Diagnostic value of Alt a 1, fungal enolase and manganese-dependent superoxide dismutase in the component-resolved diagnosis of allergy to Pleosporaceae.

BACKGROUND: Over the last decades, genomics and proteomics have contributed to the current knowledge of individualized allergenic components and their potential use in the diagnosis of IgE-mediated allergies. Recent investigations have demonstrated that Alt a 1 should be considered as a relevant allergen of the Pleosporaceae group and that enolase is the main allergen involved in the cross-reactivity to fungi. However, the real utility of these allergens as tools for the diagnosis of allergy to Alternaria is still unclear. OBJECTIVE: To demonstrate the current value of the available fungal allergen panel and the need to build an accurate mould allergen array for the diagnosis of allergy to Pleosporaceae. METHODS: Specific IgEs to individual mould allergens and allergenic mould extracts were evaluated using the ImmunoCAP™ system in 30 patients allergic to Alternaria and in 100 blood donors. Cross-reactivity studies were performed by Fluoro Enzyme ImmunoAssay (FEIA) and FEIA inhibition using individual allergens and allergenic extracts. Two-dimensional electrophoresis associated with a MALDI-TOF analysis was carried out to identify new allergen molecules. RESULTS: All allergic patients had positive specific IgE responses to several moulds from different taxonomical families. Classic and molecular diagnosis demonstrated that 23% of patients had multi-sensitization. The current commercially available fungal allergen array was not sufficient to establish an accurate diagnosis. Unexpected correlations between Alternaria or Alt a 1 and Curvularia or Cladosporium stimulated the investigation of a more accurate allergen panel. A manganese-dependent superoxide dismutase (MnSOD) homologous to Asp f 6 was identified as a new IgE-binding molecule from Alternaria alternata. CONCLUSIONS AND CLINICAL RELEVANCE: Alt a 1 is the marker for allergy to Pleosporaceae, not including Curvularia. MnSOD can explain 6.6% of allergy to Alternaria without Alt a 1 sensitization and should be included together with Alt a 1 and fungal enolase in the molecular array for the diagnosis of allergy to Pleosporaceae.

Allergic hypersensitivity to cannabis in patients with allergy and illicit drug users.

BACKGROUND: Cannabis is the illicit drug most widely used by young people in high-income countries. Allergy symptoms have only occasionally been reported as one of the adverse health effects of cannabis use. OBJECTIVES: To study IgE-mediated response to cannabis in drug users, atopic patients, and healthy controls. METHODS: Asthmatic patients sensitised to pollen, and all patients sensitised to tobacco, tomato and latex, considered as cross-reacting allergens, were selected from a data base of 21,582 patients. Drug users attending a drug-rehabilitation clinic were also included. Controls were 200 non-atopic blood donors. Specific IgE determination, prick tests and specific challenge with cannabis extracts were performed in patients and controls. RESULTS: Overall, 340 patients, mean age 26.9±10.7 years, were included. Males (61.4%) were the most sensitised to cannabis (p<0.001). All cannabis-sensitised patients were alcohol users. Eighteen (72%) of the patients allergic to tomato were sensitised to cannabis, but a positive specific challenge to cannabis was highest in patients sensitised to tobacco (13/21, 61.9%), (p<0.001). Pollen allergy was not a risk factor for cannabis sensitisation. Prick tests and IgE for cannabis had a good sensitivity (92 and 88.1%, respectively) and specificity (87.1 and 96%) for cannabis sensitisation. CONCLUSIONS: Cannabis may be an important allergen in young people. Patients previously sensitised to tobacco or tomato are at risk. Cannabis prick tests and IgE were useful in detecting sensitisation.

Identification of 2 new allergens of Phoenix dactylifera using an immunoproteomics approach.

