Evaluation of an in vitro mast cell degranulation test in the context of food allergy to wheat.

BACKGROUND: Antigenic profiles obtained by ELISA with IgE from patients with wheat food allergy (WFA) established that major allergens are albumins/globulins (AG) for children suffering from atopic eczema/dermatitis syndrome (AEDS), omega5-gliadins for adults suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA), anaphylaxis or urticaria and low-molecular-weight (LMW) glutenin subunits for patients with anaphylaxis. We aimed to characterize a new mast cell transfectant for its ability to degranulate with wheat proteins and patient sera and compare these results to those obtained by ELISA. METHODS: Thirty sera from patients with WFA were tested: 14 with AEDS (group 1) and 16 with WDEIA, anaphylaxis or urticaria (group 2). An IgE Fc receptor (FcepsilonRI) humanized rat RBL-2H3 line was established by transfection with cDNAs encoding alpha-, beta- and gamma-subunits for the human IgE receptor. RESULTS: A humanized RBL clone was selected for its capacity to express mRNA alpha-, beta- and gamma-subunits of FcepsilonRI, to bind allergen-specific human IgE and to degranulate. In group 1, sera induced enhanced degranulation with AG extract, but rarely reacted with gliadins and glutenins. In group 2, half of the sera showed degranulation with LMW glutenins whereas the AG fraction and lipid transfer proteins were rarely positive. omega5-Gliadins did not appear as a major allergen in degranulation assays, although functional allergen-specific IgE was measurable in appreciable amounts. CONCLUSION: Our data demonstrate that in wheat food allergen evaluation, correlation exists between mast cell degranulation and IgE measurements, depending on the type of allergen. Therefore, the biological activity of some allergen types may also be affected by other parameters.

H2O2 impairs inflammatory mediator release from immunologically stimulated RBL-2H3 cells through a redox-sensitive, calcium-dependent mechanism.

OBJECTIVES: In this study we examined the effects of the inflammatory agent hydrogen peroxide (H2O2) on IgE-mediated mast cell responses. MATERIALS AND METHODS: Release of preformed granular mediators and newly synthesised TNF-alpha were measured in the RBL-2H3 mast cell line stimulated through IgE receptors (FcepsilonRI) in the presence of varying concentrations of H2O2. The sensitivity of the intracellular calcium response to H2O2 exposure was investigated. RESULTS: We found that H2O2 treatment impaired the release of preformed and newly synthesised mediators. H2O2 treatment simultaneously led to a profound inhibition of the calcium response. Calcium fluxes from both intra- and extracellular sources were impaired. H2O2 action was dependent on the intracellular redox state. Receptor activation directly stimulated intracellular H2O2 production. CONCLUSION: While in many cells H2O2 induces potent inflammatory responses we show that it can be an anti-inflammatory agent by not only inhibiting the release of preformed mediators but also by affecting the secretion of newly synthesized TNF-alpha. Inhibition is a consequence of the profound effect on intracellular calcium levels. The activation of an intracellular oxidative burst by FcepsilonRI aggregation and the sensitivity of intracellular responses to redox-altering agents point to an important regulatory mechanism of mast cell responses in inflammatory tissues.

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo.

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.

The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion.

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.

Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3.

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.

Specific signaling pathways in the regulation of TNF-alpha mRNA synthesis and TNF-alpha secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor.

OBJECTIVE: In the present study, we investigated signal transduction pathways involved in TNF-alpha gene expression and TNF-alpha secretion by mast cells stimulated through the high affinity IgE receptor (FcepsilonRI). MATERIALS AND METHODS: TNF-alpha mRNA steady state levels and TNF-alpha secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcepsilonRI. Relative amounts of TNF-alpha mRNA versus beta-actin levels were quantified by RNase protection and RT-PCR assays. TNF-alpha secretion was measured by a current ELISA test. RESULTS: We show that EGTA (5 mM) prevented TNF-alpha mRNA expression and TNF-alpha secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-alpha secretion at 2 microM but had only a marginal effect on TNF-alpha mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-alpha secretion at low doses (EC50= 13 nM), but only partially affected mRNA expression. CONCLUSIONS: Our results show that in FcepsilonRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-alpha secretion while for the increased TNF-alpha mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants.

