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D Fradelizi

Publications and source records attributed to D Fradelizi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

Animals↗
PubMed1996

[Experimental gene therapy in models of inflammatory arthritis].

Gene therapy is rheumatoid arthritis is presently in an experimental phase. Genes encoding for antiinflammatory proteins can be transfected in joint cells. Therefore gene encoding for interleukin-1 receptor antagonist has been transfected into synovial cells or into chondrocytes. Gene expression can be maintained as long as 8 weeks after cell transfer. We have reported that clinical and histopathological parameters of collagen-induced arthritis (an animal model of rheumatoid arthritis) can be reduced by engraftment of CHO cells transfected with genes encoding for antiinflammatory cytokines such as interleukine-4 or interleukine-13. Some of the questions which need to be assessed before planning clinical trial are the choice of vectors and the most efficient target cells; and the genes to be transfected have to be chosen among many candidates.

Animals↗
PubMed1996

Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2.

The production of human interleukin-2 (hIL-2) local to the tumor site by engineered histoincompatible cells has been shown in various murine models to promote a strong immune response leading to tumor growth inhibition or rejection. To assess whether this strategy would be similarly applicable for treatment of primary neoplastic cells, two naturally occurring tumors were used as preclinical models; the highly metastatic melanoma of the dog and the low metastatic fibrosarcoma of the cat. We demonstrate that both cats and dogs when treated by tumor surgery, radiotherapy and repeated local injections of xenogeneic Vero cells secreting high levels of hIL-2 relapse less frequently and survive longer than control animals treated by surgery and radiotherapy alone. Local secretion of hIL-2 by the xenogeneic cells is shown to be necessary for the induction of an optimal antitumor effect. Moreover, the safety of the procedure was demonstrated in both animal models and through extensive toxicological analysis performed in rats. These results confirm for the first time to our knowledge the safety and therapeutic potential of a gene transfer strategy in animals with spontaneous metastatic and nonmetastatic tumors.

Animals↗
PubMed1995

CD82, member of the tetra-span-transmembrane protein family, is a costimulatory protein for T cell activation.

It is now well documented that full activation of T cells requires a two-signal triggering that can be mimicked, in the absence of accessory cells, by co-immobilization of mAbs directed to stimulatory/accessory molecules (CD2, CD3, CD28, adhesion molecules, etc.). In this report, we describe that engagement of CD82 can delivery such a costimulatory signal for full activation of the human T cell line Jurkat, leading to strong IL-2 production and cell differentiation. The CD82 Ag, which belongs to the new tetra-span-transmembrane family (CD9, CD37, CD53, CD63, and CD81 (TAPA-1)), has been identified originally in our laboratory for its enhanced expression on three LAK-susceptible cell lines, and has been characterized as an activation/differentiation marker of mononuclear cells. Jurkat cells, stimulated in vitro by co-immobilization of anti-CD82 and anti-CD3 mAbs, produced high levels of IL-2, became strongly adherent to plastic dishes, and developed dendritic processes. These morphologic changes, associated with a total arrest of cell proliferation, were not the result of cell death but rather of cell differentiation, as shown by an increase in their metabolic activity. Costimulation through both CD82 and CD3 induced up-regulation of both IL-2 and IFN-gamma mRNA synthesis (but not of IL-4) and an increased expression of HLA class I molecules at the cell surface, which was inhibited by anti-IFN-gamma Ab.

Antibodies↗
PubMed1995

Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin-13.

Cytokines locally delivered to the site of a tumor boost both specific and nonspecific host anti-tumor defenses. Interleukin (IL)-13 is a recently described cytokine produced by mouse type 2 helper T lymphocytes. The aim of this study was to evaluate the inhibition of tumor growth induced by IL-13 delivered locally within or around transplanted tumor cells in mice. We observed that local administration of IL-13 at the site of transplanted tumor cells in vivo had potent inhibitory effects on growth of both immunogenic (P815 mastocytoma, H-2d) or nonimmunogenic (3LL lung carcinoma, H-2b) tumor cells. Mice injected with transfected P815 cells secreting large amounts of IL-13 rejected the P815 tumor and developed systemic specific anti-tumor immunity leading to long-lasting specific anti-tumor protection. Less efficient anti-tumoral effects were obtained with the nonimmunogenic 3LL tumor model when local administration of IL-13 was achieved by co-inoculating xenogeneic chinese hamster ovary (CHO) IL-13 cells. Several local injections of CHO IL-13 cells were needed to obtain rejection of 3LL tumors and no induction of long-lasting anti-3LL memory was obtained. Several studies were performed to elucidate the IL-13 anti-tumoral effects. Experiments with nude mice indicated that Il-13 can also stimulate nonspecific anti-tumor defenses. The histological examination of P815 IL-13 cells undergoing rejection showed monocytic cells and neutrophils infiltrating the tumor. Studies indicated that IL-13 administered in vitro did not directly stimulate the cytotoxicity of peritoneal macrophages and natural killer cells. However, experiments with Boyden chemotaxis chambers indicated that IL-13 was chemotactic for macrophages. Finally, preliminary experiments in vitro suggest that IL-13 improved antigenic presentation of P815 membranes. Thus, anti-tumor effects of IL-13 in vivo most probably result from pleiotropic effects including recruitment of nonspecific cells and improved stimulation of immune-specific anti-tumor effectors.

Animals↗
PubMed1995

CD82, tetra-span-transmembrane protein, is a regulated transducing molecule on U937 monocytic cell line.

The mononuclear cell surface protein IA4, recently classified as CD82, was originally identified in our laboratory by the IA4 monoclonal antibody (mAb), because of its high expression on three lymphoblastoid, LAK-susceptible, variant cell lines. We have characterized CD82 as a new activation/differentiation marker of mononuclear cells. This protein belongs to the new family of TST proteins (tetra spans transmembrane), which includes CD9, CD37, CD53, CD63, and CD81 (TAPA-1). Here we demonstrate that cross-linking of IA4 mAbs induces an increase of intracellular free calcium in U937 cells and tyrosine phosphorylation of various proteins. Our data indicate that the intracellular calcium increase is initiated by a phospholipase C (PLC)-induced PtdIns(1,4,5)P3 second messenger followed by a more stable change, linked to extracellular calcium entry. This transducing signal was dependent on dual engagement of both CD82 and Fc receptors. Surface cross-linking of CD82 together with Fc receptors (FcRs) induces a specific long-lasting increase of intracellular calcium, whereas FcR cross-linking alone induces only a transient calcium mobilization. These results suggest that, upon cross-linking of CD82, a multimolecular complex including CD82 and FcR could be induced that is able to trigger signal transduction. We have previously shown that CD82 membrane expression is up-regulated during differentiation of human monocytes. Using U937 cells, we demonstrate here that several cytokines [interleukin-1 beta (IL-1 beta), IL-4, IL-6, IL-13, interferon-gamma, tumor necrosis factor alpha] could significantly up-regulate the surface expression of CD82 antigen, by contrast with FcR surface expression, which was up-regulated only after IFN-gamma treatments. Based on our finding of a strict dependence of CD82 activation on FcR stimulation, we suggest a putative role of CD82 in enhancing FcR-mediated activation of cells from the monocyte/macrophage lineage.

Antibodies, Monoclonal↗
PubMed1995

Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2.

Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2-producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2-secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers.

Animals↗
PubMed1995

[Treatment of inflammation with anti-inflammatory cytokines: example in models of auto-immune diseases].

Cytokines may exert anti-inflammatory properties, allowing the hypothesis of their potential therapeutic use. Targeting the cytokines balance may modify the course of subsequent inflammatory events in an autoimmune disease. Many data are available. Interferon-gamma can be blocked by an anti-interferon-gamma monoclonal antibody: if the treatment is administered in the early phase of an autoimmune disease such as collagen-induced arthritis, the course of the disease is worsened; if the treatment is given later, the disease can be improved; these results are mirrored by treatment with high doses of the cytokine itself. Pharmacologic effects of antiinflammatory cytokines such as interleukin (IL)-4, IL-10 or IL-13 are a protection against several experimental autoimmune diseases. The very short half-life of cytokines makes them difficult and expensive to use directly; in such occurrence, high quantities have to be frequently injected. In this context, gene therapy appears as an effective alternative solution: the transfection of cells with cytokines genes (e.g. either IL-4 or IL-13) then the engraftment of these vectors in animals, permit the in vivo secretion of high levels of cytokines, and result in the protection of the animals from the development of the disease.

Animals↗
PubMed1994

Macrophage-inactivating IL-13 suppresses experimental autoimmune encephalomyelitis in rats.