The date palm (Phoenix dactylifera) has a wide geographical distribution (Middle East, Mediterranean, central Africa, western Asia, Australia, and North America). Pho d 2, the major allergen of date palm pollen was recently identified as a profilin, yet little is known about the nature of the other pollen allergens from this tree. The objective of this study was to characterize clinically significant allergens other than profilins from P. dactylifera pollen using immunoproteomics. In order to reveal the proteins causing the allergy, we used serum from a patient monosensitized to date palm pollen extract who experienced asthma and rhinoconjunctivitis during the palm tree pollen season. The results revealed 2 novel immunoglobulin E-binding proteins not related to the cross-reactive allergen profilin. Individualized allergens of Pdactylifera that cause specific date palm pollen sensitization must be defined to determine the real prevalence of sensitization to this species.

Variability of Alt a 1 expression by different strains of Alternaria alternata.

BACKGROUND: While it is well known that there is significant intraspecific variation in the content an potency of Alternaria alternata allergens, little data has been published on intraspecific variability for individual allergens from moulds. OBJECTIVE: To assess the variability of Alt a 1 expression in different strains of A alternata. METHODS: Eleven strains of A alternata were cultured in a Czapek broth medium and culture filtrate extracts were obtained. A sensitive two-site enzyme-linked immunosorbent assay was used to measure Alt a 1 concentrations in medium following 3 weeks of culture and in culture filtrate extracts. RESULTS: Expression of Alt a 1 was highly variable in different strains ofA alternata (coefficient of variation > 135%). A good correlation was found between Alt a 1 concentrations at the beginning of the process and measurements at the end of extract production (r=0.940). CONCLUSIONS: The high variability of Alt a 1 expression in different A alternata strains makes it necessary to measure Alt a 1 concentrations during the first stage of allergenic extract production in order to be able to choose a suitable strain for producing extracts or purifying Alt a 1.

Barnacle allergy: allergen characterization and cross-reactivity with mites.

BACKGROUND: Barnacles are a type of seafood with worldwide distribution and abundant along the shores of temperate seas. They are particularly appreciated and regularly consumed in Portugal as well as in Spain, France and South America, but barnacle allergy is a rare condition of which there is only one reference in the indexed literature. The molecular allergens and possible cross-reactivity phenomena implicated (namely with mites) have not been established. OBJECTIVE: To demonstrate the IgE-mediated allergy to barnacle and to identify the proteins implicated as well as possible cross-reactivity phenomena with mites. METHODS: We report the clinical and laboratory data of five patients with documented IgE-mediated allergy to barnacle. The diagnosis was based on a suggestive clinical history combined with positive skin prick tests (SPT) to barnacle--prick to prick method. Two barnacle extracts were prepared (raw and cooked barnacle) and sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblotting were performed. An immunoblotting inhibition assay with Dermatophagoides pteronyssinus was also done in order to evaluate cross-reactivity. RESULTS: All patients had mite-related asthma and the allergic rhinoconjunctivitis; they all experienced mucocutaneous symptoms. All of them had positive SPT to barnacle, and the immunoblotting showed several allergenic fractions with a wide molecular weight range (19 - 94 kDa). The D. pteronyssinus extract inhibited several IgE-binding protein fractions in the barnacle extract. CONCLUSIONS: We describe five patients with IgE-mediated barnacle allergy. We also describe a group of IgE-binding+proteins between 30 and 75 kDa as the allergenic fractions of this type of Crustacea. Cross-reactivity with D. pteronyssinus was demonstrated in two cases.

Alt a 15 is a new cross-reactive minor allergen of Alternaria alternata.

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases.

Allergenic cross-reactivity between Blomia tropicalis and Blomia kulagini (Acari: Echymiopodidae) extracts from optimized mite cultures.

