Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Allergenicity assessment of Delonix regia pollen grain and identification of allergens by immunoproteomic approach.

BACKGROUND: Plantation of road-side avenue trees has become a major part of urbanization programme for megacity beautification and environmental management. Due to evergreen habit and vibrant flower colour, Delonix regia (Gulmohor/Flamboyant) is frequently selected as ornamental tree for plantation programme. However, D. regia pollen is related to IgE mediated allergic reactions and no allergen has been reported so far. OBJECTIVE: Measuring the prevalence of D. regia pollen sensitivity among the local atopic individuals and identifying the allergens using immunoproteomic tools. METHODS: Aerobiological study was conducted for a period of two years to record the D. regia pollen concentration in the outdoor ambient air. Clinico-immunological tests were performed on atopic individuals to check the prevalence of sensitivity against D. regia pollen. Allergens were detected in the pollen proteome, fractionated in 1D and 2D gel by IgE serology and finally identified by mass spectrometry. RESULT: In the study area D. regia pollen grains were present in ample amount in the air during May to July. About 38% of atopic individuals displayed positive Skin Prick Test (SPT) against D. regia pollen along with elevated level of specific IgE and histamine in the sera. Immunoproteomic analyses revealed the presence of 14 IgE reactive proteins in the 2D pollen proteome, of which 8 IgE reactive proteins were identified by MALDI TOF/TOF using homology driven proteomic approach. CONCLUSION: This study demonstrated pollen related allergy symptoms by D. regia and gave significant message regarding the plantation programme to avoid the unnecessary load of allergic pollen. Also, a panel of 8 allergens were identified for the first time from D. regia pollen. Detailed study of these allergens would help to design immunotherapeutic strategies for pollinosis management.

Identifying novel allergens from a common indoor mould Aspergillus ochraceus.

The increasing burden of respiratory disease is a rising concern in India. Although chronic colonisation is primarily caused by pathogenic fungi, the common environmental fungi also play an important role in developing sensitisation. This study aims to examine the allergenic potency of mycelial proteins of a common indoor fungus Aspergillus ochraceus to a selected atopic patient cohort as well as to identify the novel IgE-binding proteins through an immunoproteomic approach. 1-D and 2-D IgE specific western blot detected the IgE reactive proteins which were identified through MALDI-TOF/TOF and manual de novo peptide sequencing. The results revealed the detection of 10 cross-reactive IgE-binding proteins. Cluster analysis of 1-D immunoblot with individual patient sera identified NADP(+)-dependent glycerol dehydrogenase (GldB) homologous protein as a major allergen, which was further purified and the allergenicity was assessed. Other IgE-binding proteins showed homology with allergens like short-chain dehydrogenase, NAD-dependent mannitol dehydrogenase, and subtilisin-like serine protease. GldB purified under native conditions showed IgE reactivity amongst the selected patient cohort, which is reported for the first time in this study. The identified IgE-binding proteins can act as candidate molecules for developing hypoallergenic vaccines for designing specific immunotherapeutic techniques to fungal allergy. THE SIGNIFICANCE OF THE STUDY: Exposure to environmental fungal allergens is directly associated with promoting allergic response as well as complicating existing respiratory disease, leading to poor respiratory health. Amongst others, Aspergillus spp. contributes to the majority of the fungal derived atopic diseases. Aspergillus ochraceus is a common indoor mould in India, however, its allergenic potency was not explored till date. In this study, we establish A. ochraceus responsible to cause an allergic response to susceptible individuals and identified 10 IgE-binding proteins using an immunoproteomics approach for the first time. A. ochraceus being unsequenced, a homology-driven proteomics approach was used to identify the IgE-binding proteins which can be extended to identify proteins from other unsequenced species. The information on the IgE-binding proteins could be used as a step towards characterising them by molecular and structural methods to investigate the molecular basis of allergenicity. This will also help to enrich the existing database of allergenic proteins and pave a way towards developing therapeutic avenues.

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Allergenicity assessment of fungal species using immunoclinical and proteomic techniques: a study on Fusarium lateritium.

