistochemical studies showed that 40 (54%) of 74 HCCs expressed high levels of p27 (50% of the tumor cells). Of these, the Ki-67 labeling index was low (20%) in 26 (65%) and high (20%) in 14 (35%). Increased proliferative activities were closely correlated with elevated kinase activities, sequestration of p27 protein, and p16 gene methylation. The association between a loss of p16 and poor prognosis was significant when p27 expression was high (P0.01).The loss of p16 appears to be closely related to the functional inactivation of p27, and assessment of p16 status may be useful for a precise prognostic prediction of individuals with HCC expressing high levels of p27.

Clinical background and incidence of hepatocellular carcinoma (HCC) of patients with chronic hepatitis C who obtained biochemical remission without eradication of virus (biochemical response) after interferon (IFN) treatment was retrospectively analyzed for 755 patients. Annual incidence of HCC was significantly lower in the patients with biochemical response and sustained response than that of the patients that did not show these responses. Logistic regression analysis showed that only the normalization of alanine aminotransferase (ALT) value at the end of IFN treatment was a significant factor for biochemical response. Annual incidence of HCC was significantly lower in the patients who obtained normalization of ALT values at the end of treatment than those who did not. Patients who were younger, who had a lower level of activity and fibrosis indices in histology, higher platelet count, and who were given more higher total dose of IFN were more likely to attain normalization of ALT levels at the end of treatment, and this was related to biochemical response. Low incidence of HCC in patients who obtained normalization of ALT values at the end of treatment was likely because they were in the earlier stage of chronic hepatitis. Active treatment of chronic hepatitis C with interferon in the early phase of the disease may bring about a biochemical response in some patients, even if sustained virological response is not obtained.

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.

Oral or portal administration of allogeneic antigens downregulates the alloimmune response and prolongs graft survival following organ transplantation. However, the effect of donor-specific transfusion via the portal vein has been reported in rodent models, but has not been reported in human cases. We investigated whether donor-specific transfusion via the portal vein would bring up the clinical and immunological benefits in living-related donor liver transplantation.Eighteen patients who underwent living-related donor liver transplantation from March 1999 to December 2001, were investigated. Seven patients were given the Tac + steroid regimen (IP(-) group: n = 7, mean age 54 +/- 9 yo). Eleven patients had postoperative repeated donor specific transfusion performed via a portal venous catheter inserted from vena colica media besides from the Tac + steroid (IP(+) group: n = 11, mean age 45 +/- 15 yo). The clinical effects of the reduction of immunosuppression and the rejection, and the immunological analysis were studied in the two groups.Total amount of methylprednisolone and prednisolone within one month in the IP(+) group was smaller than that in the IP(-) group with statistical significance. Amount of Tac within one month and Trough level of Tac was statistically smaller in the IP(+) group than that in the IP(-) group. Minimum dose of Tac in the IP(+) group was clearly smaller than that in the IP(-) group with statistical significance. The frequency of acute cellular rejection within one month and after one month or total frequency of acute cellular rejection in the IP(+) group tended to be less than that in the IP(-) group. Macrochimerism of donor type CD56+ T cells in a graft were confirmed in patients with donor-specific transfusion via the portal vein. Conversely recipient type CD56+ T cells increased in the graft liver in patients without donor-specific transfusion. IL-10 production of the donor-specific transfusion(+) group was higher than that of the donor-specific transfusion(-) group on day 1 after living-related donor liver transplantation.The repeated donor-specific transfusion via the portal vein has brought the rapid reduction of immunosuppressants. Donor type natural killer T cells especially CD56+ T cells, may induce tolerance by Veto mechanism and anti-idiotype network mechanism. These benefits might introduce more advantages in frequency of complications and cost of transplantation.