BACKGROUND: At present, data about the cross-reactivity of Blomia spp. comes from studies made among different genera of mites, and no results have been published involving different species of the genus Blomia. OBJECTIVE: The aim of this study was to find out the level of cross-reactivity between the two main species of Blomia causing allergy, and its implication in the diagnosis of Blomia sensitization. METHODS: Using extracts from optimal growth phases of Blomia kulagini (Zakhvatkin, 1936) and Blomia tropicalis (van Bronswijk, Cock and Oshima, 1973) as allergenic material, the allergenic cross-reactivity between both house dust mites was evaluated by means of cutaneous tests, specific IgE values, ImmunoCAP-inhibition and SDS-PAGE-IgE-immunoblotting-inhibition. RESULTS: The results demonstrated that IgE-binding components belonging to both species are very similar from the immunological point of view, showing high correlations between both species when using cutaneous tests (R2=0.915) or specific IgE (R2=0.980). ImmunoCAP-inhibition and SDS-PAGE-IgE-Immunoblotting-inhibition probed with human sera, showed a total inhibition of specific IgE reactions by the heterologous antigens. CONCLUSION: The results obtained strongly suggest the great resemblance between the allergenic composition of both species.

Membranous glomerulonephritis secondary to hydatid disease.

To study the association of hydatid disease of the liver with a nephrotic syndrome we processed renal biopsy tissue for light microscopy, immunofluorescence and electron microscopy studies. These procedures disclosed the typical features of a membranous nephropathy whereas indirect immunofluorescence revealed glomerular deposits of hydatid antigen. Rabbits, previously immunized with bovine hydatid fluid, provided the specific antiserum. Clearly, the pathogenicity of this parasitosis derives, in part, from its immunologic repercussions. In this regard, our results support an etiopathogenic role for hydatid antigen in an immune-mediated glomerulonephritis.

Evaluation of whole and purified hydatid fluid antigens in the diagnosis of human hydatidosis by the immunoelectrophoresis test.

Whole sheep hydatid cyst fluid (WHF) and purified hydatid fluid antigens (PHF) were evaluated in the immunoelectrophoresis (IEP) test for the diagnosis of human hydatid disease. A higher sensitivity was obtained using the PHF than with the WHF antigen but false positive results were observed with the former in 22.4% of the 102 non-hydatid sera examined. Using the presence of arc 5 as the criterion of positivity however, no false positives were observed in non-hydatid sera with the WHF antigen. It is postulated that the number of bands other than arc 5 may be of value in the diagnosis of hydatid disease by the IEP test, in cases where this diagnostic arc is absent.

Evaluation of three immunodiagnostic tests for human hydatid disease.

The comparative sensitivity and specificity of the immunoelectrophoresis (IEP), latex agglutination (LA) and indirect hemagglutination (IHA) test for hydatidosis were evaluated using a single hydatid cyst fluid pool as antigen and the same hydatid and nonhydatid sera and were found to vary with the type of IHA test, the criterion for IHA test positively and the group of sera selected for study. The sensitivity of the LA and IEP tests was comparable, both tests correlated well and neither gave a positive result in the nonhydatid sera studied. The sensitivity of the IEP test was higher when IHA test positivity was based on diagnostically significant titers but not when all IHA test reactors were considered as positive to this test. The merits and limitations of the LA and IHA tests in diagnostic and seroepidemiologic studies of human hydatidosis are discussed. On the basis of the present findings, the use of the LA test and possibly of the tannic acid IHA test, to screen for IEP test positive sera is proposed as the most reliable immunodiagnostic approach to estimate the prevalence of human hydatidosis for seroepidemiologic purposes and also for detecting hydatidosis cases in the field.

Enzymatic analyses of house dust mite extracts from Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) during different phases of culture growth.

