Naveen Arora

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.

Enzymatic hydrolysis: a method in alleviating legume allergenicity.

Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes - kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase(®) and Flavourzyme(®). Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2u2009±u20097.7%, 87.1u2009±u20099.6% and 91.8u2009±u20097.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (pu2009<u20090.01). The release of histamine was decreased significantly when sensitized basophils were challenged with hydrolysates as compared to raw extracts. Significant reduction in the biopotency of hydrolysates was also observed in SPT where only 1/10 kidney bean-sensitive individuals, 2/6 black gram-sensitive individuals and 1/7 peanut-sensitive individuals were found positive to their respective hydrolysates. In conclusion, enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts.

Air pollution and respiratory health of school children in industrial, commercial and residential areas of Delhi

Air pollutants may cause severe respiratory problems in the children during their developmental stages. In this study, the respiratory health of school children belonging to three different localities namely industrial, commercial and residential areas of Delhi City was assessed by questionnaire survey and lung function test. The outdoor air pollution data from the National Ambient Air Quality Monitoring (NAAQM) stations was used for the study, which were located within 1-km distance from the selected school sites. Indoor air quality monitoring has also been done at classrooms for particulate matter (PM10), sulphur dioxide (SO2) and nitrogen oxides (NOx). Indoor and outdoor PM10 concentration at schools in commercial area (815u2009±u2009354.45 and 337u2009±u200985xa0μg/m3) was ten times above the NAAQM limits of 100xa0μg/m3. PM10 concentration was lower in industrial (694.6u2009±u2009322.9 and 274u2009±u200978xa0μg/m3) and residential (534.3u2009±u200994.22 and 197u2009±u200948xa0μg/m3) areas. However, levels of SO2 and NO2 were under the permissible limits. The findings also revealed that the children living in the commercial area had higher respiratory symptoms as compared to industrial and residential areas. The respiratory symptoms were found to have a statistically significant positive association with PM10 levels in the air.

Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo).

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiffs and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14-16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues DDYDNLQL showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

Immunoglobulin E (IgE)-mediated cross-reactivity between mesquite pollen proteins and lima bean, an edible legume

Immunoglobulin E (IgE)‐mediated food allergy often develops as a consequence of allergic sensitization to pollen proteins. Mesquite (Prosopis juliflora) tree pollen is reported to be cross‐reactive with other pollen species, but little has been reported on its cross‐reactivity with plant‐derived foods belonging to the same/different families. The present study investigates the in vitro cross‐reactivity of mesquite pollen and lima bean (Phaseolus lunatus), an edible seed belonging to the Leguminosae family. Of 110 patients (asthma, rhinitis or both) tested intradermally, 20 showed marked positive reactions with Prosopis pollen extract. Of these, 12 patients showed elevated specific IgE to Prosopis pollen extract alone and four to both Phaseolus and pollen extract. In vitro cross‐reactivity was investigated using inhibition assays [enzyme‐linked immunosorbent assay (ELISA) inhibition, immunoblot inhibition], histamine release and lymphoproliferation. P. lunatus extract could inhibit IgE binding to P. juliflora in a dose‐dependent manner, requiring 400u2003ng of protein for 50% inhibition in ELISA assay. Immunoblot and immunoblot inhibition demonstrated the presence of 20, 26, 35, 66 and 72u2003kDa as shared IgE binding components between the two extracts. Histamine release, peripheral blood mononuclear cells proliferation and interleukin (IL)‐4 levels also suggested allergenic cross‐reactivity. In conclusion, there is humoral and cellular cross‐reactivity between Prosopis pollen and Phaseolus seed allergens.

