Oral berotralstat for the prophylaxis of hereditary angioedema attacks in patients in Japan: A phase 3 randomized trial.

BACKGROUND: With no approved treatments in Japan for the prevention of hereditary angioedema (HAE) attacks, there is a significant unmet need for long-term prophylactic therapies for Japanese patients with HAE. Berotralstat (BCX7353) is an oral, once-daily, highly selective inhibitor of plasma kallikrein in development for prophylaxis of angioedema attacks in HAE patients. METHODS: APeX-J is a phase 3, randomized, double-blind, placebo-controlled, parallel-group, 3-part trial conducted in Japan (University Hospital Medical Information Network identifier, UMIN000034869; ClinicalTrials.gov identifier, NCT03873116). Patients with a clinical diagnosis of type 1 or 2 HAE underwent a prospective run-in period of 56 days to determine eligibility, allowing enrollment of those with ≥2 expert-confirmed angioedema attacks. Patients were randomly assigned (1:1:1) and stratified by baseline attack rate (≥2 vs. <2 expert-confirmed attacks/month between screening and randomization) to receive once-daily berotralstat 110 mg, berotralstat 150 mg, or placebo. The primary endpoint was the rate of expert-confirmed angioedema attacks during dosing in the 24-week treatment period. RESULTS: Nineteen patients were randomized to receive once-daily berotralstat 110 mg (n = 6), berotralstat 150 mg (n = 7), or placebo (n = 6). Treatment with berotralstat 150 mg significantly reduced HAE attacks relative to placebo (1.11 vs. 2.18 attacks/month, p = .003). The most frequently reported treatment-emergent adverse events (TEAEs) in berotralstat-treated patients (n = 13) were nasopharyngitis (n = 4, 31%), abdominal pain, cough, diarrhea, and pyrexia (n = 2 each, 15%). CONCLUSIONS: Orally administered, once-daily berotralstat 150 mg significantly reduced the frequency of HAE attacks and was safe and well tolerated, supporting its use as a prophylactic therapy in patients with type 1 or 2 HAE in Japan.

The combination therapy of α-galactosylceramide and 5-fluorouracil showed antitumor effect synergistically against liver tumor in mice.

α-Galactosylceramide (α-GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo-immunotherapy using α-GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α-GalCer and 5-fluorouracil (5-FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor-bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5-FU or α-GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α-GalCer treatment induced significant activation of liver NK cells in vivo, but 5-FU treatment did not. 5-FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae-1 and H60) and DNAM-1 ligands (CD112 and CD155) on MC38 cells, but α-GalCer did not. The cytolytic activity of α-GalCer-activated liver mononuclear cells against 5-FU-treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5-FU partially depended on NKG2D-Rae-1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5-FU against MC38 liver tumors, which suggested that the antitumor effect of 5-FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α-GalCer and 5-FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo-immunotherapy against metastatic liver cancer.

Interleukin-1ß enhances the production of soluble MICA in human hepatocellular carcinoma.

The production of soluble major histocompatibility complex class I-related chain A (MICA) is thought to antagonize NKG2D-mediated immunosurveillance. Interleukin-1ß (IL-1ß) is elevated in patients with chronic hepatitis C (CH), and this might contribute to the escape of hepatocellular carcinoma (HCC) cells from innate immunity. In this study, we investigated the immunoregulatory role of IL-1ß in the production of soluble MICA of HCC cells. First, we investigated the correlation between the serum IL-1ß levels and soluble MICA in CH patients. Serum IL-1ß levels were associated with soluble MICA levels in CH patients. The serum IL-1ß levels of CH patients with the HCC occurrence were significantly higher than those of CH patients without HCC. We next examined the MICA production of IL-1ß-treated HCC cells. Addition of IL-1ß resulted in significant increase in the production of soluble MICA in HepG2 and PLC/PRF/5 cells, human HCC cells. But soluble MICA was not detected in both non-treated and IL-1ß-treated normal hepatocytes. Addition of IL-1ß did not increase the expressions of membrane-bound MICA on HCC cells. These were observed similarly in various cancer cells including a gastric cancer (MKN1), two colon cancers (HCT116 and HT29) and a cervical cancer (HeLa). Addition of IL-1ß also increased the expression of a disintegrin and metalloproteinase (ADAM)9 in HCC cells, and the knockdown of ADAM9 in IL-1ß-treated HCC cells resulted in the decrease in the production of soluble MICA of HCC cells. These findings indicate that IL-1ß might enhance the production of soluble MICA by activating ADAM9 in human HCC.

Fibroblast growth factor-2 enhances NK sensitivity of hepatocellular carcinoma cells.

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1ß and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.

Altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis C virus infection.

BACKGROUND & AIMS: Natural killer (NK) cells play an important role in the immune response against virus infection. Interferon (IFN)-alpha, an essential component in therapy against hepatitis C virus (HCV) infection, regulates NK cell function. However, it remains obscure how chronic HCV infection (CHC) modifies intracellular IFN-alpha signaling in NK cells. We investigated IFN-alpha signaling in NK cells in patients with CHC. METHODS: Peripheral blood mononuclear cells were obtained from patients with CHC and healthy subjects (HS) as controls. RESULTS: The expression level of signal transducer and activator of transcription (STAT) 1, a key molecule of IFN-alpha signaling, was clearly higher in NK cells from the CHC patients than in those from HS. The phosphorylation level of STAT1 with IFN-alpha stimulation was significantly greater in NK cells from the CHC patients than in those from the HS, while that of STAT4 was significantly less. These phosphorylation levels of STAT1 and STAT4 positively and negatively correlated with the STAT1 level in NK cells, respectively. The IFN-alpha induced messenger RNA level of the suppressor of cytokine signaling 1, which is a downstream gene of phosphorylated-STAT1, was clearly greater in NK cells from the CHC patients than in those from the HS, while that of IFN-gamma, which is a downstream gene of phosphorylated-STAT4, was clearly lower. CONCLUSIONS: These results indicate altered IFN-alpha signaling in NK cells in CHC patients, suggesting that this alteration is associated with the persistence of HCV infection and resistance to IFN-alpha therapy.

Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma.

BACKGROUND & AIMS: Although CD133 expression is identified as a cancer stem cell marker of hepatocellular carcinoma (HCC), the detailed characteristics of HCC cells expressing CD133 remain unclear. METHODS: We examined the malignant characteristics of CD133-expressing HCC cells. RESULTS: CD133-expressing cells could be detected with low frequency in 5 HCC tissues. We derived two different HCC cell lines by (1) transfection of CD133 siRNA in PLC/PRF/5 cells in (CD133si-PLC/PRF/5), and (2) by a magnetic cell sorting method that allowed to divide Huh7 cells into two CD133 positive (+) and negative (-) groups. CD133 knockdown in PLC/PRF/5 cells resulted in a decrease of the mRNA and protein expressions of matrix metalloproteinase (MMP)-2 and a disintegrin and metalloproteinase (ADAM)9. We next examined the malignant characteristics related to decreasing MMP-2 and ADAM9 in HCC cells. In CD133si-PLC/PRF/5 cells and CD133- Huh7 cells, invasiveness and vascular endothelial growth factor (VEGF) production, which are both related to the activity of MMP-2, were inhibited compared CD133-expressing HCC cells. We previously demonstrated that ADAM9 protease plays critical roles in the shedding of MHC class I-related chain A (MICA) which regulates the sensitivity of tumor cells to natural killer cells (NK). Decreasing ADAM9 expression in CD133si-PLC/PRF/5 cells and CD133- Huh7 cells resulted in an increase in membrane-bound MICA and a decrease in soluble MICA production. Both CD133si-PLC/PRF/5 cells and CD133- Huh7 cells were susceptible to NK activity, depending on the expression levels of membrane-bound MICA, but CD133-expressing HCC cells were not. CONCLUSION: These results demonstrate that CD133 expression in HCC cells confers malignant potential which may contribute to the survival of HCC cells.

Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion.

