In silico tools to assess the potential allergenicity of shiitake mushrooms (Lentinula edodes).

BACKGROUND: Computational tools may have an edge over conventional methods for the preliminary evaluation of food allergenicity. In this study, the allergenic potential of Lentinula edodes was evaluated and validated using in silico tools. RESULTS: The potential cross-reactivity of mushroom proteins with fungal allergens was determined using sequence alignment - the Fast Alignment (FASTA) and Basic Local Alignment Search Tool (BLAST) algorithm. Eight L. edodes proteins were cross-reactive with allergens from fungal origin, showing 52%-89% sequence identity using FASTA algorithm-based alignment. The BLAST data were corroborated by percentage identity and query coverage. Physico-chemical property-based allergenicity was deciphered by AlgPred, Allermatch, and AllergenFP software, which predicted six out of eight proteins as potential allergens. Sequence alignment showed 66%-86% conservancy between mushroom protein and known fungal allergens. Secondary structure and amino acid composition supported structural affinity between query and fungal proteins. Three-dimensional structures of five mushroom proteins were generated, quality assessed, and superimposed with fungal allergens, suggesting possible allergenicity of mushroom proteins. An enzyme-linked immunosorbent assay (ELISA) demonstrated immunoglobulin E (IgE) binding in 13 out of 21 food-hypersensitive patients' sera. CONCLUSION: In silico tools provide preliminary indications about the potential allergenicity and cross-reactivity of mushroom proteins. This approach may be used for the prelusive allergenicity assessment of allergen sources. © 2022 Society of Chemical Industry.

Iron deficiency in non-anemic chronic obstructive pulmonary disease in a predominantly male population: an ignored entity.

Non-anemic iron deficiency has been studied in heart failure, but studies are lacking in chronic obstructive pulmonary disease (COPD). The potential clinical implications of association of iron deficiency with the severity of COPD warrant research in this direction. This was an observational, cross-sectional study on patients with COPD to compare disease severity, functional status and quality of life in non-anemic patients with COPD between two groups - iron deficient and non-iron deficient. Stable non-anemic COPD with no cause of bleeding were evaluated for serum iron levels, ferritin levels, TIBC, 6MWD, SGRQ, spirometry, and CAT questionnaire. The study patients were divided into iron replete (IR) and iron deficient (ID) groups. A total of 79 patients were studied, out of which 72 were men and seven were women. The mean age was 61.5±8.42 years. Of these, 36 (45.5%; 95% CI, 34.3-56.8%) had iron deficiency. Mean 6-minute-walk distance was significantly shorter in ID (354.28±82.4 meters vs 432.5±47.21 meters; p=0.001). A number of exacerbations in a year were more in ID group (p=0.003), and more patients in ID had at least two exacerbations of COPD within a year (p=0.001). However, the resting pO2, SaO2, and SpO2 levels did not differ significantly between the two groups (p=0.15 and p=0.52, respectively). Also, there was no significant difference in the distribution of patients of a different class of airflow limitations between the two groups. Non-anemic iron deficiency (NAID) is an ignored, yet easily correctable comorbidity in COPD. Patients with iron deficiency have a more severe grade of COPD, had lesser exercise capacity and more exacerbations in a year as compared to non-iron deficient patients. So, foraying into the avenue of iron supplementation, which has shown promising results in improving functional capacity in heart failure and pulmonary hypertension, may well lead to revolutionary changes in the treatment of COPD.

The role of fungal sensitisation in clinical presentation in patients with chronic obstructive pulmonary disease.

Atopic patients with chronic obstructive pulmonary disease (COPD) demonstrate more severe symptoms than their non-atopic counterparts. Also, Aspergillus hypersensitivity is known in COPD. However, allergic sensitisation to non-Aspergillus fungi has never been studied in COPD patients. To evaluate the prevalence of fungal sensitisation and its impact on the clinical presentation and outcome of COPD patients. Sensitisation to 17 fungi was studied in 55 COPD patients through skin prick tests, fungus-specific IgE, precipitating antibodies, total IgE and eosinophil counts. The clinical symptoms of patients were monitored thorough a patient-administered questionnaire. Overall, 5.4% (n = 3) of COPD patients were fungus sensitive. The sensitisation was noted to Alternaria alternata and Schizophyllum commune in two patients each, whereas another was sensitive to A. tamarii, Rhizopus spp. and Aspergillus fumigatus. Eosinophils were higher in fungus-sensitised patients (P = 0.001 vs. 0.003). No differences were noted in the clinical presentation of patients sensitised to fungi compared to those not sensitised to fungi or non-atopic. Although low, fungal sensitisation occurs in COPD but it is not limited to Aspergilli alone. Fungus-sensitised patients exhibit greater eosinophilia, implying more severe inflammation. Thus, such patients should be followed up regularly to recognise clinical worsening or development of ABPM.

Allergic bronchopulmonary mycosis due to fungi other than Aspergillus: a global overview.

Allergic bronchopulmonary mycosis (ABPM) is a hypersensitivity-mediated disease of worldwide distribution. We reviewed 143 reported global cases of ABPM due to fungi other than aspergilli. The commonest etiologic agent was Candida albicans, reported in 60% of the cases, followed by Bipolaris species (13%), Schizophyllum commune (11%), Curvularia species (8%), Pseudallescheria boydii species complex (3%) and rarely, Alternaria alternata, Fusarium vasinfectum, Penicillium species, Cladosporium cladosporioides, Stemphylium languinosum, Rhizopus oryzae, C. glabrata, Saccharomyces cerevisiae and Trichosporon beigelii. India accounted for about 47% of the globally reported cases of ABPM, attributed predominantly to C. albicans, followed by Japan (16%) where S. commune predominates, and the remaining one-third from the USA, Australia and Europe. Notably, bronchial asthma was present in only 32% of ABPM cases whereas its association with development of allergic bronchopulmonary aspergillosis (ABPA) is known to be much more frequent. The cases reviewed herein revealed a median IgE value threefold higher than that of ABPA, suggesting that the etiologic agents of ABPM incite a stronger immunological response than that by aspergilli in ABPA. ABPM is currently underdiagnosed, warranting comprehensive basic and clinical studies in order to elucidate its epidemiology and to devise a more effective therapy.

NCCP-ICS joint consensus-based clinical practice guidelines on medical thoracoscopy.

ABSTRACT: Medical Thoracoscopy (MT) is commonly performed by respiratory physicians for diagnostic as well as therapeutic purposes. The aim of the study was to provide evidence-based information regarding all aspects of MT, both as a diagnostic tool and therapeutic aid for pulmonologists across India. The consensus-based guidelines were formulated based on a multistep process using a set of 31 questions. A systematic search of published randomized controlled clinical trials, open labelled studies, case reports and guidelines from electronic databases, like PubMed, EmBase and Cochrane, was performed. The modified grade system was used (1, 2, 3 or usual practice point) to classify the quality of available evidence. Then, a multitude of factors were taken into account, such as volume of evidence, applicability and practicality for implementation to the target population and then strength of recommendation was finalized. MT helps to improve diagnosis and patient management, with reduced risk of post procedure complications. Trainees should perform at least 20 medical thoracoscopy procedures. The diagnostic yield of both rigid and semirigid techniques is comparable. Sterile-graded talc is the ideal agent for chemical pleurodesis. The consensus statement will help pulmonologists to adopt best evidence-based practices during MT for diagnostic and therapeutic purposes.

Clinical significance and molecular characterization of nonsporulating molds isolated from the respiratory tracts of bronchopulmonary mycosis patients with special reference to basidiomycetes.

