Shigetaka Shimodaira

Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkins B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patients spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma

Objectives In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)–based immunotherapy in combination with gemcitabine and/or S-1. Methods Dendritic cell–based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. Results Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. Conclusions Dendritic cell vaccine–based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

Treatment of HCV-Related Mantle-Cell Lymphoma with Ribavirin and Pegylated Interferon Alfa

To the Editor: We expand on the data presented by Hermine et al., who demonstrated the antitumor efficacy of interferon alfa in hepatitis C virus (HCV)–infected patients with marginal-zone lymphoma...

Therapeutic angiogenesis by bone marrow implantation for critical hand ischemia in patients with peripheral arterial disease : a pilot study

ABSTRACT Objective: Implantation of bone marrow mononuclear cells (BM-MNCs), including endothelial progenitor cells, into ischemic lower limbs has been shown to improve symptoms in patients with peripheral arterial diseases (PAD). This study investigated whether BM-MNC implantation (BMI) is also effective for the ischemic hands of these patients. Methods: Seven PAD patients with hand ischemia were enrolled: six patients had thromboangiitis obliterans and one had collagen disease. All seven had symptoms involving either resting pain or non-healing ischemic ulcers of the hand. Approximately 600 mL of MNCs were separated from BM and concentrated to a final volume of 40–50 mL, which were injected into ischemic hands. Ischemic status was evaluated by measuring the digital/brachial pressure index (DBI), visual analog pain scale, and the healing of ulcers before and 6 months after BMI. Results: The mean number of implanted MNCs, CD34-positive cells, and CD34,133-positive cells was 3.67 ± 0.53 × 109, 4.94 ± 2.45 × 107, and 2.52 ± 1.57 × 107, respectively. Mean DBI in those patients was 0.15 ± 0.30 before BMI and significantly increased to 0.67 ± 0.19 at 6 months after BMI ( p = 0.004). All patients also showed improvement of pain scale and ischemic ulcers. There was no significant correlation between the number of implanted cells and improvement in the degree of DBI or the pain scale. Conclusion: Autologous BMI could be a promising and safe method of therapeutic angiogenesis for critical hand ischemia in PAD patients.

The detection of clonal proliferation in granular lymphocyte-proliferative disorders of natural killer cell lineage.

Summary. The clonal proliferation of large granular lymphocytes can be detected in patients with T‐cell‐lineage granular lymphocyte‐proliferative disorders (T‐GLPD) by Southern blotting T‐cell receptor genes. However, this cannot be applied to patients with natural killer‐cell‐lineage GLPD (NK‐GLPD) as it lacks a clonal marker. We therefore investigated the use of two other diagnostic techniques in evaluating clonal proliferation in Japanese patients with NK‐GLPD (n = 4) and T‐GLPD (n=3) by chromosomal analysis of peripheral blood mononuclear cells (PBMC) stimulated with either interleukin‐2 or phytohaemaggluti‐nin, and Epstein‐Barr viral (EBV) genomic DNA analysis. Chromosomal analysis revealed abnormal karyotypes in the PBMC of three of four patients with NK‐GLPD, whereas EBV analysis showed a monoclonal terminal configuration in the PBMC in the fourth patient. Southern blots revealed rearrangements of the TCR genes in all three patients with T‐GLPD but in none of those with NK‐GLPD. It is suggested that these methods may be useful in detecting the abnormal proliferation of large granular lymphocytes in NK‐GLPD.

Significance of chemokine receptor expression in aggressive NK cell leukemia

Natural killer (NK) cell-type lymphoproliferative diseases of granular lymphocytes can be subdivided into aggressive NK cell leukemia (ANKL) and chronic NK cell lymphocytosis (CNKL). One reason for the poor outcome in ANKL is leukemic infiltration into multiple organs. The mechanisms of cell trafficking associated with the chemokine system have been investigated in NK cells. To clarify the mechanism of systemic migration of leukemic NK cells, we enrolled nine ANKL and six CNKL cases, and analyzed the expression profiles and functions of chemokine receptors by flowcytometry and chemotaxis assay. CXCR1 was detected on NK cells in all groups, and CCR5 was positive in all ANKL cells. Proliferating NK cells were simultaneously positive for CXCR1 and CCR5 in all ANKL patients examined, and NK cells with this phenotype did not expand in CNKL patients or healthy donors. ANKL cells showed enhanced chemotaxis toward the ligands of these receptors. These results indicated that the chemokine system might play an important role in the pathophysiology of ANKL and that chemokine receptor profiling might be a novel tool for discriminating ANKL cells from benign NK cells.

Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ibα with human platelet antigen-2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease

The −5 C/T polymorphism of platelet glycoprotein (GP) Ibα is a major determinant of the level of GP Ib/V/IX complex surface expression. We investigated the frequency of this polymorphism among Asian populations. The gene frequencies of cytosine (C) in this polymorphism were 0·283 and 0·219 in Japanese and Korean populations respectively. The C allele is linked with human platelet antigen (HPA)‐2a and smaller types of variable number of tandem repeats (VNTR). A novel allele, C‐HPA‐2a‐D of VNTR, was found. No association was observed between these alleles and coronary artery disease in this case–control study. The clinical relevance of this polymorphism in the thrombotic status remains undetermined.

Changes in angiogenesis-related factors in serum following autologous bone marrow cell implantation for severe limb ischemia

Objective: Bone marrow mononuclear cell (BM-MNC) implantation (BMI) for critical severe limb ischemia especially for Buergers disease shows excellent clinical results but the mechanism of this treatment is still unknown. In this study, we investigated the changes in serum levels of angiogenesis-related factors after BMI treatment. Research design/methods: Twelve patients whose BMI treatments were clinically very effective was selected out of ninteen cases, nine patients had Buergers disease, two patients had arteriosclerosis obliterans and one had systemic sclerosis. Venous bood from femoral vein or brachial vein of the recipient limbs of these patients. Results: Adrenomedulin (AM), soluble vascular cell adhesion molecule-1 (sVCAM-1), and C-reactive protein (CRP) serum levels 24 h after BMI treatment were significantly increased compared with those before BMI treatment (p < 0.05). Vascular endothelial growth factor (VEGF) serum levels after BMI treatment significantly increased between 1 week and 3 months after BMI treatment (p < 0.05). Nitric oxide (NO) serum levels after BMI treatment increased significantly 2 weeks after BMI treatment (p < 0.05). There was no correlation between the numbers of implanted cells and serum levels of measured angiogenesis-related factors that were significantly increased after BMI treatment. Conclusion: It was concluded that the mechanism underlying BMI treatment consists of early and late phases. The early phase involves the direct action by implanted cells, and the late phase involves indirect paracrine action. In addition, it was considered that BMI treatment is effective when we implant a sufficient level of bone marrow (600 ml) to treat severe limb ischemia.

Changes in serum thrombopoietin levels after splenectomy.

To clarify the role of thrombopoietin (c-Mpl ligand, TPO) in ‘hypersplenic’ thrombocytopenia, we used an enzyme-linked immunosorbent assay to examine changes in serum TPO levels accompanied with splenectomy in 6 patients with liver cirrhosis, 4 patients with gastric cancer, and 2 patients with lymphoid malignancies. We also measured serum levels of other thrombopoietic cytokines such as interleukin-6 (IL-6) and erythropoietin. Platelet counts reached a maximum at day 14 after splenectomy in all subjects. In patients with liver cirrhosis, a lower elevation of platelet counts was observed compared with that in patients with gastric cancer. Serum TPO levels gradually elevated after splenectomy and reached a maximum 3.5 days after splenectomy in noncirrhotic patients, whereas peak serum TPO levels were delayed until day 7 in the cirrhosis group. IL-6 and erythropoietin showed similar kinetics between cirrhotic and noncirrhotic patients. These findings suggest that transient thrombocytosis after splenectomy may be associated with an alteration in the site of TPO catabolism by platelets from spleen to the blood and that deterioration of TPO production may play a role in thrombocytopenia in liver cirrhosis.

2-bromopropane-induced hypoplasia of bone marrow in male rats

2‐Bromopropane‐lnduced Hypoplasia of Bone Marrow in Male Rats: Tamie Nakajima, et al. Department of Hygiene, Shinshu University School of Medicine—The hematotoxicity of 2 ‐bromopropane was investigated in thirty‐six male Wistar rats. The rats were put into four groups: three groups were exposed to 0, 300, and 1,000 ppm 2‐bromopropane for 8 hr per day, for 9 weeks, respectively, and the remaining group was exposed to 3,000 ppm only for 9‐11 days. Hematotoxicity was assessed by measuring peripheral blood cells as well as cellularity, the number of megakaryocytes and morphological findings in the bone marrow. Exposure to 2‐bromopropane decreased the numbers of erythrocytes in the peripheral blood at 300 ppm or higher, leukocytes at 1,000 ppm, and platelets at 300 and 1,000 ppm. Exposure to 300 ppm 2‐bromopropane did not influence the indices of bone marrow toxicity. Exposure to 1,000 ppm 2‐bromopropane or a higher dose‐dependently induced a hypoplastic profile with replacement of fatty spaces in the bone marrow, though the durations of exposure to 3,000 ppm were under one sixth. These exposures also induced dose‐dependent decreases in the number of megakaryocytes, with the maintained ratio of granulocytes to erythrocytes in the bone marrow. Residual progenitor cells showed some dysplastic or megaloblastic changes. These results suggest that exposure to 2‐bromopropane leads to a reduction in the numbers of hematopoietic cells in the bone marrow, the result being a persistent pancytopenia in male rats.