B.P. Singh

Serine Protease Activity of Cur l 1 from Curvularia lunata Augments Th2 Response in Mice

RationaleStudies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens.ObjectiveThe objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness.MethodsCur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels.ResultsMice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group.ConclusionProteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

Purification and characterization of a cross-reactive 45-kD major allergen of Fusarium solani.

Background:Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients’ sera sensitive to many fungi. Objectives: To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy. Methods: FS culture filtrate extract was separated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods. Results: The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients’ sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergic patients. Conclusion: A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.

Effect of proteolytic activity of Epicoccum purpurascens major allergen, Epi p 1 in allergic inflammation

Enzymes play an important role in inducing airway inflammation, but knowledge is limited to few proteins. This study was carried out to assess the role of Epi p 1, a serine protease of Epicoccum purpurascens, in inducing allergy and inflammation in a murine model. Balb/c mice were sensitized with Epi p 1 active protease (EAP) or Epicoccum extract. Subsequently, Epi p 1 sensitized mice were boosted on day 14 with EAP or inactivated protease (EIAP). Three intranasal challenges were given and mice were killed to obtain blood, bronchoalveolar lavage fluid (BALF), spleen and lung tissues. Cellular airways infiltration, immunoglobulin E (Ig)E titres and cytokine levels in BALF and splenocyte culture supernatant were compared. Mice immunized with EAP had higher Epi p 1‐specific serum IgE and IgG1 than EIAP immunized mice (P < 0·01). There was a twofold difference in the number of eosinophils in BALF of EAP mice and EIAP mice (P < 0·01). A similar trend was recorded for eosinophil peroxidase activity (P < 0·05), indicating the role of proteolytic activity in inducing inflammation. Further, lung histology revealed increased leucocyte infiltration and airway narrowing, with higher inflammation scores in the EAP group than in the EIAP group. The lungs of EAP mice showed increased mucus and goblet cell metaplasia. Interleukin (IL)‐4 and IL‐5 levels were higher in BALF and splenocyte culture supernatant of EAP mice than in EIAP mice (P < 0·05), indicating a T helper 2 response. Proteolytic activity of Epi p 1 plays an important role in inducing allergic inflammation. The enzymatically inactive form may be investigated for immunotherapy.

Characterization of Cogon Grass (Imperata cylindrica) Pollen Extract and Preliminary Analysis of Grass Group 1, 4 and 5 Homologues Using Monoclonal Antibodies to Phleum pratense

Background: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. Objectives: To characterize the IgE–binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. Methods: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme–linked immunosorbent assay (ELISA), ELISA–inhibition, thin–layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well–known group 1, 4, and 5 allergen homologues in Ic pollen extract. Results: Out of 303 respiratory allergies patients skin–tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver–stained bands (pI 3.5–7.0) while SDS–PAGE resolved it into 24 Coomassie–Brilliant–Blue–stained bands (MW 100–10 kD). Immunoblotting with individual patient sera recognized 7 major IgE–binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. Conclusion: Ic pollen extract was characterized for the protein profile by TLIEF and SDS–PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

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 400 ng of protein for 50% inhibition in ELISA assay. Immunoblot and immunoblot inhibition demonstrated the presence of 20, 26, 35, 66 and 72 kDa 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.

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.

Standardizing Imperata cylindrica--source material for quality allergen preparations.

BACKGROUND Tropical countries experience wide variations in daytime temperature and relative humidity. This affects the quality of the source material used for allergen extracts. The present study was undertaken to standardize the processing and preservation conditions of Imperata cylindrica grass pollen. METHODS I. cylindrica (Ic) inflorescence were freeze-dried, pollens sieved out and stored at -70 degrees C (IcA). Alternatively, the inflorescence were dried at room temperature and then at 37 degrees C, pollens sieved out and stored at 4 degrees C (IcB). The extracts prepared in PBS were analyzed in vivo by skin tests and in vitro by immunochemical methods. RESULTS Reduced SDS-PAGE revealed 37 protein bands in IcA extract and 23 in IcB extract. IgE immunoblot with a pool of sera from Ic hypersensitive patients showed 30 allergenic bands in IcA and 14 in IcB. Immunoblot using anti-Ic rabbit sera revealed 33 antigenic bands in IcA and 22 in IcB. In both blots, the IcA extract exhibited sharp bands and the IcB extract exhibited diffuse bands. ELISA, ELISA inhibition and skin test procedures showed that IcA extracts had a higher potency than IcB extracts. CONCLUSIONS Extracts prepared from -70 degrees C processed and preserved pollens (IcA) are allergenically more potent and contain a greater number of major and minor allergens than IcB extracts.