Per a 10 activates human derived epithelial cell line in a protease dependent manner via PAR-2

BACKGROUNDnProtease activity of Per a 10 has been shown to modulate dendritic cells toward Th-2 polarization and to induce airway inflammation.nnnOBJECTIVEnTo elucidate the role of serine protease activity of Per a 10 in inducing biochemical responses in epithelial cells.nnnMETHODSnPer a 10 was inactivated by heat treatment (ΔPer a 10) or AEBSF (iPer a 10). A549 cells were exposed to either enzymatically active/inactive Per a 10. The supernatant was analyzed for the secretion of proinflammatory cytokines by ELISA. Ca(2+) mobilization was analyzed by flow cytometry. A PAR-2 derived synthetic peptide 28GTNRSSKGRSLIGKVDGTSHVTGKGVTC54 was incubated with Per a 10 and the resultant cleaved products were analyzed by LC-MS. PAR-2 activation was inhibited by PAR-2 cleavage inhibiting antibody.nnnRESULTSnΔPer a 10 was completely inactivated whereas iPer a 10 showed some residual activity. nPer a 10 having protease activity increased the secretion of IL-6, IL-8 and GMCSF from A549 in a dose and time dependent manner whereas iPer a 10 has reduced cytokine secretion. ΔPer a 10 and rPer a 10 were unable to activate the cells. nPer a 10 mobilized intracellular Ca(2+). nPer a 10 cleaved the PAR-2 derived peptide between arginine and serine residues (36R-S37) to expose PAR-2 ligand SLIGKV, as determined by LC-MS. Incubating with anti-PAR-2 cleavage antibody showed diminished cytokine secretion when treated with nPer a 10.nnnCONCLUSIONnSerine protease activity of Per a 10 activates A549 cells to secrete proinflammatory cytokines by PAR-2 activation and Ca(2+)mobilization and can be exploited therapeutically.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL‐23 and OX40L

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL‐17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL‐12p70 secretion as compared to heat inactivated Per a 10. IL‐23, TNF‐α, and IL‐6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC‐T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL‐4 and IL‐13 that were reduced on blocking of either IL‐23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL‐23 secretion and OX40L expression on dendritic cells.

Airway epithelial cells: Barrier and much more

Airway epithelial cells were first considered as a barrier to the inhaled environmental bioparticles, but recent evidences show that they have a more vital role to play in the pathophysiology of Airway diseases. Many of the factors present in the inhaled air interact with the receptors expressed on the epithelial cells leading to their activation. Activated epithelial cells then secrete a range of mediators that help in mediating the inflammation. These mediators contain the chemokines that act as chemoattractants and recruit inflammatory cells like neutrophils, macrophages, mast cells, eosinophils, and Th-2 cells that further exacerbate the intensity of inflammation. Some of the inhaled substances like protease enzymes can also disrupt the barrier of epithelium and gain an entry to the immune cells of the body leading to their activation. Bronchial asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome represent a broad range of conditions involving pulmonary inflammation. This review takes into account the role of epithelial cells in initiating allergic reactions at mucosal surfaces.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte‐derived DCs from cockroach‐allergic patients were treated with proteolytically active/heat‐inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (Pu2009<u20090·05), while enhanced soluble CD40 level in the culture supernatant (Pu2009<u20090·05) compared to the heat‐inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)‐12 and interferon (IFN)‐γ secretion by DCs (Pu2009<u20090·05) compared to heat‐inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL‐12 secretion. Active Per a 10 stimulation caused low nuclear factor‐kappa B (NF‐κB) activation in DCs compared to heat‐inactivated Per a 10. Inhibition of the NF‐κB pathway suppressed the CD40 expression and IL‐12 secretion by DCs, further indicating that NF‐κB is required for CD40 up‐regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor‐associated factor 6 (TRAF6), thereby suggesting its involvement in NF‐κB activation. Protease activity of Per a 10 induced p38 mitogen‐activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF‐κB suppressed the secretion of IL‐12, IFN‐γ, IL‐6 and TNF‐α by DCs. Such DCs further reduced the secretion of IL‐4, IL‐6, IL‐12 and TNF‐α by CD4+ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down‐regulation leads to low NF‐κB levels, thereby modulating DC‐mediated immune responses.

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.