MDA-9/syntenin is essential for factor VIIa-induced signaling, migration, and metastasis in melanoma cells.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, is a novel gene that positively regulates cancer cell motility, invasion, and metastasis through distinct biochemical and signaling pathways, but how MDA-9/syntenin is regulated in response to signals with the extracellular environment and promotes tumor progression is unclear. We now demonstrate that MDA-9/syntenin is dramatically up-regulated by a combination of rFVIIa and factor F(X) in malignant melanoma. Induction of MDA-9/syntenin in melanoma was found to occur in a thrombin-independent signaling pathway and involves the PAR-1/c-Src/Rho GTPases Rac1 and Cdc42/c-Jun N-terminal kinase axis resulting in the activation of paxillin, NF-κB, and matrix metalloproteinase-2 (MMP-2). MDA-9/syntenin physically interacts with c-Src through its PDZ binding motif following stimulation of melanoma cells with rFVIIa and FX. We also document that induction of this signaling pathway is required for TF·FVIIa·Xa-induced cell migration, invasion, and metastasis by melanoma cells. The present finding uncovers a novel role of MDA-9/syntenin as an important TF·FVIIa·Xa/PAR-1-regulated gene that initiates a signaling circuit essential for cell motility and invasion of metastatic melanoma. In these contexts, targeting TF·FVIIa·Xa and its relevant downstream targets such as MDA-9/syntenin, may represent a novel therapeutic strategy to control the evolution of neoplastic cells.

Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene.

INTRODUCTION: Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford 'prope' tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. OBJECTIVES: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain. MATERIALS AND METHODS: To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice. RESULTS: Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9+/-1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6+/-1.2 days). CONCLUSIONS: IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant (P<0.001) prolongation of the survival of donor skin allografts was observed.

Gene therapy for human immunodeficiency virus infection in the humanized SCID mouse model.

BACKGROUND: The humanized SCID mouse model is an attractive tool for testing gene therapy to combat human immunodeficiency virus infection in vivo. OBJECTIVES: To devise a more specific gene therapy directed against HIV, replacing the formerly used interferon with either soluble CD4 molecule immunoadhesin (sCD4-IgG) and/or anti-gp41 monoclonal antibody (2F5), or HIV-negative transdominants genes (Tat, Rev). METHODS: Human monocytoid cell line (U937) was transfected with IFN alpha, beta or gamma genes. 3T3 murine fibroblastic cell line was transfected with sCD4-IgG or 2F5, or both genes, and a human T4 cell line (CEM) was grafted to SCID mice. Negative transdominant genes (Tat, Rev or both) were also transduced in CEM T cell line. Animals were then challenged with HIV-1, Viral load was followed. RESULTS: IFN alpha or beta were potent anti-HIV, reducing viral load in vivo and inhibiting reverse transcriptase activity in human-removed cells from animals. sCD4-IgG immunoadhesin and gp41 monoclonal antibody resulted in a dramatic reduction of HIV-1 cellular and plasmatic viral load in humanized SCID mice. The simultaneous introduction of negative Tat and Rev genes resulted in a synergistic inhibition of HIV-1 replication in vivo. CONCLUSIONS: Despite the marked reduction of HIV-1 propagation by IFN genes or by negative Tat and Rev transdominants, the gene therapy using soluble CD4 immunoadhesin or anti-gp41 was a more efficient preventive treatment against HIV infection.

Prope tolerance to heart allografts in mice associated with persistence of donor interleukin-10-transduced stem cells.

BACKGROUND: We previously reported that transduction of the human interleukin (IL)-10 gene into the total fetal liver stem cells (hIL-10-TFLs) of mice protects against their rejection in an allogeneic host. In this study, we explored the effects of these cells in two different models of organ transplantation. METHODS: Balb/c mice were sublethally irradiated before receiving skin or vascularized heterotopic heart grafts from C57Bl/6 mice. TFLs from C57Bl/6 mice transduced with hIL-10 or untransduced TFLs were injected on the day of transplantation into recipient mice once or also every 20 days thereafter. RESULTS: Skin allograft survival was prolonged for up to 17.8±0.6 days, vs. 9.0±0.4 days, in mice that received hIL-10-TFLs or untransduced TFLs, respectively. Allogeneic heart transplants survived for 86.25±13.8, 46.3±4.6, 28.1±6.1, or 11.5±0.6 days in mice that received repeated injections of hIL-10-TFLs, a single injection of hIL-10-TFLs, repeated injections of untransduced TFLs, or controls, respectively. Histological analyses of the grafts showed fewer inflammatory foci and CD8+ infiltrating cells in mice injected with hIL-10-TFLs compared with untreated mice. Expressions of H-2b and hIL-10 were found in several organs, including the thymus, liver, and the transplant, in hIL-10-TFL-injected mice. Finally, in hIL-10-TFL-injected mice, FoxP3 T cells were present inside the transplanted heart as late as 140 days after transplantation. CONCLUSIONS: In this study, we showed that repeated injections of hIL-10-TFLs are efficient in mitigating transplant rejection. This "prope" tolerance was associated with survival of donor hematopoietic cells in the host.

Transplantation tolerance induced in humans at the fetal or the neonatal stage.

Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens. Engraftment needs no conditioning regimen prior to transplantation in neonates with severe combined immunodeficiency disease or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.

HIV-1 infection: functional competition between gp41 and interleukin-2.

To determine whether the gp41 of HIV-1 could adhere to the interleukin (IL)-2 receptor at the surface of target cells in vitro, we analysed in vitro the possible functional competition between various forms of the HIV-1 gp41 molecule (i.e. peptides, trimeric or primary structures) and IL-2. This competition has been analysed in a test involving the proliferation of an IL-2-dependent cell line (CTLL2). The putative interaction between the IL-2 molecule and HIV-1 has also been assayed on MT4 cells (CD4(+) T lymphocytes) in culture. The gp41 trimeric molecule and an HIV-1 gp41 peptide (578-590 aminoacid sequence) dramatically inhibited CTLL2 cell proliferation, despite the presence of IL-2. The addition of serum, containing anti-gp41 antibodies, from HIV-1 patients resulted in a significant abolition of this inhibition. The concomitant incubation of IL-2 and HIV-1 with MT4 cells resulted in a strong decrease (70%) in HIV-1 p24 release. These data suggest that the gp41 of HIV-1 can use the IL-2 receptor during the process of HIV-1 infection and that there is some functional mimesis between gp41 and IL-2.

Endogenous retroviral genes, Herpesviruses and gender in Multiple Sclerosis.

Unexpected findings on endogenous retroviral elements expressed in cells from patients with Multiple Sclerosis appear to open a new avenue of research, after years of research dedicated to the understanding of their biological significance in human health and disease. Human endogenous retroviral family W (HERV-W) RNA present in circulating viral particles (Multiple Sclerosis associated RetroViral element, MSRV) has been associated with the evolution and prognosis of Multiple Sclerosis. HERV-W elements encode a powerful immunopathogenic envelope protein (ENV) that activates a pro-inflammatory and autoimmune cascade through interaction with Toll-Like Receptor 4 (TLR4) on antigen-presenting cells, and triggers superantigen-like dysregulation of T-lymphocytes. HERV-W/ENV antigen has further been shown to be an upstream inducer of immunopathogenicity like that in MS and has repeatedly been detected in association with MS lesions in post-mortem brain studies. ENV protein now represents a novel target in MS, in our ongoing development of a neutralising therapeutic antibody. We here review the pieces of a puzzle, which now offer a consistent picture for Multiple Sclerosis aetiopathogenesis. Interestingly, at the gene-environment interface, this picture also includes gender-related specificities through the potential interplay with endogenous retrovirus type W copies present on the X chromosome.

Dendritic cells dysfunction in tumour environment.

Several studies indicate that most tumours are immunogenic and they rarely succeed to induce an efficient immune response. Many mechanisms have been involved in the tumour escape from host immune surveillance. The tumour microenvironment has emerged as an important component contributing to dendritic cells (DCs) dysfunction. There is evidence that DCs play a key role in the induction of tumour-specific immune responses, especially via cross-priming through MHC-class I antigens presentation. In this review we will discuss the potential role of the tumour microenvironment in DCs dysfunction.