Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

Susceptibility of human pancreatic ß cells for cytomegalovirus infection and the effects on cellular immunogenicity.

OBJECTIVES: Human cytomegalovirus (HCMV) infection has been suggested to be a causal factor in the development of type 1 diabetes, posttransplantation diabetes, and the failure of islet allografts. This effect of CMV has been interpreted as an indirect effect on the immune system rather than direct infection-induced cell death. In the present study, we investigated (i) the susceptibility of ß cells to HCMV infection, (ii) regulation of immune cell-activating ligands, (iii) release of proinflammatory cytokines, and (iv) the effects on peripheral blood mononuclear cell (PBMC) activation. METHODS: CM insulinoma cells and primary ß cells were HCMV-infected in vitro using a laboratory and a clinical HCMV strain. The susceptibility to infection was measured by the expression of viral genes and proteins. Furthermore, expression levels of Major Histocompatibility Complex I, Intracellular Adhesion Molecule-1, and Lymphocyte Function Associated Antigen-3 and the release of proinflammatory cytokines were determined. In addition, PBMC activation to HCMV-infected ß cells was determined. RESULTS: ß Cells were susceptible to HCMV infection. Moreover, the infection increased the cellular immunogenicity, as demonstrated by an increased MHC I and ICAM-1 expression and an increased proinflammatory cytokine release. Human cytomegalovirus-infected CM cells potently activated PBMCs. The infection-induced effects were dependent on both viral "sensing" and viral replication. CONCLUSIONS: In vivo ß-cell HCMV infection and infection-enhanced cellular immunogenicity may have important consequences for native or transplanted ß-cell survival.

Enterovirus infection induces cytokine and chemokine expression in insulin-producing cells.

Despite evidence supporting an association between enterovirus (EV) infection and type 1 diabetes, the etiological mechanism(s) for EV-induced beta cell destruction is(are) not well understood. In this study, the effects of Coxsackievirus B (CVB) 1-6 on cell lysis and cytokine/chemokine expression in the insulinoma-1 (INS-1) beta cell line were investigated. Cytolysis was assessed using tissue culture infectious dose 50 (TCID(50)). Quantitative RT-PCR was used to measure viral RNA and mRNA of cytokines interferon (IFN)-α, IFN-ß, IFN-γ, tumor necrosis factor (TNF)-α, and chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), and chemokine (C-C motif) ligand 5 (CCL5) in infected INS-1 cells. CVB2, 4, 5, and 6 lysed and replicated in INS-1 cells; TCID(50) was lowest for CVB5 and highest for CVB6. IFN-γ, CXCL10, and CCL5 mRNA levels all increased significantly following infection with CVB2, 4, 5, and 6 (P<0.05). CCL2 mRNA increased with CVB2, 5, and 6 (P<0.05), IFN-α mRNA increased with CVB5 infection (P<0.05), while TNF-α mRNA and IFN-ß mRNA (P<0.001) increased with CVB2 infection. Dose-dependent effects of infection on cytokine mRNA levels were observed for all (P<0.01) except IFN-γ. Following inoculation of INS-1 cells with CVB1 and 3, viral RNA was not detected and cytokine/chemokine mRNA levels were unchanged. In conclusion, CVB2, 4, 5, and 6 induce dose-dependent cytokine and chemokine mRNA production from INS-1 cells suggesting that pro-inflammatory cytokine and chemokine secretion by beta cells is a potential mechanism for EV-induced beta cell damage in type 1 diabetes.

Amino acids of Coxsackie B5 virus are critical for infection of the murine insulinoma cell line, MIN-6.

It was shown recently that 15 successive passages of a laboratory strain of the Coxsackie B virus 5 in a mouse pancreas (CBV-5-MPP) resulted in apparent changes in the virus phenotype, which led to the capacity to induce a diabetes-like syndrome in mice. For further characterization of islet cell interactions with a passaged virus strain, a murine insulinoma cell line, MIN-6, was selected as an experimental model. The CBV-5-MPP virus strain was not able to replicate in MIN-6 cells in vitro but required adaptation over a few days for progeny production and the generation of cytopathic effects. In order to determine the genetic characteristics required for virus growth in MIN-6 cells, the whole genome of the MIN-6-adapted virus variant was sequenced, and critical amino acids were identified by comparing the sequence with that of a virus strain passaged repeatedly in the mouse pancreas. The results of site-directed mutagenesis demonstrated that only one residue, amino acid 94 of VP1, is a major determinant for virus adaptation to MIN-6 cells.

Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein.

High-risk human papillomaviruses (HPV) such as HPV16 are associated with the development of cervical cancer. The HPV16-E6 and HPV16-E7 oncoproteins are expressed throughout the replicative cycle of the virus and are necessary for the onset and maintenance of malignant transformation. Both these tumor-specific antigens are considered as potential targets for specific CTL-mediated immunotherapy. The adenylate cyclase (CyaA) of Bordetella pertussis is able to target dendritic cells through specific interaction with the alpha(M)beta(2) integrin. It has been previously shown that this bacterial protein could be used to deliver CD4(+) and CD8(+) T cell epitopes to the MHC class II and class I presentation pathways to trigger specific Th and CTL responses in vivo, providing protection against subsequent viral or tumoral challenge. Here, we constructed recombinant CyaA containing either the full sequence or various subfragments from the HPV16-E7 protein. We show that, when injected to C57BL/6 mice in absence of any adjuvant, these HPV16-recombinant CyaAs are able to induce specific Th1 and CTL responses. Furthermore, when injected into mice grafted with HPV16-E7-expressing tumor cells (TC-1), one of these recombinant proteins was able to trigger complete tumor regression in 100% of the animals tested. This therapeutic efficacy compared favorably to that of strongly adjuvanted peptide and was marginally affected by prior immunity to CyaA protein. This study represents the first in vivo demonstration of the antitumoral therapeutic activity of recombinant CyaA proteins carrying human tumor-associated antigens and paves the way for the testing of this vector in clinical trials.

Role of the amino-terminal domain of simian virus 40 early region in inducing tumors in secretin-expressing cells in transgenic mice.

BACKGROUND & AIMS: The early region of simian virus 40 (SV40) encodes 2 transforming proteins, large T (Tag) and small t antigen, that produce neuroendocrine tumors in the intestine and the pancreas when expressed in secretin cells of transgenic mice. METHODS: Two SV40 early-region transgenes containing a deletion that eliminated expression of the small t antigen were expressed in transgenic mice under control of the secretin gene. The 2 lines of mice, one expressing the native large T antigen and the other T antigen with a mutation in its N-terminal J domain, were examined to determine which biological activities of the SV40 early region were required for tumorigenesis. RESULTS: Most animals expressing wild-type large T antigen developed pancreatic insulinomas and lymphomas and died between 3 and 6 months of age. However, small intestinal neoplasms were extremely rare in the absence of small t antigen expression. Transgenic lines expressing the J domain mutant failed to develop tumors. CONCLUSIONS: Transformation of secretin-producing enteroendocrine cells by SV40 requires functional cooperation between intact large T and small t oncoproteins. In contrast, large T antigen alone is sufficient to induce tumors in the endocrine pancreas and thymus.

BK virus-induced osteosarcoma (Os515) as a model of human osteosarcoma.

BK virus was isolated by Gardner et al in 1971 from the urine of an immunosuppressed patient with a kidney allograft. Antibodies to this virus are ubiquitous in the general populations worldwide, but the oncogenic capacity of BKV in humans had not been reported. The virus transformed in vitro permissive human and non-permissive animal cells, and the transformed cells had the T antigen. Intracerebral and intravenous inoculation of BKV in newborn hamsters induced malignant tumours (mainly ependymomas, malignant insulinomas, and osteosarcomas). Subcutaneous and intraperitoneal routes were also effective. The virus was only rescued from a few tumours by fusion with human embryonic cells or Vero cells. Brain tumours appeared earlier and osteosarcomas developed in animals which survived for more than 6 months. Many of the osteosarcomas were bony and grew slowly with frequent lung metastases, and a few osteosarcomas were soft and grew rapidly without lung metastases. Experimental targeting chemotherapy with doxorubicin (DX)-containing immunoliposomes was performed against Os515 osteosarcoma. In in vitro experiments, DX-Lip-MoAb29 showed a more significant inhibitory effect on cultured Os515 cells than free Dx and DX-Lip. DX-Lip DNP had less effect. In in vivo experiments, DX-Lip-MoAb29 suppressed the growth of Os515 tumour isografts in hamsters and prolonged the survival of recipients more significantly than free DX.