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.

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 (AU)

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.

Uncinaria stenocephala: assessment of antigens for the immunodiagnosis of canine uncinariosis.

Although Uncinaria stenocephala is the most frequent hookworm in the intestine of dogs from Northern, Central and Southern Europe, little is known about its host-parasite relationship. Three groups of sera from dogs (Group 1: dogs naturally infected only by U. stenocephala; Group 2: helminth-free dogs at necropsy, and Group 3: dogs parasitized by other helminths) were analyzed by ELISA using U. stenocephala antigens from adult worms (somatic and excretory-secretory antigens) and from L3 larvae (somatic antigens). All three sources of antigens were found to be suitable for immunodiagnosis of canine uncinariosis with up to 90% efficacy. However, an analysis to assess the diagnostic value of the different antigens demonstrated that the adult excretory-secretory antigens had a higher diagnostic efficacy (96.7%), indicating that this is the best antigen source for the diagnosis of Uncinaria infection.

The major allergen of Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae family.

There is general consensus regarding the scarce cross-reactivity existing between Alternaria alternata and other allergenic moulds such as Aspergillus fumigatus, Penicillium notatum or Cladosporium herbarum. However, A. alternata has been shown to have a very significant level of allergenic cross-reactivity with other fungi belonging to the Pleosporaceae family. To date, no biological identity or homologies with other proteins have been described for Alt a 1, and it remains unclear whether the major allergen Alt a 1 contributes to the cross-reactivity shown for these moulds. Specific quantification of Alt a 1 in culture filtrates of Stemphylium botryosum, Ulocladium botrytis, Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum and Asp. fumigatus, and immunoblotting using culture filtrate extracts from the above-mentioned moulds and rabbit serum anti-recombinant Alt a 1 have shown significant amounts of Alt a 1 in culture filtrates as well as antigenic components ranging from 14 to 20 kDa that strongly react with the specific serum for all taxonomically related species (Pleosporaceae family). No reactions were revealed in culture filtrates of Cladosporium, Penicillium and Aspergillus. These results restrict the cross-reactivity phenomenon due to Alt a 1 to the scope of the taxonomical term of family.

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.

Characterization of excretory-secretory products from protoscoleces of Echinococcus granulosus and evaluation of their potential for immunodiagnosis of human cystic echinococcosis.

This study describes, for the first time, the characterization of excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS-PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory-secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n = 15), non-hydatidic parasitoses (n = 19), various liver diseases (n = 24), lung neoplasia (n = 16), and healthy donors (n = 18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41-43 kDa and 91-95 kDa were recognized by the majority of the non-hydatid sera studied.

Uncinaria stenocephala: antigenic characterization of larvae and adults worms using sera from naturally infected dogs.

An antigenic characterization of the larval (somatic) and adult (somatic and excretory-secretory) antigens of Uncinaria stenocephala was made, employing immunoblotting and immunoblotting-inhibition with 10 selected sera from dogs naturally infected by this hookworm. The results indicated that each one of the three parasitic extracts has different antigenic components. Sixty percent of the dog sera consistently recognised four antigens of 20, 25, 30, and 38kDa of the larval extract and the immunoblotting inhibition showed that these antigens were only observed at the larval stage. All these results indicated that these antigens can be considered as major antigens and they could be useful for the immunodiagnosis of this parasitic infection.

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.

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.

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.

Quality parameters for the production of mite extracts.

Mites present in house dust are of great etiological importance in type I hypersensitivity, with those belonging to the Dermatophagoides genus (D. pteronyssinus and D. farinae), of the Pyroglyphidae family, being the most frequent and principal source of allergens. For the production of allergenic extracts destined for specific diagnostic and treatment purposes of allergic diseases, the culture of such mites is absolutely necessary. In accordance with studies carried out in our laboratories to obtain adequate extracts, one must bear in mind the culture mite phase. Three growth phases have been distinguished for both species: latency phase (F1), growth phase (F2) in which the allergenic proteins are expressed with greater intensity, and death phase of the culture (F3). In the same study, the biological standardization of the extracts demonstrated that those produced from the maximum growth phase gave both in vitro and in vivo results, at least three times more sensitive than those from the other phases. We checked the reproducibility of the production method, obtaining different batches in similar conditions with a high homogeneity regarding allergenic activity. The sensitivity and specificity of the allergenic extracts depends just as much upon the production method as the standardization method. During the biological cycle of Dermatophagoides in culture, it is only from the maximum growth phase (F2), that allergenic extracts with an excellent diagnostic value, high sensitivity and specificity, can be obtained.

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.

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)

The diagnosis of human hydatidosis by measurement of specific IgE antibody by enzyme immunoassay.

The sensitivity and specificity of an enzyme immunosorbent assay (EIA) for measurement of anti-Echinococcus granulosus specific IgE antibody using microtiter strips was evaluated. The sensitivity was 92.2% for 90 patients with cysts in the liver or other body sites but excluding the patients with cysts in the lungs or bones. In this latter group (n = 26) a sensitivity of 61.5% was recorded. The specificity for control groups comprising 89 blood bank sera from Spain and 48 sera from atopic Swedes was 100%. Patients infected with other parasites (n = 78) were usually negative (81%). 11 of the 15 false positives were found in patients with total IgE greater than 5,000 kU/l. The microtiter EIA method employed can be considered as a very convenient method to be included in the range of immunodiagnostic tests for human hydatid disease.

Total and specific IgE levels in human hydatid disease determined by enzyme-immunoassay. I. Preoperative levels.

The levels of total IgE and specific IgE in preoperative sera from 29 adult patients with surgically confirmed hydatidosis were studied by means of enzyme-immunoassay. Sera from 88 healthy donors were likewise studied. Findings showed that in the majority of the hydatidosis cases there was an increase of specific IgE and total IgE although individual variance was significant. Determination of total IgE as the only serological diagnostic test is not valid, since 27.5% of patients showed a total IgE level within normal values. Moreover, the determination of specific IgE has more diagnostic value than that of total IgE although it must be taken into account that in 7% of human hydatidosis cases there are no detectable levels of anti-Echinococcus IgE.