Isolation and properties of mannans from Alternaria kikuchiana Tanaka and Alternaria zinniae.
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Animals↗
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The lipid composition of a species of Alternaria of the "Alternaria alternata group".
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Chromatography, Gas↗
[Isolation of fungi of the Alternaria genus in São Paulo city (Brazil). Intradermal tests with Alternaria species extracts].
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Brazil↗
Comparison of enzyme immunoassay-based assays for environmental Alternaria alternata.
BACKGROUND: Alternaria alternata-derived allergenic materials are causes of human disease. Several immunoassays exist to quantify these materials. OBJECTIVE: To compare methods for evaluating Alternaria content. METHODS: Four methods, including 1 monoclonal antibody (MAb)-based assay specific for recombinant Alt a 1, 1 MAb-based assay for chromatographically purified Alt a 1, 1 polyclonal antibody (PAb)-based assay for chromatographically purified Alt a 1, and 1 PAb-based assay for whole Alternaria extract, were evaluated. Environmental samples collected as part of the National Survey of Lead and Allergens in Housing were examined. Alternaria spore counts were determined in dust by observation. RESULTS: The MAb-based assay for recombinant Alt a 1 detected Alternaria in few samples (25%); the PAb-based assay for whole Alternaria proteins detected antigen in 97% of the samples. The PAb- and MAb-based assays for purified Alt a 1 detected antigen in 100% of the samples. There was a significant positive correlation between the 2 assays directed against purified Alt a 1. There was a positive correlation between the PAb-based assay for whole Alternaria and the PAb-based assay for Alt a 1. Nearly all the dust samples contained Alternaria spores, and there was a strong positive correlation between counts and all assays. CONCLUSION: Because of the multifaceted nature of Alternaria, the disparities between methods for quantifying Alternaria, the cross-reactivity between fungal allergens, and the documented genetic promiscuity of this fungus, enzyme immunoassays using PAbs against a range of Alternaria proteins will probably produce the most reliable estimation of overall Alternaria exposure in house dust.
Air Pollutants↗
Production of a recombinant protein from Alternaria containing the reported N-terminal of the Alt a1 allergen.
Allergen content of extract derived from Alternaria is somewhat variable. Allergenic molecules from Alternaria that appear as differing molecular size bands on IgE probed immunoblots may have a great deal of sequence homology and differ only in the length of the amino acid chain. One method to study this problem is to produce recombinant proteins from Alternaria. To explore these possibilities, the following experiments were performed. A strain of Alternaria was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration. Rapidly expanding mycelia were removed from the culture and mRNA was extracted. Purified mRNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification. Modified DNA was then spliced into lambda gt11 phage and yielded a recombinant library with 10(5) PFU. The library was screened for the presence of allergenic proteins using IgE containing human sera from Alternaria-sensitive patients. Positive plaques were cloned. PCR analysis of positive clones using an oligonucleotide from the reported N-terminal sequence of Alt a1 indicated an insert of 295 base pairs. Sequence analysis yielded a reading frame containing 84 amino acid and confirmed that this segment contained the code for the reported N-terminal amino acid sequence of Alt a1. A computer search for this sequence found no homologous proteins in the Entrez sequences. Northern blotting studies on RNA purified from nine strains of Alternaria with the radiolabeled 247 BP DNA fragment indicated that this sequence was present in all strains. The 247 BP nucleotide was spliced into the Pflag vector and clones containing insert in the proper reading frame were identified. The presence of recombinant protein in the clones was verified by SDSPAGE time studies. Protein produced in time studies was shown by immunoblotting and sandwich EIA to bind human IgE from Alternaria sensitive patients. This recombinant protein, containing amino acid sequence for Alt a1, is bound by human IgE and therefore should be useful as a model for studying allergy to the native Alternaria glycoprotein. Further research should define where this sequence occurs in the Alternaria genome and should determine the sequence of the entire protein.
Allergens↗
Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts.