The majority of clinically important allergens of Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 present enzymatic activity. The allergenic enzymes described include cysteine proteases in group 1 allergens, trypsins in group 3, amylases in group 4, and chymotrypsins in group 6. Apart from these, other possibly allergenic enzymes also have been identified. Therefore, enzymatic profiles were studied during the 3 growth periods of the mite population--latency phase, exponential growth phase, and death phase. The activity of 19 different enzymes was analyzed by means of the Api Zym system, a method that has been used to study both mite extracts and other allergenic materials. Our study has demonstrated that the extracts contain a large variety of enzymes. It has been observed that enzymatic activity is caused exclusively by mites because the control carried out on the culture medium was negative for all the enzymes studied. Generally, the levels of diverse enzymatic activity increased with the growth of the culture, and decreased later, in both species. However, proteases are the exception; they maintain a high level of activity during the death phase of the cultured mites. The ratio between trypsin and chymotrypsin activity can be used as an excellent tool for quality control parameters during obtention of allergenic mite extracts.

Cross-reactions between Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) related to the different growth phases of cultures.

The allergenic cross-reactivity of both inter- and intraspecies of house dust mites, Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961, taking into account the allergenic differences that exist throughout the growth curves, was evaluated by means of RAST-inhibition, using sera from patients allergic to these mites. The results demonstrate that extracts obtained from mite cultures during the maximum exponential growth phase are the best source of reagents to better discriminate cross-reactivity studies. The analyses obtained from this work, together with those obtained in previous reports, help to define the ideal conditions related to the allergenic diversity, avidity, and cross-reactivity of specific antibodies for the elaboration of allergenic extracts as a tool for use in diagnosis and specific treatment of IgE-mediated hypersensitivity caused by house dust mites.

Analysis of the allergen expression of Blomia tropicalis and Blomia kulagini (Astigmata: Glycyphagidae) cultures.

Laboratory cultures of the mites Blomia tropicalis (van Bronswijk, Cock & Oshima) and Blomia kulagini (Zakhvatkin) were used to study the population dynamics of the mites and the kinetics of released allergens during the growth cycle. The analysis of extracts obtained after different incubation periods, by means of immunoblotting, and quantification of the major allergen Blo t 5, allowed definition of three different growth phases, demonstrating that mite cultures during the maximum growth (end of exponential growth curve-beginning maximum growth plateau) contain the largest amount of allergenic components as well as the highest Blo t 5 concentration.

Kinetics of allergen expression in cultures of house dust mites, Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae).

Laboratory cultures of house dust mites Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 were used to study the population dynamics of the mites and the kinetics of antigen appearance. The analysis of extracts obtained after different incubation periods, carried out by SDS-PAGE, immunoblotting, and enzyme-linked immunosorbent assay, allows for the definition of 3 different growth phases: the latency phase (F1); the exponential growth phase (F2) during which the allergenic proteins, including the Der 1 and Der 2 major allergens, were expressed more intensely and in larger quantities; and a final phase (F3), death, in which the lowest rates of allergenic components with a clearly different pattern were seen. The data obtained from this work demonstrates that mite cultures during the maximum growth phase (F2) contain the largest amount of allergenic components as well as the highest major allergen concentrations.

Application of random amplified polymorphic DNA (RAPD) assay to the study of mites related to allergic diseases.

In this study, the conditions for the successful application of the random amplified polymorphic DNA (RAPD) assay to differentiate mite populations based on genetic variation were defined. Five species of mites related to allergic diseases were studied: Dermatophagoides pteronyssinus, D. farinae (2 strains), Blomia tropical is, Glycyphagus domesticus and Tyrophagus putrescentiae. The mites were isolated from pure cultures and processed according to the method described in this paper. The banding patterns obtained were different for all the species studied. When the DNA from two different strains of D. farinae were studied, the "fingerprint" banding patterns obtained showed differences between them. The random amplified polymorphic DNA assay may be a useful tool to aid the taxonomic study of mite populations.

Influence of mite growth culture phases on the biological standardization of allergenic extracts.

