Katsuaki Tanaka

Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.

We recently found that bovine lactoferrin (bLF), a milk glycoprotein belonging to the iron transporter family, prevented hepatitis C virus (HCV) infection in human hepatocyte PH5CH8 cells, that are susceptible to HCV infection, and demonstrated that the anti-HCV activity of bLF was due to the interaction of bLF and HCV. In this study we further characterized the anti-HCV activity of bLF and the mechanism by which bLF prevents HCV infection. We found that bLF inhibited viral entry to the cells by interacting directly with HCV immediately after mixing of bLF and HCV inoculum. The anti-HCV activity of bLF was lost by heating at 65 degrees C, and other milk proteins (mucin, beta-lactoglobulin and casein) did not prevent HCV infection, indicating that bLF prevented HCV infection in a rather specific manner. Furthermore, we found that bovine lactoferricin, a basic N-terminal loop of bLF that is an important region for antibacterial activity, did not exhibit any anti-HCV activity, suggesting that some other region is involved in anti-HCV activity. We confirmed that prevention of HCV infection by bLF was a general phenomenon, because bLF inhibited HCV infection with all five inocula examined, and bLF inhibited HCV infection in human MT-2C T-cells, that were susceptible to HCV infection. In addition, infection with hepatitis G virus, which is distantly related to HCV, was prevented also by bLF. In conclusion, lactoferrin is a natural glycoprotein which effectively protects against HCV infection in hepatocytes and lymphocytes by neutralizing the virus.

Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C : A pilot study

Hepatitis C virus (HCV) is associated with the development of cirrhosis and hepatocellular carcinoma. We recently found that bovine lactoferrin, a milk protein belonging to the iron transporter family, effectively prevented HCV infection in cultured human hepatocytes (PH5CH8). We tested the hypothesis that lactoferrin inhibits HCV viremia in patients with chronic hepatitis C. Eleven patients with chronic hepatitis C received an 8‐week course of bovine lactoferrin (1.8 or 3.6 g/day). At the end of lactoferrin treatment, a decrease in serum alanine transaminase and HCV RNA concentrations was apparent in 3 (75%) of 4 patients with low pretreatment serum concentrations of HCV RNA. However, 7 patients with high pretreatment concentrations showed no significant changes in these indices. This pilot study suggests that lactoferrin is one potential candidate as an anti‐HCV reagent that may be effective for the treatment of patients with chronic hepatitis.

Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus

We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70-100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32 degrees C. Moreover, we demonstrated that interferon alpha had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.

Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance

Background and Aims : The natural immunomodulator, lactoferrin, is widespread among various biological fluids and is known to exert an anti‐inflammatory effect. However, there has been only one study that examined the mode of action of lactoferrin in reducing intestinal damage. We investigated the therapeutic role of lactoferrin and its effect on the levels of pro‐inflammatory and anti‐inflammatory cytokines, by using a rat model of dextran sulfate sodium (DSS) induced‐colitis.

Clinical characteristics of Japanese reflux esophagitis patients as determined by Los Angeles classification

Background:u2002 Recent studies have shown that the number of patients with reflux esophagitis is increasing in Japan, but the prevalence and risk factors associated with reflux esophagitis in Japanese patients are not well defined.

Transarterial chemotherapy alone versus transarterial chemoembolization for hepatocellular carcinoma: A randomized phase III trial