Nonsporulating molds (NSMs), especially basidiomycetes, have predominantly been reported as human pathogens responsible for allergic and invasive disease. Their conventional identification is problematic, as many isolates remain sterile in culture. Thus, inconclusive culture reports might adversely affect treatment decisions. The clinical significance of NSMs in pulmonary mycoses is poorly understood. We sequenced the internal transcribed spacer (ITS) region and D1/D2 domain of the larger subunit (LSU) of 52 NSMs isolated from respiratory specimens. The basidiomycetes were the predominant NSMs, of which Schizophyllum commune was the most common agent in allergic bronchopulmonary mycosis (ABPM), followed by Ceriporia lacerata in invasive fungal disease. Porostereum spadiceum, Phanaerochaete stereoides, Neosartorya fischeri, and Marasmiellus palmivorus were the other molds observed. Application of ITS and LSU region sequencing identified 92% of the isolates. The antifungal susceptibility data revealed that all basidiomycetes tested were susceptible to amphotericin B and resistant to caspofungin, fluconazole, and flucytosine. Except for 3 isolates of S. commune and a solitary isolate of M. palmivorus, all basidiomycetes had low MICs for itraconazole, posaconazole, and voriconazole. Basidiomycetes were isolated from patients with ABPM, invasive pulmonary mycosis/pneumonia, or fungal balls. In addition, the majority of the basidiomycetes were isolated from patients with chronic respiratory disorders who were sensitized to one of the basidiomycetous fungi and demonstrated precipitating antibodies against the incriminating fungi, indicating an indolent tissue reaction. Thus, isolation of basidiomycetes from the lower respiratory tract could be significant, and it is important to monitor these patients in order to prevent subsequent lung damage.

Medication use in Indian children with asthma: the user's perspective.

BACKGROUND AND OBJECTIVE: Despite the high prevalence of asthma in children, there has been limited research into patient perception of medication use, particularly in the developing world. This study therefore aimed to carry out an in-depth exploration of the views of carers and children with asthma on asthma medication use. METHODS: Grounded theory approach was used to conduct semistructured qualitative interviews in a purposive convenience sample of parents and children with asthma. The participants were recruited from two specialty hospitals in New Delhi, India. Interviews were tape-recorded, transcribed verbatim and thematically analysed. RESULTS: Twenty children (7-12 years old) with asthma and their parent or carer were interviewed in July 2011. Major reported issues included poor parent and child understanding of disease and medications. Fears, misinformed beliefs and lack of self-management skills were apparent. Child self-image, resistance to medication use and lack of responsibility in medication taking were themes that emerged from child interviews. CONCLUSIONS: This is one of the first research studies exploring the viewpoint of children with asthma about their medications. Resource constraints dictate a pragmatic paternalistic approach by physicians which, in contrast to patients in westernized nations, seems to be acceptable and satisfactory to Indian patients (carers).

Immuno-informatic Prediction of B and T cell Epitopes of Cysteine Protease Allergen from Phaseolus vulgaris with Cross-reactive Potential and Population Coverage.

BACKGROUND: In-silico mapping of epitopes by immune-informatics has simplified the efforts towards understanding antigen-antibody interactions. The knowledge of allergen epitopes may help in advancing the diagnosis and therapy of allergic diseases. OBJECTIVE: This study was intended to identify B and T cell epitopes of cysteine protease allergen of Phaseolus vulgaris. METHODS: Modeller 9v20 software was used for the generation of three-dimensional model of cysteine protease and quality assessment was performed using SAVES webserver and other in silico software. Linear and conformational B and T cell epitopes were predicted via immuno-informatics based computational servers. Epitopes were synthesized and their immunoreactivity was analyzed using specific IgE ELISA with food allergy positive patient's sera. Cellular immune response of peptides was determined through basophil activation assay. Consurf and SDAP (property distance) were used to examine the evolutionary conservancy and potential cross-reactivity of predicted epitopes. MSA based positional conservancy between HDM allergen epitopes and predicted peptides was also established using IEDB epitope database. Finally, population coverage for each promiscuous T cell epitope was predicted using IEDB population coverage analysis tool. RESULTS: Cysteine protease structure was derived by homology modeling and combination of bioinformatic tools predicted three B- and three T-cell peptides by consensus method and validated computationally. ELISA with kidney bean sensitive patient's sera showed higher IgE binding of B-cell peptides as compared to T-cell or control peptides. Epitope conservancy revealed B-cell epitopes being upto 95% conserved in comparison to variable T-cell epitopes (upto 69%). B-cell peptides were crossreactive with homologous allergens based on PD values. Structural comparison of cysteine protease with Der p 1 and Der f 1 showed similar epitopic regions, validating the prediction accuracy of epitopes. Promiscuous T-cell epitopes binding to broad-spectrum class-II MHC alleles demonstrated the distribution of T-cell peptides world-wide (30-98%) and in Asian population (99%). CONCLUSION: The current approach can be applied for identification of epitopes. Analysis of crossreactive and widely-distributed specific epitopes of allergen and knowledge about their interactive surfaces will help in understanding of food allergy and related immune responses.

