Component-Resolved Diagnosis of American Cockroach (Periplaneta americana) Allergy in Patients From Different Geographical Areas.

Background: Manifestation of respiratory allergy to American cockroach (Periplaneta americana) is prominent in the subtropical and tropical areas. However, co-existing perennial indoor inhalant allergies frequently compromise clinical diagnosis of cockroach allergy, and the analysis of sensitization pattern is limited by the lack of Periplaneta allergens widely available for component-resolved diagnostics (CRD). Objective: To evaluate a collection of previously described recombinant Periplaneta allergens for CRD in cockroach allergy. Methods: A panel of nine recombinant Periplaneta allergens (Per a 1-5, 7-10) was generated, purified, and subjected to physicochemical characterization by applying circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), amino acid (AA) analysis, and mass spectrometry (MS). Patients (n = 117) from India, Korea, Venezuela, and Iran, reporting perennial respiratory indoor allergies with IgE sensitization to cockroach (P. americana and/or Blattella germanica), were included. The sensitization profile was monitored by the experimental ImmunoCAP testing. Results: ImmunoCAP testing confirmed IgE sensitization to Periplaneta and/or Blattella extract in 98 of 117 patients (r = 0.95). Five out of 117 patients were sensitized to only one of the two cockroach species. Within the whole study group, the prevalence of sensitization to individual allergens varied from 4% (Per a 2) to 50% (Per a 9), with the highest IgE values to Per a 9. Patients from four countries displayed different sensitization profiles at which Per a 3 and Per a 9 were identified as major allergens in India and Korea. Periplaneta-derived lipocalin and myosin light chain were characterized as new minor allergens, designated as Per a 4 and Per a 8. Periplaneta extract showed higher diagnostic sensitivity than all individual components combined, suggesting the existence of allergens yet to be discovered. Conclusion: Utilization of a panel of purified Periplaneta allergens revealed highly heterogeneous sensitization patterns and allowed the classification of lipocalin and myosin light chain from Periplaneta as new minor allergens.

Gene profiling and expression of major allergen Alt a 1 in Alternaria alternata and related members of the Pleosporaceae family.

BACKGROUND: Members of the Pleosporaceae family are known as important sources of airborne allergens which are responsible for asthma and allergic diseases. AIMS: The purpose of this study was to investigate the gene profiling and expression pattern of Alt a 1 in Alternaria alternata and other members of the Pleosporaceae family including Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides, and Epicoccum nigrum. METHODS: Alternaria alternata and related genera were cultured on Czapek-Dox broth medium at 25°C for 21 days. The presence of Alt a 1 was assessed in fungal culture filtrates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then confirmed by immunoblot analysis. Real-time PCR was carried out for quantitation of the Alt a 1 gene encoding corresponding protein at the transcriptional level using cDNA prepared from fungal RNA. RESULTS: SDS-PAGE showed protein bands ranging from 14 to 100kDa. A 14kDa band corresponding to Alt a 1 was present in A. alternata, S. botryosum and U. chartarum. The gene expression of Alt a 1 was reported in A. alternata and some other related genera. The Ct mean value recorded for A. alternata strains ranged from 24.70 to 27.84 while it was in the range 23.62-32.09 for other related taxa. No apparent transcription or expression was revealed in C. cladosporioides. CONCLUSIONS: The presence and efficient expression of Alt a 1 gene in A. alternata and other related taxa indicate that Alt a 1 protein is a major component of the secretory machinery of Pleosporaceae family members, and it may play a crucial role in its allergenicity.

Sensitization and Geographical Distribution of Main Aeroallergens in Iran.

N0 Abstract.

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

No Abstract.

Gene profiling and expression of major allergen Alt a 1 in Alternaria alternata and related members of the Pleosporaceae family / Perfil de expresión génica del alérgeno Alt a 1 mayoritario en Alternaria alternata y otros taxones próximos de la familia Pleosporaceae

Background: Members of the Pleosporaceae family are known as important sources of airborne allergens which are responsible for asthma and allergic diseases. Aims: The purpose of this study was to investigate the gene profiling and expression pattern of Alt a 1 in Alternaria alternata and other members of the Pleosporaceae family including Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides, and Epicoccum nigrum. Methods: Alternaria alternata and related genera were cultured on Czapek-Dox broth medium at 25°C for 21 days. The presence of Alt a 1 was assessed in fungal culture filtrates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then confirmed by immunoblot analysis. Real-time PCR was carried out for quantitation of the Alt a 1 gene encoding corresponding protein at the transcriptional level using cDNA prepared from fungal RNA. Results: SDS-PAGE showed protein bands ranging from 14 to 100 kDa. A 14kDa band corresponding to Alt a 1 was present in A. alternata, S. botryosum and U. chartarum. The gene expression of Alt a 1 was reported in A. alternata and some other related genera. The Ct mean value recorded for A. alternata strains ranged from 24.70 to 27.84 while it was in the range 23.62-32.09 for other related taxa. No apparent transcription or expression was revealed in C. cladosporioides. Conclusions: The presence and efficient expression of Alt a 1 gene in A. alternata and other related taxa indicate that Alt a 1 protein is a major component of the secretory machinery of Pleosporaceae family members, and it may play a crucial role in its allergenicity

Antecedentes: Los miembros de la familia Pleosporaceae son una fuente importante de alérgenos aéreos causantes de asma y enfermedades alérgicas. Objetivos El objetivo de este trabajo fue estudiar el perfil de expresión génica de la proteína Alt a 1 en Alternaria alternata y otros miembros de la familia Pleosporaceae, entre las cuales pueden citarse Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides y Epicoccum nigrum. Métodos: Alternaria alternata y otros géneros relacionados se cultivaron en caldo Czapek-Dox a 25°C durante 21 días. La existencia de Alt a 1 en los filtrados de los cultivos se evaluó mediante electroforesis en gel de poliacrilamida con dodecilsulfato sódico (SDS-PAGE) para después confirmarla mediante inmunotransferencia. Se realizó RCP en tiempo real para la cuantificación de la transcripción del gen responsable (Alt a 1) utilizando ADNc a partir del ARN del hongo. Resultados: Mediante SDS-PAGE se visualizaron bandas de proteínas de 14 a 100 kDa. La banda de 14 kDa, correspondiente a Alt a 1, estaba presente en A. alternata, S. botryosum y U. chartarum. Se detectó expresión génica de Alt a 1 en A. alternata y otros géneros relacionados. El valor medio de Ct registrado en los aislamientos de A. alternata varió entre 24,70 y 27,84; en otros taxones cercanos, el intervalo estuvo entre 23,62 y 32,09. No se detectó transcripción o expresión en C. cladosporioides. Conclusiones: La presencia del gen Alt a 1 y su expresión en A. alternata y otros taxones próximos indica que la proteína Alt a 1 es uno de los componentes principales del mecanismo secretorio de los miembros de la familia Pleosporaceae y puede desempeñar un papel fundamental en su capacidad alergénica

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.