Claude Auriault

Proteolytic cleavage of IgG bound to the Fc receptor of Schistosoma mansoni schistosomula

After the binding of IgG to the surface Fc receptor of Schistosoma mansoni schistosomula, the Fab portions of IgG are cleaved and small peptides are liberated in the culture medium. At least two types of proteinase activities have been demonstrated in the secretory products of schistosomula. One is an endoprotease with trypsin‐like activity, with an optimum pH of 7 and an optimum temperature of 45°C. The other is a metalloaminopeptidase with an optimum pH of 7 and temperature of 37°C.

A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice

HLA‐A2.1‐/HLA‐DR1‐transgenic H‐2 class I‐/class II‐knockout mice were created and their immunological potential evaluated in response to hepatitis B DNA vaccine. Every single immunized mouse developed hepatitis B virus‐specific antibodies, HLA‐DR1‐restricted helper, and HLA‐A2.1‐restricted cytolytic T cell responses directed at the same immunodominant epitopes as those identified in naturally infected or vaccinated humans. These mice were specifically protected against a hepatitis B‐recombinant vaccinia virus infection with a 10,000‐fold or more reduction of the virus load at day 4 post‐challenge. These mice represent a unique in vivo experimental model for human immune function studies without any interference with mouse MHC response which dwarfed the prediction of human responses. Furthermore, they enable the complete monitoring of immune adaptative responses for preclinical screening of candidate vaccines.

Induction of a protective antibody-dependent response against toxoplasmosis by in vitro excreted/secreted antigens from tachyzoites of Toxoplasma gondii

Summary Toxoplasma gondii is a worldwide protozoan parasite which causes severe disease in congenitally infected children and in immunocompromised patients. Besides the well‐defined cytoplasmic and membrane antigens of tachyzoites, we felt that excreted/secreted antigens could play a major role in the immune response. We first report the development of a well‐controlled procedure for obtaining tachyzoite excreted/secreted antigens (E/SA) in cell‐free incubation media. The E/SA immunogenic in human, rat and mouse toxoplasmosis were then characterized. The major E/SA recognized by human sera from the chronic phase of toxoplasmosis had molecular weights of 108, 97, 86, 69,60, 57, 42, 39, 28.5, 27 and 26 kD. When injected into +/+ Fischer rats, E/SA elicited high antibody titres. In addition, passive transfer of these sera to highly susceptible nu/nu littermates induced a significant degree of protection towards the virulent RH strain of T. gondii. This work, which demonstrates the key role played by E/SA in the protective immune response, suggests that these antigens should be of value both for diagnostic purposes and for the development of new strategies for immunization against toxoplasmosis.

Quantitative PCR: validation of the use of a multispecific internal control

Although RT-PCR has many advantages over RNA blotting methods, it can be difficult to obtain quantitative information, due to the enzymatic nature of the PCR amplification, where small variations in amplification conditions result in drastic changes in product yield. In addition, due to the consumption of the reaction components and generation of inhibitors, the amount of products generated plateaus during later stages of the reaction. These problems can be overcome by the use of internal controls in the PCR. In competitive PCR, a DNA fragment containing the same primer template sequences as the target is used to compete for primers annealing and amplification. PCR products are then distinguished by size, restriction sites or Southern blot analysis. Competitive PCR methods require that the target gene and competitive fragment amplify with equal efficiency; this is the case when the internal standard is as similar as possible to the sequence of interest. It had been postulated that the length or primary sequence differences between the two templates could have an effect on relative amplification efficiency. However, results obtained by different laboratories using a multispecific internal control template show that this is not the case, at least in the exponential phase of the amplification (1,2). In those reports, the analysis was limited to cycles 4 to 5 of the late exponential phase, hence further analysis to define relative amplification efficiency, at other stages of the reaction, are necessary. This is the major drawback to a routine application of the original Wang et al. technique (1). In our report we have examined whether measurements using a multispecific internal control can also be performed beyond the exponential phase of reaction, with the aim of rendering the technique independent of the number of cycles used. This possibility has already been suggested for methods employing an internal standard with the same sequence as the cellular template, apart from the presence of either a small intron (3), a small deletion (4) or a mutated restriction site (5). To address this question, we have used for our multispecific internal control two templates both different in size and nucleotides composition from the cellular template. One pair of primers, specific for mouse IFNy cDNA and multispecific internal control, was used to amplify a 280 bp and a 250 bp fragment, respectively, and the second pair of primers, specific for mouse IL10, to amplify two fragments of 400 and 250 bp, respectively. For quantitative analysis, fluorescent labeled primers were used for the PCR and individual products were separated on sequencing gels and quantified using a fluorescent automated DNA analyzer (4,6). In order to determine the quantity of products before PCR, we have taken advantage of this labeling and purified fluorescent amplified PCR products. Standard and cellular specific PCR products were mixed at different ratios (1/l and 1/10 approximatively for IL10 and IFNy) and then analyzed with the automated DNA analyzer to calculate the real ratio obtained (stars on right panels). Mixes were then diluted up to 106 or 107 times and reamplified with the same primers for different cycles numbers. Subsequently, the fluorescent intensity obtained was quantified by computer analysis and then plotted on a log scale against the number of cycles. As Fig. 1 clearly shows the amplification proceeded with the same efficiency for both templates, not only during the exponential phase, but also during the non-exponential phase just up to the plateau. Moreover, the ratios obtained over the entire amplification period, up to the plateau phase, were always identical to the initial ratios (stars on right panels). This showed that despite a difference in primary sequence, a difference in size of 150 bp for IL10 and a difference in initial quantities (up to 12 times), the two templates were amplified in an identical manner for up to 40 cycles. Thus, the amount of a specific cDNA, in the total cDNA, could be calculated by comparison with the internal standard at any cycle number. Our results support the validity of a multispecific internal control, coupled to the quantification of PCR products with fluorescent primers, for the quantification of a variety ofmRNA molecules.

