Multiple sclerosis-associated retrovirus particles cause T lymphocyte-dependent death with brain hemorrhage in humanized SCID mice model.

A retroviral element (multiple sclerosis-associated retrovirus, MSRV) defining a family of genetically inherited endogenous retroviruses (human endogenous retrovirus type W, HERV-W) has been characterized in cell cultures from patients with multiple sclerosis. Recently, MSRV retroviral particles or the envelope recombinant protein were shown to display superantigen activity in vitro, but no animal model has yet been set up for studying the pathogenicity of this retrovirus. In the present study, the pathogenicity of different sources of MSRV retroviral particles has been evaluated in a hybrid animal model: severe combined immunodeficiency (SCID) mice grafted with human lymphocytes and injected intraperitoneally with MSRV virion or mock controls. MSRV-injected mice presented with acute neurological symptoms and died within 5 to 10 days post injection. Necropsy revealed disseminated and major brain hemorrhages, whereas control animals did not show abnormalities (P <.001). In ill animals, reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed circulating MSRV RNA in serum, whereas overexpression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma was evidenced in spleen RNA. Neuropathological examination confirmed that hemorrhages occurred prior to death in multifocal areas of brain parenchyma and meninges. Further series addressed the question of immune-mediated pathogenicity, by inoculating virion to SCID mice grafted with total and T lymphocyte-depleted cells in parallel: dramatic and statistically significant reduction in the number of affected mice was observed in T-depleted series (P <.001). This in vivo study suggests that MSRV retroviral particles from MS cultures have potent immunopathogenic properties mediated by T cells compatible with the previously reported superantigen activity in vitro, which appear to be mediated by an overexpression of proinflammatory cytokines.

A new Mr 55,000 surface protein implicated in melanoma progression: association with a metastatic phenotype.

Emergence of the invasive phenotype is a key event in the progression of human melanoma from benign proliferative lesions to malignant lesions. Recently we successfully selected in vivo from a poorly metastatic M4Beu. human melanoma cell line two variants (7GP and T1P26) that generate a higher frequency of spontaneous metastases to the lungs into immune-suppressed neonatal rats. Both cell lines showed no significant differences in the integrin profile of the subunits analyzed except for beta3, which was reduced to a background level in metastatic variants. To investigate how these variant sublines of human melanomas manage to sustain growth in the absence of alpha(v)beta3, a subtractive immunization approach was used to elicit host antibody response against cell surface proteins expressed on metastatic variants. In this study, a new monoclonal antibody (MoAb), LY1, that is highly specific for the 7GP and T1P26 variants, was isolated. LY1 identifies a membrane protein of Mr 55,000 on melanoma variants with epitopes that were resistant to sugar-cleaving enzymes. Immunostaining cells from variants by LY1 showed that staining is distributed to the cell periphery with high labeling intensity at the cell-to-cell contact points. This MoAb significantly inhibited invasion of metastatic variants through a reconstituted basement membrane (Matrigel) in vitro. Moreover, tumor growth of melanoma variants was dramatically affected in vivo with this MoAb. In vitro studies indicate that the LY1 MoAb does not inhibit chemotactic migration of the metastatic variants, the adhesion of tumor cells to vitronectin, collagen IV, fibronectin, and laminin, or cell proliferation. Expression of this antigen is high in human striated muscle, heart, spleen, brain, and lung and absent in kidney, liver, and pancreas. Using 59 fixed, paraffin-embedded archival tissues of human melanomas and nevi, LY1-reactive cells were not observed in melanocytes, nevi, or radial growth phase primary melanomas. In sharp contrast, LY1 selectively stained melanocytes derived from the vertical growth phase of many primary melanomas and metastatic melanomas. These results provide evidence that the Mr 55,000 protein expressed by selected variants with increased metastatic properties in vivo plays a functionally important role in determining metastasis. This molecule may represent a new metastatic risk marker in human melanoma and may be of biological importance in the identification of fatal metastatic subpopulations that have acquired competence for metastasis production.

Gene transfer of anti-gp41 antibody and CD4 immunoadhesin strongly reduces the HIV-1 load in humanized severe combined immunodeficient mice.

