Véronique Pancré

Aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks. Diagnostic and physiopathological implications.

The pathogenesis of aspirin-sensitive asthma remains unknown. Using a new model of platelet activation, initially described as a response of platelets to IgE antibody-dependent stimuli, this study was designed to test the hypothesis of a possible involvement of platelets in aspirin-sensitive asthma. Washed platelets from 35 aspirin-sensitive asthmatics showed an abnormal in vitro response to cyclooxygenase inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs)--aspirin, indomethacin or flurbiprofen--characterized by the generation of a cytocidal supernatant and (14 patients explored) a burst of chemiluminescence; these drugs had no similar effect on platelets from 31 controls (p less than 0.0001). It was shown that the abnormal platelet response to NSAIDs was not mediated by IgE. In contrast to platelets, aspirin-sensitive asthmatic leukocytes generated neither cytocidal factors nor chemiluminescence in the presence of NSAIDs. Sodium salicylate and salicylamide, which, though structurally similar to aspirin, do not inhibit cyclooxygenase and are well tolerated by aspirin-sensitive asthmatics, did not activate their platelets to release cytocidal factors. Moreover, preincubation of platelets with sodium salicylate, salicylamide or prostaglandin endoperoxide PGH2, highly prevented their abnormal response to NSAIDs (greater than 80%; p less than 0.0001). Since several lipoxygenase inhibitors (NDGA, esculetin), including inhibitors of both cyclooxygenase and lipoxygenase (ETYA, BW755c), did not activate patient platelets and prevented the subsequent abnormal response to NSAIDs, it is suggested that the abnormal platelet activation by NSAIDs is not only the consequence of an inhibition of cyclooxygenase, but also involves generation of lipoxygenase metabolites of arachidonate. Besides, platelets from 4 aspirin-sensitive asthmatics undergoing aspirin desensitization were found to have completely lost their abnormal responsiveness to NSAIDs. These findings represent the first identification in aspirin-intolerant asthmatics of a specific abnormal cellular response to drugs inducing asthmatic attacks and open new perspectives into the pathogenesis, prevention and diagnosis of this disease. They also provide support to the concept of a role for platelets in asthma.

Platelets as Effectors in Immune and Hypersensitivity Reactions

IgE receptors have been recently characterized on human blood platelets. These receptors share common properties within the Fc epsilon R2 previously described on macrophages and eosinophils with a Ka of 3 X 10(7) M-1 and a mean number of 600-1,000 binding sites for IgE per platelet. The production of an anti-Fc epsilon R2 monoclonal antibody has allowed the identification on platelet membrane preparations of two major bands of 43-45 and 31 kD. In parasitic infections (schistosomiasis, filariasis) IgE-dependent killing by platelets has been demonstrated. In allergic asthma and in Hymenoptera venom sensitivity patients, IgE-dependent activation of platelets expressed by the release of cytocidal mediators and oxidative burst can be specifically triggered by the corresponding allergen. In aspirin-sensitive asthma, a direct, non-IgE-dependent platelet activation by nonsteroidal anti-inflammatory drugs has been demonstrated. The platelet abnormality apparently involved a defect of the prostaglandin H2 binding to its specific receptor and a possible imbalance in the regulatory functions of the lipoxygenase metabolites. Platelet effector functions have been recently shown to be regulated by T cell factors. A novel suppressive lymphokine (PASL) produced by OKT8 T cell subset inhibits platelet activation and killing whereas IFN-gamma has been identified among T cell factors produced by OKT4+ cells able to trigger platelet activation. These observations open original perspectives into the pathogenesis, the diagnosis and the prevention of allergic and pseudoallergic disorders, and they provide support to the concept of a role for platelets in various immune and hypersensitivity reactions.

Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies

The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4+ T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4+ T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.

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.

Novel Hyperbranched Glycomimetics Recognized by the Human Mannose Receptor: Quinic or Shikimic Acid Derivatives as Mannose Bioisosteres

The mannose receptor mediates the internalization of a wide range of molecules or microorganisms in a pattern recognition manner. Therefore, it represents an attractive entry for specific drug, gene, or antigen delivery to macrophages and dendritic cells. In an attempt to design novel effective synthetic mannose receptor ligands, quinic and shikimic acid were selected as putative mannose mimics on the basis of X‐ray crystallographic data from the related rat mannose‐binding lectin. As the mannose receptor preferentially binds to molecules displaying several sugar residues, fluorescein‐labeled cluster quinic and shikimic acid derivatives with valencies of two to eight were synthesized. Their mannose receptor mediated uptake was assayed on monocyte‐derived human dendritic cells by cytofluorimetric analysis. Mannose‐receptor specificity was further assessed by competitive inhibition assays with mannan, by confocal microscopy analysis, and by expression of the mannose receptor in transfected Cos‐1 cells. Constructs derived from both quinic and shikimic acid were efficiently recognized by the mannose receptor with an optimum affinity for the molecules with a valency of four. As a result, commercially available quinic and shikimic acids appear as stable mannose bioisosteres, which should prove valuable tools for specific cell delivery.

IgE-dependent killing of Brugia malayi microfilariae by human platelets and its modulation by T cell products

Platelets isolated from patients infected with filariasis were cytotoxic for microfilariae in vitro. Moreover, platelets from normal donors acquired killing properties in the presence of serum from infected individuals. The humoral factor involved in this cytotoxic process was shown to be IgE. This IgE-dependent cytotoxicity of platelets was strongly inhibited by antigen-stimulated T lymphocyte supernatants from filarial patients.

Schistosoma mansoni-infected mice show augmented hepatic fibrosis and selective inhibition of liver cytokine production after treatment with anti-NK1.1 antibodies

Gamma interferon (IFN-gamma) plays an immunoregulatory role at different stages of the experimental Schistosoma mansoni-driven processes in mice through its ability to induce cell cytotoxicity against the parasite larvae and to reduce established hepatic fibrosis. The role of Natural Killer (NK) cells, as possible major source of IFN-gamma, has never been studied during the entire course of murine schistosomiasis. In this paper, we investigated the consequences of in vivo NK cell depletion, maintained during 17 weeks of infection, on both hepatic granuloma development and immunological parameters. We found that NK cell depletion following anti-NK1.1 monoclonal antibody (mAb) injections led to an increase of hepatic collagen content in the late stages of granuloma formation and to the diminution of interleukin 12 (IL-12) p40 and IL-7 mRNA expression in the livers. The hepatic mRNA expression of other cytokines (IFN-gamma, tumor necrosis factor alpha [TNF-alpha] and IL-4), as well as humoral and cytokine responses in sera, were not significantly different between control monoclonal antibody (CmAb) and anti-NK1.1-treated mice. Thus, we demonstrate that the anti-NK1.1 treatment might induce alterations of regulatory mechanisms, detectable at a late stage of a chronic process in immunocompetent mice.

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 glutathion‐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 lipopeptidic 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.

IgE-dependent platelet cytotoxicity against helminths.

During the last decade, a growing interest has been devoted to parasite immunology, and, more precisely, to the analysis of effector mechanisms against helminths. These works have led to entirely new concepts in cellular immunology, among which the promotion of the eosinophils to the full status of cytotoxic effectors toward several species of trematodes and nematodes, or the description of receptors for IgE on cells other than mast cells and basophils, such as lymphocytes, eosinophils, macrophages and monocytes, the interaction of IgE with its specific receptor on the cell membrane being associated, in most cases, with highly efficient activation processes.

Activation of a helper and not regulatory human CD4+ T cell response by oncolytic H-1 parvovirus

Background H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment. Methodology/Principal findings Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-γ and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. Conclusions/Significance Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment.