A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content, and Allergenicity.

This review summarizes the available data related to the effects of air pollution on pollen grains from different plant species. Several studies carried out either on in situ harvested pollen or on pollen exposed in different places more or less polluted are presented and discussed. The different experimental procedures used to monitor the impact of pollution on pollen grains and on various produced external or internal subparticles are listed. Physicochemical and biological effects of artificial pollution (gaseous and particulate) on pollen from different plants, in different laboratory conditions, are considered. The effects of polluted pollen grains, subparticles, and derived aeroallergens in animal models, in in vitro cell culture, on healthy human and allergic patients are described. Combined effects of atmospheric pollutants and pollen grains-derived biological material on allergic population are specifically discussed. Within the notion of "polluen," some methodological biases are underlined and research tracks in this field are proposed.

IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells.

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10-secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti-IFN-α Ab immunotherapy in lupus patients.

Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis.

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one-dimensional gel electrophoresis (D1-DE) as an alternative to the 2-DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one-dimensional combination of IEF and SDS-PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1-DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one-dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin-based protein-protein interactions. Therefore, D1-DE could be used in routine work as a convenient alternative to 2-DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.

IFNalpha kinoid vaccine-induced neutralizing antibodies prevent clinical manifestations in a lupus flare murine model.

A major involvement of IFNalpha in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNalpha from a recombinant adenovirus (IFNalpha Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) x New Zealand White (NZW)F(1) mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNalpha immunogen, termed IFNalpha kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNalpha Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNalpha inhibitory capacity was present in the serum.

Ability of pollen cytoplasmic granules to induce biased allergic responses in a rat model.

BACKGROUND: Grass pollen is one of the most important aeroallergens in Europe. It highly contributes to respiratory allergic diseases, mainly allergic rhinitis. In contact to water or airborne pollutants, pollen grains can release pollen cytoplasmic granules (PCGs) containing allergens. Because of their size (<5 µm), PCGs may penetrate deeper into the lungs to induce higher allergic responses, such as asthma. They have been associated with thunderstorm-related asthma. The aim of this study was to evaluate, with Brown Norway rats, the allergenic potential of isolated PCGs and to compare it with the allergenicity of whole timothy grass pollen. METHODS: Rats were sensitized (day 0) and challenged (day 21), in controlled comparative conditions, with pollen grains (0.5 mg) or PCGs (4.5 × 107 and 0.5 mg). At day 25, blood samples, bronchoalveolar lavage fluid (BALF) and bronchial lymph node were collected. IgE and IgG1 levels in sera were assessed by ELISA. Alveolar cells, protein and cytokine concentrations were quantified in BALF. T cell proliferation, in response to pollen or granules, was performed by lymph node assay. RESULTS: The results showed that proliferative responses of lymph node cells were similar in PCG- and pollen-sensitized rats. IgE and IgG1 levels were higher in pollen- than in PCG-sensitized rats. However, eosinophils, lymphocytes and pro-allergy cytokines in BALF were higher in PCG- than in pollen-sensitized rats. CONCLUSIONS: Thus, PCGs, able to deeply penetrate in the respiratory tract, induced local and strong allergic and inflammatory responses more linked with asthma- than rhinitis-related allergic symptoms.

Micropillar array chips toward new immunodiagnosis.

In this paper, we demonstrate the possibility to use a micropillar array to perform molecular immunodiagnosis. A polydimethylsiloxane (PDMS) microdevice consisting of a rectangular array of micropillars (45 µm in height, 100 × 100 µm square cross section) was used to replace microchannels or gels (polyacrylamide or agarose) to perform electrokinetic separation. This microarray was used to mimic highly diluted gel and to maintain electrolyte within the pillar zone by capillary effect. The electrolyte composition (glycerol and agarose content) was investigated in order to improve protein separation by isoelectric focusing (IEF). The influence of glycerol on focusing time and on the different evaporative contributions was further evaluated. In order to perform an immunodiagnostic of milk allergy, different surface treatments were optimized to prevent milk allergen adsorption on PDMS surface. Poly(dimethylacrylamide)-co-allyl glycidyl ether (PDMA-AGE) as well as gelatin led to a satisfactory signal to noise ratio. Finally the possibility to perform protein mixture separation using this micropillar array chip followed by immunoblotting was demonstrated by using the serum from an allergic individual, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Evaluation of microchip material and surface treatment options for IEF of allergenic milk proteins on microchips.

The use of glass and PDMS microchips has been investigated to perform rapid and efficient separation of allergenic whey proteins by IEF. To decrease EOF and to limit protein adsorption, two coating procedures have been compared. The first one consists in immobilizing hydroxypropyl cellulose (HPC) and the second one poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE). EOF limitation has been evaluated using frontal electrophoresis of a fluorescent marker of known effective mobility. EOF velocity was decreased by a factor about 100 and 30, respectively. pH gradient formation has been evaluated for each microchip using fluorescent pI markers. It was demonstrated that as expected a coating was essential to avoid pH gradient drift. Both coatings were efficient on glass microchips, but only PDMA-AGE allowed satisfying focusing of pI markers on PDMS microchips. Fluorescent covalent and noncovalent labelings of milk proteins have been compared by IEF on slab-gels. IEF separation of three major allergenic whey proteins [beta-lactoglobulin A (pI 5.25) and B (pI 5.35) and alpha-lactalbumin (pI 4.2-4.5)] was performed in both microchips. Milk proteins were separated with better resolution and shorter analysis time than by classical CIEF. Finally, better resolutions for milk allergens separation were obtained on glass microchips.

