Fungal Allergen and Mold Allergy Diagnosis: Role and Relevance of Alternaria alternata Alt a 1 Protein Family.

Alternaria is a genus of worldwide fungi found in different habitats such as soil, the atmosphere, plants or indoor environments. Alternaria species are saprobic-largely involved in the decomposition of organic material-but they can also act as animal pathogens, causing disease in humans and animals, developing infections, toxicosis and allergic diseases. A. alternata is considered one of the most important sources of fungal allergens worldwide and it is associated with severe asthma and respiratory status. Among the A. alternata allergens, Alt a 1 is the main sensitizing allergen and its usefulness in diagnosis and immunotherapy has been demonstrated. Alt a 1 seems to define a protein family that can be used to identify related pathogenic fungi in plants and fruits, and to establish taxonomic relationships between the different fungal divisions.

The Quantification of IgG Specific to α-Gal Could Be Used as a Risk Marker for Suffering Mammalian Meat Allergy.

The alpha-Gal Syndrome is a delayed meat allergy characterized by the presence of sIgE against α-Gal epitope. It is known that the α-Gal present in tick saliva induces the sensitization to this epitope ending in the production of sIgG and sIgE to α-Gal. It could be considered that the more times a person is bitten by tick species, the higher the probability of making the switch from sIgG to sIgE to α-Gal and developing allergy, but it is no clear when the switch occurs. To determine the likelihood that a subject bitten by ticks but without AGS be at risk of developing this allergy, we quantified the levels of sIgG to α-Gal by an automated system (ImmunoCap). To stablish a cut-off value for sIgG to α-Gal, a receiving operating curve (ROC) was constructed. The statistical analysis demonstrated that the risk of suffering AGS in individuals bitten by ticks was 35% when the sIgG to α-Gal was greater than or equal to 40 µg/mL. Our data indicate that the sIgG values against α-Gal could be used as a prognostic marker for developing mammalian meat allergy.

Purified Native and Recombinant Major Alternaria alternata Allergen (Alt a 1) Induces Allergic Asthma in the Murine Model.

Aeroallergens such us the spores of Alternaria alternata are described as the most important agents associated with respiratory allergies and severe asthma. Various experimental models of asthma have been developed using A. alternata extracts to study the pathogenesis of asthma, establishing the main parameters that trigger the asthmatic response. In this study, we describe a mouse model of asthma induced only by Alt a 1. To induce the allergic response, mice were challenged intranasally with the major allergen of A. alternata, Alt a 1. The presence of eosinophils in the lungs, elevated concentrations of Th2 family cytokines, lymphocyte proliferation and elevated IgE total serum levels indicated that the sensitisation and challenge with Alt a 1 induced the development of airway inflammation. Histological studies showed an eosinophilic cellular infiltrate in the lung tissue of mice instilled with Alt a 1. We demonstrate that Alt a 1 alone is capable of inducing a lung inflammatory response with an increase in IgE serum levels mimicking the allergic asthma immunoresponse when it is administered into BALB/c mice. This model will allow the evaluation of the immunoregulatory or immunotolerant capacity of several molecules that can be used in targeted immunotherapy for fungal allergic asthma.

The Aspergillus niger Major Allergen (Asp n 3) DNA-Specific Sequence Is a Reliable Marker to Identify Early Fungal Contamination and Postharvest Damage in Mangifera indica Fruit.

The aim of this work was to study the value of the main allergen Asp n 3 of Aspergillus niger as a molecular marker of allergenicity and pathogenicity with the potential to be used in the identification of A. niger as a contaminant and cause of spoilage of Mangifera indica. Real-time polymerase chain reaction (RT-PCR) was used for the amplification of Asp n 3 gene. Two pairs of primers were designed: one for the amplification of the entire sequence and another one for the amplification of the most conserved region of this peroxisomal protein. The presence of A. niger was demonstrated by the early detection of the allergenic protein Asp n 3 coding gene, which could be considered a species-specific marker. The use of primers designed based on the conserved region of the Asp n 3 encoding gene allowed us to identify the presence of the closely related fungal species Aspergillus fumigatus by detecting Asp n 3 homologous protein, which can be cross-reactive. The use of conserved segments of the Asp n 3 gene or its entire sequence allows us to detect phylogenetically closely related species within the Aspergilaceae family or to identify species-specific contaminating fungi.

