Geriatric nutritional risk index as a prognostic indicator in elderly patients with early colorectal cancer undergoing endoscopic submucosal dissection.

OBJECTIVES: Data on the long-term outcomes of endoscopic submucosal dissection (ESD) performed in elderly patients with early colorectal cancer (CRC) are limited. We analyzed the prognosis of elderly CRC patients, not only from the viewpoint of treatment curability but also from the patients' baseline physical condition assessed by several indexes. METHODS: A retrospective analysis of 729 patients aged ≥75 years who underwent ESD for Tis/T1 CRC in 16 institutions was conducted. The patients were classified into three groups based on curability: curative ESD (Group A, n = 582), non-curative ESD with additional surgery (Group B, n = 60), and non-curative ESD without additional surgery (Group C, n = 87). Overall survival (OS) was compared among the groups, and factors associated with reduced OS were investigated. RESULTS: The median follow-up periods in Groups A, B, and C were 41, 49, and 46 months, respectively (P = 0.62), during which 92 patients died. Two patients (0.3%) in Group A, none (0%) in Group B, and three (3.4%) in Group C died of CRC. Three-year OS rates in Groups A, B, and C were 93.9%, 96.1%, and 90.1%, respectively, without a significant difference (P = 0.07). Multivariate analysis indicated low (<96.3) geriatric nutritional risk index (GNRI) as the sole independent predictor for reduced OS (hazard ratio 3.37; 95% confidence interval 2.18-5.22; P < 0.0001). CONCLUSIONS: Low GNRI, but not the curability attained by ESD, was independently associated with reduced OS in patients with early CRC aged ≥75 years.

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.

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.

Activated liver dendritic cells generate strong acquired immunity in alpha-galactosylceramide treatment.

BACKGROUND/AIMS: Alpha-galactosylceramide (alpha-GalCer) presented by dendritic cells (DCs) activates NKT cells that in turn drive DC maturation. However, the potential of generating acquired immunity of liver DCs in alpha-GalCer treatment remains unclear. METHODS: We examined the activation of acquired immunity in the alpha-GalCer treatment against liver or spleen tumor and the ability of liver and spleen DCs in the generation of acquired immunity. RESULTS: Administration of alpha-GalCer resulted in generation of p53 peptide-specific cytotoxic T lymphocytes (CTLs) in mice bearing liver CMS4 tumor, aberrantly expressing p53, but not in mice bearing spleen CMS4 tumor. The growth of rechallenged CMS4 subcutaneous tumor was inhibited in alpha-GalCer-treated mice against liver CMS4 tumor, but not in alpha-GalCer-treated mice against CMS4 spleen tumor. The antigen presenting related functions of liver DCs were significantly higher than those of spleen DCs in alpha-GalCer-treated mice. Vaccination of normal mice with p53 peptide pulsed liver DCs isolated from alpha-GalCer treated mice resulted in generation of p53 peptide-specific CTLs, but that with p53 peptide pulsed spleen DCs did not. CONCLUSIONS: These results demonstrated that alpha-GalCer treatment induced unique immunologic activation of liver DCs in comparison with spleen DCs, which might be favorable to generate liver acquired immunity.

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.

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.

α-Galactosylceramide activates antitumor immunity against liver tumor.

AIM: α-Galactosylceramide (α-GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α-GalCer treatment in the mouse liver tumor model. METHODS: BALB/c mice bearing CMS4 liver tumor (p53 peptide-expressing tumor) were treated by α-GalCer. We evaluated the activation of liver DC and immune cells after α-GalCer treatment. Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide-specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α-GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re-challenged s.c. RESULTS: The liver weights of α-GalCer-treated mice were significantly lighter than those of vehicle-treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α-GalCer. α-GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co-stimulatory molecules on liver DC significantly increased with the peak at 1 day after α-GalCer administration. IFN-γ ELISPOT assay demonstrated that p53 peptide-specific CTL was generated efficiently in α-GalCer-treated mice. (51) Cr-release assay revealed that CD8(+) , not CD4(+) , CTL against CMS4 cells were generated in α-GalCer-treated mice. The mice that had been protected from CMS4 liver tumor by α-GalCer injection became resistant against s.c. CMS4 re-challenge, but not against Colon26 re-challenge. CONCLUSION: These results demonstrated the therapeutic potential of α-GalCer against liver cancer through activating liver DC and immune cells in the liver.

Absence of invariant natural killer T cells deteriorates liver inflammation and fibrosis in mice fed high-fat diet.

BACKGROUND: Invariant natural killer T (iNKT) cells have been suggested to play critical roles in a wide range of immune responses by acting in a proinflammatory or anti-inflammatory manner. Nonalcoholic steatohepatitis (NASH) is a chronic liver disease progressing to advanced cirrhosis and hepatocellular carcinoma. Despite the abundance of iNKT cells in the liver, their role in the pathogenesis of NASH remains obscure. Here, we investigated their role in the development of diet-induced steatosis/steatohepatitis. METHODS: We used BALB/c wild-type mice and Jα18-deficient (KO) mice lacking iNKT cells fed either a normal diet or a high-fat diet (HFD). The liver and blood were collected from these mice to examine liver inflammation, steatosis, and fibrosis at the indicated time points. RESULTS: KO mice fed the HFD, compared with control mice fed the HFD, exhibited a clearly higher serum alanine aminotransferase level and a greater number of hepatic inflammatory foci, although there was no significant difference in hepatic lipid retention between these groups of mice. The HFD enhanced hepatic messenger RNA expression of inflammatory cytokines and chemokines in KO but not in control mice. The HFD also increased the proportion of hepatic CD4 T cells and CD8 T cells that composed hepatic inflammatory foci in KO mice, but not in the controls. Prolonged feeding with the HFD augmented liver fibrosis in KO but not in control mice. CONCLUSIONS: These findings indicate that iNKT cells play a protective role against liver inflammation progressing to fibrosis, but not against steatosis, enhanced by dietary excess fat, suggesting a key role of these cells in NASH pathogenesis.

Decreased expressions of CD1d molecule on liver dendritic cells in subcutaneous tumor bearing mice.

BACKGROUND/AIMS: Alpha-Galactosylceramide (alpha-GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. However, the activation of liver innate immunity by alpha-GalCer should be examined because clinical trials of alpha-GalCer resulted in limited clinical responses. METHODS: We examined the activation of liver innate immunity by alpha-GalCer in subcutaneous Colon26 tumor bearing-mice (C26s.c.TB-mice). RESULTS: The expressions of CD1d molecule on liver dendritic cells (DCs) were significantly lower in C26s.c.TB-mice than those in tumor-unbearing normal mice. Although liver NK cells and NKT cells activated in normal mice after alpha-GalCer treatment, the activation of these cells were significantly inhibited in C26s.c.TB-mice. Alpha-GalCer treatment resulted in significant antitumor effect against Colon26 metastatic liver tumor in normal mice, but not in C26s.c.TB-mice. The serum levels of TGF-beta, known to suppress the CD1d expressions on DCs, in C26s.c.TB-mice were significantly higher than those in normal mice. Surgical subcutaneous tumor mass reduction resulted in the reduction of serum TGF-beta, the recovery of CD1d expressions on liver DCs and the improvement of antitumor effect of alpha-GalCer against metastatic liver tumor. CONCLUSIONS: These results suggested that tumor burden reduces CD1d expressions on liver DCs, thus impeding alpha-GalCer-mediated NK cell activation and antitumor activity in the liver.

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.

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.

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.