MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes.

BACKGROUND: It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability. OBJECTIVE: The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent. METHODS: Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis. RESULTS: ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage. CONCLUSIONS: Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.

[Hypersensitiviy to Trichophyton rubrum antigens in atopic and non-atopic podiatrists]. / Hipersensibilidad a los antígenos del Trichophyton rubrum en podólogos atópicos y no atópicos.

BACKGROUND: Podiatrists as well as manicurists are exposed to inhale nail dust contaminated with mycotic particles when filing and burring the onychomycotic nails of their patients. As some of them with atopic background suffered worsening of their symptoms we decided to study the immune response to the fungus Trichophyton rubrum (Tr) that was isolated from the nail dust obtained from the podiatrists office. METHODOLOGY AND RESULTS: A detailed clinical record, intracutaneous skin tests with a extract of Tr and the serum levels of total and specific IgE were performed in rhinitis-asthma as well as control patients. As the asthmatic group refused to perform the challenge bronchial test with Tr we developed a guinea pig experimental model with daily aerosolization of Tr during 12 weeks studying the levels of specific IgE and IgG as well as the lung's histopathology. Atopic patients showed positive immediate skin tests with Tr and both groups revealed delayed hypersensitivity to the antigen.RAST-IgE-anti-Tr and RAST-inhibition confirmed the specificity of the antibodies. Guinea pigs also synthetized IgG and IgE anti-Tr and suffered different degrees of lung lesions similar to those of hypersensitivity pneumonitis. CONCLUSION: Atopic podiatrists are exposed to fungal allergens that may participate or aggravate their previous respiratory conditions.

Immunological changes during cockroach immunotherapy.

Data obtained in a 3-year survey of specific immunotherapy (SIT) with a Periplaneta americana antigen (Pa-1) are presented. Parameters such as serum IgE-paperadioimmunosorbent test, specific IgE and IgG, interleukin (IL)-2, IL-4 and IL-4R levels were recorded before and after SIT. While serum IgE levels and IgE-RAST-anti-Pa-1 did not change throughout SIT (p = not significant), IgG-RAST-anti-Pa-1 showed a marked increase from the first year (p < 0.002 to p < 0.001). Only after 3 years of SIT did the serum levels of IL-2, IL-4 and IL-4R show lower values than before this period (p = 0.05, p = 0.05, p = 0.01, respectively). The comparative statistical analysis of the cytokine data between the nonatopic subjects and the atopic treated patients revealed no significant differences (p = 0.02). The symptomatic scores showed significant results at the third year of SIT in sneezing attacks, nose blowing and nasal obstruction (p = 0.001, p < 0.001, p = 0.001, respectively).

Proteinase and gelatinolytic activities of house dust mite and cockroach extracts.

The presence of gelatinolytic activity in dust mite and Periplaneta americana allergenic crude extracts were studied. The former presented major activity in a broad band between 45 and 66 kDa and minor activity at 32 kDa, while the latter showed a more complex pattern with gelatinolytic activity at 90, 78, 65, 34, 32 and 24 kDa. When the proteolytic activity patterns of dust mites and cockroach crude extracts were analyzed at three different pH levels, the proteases in both cases were optimally active at pH 6, showed no activity at pH 3.5 and little activity at pH 8.5. The susceptibility of both extracts to a set of well-known protease inhibitors suggested that they are composed of cysteine and serine proteinases, the latter probably being a trypsin-like type. When immunochemical properties were studied, dust mite bands of about 200, 110, 65, 60 and 43 kDa showed immunoreactivity against a polyclonal human anti-dust mite serum, with the band of approximately 200 kDa presenting the highest antigenicity. A similar analysis was applied to the cockroach extract, which exhibited immunoreactive bands at 90, 78, 65 and 34 kDa when incubated with a polyclonal rabbit anti-Blatta serum. Only those of 90, 78 and 65 kDa reacted against a polyclonal human anti-Blatta serum. These results suggested a correlation between some proteases with gelatinolytic activity and the allergenicity of both extracts.

Hypersensitivity pneumonitis induced by Penicillium notatum antigens in guinea pigs.

Data concerning the experimental induction of hypersensitivity pneumonitis in guinea pigs using a glycoprotein derived from Penicillium notatum are presented. This antigen was obtained from the mycelial and metabolic products of the cultures passed through a Sephadex G-50 column. It was aerosolized for inhalation by adult guinea pigs for 12 weeks to detect specific serum IgM, IgG and IgE antibodies as well as sensitized leukotriene CD4 cells. Histopathological studies of the lungs showed interstitial infiltrates of macrophages and leukotriene cells, cellular bronchiolitis and single non-necrotizing granulomas. The results from this animal model suggest that this hypersensitivity pneumonitis is a typical delayed-type reaction due to chronic contact with the heterologous glycoprotein of Penicillium.

Bat feces as an indoor allergen.