Urticaria and silent parasitism by Ascaridoidea: Component-resolved diagnosis reinforces the significance of this association.

Urticaria remains a major problem in terms of aetiology, investigation, and management, and although parasitic diseases are considered potential causes, the absence of a consistent link between parasitic infections and skin allergy symptoms leads to the need for a deeper study of parameters that support this association. The objectives of this study were to analyse a possible relationship between parasitism by Ascarididae (Toxocara canis and Anisakis simplex) and the clinical expression of urticaria and to identify possible parasitic molecular markers for improving the diagnosis of unknown urticaria aetiology. The prevalence of Toxocara and Anisakis infestations was evaluated by measuring the levels of specific IgG (sIgG) and IgE (sIgE) antibodies against crude extracts and isolated components from whole larvae of Anisakis simplex (Ani s 1, Ani s 3 and Ani s 7) and Toxocara canis (TES-120, TES-70, TES-32 and TES-26) using immunologic and molecular diagnostic methods. A cross-sectional study was performed in a group of 400 individuals. The study group consisted of 95 patients diagnosed with urticaria (55 with chronic urticaria and 40 with acute urticaria). A control group consisted of 305 subjects without urticaria (182 diagnosed with respiratory allergy and 123 without allergy). Statistically significant differences were demonstrated in the seroprevalence of specific IgG and IgE antibodies between the urticaria patients and the healthy general population when isolated ascarid antigens were evaluated. The prevalence of IgG antibodies against Ani s 1, IgE antibodies against TES-120 and IgE antibodies against TES-70 were significantly different between the control individuals (healthy general population) and patients with urticaria. Moreover, the urticaria patient group demonstrated a higher seroprevalence of antibodies (sIgE and sIgG) against Anisakis simplex larva whole extract than the control group but just with statistically diferences when sIgE was evaluated. The presence of IgE and/or IgG antibodies against Ani s 3 (tropomyosin) can help to discriminate between patients with and without urticaria. Both ascarids seem to be associated with urticaria, although in our region, Anisakis seems to have greater involvement than Toxocara in this relationship. Molecular diagnostics can be used to associate urticaria with parasite infestations. Tropomyosin and Ani s 1 were the most relevant markers to demonstrate the association between urticaria and the most relevant Ascarididae parasites in our region.

Sensitization and Geographical Distribution of Main Aeroallergens in Iran.

N0 Abstract.

Cocaine Allergy in Drug-Dependent Patients and Allergic People.

BACKGROUND: Adverse reactions to local anesthetics (LAs), especially esters, are not uncommon, but true allergy is rarely diagnosed. To our knowledge, currently there is no reliable method of determining IgE-mediated hypersensitivity to LAs and cocaine. OBJECTIVE: To assess the clinical value of allergy tests (prick, IgE, challenges, and arrays) in people suffering hypersensitivity reactions (asthma and anaphylaxis) during local anesthesia with cocaine derivatives and drug abusers with allergic symptoms after cocaine inhalation. METHODS: We selected cocaine-dependent patients and allergic patients who suffered severe reactions during local anesthesia from a database of 23,873 patients. The diagnostic yield (sensitivity, specificity, and predictive value) of allergy tests using cocaine and coca leaf extracts in determining cocaine allergy was assessed, taking a positive challenge as the criterion standard. RESULTS: After prick tests, specific IgE, and challenge with cocaine extract, 41 of 211 patients (19.4%) were diagnosed as sensitized to cocaine. Prick tests and IgE to coca leaves (coca tea) had a good sensitivity (95.1% and 92.7%, respectively) and specificity (92.3 and 98.8%, respectively) for the diagnosis of cocaine allergy and LA-derived allergy. CONCLUSIONS: Cocaine may be an important allergen. Drug abusers and patients sensitized to local anesthesia and tobacco are at risk. Both prick tests and specific IgE against coca leaf extract detected sensitization to cocaine. The highest levels were related to severe clinical profiles.

Learning from the Molecular Profile of Blattella Germanica Allergens Identified from Allergic Patients in Tehran, Iran.

No Abstract.

Alternaria alternata allergens: Markers of exposure, phylogeny and risk of fungi-induced respiratory allergy.

