Expression levels of vascular endothelial growth factors A and C in patients with peptic ulcers and gastric cancer

Purpose: Vascular endothelial growth factor (VEGF) is one of the most important growth factors for metastatic tumors. To clarify the role of VEGF-A and C in patients with peptic ulcer disease (PUD) or gastric cancer (GC), we evaluated the expression levels of these two molecules. We also analyzed the effect of Helicobacter pylori infection on VEGF-A and C expression levels

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

Production and Characterization of Monoclonal Antibody against Vit v1: A Grape Allergen Belonging to Lipid Transfer Protein Family

Allergy to non-specific lipidtransfer protein (nsLTP), the major allergen of grape (Vit v1), is considered as one of the most common fruit allergies in Iran. Therefore, a specific monoclonal antibody (mAb) can be used for the characterization and assessment of. Accordingly, this study aimed to generate and characterize a mAb against Vit v1 with a diagnostic purpose. To this end, Vit v1 allergen (9 kDa) was extracted using a modified Bjorksten extraction method. Natural Vit v1-immunized mouse splenocytes were fused with SP2/0Ag-14 myeloma cells for generating hybridoma cells. Specific antibody-secreting Hybridoma cells were selected using ELISA. Finally, anti-Vit v1 mAb was characterized by western blotting, ELISA, and isotyping methods. In the current study, a 9 kDa (Vit v1) protein was attained fromcrude and fresh juice of grape extracts and the isotype of desired anti-Vit v1 mAb was determined as IgM with K light chain. In addition, The ELISA results demonstrated that anti-Vit v1 mAb was specified against natural Vit v1 in the grape cultivar and related LTP allergens, such as Pla or 3 (p<0.0001). In the present study, a specific mAb was produced for detecting the LTP allergen. This mAb with a confirmed specificity can be utilized for evaluating the LTP allergens and their allergenicity in different grape cultivars. © 2020 Tehran University of Medical Science

Molecular Cloning and Expression of Cro s 1: an Occupational Allergen from Saffron Pollen (Crocus sativus)

Background: The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. Methods: The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. Results: The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. Conclusion: We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron

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

An approach for detection and quantification of fruits' natural profilin: Natural melon profilin as a model

Anaphylactic reactions are of great importance in clinical allergology. Measurement of allergen concentration in foods is helpful to estimate their allergenicity. In this study, a sandwich ELISA for the evaluation of profilin concentration in fruits was designed. A mixture of monoclonal antibodies against saffron profilin was used as a capture antibody. The recombinant melon profilin was expressed in Escherichia coli BL-21 and applied as a standard antigen along with different fruit extracts to measure their profilin concentration. Furthermore, a polyclonal antibody against the recombinant melon profilin was produced and applied as the secondary antibody. In this way, the profilin concentration of various fruits including banana, tomato, kiwi, mulberry, cantaloupe, peach, watermelon and zucchini was measured. The results showed that the designed ELISA had a sensitivity threshold of about 1 ng/ml for the melon profilin and it is therefore suggested for determination of profilin concentration in other fruits. © 2011 Taylor & Francis

Carvacrol ameliorates experimental autoimmune encephalomyelitis through modulating pro- and anti-inflammatory cytokines

Aim: The inflammatory process is a key step in multiple sclerosis (MS) development. Carvacrol exhibits various anti-inflammatory properties. We aimed to assess the Carvacrol effects on clinical manifestations and production of pro-inflammatory (IFN-γ, IL-6 and IL-17) and anti-inflammatory (TGF-β, IL-4, and IL-10) cytokines in experimental autoimmune encephalomyelitis (EAE) as MS animal model. Main methods: EAE mice were treated with 5, 10 mg/kg dose of Carvacrol or vehicle, as the control EAE group, every other day until day-21 post EAE induction. On day22, the leukocyte infiltration within the CNS was estimated using hematoxylin-eosin staining. The cytokine production by splenocytes was determined after in vitro stimulating with myelin oligodendrocyte protein (MOG). Key findings: The EAE clinical scores in 5 and 10 mg/kg Carvacrol-treated mice were lower than untreated group (P < 0.001 and P < 0.01, respectively). The amounts of IFN-γ and IL-6 production by splenocytes of 5 and 10 mg/kg Carvacrol-administered mice were lower than control group (P < 0.001, and P < 0.01 for IFN-γ respectively; P ˂ 0.05 for IL-6). Splenocytes of 5 and 10 mg/kg Carvacrol-treated mice produced higher levels of TGF-β than untreated mice (P < 0.001). in splenocytes of 5 mg/kg Carvacrol-treated group the IL-10 production was higher while IL-17 secretion was lower than control group (both with P < 0.01). Significance: Carvacrol exhibits modulatory effects on expression of pro- and anti-inflammatory cytokines. It ameliorates EAE clinical and pathological consequences and therefore its potentials may be considered in treating MS patients

Immune responses modulation by curcumin and allergen encapsulated into PLGA nanoparticles in mice model of rhinitis allergic through sublingual immunotherapy

The purpose of this study was the combination of curcumin and ovalbumin in free form or encapsulated into PLGA NPs (polylactic co-glycolic acid nanoparticles) to enhance their sublingual immunotherapy (SLIT) efficiency in mouse model of rhinitis allergic. PLGA NPs containing curcumin (CUR), ovalbumin (OVA) or both were prepared by emulsion-solvent evaporation method and characterized. After sensitization of BALB/C mice with ovalbumin, SLIT with free or encapsulated formulations was carried out and immunological profiles were evaluated. SLIT treatment with all synthesized PLGA formulations lead to significantly decreased total IgE. The combination immunotherapy in the present of free form of curcumin or ovalbumin with encapsulated forms of the another substance (P.OVA-CUR 10 and P.CUR 5-OVA), showed the highest level of IFN-γ:IL-4 compared to other target groups. On the other hands, a significant increasment was observed in this ratio between these optimal groups and treated group with subcutaneous administration of OVA as the most commonly used method for immunotherapy. The study of nasal lavage fluid (NALF) showed significant decreased levels of total and eosinophil cell count in the traeted nano-formulation groups. The histopathological results of NAL were also like normal with no cellular infiltration and no inflammation in the optimal formulations. Therefore, using curcumin and nanoparticles with allergen can be considerd as potential immune modulatory agents. © 2020 Elsevier B.V

Apoptosis of Human Lymphocytes after Exposure to Hydatid Fluid

Background: Modulation of the immune response is an important strategy by which establish­ment and growth of hydatid cyst in the internal organs of human is warranted. Induction of apop­tosis in the lymphocytes might be a considerable component. This study was designed to evaluate apoptotic impact of hydatid fluid (HF) on human lymphocytes.Methods: Human lymphocytes were treated with hydatid fluid. After 6 hours of exposure, cas­pase-3 activity, the central enzyme of apoptosis cascade, was measured by fluorometric assay in the HF-treated lymphocytes and control cells. In addition, the expression of Bax (a pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) mRNA was assessed by RT-PCR after 12 hours of exposure.Results: Both the ratio of Bax/Bcl-2 mRNA expression and Caspase-3 activity were higher in the HF-treated lymphocytes relative to the control group.Conclusion: Apoptosis could be as a possible mechanism by which Echinococcus granulosus overwhelms host defenses

Supplementary Material for: Immunotherapy with a Recombinant Hybrid Molecule Alleviates Allergic Responses More Efficiently than an Allergenic Cocktail or Pollen Extract in a Model of <b><i>Chenopodium album</i></b> Allergy

<b><i>Background:</i></b> The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on <i>Chenopodium album</i> allergy, using a BALB/c mouse model. <b><i>Methods:</i></b> The BALB/c mice had already been sensitized to <i>C. album</i> via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR. <b><i>Results:</i></b> Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or <i>C. album</i> pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased. <b><i>Conclusion:</i></b> Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response