Magnetic beads based immunoaffinity capillary electrophoresis of total serum IgE with laser-induced fluorescence detection.

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.

Specific immunotherapy for common grass pollen allergies: pertinence of a five grass pollen vaccine.

Patients throughout Europe are concomitantly exposed to multiple pollens from distinct Pooideae species. Given the overlap in pollination calendars and similar grain morphology, it is not possible to identify which grass species are present in the environment from pollen counts. Furthermore, neither serum IgE reactivity nor skin prick testing allow the identification of which grass species are involved in patient sensitisation. Due to their high level of amino acid sequence homology (e.g., >90% for group 1, 55-80% for group 5), significant cross-immunogenicity is observed between allergens from Pooideae pollens. Nevertheless, pollen allergens also contain species-specific T or B cell epitopes, and substantial quantitative differences exist in allergen (e.g., groups 1 and 5) composition between pollens from distinct grass species. In this context, a mixture of pollens from common and well-characterised Pooideae such as Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis is suitable for immunotherapy purposes because (1) it has been validated, both in terms of safety and efficacy, by established clinical practice; (2) it reflects natural exposure and sensitisation conditions; (3) it ensures a consistent and well-balanced composition of critical allergens, thus extending the repertoire of T and B cell epitopes present in the vaccine.

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis.

Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by field-amplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached.

A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins.

A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l(-1) thiourea, 7 mol l(-1) urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60-500 ng ml(-1)) of water-insoluble proteins extracted with high concentrations of TUC.

Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution.

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.

Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

Immune Aspects of Female Infertility.

Immune infertility, in terms of reproductive failure, has become a serious health issue involving approximately 1 out of 5 couples at reproductive age. Semen that is defined as a complex fluid containing sperm, cellular vesicles and other cells and components, could sensitize the female genital tract. The immune rejection of male semen in the female reproductive tract is explained as the failure of natural tolerance leading to local and/or systemic immune response. Present active immune mechanism may induce high levels of anti-seminal/sperm antibodies. It has already been proven that iso-immunization is associated with infertility. Comprehensive studies with regards to the identification of antibody-targets and the determination of specific antibody class contribute to the development of effective immuno-therapy and, on the other hand, potential immuno-contraception, and then of course to complex patient diagnosis. This review summarizes the aspects of female immune infertility.

Allergome: the characterization of allergens based on a 2D gel electrophoresis approach.

Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcepsilon receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.

Carrier ampholytes-based capillary electrophoresis as an alternative to capillary zone electrophoresis in classical background electrolytes.

Carrier ampholytes (CAs), originally designed for isoelectric focusing (IEF), have been used as background electrolytes (BGE) in capillary zone electrophoresis (CZE). Their main electrophoretic properties, relatively high buffering capacity and low electric conductivity allowed fast (less than 2 min) and high efficient (500,000 theoretical plates/m) separation of a test mixture of proteins under very high electric field strength (more than 1000 V/cm). The results obtained in such buffers have been compared to those obtained in more classical sodium--phosphate and sodium--N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonate) background electrolytes. High ionic strength classical buffers were necessary to achieve the separation of the proteins contained in the test mixture. This induced a significant Joule heating and temperature increase inside the capillary whereas a negligible Joule heat was produced in carrier ampholyte buffers even at the above electric field strength (higher than 1000 V/cm).

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study.

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 "narrow pH cuts" of wide pH range (3-10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that "narrow pH cuts" of CA might be well suited buffers for electrophoretic separations.

Analysis of water extracts from airborne dust samples by capillary isotachophoresis.

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM beta-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.

Analysis of liquid extracts from tree and grass pollens by capillary electromigration methods.

Capillary electromigration methods, zone electrophoresis (CZE), micellar electrokinetic chromatography (CMEKC) and isotachophoresis (CITP), have been used for analysis of water and water-buffer extracts from tree-common birch (Betula verrucosa) and grass-orchardgrass (Dactylis glomerata) pollen samples. Water extracts were analyzed by CZE using acetic acid as background electrolyte (BGE), by CMEKC in tris-phosphate BGE with anionic detergent sodium dodecyl sulfate (SDS) micellar pseudophase (TP-SDS) and by CITP in cationic mode with leading/terminating cations K+/BALA+ (beta-alanine (BALA)) and in anionic mode with leading/terminating anions Cl-/MES- (2-(N-morpholino)ethanesulphonic acid (MES)). Moreover, acetic acid extracts were analyzed by CZE using acetic acid as BGE, and alkaline water-SDS-buffer extracts were analyzed by CMEKC using TP-SDS as BGE. Extracted amounts of pollen allergens and other UV-absorbing compounds and the number of resolved components were evaluated from CZE, CMEKC and CITP analyses of the liquid extracts. Larger amounts of UV-absorbing material were found in the water-buffer pollen extracts than in the water extracts. More UV-absorbing material was found in all extracts from D. glomerata pollen than in relevant extracts from B. verrucosa pollen. It was found by CITP that the extracted amounts of anionic components and their number were much higher than those of cationic components. Concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in pollen samples were also determined by CITP.