Alternaria alternata spores are considered a well-known biological contaminant and a very common potent aeroallergen source that is found in environmental samples. The most intense exposure to A. alternata allergens is likely to occur outdoors; however, Alternaria and other allergenic fungi can colonize in indoor environments and thereby increase the fungal aeroallergen exposure levels. A consequence of human exposure to fungal aeroallergens, sensitization to A. alternata, has been unequivocally associated with increased asthma severity. Among allergenic proteins described in this fungal specie, the major allergen, Alt a 1, has been reported as the main elicitor of airborne allergies in patients affected by a mold allergy and considered a marker of primary sensitization to A. alternata. Moreover, A. alternata sensitization seems to be a triggering factor in the development of poly-sensitization, most likely because of the capability of A. alternata to produce, in addition to Alt a 1, a broad and complex array of cross-reactive allergens that present homologs in several other allergenic sources. The study and understanding of A. alternata allergen information may be the key to explaining why sensitization to A. alternata is a risk factor for asthma and also why the severity of asthma is associated to this mold. Compared to other common environmental allergenic sources, such as pollens and dust mites, fungi are reported to be neglected and underestimated. The rise of the A. alternata allergy has enabled more research into the role of this fungal specie and its allergenic components in the induction of IgE-mediated respiratory diseases. Indeed, recent research on the identification and characterization of A. alternata allergens has allowed for the consideration of new perspectives in the categorization of allergenic molds, assessment of exposure and diagnosis of fungi-induced allergies.

From respiratory sensitization to food allergy: Anaphylactic reaction after ingestion of mushrooms (Agaricus bisporus).

We report a case of a 38-year-old mold-allergic patient who developed episodes of generalized urticaria and systemic anaphylactic shock immediately after ingesting button mushrooms. A manganese-dependent superoxide dismutase (MnSOD) and a NADP-dependent mannitol dehydrogenase (MtDH) from Agaricus bisporus mushroom were identified as patient-specific IgE-binding proteins. Cross-reactivity between A. bisporus MnSOD and mold aeroallergens was confirmed. We conclude that prior sensitization to mold aeroallergens might explain severe food reactions to cross-reacting homologs mushroom proteins.

Characterisation of Alternaria alternata manganese-dependent superoxide dismutase, a cross-reactive allergen homologue to Asp f 6.

It is well known that Alternaria alternata presents a significant level of allergenic cross-reactivity with several other phylogenetically related and non-related allergenic moulds. To improve the molecular diagnosis, the identification and characterisation of all clinically relevant allergens, including both species-specific and cross-reacting proteins, is required. In this study we report the molecular and immunological characterisation of the A. alternata manganese-dependent superoxide dismutase (Alt a MnSOD) and its cross-reactivity with Asp f 6, a diagnostic marker allergen in allergic bronchopulmonary aspergillosis (ABPA). The cDNA coding for Alt a MnSOD sequence was isolated by RACE and PCR. Alt a MnSOD is a protein of 191 amino acids that presented significant homology and potential cross-reactive epitopes with Asp f 6. The recombinant protein was produced in Escherichia coli and the immunoreactivity was evaluated in patient sera. Immunoblotting analyses showed that seven of sixty-one A. alternata-sensitised patient sera and two ABPA patient sera reacted with the recombinant Alt a MnSOD. The native counterpart contained in both A. alternata and Aspergillus fumigatus extracts inhibited IgE binding to the recombinant molecule. The allergen was named Alt a 14 by the official Allergen nomenclature subcommittee. Thus, Alt a 14 is a relevant allergen in A. alternata sensitisation that may be used to improve diagnostic procedures. Evidence of cross-reactivity between Asp f 6 and Alt a 14-recognition by ABPA patient sera suggest the existence of an Alt a 14-mediated mechanism that, similar to Asp f 6, may be related to the pathogenesis of ABPA.

Identification of the main allergen sensitizers in an Iran asthmatic population by molecular diagnosis.

