Identification of B cell epitopes of Per a 5 allergen using bioinformatic approach.

BACKGROUND: Immune epitopes of allergens are pivotal for development of novel diagnostic and therapeutic modalities. Present study aims to identify antigenic determinants of Per a 5, a clinically relevant cross reactive cockroach allergen. METHODS: The three dimensional structure of Per a 5 was modelled using Modeller 9v11 software. A combination of sequence and structure based computational tools were employed for predicting B cell epitopes. Epitopes were synthesized and immunoreactivity was assessed by ELISA using cockroach hypersensitive patient's sera. Cross-reactivity potential of predicted epitopes was assessed with SDAP and ConSurf and validated by IgE ELISA with fungal and mite hypersensitive patient's sera. RESULTS: Per a 5 structure exhibited good quality factor in ERRAT and high stereochemical stability. In silico analysis revealed six B cell epitopes (BC-P1 to P6). BC-P3 demonstrated significant IgE binding followed by BC-P2 and BC-P1 with cockroach hypersensitive patient's sera. Per a 5 epitopes demonstrate considerable similarity with broad spectrum of allergens from fungal, mites, helminths, fruits and nuts. Analysis of PD values indicate BC-P4 to be well conserved among dust mite and helminth GSTs (8.89, 10.63 and 10.69 with D. pteronyssinus, W. bancrofti and F. hepatica respectively). ConSurf analysis of Per a 5 revealed specific enrichment of evolutionarily similar amino acid residues in BC-P2 (with fungal and mite GSTs) and BC-P4 (with mite and helminth GSTs). Further, IgE binding analysis of epitopes demonstrate BC-P2, BC-P3 and BC-P5 as high IgE binders in fungal hypersensitive sera while BC-P1, BC-P2, BC-P4 and BC-P5 demonstrated significant IgE binding with mite hypersensitive sera. CONCLUSIONS: Among the predicted epitopes, BC-P3 demonstrates maximal IgE binding ability. Computational analysis suggests strong evolutionary conservation and cross reactive potential of BC-P4 with allergens in dust mite and helminths. ELISA highlights predictive potential of analysing evolutionarily conserved residues for uncovering potentially cross reactive antigenic determinants. GENERAL SIGNIFICANCE: Immune epitopes of Per a 5 were identified for aiding molecular diagnosis and potential cross reactivity.

Guidelines for diagnostic flexible bronchoscopy in adults: Joint Indian Chest Society/National College of chest physicians (I)/Indian association for bronchology recommendations.

Flexible bronchoscopy (FB) is commonly performed by respiratory physicians for diagnostic as well as therapeutic purposes. However, bronchoscopy practices vary widely across India and worldwide. The three major respiratory organizations of the country supported a national-level expert group that formulated a comprehensive guideline document for FB based on a detailed appraisal of available evidence. These guidelines are an attempt to provide the bronchoscopist with the most scientifically sound as well as practical approach of bronchoscopy. It involved framing appropriate questions, review and critical appraisal of the relevant literature and reaching a recommendation by the expert groups. The guidelines cover major areas in basic bronchoscopy including (but not limited to), indications for procedure, patient preparation, various sampling procedures, bronchoscopy in the ICU setting, equipment care, and training issues. The target audience is respiratory physicians working in India and well as other parts of the world. It is hoped that this document would serve as a complete reference guide for all pulmonary physicians performing or desiring to learn the technique of flexible bronchoscopy.

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 (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) 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 (P < 0·05) 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.

Immunotherapy with B cell epitopes ameliorates inflammatory responses in Balb/c mice.

Osmotin, a protein from the pathogenesis-related family (PR-5), has been identified as an allergen based on in-silico and in-vitro studies. In the present study, three B cell epitopes of osmotin with single and double amino acid modifications were studied for immunotherapy in a murine model. The single-modification peptides (P-1-1, P-2-1 and P-3-1) and double-modification peptides (P-1-2, P-2-2 and P-3-2) showed significantly lower immunoglobulin (Ig)E binding with patients' sera compared to osmotin (P < 0·01). These peptides showed reduced IgE binding compared to the unmodified peptides (B cell epitopes) P-1, P-2 and P-3. Among the modified peptides, P-2-1, P-3-1, P-2-2 and P-3-2 showed significant reduction in IgE binding and were used for immunotherapy in mice. The sera of mice group treated with peptides showed a significant increase in IgG2a level and a significant decrease in IgE and IgG1 levels (P < 0·05). The mice that received peptide immunotherapy showed a shift from a T helper type 2 (Th2) to Th1 type where interferon (IFN)-γ and interleukin (IL)-10 levels were elevated, with a significant increase in groups treated with peptides P-3-1 and P-3-2 (P < 0·05). There was a reduction in the IL-4 and IL-5 levels in bronchoalveolar lavage fluid (BALF) in the peptide-treated mice groups. Total cell count and eosinophil count in BALF of the peptide-treated groups was also reduced compared to the phosphate-buffered saline (PBS)-treated group. Lung histology showed a significant reduction in cellular infiltrate in mice treated with P-2-2 and P-3-2 compared to PBS. In conclusion, peptides P-2-2 and P-3-2 lowered inflammatory responses and induced a Th1 response in mice.

An Unusual Cause of a Pulmonary Mass: Actinomycosis.

We present the case of a 62-year-old male with chronic obstructive pulmonary disease and poorly controlled diabetes mellitus who presented with haemoptysis. A radiograph of the chest showed a right lower parahilar opacity which on the contrast enhanced computed tomography was seen to be an irregular, spiculated mass localised to the middle lobe. Considering malignancy as the most probable diagnosis, a bronchoscopic endobronchial biopsy was performed which surprisingly established pulmonary actinomycosis as the diagnosis. The patient was successfully managed with amoxicillin and clavulanic acid and glycaemic control.

Sarcoidosis in north Indian population: a retrospective study.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown origin most commonly involving the lungs. Sarcoidosis is frequently misdiagnosed due to its clinico-radiological resemblance to tuberculosis (TB). Hence, the present study was undertaken with the aim of studying the clinico-radiological profile of sarcoidosis in the Indian context. METHODS: We retrospectively studied 146 patients diagnosed to have sarcoidosis during the period 2001-2010 at one of the respiratory units at Vallabhbhai Patel Chest Institute. RESULTS: Majority of them (70%) were more than 40 years of age; females comprised 58.2% of the patients. Before coming to our clinic, 30% patients had been misdiagnosed to have TB. Cough (89.7%) was the most common presenting symptom; joint symptoms (28.8%) and end inspiratory crepitations at lung bases (49.3%) were other salient manifestations. Cutaneous involvement and digital clubbing were rarely seen. Pulmonary function testing showed restriction with impaired diffusion in 72.7% patients. The most common radiological feature was bilaterally symmetrical hilar lymphadenopathy. Transbronchial lung biopsy (TBLB) had a very high diagnostic yield (90.8%). CONCLUSIONS: Sarcoidosis is often misdiagnosed as TB in India. Transbronchial lung biopsy has high diagnostic yield in sarcoidosis.

First human case of pulmonary fungal ball due to a Perenniporia species (a basidiomycete).

Perenniporia species are basidiomycetes, resupinate shelf fungi responsible for white rot decay of wood. Here, we report for the first time an intracavitary pulmonary fungal ball due to a species of Perenniporia that has not been recognized so far as a human pathogen. The fungus was identified by sequencing of the partial ribosomal operon of a culture from a clinical specimen.

A rare case of allergic bronchopulmonary mycosis caused by Alternaria alternata.

A rare case of allergic bronchopulmonary mycosis (ABPM), caused by Alternaria alternata, is reported in an immunocompetent resident of Delhi. Her complaints included a generalized, urticarial skin rash and occasional pain in the right lower chest. Her differential count showed eosinophils, 22%; absolute eosinophil count (AEC), 2400 cells/µl; and total IgE, 4007 IU/ml. The computerised tomogram (CT) scan of her thorax showed an enhancing lesion with surrounding ground glass haziness in the right lower lobe. Histopathologic examination of the resected lung revealed a necrotizing granulomatous inflammation, parenchymal infiltration by eosinophils, lymphocytes, neutrophils, plasma cells and some exudative bronchiolitis suggestive of ABPM. Observation of KOH wet mounts of repeat sputum and BAL samples demonstrated the presence of septate, brownish hyphae and cultures of these specimens yielded A. alternata (identified by sequencing of the ITS region). Her serum showed a three-fold higher specific IgE to A. alternata antigens than control levels, and the type I cutaneous hypersensitivity response to antigens of A. alternata was strongly positive. She was treated successfully with oral glucocorticoids and itraconazole. To our knowledge, ABPM due to Alternaria alternata has not been reported previously.