We have previously identified a Plasmodium falciparum liver stage-specific Ag (LSA-1) found to encode tandem 17 amino acid repeats harboring B cell determinants. Here we extend this study in terms of sequence analysis, protein localization, and immunologic properties. Analysis of the N- and C-terminal regions of LSA-1 from the T9/96 clone reveals high sequence conservation with LSA-1 from NF54. This 200-kDa protein is detected throughout liver schizogony and accumulates in the parasitophorous vacuole space. In our investigation of T and B cell responses to LSA-1, we have focused on both the area of the C-terminal, nonrepetitive "hinge" region and the conserved repetitive region and derived synthetic peptides. These were found to contain major B and T cell determinants. High prevalences and elevated Ab levels to LSA-1, directed primarily, although not exclusively, to the repetitive region, were detected in sera of individuals from one moderately high and two low transmission malaria-endemic areas (prevalences of 97%, 75, and 77%, respectively). In one of these low transmission areas, secretion of the cytokine IFN-gamma, known to inhibit malaria liver stages, and T cell proliferation were detected in PBMC of 22 to 48% and 6 to 20%, respectively, of individuals in response to separate LSA-1 peptides. These results complement the recent finding of conserved CTL epitopes in LSA-1 and support the assertion that immune responses to LS Ag are involved in protection against malaria pre-erythrocytic stages.

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning.

The liver phase of development of malaria parasites has been studied only recently and remains poorly understood compared to the other stages such as sporozoïtes, merozoïtes and gametes. Access to liver forms of Plasmodium falciparum has been improved by the development of in vivo and in vitro propagation methods, but the yield of mature schizonts remains limited and does not allow a detailed antigenic analysis. To date, only immunofluorescence assays (IFA) have permitted a description of a species and liver-stage-specific antigen(s) (LSA; ref. 3). Monospecific antibodies to these antigens have not been obtained due either to difficulty in immunizing mice (against LSA), or to poor stability of human monoclonal antibodies. Therefore, as a means of characterizing the LSA, we used an alternative immunological approach to identify clones of the corresponding LSA genes. We describe here the isolation of a DNA sequence coding for a P. falciparum liver-stage-specific antigen composed of repeats of 17 amino-acids, which is immunogenic in man.

The genome of Theileria parva: some structural properties.

The DNA of the protozoan Theileria parva, the causal agent of the bovine East Coast Fever, has been prepared at least 99% pure from the intra-erythrocytic form of the parasite. Its buoyant density was found to be 1.696 g/cm3 and its calculated G + C content was 36.7%. Fragmentation of T. parva by the restriction enzyme EcoRI provides some evidence of the presence of repetitive DNA sequences.

Inhibition of murine plasmocytoma tumours with antibody activity by their respective specific antigens.

Three murine plasmocytoma tumour which secrete specific antibodies have been studied for the effect of specific antigens (pneumococcal C polysaccharide and dinitrophenylated bovine gamma globulin) on the growth of these tumours in vivo. In each case, the effect of the specific antigen was to inhibit the growth of that tumour which synthesized the specific antibody. Low molecular weight haptens had no effect on tumour growth. We suggest that this antigen specific growth inhibitory effect is a function of the antigen's binding to membrane bound antibody resulting in defective membrane function.

Mechanism of immune stimulation and tolerance based on an hypothesis on the cell division mechanism directed by the cell membrane.

The binding of the antigen to membrane receptors is suggested to modify the structure of the membrane and consequently the activity of some of its enzymes and the flow of metabolites. The nature of the effects may be a function of the quantity of the interacting antigen.