Hepatitis B virus (HBV) mutants with deletions in the preS region have not been evaluated for association with viral genotypes. In a case-control study, HBV DNA samples collected from 80 each of carriers infected with HBV genotype B or C were examined for preS deletions. PreS deletion mutants were found in a total of 37 of 160 (23%) HBV carriers. Carriers with preS deletion mutants were older (56.0 ± 12.7 vs 49.3 ± 16.9 years, P

Reduced sensitivity of HBV to lamivudine, which causes a viral breakthrough during treatment, is attributed to mutations within the tyrosine-methionine-aspartate(YMDD) locus in the reverse transcriptase(rt) domain of HBV polymerase, mainly a methionine(rtM204) substitution. The sensitive detection of such mutations before or early in treatment could assist in optimizing antiviral treatment. For this purpose, we developed peptide nucleic acid(PNA) mediated polymerase chain reaction(PCR) clamping(PMPC) with a PNA probe targeting the YMDD locus. We first tested this method for its sensitivity and specificity in detecting a mutant on HBV DNA standards consisting of serial copy number ratios of a known lamivudine-resistant, mutant clone with rtM204I(ATT) to a wild-type clone with rtM204(ATG). The sensitivity was 0.1 to 0.01% in the coexistence of wild-type clones and the specificity was guaranteed by direct sequencing of the products. We next applied this method to HBV DNA specimens extracted from serum from 4 chronic hepatitis B patients treated with lamivudine. Two of these exhibited a break-through of the HBV mutant with rtM204I(ATT), while the other 2 did not. Before treatment, all 4 patients showed HBV with rtM204I encoded by ATA. During treatment, HBV with the rtM204I(ATT) emerged in the 2 breakthrough patients more than 3 months before the breakthrough, whereas this and other known lamivudine-resistant viruses did not appear in the 2 non-breakthrough patients. Thus, our PMPC-direct sequencing method is highly sensitive and reliable for the early identification of lamivudine-resistant HBV that causes a viral breakthrough.

BACKGROUND AND AIM: Thymosin-alpha1 (T-alpha1) influences T-cell maturation, production of Th1-type cytokines, and activity of NK cell-mediated cytotoxicity. The aim of this study is to evaluate the types of lymphocytes that contribute to the reduction in viral load in patients with chronic hepatitis B (CHB) following T-alpha1 treatment. METHODS: Seven patients with CHB were treated with 1.2 mg of T-alpha1 for 24 weeks. Peripheral blood lymphocytes (PBL) and intrahepatic lymphocytes were analyzed by flow cytometry. Serum cytokines (IL-4 and IFN-gamma) were measured by ELISA. RESULTS: Forty-eight weeks after T-alpha1 treatment, two patients (28.6%) showed normalized alanine aminotransferase and decreased HBV-DNA to undetectable level from serum. The histology activity index score significantly decreased (P

In this report, we examine two patients with chronic hepatitis B virus (HBV) infection that had been diagnosed as precirrhosis or liver cirrhosis more than a decade previously. These patients had been cleared of HBsAg and had developed anti-HBs at a later time, yet hepatocellular carcinoma (HCC) eventually occurred. Both patients had been found negative for HBV DNA, using sensitive methods. Interestingly, a nontumor specimen of the liver obtained at surgical resection showed a marked reduction of fibrosis when compared to the histology observed when the patient was diagnosed as precirrhosis. Our findings suggest that the fibrosis from liver cirrhosis had been absorbed to a large extent during the long-term absence of active viremia and the normalization of alanine aminotransferase (ALT) levels. However, the cancer-prone biological characteristics of liver cirrhosis remained. Thus, patients with liver cirrhosis due to past chronic hepatitis B should be monitored carefully for the development of HCC even if HBV infection has been serologically resolved.

We wanted to differentiate small hypervascular hepatocellular carcinoma (HCC) from hypervascular pseudolesion (HPL) on magnetic resonance imaging (MRI).We reviewed small hypervascular foci (or = 2 cm in diameter) on dynamic MRI in patients with chronic liver disease, which were followed-up with serial MRI examinations.Twenty of 34 hypervascular foci were larger at follow-up; 19 of 20 foci had characteristics suggesting HCC; and 14 foci did not grow or disappeared and were judged to be HPLs. There were no differences in the initial sizes and follow-up periods between HCCs and HPLs. On initial MRI, nine of 19 HCCs (47%) and one of 14 HPLs (7%) appeared hyperintense on T2-weighted images. The difference between HCCs and HPLs on T2-weighted images was statistically significant (p = 0.039).HPLs are seen frequently as small hypervascular foci on dynamic MRI in patients with chronic liver disease. Hyperintensity of the foci on T2-weighted images differentiates HCCs from HPLs.