Virological features of fulminant liver disease-causing hepatitis B virus (HBV) have not been fully elucidated. We studied longitudinally the viruses obtained before and after fulminant liver disease in a patient with chronic HBV infection showing fatal exacerbation. HBV strains were obtained before and after exacerbation (designated as FEP1 and FEP2). Their virological features were investigated by in vitro transfection. FEP1 and FEP2 possessed higher activity of overall HBV DNA synthesis than the wild-type. FEP1 lacked competence for relaxed circular (RC) HBV DNA synthesis and RC HBV DNA-containing virion secretion, but FEP2 maintained it. Chimeric analysis revealed that the preS/S gene, where FEP1 had a considerable number of mutations and deletions but FEP2 did not, was responsible for impaired RC HBV DNA synthesis and virion secretion. Furthermore, incompetence of FEP1 strain was transcomplemented by the preS/S protein of wild-type strain. In conclusion, the viral strain after exacerbation showed resurgent RC HBV DNA synthesis and virion secretion, which was caused by conversion of the preS/S gene from a hypermutated to hypomutated state. This may have been responsible for disease deterioration in the patient. This is a novel type of HBV genomic variation associated with the development of fulminant liver disease.

Sorafenib inhibits the shedding of major histocompatibility complex class I-related chain A on hepatocellular carcinoma cells by down-regulating a disintegrin and metalloproteinase 9.

UNLABELLED: The ectodomain of major histocompatibility complex class I-related chain A (MICA) is shed from tumor cells, and may be an important means of evading antitumor immunity. This study investigated the roles of a disintegrin and metalloproteinase 9 (ADAM9) in the shedding of MICA in human hepatocellular carcinoma (HCC). Small interfering RNA-mediated knockdown (KD) of ADAM9 resulted in up-regulation of membrane-bound MICA expression on the HepG2 and PLC/PRF/5 cellular surfaces and down-regulation of soluble MICA levels in their culture supernatant. ADAM9 was cleaved at a site between Gln347 and Val348 of MICA in vitro. We constructed a plasmid of the MICA gene with mutation or deletion of the ADAM9 cleavage site to examine the detailed mechanism of MICA shedding by ADAM9 protease. The results suggested that MICA might be cleaved at the intracellular ADAM9-recognized cleavage site and was further cleaved at the extracellular ADAM9-independent cleavage site in HCC cells, resulting in the production of soluble MICA. Immunohistochemical analysis revealed that ADAM9 was overexpressed in human HCC compared to normal liver tissues. The cytolytic activity of natural killer (NK) cells against ADAM9KD-HCC cells was higher than that against control cells, and the enhancement of this cytotoxicity depended on the MICA/B and NK group 2, member D pathway. Sorafenib treatment resulted in decreased expression of ADAM9, increased expression of membrane-bound MICA expression, and decreased levels of soluble MICA in HCC cells. Adding sorafenib enhanced the NK sensitivity of HCC cells via increased expression of membrane-bound MICA. CONCLUSION: ADAM9 is involved in MICA ectodomain shedding in HCC cells, and sorafenib can modulate ADAM9 expression. Sorafenib therapy may have a previously unrecognized effect on antitumor immunity in patients with HCC.

EphA2-derived peptide vaccine with amphiphilic poly(gamma-glutamic acid) nanoparticles elicits an anti-tumor effect against mouse liver tumor.

The prognosis of liver cancer remains poor, but recent advances in nanotechnology offer promising possibilities for cancer treatment. Novel adjuvant, amphiphilic nanoparticles (NPs) composed of L: -phenylalanine (Phe)-conjugated poly(gamma-glutamic acid) (gamma-PGA-Phe NPs) having excellent capacity for carrying peptides, were found to have the potential for use as a peptide vaccine against tumor models overexpressing artificial antigens, such as ovalbumin (OVA). However, the anti-tumor potential of gamma-PGA-Phe NPs vaccines using much less immunogenic tumor-associated antigen (TAA)-derived peptide needs to be clarified. In this study, we evaluated the effectiveness of immunization with EphA2, recently identified TAA, derived peptide-immobilized gamma-PGA-Phe NPs (Eph-NPs) against mouse liver tumor of MC38 cells (EphA2-positive colon cancer cells). Immunization of normal mice with Eph-NPs resulted in generation of EphA2-specific type-1 CD8+ T cells. Immunization with Eph-NPs tended to provide a degree of anti-MC38 liver tumor protection more than that observed for immunization with the mixture of EphA2-derived peptide and complete Freund's adjuvant (Eph + CFA). Neither Eph-NPs nor Eph + CFA vaccines inhibited tumor growth of BL6, EphA2-negative melanoma cells. Splenocytes isolated from MC38-bearing mice treated with Eph-NPs showed strong and specific cytotoxic activity against MC38 cells. Immunization with Eph + CFA induced liver damage as evidenced by elevation of serum alanine aminotransferase, while Eph-NPs vaccination did not exhibit any toxic damage to the liver. These results demonstrated that immunization with Eph-NPs displayed anti-tumor effects against liver tumor by generating acquired immunity equivalent to the toxic adjuvant CFA, suggesting that safe gamma-PGA-Phe NPs could be applied clinically for the vaccine treatment of liver cancer.