New Functions for Platelets and Their Pathological Implications

We have recently demonstrated in Schistosoma mansoni infection that rat and human platelets could very efficiently kill parasite larvae, both in vivo and in vitro. The study of this IgE-dependent platelet effector function has led us to several subsequent findings. They concern: (1) the existence of a specific receptor for IgE on the platelet surface; (2) its close association with a platelet membrane glycoprotein of essential functional importance, the GPIIb-IIIa complex; (3) the observation, in extrinsic allergic asthma, of an allergen-specific IgE-dependent platelet activation; (4) the identification, in aspirin-sensitive asthma, of a similar, but non-IgE-dependent, platelet activation selectively induced by cyclo-oxygenase inhibitors, and prevented by salicylate. Beyond their implication in anti-parasite immunity, these findings provide a basis for new insights on the participation of platelets in disease.

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.

Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene

Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin‐10 (IL‐10) is an anti‐inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL‐10 adenovirus (Ad‐vIL‐10)‐mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S‐antigen (S‐Ag). B10‐A mice that received a single unilateral injection of Ad‐vIL‐10 in the retro‐orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL‐10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN‐γ and IL‐2 were found in cellular supernatants from IRBP‐stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL‐10 was neutralized completely by injection of a monoclonal anti‐vIL‐10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL‐10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad‐vIL‐10 was performed in Lewis rats simultaneously with S‐antigen in the footpads. This injection determined in situ vIL‐10 expression with very low circulating vIL‐10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad‐mediated gene transfer resulting in systemic and local expression of vIL‐10 provide a promising approach for the treatment of uveitis.

Characterization and synthesis of a macrophage inhibitory peptide from the second constant domain of human immunoglobulin G

We have shown that IgG hydrolysed by Schistosoma mansoni schistosomula inhibited various macrophage functions, especially phagocytosis and anti‐schistosome cytotoxicity. Here we show that a tripeptide, Thr289‐Lys‐Pro291, of the second constant domain of human immunoglobulin G (peptide 286–292) reproduced the inhibitory effect of a total hydrolysate. Indeed the β‐glucuronidase release from IgE‐anti‐IgE‐stimulated rat and human macrophages decreased and its intracellular level did not rise after a prior incubation of the cells with Thr‐Lys‐Pro (500 nmol/ml). Moreover, the cell migration as well as the superoxide anion O− 2 generation were 50–80% reduced by the tripeptide. These results suggest that a single peptide set may be responsible for the decrease of the macrophage functions at the early stage of the parasite infection in the mammalian host. The pharmacologic properties of this tripeptide are under investigation.

Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis

P30, also referred to as SAG-1, is now recognized as a major Toxoplasma gondii antigen potentially important for both diagnosis and immunoprophylaxis of toxoplasmosis. By using predictive algorithms, five synthetic peptides (48-67, 82-102, 213-230, 238-256 and 279-285) derived from P30, were investigated for B- and T-cell determinants in mouse and rat experimental models. Antibody recognition appeared more broadly distributed along the P30 sequence, whereas T-cell recognition was mainly targeted on the 238-256 peptide. In the absence of any carrier protein, this peptide induced a B- and T-cell immune response independent of the route of immunization (oral route or subcutaneous injection). This peptide (238-256) induced multiple antibody isotypes. In contrast with the 238-256 peptide, the 48-67 peptide, either free or in the form of a multiple antigenic peptide (MAP) construct or the 279-295 peptide, elicited antibodies associated with a TH2 response. This study reports for the first time the analysis of the antigenic and immunogenic properties of P30-derived peptides and are potentially useful for vaccinal strategies incorporating the P30 Toxoplasma gondii antigen.

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.