BACKGROUND: There has been a significant growth in the prevalence of allergy, mainly associated to IgE-mediated disorders such as asthma and rhinitis. The identification of atopy in asthmatic patients through the measurement of specific IgE can help to identify risk factors that cause asthmatic symptoms in patients. The development and use of individualized allergen-based tests by the Component Resolved Diagnosis has been a crucial advance in the accurate diagnosis and control of allergic patients. The objective of this work was to assess the usefulness of molecular diagnosis to identify environmental allergens as possible factors influencing the development and manifestation of asthma in a group of asthmatic patients from Iran. STUDIED POPULATION: 202 adult asthmatic patients treated at the Loghman Hakim Hospital and Pasteur Institute of Teheran (Iran) from 2011 to 2012. Specific IgE determined by the ImmunoCAP system were used to both evaluate the patients' atopic condition and the molecules involved in the allergic sensitization. SDS-PAGE IgE-immunoblotting associated with mass spectrometry was carried out to study the cockroach IgE-binding sensitizing proteins. RESULTS: Forty-five percent of all patients could be considered atopic individuals. Eighty-two percent of atopic patients were sensitized to pollen allergens. The Salsola kali (Sal k 1) and the Phleum pratense (rPhl p 1 and/or rPhl p 5) major allergens were the most common sensitizers among pollens (71% and 18%, respectively). Thirty-five percent of the atopic population was sensitized to cockroach. Four different allergens, including a previously unknown alpha-amylase, were identified in the cockroach extract. No significant associations could be demonstrated between the severity of asthma and the specific IgE levels in the atopic population. Statistical analysis identified the Sal k 1 as the main protein allergen influencing the development and expression of asthma in the studied population. CONCLUSIONS: Pollen and cockroach were the most relevant allergen sources in the asthmatic population. The Salsola kali major allergen was the main cause for sensitization in the atopic patients suffering asthma. Using the Component Resolved Diagnosis, it was possible to identify a new Blattella germanica cockroach allergen (Blattella alpha amylase 53 kDa) that could sensitize a relevant percentage of this population.

IgE-mediated allergic reactions after the first administration of glatiramer acetate in patients with multiple sclerosis.

BACKGROUND: Immediate adverse reactions to glatiramer acetate (GA), a drug used in the treatment of patients with multiple sclerosis (MS), have been poorly investigated. We studied 3 MS patients who presented adverse reactions following GA administration. Two of them experienced severe anaphylactic reactions after the first administration and the other an eyelid edema upon reintroduction 6 months after GA withdrawal. METHODS: Skin prick tests (SPT) to GA and mannitol were performed on all 3 patients and in 10 atopic controls. Specific IgE (sIgE) levels to GA, myelin basic protein (MBP) and MBP fragments were assessed in all 3 patients, 6 MS patients treated with GA for more than 6 month and 10 healthy donors. Specific IgG (sIgG) to GA was also quantified in the three study groups. Both sIgE and sIgG were determined by means of the UniCAP 100 assay. RESULTS: SPT and sIgE to GA were positive only in the 3 patients with adverse reactions while sIgE to mannitol was negative in all. sIgE tests against MBP and its fragments were negative in all individuals. Similar levels of sIgG to GA were found in all studied subjects. CONCLUSION: These results demonstrate the significance of sIgE in allergic reactions to GA presented by these patients and suggest the importance of strict surveillance during administration of the first GA doses.

Profilin cross-reactive panallergen causes latex sensitization in the pediatric population allergic to pollen.

BACKGROUND: Component-resolved diagnostics (CRD) has been demonstrated to be an excellent new tool for improving the current diagnosis of allergies, and it allows differentiation between polysensitization and cross-reactivity. OBJECTIVE: To demonstrate the role of cross-reactive pollen allergens in pediatric patients living in areas with large amounts of airborne grass pollen grains who are sensitive to grass pollen and latex. METHODS: Serum samples were obtained from 106 children between 3 and 14 years of age diagnosed with allergies to pollen based on clinical history, skin prick tests, and specific immunoglobulin E (IgE). None of them had allergy symptoms to latex or fruits. From these 106 children, 56 patients revealed positive results to Phleum-specific major allergens but not to cross-reactive allergens. The other 50 patients who showed positive specific IgE to Phleum-specific major allergens and to cross-reactive pollen allergens also showed positive results to latex allergens. CRD was carried out by specific IgE quantification using a fluoro-enzyme immunoassay (ImmunoCAPT System). RESULTS: Results demonstrated a positive significant relationship between the specific IgE to Hev b 8 and Phl p 12 and also between the specific IgE to Hev b 8 and latex extract in the group of patients sensitized to species-specific and cross-reactive Phleum allergens. Positive significant relationships were also found between profilin and avocado or peach sensitizations. No other latex allergens gave positive results. CONCLUSION: The apparent sensitization to latex in pediatric patients allergic to grass pollen is caused by the cross-reactive profilin panallergen; however, it is appears not to be clinically relevant.

Identification of allergens homologous to Alt a 1 from Stemphylium botryosum and Ulocladium botrytis.