Choline supplementation reduces oxidative stress in mouse model of allergic airway disease.

BACKGROUND: Asthma is a multi-factorial inflammatory disease associated with increased oxidative stress and altered antioxidant defences. We have evaluated the effect of choline on oxidative stress in a mouse model of airway disease. MATERIALS AND METHODS: Balb/c mice were sensitised with 100 microg of ovalbumin on days 0 and 14, and challenged with aerosolized ovalbumin on days 25-27. Mice were administered 1 mg kg(-1) of choline via oral gavage or intranasal route on days 14-27. Mice were also administered 100 mg kg(-1) of alpha-lipoic acid as standard antioxidant. Total cell counts, eosinophils and eosinophil peroxidase (EPO) activity were determined in bronchoalveolar lavage (BAL) fluid. Reactive oxygen species (ROS), lipid peroxidation and isoprostanes levels were measured in BAL fluid. IL-13 and tumour necrosis factor-alpha (TNF-alpha) levels were also measured in BAL fluid and spleen cell culture supernatant. Nuclear factor kappaB (NFkappaB) p65 protein expression was measured after last ovalbumin challenge in nuclear and cytosolic extracts of lungs. RESULTS: Compared with ovalbumin-challenged mice, choline and alpha-lipoic acid treated mice had significantly reduced eosinophilic infiltration and EPO activity in BAL fluid. Choline and alpha-lipoic acid treatment reduced ROS production and isoprostanes level significantly in BAL fluid and thus suppressed oxidative stress. Choline and alpha-lipoic acid administration by either route decreased lipid peroxidation levels and down regulated NFkappaB activity. Further, choline and/or alpha-lipoic acid treatment suppressed TNF-alpha level significantly as compared with that of ovalbumin-challenged mice. CONCLUSIONS: Choline administration reduces oxidative stress possibly by modulating the redox status of the cell and inhibits inflammatory response in a mouse model.

Indoor air pollution and respiratory function of children in Ashok Vihar, Delhi: an exposure-response study.

The objective of this study was to investigate the effects of indoor air pollution on respiratory function of children (aged 7-15 years). The study took place at Ashok Vihar, an urban locality in the northwest part of Delhi during the summer months of June and July 2004. The team did house visits. The questionnaire, administered at the house itself, asked about the history of smoking in the family, type of cooking fuel used, duration of cooking, ventilation and lighting at the cooking place, and other confounders. In total, 441 children (59% male, 41% female) between ages 7 and 15 years were considered for the study, and a detailed profile was collected. Clinical examination with special reference to respiratory system was done. Pulmonary function tests/peak expiratory flow rates of each child were measured. Indoor air pollutant (suspended particulate matter, SO(2), NO(2)) was measured, and the effect of these pollutants on the children's respiratory function was analyzed. The respiratory health profile suggests that children had cough, sputum production, shortness of breath, wheezing, common cold, and throat congestion. Indoor SO(2) , NO(2), and suspended particulate matter levels were high in houses where there was a family history of smoking. SO(2) level was significantly high according to occupancy per room. NO(2) and suspended particulate matter levels were significantly high in houses where children had respiratory problems. It is concluded that indoor air pollution had an association with respiratory function of children.

Zygomycosis--a case report and overview of the disease in India.

A case of zygomycosis caused by Rhizopus oryzae in a diabetic patient previously misdiagnosed as invasive pulmonary aspergillosis and an overview of the disease in India are presented. The case was diagnosed by direct microscopy, histopathologic examination and culture. Following surgical resection of pulmonary cavity under cover of amphotericin B administration, the patient recovered completely. Of 461 cases reported to-date, approximately 70% had been diagnosed at the Postgraduate Institute of Medical Education and Research, Chandigarh, in north India. This may be attributed to better awareness, expertise and infrastructural facilities for mycological diagnosis than to any particular regional preponderance of the disease. Rhino-orbito-cerebral manifestations were the most common feature of zygomycosis (269 cases), followed by cutaneous disease (66 cases), which is in conformity with the pattern prevalent worldwide. The etiologic agents encountered were Rhizopus oryzae, Apophysomyces elegans, Saksenaea vasiformis, Cunninghamella bertholletiae, Absidia corymbifera, Basidiobolus ranarum and Conidiobolus coronatus. In contrast to cases from the developed world where transplant recipients and patients with haematological malignancies seem to be most vulnerable to zygomycosis, the most common risk factor in India was uncontrolled diabetes mellitus. Amphotericin B was the mainstay of various treatment modalities employed. The relevance of a strong clinical suspicion and early diagnosis of zygomycosis for favourable prognosis can hardly be over-emphasised.