Anticancer chemotherapy inhibits MHC class I-related chain a ectodomain shedding by downregulating ADAM10 expression in hepatocellular carcinoma.

MHC class I-related chain A (MICA) is a ligand for the NKG2D-activating immunoreceptor that mediates activation of natural killer (NK) cells. The ectodomain of MICA is shed from tumor cells, which may be an important means of evading antitumor immunity. We previously reported that patients with hepatocellular carcinoma (HCC) display high levels of soluble MICA in circulation, which could be downregulated by chemotherapy. The present study shows that anti-HCC drugs suppress MICA ectodomain shedding by inhibiting expression of a disintegrin and metalloproteinase 10 (ADAM10). Both ADAM10 and CD44, a typical substrate of the ADAM10 protease, were expressed in human HCC tissues and HCC cells but not in normal liver tissues or cultured hepatocytes. Small interfering RNA-mediated knockdown experiments revealed that ADAM10 is a critical sheddase for both MICA and CD44 in HCC cells. Of interest is the finding that epirubicin clearly downregulated ADAM10 expression and MICA shedding in HCC cells; its suppressive effect on MICA shedding was abolished in ADAM10-depleted cells. Epirubicin treatment also enhanced the NKG2D-mediated NK sensitivity of HCC cells. Patients with HCC had significantly higher levels of serum-soluble CD44, which correlated well with serum-soluble MICA levels, thus suggesting a close link between ADAM10 activity and MICA shedding in these patients. Soluble MICA and CD44 levels were downregulated with a significant correlation in patients treated by transarterial chemoembolization using epirubicin. In conclusion, anticancer drugs can modulate expression of ADAM10, which is critically involved in MICA ectodomain shedding. Epirubicin therapy may have a previously unrecognized effect on antitumor immunity in HCC patients.

Mutations associated with the therapeutic efficacy of adefovir dipivoxil added to lamivudine in patients resistant to lamivudine with type B chronic hepatitis.

Factors influencing the therapeutic efficacy of adefovir dipivoxil added to continuing lamivudine have not been elucidated in lamivudine-resistant patients with type B chronic hepatitis. The viral mutations influencing the efficacy of treatment with adefovir dipivoxil were investigated by sequencing analysis of the whole virus genome. Thirty patients resistant to lamivudine receiving adefovir dipivoxil therapy added to lamivudine were studied. From serum samples obtained before the administration of adefovir dipivoxil, full-length viral DNA sequences were determined by PCR-direct sequencing. Susceptibility of the virus to adefovir was examined further using in vitro transfection analysis. By screening the whole viral genome, the presence of two mutations, a T-to-C/G/A mutation at nt1753 (V1753) and an A-to-C mutation at nt2189 (C2189), correlated with the higher incidence of sustained viral DNA clearance during therapy (P < 0.005 and P < 0.05). In multivariate analysis, the V1753 (P = 0.001) and the C2189 (P = 0.007) mutations, and elevated transaminase (P = 0.011) and low viral load (P = 0.008) at the baseline were selected as significant independent factors associated with improved antiviral efficacy. In vitro transfection analysis showed no differences in susceptibility to adefovir among wild-type virus and C1753 and C2189 mutant viruses, suggesting that the virus possessing these mutations may be eradicated more efficiently than the wild-type virus by treatment regardless of a direct antiviral effect of adefovir.

Supportive role played by precore and preS2 genomic changes in the establishment of lamivudine-resistant hepatitis B virus.