Several studies have demonstrated that proteins homologous to the Alt a 1 major allergen of Alternaria alternata are expressed in other members of the Pleosporaceae family. However, since no direct biochemical data have been reported concerning the presence of Alt a 1 allergen homologues capable of binding IgE in the excretion-secretion products of Stemphylium and Ulocladium, our objective was to explore their presence in Stemphylium botryosum and Ulocladium botrytis. S. botryosum and U. botrytis culture filtrate extracts were analyzed by two-dimensional (2D)-electrophoresis and 2D-immunoblotting using polyclonal rabbit antibodies raised against recombinant Alt a 1, as well as five human sera from patients allergic to Alternaria. Cross-reactivity immunoassays were performed by ImmunoCAP inhibition and 2D-immunoblotting inhibition. IgE-binding proteins recognized by the rabbit antiserum raised against Alt a 1, with apparent molecular weights of 17-18 kDa and isoelectric points of 4, were identified as Alt a 1-like proteins. Alt a 1 inhibited IgE-specific binding to the Alt a 1 homologues from S. botryosum and U. botrytis. In conclusion, it was demonstrated that allergens which are homologous to Alt a 1 are expressed in the excretory-secretory materials of the phylogenetically-related species S. botryosum and U. botrytis.

A new application of Streptavidin ImmunoCAP for measuring IgG antibodies against non-available commercial antigens.

BACKGROUND: Enzyme immunoassay has been successfully used for the diagnosis of hypersensitivity pneumonitis (HP), but there are difficulties in standardising this method because of the difficulty in obtaining stable solid phases from a huge variety of heterogeneous antigenic sources. With this in mind, the aim of this work was to demonstrate the usefulness of the standard available fluoro-enzyme-immuno-assay (FEIA) to measure specific IgG to uncommon, commercially unavailable HP-causing antigens. METHODS: Serum samples from patients suffering from HP caused by canary and pigeon protein exposure with positive immunoprecipitation values, as well as cystic fibrosis patients, patients with respiratory allergies, asymptomatic subjects exposed to canary proteins (canary breeders) and healthy unexposed individuals were evaluated by FEIA using biotinylated serum proteins coupled to streptavidin-activated solid phases. Statistical analyses were performed to compare the different groups and to evaluate the performance of this technique. RESULTS: The group of patients with hypersensitivity pneumonitis caused by exposure to canary proteins showed the highest value of specific IgG (35.70 mg IgG/mL). ROC analysis demonstrated an optimal cut-off value of 8.44 mg IgG/mL with a sensitivity of 100% and a specificity of 91.76%. Comparisons with other groups revealed statistically significant differences. Comparison between ELISA and FEIA results revealed a strong positive relationship between values obtained in both assays. CONCLUSION: The results demonstrate that Streptavidin ImmunoCAP is a sensitive, specific and efficient laboratory method for routine diagnostic testing of HP caused by protein antigens that are not commercially available.

Bovine serum albumin contained in culture medium used in artificial insemination is an important anaphylaxis risk factor.

OBJECTIVE: To analyze the cause of the anaphylactic reaction after a standard artificial insemination process in a patient diagnosed with asthma. DESIGN: Case report. SETTING: Residencia Sanitaria Virgen de la Arrixaca (Murcia, Spain) and University of the Basque Country (Vitoria, Spain). PATIENT(S): A 30-year-old woman with a previous medical history compatible with respiratory allergy who suffered an anaphylactic reaction after an artificial insemination with spermatozoids in capable medium (Upgraded B2 INRA medium; Laboratories CCD, Paris, France). INTERVENTION(S): Cutaneous tests and specific IgE levels to inhalant allergens, grass and Olea pollens, and insemination medium were performed. MAIN OUTCOME MEASURE(S): Specific IgE levels to mammal epithelia and bovine serum albumin (BSA). RESULT(S): Skin prick tests were positive for inhalant allergens such as mites, cat, dog, horse, and rabbit epithelia, grasses and Olea pollens, and the insemination medium. The beta-lactamic tests were negative. The determination of specific IgE demonstrated positive values to mammal epithelia and mammal serum albumins including BSA. CONCLUSION(S): We report a case of an anaphylactic reaction to the BSA included in the insemination culture medium induced by a subclinical sensitivity to serum albumins of mammal epithelia. A previous testing with the medium is recommended and specific testing might be needed in women who have a history of animal epithelium allergies.