Sharad V. Gangal

Immunobiochemical Characterization of Brassica campestris Pollen Allergen

Brassica campestris (BC), Eng. Mustard, is an important source of pollen allergen, responsible for type I hypersensitivity disorders. In the present study, BC pollen extract was characterized by TLIEF, SDS-PAGE and immunoprinting. The extract separated into 50 silver stained bands of pI 3-9 on isoelectric focusing whereas it resolved into 14 Coomassie blue stained protein bands of 14-100 kD on SDS-PAGE. Immunoblot analysis with individual patient sera detected four allergenic proteins of 90, 67, 60 and 14 kD. BC separated into 8 peaks (Bras 1-8) on DEAE Sephadex A-50 column. Bras 2 was found to be most potent by IgE specific ELISA, hence further fractionated on Sephadex G-200. A protein of 90 kD (Bras 2a) isolated by gel filtration was found to be most allergenic protein by ELISA inhibition. The findings shall be applicable in standardization of future batches of BC pollen extract to be used for allergy diagnosis and immunotherapy.

Immunomodulation by liposome entrapped allergen.

Liposomes are non-toxic, biodegradable and feebly immunogenic lipid vesicles made from natural and synthetic lipids. They are known to act as immunopotentiating agents and can be used to formulate sustained release preparation by encapsulation. In the present study, liposome entrapped allergen and free allergen were used to inject in Balb/C mice at different time intervals and their immune response in terms of specific IgG and specific IgE levels was quantitated by ELISA (Enzyme Linked Immuno sorbent Assay). The results indicated that specific IgE response was significantly higher in mice injected free allergen as compared to that of mice given liposome entrapped allergen. However, the specific IgG response was not statistically significant. Experiments carried out with liposome entrapped allergen and liposome coupled allergen showed no statistically significant difference in specific IgE and specific IgG titre between the two groups of mice. This type of immunomodulatory effect of liposomes in reducing IgE levels and without affecting IgG levels may be useful in Type I allergic disorders.

Purification and characterization of allergens from Xanthium strumarium pollen.

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic componenets. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103 000 daltons. Xan VIa was a glycoprotein of molecular weight 17 000 daltons. The carbohydrate moiety of Xan Vla was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.

Airborne fungi in the hospitals of metropolitan Delhi

SummaryThe fungal airspora of a large hospital in Delhi Metropolis was studied from May 1989 – April 1991, using Andersen Six Stage Volumetric Sampler and Burkard Personal Slide Sampler. Simultaneously, samples were also collected from outside the hospital to act as a control. Samplers were operated for 10 min. each time, at 10 - day intervals. Additional samples were also collected from different sections of 3 other hospitals. Some of the dominant forms encountered wereCladosporium spp.,Aspergillus flavus, Smut,Fusarium spp.,Aspergillus niger, Alternaria spp.,Penicillium citrinum, Aspergillus versicolor, andPenicillium oxalicum. Aspergillus flavus showed significantly high concentration inside hospital (n=66, x=53 CFU m−3, p<0.05) as compared to outside air. The peak period for fungi was observed to be from June – September. The spore concentration was much lower in hospital units receiving filtered air as compared to control environment, but in naturally ventilated hospitals the concentration was similar to that of outside air.

Immunobiochemical Characterization of Putranjiva roxburghii Pollen Extract and Cross-Reactivity with Ricinus communis

Putranjiva roxburghii (PR) pollen has been found to be an important aeroallergen for type I hypersensitivity. In the present study, the IgE binding proteins of PR pollen have been characterized and compared with pollen allergens of Ricinus communis (RC) belonging to family Euphorbiaceae. On isoelectric focusing, PR pollen extract resolved into 35 bands (pI 3-9), whereas SDS-PAGE separated it into 18 protein components (MW 14-100 kD). Pooled patients sera (ID +ve to PR) recognized 12 allergenic proteins in Putranjiva and five of them (MWs 92, 80, 55, 43 and 30 kD) showed immunologic reactivity to most of the sera samples tested individually by immunoblot. A number of shared allergenic proteins (MWs 92, 80, 66, 50, 43 and 14 kD) were observed between PR and RC pollen extracts on immunoblot using Putranjiva allergic serum pool. Inhibition in the binding for most of PR pollen allergenic proteins was obtained with higher concentration of RC extract than PR itself, depicting the presence of cross-reacting allergens in both. Putranjiva pollen extract was fractionated by a combination of DEAE Sephadex-A 50 and Sephadex-G 200 column chromatography. Periodate deglycosylation of western blotted PR extract and Put I fraction indicated the involvement of carbohydrate moieties in the allergenic activity. Of the two fractions from Put I (Ia and Ib), Put Ib was found to be the most allergenic protein by ELISA inhibition. Dot blot analysis with individual patients sera identified it as a major allergen of PR.