BACKGROUND: Hepatitis B virus (HBV) establishes lamivudine resistance via the resistance-causative rtM204V/I mutation and the replication-compensatory rtL180M mutation. However, both lamivudine-resistant viruses with and those without rtL180M can exist in clinical settings. To elucidate the differences between viruses with and those without rtL180M, we conducted full-length sequencing analysis of HBV derived from patients with type B chronic hepatitis showing lamivudine resistance. METHODS: The full-length HBV DNA sequences derived from 44 patients showing lamivudine resistance were determined by polymerase chain reaction direct sequencing. Viral replicative competence was examined by in vitro transfection analysis using various HBV-expressing plasmids. RESULTS: Throughout the HBV genome, a precore-defective A1896 mutation and a short deletion in the preS2 gene were detected more frequently in viruses without rtL180M than in those with it (64% vs. 17% [P < .005] and 50% vs. 10% [P < .01], respectively). In vitro transfection analysis revealed that the level of reduction in intracellular viral replication caused by the introduction of lamivudine resistance-associated mutations was lower in precore-defective and preS2-deleted viruses than in wild-type virus. CONCLUSIONS: Both the precore-defective mutation and the preS2 deletion may play a supportive role in the replication of lamivudine-resistant HBV, which may be a reason for there being no need for the compensatory rtL180M mutation in lamivudine-resistant HBV possessing the precore and preS2 genomic changes.

Serum levels of soluble major histocompatibility complex (MHC) class I-related chain A in patients with chronic liver diseases and changes during transcatheter arterial embolization for hepatocellular carcinoma.

Soluble forms of major histocompatibility complex (MHC) class I-related chain A and B (MICA/B) are increased in the sera of patients with malignancy and impair the antitumor immune response by downregulating expression of their cognate immunoreceptor natural killer group 2, member D (NKG2D). Recently, soluble MICA/B were reported to appear even in some premalignant diseases, raising questions about the impact of soluble MICA/B produced from tumors on the expression of NKG2D. The present study examined soluble MICA/B in chronic liver disease and hepatocellular carcinoma (HCC) and their involvement in the immune-cell expression of NKG2D during transcatheter arterial embolization for HCC. The levels of soluble MICA/B were significantly higher in chronic liver disease and HCC patients than in healthy volunteers. The progression of liver disease and that of the tumor were independent determinants for soluble MICA/B levels. Immunohistochemistry revealed that MICA/B were expressed not only in HCC tissue but also on hepatocytes in cirrhotic livers. The transcatheter arterial embolization therapy significantly decreased serum levels of soluble MICA, but not soluble MICB, and increased the NKG2D expression on natural killer cells and CD8-positive T cells; there was an inverse correlation between changes in soluble MICA levels and in NKG2D expression. In conclusion, although soluble MICA/B are produced from both HCC and premalignant cirrhotic livers, therapeutic intervention for HCC can reduce the levels of soluble MICA and thereby upregulate the expression of NKG2D. Cancer therapy may have a beneficial effect on NKG2D-mediated antitumor immunity.

Dendritic cell-based vaccines suppress metastatic liver tumor via activation of local innate and acquired immunity.

BACKGROUND: Dendritic cell (DC)-based vaccines have been applied clinically in the setting of cancer, but tumor-associated antigens (TAAs) have not yet been enough identified in various cancers. In this study, we investigated whether preventive vaccination with unpulsed DCs or peptide-pulsed DCs could offer anti-tumor effects against MC38 or BL6 liver tumors. METHODS: Mice were subcutaneously (s.c.) immunized with unpulsed DCs or the recently defined TAA EphA2 derived peptide-pulsed dendritic cells (Eph-DCs) to treat EphA2-positive MC38 and EphA2-negative BL6 liver tumors. Liver mononuclear cells (LMNCs) from treated mice were subjected to (51)Cr release assays against YAC-1 target cells. In some experiments, mice were injected with anti-CD8, anti-CD4 or anti-asialo GM1 antibody to deplete each lymphocyte subsets. RESULTS: Immunization with unpulsed DCs displayed comparable efficacy against both MC38 and BL6 liver tumors when compared with Eph-DCs. Both DC-based vaccines significantly augmented the cytotoxicity of LMNCs against YAC-1 cells. In vivo antibody depletion studies revealed that NK cells, as well as, CD4+ and CD8+ T cells play critical roles in the anti-tumor efficacy associated with either DC-based modality. Tumor-specific cytotoxic T lymphocyte (CTL) activity was generally higher if mice had received Eph-DCs versus unpulsed DCs. Importantly, the mice that had been protected from MC38 liver tumor by either unpulsed DCs or Eph-DCs became resistant to s.c. MC38 rechallenge, but not to BL6 rechallenge. CONCLUSIONS: These results demonstrate that unpulsed DC vaccines might serve as an effective therapy for treating metastatic liver tumor, for which TAA has not yet been identified.