Alternaria and Stemphylium extracts were compared by means of various immunochemical and biologic methods. Skin-test responses to Alternaria and Stemphylium in allergic patients were positively correlated, and RAST binding values with the sera of 30 patients utilizing solid-phase Alt-I, Alternaria (70% ammonium sulfate--precipitable fraction), and Stemphylium allergens showed significant correlation, giving evidence for the presence of common allergenic determinants among these three extracts. In RAST inhibition assays, Alternaria and Stemphylium extracts exhibited dose-related inhibiton with solid-phase Stemphylium, Alternaria, and Alt-I, confirming the presence of Alt-I as the major shared allergenic fraction in the two extracts. In repetitive absorption experiments, both Alt-I and Alternaria solid-phase allergens absorbed Stemphylium-specific IgE antibodies from a 12-person serum pool. Similarly, Stemphylium solid-phase allergen absorbed both Alternaria- and Alt-I--specific IgE antibodies. Double-antibody radioimmunoassay for Alt-I showed higher Alt-I activity in Stemphylium than in Alternaria extract. In double-immunodiffusion experiments with rabbit anti--Alt-I antibodies, both Stemphylium and Alternaria extracts produced precipitin lines of identity with Alt-I. The antigenic relationship of the two crude extracts was further confirmed by crossed and crossed-line immunoelectrophoresis experiments. Our results showed that Alternaria and Stemphylium extracts contain multiple shared allergenic and antigenic determinants, including Alt-I.
Absorption↗
Dustborne Alternaria alternata antigens in US homes: results from the National Survey of Lead and Allergens in Housing.
BACKGROUND: Alternaria alternata is one of the most common fungi associated with allergic disease. However, Alternaria exposure in indoor environments is not well characterized. OBJECTIVE: The primary goals of this study were to examine the prevalence of Alternaria exposure and identify independent predictors of Alternaria antigen concentrations in US homes. METHODS: Data for this cross-sectional study were obtained from the National Survey of Lead and Allergens in Housing. A nationally representative sample of 831 housing units in 75 different locations throughout the United States completed the survey. Information on housing and household characteristics was obtained by questionnaire and environmental assessments. Concentrations of A alternata antigens in dust collected from various indoor sites were assessed with a polyclonal anti-Alternaria antibody assay. RESULTS: Alternaria antigens were detected in most (95% to 99%) of the dust samples. The geometric mean concentration, reflecting the average Alternaria concentration in homes, was 4.88 microg/g (SEM, 0.13 microg/g). In the multivariable linear regression analysis, the age of the housing unit, geographic region, urbanization, poverty, family race, observed mold and moisture problems, use of de-humidifier, and presence of cats and dogs were independent predictors of Alternaria antigen concentrations. Less frequent cleaning and smoking indoors also contributed to higher Alternaria antigen levels in homes. CONCLUSION: Exposure to A alternata antigens in US homes is common. Antigen levels in homes are influenced not only by regional factors but also by residential characteristics. Preventing mold and moisture problems, avoiding smoking indoors, and regular household cleaning may help reduce exposure to Alternaria antigens indoors.
Air Pollution, Indoor↗
Association of sensitization to Alternaria allergens with asthma among school-age children.
BACKGROUND: Molds in the Alternaria genus, normally found on outdoor vegetation, produce some of the most common fungal allergens to elicit a skin test response. OBJECTIVES: The objectives of this study were to evaluate a serum assay for IgE antibodies to Alternaria allergens and to establish the prevalence of sensitization to Alternaria allergens among children and adults enrolled in epidemiologic studies of asthma. In addition, the significance of sensitization to Alternaria allergens as a risk factor for asthma was compared with that of sensitization to indoor allergens or pollens. METHODS: Using the Pharmacia Capsulated Hydrophobic Carrier Polymer (CAP) system, we have evaluated the significance of Alternaria allergens by using sera from several epidemiologic studies of asthma. RESULTS: Comparisons between serum assays and skin test results suggest that this in vitro assay yields results similar to those for traditional RASTs and is as sensitive as skin prick testing. In each of the groups studied, sensitization to Alternaria allergens was more common among asthmatic than control subjects, and in two studies the relationship was highly significant. Alternaria allergens were significantly associated with asthma in middle schools in Charlottesville, Virginia and Los Alamos, New Mexico but not in Albemarle County, Virginia. Logistic regression analysis of the results for the three schools identified an association between sensitization to Alternaria allergens and asthma independent of, but not as strong as, that found between sensitization to indoor allergens and asthma (p < 0.001). CONCLUSIONS: The Pharmacia CAP system is a useful tool for measuring specific IgE to Alternaria allergens. Although not as important as sensitization to dominant local indoor allergens, sensitization to Alternaria allergens appears to be a significant independent risk factor for asthma in children in some locations of the United States.
Adolescent↗
Natural exposure to Alternaria spores induces allergic rhinitis symptoms in sensitized children.
Allergic rhinitis is associated with exposure to inhaled allergens. Allergic sensitization to the fungus Alternaria is common, especially in warm climates, but the relevance of sensitization to Alternaria in the generation of rhinitis has not been elucidated. The aim of this study was to determine whether natural exposure to Alternaria induces rhinoconjuctivitis symptoms in Alternaria-sensitized children. Symptoms of rhinoconjuctivitis in 132 atopic (measured by skin prick tests) children (aged 7-12 years) in inland New South Wales Australia were measured using parent-completed questionnaires in summer and winter 1998. Airborne concentrations of Alternaria spores and grass pollen were measured throughout the study period using a Burkard volumetric trap. The relation between airborne concentrations of Alternaria spores and symptoms of rhinoconjunctivitis was examined using Wilcoxon's matched pairs signed ranked test and generalized estimating equations. The proportion of children atopic to Alternaria reporting symptoms of rhinitis was significantly higher in the summer, when airborne concentrations of Alternaria were high, than in the winter, when airborne concentrations were low (66.2% vs. 38.2% for nasal symptoms at night, p = 0.0001, 70.6% vs. 51.52% for nasal symptoms during the day, p = 0.02). The proportion of children using anti-allergic treatment was also higher in summer than in winter (anti-allergic medication for the nose 39.7% vs. 17.7%, p = 0.0003). A strong correlation between airborne concentrations of grass pollen and Alternaria was found (R = 0.94, p < 0.001). However, when all children atopic to ryegrass were excluded from the analyses, significant associations between symptoms and season remained. We conclude that exposure to Alternaria spores may be an important cause of allergic rhinoconjunctivitis.
Allergens↗
Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay.
Alternaria sp. are important fungal contaminants of grain products; they secrete four structural classes of compounds that are toxic or carcinogenic to plants and animals and cause considerable economic losses to growers and the food-processing industry. Alternaria toxins have been detected by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, and other techniques. Here, we report the development of a polymerase chain reaction (PCR)-based method for the detection of Alternaria DNA. PCR primers were designed to anneal to the ITS1 and ITS2 regions of the 5.8S rDNA gene of Alternaria alternata or Alternaria solani but not to other microbial or plant DNA. We compared the sensitivity of PCR in detecting Alternaria DNA, that of the HPLC method in detecting Alternaria alternariol and alternariol methyl ether toxins, and that of the morphological examination of mycelia and conidia in experimentally infested corn samples. The sensitivity of toxin detection for HPLC was above the level of contamination in a set of commercially obtained grain samples, resulting in negative scores for all samples, while the PCR-based method and mold growth plating followed by morphological identification of Alternaria gave parallel, positive results for 8 of 10 samples. The PCR assay required just 8 h, enabling the rapid and simultaneous testing of many samples at a low cost. PCR-based evidence for the presence of Alternaria DNA followed by positive assay results for Alternaria toxins would support the rejection of a shipment of grain.
Alternaria↗
Alternaria aerosol during a bovine respiratory syncytial virus infection alters the severity of subsequent re-infection and enhances IgE production.
BACKGROUND: Previous studies with cattle and rodent models have shown that bovine and human RSV infections influence the immune response to inhaled allergen. In the present study, we extended these observations to examine the effect of fungal allergen Alternaria alternata aerosol exposure (prior to and during BRSV infection) on the immune response and clinical outcome of a secondary BRSV infection. METHODS: Calves were either Alternaria (Alt)/mock Alt (mAlt) and BRSV/mBRSV exposed. Exposures began on day -6 and continued every other day until day 6 post infection. A second set of aerosols/infection began on day 103 and continued as before. Clinical outcome during infections was measured in each group. IgG1, IgA, and IgE responses to Alternaria were measured in serum or bronchiolar alveolar lavage fluid (BALF). Cytokine responses, including IL-4, were also measured. RESULTS: Alternaria did not influence primary infection; however, the Alt/BRSV group had less disease than mAlt/BRSV group (median clinical score 8 vs 476.5; p<or=0.01) after secondary infection. Exposure to Alternaria facilitated IgE antibody production in BRSV infected calves. IgE responses to Alternaria were higher in Alt/BRSV than Alt/mBRSV animals on day 10 (mean baseline fold increase 1.97 vs 1.06; p=0.013) and 109 (1.40 vs 0.810; p=0.008). Comparatively, Alt/BRSV calves had less Alternaria specific IgG1 than Alt/mBRSV calves on days 0, 107, 109, 113, 115, and 120 (p<or=0.05) with more lung eosinophils and IL-4 secreting PBMCs. CONCLUSION: Alternaria aerosols during primary and secondary BRSV infections decreased disease in secondary infections; however, BRSV infection enhanced Th2 responses against inhaled Alternaria.
