Cloning and Expression of Ama r 1, as a Novel Allergen of Amaranthus retroflexus Pollen

Sensitisation to Amaranthus retroflexus pollen is very common in tropical and subtropical countries. In this study we aimed to produce a recombinant allergenic Ole e 1-like protein from the pollen of this weed. To predict cross-reactivity of this allergen (Ama r 1) with other members of the Ole e 1-like protein family, the nucleotide sequence homology of the Ama r 1 was investigated. The expression of Ama r 1 in Escherichia coli was performed by using a pET-21b(+) vector. The IgE-binding potential of recombinant Ama r 1 (rAma r 1) was evaluated by immunodetection and inhibition assays using 26 patients' sera sensitised to A. retroflexus pollen. The coding sequence of the Ama r 1 cDNA indicated an open reading frame of 507 bp encoding for 168 amino acid residues which belonged to the Ole e 1-like protein family. Of the 26 serum samples, 10 (38.46) had significant specific IgE levels for rAma r 1. Immunodetection and inhibition assays revealed that the purified rAma r 1 might be the same as that in the crude extract. Ama r 1, the second allergen from the A. retroflexus pollen, was identified as a member of the family of Ole e 1-like protein. © 2016 Payam Morakabati et al

Cloning and expression of Aca f 1: A new allergen of Acacia farnesiana pollen

Acacia farnesiana is the main source of allergenic pollen and one of the most important causes of respiratory allergic disease in tropical and subtropical regions of the world. The purpose of this study was to produce a recombinant variety of allergenic Ole e 1-like protein from the pollen of this tree. To predict its allergenic cross-reactivity with other members of the Ole e 1-like protein family of common allergenic plants, the nucleotide sequence homology of the Acacia Ole e 1-like protein was evaluated. Amplification of cDNA strands encoding Acacia Ole e 1-like protein was performed by polymerase chain reaction (PCR) and sequenced. Following expression in Escherichia coli using the pET-21b(+) vector, the recombinant protein was purified using metal-affinity chromatography. IgE-binding competence of purified recombinant Ole e 1- like protein (rAca f 1) was analysed by immunoassay using 25 sera collected from Acacia pollen-sensitised patients. Nucleotide sequencing revealed an open reading frame of 453 bp encoding 150 amino acid residues that belonged to the Ole e 1-like protein family, and 11 patients (44) had considerable specific IgE levels for the rAca f 1. Immunodetection and inhibition assays indicated that the purified rAca f 1 may be the same as that in the crude extract. Aca f 1, the second allergen from Acacia pollen, was identified as a member of the family of Ole e 1-like protein. A high degree of homology was found among amino acid sequences of Aca f 1 and several allergenic members of Ole e 1-like protein family

Identification of methionine synthase (Sal k 3), as a novel allergen of Salsola kali pollen

Salsola kali pollen is a common cause of pollinosis during summer and early fall in desert and semi-desert regions. The aim of this study was the identification and characterization of Sal k 3, a new allergen from S. kali pollen. S. kali pollen extract was fractionated by SDS-PAGE and the allergenic profile was determined by IgE-immunoblotting using twelve S. kali allergic patients. Protein identification was carried out by the means of mass spectrometry. Using degenerated primers, two DNA fragments encoding N- and C-terminal domain of Sal k 3 were amplified by PCR, then cloned into the PTZ57R/T vector and sequenced. The open reading frame of Sal k 3 fragments were subcloned in the pET-32b(+) vector, expressed in E. coli, and purified by Ni2+ affinity chromatography. The IgE-binding capacity of rSal k 3 fragments was then studied by IgE-immunoblotting, inhibition assays, and skin prick tests. A 45-kDa allergen was identified as a fragment of the cobalamin-independent methionine synthase (MetE) by mass spectrometry and was detected in the sera of 8/12 (66.6) of S. kali allergic patients. Moreover, inhibition assays demonstrated that the purified rSal k 3 fragments were similar to their counterparts in the crude extract. Sal k 3 represents a new allergen of S. kali pollen and seems to be an important allergenic compound in S. kali pollen. © 2010 Springer Science+Business Media B.V