Molecular and immunological characterization of cysteine protease from Phaseolus vulgaris and evolutionary cross-reactivity.

A commonly consumed legume in India, the kidney bean (Phaseolus vulgaris) is associated with allergy. We report molecular and immunological characterization of cysteine protease allergen and its cross-reactivity. In silico allergenicity assessment and phylogenetic analysis of kidney bean cysteine protease showed significant sequence homology (upto 67%) with allergens from kiwi, papaya, soybean, ragweed pollen and mites. Physicochemical properties and motif-analysis depicted cysteine protease as probable allergen. Multiple sequence alignment and phylogenetic analysis indicated structural conservation between kidney bean and homologous cysteine protease sequences. The gene was cloned, expressed and affinity purified. Cysteine protease was resolved at 42 kDa and exhibited high IgE binding (up to 89%) with hypersensitive sera. Cysteine protease showed functional property on cross-linking IgE receptors and upregulated expression of CD203c on activated basophils. In inhibition studies, 8.4 ng of cysteine protease was required for 50% self-inhibition, whereas significant inhibition was also observed with kidney bean (52 ng), black gram (155 ng), chick pea (437 ng), mesquite pollen (36 ng), house dust mite (64.85 ng), Alternaria alternata (78.8 ng) and Curvularia lunata (73.6 ng) extracts. ConSurf analysis indicated conserved active site and catalytic residues in mature domain among proteases from legumes, fruits, pollens, mites and fungus. In summary, P. vulgaris cysteine protease was molecularly characterized having functional activity. This study demonstrated, cross-reactivity between food and aeroallergens based on evolutionary conservancy that showed its clinical importance as cross-reactive allergen. PRACTICAL APPLICATIONS: Adaptation of sustainable lifestyle has led to a surge in consumption of plant-based foods especially legumes. Their high nutritional content lowers the risk of developing cardiovascular diseases, diabetes, obesity, and stroke. Kidney beans, a commonly consumed legume in Indian subcontinent, have a potential to be used as nutraceutical and functional food. Despite its alimentary nature, it elicits allergic reactions. Being a major sensitizer, trivial information regarding its allergic components has led to an urgent need for exploring its allergen repertoire. Our study reported biochemical and immunological characterization of its major cysteine protease allergen. Cysteine proteases are major cross-reactive allergens from insects, fruits and fungal sources. Identification and molecular characterization of such immunodominant allergens by RDT offers the prospect of using recombinant proteins for accurate diagnosis and therapeutic purposes. This study suggests that a potential major cross-reactive allergen may aid in developing allergy management interventions for a wide range of allergenic sources.

Clinico-immunological changes post-immunotherapy with Periplaneta americana.