Mutated glutathione-S-transferase reduced airway inflammation by limiting oxidative stress and Th2 response.

Oxidative stress is an important factor in the pathogenesis of asthma. Furthermore, antioxidants like GST are reduced in asthma patients. In the present study, the therapeutic effects of exogenous GST and mGST were evaluated in a mice model. GST mutated at residues 21/27 has reduced IgE binding with similar enzyme activity as that of GST. To evaluate the therapeutic effects of GST, BALB/c mice were immunized and challenged with ovalbumin. Mice were given GST, mGST, and alpha-lipoic acid by inhalation and sacrificed on Day 31 to evaluate inflammation and oxidative stress. Mice treated with mGST showed significantly reduced total cell count (P<0.01) and eosinophils (P<0.01) in BALF compared to GST- or PBS-treated groups. The lung inflammation score was lowest for the mGST-treated group along with reduced IL-4 (P<0.01) and OVA-specific IgE than other groups. Oxidative stress as per the lipid peroxidation level in BALF of mGST-treated mice was reduced significantly in comparison to PBS- or GST-treated mice. In conclusion, inhalation of mGST reduced airway inflammation in mice. Mutated GST with reduced allergenicity has better therapeutic potential and can be explored as an adjunct therapy in asthma.

Outcome of management of distal humerus fractures by locking compression plate

Background: Distal humerus fracture is one of the commonest fractures among young adults and which accounts for about 30% of all elbow fractures. The treatment of these fractures continues to the challenges for orthopedics despite of many advances in technique and implants. Different modalities like 1/3rd tubular plate, reconstruction plate, K wires, double tension band wiring, etc, have been tried. The new distal humerus locking compression plate (LCP) system allows angular stable fixation of these complex fractures with anatomically pre-shaped plates. Aims: Aim of this study is to assess the benefits of using locking compression plate (LCP) in the management of distal humerus fractures clinically and as well as functionally. Materials and Methods: A prospective study of 30 adult patients with closed distal humerus fractures were treated by locking compression plate in the department of Orthopaedics, RIMS, Ranchi, between June 2014 to Sep 2015. Variables of each patient were recorded and analysed with respect to age, sex, fracture type, mode of injury, limb involvement, associated injuries, timing and duration of operation, duration of hospital stay, follow up, complications and final outcomes. These patients were followed up at different intervals i.e. at 3 weeks for first 3 months, then at 6-weeks interval for next 6 months and then at 3-month interval. Results: The average age was 38.5 years and majority patients were men (60%). The right humerus was involved in majority (70%) of patients. The complete union was achieved in all patients which was confirmed by radiographically. Average time interval between admission of patients and surgery was 7.8 days (range 4-13 days). The average operative time was 82 minutes (range 70-100 min). All the fractures as well as the olecranon osteotomies united at 12-18 weeks (average 13.80 weeks). There were no any case of primary malposition or secondary dislocation was observed. Using the Mayo elbow performance score, the majority (53.33%) of patients were graded as excellent. There was no any patient reported with deep infection, implant failure, non-union of fracture site or olecranon osteotomy site. There were only three patients those reported superficial wound infection, which was treated with antiseptic dressing and antibiotics. Transient ulnar nerve palsy developed in only 2 (6.66%) cases and both were recovered with conservative treatment. Conclusion: Findings can be concluding that the treatment of distal humerus fractures is a challenging task. Anatomically preshaped distal humeral locking compression plate system facilitates operative reduction and stabilization of the fracture and may allow with good range of motion, and flexion and extension force.