Influence of high-fat feeding on both naive and antigen-experienced T-cell immune response in DO10.11 mice.

Obesity is becoming one of the most serious public health problems in industrialized societies, due to the profound changes in lifestyle, and notably in nutrition. Beside diabetes, cardiovascular diseases or hypertension, increased susceptibility to infection is one of the pathological consequences of being overweight. In this paper, we have assessed the influence of a high-fat diet (HFD) rich in saturated fatty acids on the immune system of DO11.10 mice, which are transgenic for a T-cell receptor specifically recognizing a peptide of ovalbumin. We showed that the specific T-cell immune response was impaired by high-fat feeding, and that the expression of this defect is different depending on whether T cells are naive or Ag experienced. Indeed, on in vitro ovalbumin stimulation, spleen T cells from naive HFD-fed transgenic mice showed proliferation similar to that of cells from standard diet (SD)-fed mice, but exhibited a strong inflammatory profile as shown by the markedly increased IFN-gamma/IL-4 ratio. Inversely, spleen T cells from ovalbumin-immunized HFD mice were impaired in their Ag-dependent proliferation compared to cells from SD mice. By co-culture experiments, we showed that both T cells and antigen-presenting cells were involved in this impairment. Moreover, in ovalbumin-immunized HFD animals, a trend towards Th2 response was noted, compared to immunized SD mice. This data implies that naive T cells could participate actively in the low-grade systemic inflammation observed in overweight patients. Moreover, the impaired activity of Ag-experienced T cells could have major consequences both in defence against infection and/or in vaccination protocols.

Genes involved in obesity: Adipocytes, brain and microflora.

The incidence of obesity and related metabolic disorders such as cardiovascular diseases and type 2 diabetes, are reaching worldwide epidemic proportions. It results from an imbalance between caloric intake and energy expenditure leading to excess energy storage, mostly due to genetic and environmental factors such as diet, food components and/or way of life. It is known since long that this balance is maintained to equilibrium by multiple mechanisms allowing the brain to sense the nutritional status of the body and adapt behavioral and metabolic responses to changes in fuel availability. In this review, we summarize selected aspects of the regulation of energy homeostasis, prevalently highlighting the complex relationships existing between the white adipose tissue, the central nervous system, the endogenous microbiota, and nutrition. We first describe how both the formation and functionality of adipose cells are strongly modulated by the diet before summarizing where and how the central nervous system integrates peripheral signals from the adipose tissue and/or the gastro-intestinal tract. Finally, after a short description of the intestinal commensal flora, rangingfrom its composition to its importance in immune surveillance, we enlarge the discussion on how nutrition modified this perfectly well-balanced ecosystem.

Ocular transfer of retinal glial cells transduced ex vivo with adenovirus expressing viral IL-10 or CTLA4-Ig inhibits experimental autoimmune uveoretinitis.

Gene transfer using immunomodulatory molecules is a promising tool for in vivo regulation of immune responses. Experimental autoimmune uveitis (EAU), which serves as a model for human ocular inflammation, is induced by systemic immunization with autoantigens, but its expression is restricted to the eye. Previously, we reported protection of rodents against EAU by intravenous or/and periocular injection of vIL-10-expressing adenovirus. Here, the expression of vIL-10 was targeted into the rat Lewis eye, by intravitreal injection of either the free virus or ex vivo transfected retinal Müller glial cells (RMG-vIL-10). As shown using GFP-expressing adenovirus, a longer expression of transgene was observed in the eye after transfer of transfected syngeneic RMG cells than was seen after injection of free virus. Intravitreal injection of RMG-vIL-10 led to significant decrease in ocular pathological manifestations, compared to control RMG cells. This was observed when cells were injected simultaneously with autoantigen, but also after a delayed administration of transfected cells. Finally, injection of RMG cells transfected with adenovirus expressing CTLA4 had a strongly protective effect. In conclusion, inhibition of antigen presentation at the site of expression of the autoimmune disorders represents an attractive alternative to treat ocular inflammation, and the transfer of ex vivo genetically modified cells provides a promising method to target the factor of interest into the eye.

Novel promiscuous HLA-DQ HIV Nef peptide that induces IFN-gamma-producing memory CD4+ T cells.