BACKGROUND: Cockroach proteins induce allergies including asthma in predisposed individuals. Well-designed controlled studies are required to show the effect of cockroach immunotherapy (IT). This study is aimed to assess changes in clinical and immunological parameters post-IT with Periplaneta americana extract. MATERIALS AND METHODS: A double-blind, placebo-controlled trial of cockroach IT was performed for 1year in 50 patients of asthma, rhinitis or both. The efficacy of IT was assessed by change in skin reactivity and clinical parameters such as symptom/drug score, airway reactivity and immunological parameters namely IgE, IgG1 and IgG4, IL-4 and IFN-γ by enzyme-linked immunosorbent assay and western blotting using patients' sera at baseline and after 1year of treatment. RESULTS: Immunotherapy with cockroach extract demonstrated significant improvement in clinical parameters of active group patients compared with baseline values and placebo group. Specific IgE levels showed a modest reduction, while IgG4 levels increased significantly in active IT group after 1year. IgE immunoblotting demonstrated reduction in intensity and number of specific bands, whereas IgG4 binding showed more number and distinct bands following IT. Active group patients showed correlation between increase in IgG4/IgG1 ratio and reduction in symptom score post-IT. CONCLUSIONS: Immunotherapy with cockroach extract improved clinical and immunological status of asthma and rhinitis patients. Clinical improvement in patients after IT is associated with immunological changes.

Proteolytically inactive per a 10 allergen of Periplaneta americana modulates Th2 response and enhances IL-10 in mouse model.

BACKGROUND: Purified allergens with reduced IgE reactivity are required to improve the safety and efficacy of allergen-specific immunotherapy (IT). OBJECTIVE: The present study investigates the efficacy of purified cockroach allergen immunotherapy with proteolytically active and inactive Per a 10 in allergic mouse model. METHODS: Balb/c mice were sensitized intraperitoneally with cockroach extract (CE) and purified allergen Per a 10 in separate groups. Mice were treated subcutaneously with phosphate-buffered saline (PBS), CE, active and inactive Per a 10 and challenged intranasally. Antigen specific IgE, IgG1 and IgG2a in serum and cytokines IL-4, IL-13, IFN-gamma, IL-10, TGF-beta in bronchoalveolar lavage (BAL) fluid and spleen culture supernatant (CS) were estimated by enzyme-linked immunosorbent assay. Lung histology was analyzed by hematoxylin and eosin staining. RESULTS: IT with Per a 10 demonstrated significant reduction in IgE levels in serum, IL-4 levels in BAL fluid, CS, and eosinophilic infiltration in lungs than PBS-treated mice. This was associated with significantly increased IL-10 secretion in BAL fluid and CS. IT with Per a 10 effectively suppressed T-helper type 2 (Th2) response in mice sensitized with Per a 10 than CE group. Further, IT with inactive Per a 10 showed maximum reduction in systemic and airway inflammation and induced maximum IL-10 release in BAL fluid and CS than other antigens. CONCLUSIONS: IT with Per a 10 effectively suppressed Th2 response and lung inflammation in Per a 10- or CE-sensitized mice. The beneficial effects of IT with inactive Per a 10 are more pronounced than active Per a 10.

Clinico-immunologic study on immunotherapy with mixed and single insect allergens.

BACKGROUND: Immunotherapy (IT) is practiced mainly with mixed and single allergen vaccines. But studies are rare with mixed allergen preparations. OBJECTIVE: The objective of this study is to study mix and single insect allergen IT in patients of allergic rhinitis and asthma. METHODS: We performed a double-blind placebo-controlled trial of mix and single allergen IT for 1 year in 99 patients of asthma or rhinitis or both. There were two groups, (1) active allergen IT (n = 61) with three subgroups single insect extract (cockroach, housefly, or mosquito) and mix allergen IT (two or three insect extracts) and (2) placebo (n = 38). Clinical (skin reactivity, airway reactivity, and symptom score) and immunological (IgE/IgG4 and IgG1/IgG4 ratio) parameters were assessed at baseline and after 1 year of IT. RESULTS: Eighty-five patients completed 1 year of IT. The active allergen IT group patients showed a significant improvement compared to baseline values (p < 0.05) and placebo group patients (p < 0.05) with regard to symptom scores, FEV1 values, and immunological parameters (IgG4). No significant difference was found between mixed and single IT group patients for changes in clinical and immunological parameters. Positive correlation was observed between increase in IgG4 and clinical improvement. The changes in above parameters in placebo group were nonsignificant after 1 year of treatment. CONCLUSION: IT with two to three mix extract from the same allergen group is effective for insect hypersensitivity.

