Shinwa Shibata

Inhibition of B16 melanoma experimental metastasis by interferon-γ through direct inhibition of cell proliferation and activation of antitumour host mechanisms

Interferon‐γ (IFN‐γ) has pleiotropic activities other than its antivirus action, including cell growth inhibition, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation, and angiogenesis inhibitory activity, and these activities are supposed to be involved in its antitumour activity. However, it has not been completely elucidated which activity is mainly involved in the tumour suppression in vivo. In this study, we analysed inhibitory mechanisms of endogenous IFN‐γ against B16 melanoma experimental metastasis. After intravenous injection of tumour cells, tumour deposits in the lungs and liver were increased and life span was shorter in IFN‐γ−/− mice, indicating important roles for IFN‐γ in antitumour mechanisms. Interestingly, tumour deposits were not increased in IFN‐γ receptor (R)−/− mice. Furthermore, only low levels of cell‐mediated immunity against the tumour and activation of NK cells were observed, indicating that antimetastatic effects of IFN‐γ is not mediated by host cells. The survival period of B16 melanoma‐bearing IFN‐γR−/− mice was, however, shorter than wild‐type mice. These observations suggest that IFN‐γ prevents B16 melanoma experimental metastasis by directly inhibiting the cell growth, although antitumour host functions may also be involved in a later phase.

SCID-bg mice as xenograft recipients

SCID-bg (scid/scid, beige/beige) is a strain of double-mutant mice with impaired lymphoid development and reduced natural killer (NK) cell activity. The present study was undertaken to evaluate the usefulness of SCID-bg mice as xenograft recipients. Fetal guineapig tissues (liver, thymus, spleen) were transplanted under the kidney capsule of the mice and their serum guineapig IgG levels were measured weekly thereafter. C.B.-17-scid and anti-asialo GM1 antiserum-treated (NK-depleted) C.B.-17-scid (C.B.-17-scid-AGM1) mice that received the identical transplants were used as controls. Throughout the experimental period (1, 2, and 3 weeks after transplantation), the average serum guineapig IgG concentrations was highest in C.B.-17-scid-AGM1 mice followed by SCID-bg mice and lowest in C.B.-17-scid mice without antiserum treatment, though we could not find any statistical significance among these groups. However, SCID-bg mice always showed the smallest within-group variance (individual differencel in the serum guineapig IgG concentrations (P <0.05, versus C.B.-17-scid-AGM1 mice at 1,2, and 3 weeks and versus C.B.-17-scid mice at 2 weeks). The graft size was not significantly different among these three groups, but the spleen grafts in C.B.-17-scid mice contained fewer nucleate cells than the other two groups. These results indicate that the reduced NK cell activity by beige mutation is not crucial for the success of xenogenic transplantation, though SCID-bg mice may be useful as xenograft recipients with a consistent potential to retain the viability and function of engrafted tissues.

Acute hepatic failure in IFN-γ-deficient BALB/c mice after murine coronavirus infection

Abstract We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-γ (IFN-γ)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-γ-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-γ to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.

MHV-Induced Fatal Peritonitis in Mice Lacking IFN-γ

IFN-gamma gene was disrupted by homologous recombination in A3-1 embryonic stem cells. Germinally transmitted chimeric mice were successfully obtained and backcrossed with C57BL/6 (B6) mice 5 or 6 times. Deficiency of IFN-gamma in homozygous mice was confirmed by northern blot analysis of spleen cells stimulated with phorbor esther and calcium ionophore and also by IFN-gamma production in the culture supernatant of spleen cells stimulated with the same reagents. B6 mice lacking IFN-gamma were infected intraperitoneally (ip) with 10(6) PFU of JHMV and monitored for their survival. Approximately 90% of the mice died at 50 days post-infection (pi) and the mean survival time was 28 days. Mice sacrificed at 3 weeks pi showed severe peritonitis accompanying the accumulation of a viscous fluid in the abdominal and thoracic cavities. Microscopically, the disease was characterized by disseminated granulomatous inflammation and exudative fibrinous serositis in the abdominal cavity. Infectious virus was isolated in most tissues including the liver, spleen, kidney, pancreas and lung during the experimental periods. The disease was not observed in wild-type or heterozygous littermates infected i.p. with JHMV. These results suggest that IFN-gamma plays a critical role in MHV infection in mice. This experimental model may provide a unique opportunity to address the pathogenesis of virus-induced peritonitis such as feline infectious peritonitis in cats.

Localization of HIV-1 in human thymic implant in SCID-hu mice after intravenous inoculation

Human immunodeficiency virus type 1 (HIV‐1) was immunohistochemically and ultrastructurally localized in human thymus implants in SCID‐hu mice 3 weeks after intravenous (i.v.) inoculation of the virus. A viral antigen (gp120) was predominantly distributed in and around the epithelial cells in Hassall’s corpuscles as demonstrated by fluorescence immunohistochemistry. Occasional solitary round cells positive for the viral antigen but negative for cytokeratin were detected in the perivascular areas. Ultrastructural examinations clearly revealed a number of mature viral particles in the intercellular spaces of the Hassalls corpuscles. Thus the present study indicates the possibility that thymic epithelial cells in Hassalls corpuscles act as a target and/or reservoir in an early stage of HIV infection.

The Severity of Hepatic Lesion after Intraperitoneal JHMV Infection in IFN-gamma Deficient Mice is Parallel to Viral Replication in Hepatocytes in Vitro

Several factors affect MHV infection in mice (Table 1). They can be classified into two groups, viral factors and host factors. Although JHMV induces a fatal encephalitis in mice after intracerebral infection, it does a mild hepatitis when it is inoculated intraperitoneally. On the other hand, host genetic factor is a critical determinant. Among MHV researchers, it is well known that SJL mice are relatively resistant to intracerebral infection with JHMV (Stohlman & Frelinger 1978). In addition, the immune system is one of the key players that determine MHV infection in mice.

Mouse Hepatitis Virus-Specific CD8+ Cytotoxic T Lymphocytes Induce Apoptosis in Their Target Cells

CD8+ cytotoxic T lymphocytes (CTL) have been suggested to play an important role in virus clearance and development of demyelination during mouse hepatitis virus (MHV) infection in mice1. Production of antiviral cytokines such as γ-IFN and direct cytolysis of virus-infected cells have been considered to be the major role of CTL, but their precise mechanisms in MHV infection remain unclarified. We have previously established CTL line, P1 1D which specifically lyses cells infected with MHV, strain JHM (JHMV)2 and saves mice from lethal JHMV infection3. To clarify the detailed mechanism of CTL during MHV infection, we examined morphological changes of JHMV-infected target cells in vitro cocultured with anti-MHV CTL line, P1 1D.