Assessment of IgE- and cell-mediated immunity in pediatric patients with eosinophilic esophagitis.

Summary: Eosinophilic esophagitis (EoE) is a chronic allergen/immune-mediated disease leading to esophageal dysfunction. Food allergens play critical roles in the pathogenesis and treatment of EoE via different mechanisms. This study aimed to present the characteristics and evaluate the ability of skin prick test (SPT), skin prick to prick test (SPP) (IgE-mediated), and atopic patch test (APT) (cell-mediated) individually or simultaneously to diagnose food allergy in patients suffering from EoE. This prospective study was conducted on 58 patients with EoE. Seven patients (12.1%) were positive to only one, 3 (5.2%) were simultaneously positive to two, and 32 (55.2%) were simultaneously positive to three tests. Single and double sensitizations were totally 10.4% in IgE-mediated reactions, while 36.5% in cell-mediated reactions. In contrast, poly sensitization (> 2 allergens) was 51.7% in IgE-mediated tests and 20.7% in the cell-mediated test. Multiple sensitization findings showed egg white, milk, yolk, and soy were the most frequent allergens. Our findings indicate that EoE is early onset and associated with multiple food sensitizations, particularly via IgE-mediated mechanisms. These immune-mediated responses encompass both IgE-mediated (SPT and SPP) and cell-mediated (APT) reactions simultaneously not individually. Therefore, employing multiple assays may strengthen the diagnosis of food sensitization.

Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen

Introduction: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. Methods: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. Results: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10 kDa protein in crude extract. These results were confirmed by inhibition methods, too. Conclusion: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart

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Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen.

INTRODUCTION: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. METHODS: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. RESULTS: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. CONCLUSION: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

Iranian HIV/AIDS patients with oropharyngeal candidiasis: identification, prevalence and antifungal susceptibility of Candida species.

Oropharyngeal candidiasis is the commonest mucocutaneous infection in HIV-positive individuals. Herein, samples were taken from oral cavities of 150 HIV-infected patients and cultured on Sabouraud-dextrose agar; 89 (59·3%) of 150 patients had positive culture for Candida and presented clinical sign of classical oral candidiasis. Totally, 102 morphologically distinct colonies were isolated from Candida positive cultures and subsequently identified by polymerase chain reaction and sequencing assay, presenting the following frequency: 54 C. albicans (52·9%), 16 C. dubliniensis (15·7%), 12 C. tropicalis (11·8%), 9 C. glabrata (8·8%), 7 C. kefyr (6·9%) and 4 C. africana (3·9%). Additionally, multiple Candida species were co-isolated from 13·5% (12/89) patients. Regarding the antifungal susceptibility test, which was performed by CLSI protocol (M27-A3/M27-S3), all Candida isolates were susceptible to amphotericin B and caspofungin, while some of them were resistant to fluconazole (17·6%; 16 C. albicans, 1 C. dubliniensis and 1 C. glabrata), itraconazole (16·7%; 15 C. albicans, 1 C. dubliniensis and 1 C. tropicalis) and voriconazole (5·9%; 5 C. albicans and 1 C. tropicalis). Collectively, our findings reinforce the urgent necessity to find new therapeutic agents to treat oral candidiasis in HIV-positive patients, especially due to the high incidence of azole-resistant Candida strains and the increased frequency of non-C. albicans species. SIGNIFICANCE AND IMPACT OF THE STUDY: The Candida species recovered from oral cavity of 150 Iranian HIV/AIDS patients and their antifungal susceptibility profiles were reported. Candida albicans was the commonest Candida species, followed by C. dubliniensis, C. tropicalis, C. glabrata, C. kefyr and C. africana. All Candida isolates were susceptible to amphotericin B and caspofungin, while resistance to azoles was detected. The growing drug-resistance profile reported in clinical isolates of C. albicans and non-C. albicans strains is a serious problem in hospitals worldwide. Consequently, the suitable antifungal choice to treat the HIV/AIDS population with oral candidiasis needs to be rethought and new therapeutic options must urgently arise.