Acute toxicity assessment of choline by inhalation, intraperitoneal and oral routes in Balb/c mice.

Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined.

Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation.

Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces.

Activities-specific balance confidence scale for predicting future falls in Indian older adults.

BACKGROUND: Activities-specific balance confidence (ABC) scale is a subjective measure of confidence in performing various ambulatory activities without falling or experiencing a sense of unsteadiness. OBJECTIVE: This study aimed to examine the ability of the Hindi version of the ABC scale (ABC-H scale) to discriminate between fallers and non-fallers and to examine its predictive validity for prospective falls. DESIGN: This was a prospective cohort study. MATERIALS AND METHODS: A total of 125 community-dwelling older adults (88 were men) completed the ABC-H scale. The occurrence of falls over the follow-up period of 12 months was recorded. Discriminative validity was analyzed by comparing the total ABC-H scale scores between the faller and non-faller groups. A receiver operating characteristic curve analysis and a logistic regression analysis were used to examine the predictive accuracy of the ABC-H scale. RESULTS: The mean ABC-H scale score of the faller group was significantly lower than that of the non-faller group (52.6±8.1 vs 73.1±12.2; P<0.001). The optimal cutoff value for distinguishing faller and non-faller adults was ≤58.13. The sensitivity, specificity, area under the curve, and positive and negative likelihood ratios of the cutoff score were 86.3%, 87.3%, 0.91 (P<0.001), 6.84, and 0.16, respectively. The percentage test accuracy and false-positive and false-negative rates were 86.87%, 12.2%, and 13.6%, respectively. A dichotomized total ABC-H scale score of ≤58.13% (adjusted odds ratio =0.032, 95% confidence interval =0.004-0.25, P=0.001) was significantly related with future falls. CONCLUSION: The ABC-H scores were significantly and independently related with future falls in the community-dwelling Indian older adults. The ability of the ABC-H scale to predict future falls was adequate with high sensitivity and specificity values.

Engineered hypoallergenic variants of osmotin demonstrate hypoallergenicity with in vitro and in vivo methods.

The molecular structure of protein and epitope mapping strategies are required to engineer the epitopes of a protein. In the present study, IgE binding regions of osmotin were identified and mutated to obtain hypoallergenic variant. The three dimensional (3-D) model of osmotin obtained by homology modeling comprised of a characteristic thaumatin-like fold. This model was used to predict IgE binding regions of osmotin. These regions were mutated and three mutant proteins with four mutations (Ma, Mb and Mc) and one with six mutations (Mabc) were expressed and purified to homogeneity. IgE binding of the mutant proteins was evaluated by in vitro studies using patients' sera. Ma, Mb and Mc demonstrated reduction in IgE binding of 73%, 83% and 77%, respectively, whereas Mabc showed complete abrogation of IgE binding. Ma, Mb and Mc showed inhibition of 48%, 44% and 38%, respectively to osmotin, while Mabc showed 24% inhibition at 10 µg with pooled patients' sera. Osmotin reached effective concentration at 50% inhibition (EC50) at 3 ng and none of the mutant proteins reach the EC50 value. The immunological response to mutant proteins was examined in mice. Blood, bronchoalveolar lavage fluid spleen and lung tissue were excised from mice for analysis. The mice treated with mutant proteins showed significant reduction in IgE and IgG1 levels as compared to mice given osmotin (p<0.001). Th2 cytokines level in splenocyte supernatant and BALF of mice given mutant proteins were significantly lower (p<0.001), accompanied with significant reduction in cellular infiltration in lungs (p<0.001). In conclusion, osmotin structure was predicted by homology modeling and IgE binding regions predicted were mutated to obtain a hypoallergenic protein.

Epi p 1, an allergenic glycoprotein of Epicoccum purpurascens is a serine protease.