OBJECTIVE: To study the anti-HIV-1 effects of the delivery of anti-gp41 monoclonal antibody (mAb) and soluble CD4 (sCD4) immunoadhesin by genetically modified cells in HIV-1-infected, humanized severe combined immunodeficient (SCID) mice. DESIGN: The complementary DNA of mAb 2F5, an anti-HIV-1 gp41 antibody, and of sCD4-IgG chimeric immunoadhesin were transferred into 3T3 cells using Moloney murine leukaemia virus vectors. The cells were then incorporated into a collagen structure called the neo-organ, which allowed the continuous production of the therapeutic molecules. METHODS: The antiviral effects in vivo of 2F5 or sCD4-IgG or both compounds were evaluated in neo-organ-implanted SCID mice that were grafted with human CD4 CEM T cells and challenged with HIV-1 Lai or MN. RESULTS: In SCID mice implanted with 2F5 neo-organs, antibody plasma levels reached 500-2000 ng/ml. Viral loads after HIV-1 challenge were significantly reduced in neo-organ-implanted HIV-infected mice. Although 29 x 10(7) and 13 x 10(8) HIV-1-RNA copies/ml were detected at 12 days in the controls (mice injected with Lai and MN, respectively) less than 16.5 x 10(3) HIV-1-RNA copies/ml were observed in all implanted mice injected with either Lai or MN. The intracellular viral load was also reduced in CD4 cells recovered from the implanted mice. Comparable antiviral effects were obtained with CD4-IgG neo-organs. CONCLUSION: Our results confirm the anti-HIV properties of 2F5 and sCD4-IgG continuously produced in vivo after ex-vivo gene therapy in SCID mice.

[In vitro and in vivo inhibition of HIV1 replication by retroviral transfer of interferon alpha, beta, or gamma genes: application to gene therapy of AIDS]. / Inhibition in vitro et in vivo de la réplication du VIH1 par transfert rétroviral des gènes d'interféron alpha, beta ou gamma: application à la thérapie génique du sida.

Somatic gene therapy is defined as the transfer of a heterologous gene into an organism for the purpose of correcting a genetic defect or providing a new therapeutic function to the target cell and thus inducing a cure or improving associated symptoms. While encouraging results have been generated by recent clinical evaluation of combination of anti-viral drugs, Aids still constitute an obvious candidate among the infectious diseases which might be treated by gene therapy. We have therefore chosen to develop and evaluate a gene therapy strategy based on the transfer into human target cells of HIV1-inducible interferon (IFN) alpha, beta or gamma genes. In a preliminary study, myeloïd U937 cell lines transfected with expression vectors containing the IFN alpha, beta or gamma genes under the control of the long terminal repeat (LTR) sequences of HIV1 were shown to be strongly resistant against an in vitro and in vivo (in HIV1 challenged SCID mice model) HIV1 infection. This cellular resistance was correlated with a strong induction of transgenic IFN synthesis and for IFN gamma, with a defect of HIV particles maturation. Secondly, construction and production of high titer retroviral vectors containing Tat-inducible IFN genes allowed efficient transduction of lymphoïd cell lines and human primary lymphocytes. These transduced cells were shown to be highly resistant against laboratory and primary HIV isolates. Taken together, our in vitro and in vivo results suggest that HIV1 inducible IFN gene therapy can be beneficial to HIV-infected individuals provided the fact that methods are developed that allow the efficient transduction of human hematopoïetic stem cells.

Experimental gene therapy: the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice.

OBJECTIVES: To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer on interferon (IFN)-alpha, -beta and -gamma genes to human cells. DESIGN: Human U937 promonocytic cells were stably transfected with Tat-inducible IFN expression vectors conferring an antiviral state against infection with HIV. METHODS: Transfected cells were either infected by HIV-1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo. RESULTS: U937 cell lines stably carrying IFN transgenes under the positive control of the HIV-1 Tat protein were highly resistant to HIV-1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV-1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN-U937 cells permitted full HIV-2 replication. Transfected cells injected into SCID mice and challenged against HIV-1 were strongly resistant to infection when cells were transduced with IFN-alpha of IFN-beta genes. However, IFN-gamma-transfected cells permitted HIV-1 infection in vivo despite the induction of a high level of IFN-gamma secretion. The quantity of proviral DNA was 10(5)-fold lower in IFN-alpha- or IFN-beta-transfected U937 cells collected from these SCID mice than that in non-transfected cells. CONCLUSIONS: Our results substantiated the validity of a strategy, bases on the transfer of HIV-1-inducible IFN-alpha or IFN-beta genes, to confer antiviral resistance to human cells.

Loss of T-cell cytotoxic responses in the course of HIV-1 infection.

