Publications by authors named "Abdol-Reza Varasteh"

36 Publications

A novel formulation of Mtb72F DNA vaccine for immunization against tuberculosis.

Iran J Basic Med Sci 2020 Jun;23(6):826-832

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Objectives: (), an intracellular pathogen, causes 1.5 million deaths globally. Bacilli Calmette-Guérin (BCG) is commonly administered to protect people against infection; however, there are some obstacles with this first-generation vaccine. DNA vaccines, the third generation vaccines, can induce cellular immune responses for tuberculosis (TB) protection. In this study, optimized DNA vaccine (pcDNA3.1-Mtb72F) entrapped in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was used to achieve higher immunogenicity.

Materials And Methods: Plasmid Mtb72F was formulated in PLGA NPs using double emulsion method in the presence of TB10.4 and/or CpG as an adjuvant. Female BALB/c mice were immunized either with NP-encapsulated Mtb72F or naked Mtb72F with or without each adjuvant, using the BCG-prime DNA boost regimen.

Results: These NPs were approximately 250 nm in diameter and the nucleic acid and protein encapsulation efficiency were 80% and 25%, respectively. The NPs smaller than 200 nm are able to promote cellular rather than humoral responses. The immunization with the formulation consisting of Mtb72F DNA vaccine and TB10.4 entrapped in PLGA NPs showed significant immunogenicity and induced predominantly interferon-ɣ (IFN-ɣ) production and higher INF-ɣ/interleukin-4 (IL-4) ratio in the cultured spleen cells supernatant.

Conclusion: PLGA NPs loaded with Mtb72F DNA-based vaccine with TB10.4 could be considered as a promising candidate for vaccination against TB. These results represent an excellent initial step toward development of novel vaccine for TB protection.
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http://dx.doi.org/10.22038/ijbms.2020.41806.9881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351443PMC
June 2020

Investigation of the Effects of Apple Trees Infection by on the Expression of Pathogenesis-Related Proteins Homologous to Allergens.

Rep Biochem Mol Biol 2020 Jan;8(4):376-382

Varastegan Institute for Medical Sciences, Mashhad, Iran.

Background: Pathogenesis-related (PR) proteins are induced in response to biotic and abiotic stresses. Some plant proteins, including Mal d 1, Mal d 2, and Mal d 3 in apple, are allergens. In this study, the effects of infection of two apple cultivars, Red and Golden Delicious, on the expression of PR proteins homologous to Mal d 1, 2, and 3 were investigated.

Methods: In natural conditions trees with or without disease symptoms were sampled. In addition, seeds of the cultivars were grown in a greenhouse and seedlings were examined in three groups: 1) those inoculated by , 2) those inoculated by sterilized distilled water, and 3) uninoculated. Real-time PCR was used to determine expression of the Mal d 1, 2, and 3 genes (, and 3) in infected and uninfected samples. Statistical analyses were performed using SPSS and graphs were produced by Excel. P values < 0.05 were considered significant.

Results: The analysis of variance showed that in natural conditions the effect of infection on the mean relative expression of and 3 was significant, and more so in Red than in Golden Delicious. The analysis of variance of the greenhouse samples showed that the effect of infection on the mean relative expression of , and in both cultivars was significant.

Conclusion: Our results suggest that Mal d 2 is more related to plant defense than Mal d 1 or Mal d 3, and is more highly expressed in -resistant than in -sensitive cultivars.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275835PMC
January 2020

Sublingual dendritic cells targeting by aptamer: Possible approach for improvement of sublingual immunotherapy efficacy.

Int Immunopharmacol 2020 Aug 30;85:106603. Epub 2020 May 30.

Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

The efficacy improvement of current sublingual immunotherapy (SLIT) for preventing and treating respiratory airway allergic diseases is the main purpose of many investigations. In this study, we aimed to assess whether ovalbumin (Ova) encapsulated poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) decorated with dendritic cells (DCs)-specific aptamer could be applied for this purpose.The nanoparticles containing Ova were synthesized by emulsion/solvent evaporation method and attached to DCs-specific aptamer. Ova-sensitized BALB/c mice have been treated in five ways: subcutaneously with free Ova (SCIT), sublingually either with free Ova, Ova-PLGA NPs (two doses), Apt-Ova-PLGA NPs (two doses) and placebo/control Apt-Ova-PLGA NPs. For assessment of immunologic responses, IL-4, IFN-γ, IL-17, IL10, and TGF-β and IgE antibody levels were measured by ELISA and T cell proliferation were evaluated by MTT. In addition, lung and nasal histological examinations, NALF cells counting were carried out. Results declared that the lowest IgE and IL- 4 levels were observed in Apt-Ova-PLGA NPs (both doses). In the other hands, Apt-Ova-PLGA NPs (high dose) showed the highest increase of IFN- γ and TGF- β, decrease of IL-17 levels, total cell count and T-cell proliferation. IL-10 levels showed more decrease in SCIT, Apt-Ova-PLGA NPs (high dose) and Ova-PLGA NPs (high dose) than other groups. Histopathological examinations also confirmed in vitro results. Our findings suggest SLIT with this functionalized delivery system could be a promising approach for promoting the SLIT efficiency by decreasing the required allergen doses through specific delivery of allergen to sublingual DCs and enhancing the suppression of allergic responses.
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http://dx.doi.org/10.1016/j.intimp.2020.106603DOI Listing
August 2020

Dc-specific aptamer decorated gold nanoparticles: A new attractive insight into the nanocarriers for allergy epicutaneous immunotherapy.

Int J Pharm 2020 Jun 5;584:119403. Epub 2020 May 5.

Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Recently, the main goal of many allergy epicutaneous immunotherapy (EPIT) studies is to enhance the allergen delivery through the intact skin. Therefore, applying new strategies for tackling this issue are inevitable. For this purpose, ten groups of Che a 2-sensitized BALB/c mice were epicutaneously treated for a 6-week period with the rChe a 2-GNPs-Aptamer, rChe a 2-GNPs-Aptamer + skin-penetrating peptides (SPPs), rChe a 2-GNPs, rChe a 2, GNPs, and PBS. Afterward, the serum IgE and IFN-γ, TGF-β, IL-10, IL-4, IL-17a cytokine production, NALF analysis, and lung/nasal histological examinations were performed. The present study results demonstrate that, EPIT in aptamer treated groups had a significant increase of IFN-γ, TGF-β, and IL-10 concentrations and a significant decrease of IgE, IL-4, and IL-17a concentrations as well as NALF infiltrated immune cell count compared to the non-targeted ones. In addition, SPPs led to more significant improvement of immunoregulatory parameters, especially IL-10 cytokine. Accordingly, the targeted-GNPs with DC-specific aptamers could act as an efficient approach for the improvement of EPIT efficacy compared to the free allergen. Moreover, the application of SPPs might be considered as a useful tool in achieving a successful EPIT with lower doses of allergen at a shorter duration of the treatment.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119403DOI Listing
June 2020

Determination of Optimum Excipients for Platanus orientalis Pollen Extract by Accelerating Chemical Stability Test and Their Synergistic Effect.

Rep Biochem Mol Biol 2019 Jan;7(2):189-195

Allergy Research Center, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: The quality of extracts used in the skin prick test directly influences the interpretation of the test. Accordingly, the outcomes and effectiveness of immunotherapy for the management of IgE-mediated allergies depend on the quality of the extracts used. Excipients, which are pharmacologically inert ingredients, are intentionally added to the active ingredients. The aim of this study was to address optimum excipients for stability extract.

Methods: In this study the excipients examined were l-lysine (20 mM), l-cysteine (20 mM), albumin (0.5%), sorbitol (2%), sucrose (750 mM), trehalose (20 mM), D-mannitol (2% w/v), urea (100 mM) and Tween-20 (0.1%). Their effects on P. orientalis extract stability were analyzed using an inhibition enzyme linked immune assay at 37 °C.