Sarcoidosis presenting as acute bilateral parotid swelling.

A case of bilateral parotid swelling of short duration, which turned out to be sarcoidosis confirmed by fine needle aspiration cytology, is presented here. The patient also had asymptomatic bilateral hilar lymphadenopathy and dryness of the eyes suggesting sarcoid involvement of the lungs and lacrimal glands. Sarcoidosis of the parotids, although rare, should be considered in the differential diagnosis of acute bilateral parotid swelling. Clinico-radiological findings of multi-organ involvement suggest the diagnosis in such cases.

Malignant pleural mesothelioma without asbestos exposure.

A 45-year-old male, non smoker and lecturer by profession was diagnosed as an advanced case of bilateral mesothelioma involving lung and pleura. He was never exposed to asbestos, which makes it a rare case.

Clinico-pathological studies in cattle experimentally infected with Taenia saginata eggs.

Calves 1-2 months old were experimentally infected with eggs of Taenia saginata and clinical and haematological deviations, development and distribution of cysticerci and pathological changes were recorded. The calves infected with 5,000, 10,000 or 50,000 eggs showed an increase in pulse and respiratory rates. The animals that received 50,000 eggs had significantly increased pulse (p < 0.05) and respiratory rates (p < 0.005). The symptoms were more severe in young, 30-day-old calves infected with 50,000 eggs. Haemoglobin concentration, haematocrit values and red blood cell count decreased, but white blood cell count increased slightly. Lymphocytes and eosinophils also increased up to 88% and 14% (p < 0.05), respectively. Most of the cysticerci were not fully formed 1 month post-infection, but at 2 months the cysts were fully mature and at 4 months, some cysts had degenerated. There was no uniform pattern of distribution of cysticerci in the body of infected calves, but the most commonly affected sites were masseter and heart muscles, followed by diaphragm, tongue and other skeletal muscles. The maximum concentration of 8-14 cysticerci per 10 g of tissue was recorded in masseter muscles and heart. The affected parts revealed tissue reactions that included pressure atrophy, necrosis and fibrosis. Microscopically, the lesions comprised infiltration with lymphocytes, plasma cells, eosinophils and macrophages, fibrosis, necrosis and calcification. The tissue reaction was severe in calves infected with 50,000 eggs. The severity of clinical signs, haematological and pathological changes depended mostly on the age of the animals and dose of infection.

Taenia saginata cysticercosis in cattle with special reference to its prevalence, pathogenesis and economic implications in Fars Province of Iran.

The study was carried out during a 3-year period. Of 9501 cattle examined, 736 (7.7%) were infected with cysticerci of Taenia saginata. The endemic foci were identified and prevalence was significantly higher (P < 0.005) in animals from Noorabad Mammasani (10.7%), Kenareh (10.0%) and Shiraz area (8.5%) than elsewhere. The prevalence was significantly higher (P < 0.005) during spring and autumn seasons. There was no variation in the infection rate in animals of different age groups, suggesting that immunity was acquired to super-infection. The most common sites were muscle of the shoulder (26.3%) and masseter (23.7%), tongue (24.9%) and heart (23.4%). Pharynx, oesophagus and diaphragm showed 0.9, 0.5 and 0.4% infection, respectively. The metacestode caused extensive damage resulting in infiltrative, degenerative changes, haemorrhages, necrosis and exudation mainly in the vicinity of cysts. The infection was the cause of condemnation of 34.6% of infected carcases. The rejected carcases and infected organs were valued at 100.1 million Rials over the 3-year period.

Metacestodes of sheep with special reference to their epidemiological status, pathogenesis and economic implications in Fars Province, Iran.