Thrombocytopenia is one of the major complications of liver cirrhosis. Except for hypersplenism associated with portal hypertension, it is not known which abnormalities of thrombopoiesis cause thrombocytopenia. To evaluate thrombopoiesis in liver cirrhosis, we analyzed thrombopoietin (TPO) and its receptor c-Mpl levels in cirrhotic patients. Expression of c-Mpl on platelets and the serum level of TPO were investigated from 38 patients with various stages of liver cirrhosis by flow-cytometric analysis and enzyme-immuno assay. Samples obtained from 22 individuals without evidence of liver disease were used as controls. Neither platelet counts nor TPO levels correlated with disease progression defined by the Child-Pugh classification. c-Mpl was constitutively expressed on the platelets of cirrhotic patients, and its expression level was reduced with disease progression defined by the Child-Pugh classification. In this study, serum TPO did not fluctuate according to the grade of cirrhosis. However, its receptor c-Mpl, which is expressed on platelets, was decreased significantly in severely cirrhotic patients with thrombocytopenia. Thus, a correlation between reduced c-Mpl expression and the progression of liver cirrhosis was demonstrated. We conclude that, in addition to hypersplenism, the reduced expression of c-Mpl may play a significant role in the thrombocytopenia observed in severe liver cirrhosis.

The entire nucleotide sequences of 70 hepatitis B virus (HBV) isolates of genotype B (HBV/B), including 38 newly determined and 32 retrieved from the international DNA database (DDBJ/EMBL/GenBank), were compared phylogenetically. Two subgroups of HBV/B were identified based on sequence divergence in the precore region plus the core gene, one with the recombination with genotype C and the other without it. The analysis over the entire genome of HBV/B by the SimPlot program located the recombination with genotype C in the precore region plus the core gene spanning nucleotide positions from 1740 to 1838 to 2443 to 2485. Within this genomic area, HBV/B strains with the recombination had higher nucleotide and amino acid homology to genotype C than those without the recombination (96.9 versus 91.1% in nucleotides and 97.0 versus 92.9% in amino acids). There were 29 HBV/B strains without the recombination, and they were all recovered from carriers in Japan. The remaining 41 HBV/B isolates having the recombination with genotype C were from carriers in China (12 strains), Hong Kong (3 strains), Indonesia (4 strains), Japan (3 strains), Taiwan (4 strains), Thailand (3 strains), and Vietnam (12 strains). Due to the frequency of the distribution of HBV/B without the recombination (29 of 32 isolates, or 91%) and the fact that it was exclusive to Japan, it was provisionally classified into the Bj (j standing for Japan) subgroup, and HBV/B with the recombination was classified into the Ba (a for Asia) subgroup. Virological differences between HBV/Bj and HBV/Ba may be reflected in the severity of clinical disease in the patients infected with HBV of genotype B, which seems to be under strong geographic influences in Asia., published_or_final_version

In Japan, most cases of gastric carcinoid tumor (GCT) are unassociated with either autoimmune gastritis (AIG) showing type-A chronic atrophic gastritis (CAG-A) or Zollinger-Ellison syndrome (ZES). However, the pathogenesis of this tumor remains unknown. Recent studies have determined that Helicobacter pylori infection induces gastric carcinoid in Mongolian gerbils and that H. pylori lipopolysaccharide exerts a mitogenic effect on ECL cells. We examined five patients with histologically diagnosed GCT, 40 patients with H. pylori-positive gastric ulcer (Hp+GU), 24 patients with H. pylori-positive duodenal ulcer (Hp+DU), and 12 patients with AIG showing CAG-A topographically. We compared the prevalence of H. pylori infection, and the levels of gastrin and pepsinogen (PG) in the serum of patients with GCT with those of patients with Hp+GU, or Hp+DU, and AIG. We also investigated the histological characteristics of the tumor and the gastric corpus mucosa in the GCT patients. The levels of serum gastrin and PG I and II were measured using an RIA kit. In all five (100%) patients with GCT, H. pylori infection was present, without any evidence of AIG or ZES. The serum levels of gastrin in the GCT patients were higher than those in either Hp+GU or Hp+DU patients and lower than those in the AIG patients. In contrast, serum PG I levels and the PG I/II ratio were lower in the GCT group than in the Hp+GU or Hp+DU groups. Histologically, all GCTs were ECL cell tumors and peritumoral corporal mucosal atrophy was observed in four of the five patients with GCT. In conclusions, H. pylori infection and hypergastrinemia were found in the patients with GCT without AIG. This finding suggests that H. pylori infection may induce corporal mucosal atrophy and hypergastrinemia that can produce a GCT with time.