Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.     

Method for In Situ Evaluation of Superoxide Production by Pulmonary Macrophages in the Rat

In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)₂, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.     

Granzyme B-positive primary gastric T-cell lymphoma: gastric T-cell lymphoma with the possibility of extrathymic T cell origin

A case of primary gastric T-cell lymphoma, which was positive for granzyme B, is reported. The patient was a 47-year-old Japanese female who complained of a dull upper abdominal pain. Radiographic and endoscopic examinations revealed an ulcerative infiltrative lesion in her stomach. Following the confirmation of a high-grade malignant lymphoma, a distal gastrectomy with regional lymph nodal dissection was performed. The histology of the gastric lesion revealed a malignant lymphoma of the diffuse pleomorphic type without lymph nodal involvement. Immunohistochemistry revealed that the tumor cells were positive for LCA, CD3, TIA-1 and granzyme B, but were negative for CD4, CD8, CD56, CD30, L-26, EMA, TCR alpha/beta and TCR gamma/delta. Because the tumor cells showed T cell nature with cytotoxic activity proved by TIA-1 and granzyme B, and without evidence of further maturation of T cell, a malignant lymphoma originating from extrathymic-derived T cells was suggested.     

Calcium pump expression in human bone and soft tissue tumours

Ninety-one cases of human bone and soft tissue tumours were studied for calcium pump expression by strepto-avidin-biotin immunohistochemical staining with a monoclonal antibody against sarcoplasmic reticulum calcium-ATPase (mAb6F5). Two out of 5 cases of embryonal rhabdomyosarcoma, 1 out of 5 cases of biphasic synovial sarcoma, 4 of 4 cases of chordoma and all of 3 chondrosarcoma cases were positive for mAb6F5. Although this novel monoclonal antibody can be used as a marker of myogenic tumours, the present positive result for endoplasmic reticulum calcium-ATPase (calcium pump) in other tumours including chordoma, chondrosarcoma and synovial sarcoma indicates a wider immunoreactivity. The findings further suggest that intracellular calcium may play an important role in cell proliferation and/or differentiation.     

A PKC epsilon-ENH-channel complex specifically modulates N-type Ca2+ channels

Multiple protein kinase C (PKC) isozymes are present in neurons, where they regulate a variety of cellular functions. Due to the lack of specific PKC isozyme inhibitors, it remains unknown how PKC acts on its selective target(s) and achieves its specific actions. Here we show that a PKC binding protein, enigma homolog (ENH), interacts specifically with both PKCepsilon and N-type Ca2+ channels, forming a PKCepsilon-ENH-Ca2+ channel macromolecular complex. Coexpression of ENH facilitated modulation of N-type Ca2+ channel activity by PKC. Disruption of the complex reduced the potentiation of the channel activity by PKC in neurons. Thus, ENH, by interacting specifically with both PKCepsilon and the N-type Ca2+ channel, targets a specific PKC to its substrate to form a functional signaling complex, which is the molecular mechanism for the specificity and efficiency of PKC signaling.     

Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells

To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161c-treated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1(+) NK cells.     

Evaluation of a Membrane Filter Assay System, Ortho HCV Ab Quik Pack, for Detection of Anti-Hepatitis C Virus Antibody

A simple membrane immunoassay assay system, Quik Pack, for the detection of hepatitis C virus antibody was compared with two enzyme-linked immunosorbent assays (ELISAs) in a study of 600 serum samples. Quik Pack exhibited excellent sensitivity and specificity: 96.0 and 99.7%, respectively, versus the ELISA-2 and 99.7 and 99.4%, respectively, versus the ELISA-3.