This study was undertaken over a period of 2 years. Of 7992 sheep examined, 2088 (26.12%), 2266 (28.36%), 15 (0.19%) and 738 (9.8%) were infected with Echinococcus granulosus, Taenia hydatigena, Taenia ovis and Taenia multiceps metacestodes, respectively. The prevalence was higher in animals belonging to migratory tribal people than in those owned by the permanent residents of Fars Province. Dogs in the former group also showed significantly higher levels of infection with E. granulosus (33.3%) and Taenia spp. (40.7%) than those in the latter group. The metacestodes caused tissue damage in different organs, resulting in condemnation of meat or viscera valued at 10.2 million rials. The migration of tribal people has a direct bearing on the transmission of these parasites and must be taken into account in control planning.

Countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats.

The procedure of countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats was carried out for antemortem diagnosis of T. hydatigena cysticercosis in experimentally and naturally infected goats. The antigens of cyst fluid, scolex and membrane of T. hydatigena metacestodes were purified and compared. The sensitivity of the test in experimentally and naturally infected goats was 57.1 and 52.5%, respectively, whereas its specificity using antisera raised against T. solium cysticercosis, hydatid cyst and Fasciola gigantica was 66.7 and 83.4% with partially purified and fractionated antigens, respectively. Of all three antigens, the cyst fluid antigen was found to be most reactive. The test could be employed for antemortem diagnosis of T. hydatigena cysticercosis using purified antigen.

Fibrebronchoscopy in pulmonary sarcoidosis--an Indian experience.

Twenty-four patients suspected to have sarcoidosis were subjected to fibrebronchoscopy. Histopathological support for the diagnosis was ultimately obtained in 20 patients. Fibrebronchoscopy provided the diagnosis in 17 patients, while histopathological confirmation was obtained from extrapulmonary biopsy sites in 3 patients. Transbronchial lung biopsy, attempted without fluoroscopic guidance, revealed non-caseating granulomata in 15 patients. The only complication encountered was a small pneumothorax, not requiring intervention, in one patient. Lack of fluoroscopic guidance did not compromise the diagnostic yield or increase the complication rate of the procedure. Bronchial biopsy confirmed the diagnosis in 2 patients with a non-specific lung biopsy. It was positive in 6 of 8 patients with an abnormal appearing mucosa and in 5 of 12 patients with a normal bronchial tree. Random bronchial biopsy in all patients, irrespective of mucosal changes, made an important contribution to the yield of fibrebronchoscopy. Fibrebronchoscopy confirmed the diagnosis of tuberculosis in 2 patients with an atypical radiological picture, thereby differentiating the two conditions which occasionally mimic each other.

Immunization of pigs with culture antigens of Taenia solium.

An evaluation has been made of the protective effect of immunizing pigs with excretory-secretory homologous antigens on Taenia solium infections. This procedure reduced the number of cysticerci established from a challenge infection.

Shared and specific allergenic and antigenic components in the two sexes of American cockroach--Periplaneta americana.

The whole body extracts (WBEs) of female and male cockroaches (Periplaneta americana; Pa) were prepared separately to study the specific and/or shared allergenic and antigenic components in the two sexes. These two extracts were skin-tested on 170 respiratory allergy patients and 52 (30.6%) of them elicited a markedly positive cutaneous reaction (2+ to 4+) to any one or both the WBEs. Of these 52 patients, 32 (61.5%) produced a 2+ to 4+ response to only one, and the remaining 20 (38.5%) to both the extracts. In female and male RASTs to Pa both the extracts produced dose-related inhibition. Using rabbit anti-Pa female serum: (1) immuno-diffusion experiments resulted in lines of identity with the two extracts, and (2) the two-dimensional immunoelectrophoresis of Pa female and Pa male WBEs elicited 12 and nine precipitin peaks, respectively. In partial purification studies, only fraction Pa(F)III (approximately 50 kD) of Pa female WBE and Pa(M)I (greater than or equal to 600 kD) and Pa(M)III (approximately 50 kD) fractions of Pa male WBE revealed significant allergen activity both on skin testing and also in RAST inhibition studies. These results provide evidence for the presence of shared as well as specific allergenic and antigenic components in the two sexes of American cockroach.