We describe the highly conserved sequence 56-68 of the HIV Nef protein as the first promiscuous HLA-DQ HIV-derived peptide. The Nef peptide exhibits an albeit rare capacity to bind 6 different HLA-DQ molecules whereas no binding is observed with the 10 HLA-DR molecules tested. In agreement with these data, after immunization with the Nef peptide, HLA-DQ transgenic Abeta degrees mice display a vigorous cellular and humoral response while the specific immune response of HLA-DR expressing mice is minimal. The promiscuous potentiality of the Nef 56-68 peptide in humans has been confirmed by ex vivo immunization experiments with CD4+ T cells from 14 healthy donors expressing different HLA genotypes. Nef 56-68 specific CD4+ T cells rapidly acquire a memory cell phenotype and are characterized by the preferential usage of the TCR Vbeta 6.1 gene segment and predominant production of IFN-gamma. Taken together, these data indicate that the Nef 56-68 peptide constitutes an attractive component of vaccines aiming at inducing or enhancing HIV-specific T cell immunity.

HLA class II polymorphism influences onset and severity of pathology in Schistosoma mansoni-infected transgenic mice.

Genetic factors that might influence susceptibility or resistance in naive individuals and early-stage pathology in schistosomiasis are difficult to study in clinical trials, since in areas where the disease is endemic the first contact with the parasite occurs most often at very early ages. Therefore, four strains (DR1.Abeta degrees, DR2.Abeta degrees, DQ8.Abeta degrees, and DQ6.Abeta degrees ) of major histocompatibility complex class II-deficient mice (Abeta degrees ), transgenic for different HLA alleles, have been used to evaluate the potential role of HLA class II polymorphism in the onset of the infection by Schistosoma mansoni. The survival rates and parasitological and immunological parameters after infection were evaluated and compared against the control values obtained with Abeta degrees mice. All four mouse strains used in this study were able to generate a specific immune response against S. mansoni antigens (cytokine production and antibody production). However, only mice expressing DR alleles survived until the chronic stage of the infection and were able to mount protective granulomatous response avoiding hepatic damage, presenting predominant gamma interferon production. In contrast, strains expressing DQ alleles revealed an impairment in generating effective granulomas, resulting in earlier death, which was associated with an impaired hepatic granulomatous response and liquefactic necrosis, reflecting the influence of HLA polymorphism in the establishment of protective response in the early stage of infection.

Systemic delivery of an adenovirus expressing EBV-derived vIL-10 in mice infected with Schistosoma mansoni or Leishmania amazonensis: controversial effects on the development of pathological parameters.

Within the context of microorganism/host interactions, those which last over weeks are expected to be sensitive to more or less sustained and targeted immuno-intervention, such as delivery of cytokines known to operate as down-regulators of acute inflammatory processes. IL-10 has received growing attention as a potential tool in immunotherapy, due to its anti-inflammatory and immunosuppressive properties. Therefore, using two experimental models of long-term interactions between parasites and laboratory mice, we monitored some effects of the systemic delivery of an adenovirus (Ad) expressing EBV-derived IL-10 (vIL-10) designated Ad-vIL-10. We first monitored the vIL-10 serum level following intranasal, intraperitoneal, intramuscular and intravenous administration. The i. p. and i.v. delivery of Ad-vIL-10 allowed a high serum level of vIL-10 (= 100 ng/ml), the i.v. route leading to a more sustained expression (up to 3 weeks). As a first model of parasite/mouse interaction, Schistosoma mansoni/C57Bl/6 mouse was selected. Ad-vIL-10 delivery was performed 4 weeks after S. mansoni infection i.e. at the time of egg-laying, and several parameters were monitored: (i) number of adult worms in the mesenteric vein, (ii) number of eggs trapped in the liver and intestine, (iii) liver fibrosis, (iv) serum levels of egg-reactive antibody subclasses, (v) serum content of cytokines, and (vi) cytokine production in the supernatant of antigen-stimulated mesenteric lymph node cells. No apparent effect was observed, either on the different parasitological parameters or on fibrosis development at day 70 of infection. Surprisingly, a marked increase in both Th1 and Th2 type cytokines was observed in the sera of the Ad-vIL-10 injected animals, as well as in the supernatants of their Ag-stimulated mesenteric lymph node cells. Nevertheless, polarization of the humoral response towards a Th2 profile was demonstrated by an increase in the IgE level in the Ad-vIL-10-injected animals. As far as the second model is concerned, namely the Leishmania amazonensis /C57Bl6 mouse interactions, Ad-vIL-10 was delivered intravenously one day before subcutaneous injection of stationary promastigotes and footpad swelling was monitored over 110 days. Under these conditions, vIL-10 exhibited a biphasic effect, decreasing the lesion size at the early stages of infection, but leading to a more pronounced lesion size during the chronic phase. This observation suggests a deactivation of the macrophage host cells under the influence of vIL-10. The results are discussed in the context of immunotherapy and the paradoxical effects observed in immunointervention with vIL-10.