Airborne fungal spores are extensively reported as the elicitors of respiratory allergies in human. Fusarium lateritium is one such fungal species reported for eliciting significant skin prick results from India. The present study aims to analyze the allergenic potential of F. lateritium followed by the identification of allergens. The total protein of F. lateritium was subjected to 1dimensional (1D) and 2D gel electrophoresis followed by corresponding IgE-specific immunoblots. We found 8 immunoreactive bands/zones in (1D) immunoblot using 11 F. lateritium-sensitised patient sera. In 1D immunoblot, a 34 kDa band was detected in >80% of the patients and hence considered as a potential allergen of F. lateritium. Corresponding 34 kDa spot in 2D-immunoblot was analyzed by mass spectrometric analysis and identified as Glyceraldehyde 3-phosphate dehydrogenase. The identified F. lateritium allergen holds the potential to instigate vaccine development for immunotherapy of F. lateritium sensitized patients.

Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma.

BACKGROUND: The pathobiology of atopic asthma is complex and the symptoms similar to other respiratory diseases. As such, identification of biomarkers of atopic asthma is of prime importance for better diagnosis and control of the disease. OBJECTIVES: We sought to study the changes in plasma proteome and cytokine-expression profile across healthy and atopic asthmatics for identifying biomarkers and exploring aberrant pathways for atopic asthma. METHODS: A pilot-scale study in humans was performed to identify differentially expressed proteins in blood plasma of healthy controls (n=5) and treatment-naïve atopic asthma patients (n=5) using quantitative label-free liquid chromatography-tandem mass spectrometry proteomics and ELISA. RESULTS: Mass spectrometry-based proteomic analysis revealed ApoE to be significantly downregulated in atopic asthmatics compared to healthy volunteers. Decreased expression of ApoE in atopic asthmatics was validated by immunoblotting (50.74% decrease). Comparison with atopic asthmatics and COPD patients showed that ApoE was decreased (36.33%) in atopic asthma compared to COPD. IL33 was significantly upregulated in atopic asthmatics compared to healthy subjects (3.84-fold). CONCLUSION: ApoE was downregulated and IL33 upregulated in atopic asthma patients compared to healthy volunteers. These two proteins' profiles were distinct in atopic asthma from healthy and COPD plasma samples. Differential expression of these proteins could serve as a probable candidate for a two-protein classifier-based prognostic biomarker of atopic asthma.

Spectrum of Allergens and Allergen Biology in India.

The growing prevalence of allergy and asthma in India has become a major health concern with symptoms ranging from mild rhinitis to severe asthma and even life-threatening anaphylaxis. The "allergen repertoire" of this subcontinent is highly diverse due to the varied climate, flora, and food habits. The proper identification, purification, and molecular characterization of allergy-eliciting molecules are essential in order to facilitate an accurate diagnosis and to design immunotherapeutic vaccines. Although several reports on prevalent allergens are available, most of these studies were based on preliminary detection and identification of the allergens. Only a few of these allergen molecules have been characterized by recombinant technology and structural biology. The present review first describes the composition, distribution pattern, and natural sources of the predominant allergens in India along with the prevalence of sensitization to these allergens across the country. We go on to present a comprehensive report on the biochemical, immunological, and molecular information on the allergens reported so far from India. The review also covers the studies on allergy- related biosafety assessment of transgenic plants. Finally, we discuss the allergen-specific immunotherapy trials performed in India.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing protective antibody response for immunotherapeutic applications.

Clinical and immuno-proteomic approach on Lantana camara pollen allergy-a major health hazard.

