Kazuki Ando

Prevalence of hepatitis B and C virus infection in haematological malignancies and liver injury following chemotherapy

Abstract:  The aim of this study was to determine the carrier rate of hepatitis virus in patients with haematological malignancies and the incidence of liver injury in these patients following chemotherapy. From January 1996 to September 2002, we studied 601 consecutive, unselected series of patients with haematological malignancies admitted in our hospital unit (Japan). They consisted of 246 cases of acute leukaemia, 218 non‐Hodgkins lymphoma (NHL), 13 adult T‐cell leukaemia, and 124 multiple myeloma. Of these 601 patients, 373 were men and 228 were women; their mean age was 61 yr, with a range from 18 to 89 yr. The prevalences of hepatitis B virus (HBV) and hepatitis C virus (HCV) were 7.3% and 10.1%, respectively, in NHL, both higher than those in acute leukaemia (1.7% and 2.9%, P < 0.005) and in general Japanese population (1.2% and 2.6%). The incidence of post‐chemotherapy liver injury in 25 HBV carriers (36.0%) was significantly higher than that in 539 non‐hepatitis virus carriers (12.6%, P = 0.003) and 37 HCV carriers (10.8%, P = 0.026). Liver injury in HBV carriers was more often present in patients who had been treated with steroids than in those without steroids (72.7% and 0%, P = 0.013). After lamivudine became available in our institution, the incidence of liver injury in HBV carriers was reduced from 53.3% to 10.0% (P = 0.041). The therapeutic strategy for haematological malignancies in hepatitis virus carriers should be further investigated.

Lack of Tumor Necrosis Factor Alpha Induces Impaired Proliferation of Hepatitis B Virus-Specific Cytotoxic T Lymphocytes

ABSTRACT Recent studies have shown that tumor necrosis factor alpha (TNF-α) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-α knockout (TNF-α−/−) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-α−/− mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-α−/− mice after two sessions of immunization with the HBV plasmid; however, TNF-α was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-α−/− mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-α enhanced the proliferation of a TNF-α−/− clone but suppressed that of a TNF-α+/+ clone in vitro, TNF-α also has a direct effect on the proliferation of CTLs. In conclusion, TNF-α is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.

Intrahepatic cholangiocarcinoma arising 10 years after the excision of congenital extrahepatic biliary dilation

A 52-year-old woman was found to have a liver tumor during treatment for a liver abscess. The tumor was diagnosed as intrahepatic cholangiocarcinoma by closer examinations, including a percutaneous needle biopsy. Ten years previously, she had undergone excision of a choledochal cyst, with reconstruction by Roux-en-Y hepaticojejunostomy, as treatment for Todanis type Ia congenital biliary dilation, which had been confined only to the extrahepatic bile duct. The significant association between congenital biliary dilation and hepatobiliary malignancies is well known. Some patients have been reported to develop biliary cancer long after the excision of the entire extrahepatic bile duct and hepaticoenterostomy. However, in these patients, the development mostly took place in the remnant choledochal cyst, the anastomotic site, or in the dilated intrahepatic bile duct of Todanis type IV-A congenital biliary dilation. The development of intrahepatic cholangiocarcinoma after operation has not been reported previously in a patient with Todanis type I congenital biliary dilation. This case suggests that the entire biliary tree may have a high risk of field cancerization, even in extrahepatic congenital biliary dilation.

Role of Vα14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of Vα14+ NKT cells promotes the breakage of CTL tolerance

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or alpha-GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and alpha-GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40-CD40L interaction mediate the enhancement of CTL induction caused by alpha-GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection.

Excessive production of tumor necrosis factor-alpha by bone marrow T lymphocytes is essential in causing bone marrow failure in patients with aplastic anemia

Abstract:  Aplastic anemia (AA) is regarded as an immunological disorder because of the clinical effect of immunosuppressive therapy. Recent studies have reported that cytokines play an important role in the development of AA. In the present study, we measured levels of T‐cell derived intracellular cytokine production in peripheral blood and bone marrow (BM) of patients with AA. We demonstrated that BM lymphocytes, particularly CD4+ and CD8+ T cells, in patients with AA produced significantly higher amounts of tumor necrosis factor‐alpha (TNF‐α), compared with lymphocytes in normal controls. We have previously reported that expression of TNF receptor (R)1 and TNFR2 in the CD34+ CD38− and CD34+ CD38+ fractions of patients with AA is significantly higher than those in normal control. These results indicate that BM stem cells in patients with AA may possess high sensitivity to TNF‐α. This in turn suggests that TNF‐α affects hematopoiesis at an earlier stage in AA patients than in normal controls. We strongly support the hypothesis that a simultaneous increase in TNF‐α production by BM lymphocytes and sensitivity of stem cells to TNF‐α leads to BM failure in AA.

Ability of IDO To Attenuate Liver Injury in α-Galactosylceramide–Induced Hepatitis Model

IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in α-galactosylceramide (α-GalCer)–induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered α-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after α-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-d-tryptophan also exacerbated liver injury in this murine hepatitis model. In α-GalCer–induced hepatitis models, TNF-α is critical in the development of liver injury. The mRNA expression and protein level of TNF-α in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with α-GalCer were analyzed by flow cytometry, and the numbers of CD49b+ and CD11b+ cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with α-GalCer, TNF-α secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in α-GalCer–induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-α–producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the α-GalCer–induced hepatitis model.

Role of TNF-α Produced by Nonantigen-Specific Cells in a Fulminant Hepatitis Mouse Model

In previous studies, the mechanisms of acute liver injury and virus exclusion have been examined using a model wherein HBsAg-specific CTL are injected into HBsAg transgenic (Tg) mice. The importance of the role of TNF-α in virus exclusion was shown, but its role in liver injury was unclear. We crossed the TNF-α knockout mouse and HBsAg-Tg mouse to establish the HBsAg-Tg/TNF-α KO mouse, and examined the influence of TNF-α on liver injury. The severity of liver damage, as determined by serum alanine aminotransferase activity, was ∼100 times greater in HBsAg-Tg/TNF-α+/+ than in HBsAg-Tg/TNF-α−/− mice after i.v. administration of 5 × 106 CTLs. This liver damage reached the peak of its severity within 24–48 h, and was restored 7 days later. Histopathological examination showed hepatocellular necrosis and inflammatory cell infiltrate 24 h after the CTL injection in HBsAg-Tg/TNF-α+/+ mice but not in HBsAg-Tg/TNF-α−/− mice. The liver damage was fatal for all HBsAg-Tg/TNF-α+/+ mice that received 1.5 × 107 CTLs. In contrast, 1.5 × 107 CTLs could not kill the HBsAg-Tg/TNF-α−/− mice. The TNF-α production level was enhanced after the CTL injection in not only intrahepatic macrophages but also other types of mononuclear cells from non-HBsAg-Tg/TNF-α+/+ mice. An adoptive transfer examination revealed that severe liver damage occurred in HBsAg-Tg/TNF-α−/− mice that had received mononuclear cells from TNF-α+/+ mice. In conclusion, the present study provides evidence that TNF-α produced by intrahepatic non-Ag-specific inflammatory cells is critical in the development of lethal necroinflammatory liver disease.

Upregulation of indoleamine 2,3-dioxygenase in hepatocyte during acute hepatitis caused by hepatitis B virus-specific cytotoxic T lymphocytes in vivo.

Background/Aims: Indoleamine‐2,3‐dioxygenase (IDO) is a tryptophan‐catabolizing enzyme inducing suppression of T‐cell function and immune tolerance. In hepatitis B virus (HBV) transgenic (Tg) mice, the adoptive transfer of HBV‐specific cytotoxic T lymphocytes (CTL) causes a necroinflammatory liver disease that is histologically similar to acute viral hepatitis in man. The present study aimed to determine IDO expression in the liver and hepatocytes during an acute hepatitis model.

Interferon-β reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects

Type I interferons (IFNs), IFN‐α and IFN‐β, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0·3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN‐β pretreatment. IFN‐β suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN‐β reduced the expressions of transforming growth factor‐β, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN‐β reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Elevated intracellular IFN-γ levels in circulating CD8+ lymphocytes in patients with fulminant hepatitis

BACKGROUND/AIMS Fulminant hepatitis usually takes a rapidly progressive course, terminating in death within a short period. Experimental studies have demonstrated that immunological mechanisms play an important role, especially those involving virus-specific CD8+ cytotoxic T lymphocytes and their production of interferon-gamma (IFN-gamma). However, there are no immunological markers for prediction of the development of fulminant hepatitis in man. METHODS Peripheral blood lymphocytes from four patients with fulminant hepatitis, six with acute hepatitis and 11 healthy volunteers as normal controls were analyzed. Intracellular IFN-gamma production in both CD8 positive and negative T lymphocytes was assessed by flow cytometry. RESULTS Populations of CD8+ IFN-gamma+ T lymphocytes were significantly increased in patients with fulminant hepatitis, as compared with those with acute hepatitis and normal controls. Production of IFN-gamma in CD8+ T lymphocytes of patients with fulminant hepatitis was also elevated, furthermore significantly correlating with the prothrombin time (r=-0.64, p<0.01). CONCLUSIONS The capacity for IFN-gamma production by CD8+ lymphocytes is up-regulated in fulminant hepatitis, and this may be important for the development of fulminant hepatitis.