Keiko Mochida

Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.

Objectives:  Macrophages (Mφs) have various functions and play a critical role in host defense and the maintenance of homeostasis. Mφs exist in every tissue in the body, but Mφs from different tissues exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression and function, and are called by different names. However, the precise mechanism of the generation of macrophage heterogeneity is not known. In the present study, the authors examined the functional heterogeneity of Mφs generated from human monocytes under the influence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and macrophage‐CSF (M‐CSF).

Enhanced expression of lymphomagenesis-related genes in peripheral blood B cells of chronic hepatitis C patients.

Epidemiological data indicate a close relationship between chronic hepatitis C virus (HCV) infection and B-cell non-Hodgkins lymphoma (B-NHL), suggesting that chronic HCV infection is, at least in part, associated with B-lymphomagenesis. However, experimental data concerning these conditions remains elusive. In this study, we confirmed that peripheral blood B cells of chronic hepatitis C (CHC) patients were infected with HCV. Expression levels of activation-induced cytidine deaminase (AID) which are thought to be associated with occurrence of B-NHL were analyzed in these CHC B cells. It was demonstrated that AID mRNA/protein levels in CHC B cells were dramatically increased compared with those of healthy subjects. Furthermore, expression levels of several previously reported prognostic B-NHL marker genes in the B cell subset of CHC patients were increased. These results suggest a possible relationship between chronic HCV infection and B-lymphomagenesis.

Peripheral B Cells May Serve as a Reservoir for Persistent Hepatitis C Virus Infection

A recent study by our group indicated that peripheral B cells in chronic hepatitis C (CHC) patients are infected with hepatitis C virus (HCV). This raised the logical question of how HCV circumvents the antiviral immune responses of B cells. Because type I interferon (IFN) plays a critical role in the innate antiviral immune response, IFNβ expression levels in peripheral B cells from CHC patients were analyzed, and these levels were found to be comparable to those in normal B cells, which suggested that HCV infection failed to trigger antiviral immune responses in B cells. Sensing mechanisms for invading viruses in host immune cells involve Toll-like receptor-mediated and retinoic acid-inducible gene-I (RIG-I)-mediated pathways. Both pathways culminate in IFN regulatory factor-3 (IRF-3) translocation into the nucleus for IFNβ gene transcription. Although the expression levels of RIG-I and its adaptor molecule, IFN promoter-stimulator-1, were substantially enhanced in CHC B cells, dimerization and subsequent nuclear translocation of IRF-3 were not detectable. TANK-binding kinase-1 (TBK1) and IĸB kinase Ε (IKKΕ) are essential for IRF-3 phosphorylation. Constitutive expression of both kinases was markedly enhanced in CHC B cells. However, reduced expression of heat shock protein of 90 kDa, a TBK1 stabilizer, and enhanced expression of SIKE, an IKKΕ suppressor, were observed in CHC B cells, which might suppress the kinase activity of TBK1/IKKΕ for IRF-3 phosphorylation. In addition, the expression of vesicle-associated membrane protein-associated protein-C, a putative inhibitor of HCV replication, was negligible in B cells. These results strongly suggest that HCV utilizes B cells as a reservoir for persistent infection.

HCV Infection and B-Cell Lymphomagenesis

Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkins lymphoma (NHL). Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.

Enhanced RIG-I expression is mediated by interferon regulatory factor-2 in peripheral blood B cells from hepatitis C virus-infected patients

Chronic hepatitis C patients carry the risk of developing into B-cell non-Hodgkins lymphoma (B-NHL). To clarify the mechanisms underlying this association, we first investigated the molecular markers of B cells from hepatitis C virus (HCV)-infected patients. CD19-positive cells were isolated as B cells from the peripheral blood mononuclear cells of patients infected with the hepatitis C virus and IFN-related gene expression was analyzed. We found that RIG-I and IRF-2 expression were up-regulated in CD19-positive cells from the infected patients. In vitro luciferase reporter analysis using human cell lines indicated that IRF-2 activates the human RIG-I promoter. IRF-2 expression levels were enhanced by HCV cDNA transfection in Huh7 cells. In addition, we observed much less induction in the interferon stimulated gene 15 (ISG15) after Sendai virus (SenV) stimulation of CD19-positive cells from infected patients versus healthy controls, thereby suggesting an impairment of RIG-I downstream signaling in HCV-infected patients. Hence, we found that the failure of the anti-viral response with enhanced IRF-2 oncogenic protein expression in blood B cells from HCV-infected patients. Our results provide important information to better understand the role of IRFs in the cause of HCV chronic infection.

Genetic verification of Bordetella pertussis seed strains used for production of Japanese acellular pertussis vaccines

In Japan, the Bordetella pertussis strain Tohama provided by the National Institute of Health, Japan has been used for the production of acellular pertussis (aP) vaccines since 1981. In the present study, in order to verify the genetic consistency of B. pertussis vaccine seed strains, we analyzed the genetic properties of the working seeds obtained from five Japanese vaccine manufacturers, and compared them with those of B. pertussis Tohama reference strains (NIID L-7 and ATCC BAA-589). Genetic analyses with pulsed-field gel electrophoresis and allele typing showed 100% genetic identity among the five seed strains and the Tohama reference strains. In addition, Southern blot analyses revealed the absence of four orthologous genes (BB0537, BB0920, BB1149 and BB4885), which are specifically absent in the strain Tohama, and in the genome of all seed strains tested, suggesting that the regions of difference (RD11-RD14) are absent in their genomes. Consequently, no genetic difference was observed among the working seeds and Tohama reference strains. Our observations indicate that B. pertussis seed strains for Japanese aP vaccine production are genetically comparable with B. pertussis Tohama.

Development of an Infectious Surrogate Hepatitis C Virus Based on a Recombinant Vesicular Stomatitis Virus Expressing Hepatitis C Virus Envelope Glycoproteins and Green Fluorescent Protein

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.