Unrestricted agonist activity on murine and human cells of a lipopeptide derived from IFN-gamma.

We describe the isolation of a synthetic agonist of IFN-gamma (L-mIFN-gamma-CT) active on mouse and human cells. Its biological activity is the result of the ability of the C-terminal extremity of murine IFN-gamma to interact with the intracellular part of IFNgamma-R and the observation that the modification of peptides by a palmitic acid enables their cytoplasmic delivery. L-mIFN-gamma-CT stimulated murine cells exhibited an increase in MHC class II molecules and FcgammaRII/III expression and conferred protection against viral lysis. Unresponsiveness to L-mIFN-gamma-CT of cells recovered from IFNgamma-R alpha-chain knockout mice indicated the involvement of IFNgamma-R in the biological activities observed. Induction of VCAM-1, ICAM-1, and HLA-DR expression on human cells stimulated with L-mIFN-gamma-CT demonstrated an abrogation of species specificity. These results describe the development of a new synthetic agonist of IFN-gamma, which substitutes for the native cytokine in any IFN-gamma responsive cells, by acting intracellularly on IFN-gammaR.

Induction of cytotoxic T-cell activity by the protective antigen of Schistosoma mansoni Sm28GST or its derived C-terminal lipopeptide.

In a previous work the authors demonstrated that immunization with Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) was able to reduce hepatic damage in infected mice and that the adoptive transfer of Sm28GST-specific T cells reproduced the protective effect obtained with the recombinant molecule. In the present paper, the authors show that Sm28GST is also able to stimulate an antigen-specific, cytotoxic T-cell response against Sm28GST-pulsed P815 target cells in normal mice and that effector cells induced in vivo were classical Class I MHC-restricted CD8+ lymphocytes. The authors found no spontaneous CTL activity against Sm28GST-pulsed target cells during the course of the infection by S. mansoni although Sm28GST is expressed at different developmental stages of the parasite. It was observed, however, that immunization with Sm28GST is sufficient to elicit a significant level of CTL response for 6 weeks in infected mice. The role of these Class I MHC-restricted CD8+ lymphocytes in the protection observed precisely at the same period in immunized mice remains to be elucidated. The authors also observe that immunization with the lipopeptide form of the C-terminal peptide of the molecule (190-211 peptide) led to a CTL activation comparable to that observed after immunization with the whole molecule demonstrating the feasibility of using a synthetic lipopeptide as immunogen for a CTL response against Sm28GST epitopes. Moreover, like Sm28GST-specific CTLs, 190-211 lipopeptide-specific cells were also Class I MHC-restricted lymphocytes.

Assessing delivery of lipopeptides into the cytoplasm of intact cells by a functional assay based on PKC inhibition. I. The Jurkat model.

We have developed a functional assay to verify the delivery into the cytoplasm of a 14-amino acid hydrophilic peptide, modified by the incorporation of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudo-substrate sequence for the protein kinase C (PKC) isoenzymes of alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell activation by phorbol ester and anti-CD3 MAb. This cellular assay is simple, and it allows for a rapid and comparative study of several peptides. The lipidic analogues of the pseudo-substrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC50 in the 40-60 microM range, whereas the unmodified peptide was not active. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms that the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allow entry into intact cells.

Evaluation of the effect of Sm28GST-derived peptides in murine hepatosplenic schistosomiasis: interest of the lipopeptidic form of the C-terminal peptide.

Among the synthetic peptides derived from the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST), immunization with the C-terminal peptide comprising amino acid residues 190-211 induced a reduction in splenomegaly, in the number of hepatic eggs and in hepatic fibrosis in mice infected by Schistosoma mansoni. The absence of antibodies specific for the Sm28GST or for the 190-211 peptide observed in our conditions of immunization with this peptide argued in favour of the involvement of cellular-dependent mechanisms in the reduction in hepatic pathology. This was confirmed by the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells which reproduced the protective effect conferred by immunization with the 190-211 peptide. These 190-211 peptide-specific cells produced little IL4 and high levels of IFN-gamma, a potent inhibitor of collagen synthesis. Furthermore, the use of a lipopeptidic form of the 190-211 peptide significantly improved the reduction in hepatic pathology obtained with the uncoupled peptide and induced a durable protective response. These results provide encouraging information for the possible use of synthetic peptides in the immunoprophylaxis of Schistosomiasis.