BACKGROUND/AIMSnTranscatheter arterial chemoembolization (TACE) is a combination of transarterial infusion chemotherapy (TAI) and embolization, and has been widely used to treat patients with hepatocellular carcinoma (HCC). However, since the impact of adding embolization on the survival of patients treated with TAI had never been evaluated in a phase III study, we conducted a multi-center, open-label trial comparing TACE and TAI to assess the effect of adding embolization on survival.nnnMETHODSnPatients with newly diagnosed unresectable HCC were randomly assigned to either a TACE group or a TAI group. Zinostatin stimalamer was injected into the hepatic artery, together with gelatin sponge in the TACE group and without gelatin sponge in the TAI group. Treatment was repeated when follow-up computed tomography showed the appearance of new lesions in the liver or re-growth of previously treated tumors.nnnRESULTSnSeventy-nine patients were assigned to the TACE group, and 82 were assigned to the TAI group. The two groups were comparable with respect to their baseline characteristics. At the time of the analysis, 51 patients in the TACE group and 58 in the TAI group had died. The median overall survival time was 646 days in the TACE group and 679days in the TAI group (p=0.383).nnnCONCLUSIONSnThe results of this study suggest that treatment intensification by adding embolization did not increase survival over TAI with zinostatin stimalamer alone in patients with HCC.

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMSnThe aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia.nnnMETHODSnHLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B.nnnRESULTSnAn HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively.nnnCONCLUSIONSnWe identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.

Very-Low-Calorie Diet-Induced Weight Reduction Reverses Impaired Growth Hormone Secretion Response to Growth Hormone-Releasing Hormone, Arginine, and L-Dopa in Obesity

To determine whether impaired growth hormone (GH) secretion in obese subjects is a consequence of obesity or a pre-existing pituitary-hypothalamic disorder, we measured (1) plasma GH response to growth hormone-releasing hormone (GRH; 1 microgram/kg body weight [BW]), arginine (0.5 g/kg BW), and L-dopa (500 mg); and (2) plasma glucose, insulin, and free fatty acids (FFA) in obese subjects before and after weight reduction due to very-low-calorie diet therapy using Optifast (240 kcal/d for 8 to 12 weeks). Body weight and body mass index (BMI) values before and after weight reduction were 87.2 +/- 4.1 kg and 34.5 +/- 0.9 kg/m2, and 67.8 +/- 2.7 kg and 27.0 +/- 0.4 kg/m2, respectively. GH response to GRH, arginine, and L-dopa in obese subjects was markedly impaired before weight reduction, whereas significantly increased responses were noted after weight reduction (P less than .01). Impaired integrated GH response to GRH, arginine, and L-dopa in obese subjects was significantly restored after weight reduction (P less than .01). Plasma glucose levels did not change, while plasma insulin and FFA levels decreased significantly after weight reduction (P less than .01, P less than .05). There was no significant correlation between integrated GH response to these three stimuli and plasma levels of glucose, insulin, and FFA, respectively. The reversibility of GH response to all three stimuli after weight reduction suggests that impaired GH secretion is a consequence of obesity rather than a pre-existing pituitary-hypothalamic disorder.

Detection of vascular endothelial growth factor and its receptor expression in human hepatocellular carcinoma biopsy specimens.

Background : Vascular endothelial growth factor (VEGF) exerts its actions on the microvasculature, by interacting with specific endothelial cell receptors, and thus, contributes to angiogenesis and growth in many tumours.

Analysis of the cell tropism of HCV by using in vitro HCV-infected human lymphocytes and hepatocytes.

BACKGROUND/METHODSnWe recently established two hepatitis C virus (HCV) replication systems, using MT-2, a human T-cell leukemia virus type I-infected human T-cell line, and PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. These HCV replication systems were used to assess the infective potencies of seven sera containing more than 10(6) HCV genomes per ml obtained from HCV-positive blood donors.nnnRESULTSnThe results showed that these sera had different infectivities for MT-2 and PH5CH cells. One of the seven sera, 1B-1, was more infective for MT-2 cells than PH5CH cells, whereas all the sera except serum 1B-1 were more infective for PH5CH cells than for MT-2 cells. Intracellular HCV RNA could be detected at least 30 days after inoculation with three of the sera. These findings suggested that the infective potency of each serum depends on the type of target cells. To further investigate HCV replication in these cells, we examined the hypervariable region 1 (HVR1) populations of HCV recovered from both MT-2 and PH5CH cells at 8 days postinoculation. The results revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation. Furthermore, in two of four sera, the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different.nnnCONCLUSIONnThese results suggest that HCV exhibits cell tropism.