Aerosols↗
IgE and IgG antibody responses to recombinant Alt a 1 as a marker of sensitization to Alternaria in asthma and atopic dermatitis.
BACKGROUND: Sensitization to Alternaria alternata is a risk factor for the development of wheezing and asthma. Alt a 1 is the major Alternaria allergen causing sensitization in asthmatics. Some atopic dermatitis (AD) patients have very high immunoglobulin (Ig)E antibody (ab) to Alternaria as analysed by Pharmacia CAP, however, it is not clear whether these are specific responses or whether Alt a 1 is involved in disease symptoms. OBJECTIVE: The aim of this study was to analyse specific IgE and IgG ab responses to recombinant Alt a 1 in asthmatic and AD patients and to compare these results to IgE ab against Alternaria measured by CAP. METHODS: Sera from individuals who were IgE positive to Alternaria by CAP were obtained from 58 patients with asthma/rhinitis, 19 patients with AD, and 20 patients with cystic fibrosis (CF) who were included as specificity controls. IgE and IgG ab to recombinant Alt a 1 were measured by radioimmunoassay (RIA). RESULTS: Of 43 asthma/rhinitis patients having an Alternaria CAP score > 2, a high percentage (93%) had both IgE and IgG ab to Alt a 1, emphasizing its importance as a major allergen. Only, 47% of AD patients with CAP score greater than 2 had ab to Alt a 1, and their levels were low when compared to the asthmatics. For CF controls, 75% of these patients had no IgE ab to Alt a 1, and those which were positive to Alt a 1 by RIA were also positive by CAP. Overall, patients with a low CAP (1-2) had a low prevalence (20-30%) of IgE or IgG ab to Alt a 1. CONCLUSION: IgE and IgG ab to Alt a 1 in asthmatics are good markers for sensitization to Alternaria. Although AD patients gave high Alternaria CAP scores, they had low or undetectable levels of IgE to Alt a 1, suggesting that other Alternaria allergens may be important in AD or that the CAP results are non-specific. Recombinant allergens may provide more specific measures of sensitization to fungi.
Allergens↗
Immunological studies on Alternaria sensitivity. Use of crossed radioimmunoelectrophoresis, precipitins and enzyme-linked immunosorbent assay.
Characterization of the immunological response to Alternaria in sensitive subjects is not complete. We used crossed radioimmunoelectrophoresis (CRIE) to identify antigens in Alternaria extracts reacting with IgE antibody in five patients with Alternaria-sensitive asthma, four with Alternaria-induced rhinitis, three non-allergic asthmatics, and three normal controls. All five Alternaria-asthma patients and three of four Alternaria-rhinitis patients showed IgE binding to a third antigen. These results suggest an analogy of Alternaria antigens with that found in ragweed pollen extracts, i.e. that IgE antibody is directed against more than one antigen. Using the enzyme-linked immunosorbent assay (ELISA), we found a significant difference (P less than 0.05, unpaired Student's t-test) in IgG binding between Alternaria-sensitive asthmatics and normal controls. There was no apparent difference in IgG binding between untreated Alternaria-sensitive asthmatics and those receiving high-dose immunotherapy.
Alternaria↗
Alternaria spores in the atmosphere of Sydney, Australia, and relationships with meteorological factors.
Alternaria spores are found in the atmosphere in many locations around the world. They are significant from a human health perspective because they have been known to trigger allergic respiratory disease such as asthma and hay-fever. The presence of Alternaria spores in the atmosphere has been related to meteorological factors in past studies, but this has not been done previously in Sydney, Australia. This paper reports the results of such a study in Sydney. Alternaria spore concentration data for the period 19 August 1992 to 31 December 1995 were examined with meteorological data for the same period. The daily Alternaria spore concentration was compared to the meteorological data for the same day and for up to 3 days previously. The analysis methods were Spearman's rank correlation and multiple regression. Alternaria spores appear in the atmosphere of Sydney year-round, although they peak over spring, summer, and autumn. A number of meteorological factors, including mean, minimum, and maximum, temperature, dew point temperature, and air pressure, are significantly correlated with the atmospheric concentration of Alternaria spores. Some of these meteorological variables (temperature and dew point temperature) show significant correlations with a 1, 2, and 3 day lag, as well as for the same day. Regression models indicate that up to 31.1% of the variation in Alternaria spore concentration can be explained by meteorological factors. There is potential for the results of this study to be used by public health authorities in the prediction of Alternaria spore concentrations in Sydney.
Alternaria↗
In vitro synthesis of Alternaria allergens and their recognition by murine monoclonal and human IgE antibodies.
BACKGROUND: Allergic reactions to fungal allergens, including Alternaria, are common clinical problems. Reagents used for diagnosis and therapy of fungal allergy are complex and variable mixtures. Standardized reagents are difficult to achieve due to batch to batch variability in these biologic products. Although several Alternaria allergens have been isolated, their production by current methods is laborious. The introduction of molecular biology into allergy research has led to the molecular cloning of several allergens which may culminate in improved methods of treatment. OBJECTIVE: This report describes the development of techniques that will lead to the molecular cloning of Alternaria allergens. METHODS: We isolated the poly (A)(+)-messenger RNA from Alternaria, produced proteins in vitro and probed them for binding to murine monoclonal antibodies directed against Alternaria. In addition, we examined the ability of the translated proteins to bind IgE from the sera of Alternaria-sensitive individuals. RESULTS: We synthesized at least 20 proteins ranging in molecular weight from 2 to 90 kD using in vitro techniques. The translated proteins were detected by both murine monoclonal and human IgE antibodies. CONCLUSIONS: Alternaria allergens can be synthesized in vitro by molecular biology techniques. Such techniques will be used in the development of cDNA libraries for the production of Alternaria allergens by recombinant DNA methods.
Allergens↗
Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins.
There are at least ten plant diseases caused by Alternaria species in which host-specific toxins (HSTs) are responsible for fungal pathogenicity. Of these HST-producers, seven are considered distinct pathotypes of the species Alternaria alternata, and the remaining three are among other species of pathogenic Alternaria. Inter- and intra-specific variation among Alternaria taxa, including HST-producers, was determined by electrophoretic karyotyping using pulsed-field gel electrophoresis. A. alternata including seven pathotypes of A. alternata and eight non-pathogenic strains had 9-11 chromosomal bands with estimated sizes ranging from 0.4 to 5.7 Mb. In contrast, Alternaria species that are morphologically distinct from A. alternata had 8-10 bands with sizes between 0.9 and 5.7 Mb. Estimated genome sizes of A. alternata and other Alternaria species ranged from 28.8 to 33.6 Mb and 25.1 to 30.7 Mb, respectively. Other species of pathogenic Alternaria were difficult to differentiate from A. alternata on the basis of chromosome-size polymorphisms alone, but Southern analysis using rDNA as a probe could, in some cases, differentiate between them. These results were cytologically confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ hybridization with a rDNA probe for mitotic metaphase chromosomes prepared by the germ-tube burst method.
Alternaria↗
Transgenic Indian mustard (Brassica juncea) expressing tomato glucanase leads to arrested growth of Alternaria brassicae.
Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.
Alternaria↗
Alternaria IgG precipitins and adverse reactions.
Late reactions consisting of fever, malaise, and swelling at the site, 4 to 6 hr after injections of Alternaria extract occurred in several patients receiving immunotherapy with Alternaria. These patients had in common serum IgG precipitins and exquisite leukocyte histamine release sensitivity to Alternaria. Such precipitins were 3 times more frequent in patients receiving Alternaria immunotherapy than a control group of patients receiving immunotherapy with other antigens. A prospective study revealed that 5 of 23 Alternaria-sensitive persons had precipitins before immunotherapy and another 6 developed precipitins during therapy. Only one of the 23 experienced a late Alternaria reaction. Thus, precipitins to Alternaria are common and do not seem to be the basis for the late reactions we observed. The finding of precipitins does not contraindicate immunotherapy.
Alternaria↗