Protective effect of rSm28GST-specific T cells in schistosomiasis: role of gamma interferon.

Immunization with a single dose of 50 micrograms of recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (rSm28GST) was able to induce a reduction in the worm burden, the number of eggs, and the degree of hepatic fibrosis as quantified by the measurement of collagen content in the liver of S. mansoni-infected mice. No relationship was found between anti-Sm28GST immunoglobulin G and immunoglobulin A titers and the levels of protection obtained. Adoptive transfers of Sm28GST-specific total, CD4+, or CD8+ T cells reproduced the protective effect obtained with the recombinant molecule. Moreover, experiments studying in vivo T-cell depletion demonstrated that anti-CD4- or anti-CD8-treated mice showed a significant decrease in the protective effect conferred, suggesting a role of the two T-cell subpopulations in the expression of Sm28GST-mediated protection against hepatic damage. Sm28GST-specific cells produced little interleukin-4 and high levels of gamma interferon. Treatment of immunized mice with anti-gamma interferon antibody totally suppressed the Sm28GST-induced protective effect and led to the rapid death of infected animals, suggesting a role for this cytokine in the expression of the protective immunity obtained after immunization with rSm28GST.

A radioimmunoassay for the quantification of human ubiquitin in biological fluids: application to parasitic and allergic diseases.

Purified ubiquitin has been shown to share similar biological and physicochemical properties with a previously characterized lymphokine, platelet activity suppressive lymphokine (PASL). This lymphokine, which inhibits the cytotoxic function of activated platelets, is produced during schistosomiasis and Hymenoptera venom hypersensitivity (HVH). We have developed a radioimmunoassay specific for ubiquitin, in order to determine the ubiquitin levels in human sera and plasma from patients with these pathologies. The working range of the assay was between 60 and 500 ng/ml, and the sensitivity was 8-10 ng/ml. The reproducibility, specificity and accuracy were determined under standard condition (PBS-0.3% BSA) and using different biological fluids (human serum, plasma and T lymphocyte supernatant). Using this assay, we found that the ubiquitin concentrations were higher in both schistosomiasis and HVH (up to 150-300 ng/ml) compared with sera and plasma from healthy donors where the ubiquitin levels did not exceed 50 ng/ml.

Determination of B-cell epitopes of nef HIV-I protein: immunogenicity related to their structure.

Determination of the B-cell epitopes of the nef molecule encoded by the human immunodeficiency virus type 1 (HIV-1) was undertaken using a set of six synthetic peptides. Sequences that were both antigenic and immunogenic and stimulated the production of antibodies recognizing the full length molecule, were considered as B-cell epitopes. Two peptidic sequences were antigenic both in rodents (mice and rats) and in non-human primates (chimpanzee). They were located in the regions 45-69 and 176-206 of the nef molecule. Two additional antigenic sequences were determined, one in chimpanzees, region 79-94, and the second in rodents, region 148-161. Immunogenicity was investigated in the rodents. Only the 45-69 and 176-206 sequences were immunogenic, and specific antibodies present in the sera of the immunized animals reacted with the nef protein. Therefore, each of these two sequences could be considered as containing at least one B-cell epitope. The fine epitopic specificity was determined using subfragments of these two sequences and it was shown that the antigenic determinants were contained in the C-terminal region of each sequence overlapping with the T-cell epitopes. These results raised the importance of vicinity of B- and T-cell determinants and their immunogenicity.

Effect of ubiquitin on platelet functions: possible identity with platelet activity suppressive lymphokine (PASL).

In an attempt to clone a suppressive lymphokine of platelet function (PASL), we have obtained a cDNA clone coding for the previously described human ubiquitin-80 amino acid fusion protein. Our clone differs from the described sequence in that it contains the complete amino acid sequence of ubiquitin as well as a short (25 bp) 5' noncoding region. In addition the 3' untranslated region is slightly longer than that previously shown. Like PASL, purified ubiquitin can inhibit the cytotoxic properties of platelets and the production of oxygen metabolites by these cells. Moreover, this molecule is able to act as a proaggregating factor and seems of a great interest in pathologies involving defects in platelet aggregation. Ubiquitin could also have a potential use in the regulation of immunological disorders in which platelets seem to be implicated such as hymenoptera venom hypersensitivity and aspirin-sensitive asthma, since in both situations, ubiquitin is able, as is PASL, to inhibit the cytotoxic function of platelets. Indeed ubiquitin possesses important pharmacological potentialities which have not been previously described. This molecule and PASL share several similarities in their functional and physicochemical properties. PASL could therefore belong to the family of ubiquitins.

Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni.

We demonstrate the existence of a cooperation between monocytes and platelets for the killing of Schistosoma mansoni. Indeed, supernatants obtained after a 24 hr adherence of normal human monocytes were able to induce, in a dose dependent manner, the cytotoxicity of normal human platelets towards the young larvae of S.mansoni in vitro. The physicochemical analysis of the supernatants showed that a factor exhibiting a pl of 4.8-4.9 was responsible of this effect, suggesting a role of IL-6, detected in the supernatants, in this induction. This was confirmed by the neutralization of the cytotoxic effect by a polyclonal serum against IL6 whereas polyclonal sera against IL-1 beta or TNF-alpha, the other cytokines present in the supernatants, did not modify the cytotoxicity observed. Finally human recombinant IL-6 induces the platelet cytotoxic function, demonstrating a direct effect of IL6 on blood platelets.

Biological effect of interferon-gamma during the course of experimental infection of rat by Schistosoma mansoni.

We demonstrate here that a second mechanism of platelet activation dependent on lymphokine could also take place in the expression of platelet cytotoxicity against Schistosoma mansoni in vitro. Indeed, IgE, as previously described, but also IFN-gamma, present in the sera of infected rats, together induce platelets from normal rats into cytotoxic effectors for the parasitic larvae. This second mechanism appears also effective in vivo since the passive transfer of normal platelets treated by recombinant IFN-gamma (rIFN-gamma) and the administration of rIFN-gamma to rats conferred a protective immunity to S. mansoni.

Characterization of a suppressive factor of platelet cytotoxic functions in human and rat schistosomiasis mansoni.

In a previous study we demonstrated that mitogen-stimulated CD8+ CD4-T cells from normal donors produce a suppressive lymphokine (PASL) of IgE-dependent platelet cytotoxicity. Here we demonstrate the production, after antigenic-stimulation, of this suppressive factor during ongoing infections by Schistosoma mansoni in man and in the rat. The T lymphocyte subpopulation producing this factor was also identified as expressing the marker of the suppressive subset. Because of the absence of species restriction, the relevance in vivo of PASL was determined in the rat model. In these conditions we observed a complete abolition of the protection normally conferred against a challenge infection by the passive transfer of platelets from immune to normal rats after treatment of transferred platelets with T lymphocyte supernatants.

Recombinant tumor necrosis factors mediate platelet cytotoxicity to Schistosoma mansoni larvae.

The involvement of platelets in the immunity directed toward the parasite Schistosoma mansoni has been demonstrated in vitro and in vivo. The killing properties of platelets were shown previously to involve specific IgE after their binding to a membrane receptor. More recently, we have demonstrated that lymphokines released by S. mansoni Ag- or lectin-stimulated CD4+/CD8-T cells were able to induce the platelet cytotoxicity. The isoelectric focusing of the T lymphocyte supernatants showed that two factors were able to stimulate the platelet killing functions. One of them was clearly identified as being IFN-gamma. The present work demonstrates that the second lymphokine was TNF. Indeed the rTNF-beta and, to a lesser extent, rTNF-alpha, induce normal platelets into killer cells for the young larvae of schistosome. Moreover, an additive effect of the TNF-alpha and IFN-gamma has been observed.

Protection against experimental Schistosoma mansoni schistosomiasis achieved by immunization with schistosomula released products antigens (SRP-A): role of IgE antibodies.

Schistosomula-released products (SRP-A) have been shown to induce preferentially a significant IgE response against Schistosoma mansoni schistosomula when injected into rats, in the absence of adjuvant. The present work provides additional evidence of the in vivo relevance of the anti-SRP-A target antigens. Two strains of rat (Brown Norway and Fischer) were immunized with SRP-A and infected percutaneously. A significant level of protection (up to 83% reduction in worm burden) was observed. Passive transfer experiments carried out with anti-SRP-A or IgE-depleted anti-SRP-A sera suggested the preponderant role of antibodies and particularly of IgE in the protective immunity developed by Fischer rats. Platelets and macrophages recovered from such immunized rats had surface IgE as demonstrated by immunofluorescence analysis with FITC anti-IgE, and have been shown to be directly cytotoxic for schistosomula. The chemiluminescence observed when the macrophages were incubated with anti-IgE suggested the presence of IgE on the surface of these cells.