Prosopis juliflora pollen allergy

Prosopis is a genus of flowering plants belonging to the Fabaceae family which is found in warm climate in arid and semi-arid regions. Prosopis juliflora (Mesquite), one of the most allergenic species of Prosopis, has been identified as an important source of pollen allergens. The mesquite is native from arid regions of Southwestern United States and Mexico but has now acclimatized to various regions in Asia and the States of the Indian sub-continent. Prosopis juliflora is commonly planted as a roadside and an ornamental shade tree in parks and gardens. Flowering occurs twice a year, primarily in spring and early summer. To date, several researches from various countries have reported that mesquite pollen is one of the most important sources for triggering respiratory allergies such as seasonal allergic rhinitis and asthma. It has been documented that the frequency of sensitization to mesquite pollen among allergic patients in different countries ranges from 24 to 66. This chapter reviews the previous studies on prevalence of sensitivity to Prosopis juliflora pollen in the world. Moreover, the results of the studies on immunochemical properties, molecular characterization and cross-reactivity of Prosopis juliflora pollen grains are also presented. Few studies on the analysis of allergenic proteins of Prosopis juliflora pollen have shown several components with molecular weights ranging from 10 to 99 kDa with IgE-binding properties. In addition, Prosopis juliflora tree pollen proteins exhibit significant IgE cross-reaction with those of other plants such as Acacia farnesiana (Needle bush), Ailanthus excelsa (Tree of heaven), Cassia siamea (Kassod tree) and Salvadora persica (Mustard tree). Up to date, Pro j 2, the first reported allergen from Prosopis juliflora pollen, was identified as belonging to the family of profilins. Moreover, another allergen from Prosopis juliflora pollen was named Pro j 1 in accordance with the International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee. Pro j 1 is a member of the Ole e 1-like protein family. Regarding the extensive cross reactivity among Prosopis juliflora and the other plants and the abundance of them in arid and semi-arid regions, the recognition of allergenic components of pollen grains is essential for exploration of new guidelines for diagnostic, therapeutic, and preventive purposes. © 2021 Nova Science Publishers, Inc

Bevacizumab regulates inflammatory cytokines and inhibits VEGFR2 signaling pathway in an ovalbumin-induced rat model of airway hypersensitivity

Background: Bevacizumab with anti-angiogenesis properties reduces the vascular endothelial growth factor (VEGF) level and has widely been used to treat various diseases such as lung diseases and chronic obstructive pulmonary disease (COPD). This study, therefore, aimed to consider the effects of bevacizumab on VEGF receptor 2 (VEGFR2) and lung inflammation of the ovalbumin-induced rat model of airway hypersensitivity. Materials and methods: Twenty-one male Wistar rats were randomly divided into 3 groups (n = 7 in each group): (1) control, (2) ovalbumin (OVA)-sensitized, and (3) OVA-sensitized with bevacizumab (OVA + Bmab). Groups 2 and 3 were sensitized with ovalbumin (OVA) and aluminum hydroxide on days 1, 8 and challenged with OVA on day 15 by atomization for 10 days (inhalation). After OVA sensitization, the OVA + Bmab was treated with bevacizumab for 2 weeks. VEGFR2 was semiquantitatively analyzed in the lungs by immunohistochemistry. VEGF was measured in the lung tissue by ELISA method. The mRNA of IL-10 and IL-6 lung tissue were measured by real-time PCR. Results: Ovalbumin exposure promoted the expression of VEGF and resulted in inflammatory factors overexpression (p � 0.05). However, rats in OVA + Bmab group showed significantly a decrease in VEGFR2 and IL-1β, IL-6, TNFα, and an increase in IL-10 (p � 0.05). Conclusion: The results show that bevacizumab efficiently diminishes bronchial inflammation via reducing the expression of VEGFR2, and IL-6 genes and enhancing the expression of IL-10 gene. Hence, bevacizumab could be considered as a potential candidate drug to control pathological conditions relevant to airway hypersensitivity. © 2021, The Author(s), under exclusive licence to Springer Nature Switzerland AG

Immunochemical characterization of Amaranthus retroflexus pollen extract: Extensive cross-reactive allergenic components among the four species of Amaranthaceae/Chenopodiaceae