BACKGROUND: The incidence of allergic diseases is increasing gradually and is a global burden affecting the socio-economic quality of life. Identification of allergens is the first step towards paving the way for therapeutic interventions against atopic diseases. Our previous investigation figured out that total pollen load correlated significantly with the rise of respiratory allergy in a subtropical city in India. The most dominant pollen responsible for IgE sensitivity in most patients emerged to be from Lantana camara (LC) an obnoxious weed growing in and around suburban areas of West Bengal. In this study, we identified allergenic components from this shrub using an immunoproteomic approach. METHODS: Determination of dominant pollen species was done using aerobiological sampling during two consecutive years and correlated with hospitalization and skin prick test. Serum was collected from LC positive patients and checked for in vitro allergenicity using ELISA and Histamine assay. Total proteome was profiled in SDS-PAGE, 2D PAGE and immunoblotted to detect IgE binding proteins which were further identified using mass spectrometry. RESULTS: Lantana camara pollen emerged as a significant contributor from the correlation study with hospital admission of the respiratory allergy sufferers and its extract demonstrated an elevated IgE response in ELISA and histamine release assay tests. Five IgE reactive bands/zones were observed in 1D blot which resolved to 12 allergo-reactive spots in the 2D blot. Mass spectrometric analysis identified nine spots that grouped into four diverse proteins. Pathogenesis-related Thaumatin-like protein was found to be one of the major allergens in Lantana camara. CONCLUSIONS: This is to our knowledge the first attempt to identify allergens from Lantana camara using a proteomic approach. The allergens identified thereof can be used to prepare hypoallergenic vaccine candidates and design immunotherapy trials against LC pollen and other aeroallergen carriers which are cross-reactive and harbor similar proteins.

Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae.

BACKGROUND: Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation 'Rhi o 1'. METHOD: The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. RESULTS: The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens. CONCLUSION/SIGNIFICANCE: The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients.

BACKGROUND: Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. METHODOLOGY: Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry. RESULTS: Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease. CONCLUSION: Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.

A hospital-based survey on food allergy in the population of Kolkata, India.

BACKGROUND: Food allergy is increasing worldwide, and Asian countries are not the exception. Still, ample data are lacking in India. We conducted a cross-sectional study in a metropolis of Eastern India to record the presence of food allergy among the local population. METHODS: The prevalence of food allergy was investigated among patients reporting to The Institute of Child Health and Mediland Diagnostics in Kolkata, India. A total of 5,161 patients were subdivided into 3 age groups and surveyed accordingly. The evaluation was conducted via a questionnaire and a skin prick test. RESULTS: Among the 5,161 patients tested, 4,160 showed a positive response to one or more food items. Banana (32%), brinjal (29%), wheat (22%), and egg (23%) were found to be dominant allergens. Sixty-three percent of patients with a family history of allergy showed either a sudden or an insidious mode of onset, whereas the remaining 37% suffered insidious allergic symptoms with no record of a family history of allergy. Skin rashes, cough, and sneezing were the major symptoms observed. Patients in the age group of 15-40 years were the most susceptible. CONCLUSION: It has been observed that certain specific foods consumed in specific regions cause allergies that are unique to their respective populations. In the present study, the most commonly consumed foods in the studied area, e.g. banana, brinjal, wheat, and egg, had severe effects on the local population. Complementary studies in other countries as well as in other parts of India will allow us to gain further insight into this fact. Some other influencing factors were found to be genetics, cultural habits, and occupation. Avoidance of the allergy-causing food is the best way to deal with food allergy.

Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1.

Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼ 91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact ß-barrel made up of seven anti-parallel ß-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources.

Identification of aero-allergens from Rhizopus oryzae: an immunoproteomic approach.

Airborne fungal spores bearing allergens are the causative agent for inducing immediate hypersensitive reaction in sensitive individuals. In this study the potential aeroallergens have been reported for the first time from Rhizopus oryzae a common airborne mold. Clinical data based on SPT was further confirmed by ELISA. IgE reactive bands were revealed by one-dimensional immunoblotting. A 44 kDa major reactive band was found in all immunoblots. For precise identification of allergens, an immuno-proteomic approach was taken with a combination of 2-Dimensional gel electrophoresis and Mass-spectrometry. 2D map of spore-mycelial protein was confronted with pooled sera and several IgE reactive spots were detected, most of which were glycoproteins and except for one, which has no antigenic determinacy after metaperiodate modification. Each of those spots was identified by MALDI-TOF-TOF. Some bioinformatic approaches were taken to predict the signal peptide and subcellular localization of each protein. Major 44 kDa allergen was identified as Aspartyl endopeptidase. Sequence information was extracted from MS/MS spectra of two tryptic peptides generated from the 44 kDa endopeptidase. Multiple alignments with other reported aspartyl protease allergens showed significant homology. Allergenicity assessment of this protein was performed in silico and identified as a potential putative allergen.

Outdoor airborne fungal spora load in a suburb of Kolkata, India: its variation, meteorological determinants and health impact.