Role of liposomes in selective proliferation of splenic lymphocytes

The effects of free and encapsulated allergens of Artemisia scoparia pollen on lymphocyte proliferation and immunoglobulin production in BALB/c mice were investigated. Splenic lymphocytes from mice immunized with liposome entrapped allergen (LEA) elicited a marked proliferative response upon in vitro stimulation with both free and encapsulated allergen in comparison to mice immunized with free allergen (FA). The serum immunoglobulin profile of mice administered LEA revealed a predominance of IgG1 antibodies concomitant with an enhancement of IgG2a, IgG2b, IgG3 and IgM responses and suppression of IgE responses. However immunization with FA resulted in significant production of IgE responses and low levels of IgG antibodies. The differential ability of free and encapsulated allergens to selectively induce immunoglobulin isotypes suggests that different presentation and T cell differentiation pathways may be followed by FA and LEA in the immune system. Proliferation studies involving macrophage depletion demonstrated that macrophages play an obligatory role in the processing of LEA. Analysis of cytokine production in sera of immunized mice (FA/LEA) revealed that LEA induced significant IFN-γ responses and lower IL-4 responses than mice immunized with FA. The results of the present study indicate that liposomes synergise the proliferation by the antigen incorporated in it and polarizes the response towards Thl type of cytokine production. The immunoadjuvant and immunomodulation property of liposomes make it an effcient vehicle for effective immunotherapy.

Immunochemical characterization of Cocos nucifera pollen

The Cocos nucifera pollen, as one of the sources of allergen responsible for immediate hypersensitivity reaction, was confirmed by skin prick test, bronchial provocation test, and RAST. The whole pollen extract (WPE) of C. nucifera was fractionated by combination of gel filtration and ion-exchange columns with fast protein liquid chromatography (Pharmacia, Uppsala, Sweden). Three protein peaks designated Cocos II, Cocos VI, and Cocos VII exhibited allergenic properties, as tested by skin prick test, direct IgE ELISA, bronchial provocation test, and immunoblot analysis. In RAST inhibition, Cocos IIa (a high-molecular-weight protein) obtained by fractionation of Cocos II on Mono Q column (fast protein liquid chromatography) (Pharmacia) was found to be the most potent allergen in Cocos WPE, followed by Cocos VI and Cocos VII, which are low-molecular-weight proteins. The reference patterns of Cocos WPE on crossed immunoelectrophoresis and thin-layer isoelectric focusing were established for future standardization of Cocos WPE to be used in the diagnosis and immunotherapy of allergic patients.

Purification and Partial Characterization ofa 67-kD Cross-React ive Allergen from Imperata cylindrica Pollen Extract

Background: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85–16 kD. Objectives: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. Methodology: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. Results: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. Conclusion: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.

Purification and characterization of Fus sI3596, a 65 kd allergen of Fusarium solani

A component ofFusarium solani (F. solani), identified as the major allergen,Fus sI3596* was purified to homogeneity from culture filtrate (CF) by means of anion-exchange column chromatography, gel filtration and FPLC. The homogeneity ofFus sI3596* was assessed by IEF, PAGE, SDS-PAGE (non-reducing), immunoblot and HPLC.Fus sI3596* was isolated as a glycoprotein of MW 65 kd and pI 3.6. The IgE ELISA-inhibition assay after periodate treatment of the fraction showed a lower IgE binding capacity suggesting involvement of carbohydrate moiety in IgE binding reactions of the allergen. Peptide fragments ofFus sI3596* obtained after CNBr and trypsin treatment were analysed by immunoblotting for their allergenicity. This study indicated that there could be at least 3 allergenic determinants in the major allergen,Fus sI3596* ofF. solani CF.

Basis of Rise in Intracellular Sodium in Airway Hyperresponsiveness and Asthma

The aim of this study was to investigate the basis of disturbances in sodium transport in asthma and in airway hyperresponsiveness without symptoms of asthma (asymptomatic AHR). We measured the intracellular sodium (Nai); activity of Na+/K+-ATPase in unstimulated cells (resting activity) and in cell homogenate under optimal conditions (maximal activity); and sodium influx, in mixed leukocytes of 15 normal subjects, 12 subjects with asymptomatic AHR, and 26 asthmatics with or without active symptoms. Resting Na+/K+-ATPase activity was the same as sodium influx, consistent with homeostasis. Compared with normal subjects, those with asymptomatic AHR or asthma with controlled symptoms had a twofold increase in sodium influx and Nai. Symptomatic asthmatics also had a twofold increase in sodium influx but a fourfold elevation of Nai. Maximal Na+/K+-ATPase activity was reduced by half in symptomatic asthmatics compared with normal subjects. The reduction of maximal Na+/K+-ATPase activity was associated with a significant decrease in ATP turnover per Na+/K+-ATPase molecule but not number of Na+/K+-ATPase molecules per cell. In summary, airway hyperresponsiveness with or without asthma is associated with increased sodium influx and Na in leukocytes. Resting activity of Na+/K+-ATPase is also increased as a compensatory response to the increased sodium influx, but it is achieved at the expense of higher Nai. Symptomatic asthma is additionally associated with reduction in maximal activity of Na+/K+-ATPase, resulting in reduced capacity to handle the increase in sodium influx and consequent severe elevations in Nai.