Results: A mixture of lysine (20 mM), trehalose (20 mM), and D-mannitol (2% w/v) conferred the greatest stability on the extract.

Conclusion: The extract stability was increased by a mixture of lysine (20 mM), trehalose (20 mM), and D-mannitol.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374054PMC
January 2019

The Influence of Gamma Radiation Processing on the Allergenicity of Main Pistachio Allergens.

Rep Biochem Mol Biol 2019 Jan;7(2):150-155

Immunology Research Center, Bu-Ali Research Institute, University of Medical Sciences, Mashhad, Iran.

Background: Gamma irradiation is a form of processing with an array of applications in medical sciences such as microbial decontamination, viruses inactivation, cervical carcinoma and breast cancer treatment. One of the ways in which gamma irradiation has the potential to be used is in reducing the allergenicity of food allergens.

Methods: In the present study, pistachios were irradiated with either a 1, 10, or 100 kGy dose of gamma irradiation. The binding rate of mice and human antibodies to the allergens of the pistachio extracts were examined via Western blot analysis.

Results: Our findings show an inverse dose-response relationship between the binding rate of antibodies to the pistachio allergens and the gamma irradiation dose. Despite these promising findings, the results of our sensory evaluation indicate that gamma irradiation causes undesirable changes to the sensory characteristics of pistachios, especially at the dose of 100 kGy.

Conclusion: Gamma irradiation appears to be an effective method in reducing the allergenicity of pistachios. Thus, this form of processing has the potential to prevent adverse allergic reactions to the major pistachio allergens in sensitized subjects. However, further research must be dedicated to examining the dose sufficient in reducing allergencity, while maintaining adequate sensory quality for satisfactory consumption.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374055PMC
January 2019

Diagnostic methods for Lysosomal Storage Disease.

Rep Biochem Mol Biol 2019 Jan;7(2):119-128

Department of Medical Laboratory Sciences, Varastegan Institute for Medical Sciences, Mashhad, Iran.

Lysosomal storage disorders (LSD) are a class of metabolic disturbance in which manifested by the accumulation of large molecules (complex lipids, glycoproteins, glycosaminoglycans, etc.) in lysosomes. LSDs have a wide range of clinical symptoms that may contain organ dysfunction, neurological and skeletal disorders. The first stage of diagnosis is clinically suspected by a physician. Next stage is enzyme activity assays including Fluorometry and MS/MS methods. These methods usually placed in newborn program screening. The second laboratory diagnostic stage is molecular examination (RFLP-PCR and ARMS-PCR, Mutations Scanning Methods, DNA sequencing, MLPA and NGS methods) that is confirmation of the enzyme assays. In this article, routine diagnostic methods for LSDs were discussed. The gold standard for enzyme activity assay and molecular diagnosis is TMS and NGS, respectively.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374068PMC
January 2019

Type IV chitinase quantification in four different grape cultivars (Vitis vinifera) in northeast of Iran by an indirect sandwich enzyme-linked immunosorbent assay.

J Immunoassay Immunochem 2019 22;40(2):139-148. Epub 2018 Oct 22.

e Immunology, Buali Research center , School of Medicine Mashhad University of Medical Sciences , Mashhad , Iran (the Islamic Republic of).

The incidence of grape (Vitis vinifera) allergy in the northeast of Iran is second to melon allergy. Type IV chitinase is one of the major grape allergens. The current study investigates the level of type IV chitinase in four grape variants for the first time in Khorasan Razavi Province using a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA). This assay was developed using a polyclonal antibody as a capture antibody and monoclonal antibody as a secondary one. Finally, the amount of type IV chitinase was measured by the validated ELISA test. The sensitivity of the developed sandwich ELISA is 16 ± 0.05 ng/ml, and its mean coefficients of intraday and interday variations are <5% and <15%, respectively. The recovery of the designed ELISA is 64 ± 0.9 %. The assessments showed that the highest level of type IV chitinase was 39.7 ± 2.3 μg/g in Peykani grape, whereas in the Sultana cultivar, it was 1.76 ± 0.1 μg/g. According to the data, the level of type IV chitinase is variable in different cultivars, and hence, it will be helpful for clinicians to recommend a less allergenic variety to the patient.
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http://dx.doi.org/10.1080/15321819.2018.1535439DOI Listing
May 2019

The immunomodulatory role of probiotics in allergy therapy.

J Cell Physiol 2019 03 7;234(3):2386-2398. Epub 2018 Sep 7.

Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

The increased incidence of allergic disorders may be the result of a relative fall in microbial induction in the intestinal immune system during infancy and early childhood. Probiotics have recently been proposed as viable microorganisms for the prevention and treatment of specific allergic diseases. Different mechanisms have been considered for this probiotic property, such as generation of cytokines from activated pro-T-helper type 1 after bacterial contact. However, the effects of its immunomodulatory potential require validation for clinical applications. This review will focus on the currently available data on the benefits of probiotics in allergy disease.
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http://dx.doi.org/10.1002/jcp.27263DOI Listing
March 2019

Sublingual Immunotherapy with Sal k1 Expressing Lactococcus lactis Down-regulates Th2 Immune Responses in Balb/c Mice.

Iran J Allergy Asthma Immunol 2018 Jun;17(3):281-290

Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Sublingual immunotherapy (SLIT) has been introduced as a noninvasive and safer approach for allergen-specific immunotherapies. In this study we investigated the efficacy of oral immunotherapy with recombinant Salsola kali 1 protein (Sal k 1) on Th1/Th2 balance in a mouse model of allergy. Female Balb/c mice were intraperitoneally sensitized with rSal k1, followed by a respiratory challenge with 1% (w/v) rSal k1. The sensitized mice were subjected to SLIT using rSal K1 expressing Lactobacillus lactis strain for three weeks. Each week the experimental group underwent SLIT protocol twice. Finally, serum levels of specific immunoglobulins including IgE, IgG1 and IgG2a, as well as secretion of different cytokines from splenocytes including IL-2, IL-4, IL-10, IFNγ and TGFβ into culture media were measured by ELISA. Following immunotherapy, the levels of specific IgE and IgG1 in mice sera as well as IL-4 level in supernatant of splenocytes were significantly lower than allergic controls. While serum IgG2a, IgG2a/IgG1 ratio as well as concentration of IL-2, IL-10, IFNγ, and TGFβ were higher in the SLIT group compared to the controls. The histopathological examination of intestinal tissues revealed no sign of inflammatory response following SLIT. This study revealed that Th2 immune responses are reduced in allergic mice after feeding them with allergen expressing probiotic bacteria as a SLIT approach. Since the safety of this procedure was previously approved, thus, it seems that a similar protocol using human based probiotics could be applied for Salsola kali sensitive patients.
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June 2018

Interaction Between Air Pollutants and Pollen Grains: The Role on the Rising Trend in Allergy.

Rep Biochem Mol Biol 2018 Apr;6(2):219-224

Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Asthma and allergic diseases cases have risen in recent decades. Plant pollen is considered as the main aeroallergen causing allergic reactions. According to available data, urban residents experience more respiratory allergies than rural residents mainly due to the interaction between chemical air pollutants and pollen grains.

This interaction can occur through several mechanisms; chemical pollutants might facilitate pollen allergen release, act as adjuvants to stimulate IgE-mediated responses, modify allergenic potential, and enhance the expression of some allergens in pollen grains. This review focuses on the most recent theories explaining how air pollutants can interact with pollen grains and allergens.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941124PMC
April 2018

Production of Recombinant Protein of Salsola Kali (Sal k1) Pollen Allergen in Lactococcus Lactis.