Our objective was to conduct an aeromycological and health survey (2002-2007) in a suburban area near Kolkata, India, with the aim of achieving the following goals: (i) to prepare a fungal spore calendar, (ii) to determine the influence of different meteorological parameters, and (iii) to study the respiratory health status of local population in relation to allergy. Airborne fungal spores from more than 50 taxa were found, of which at least 15 were allergenic. The spore-concentration increased during early-winter and rainy season, and diminished during late-winter and mid-summer. Species-specific fluctuations had substantial influences from several meteorological parameters. The suburban area was found to be considerably contaminated with numerous allergenic air-spora, which caused health risk to the local population. Males were more susceptible to respiratory disorders irrespective of their age. In general, respiratory allergic patients in the 20-40 year age-group showed more frequent health problems due to aeroallergens. A positive correlation was found between the respiratory allergy cases and the air-spora concentrations.

Monitoring and assessment of airborne fungi in Kolkata, India, by viable and non-viable air sampling methods.

The composition and variability of airborne fungal spores were studied using two complementary sampling methods in an outdoor environment in Kolkata suburb for 2 years, from November 2002 to October 2004. For monitoring the total fungal spore burden in the air, Burkard 7-day volumetric sampler was used, whereas Andersen two-sage viable sampler was used for isolating the cultivable airborne fungi. Among the 37 fungal spore types identified in the air samples, the predominant ones were Cladosporium, unidentified ascospores, unidentified basidiospores, Aspergilli/Penicilli, Nigrospora, Periconia, Chaetomium, Drechslera, Alternaria, Coprinus, Ganoderma, Pithomyces, and rust spores. Only six fungal spore types (Alternaria, Aspergilli/Penicilli, Cladosporium, Curvularia, Drechslera, and Nigrospora) were recovered in common by the two samplers. For Aspergilli/Penicilli, Drechslera, and Nigrospora, the spore concentration was underestimated in the non-viable sampling method (Burkard sampler). In general, higher spore count was recorded in winter. The highest fungal species variability was observed in early monsoon (June). Relative humidity could significantly predict the seasonal periodicity of the maximum number of airborne spores. The total airborne fungi concentration recorded in the study (15-16 × 10(3) spores m(-3) of air) was lower than the proposed threshold limit value for clinical significance, suggesting apparently no or less airborne-fungi-exposure-related health risk in the sampling area. Cladosporium cladosporioides was recorded beyond the proposed threshold limit value in January 2003 and March 2004; Aspergillus fumigatus and Aspergillus nidulans in winter that might have posed considerable health risk to sensitized individuals.

Clinical and immunobiochemical characterization of airborne Peltophorum pterocarpum (yellow gulmohar tree) pollen: a dominant avenue tree of India.

BACKGROUND: Peltophorum pterocarpum (yellow gulmohar, PP) pollen is an important aeroallergen for type I hypersensitivity in the tropics. OBJECTIVE: To isolate and characterize the IgE-binding proteins of PP pollen for the first time. METHODS: Pollen extract was fractionated by a combination of Sephacryl S-200 column and diethylaminoethyl-Sephadex column. Allergen characterization was done by sodium dodecyl sulphate polyacrylamide gel electrophoresis, periodic acid-Schiff staining, enzyme-linked immunosorbent assay, and western blotting. Allergenic activities were determined by in vivo (skin prick test) and in vitro (enzyme-linked immunosorbent assay and histamine release) analyses. To determine whether the carbohydrate chains are involved in immunoreactivity, deglycosylation of PP pollen proteins was performed. RESULTS: SPT results on the respiratory allergic patients of Calcutta showed that 32.77% showed positivity with PP pollen. Eight IgE-reactive protein components were found in crude extract. Optimum IgE-reactive fraction 1 was resolved into five subfractions. The subfraction 1a showed maximum IgE reactivity containing the 28 kDa IgE-reactive component. Periodate oxidation showed that protein component was involved in its IgE binding. Twenty-eight kilodalton IgE reactive protein component was recognized by 75% of PP-sensitive patients in Western blotting. It also induced significant histamine release in sensitive patient sera. CONCLUSIONS: The purified 28 kDa protein is a clinically relevant allergen with a potential for diagnosis and therapy of patients susceptible to PP pollen.