Experimental autoimmune encephalomyelitis (EAE) is initiated by myelin basic protein (MBP)-specific CD4+ T cells of the Th1 phenotype that subsequently trigger the invasion of monocytes/macrophages into the brain. In this study, we evaluated the potential of human recombinant (hr) IL-13 to exert a protective effect on the development of EAE in Lewis rats. hrIL-13 is found to be a potent in vitro modulator of various rat macrophage functions, including an inhibition of the production of the proinflammatory cytokines IL-1 beta and TNF, and a simultaneous enhancement of MHC class II and CD4 receptor expression. Furthermore, hrIL-13 displayed a slight, but highly reproducible, inhibitory effect on the in vitro proliferative responses of encephalitogenic MBP-specific T cells stimulated in the presence of thymic APCs. Upon in vivo application of hrIL-13-secreting vector cells into MBP-immunized animals, the cytokine was capable of markedly suppressing the development of EAE, as assessed by a reduction of the mean duration, severity, and incidence of disease. This suppression of disease coincided with an only minimal reduction of MBP-directed T cell autoreactivity and no alteration in MBP-specific autoantibody production. We infer from these results that a strictly Th1-initiated immune disease can be attenuated efficiently by the administration of a cytokine that primarily targets cells of the macrophage/monocyte lineage and seems to exert no undesirable general suppression on either T cell or B cell immunoreactivity in vivo.

Animals↗
PubMed1994

Further characterization of CD82/IA4 antigen (type III surface protein): an activation/differentiation marker of mononuclear cells.

The mononuclear cell surface protein IA4 was originally identified in our lab using a mAb selected because of its strong reactivity with three lymphoblastoid variant cell lines which are HLA class I deficient, are LAK susceptible, and form a high number of conjugates with LAK effectors. We previously cloned the cDNA of the IA4 protein, coding for a 267-amino-acid type III integral membrane protein, with four transmembrane domains and three possible N-glycosylation sites. The IA4 protein belongs to the tetra span transmembrane (TST) new family of surface molecules, which also includes CD9, CD37, CD53, CD63, and TAPA-1. IA4 antigen was recently recognized as belonging to a new cluster of differentiation CD82 (International CD Workshop, Boston 1993). The IA4 antigen expression pattern at the surface of immune cells from normal donors was studied. On T lymphocytes, IA4 was barely detectable on resting cells and increased 3.5- to 7-fold following PHA or PHA+PMA stimulation. This IA4 increased expression is correlated with the morphologic change in blast cells and with the expression of activation markers such as CD2 and MHC class II antigens, therefore suggesting that IA4 is an activation marker on T lymphocytes. The expression of IA4 was low on circulating resting monocytes collected by elutriation. However, these monocytes, cultured in medium alone or with GM-CSF, acquired the morphology of macrophage and simultaneously overexpressed MHC Class II, CD14, and IA4 antigens, suggesting that IA4 is a differentiation marker for macrophages, whatever the culture conditions, either adherent (plastic culture dishes) or nonadherent (Teflon culture bags). IA4 stable transfectants of the murine mastocytoma cell line P815 were obtained and used to generate a new mAb. Competitive epitope binding studies have shown that IA4 antigen presents a dominant epitope recognized by most of the mAb prepared either in our lab or elsewhere. This dominant epitope is not shared by any of the other antigens of the TST family. Using this new mAb we were able to biochemically characterize the IA4 antigen as a 28-kDa protein, highly N-glycosylated with different patterns on various cells.

Animals↗
PubMed1994

Genomic cloning of human thioredoxin-encoding gene: mapping of the transcription start point and analysis of the promoter.

Thioredoxin (TR) is a small ubiquitous dithiol-reductase enzyme first identified in bacteria and plants. In recent years, this protein has been recognized as playing an important role in the growth control of eukaryotic cells, especially in lymphocytes. It was first cloned from a human Epstein-Barr virus-transformed lymphoblastoid B-cell line by our group in 1988 [Wollman et al., J. Biol. Chem. 263 (1988) 15506-15512] and localized on chromosome 3 p11-p12 by in situ hybridization [Lafage-Pochitaloff-Huvalé et al., FEBS Lett. 255 (1989) 89-91]. The present work was performed to study the genomic organization of the human thioredoxin (hTR)-encoding gene (hTR). The screening of a human genomic library in lambda EMBL4 phage led to the identification of two genomic clones which encompassed the entire gene, including the promoter region. The coding region of hTR spans over 13 kb and is organized into five exons separated by four introns which were 60% sequenced. We determined the transcription start point (tsp) by primer extension. This tsp located, in lymphocytes, 22-bp downstream from a TATA box (TATAA) defines a 5' untranslated region of 74 bp. We analyzed 2149-bp upstream from the promoter for sequence motifs which could bind regulatory proteins. This promoter contains many possible regulatory elements compatible with both a basal constitutive expression and a regulated inducible transcription, especially by cytokines such as interleukin-6 and interferons. Finally, Southern hybridization of genomic DNAs from several donors detected only one active gene encoding hTR.