Epicoccum purpurascens (EP) is a ubiquitous saprophytic mould, the inhalant spores and mycelia of which are responsible for respiratory allergic disorders in 5-7% of population worldwide. The diagnosis/therapy of these disorders caused by fungi involves the use of standardized and purified fungal extracts. A 33.5 kDa glycoprotein, Epi p 1 released histamine from whole blood cells of EP allergic patients at a concentration of 50-ng protein. The high specific IgE values detected in EP hypersensitive sera indicated that Epi p 1 is capable of mediating type I hypersensitive reaction in predisposed individuals. It also showed protease activity by virtue of its dose dependent cleavage of serine protease specific synthetic substrate, N-benzoyl arginine ethyl ester hydrochloride (BAEE). The serine protease nature of Epi p 1 was confirmed by its N-terminal sequence (ADG/FIVAVELD/STY) homology to a subtilisin like serine protease. The protease activity of Epi p 1 may be responsible for making its way into the system of pre-disposed individuals through epithelial cell detachment and the histamine releasing ability by cross-linking of IgE antibodies on cell surface is the cause of its allergenic nature.

In silico identification of IgE-binding epitopes of osmotin protein.

The identification of B-cell epitopes is an important step to study the antigen- antibody interactions for diagnosis and therapy. The present study aimed to identify B- cell epitopes of osmotin using bioinformatic tools and further modify these regions to study the allergenic property. B-cell epitopes were predicted based on amino acid physicochemical properties. Three single point mutations M1, M2, and M3 and a multiple point mutant (M123) were selected to disrupt the IgE binding. These mutants were cloned, expressed and proteins purified to homogeneity. The IgE binding of the purified proteins was evaluated by ELISA and ELISA inhibition with patients' sera. Three regions of osmotin M1 (57-70 aa), M2 (72-85 aa) and M3 (147-165 aa) were identified as potential antibody recognition sites using in silico tools. The sequence similarity search of the predicted epitopes of osmotin using Structural Database of Allergenic proteins (SDAP) showed similarity with known allergens from tomato, kiwifruit, bell pepper, apple, mountain cedar and cypress. Mutants M1, M2 and M3 showed up to 72%, 60% and 76% reduction, respectively in IgE binding whereas M123 showed up to 90% reduction with patients' sera. The immunoblot of M123 mutant showed 40% reduction in spot density as compared to osmotin. All mutants showed decreased inhibition potency with M123 exhibiting lowest potency of 32% with osmotin positive pooled patients' sera. The three B- cell epitopes of osmotin predicted by in silico method correlated with the experimental approach. The mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.

Characterization of recombinant per a 10 from Periplaneta americana.

Cockroach allergen is a major risk factor for IgE-mediated allergic response and asthma in sensitized individuals. Serine proteases have been identified from various sources and characterized as major allergens. The present study was aimed to express and characterize recombinant allergen Per a 10 (rPer a 10) from Periplaneta americana. rPer a 10 was expressed in Escherichia coli and purified in soluble form, yielding 0.75 mg/liter of culture. Homology of the Per a 10 protein sequence exhibited 27 to 38% similarity to the mite serine protease and 41 to 52% similarity to other insect trypsins. The purified rPer a 10 protein resolved at 28 kDa on SDS-PAGE and was recognized by cockroach-hypersensitive patients' sera by immunoblotting and enzyme-linked immunosorbent assay (ELISA). In competitive ELISA, rPer a 10 required 96 ng of purified protein for 50% inhibition of IgE binding, whereas 34 ng of native protein (nPer a 10) was required for the same inhibition. rPer a 10 and nPer a 10 induced basophil histamine release in the range of 47 to 64% and 60 to 85%, respectively, when sensitized with cockroach-hypersensitive patients' sera. In conclusion, Per a 10 was subcloned, and the protein was purified to homogeneity. rPer a 10 showed reduced IgE binding and histamine release and showed no proteolytic activity. These data suggest that rPer a 10 has potential for immunotherapy.