The importance of Amaranthus retroflexus pollen in causing respiratory allergy has been well ascertained in many countries including Iran with a high positive rate (69) among Iranian allergic patients. The aim of the present study is to identify the allergenic properties of A. retroflexus pollen. Sixteen patients with allergy to A. retroflexus pollen were selected for the study. The antigenic and allergenic profiles of the A. retroflexus pollen extract as well as pollen extracts from other species of the Amaranthaceae/Chenopodiaceae family, including Chenopodium album, Kochia scoparia, and Salsola kali, were evaluated by ELISA, immunoblotting, and immunoblot inhibition assays. The resolved protein fractions on SDS-PAGE ranged from 10-85 kDa. Several allergenic components (MW 85, 45, 39, 18, 15, and 10 kDa) of the A. retroflexus pollen extract were recognized by using patients' sera by specific antibody of IgE class using ELISA and immunoblot assays. The IgE reactivity of the A. retroflexus pollen extract was partially inhibited by all three pollen extracts tested. the inhibition by the S. kali pollen extract was more than those by other pollen extracts. Moreover, the wheal diameters by the A. retroflexus pollen extract were highly correlated with those by C. album, K. scoparia and S. kali pollen extracts. In conclusion, three proteins with apparent MWs of 39, 45, and 66 kDa are suggested as the common allergenic components among the four pollens from the Amaranthaceae/Chenopodiaceae family. It appears that there are some common (similar) epitopes among the four common allergenic pollens. Copyright© 2010, Iranian Journal of Allergy, Asthma and Immunology. All rights reserved

Induction of IL-12 from human monocytes after stimulation with Androctonus crassicauda scorpion venom

The objective of this study was to evaluate the capacity of venom from Androctonus crassicauda to induce expression/production of interleukin (IL)-12 by isolated human monocytes. For this purpose, isolated human monocytes were exposed to different concentrations of the venom (0.16-20 μg/ml) for varying periods (6, 12, and 24 h). Apart from measures of venom cytotoxicity (i.e., lactase dehydrogenase activity LDH release), measures of IL-12 p40 mRNA (by Real-time PCR) of IL-12 release (by ELISA) were performed. The results showed that the venom produced significant concentration- and duration of incubation-dependent cytotoxicity. Expression of IL-12 p40 mRNA was significantly increased at all exposure timepoints relative to that in unexposed cells, but was maximal after 6 h of exposure. At that timepoint, the effect from a dose of 2.5 μg venom/ml provided the maximal increase among all doses tested. At the level of the protein itself, IL-12 production remained almost consistently elevated (vs. unexposed control values) across all exposure timepoints, with the greatest formation again occurring after 6 h of incubation at a dose of 2.5 μg venom/ml. The findings from this study demonstrated that venom from the A. crassicauda scorpion contained active constituents that could induce a sustained activation of human monocytes that was manifested, in part, as promotion of the expression/production of IL-12. © 2015 Published by Elsevier Ltd

Molecular Cloning and Expression of Pro j 1: A new Allergen of Prosopis juliflora Pollen

Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family. © Spring 2016, Iran J Allergy Asthma Immunol. All rights reserved

The Impact of Interleukin (IL)-33 Gene Polymorphisms and Environmental Factors on Risk of Asthma in the Iranian Population

Background: Airway epithelial cells secrete Interleukin-33 in response to the different allergens. Several single nucleotide polymorphisms (SNP) of this cytokine have been reported to be involved in the development of asthma. We conducted this study to evaluate the impact of the two most common SNPs of the IL-33 gene (rs1342326 and rs3939286) and environmental factors on the susceptibility to asthma in the Iranian population. Subjects and Methods: In this study, we enrolled 126 asthmatics patients and 300 age, sex-matched controls. Genotyping was performed by real-time PCR using the TaqMan SNP genotyping assay. Moreover, total serum IgE level, eosinophil count, and skin prick test were accomplished and complete history was taken from all the participants. Results: The frequencies of mutant genotypes in both SNPs were significantly higher in asthmatics than controls. C/C genotype of rs1342326 OR (95% CI) 2.50 (1.33�4.69) and A/A genotype of rs3939286 OR (95% CI) 2.18 (1.05�4.52) were associated with higher risk of asthma development. While A/C+C/C genotype of rs1342326 was more prevalent in mild asthma OR (95% CI) 2.36 (1.14�4.89), G/A+A/A genotype of rs3939286 was associated with increased risk of moderate and severe asthma OR (95% CI) 2.53 (1.30�4.94). Conclusion: This study revealed that both IL-33 SNPs were associated with an increased risk of asthma. The rs1342326 was associated with atopic, mild and adult-onset asthma and a higher level of eosinophils in peripheral blood. However, rs3939286 was more frequent in moderate and severe asthma. Moreover, rs3939286 was associated with non-atopic and childhood-onset asthma. © 2019, Springer Science+Business Media, LLC, part of Springer Nature