Amino Acid Sequence↗
PubMed1992

Peripheral macrophage abnormalities in mutant mice with spinocerebellar degeneration.

We recently reported hyperproduction of interleukin-1 (IL1) and hyperexpression of IL1 beta mRNA, after in vitro activation by lipopolysaccharide (LPS) in peripheral macrophages of several neurological mutant mice, i.e. staggerer, lurcher, pcd and reeler, that exhibit patterns of neuronal degeneration in the cerebellum; in the present study, we investigated the expression of several cytokine mRNA in peripheral macrophages of other mutants with neuronal degeneration in the cerebellum or in the spinal cord to determine whether this genetic dysregulation is specific for IL1 beta or whether it reflects a generalized hyperexcitability of these macrophages. Hyperexpression of IL1 beta mRNA was present in the cerebellar mutants nodding and nervous, but not in weaver. A similar phenomenon was found, but to a lesser extent, in the spinal mutants dystonia musculorum, wobbler and motor neuron degeneration. On the contrary, no hyperexpression of IL1 beta mRNA was found in non-genetic models of neuronal degeneration (Wistar rats treated with X irradiation or with 3-acetyl-pyridine). In the heterozygote staggerer +/sg, which exhibits a late onset of cerebellar neuronal loss, hyperexpression was found not only in 12-month old animals but also in 2-month old ones, i.e. when the number of cerebellar neurons is still normal. Synthetic molecules (muramyl dipeptides) like MDP or murabutide (Mu), known as macrophage activators, were also efficient in inducing IL1 hyperexpression in sg/sg macrophages. Hyperexpression of two other cytokine mRNA, i.e. IL1 alpha and tumour necrosis factor alpha mRNA, was also detected in LPS-stimulated macrophages of staggerer and lurcher mutant mice. These data led us to conclude that the macrophages of spinal and cerebellar mutants are in a state of general hyperexcitability. Work is in progress to establish whether the cytokine abnormalities result from a defect intrinsic to the macrophages of the mutant mice or are secondary to the degenerative process ultimately leading to neuronal loss.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.

Animals↗
PubMed1992

Evidence for a hyperexcitability state of staggerer mutant mice macrophages.

We recently reported an abnormal production of interleukin-1 (IL-1) in peripheral macrophages of several neurological mutant mice that exhibit patterns of neuronal degeneration, especially in the cerebellum. After in vitro activation by lipopolysaccharide acid (LPS), these macrophages hyperexpress IL-1 beta mRNA and hyperproduce IL-1 protein in comparison with +/+ controls. In the present study, focused on the staggerer mutant mice, we investigate if this genetic dysregulation is specific for IL-1 beta or if it reflects a generalized hyperexcitability of these macrophages. The hyperexpression of IL-1 beta mRNA in sg/sg macrophages is present whatever the duration of LPS stimulation, even for periods as short as 15 min, although it reaches a maximum after 4 h of stimulation. The hyperinducibility of sg/sg macrophages is observed even when very low doses of LPS are used (0.01 microgram/ml) and reaches its maximum for 5 micrograms/ml LPS. Synthetic molecules (muramyl dipeptides), such as N-acetylmuramyl-L-alanyl-D-isoglutamine or murabutide, known as macrophage activators, are also efficient in revealing the cytokine hyperexpression in sg/sg macrophages. In addition, hyperexpression of two other cytokines, i.e., tumor necrosis factor-alpha and IL-1 alpha mRNAs, is also detected in LPS-stimulated macrophages of mutant mice. Finally, the effect of an inhibitor of protein synthesis, cycloheximide, is similar in +/+ and sg/sg macrophages. As a whole, these data lead us to conclude that the sg/sg macrophages are in a state of general hyperexcitability when compared with +/+ ones.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

[Potentiation of the antitumoral effect of electrochemotherapy by immunotherapy with allogeneic cells producing interleukin 2].

Electrochemotherapy (ECT) is a new antitumour treatment which consists of delivering electric pulses to the tumour a few minutes after an intravenous injection of bleomycin. The antitumour efficacy of ECT is increased by local injections of interleukin 2 (IL2) in the oedema which appears at the site of the treated tumours. We have shown that tumour cells inoculated in syngeneic mice are rejected if IL2 secreting allo- or xenogeneic cells are co-injected with tumour cells. We report here the large increase of ECT therapeutical efficacy when allogeneic cells secreting IL2 are injected into the peri-tumoural oedema.

Animals↗