We have demonstrated in an animal model (specific IgG) as well as in atopic patients suffering from rhinitis/asthma (specific IgE) that bat feces have antigenic properties. A single peak with high glycoprotein content was obtained by chromatography, while SDS-PAGE revealed several proteins between 29 and 116 kDa which showed immune serum blotting at 43.6 and 66 kDa. Positive specific skin tests with bat feces and IgE-RAST anti-bat feces were detected in atopic patients living in tall buildings and old houses in Buenos Aires. As bat feces did not cross-react with bat epithelium, studies evaluating rat serum and epithelium and pigeon feces in order to analyze the role of bat serum proteins, such as IgA, are encouraged.

Interstitial pneumonitis induced in guinea pigs by the antigens of Rhizopus nigricans.

Rhizopus nigricans (Rn) is one of the most common members of the Mucorales that produces opportunistic infections and hypersensitivity states. Data concerning experimental induction in guinea pigs of hypersensitivity pneumonitis with a glycoprotein antigen are presented. This antigen was obtained from the mycelial and metabolic products of the cultures and was aerosolized during 12 weeks. The presence of specific antibodies (IgG and/or IgE) was detected by serological techniques; histopathological studies of the lungs showed interstitial infiltrates of macrophages and LTCD8+ cells, as revealed by the MoAb used. Single non-necrotizing granulomas were characteristic from the tenth week to the end of the experiment. The results from this animal model suggest that hypersensitivity pneumonitis is a typical delayed-type reaction due to chronic contact with the heterologous glycoprotein of Rn. The relation of Rn antigen and the development of occupational diseases of the lung such as malt-worker's lung and wood-trimmer's disease is proposed and discussed.

A citrus fruit-exclusion diet in sensitive patients and its influence on specific antibodies.

The presence and kinetics of specific IgE and IgG antibodies against the oranges Citrus aurantium sinensis (CAS) and Citrus silension (CS) were studied in 41 atopic and 20 non-atopic children aged 8-12 years. Diagnostic procedures such as intracutaneous skin tests, IgE PRIST and anti-CAS and anti-CS IgE and IgG RAST were performed in both groups. A citrus fruit-exclusion diet was maintained for 180 days. The comparison of the results before and after the diet showed no significant changes in skin reactivity and RAST values, and even a slight increase was recorded in the latter. We have attempted to explain these puzzling findings.

Antigenicity of Penicillium notatum in animals and in atopic patients.

A mycelial and metabolic extract of Penicillium notatum (PN) was passed through Sephadex G-50 and DEAE-cellulose columns in order to separate soluble fractions that revealed a complex composition. Their proteins and hexoses were recorded with a LKB Uvicord spectrophotometer and quantified by the Lowry and the indol techniques. On the other hand an animal model was developed in rabbits and an IgG precipitin and hemagglutinating antibodies were obtained. The PN extract as well as those fractions with the higher protein content appeared positive when they were checked against the antiserum by means of Ouchterlony, Boyden and immunoelectrophoresis. The molecular weight of PN was established in 52,000 daltons approximately in comparison with well known marker proteins. Adult human beings suffering perennial rhinitis and bronchial asthma showed positive type I skin tests with PN and its most conspicuous fractions (glycoproteins). A RAST-IgE-anti-PN was prepared following Ceska's procedure and challenged against all human sera. Only 40% of the patients revealed a positive RAST IgE-anti-PN which correlated significatively with the protein fractions (35%) and with the skin tests (43% and 39%, respectively). These results reinforced the idea that PN is a potent antigenic mold both in animals and in humans in whom we detected an IgE specific antibody presumably related to their atopic clinical condition.

Induction of anti-orange antibodies in rabbits.

The capacity of certain foods to cause allergic reactions is well known. The four types of mechanisms that Gell and Coombs described in 1968, are involved in these reactions, although the reaginic antibodies retain the paramount attention of the immunologists. The physiochemical composition of the allergen molecule is the goal of investigators with the purpose to clarify the intrinsic kinetics of antibody synthesis. This paper contributes to the conflicting data about orange allergens especially those obtained from Citrus Aurantium Sinensis and Citrus Silension (CAS and CS, respectively). Glycoproteins were separated by gel filtration through a Sephadex G-50 column. A definite protein peak was obtained meanwhile several hexoses appeared throughout the fractionation procedure. These molecules have adequate physiochemical properties that make them able to trigger the immunological response (molecular weight, definite chemical composition and glycoprotein content). Although CAS and CS have a similar chemical composition a slight inverse proportion of proteins and hexoses was demonstrated between the two classes. Molecular weights were different for CAS (51.500) and for CS (37.000) in comparison with well established protein makers. Ouchterlony revealed two precipitin lines in the CAS-anti-CAS system but none in the CS-anti-CS one. The Boyden technique showed a titre of 1/256 in the first case and only of 1/64 in the second of specific anti-orange antibodies. All the eluted fractions gave negative results although they were concentrated ten times by pre-evaporation. This animal model reinforced the statement that after a long and continuous exposure to orange antigens it was possible to develop specific antibodies. It is assumed that this phenomenon happens in atopic children with it's diagnostic and therapeutic importance.