T-cell mediated cytotoxicity play an important role in the control of human immunodeficiency virus (HIV) infection. The polyclonal cytotoxic T lymphocyte (CTL) response against target cells infected with a recombinant vaccinia virus expressing Env, Gag, Nef or reverse transcriptase (RT) proteins has been studied in four groups of individuals: acquired immune deficiency syndrome (AIDS) patients, AIDS-related complex (ARC) patients, HIV-1 seropositive subjects and seronegative controls. CTL lines have been generated by non-specific stimulation with phytohemagglutinin and interleukin-2 and target cells have been prepared from autologous B lymphocytes. CTL from asymptomatic and ARC individuals recognized most of the various proteins of HIV-1 but those from AIDS patients had very low or absent responses to the majority of proteins, with the anti-Nef cytotoxic activity decreasing first. Two of 10 AIDS patients had demonstrable recognition of Gag p24, one of RT and eight patients had no recognition of any of the proteins. The effector cells were demonstrated to be predominantly of the CD8+ phenotype, using the appropriate monoclonal antibodies. When heterologous target cells were substituted for autologous cells, the cytotoxic response was abrogated in the vast majority of cases demonstrating its human leucocyte antigen (HLA) class I restriction. Among the 10 HIV-seronegative subjects, nine had no CTL activity against the various HIV-1 proteins but one subject was able to recognize Env and RT. In the evolution of HIV infection from the seropositive stage to AIDS, CTL polyclonal activities progressively decrease, with Nef responses disappearing first, then Env and Gag p55, followed by RT and Gag p24.

Anti-NK antibodies injected into recipient mice enhance engraftment and chimerism after allogeneic transplantation of fetal liver stem cells.

Natural killer (NK) cells have been shown to play a role in the phenomenon of resistance to transplantation of allogeneic stem cells. To explore and prevent such resistance, we treated severe combined immunodeficiency mice (SCID) with anti-NK antibodies and analysed the improved engraftment of stem cells induced by this treatment. Two groups of nine SCID mice (H-2d) were compared: group A received two injections of anti-asialo GM1 rabbit antibodies (anti-NK) on days 1 and 4; group B received two injections of normal rabbit serum. All mice were injected intravenously with 7 x 10(6) fetal liver cells from B6 mice (H-2b) on day 2. One month after fetal liver cell transplantation, all mice from group A demonstrated engraftment and chimerism; at this time, donor cells accounted for more than 50% of peripheral blood mononuclear cells (PBMC). In contrast, in group B, only one mouse had 26% of donor cells among PBMC and all other mice had less than 10%. At two months, results were virtually identical in group A (over 72% of donor cells among PBMC from all mice) and slightly improved in group B (0-38% of donor cells). After the third month and continuously until the 12th month, the stability of chimerism was established in group A (over 55% of donor cells in 7 of the 9 mice) but had virtually disappeared in group B (0-2% of donor cells in all mice). Tissue analysis demonstrated the improved reconstitution of the thymus and the spleen in mice from group A. The proliferative responses of spleen cells to phytomitogens were significantly higher in all mice from group A than in any mouse from group B. Skin allografts from a third party (H-2k) were rejected within 10 days by group A mice but not by group B mice, one year after fetal liver cell transplantation. On the whole, anti-NK antibodies were able to improve engraftment, chimerism and stability of allogeneic stem cell transplants.

Interleukin (IL)-2 deficiency aggravates the defect of natural killer cell activity in AIDS patients.

A decrease in natural killer (NK) cell activity is a common feature of the immune dysfunction found in patients with human immunodeficiency virus (HIV)-induced acquired immune deficiency syndrome (AIDS). The present study was aimed at exploring the NK and the lymphokine-activated killer (LAK) cell activities of lymphocytes from HIV-seropositive subjects. The in vitro production of interleukins (IL-2 and IL-10) in response to mitogens was also studied. Two groups of HIV-seropositive subjects were studied: asymptomatic and AIDS patients. Controls were normal blood donors. The NK cell activity in peripheral blood mononuclear cells (PBMC) from AIDS patients was significantly lower than that in PBMC from both HIV-seronegative subjects and asymptomatic patients. There was no significant difference between asymptomatic patients and controls. Exposure of PBMC from all three groups of individuals to an optimal dose of IL-2 in vitro enhanced LAK cell activity. At all three effector: target cell ratios, the LAK activity in AIDS patients remained below that in normal subjects. However, the proportional increase of lytic activity with IL-2 was slightly higher in AIDS patients than in HIV-seronegative subjects. The mitogen-induced production of IL-2 was especially reduced in AIDS patients. In contrast, very high levels of mitogen-induced production of IL-10 were found in the AIDS group, as compared to asymptomatic subjects or to controls. We therefore conclude that the alteration of NK cell activity occurs at an advanced stage of HIV infection, that the reduction of cytotoxic activity is partially restored by exogenous IL-2, and that decreased production of IL-2 and increased production of IL-10 may account for part of this reduction in cytotoxicity.