Early emergence of entecavir-resistant hepatitis B virus in a patient with hepatitis B virus/human immunodeficiency virus coinfection.

The efficacy of entecavir for patients with hepatitis B virus/human immunodeficiency virus coinfection has not been fully elucidated. Here we examined a patient coinfected with both viruses in whom entecavir-resistant hepatitis B virus appeared. The 60-year-old Japanese male with the coinfection received antiretroviral therapy including lamivudine. The therapy initially suppressed replication of both viruses, followed by reactivation of the hepatitis B virus alone by 2 years of therapy. He subsequently received entecavir therapy in addition to the antiretroviral regimen. After entecavir administration, the hepatitis B virus DNA level was slightly reduced, but then increased after 6 months of entecavir therapy. In the sequencing analysis of hepatitis B virus, no drug resistance-associated amino acid substitutions were observed in the reverse transcriptase (rt) domain before antiretroviral therapy. The lamivudine-resistant amino acid substitutions at rt173, rt180 and rt204 were detected before entecavir administration, and further the entecavir-resistant rt202 substitution was observed after 6 months of entecavir therapy. The full-length hepatitis B sequences showed that the viral strain derived from the patient belonged to genotype H. In summary, this report describes a patient with hepatitis B virus/human immunodeficiency virus coinfection who received entecavir therapy in addition to an antiretroviral regimen and showed the early emergence of entecavir-resistance hepatitis B virus. In entecavir therapy for patients infected with both viruses, great care should be taken with respect to the emergence of entecavir-resistant hepatitis B virus, especially in patients with pre-existing lamivudine-resistant virus.

Type B fulminant hepatitis is closely associated with a highly mutated hepatitis B virus strain.

OBJECTIVE: Genome-wide sequences of hepatitis B virus strain associated with type B fulminant hepatitis have not been compared with those of acute self-limited hepatitis. We carried out full-length sequencing analysis of viral strains derived from patients with type B acute liver injury. METHODS: Nine acute self-limited hepatitis and 6 fulminant hepatitis patients were the subjects of this study. Full-length sequencing analysis of viral DNA was done by PCR-direct sequencing. RESULTS: Higher frequencies in fulminant hepatitis strains compared with acute hepatitis ones were observed in the T1762/A1764 (p < 0.05), A1896 (p = 0.09) and M1753 (M = C or A) (p = 0.09) mutations. Viruses related to fulminant hepatitis possessed the higher number of nucleotide substitutions than those related to acute hepatitis in the whole virus genome (p < 0.01) and various regions including preS/S gene (p < 0.05), precore/core gene (p < 0.01), polymerase gene (p < 0.05) and basic core promoter/core upstream regulatory sequence (p < 0.01). The high number of nucleotide substitutions in viruses related to fulminant hepatitis was predominantly non-synonymous in the preS/S and precore/core genes. CONCLUSION: Development of type B fulminant hepatitis may be associated with a highly mutated hepatitis B virus strain.

Intrahepatic delivery of alpha-galactosylceramide-pulsed dendritic cells suppresses liver tumor.

UNLABELLED: Alpha-galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC-based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of alpha-Galactosylceramide-pulsed DCs (alphaGCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of alphaGCDC administration in comparison with p53 peptide-pulsed DCs using a well-established murine CMS4 tumor model. Injection of alphaGCDC into CMS4 liver tumors resulted in complete tumor rejection and established long-term survival of the animals, while injection of p53(232-240) peptide-pulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN-gamma in sera of alphaGCDC-treated mice were significantly higher than those of pepDC-treated mice. Hepatic NK cells were efficiently activated by alphaGCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody-mediated NK cell depletion study. Injection of alphaGCDC into liver tumor led to higher p53(232-240) peptide-specific CD8+ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by alphaGCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. CONCLUSION: These results demonstrate that alphaGCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor.