Shigenori Tamaki

Thrombin in the airways of asthmatic patients.

Abstract. The mechanism of airway remodeling in asthmatic patients is poorly understood. Thrombin is a multifunctional protease that, in addition to its critical role in thrombotic processes, has also been described as inducing cellular and molecular events relevant to tissue remodeling. The present investigation was undertaken to evaluate the activity of thrombin in the sputum of asthmatic patients and its potential role in airway remodeling. The study population comprised 8 healthy subjects and 14 stable patients with bronchial asthma. The concentrations of thrombin, thrombin-antithrombin complex (TAT), and tissue factor were measured in the sputum of all subjects. The concentrations of thrombin (p= 0.007), TAT (p= 0.01), and tissue factor (p= 0.02) in sputum were significantly higher in asthmatic patients than in healthy controls. The proliferative effects that sputum from asthmatic patients (p= 0.01) and thrombin (p= 0.03) have on cultured human smooth muscle cells was inhibited significantly in the presence of recombinant hirudin, a specific thrombin inhibitor. Significant statistical correlation was observed between the degree of bronchial responsiveness and the sputum concentrations of thrombin (r=−0.8; p= 0.02) and TAT (r=−0.9; p= 0.01). The results of this study showed that increased thrombin generation occurs in the airway of patients with asthma and that it may play a role in the pathogenesis of airway remodeling. Further studies should be carried out to assess whether these findings are also observed in other airway diseases.

Genetic polymorphisms of bilirubin uridine diphosphate‐glucuronosyltransferase gene in Japanese patients with Crigler–Najjar syndrome or Gilbert's syndrome as well as in healthy Japanese subjects

Background and Aim:  Numerous mutations of bilirubin uridine diphosphate‐glucuronosyltransferase gene (UGT1A1) have been reported in patients with familial unconjugated hyperbilirubinemia. The UGT1A1 mutation appears to be considerably different among ethnic groups. To clarify the incidence of this gene mutation in the Japanese population, the presence of UGT1A1 mutation was investigated in a group of Japanese patients with Crigler–Najjar syndrome type 2 (CNS2) and Gilberts syndrome (GS), as well as in healthy anicteric subjects.

Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against hepatitis C virus

The capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) was assessed. BALB/c mice were immunized with peptides based on the CTL and helper T cell (Th) epitopes of the HCV core, with a mixture of CTL and Th peptides (CTL+Th) or with a conjugated Th-CTL peptide. Mice immunized with CTL, CTL+Th and Th-CTL peptides, but not those immunized with Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic activity induced by immunization with Th-CTL was much higher than that induced by immunization with CTL+Th or CTL alone. However, rapid and high cytotoxic activities against HCV core were not only detected after immunization with peptides containing the CTL epitope but also as a result of infection with recombinant vaccinia virus carrying the HCV core gene after immunization with the Th epitope alone. Immunization with peptides containing the Th epitope also elicited spleen cell proliferation. This study demonstrates the capacity of both Th and CTL activated peptide vaccines to elicit CD8+, MHC class I-restricted CTLs. The capacity of such CTLs to contribute towards a protective and/or pathogenic immune response against HCV can now be assessed in mouse models.

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-α promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL)

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.

A single administration of interleukin-4 antagonistic mutant DNA inhibits allergic airway inflammation in a mouse model of asthma.

Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor α and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.

Role of nitric oxide in airway remodelling

Airway remodelling, which is manifested by thickening of bronchial wall, is an important causative factor of bronchial hyper-responsiveness in asthma. The pathophysiological mechanism of airway remodelling is not clear. In the present study we evaluated the relationship between nitric oxide (NO) generation and airway wall thickening in patients with chronic asthma. As a marker of NO production, the levels of nitrite/nitrate were measured in induced sputum, and bronchial wall thickening was measured by high-resolution computed tomography. Sputum concentrations of nitrite/nitrate were significantly increased in asthmatic patients compared with controls. The ratio of airway wall thickness to lumen diameter was significantly correlated with the sputum concentration of nitrite/nitrate. Although statistical correlation does not prove causation, this finding suggests that NO may play a key role in the pathogenesis of airway remodelling.

Immunization with recombinant Calmette-Guerin bacillus (BCG)-hepatitis C virus (HCV) elicits HCV-specific cytotoxic T lymphocytes in mice.

Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), an effective HCV vaccine should be capable of eliciting HCV-specific CTLs. In the present study, we assessed the capability of a novel recombinant vaccine using an attenuated tuberculosis bacillus, Calmette-Guerin bacillus (BCG), as a vaccine vehicle to elicit HCV-specific CTLs. BCG was engineered to express the CTL epitope of HCV-non-structure protein 5a (NS5a) as a chimeric protein with alpha antigen of mycobacteria. Immunization with this recombinant BCG elicited major histocompatibility complex class I-restricted CD8(+) HCV-NS5a-specific CTLs in mice. Immunized mice showed a substantial reduction in the vaccinia virus titer compared with control mice when the immunized mice were challenged with a recombinant vaccinia virus expressing HCV-NS5a genes. These findings provide evidences for the possibility of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HCV-specific immunity.

Ineffective interferon treatment of chronic hepatitis C-associated porphyria cutanea tarda, but with a transient decrease in HCV RNA levels

Abstract: Many patients with porphyria cutanea tarda (PCT) have been reported to be hepatitis C virus (HCV) carriers, suggesting that HCV infection plays a role in the pathogenesis of this type of porphyria. In this study, we report a patient with chronic hepatitis C-associated PCT. Therapy with interferon (IFN) transiently decreased HCV RNA levels, but levels of urinary porphyrins and serum transaminases and ferritin remained unchanged. Serum ferritin and urinary porphyrin levels improved after phlebotomy, but this therapy was not effective in improving serum transaminase levels. Although a physiopathological association between HCV infection and PCT has been suggested previously, IFN was not effective in this patient. The transient decrease in HCV RNA levels was a factor independent of porphyrin metabolism.

Induction of apoptotic cell death in human hepatocellular carcinoma SK-HEP-1 cells by a polyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone).

The antitumor effects of a polyamine biosynthetic pathway inhibitor methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP) on the human hepatocellular carcinoma SK-HEP-1 cell line have been investigated. The growth of these cultured hepatocellular carcinoma cells was inhibited by MGBCP in a dose-dependent manner. Spermidine and spermine levels were dose-dependently depressed, and morphological changes due to programmed cell death (apoptosis) were observed in these MGBCP-treated hepatocellular carcinoma cells. These results suggest that in addition to reducing the growth rates, MGBCP can induce apoptotic cell death in this human hepatocellular carcinoma cell line.

Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with hyperbilirubinemic hepatobiliary disease.

Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme that catalyzes hydrolysis of PAF and is important for regulation of serum PAF concentration. Twenty-seven patients with hyperbililubinemic hepatobiliary diseases (eight patients with liver cirrhosis, five with acute hepatitis and 14 with obstructive cholestasis) were studied. Serum PAF-AH activity was elevated in hyperbilirubinemic condition associated with liver cirrhosis, obstructive cholestasis or acute hepatitis. There was a significant correlation of serum PAF-AH activity with serum cholesterol (r=0.891, P=0.014 in liver cirrhosis, r=0.813, P=0.002 in obstructive cholestasis) or low density lipoprotein (LDL) cholesterol (r=0.771, P=0.007 in obstructive cholestasis). Serum PAF-AH activity/cholesterol ratio and PAF-AH activity/LDL cholesterol ratio were elevated in the three types of hyperbilirubinemic liver disease. LDL cholesterol increased and PAF-AH/LDL cholesterol ratio decreased in acute hepatitis after remission. On the other hand, LDL cholesterol decreased and PAF-AH/LDL cholesterol ratio did not change after treatment in obstructive cholestasis. These findings suggest that, though serum LDL is a factor influencing serum PAF-AH activity in obstructive jaundice, other factors, i.e. reduced biliary PAF-AH excretion, may also influence serum PAF-AH activity in hyperbilirubinemic hepatobiliary diseases.