MHC-I non-restricted cytotoxic activity in Macaca sylvana experimentally inoculated with HIV2 and SIV/mac.

The anti-retrovirus cell-mediated immunity was repeatedly investigated in seven monkeys (Macaca sylvana). Four of these animals were injected with cell-free supernatants containing human immunodeficiency viruses: two monkeys received HIV1 Bru (2.5 x 10(6) cpm), two received HIV2 Rod (1.5 x 10(6) cpm). Two additional animals were injected with a cell-free supernatant containing simian immunodeficiency virus SIV/mac 251 (1.5 x 10(6) cpm) and the last animal served as control. The four macaques infected with HIV2 Rod and SIV/mac 251 seroconverted. Freshly isolated and non stimulated peripheral blood mononuclear cells from these infected macaques and from the uninfected control were repeatedly assessed for cytolytic activity. Target cells consisted of heterologous human cell lines expressing HIV1 Bru, HIV2 Rod or SIV/mac proteins. A significant cytotoxic activity, non-restricted at the major histocompatibility complex class I (MHC-I), was demonstrated in one HIV2 Rod-infected animal (F8) and in one SIV/mac 251-infected animal (M1). This last animal showed progressively diminishing cytolytic activity that was correlated with a pronounced decrease in CD4+ lymphocytes. An AIDS-like disease developed in M1, with presence of lymphadenopathy, weight loss, diarrhea and opportunistic infections. Cytotoxic activity was active against SIV and HIV2-infected target cells in an MHC-unrestricted manner; it was specific to virus-infected cells and there was cross-reactivity between HIV2 and SIV. Cytotoxic effectors appeared to be mainly CD8+ cells. This model may prove to be very useful in evaluating the capacity of candidate AIDS vaccines to elicit effective cell-mediated immune responses.

Fetal liver cell transplantation in various murine models.

Allogeneic fetal liver cell transplantation has been shown to be able to reconstitute lymphopoietic systems of mice when these systems are defective or destroyed. Lethally irradiated mice or mice with inherited severe combined immunodeficiency disease (SCID) were grafted with 14 days gestation allogeneic fetal liver cells, then subjected to a follow-up for the immune tolerance to the donor and the normal or subnormal immune reconstitution allowing prevention of diabetes in NOD mice or cure of leukemia in AKR mice and of immunodeficiency in SCID mice. Briefly, when normal CBA mice were lethally irradiated and then grafted with allogeneic fetal liver cells from Balb/c mice, a specific immune tolerance was induced to donor skin grafts. Unrelated skin grafts were rejected and a response to antigens was observed in these chimeras. However, despite the capacity to develop hyperacute rejection of skin allografts, following hyperimmunization, these chimeric mice did not produce anti-H2 cytotoxic antibodies. In SCID mice (CB17), the immune reconstitution occurred when mice were grafted with allogeneic (C57/B16) as well as with syngeneic fetal liver cells. Human cells were found in SCID mice following implantation of human fetal liver and thymus cells. When NOD mice were irradiated, then grafted with allogeneic fetal liver cells, a large part of donor cells were found in NOD recipients, correlating with a low incidence of diabetes. Leukemic AKR mice grafted with allogeneic fetal liver cells had virtually no leukemia relapse, suggesting a strong graft-versus-leukemia effect following such a transplant.

Fetal liver cell transplantation in scid mice.

Mice with severe combined immunodeficiency (CB 17 scid) received isogeneic and allogeneic fetal liver cell transplantation. Immunological reconstitution was followed by immunoglobulin production, mitogen-induced proliferation, spleen and thymus lymphoid recolonization. Scid mice injected with isogeneic fetal liver cells showed normal IgM, IgG production and subnormal Con-A induced proliferation. Lymphoid organs were gradually repopulated. Mice having received allogeneic stem cells presented normal IgM and IgG secretion. They still had no mitogen-induced lymphocyte stimulation, two months after fetal cell transplantation, despite satisfactory repopulation of spleen and thymus. Reconstitution of cell-mediated immunity may be somewhat slower following allogeneic than isogeneic stem cell transplantation.

HTLV-I antibody and cell-mediated immunity status in sickle cell anemia.

Patients with sickle cell anemia (SCA) had an abnormal susceptibility to infections. In Martinique (French West Indies), a human T-cell leukemia/lymphoma virus type I (HTLV-I) endemic area, we found that 17 (10%) of 173 SCA patients had antibodies to HTLV-I. The possible relationship between HTLV-I seropositivity and altered immunity was studied in 13 SCA patients with HTLV-I antibodies compared with 13 matched SCA patients without HTLV-I antibodies. The immunological results, as evaluated by the T-cell subsets analysis, the lymphocyte proliferation responses analyzed after activation with concanavalin A, phytohemagglutinin, or pokeweed mitogen, and the natural killer activity were not statistically different in these two groups of patients (SCA HTLV-I positive vs SCA HTLV-I negative). These data suggest that HTLV-I infection did not result in a major alteration of cellular immunity in this population.

Endemic tropical spastic paraparesis associated with human T-lymphotropic virus type I: a clinical and seroepidemiological study of 25 cases.

Tropical spastic paraparesis (TSP) is a common myeloneuropathy with primary and predominant involvement of the pyramidal tract and minimal sensory loss. The epidemic form of TSP is related to toxic nutritional factors, but the endemic form occurs in clusters in tropical areas, especially in India, Africa, the Seychelles, Colombia, and areas of the Caribbean. We describe the clinical and epidemiological features of 25 TSP patients from Martinique (French West Indies) with serum antibodies to human T-lymphotropic virus type I (HTLV-I). Furthermore, all 11 patients who were seropositive for HTLV-I had specific HTLV-I antibodies in their CSF. All were women. The age of onset varied from 25 to 60 years (mean, 45 years). The main clinical features are spastic paraparesis or paraplegia with spasticity of the upper limbs, minimal sensory loss, and bladder dysfunction. Minimal estimated incidence and prevalence are 1 per 100,000 inhabitants per year and 8 per 100,000, respectively. Seventeen percent of the relatives of patients with HTLV-I-associated TSP have HTLV-I antibodies (1 husband and 7 children). In Martinique, the prevalence of HTLV-I antibodies in the general population is about 2% and reaches 10% for neurological disorders other than TSP. Since our initial report, the association between spastic paraparesis and HTLV-I has been confirmed in Jamaica, Colombia, and Japan, suggesting the neurotropism of this lymphotropic human retrovirus.

[Adult T-cell lymphoma/leukemia associated with HTLV-I virus in Martinique: apropos of 2 cases]. / Leucémie/lymphome T de l'adulte associé au virus HTLV-I en Martinique: a propos de deux cas.

Two HTLV-I associated adult T cell leukemia cases were observed in patient from Martinique (French West Indies). These case are similar to the clinical entity, described by Takatsuki in 1977 in Japan and by Catovsky in Caribbean patients, characterized by a lymphadenopathy, skin lesions and visceral involvement, hypercalcemia, an aggressive course, and poor prognosis. The malignant cells with T4 phenotype and often suppressive function, were pleomorphic, mature, with prominent nuclear irregularities. Systematic research of HTLV-I virus or antibodies in patients with this clinical picture, to measure the influence of this virus in T cell lymphoproliferative diseases in France and in French West Indies is required.

A new immunomodulator, LF 1695--I. In vitro effects on the T lymphocyte lineage in man.

A new immunomodulator, LF 1695, was analyzed in vitro for its capacity to induce T cell markers (HTLA and OKT3, 4 and 8 antigens), to enhance the mitogen-induced lymphocyte proliferation, and to increase the Con A-induced suppressor activity. The inductive activity was compared with that of thymic hormone (thymosin fraction V). LF 1695 was capable of significantly augmenting the percentages of HTLA+ cells (10-15%) and of OKT 3+,T4+, or T8+ cells (13-28%) in human bone marrow prothymocytes. Maximum induction was observed at the concentration of 0.5 microgram/ml. In addition, LF 1695 significantly augmented the proliferation of human peripheral blood lymphocytes stimulated by mitogens (Concanavalin A, phytohemagglutinin, pokeweed mitogen and phorbol myristate acetate). LF 1695 also increased the Con A-induced suppressor activity of human lymphocytes. These data indicate that LF 1695 is active both on T cell precursors and on more mature T cells.