Bodhisattwa Saha

Allergic asthma biomarkers using systems approaches

Asthma is characterized by lung inflammation caused by complex interaction between the immune system and environmental factors such as allergens and inorganic pollutants. Recent research in this field is focused on discovering new biomarkers associated with asthma pathogenesis. This review illustrates updated research associating biomarkers of allergic asthma and their potential use in systems biology of the disease. We focus on biomolecules with altered expression, which may serve as inflammatory, diagnostic and therapeutic biomarkers of asthma discovered in human or experimental asthma model using genomic, proteomic and epigenomic approaches for gene and protein expression profiling. These include high-throughput technologies such as state of the art microarray and proteomics Mass Spectrometry (MS) platforms. Emerging concepts of molecular interactions and pathways may provide new insights in searching potential clinical biomarkers. We summarized certain pathways with significant linkage to asthma pathophysiology by analyzing the compiled biomarkers. Systems approaches with this data can identify the regulating networks, which will eventually identify the key biomarkers to be used for diagnostics and drug discovery.

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.

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.

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.

Mass spectrometry-based identification of allergens from Curvularia pallescens, a prevalent aerospore in India.

The worldwide prevalence of fungal allergy in recent years has augmented mining allergens from yet unexplored ones. Curvularia pallescens (CP) being a dominant aerospore in India and a major sensitiser on a wide range of allergic population, pose a serious threat to human health. Therefore, we aimed to identify novel allergens from CP in our present study. A cohort of 22 CP-sensitised patients was selected by positive Skin prick grade. Individual sera exhibited elevated specific IgE level and significant histamine release on a challenge with antigenic extract of CP. First gel-based profiling of CP proteome was done by 1- and 2-dimensional gel. Parallel 1- and 2-dimensional immunoblot were performed applying individual as well as pooled patient sera. Identification of the sero-reactive spots from the 2-dimensional gel was found to be challenging as CP was not previously sequenced. Hence, mass spectrometry-based proteomic workflow consisting of conventional database search was not alone sufficient. Therefore, de novo sequencing preceded homology search was implemented for further identification. Altogether 11 allergenic proteins including Brn-1, vacuolar protease, and fructose-bis-phosphate aldolase were identified with high statistical confidence (p<0.05). This is the first study to report on any allergens from CP. This kind of proteome-based analysis provided a catalogue of CP allergens that would lead an improved way of diagnosis and therapy of CP-related allergy.

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

BackgroundThe 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.MethodsDetermination 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.ResultsLantana 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.ConclusionsThis 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.

Data on mass spectrometry based identification of allergens from sunflower (Helianthus annuus L.) pollen proteome

Allergy is a type of abnormal immune reactions, which is triggered by environmental antigens or allergens and mediated by IgE antibodies. Now-a-days mass spectrometry is the method of choice for allergen identification based on homology searching. Here, we provide the mass spectrometry dataset associated with our previously published research article on identification of sunflower pollen allergens (Ghosh et al., 2015 [1]). In this study allergenicity of sunflower (Helianthus annuus) pollen grains were primarily investigated by clinical studies followed by detailed immunobiochemical and immunoproteomic analyses. The mass spectrometry data for the identification of allergens were deposited to ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier PXD002397.

In Silico Prediction of Allergenic Proteins

Currently, the prediction of new allergens is becoming important due to use of genetically modified (GM) foods and biopharmaceuticals. In this chapter, we describe how to use four popular allergenic prediction servers: (1) Structural Database of Allergenic Proteins (SDAP), (2) Allermatch, (3) Evaller 2, and (4) AlgPred. The first two prediction servers are based on traditional approaches, whereas Evaller 2 and AlgPred use sophisticated machine learning techniques.

222 Aerobiological and Immunological Studies on Coconut Pollen Allergy.

Background Pollen grains constitute a significant portion of the aerobiological flora. The plant Coccos nucifera (commonly known as coconut) is found in huge quantities in the tropical coastal areas of the world and is very common in Kolkata, India. A 2 years aerobiological survey was carried out using Burkard Volumetric Sampler to know the seasonal variation of Cocos nucifera pollen. The plant flower through out the year but maximum concentration was found in the month of August. Allergenicity of Cocos nucifera pollen has been reported from the Skin Prick Test, Lung function test, ELISA from a 400 susceptible patients in and around West Bengal in India. An immunobiological study was conducted to identify major allergens from Coccos nucifera pollen causing hay fever, skin allergy and allergic asthma in Kolkata population. Methods Proteins from pollen grains were obtained by initially defatting and then extracted with sodium phosphate buffer with 10 mM PMSF. Total protein was divided into 4 fractions by ammonium sulfate at 25%, 50 %, 75% and 100% respectively. SDS PAGE was done with the 25% fraction (result obtained from dot blotting) and subsequently western blotting was performed. Two dimensional gel electrophoresis and immunoblotting was also done from the crude protein. Results The total protein was separated on a SDS PAGE gel showed 21 prominent bands by Coomassie Blue staining. Dot -blotting the different fractions from ammonium sulfate cut, showed a positive result in the 25% fraction. Western blot with patient specific sera gave 3 bands out of which a major band was obtained at 60Kd. This result was obtained in more than 65% of the patients from whom Sera was isolated. 2D gel electrophoresis of the crude protein sample was performed which showed 120 protein spots in the PI range of 3 to 10 and molecular weight 14Kd to 97Kd. Immunoblotting the 2D gel with pooled patient specific sera showed 20 spots thus implying IgE reactivity. Conclusions It can thus be inferred that Coccos nucifera pollen grains are very common in the air and are important to cause allergy to susceptible persons. More over the 60 Kd protein is responsible for allergenicity.