House dust mites are a well known cause of asthma and other respiratory allergies. In order to improve the standardization of allergenic extracts for diagnosis and immunotherapy, it is important to determine the frequency and concentration of the components, both the major and the minor allergens during the growth period of the mite population. In a previous paper we demonstrated that the laboratory cultures of Dermatophagoides pteronyssinus and Dermatophogoides farinae exhibited three well differentiated growth phases: latency, exponential growth, and death of the culture. Biological standardization of extracts from the two mite species were carried out by skin prick tests in a group of 20 patients, using different concentrations of the extracts at the three growth phases. The patient sera were also studied by means of the RAST technique to determine the levels of specific IgE for each phase. The extracts produced from the exponential growth phase of the cultures revealed six times more relative allergenic activity in in vivo studies, and average RAST values were approximately three times higher than those extracts from latency and death phases. The reproducibility of the extract production method was assessed by comparing different batches obtained in similar conditions. The results showed batch-to-batch homogeneity allergenic activity. In conclusion, it was demonstrated that extracts obtained from cultures with the highest concentration of live mites (maximum growth phase) render the best diagnostic results in vivo and in vitro.

Total and specific IgE levels in human hydatid disease determined by enzyme immunoassay: serological follow-up after surgery.

The evolution of both specific and nonspecific IgE in a long-term follow-up after surgery in patients with human hydatid disease was studied. Enzyme immunoassays using cyanogen bromide-activated cellulose discs as solid phase were employed. One hundred and nine postoperative serum samples from 26 patients undergoing surgery for hydatid disease were studied. Imaging studies were also carried out during the follow-up. In 8 of 26 patients, remaining cysts were detected during the follow-up. One year after surgery, total IgE levels decreased to normal values in 84.6% of the total number of patients, in 94.5% of the cases in which no remaining cysts were detected, and in 62.5% of the patients with remaining hydatid cysts. These data highlight the poor value of an isolated postoperative IgE determination as a diagnostic marker for remaining hydatidosis. On the contrary, 1 year after surgery, the levels of anti-Echinococcus IgE decreased in 55% of the patients without residual cysts and in 50% of the total number of patients. In six patients without remaining hydatid cysts, the levels of specific IgE increased 1 year after the surgery. In the group of patients with remaining cysts only in three patients did the values of specific IgE decrease, although they remained significant. Thus, 1 year after surgery, anti-Echinococcus IgE levels were still evident in all patients, although in those without remaining cysts there was a predominance of decreasing values.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracts from various mite species contain cross-reactive and noncross-reactive IgE epitopes. A RAST inhibition study.

Previous studies have demonstrated the high incidence of Dermatophagoides, Euroglyphus, Blomia, Lepidoglyphus and Chortoglyphus spp. sensitizations in a mite-allergic population. The aim of this study was to evaluate immunological cross-reactivity among the above mentioned groups, using sera from a nonrural population allergic to mites, from a subtropical area (Canary Islands). RAST inhibition studies demonstrated significant cross-reactivity among Dermatophagoides and Euroglyphus species (> or = 65% of maximum theoretical inhibition), as also noted by other authors. Blomia kulagini demonstrated scarce cross-reactivity with Dermatophagoides, Lepidoglyphus and Chortoglyphus species (< or = 30% of maximum theoretical inhibition) and medium level with Euroglyphus maynei (45% of maximum theoretical inhibition). Chortoglyphus arquatus demonstrated high cross-reactivity levels with the other species studied. The results obtained in this study demonstrated the scarce immunological cross-reactivity between Pyroglyphidae and non-Pyroglyphidae mites, thus suggesting the polysensitization of the studied population to different mite species.

Chemical and biological characterization of an active substance of the human placenta.

Acetic acid extracts of human term placenta have been fractionated by pH and salt precipitations and by exclusion chromatography on a Sephadex G-75 column. A partially purified fraction (F-II) possessing uterotropic activity in immature and young mice was obtained. This active fraction was submitted to the action of protein denaturating agents (heat, 8 M urea) and of specific proteolytic enzymes (trypsin, alpha-chymotrypsin and pronase). These treatments completely destroy the uterotropic activity showing that the active substance is of protein nature. The administration of F-II to spayed mice did not produce any increase in their uterine weight suggesting that the uterotropic activity would be due to stimulation of the female gonad.