Iran J Allergy Asthma Immunol 2018 Apr;17(2):134-143

Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

The Salsola kali pollen is considered the main cause of allergic sensitization in desert and semi-desert regions. We have constructed recombinant Lactococcus lactis producing Sal k1 protein with the aim of using it as a mucosal vaccine for specific immunotherapy. The Sal k1 gene was amplified, and transferred into a PNZ 8148 plasmid. The PNZ8148-Sal k1 recombinant plasmid was transformed into competent E.coli strain MC1061 for replication, and then was isolated and cloned into competent L. lactis by electroporation. The cloning was verified by PCR and gene sequencing. The production of recombinant Sal K1 (rSal K1) protein was induced by nisin. The rSal K1 protein was purified by affinity chromatography and dialysis, and confirmed by SDS-PAGE and western blot analyses. The recombinant L. lactis was successfully constructed. Production of a 40-kDa rSal k1 protein with the L. lactis was shown by sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) analysis. In addition, western blot analysis using specific mouse anti-Sal k1 polyclonal antibodies and sensitive human sera verified the 40-kD protein as rSal k1 allergen. This study demonstrated that L. lactis may be used as a promising live delivery system for recombinant Sal k1 protein without altering its immunoreactivity; however, its efficacy in the context of the immune system is suggested to be pursued in future studies.
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April 2018

Enhanced sublingual immunotherapy by TAT-fused recombinant allergen in a murine rhinitis model.

Int Immunopharmacol 2017 Jul 11;48:118-125. Epub 2017 May 11.

Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100μg/dose) or rTAT-Che a 3 (100μg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-β secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-β- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.
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http://dx.doi.org/10.1016/j.intimp.2017.04.011DOI Listing
July 2017

Quantification of Pla or 3, a Allergen, Grown under Different Environmental Conditions, by Sandwich ELISA.

Rep Biochem Mol Biol 2016 10;5(1):40-45

Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: species are widely cultured around the world and considered an important cause of allergic reactions. In the present study, we developed a sandwich ELISA to quantify Pla or 3 allergen in pollen extracts grown near high-traffic roads and compared it to pollen extracts collected from rural areas as control.

Methods: Pollen samples were collected from three polluted and two unpolluted sites in Mashhad, northeast Iran. Recombinant Pla or 3 was expressed and used for polyclonal antibody production in rabbit. A sandwich ELISA was developed and validated to quantify Pla or 3 levels in pollen extracts from the different sites.

Results: The coefficients of variation (CVs) for the intra- and inter- day assays were less than 5 and 18%, respectively. The working range of the standard curve was between 0.1 and 25 ng/ml, with the detection limit being 0.037 ng/ml. The recovery percentage was 88-106.4% at working concentrations from 0.31 to 26.5 ng/ml. Pla or 3 levels were significantly greater in pollens grown near high-traffic roads than in those grown in rural regions (p < 0.0001).

Conclusion: A sandwich ELISA was developed and validated to quantify Pla or 3 in pollen extracts. Using this validated ELISA, we showed a substantial difference between the amounts of Pla or 3 in pollens grown in different environments. This finding should be considered in developing public policies to reduce traffic pollution, which leads to reduced allergic reactions in atopic subjects.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214682PMC
October 2016

Methylmalonic Acidemia Diagnosis by Laboratory Methods.

Rep Biochem Mol Biol 2016 10;5(1):1-14

Pardis Clinical and Genetic Laboratory, Mashhad, Iran; Immunobiochemistry Lab, Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Varastegan Institute for Medical Sciences, Mashhad, Iran.

Methylmalonic acidemia (MMA) is usually caused by a deficiency of the enzyme methylmalonyl-CoA mutase (MCM), a defect in the transport or synthesis of its cofactor, adenosyl-cobalamin (cblA, cblB, cblC, cblF, cblD, and cblX), or deficiency of the enzyme methylmalonyl-CoA epimerase. A comprehensive diagnostic approach involves investigations of metabolites with tandem mass spectrometry, organic acid analysis with gas chromatography, enzymatic studies with fibroblast cell culture, and finally, mutation analysis. With biochemical techniques and enzymatic assay the reliable characterization of patients with isolated MMA for mutation analysis can be achieved. Reliable classification of these patients is essential for ongoing and prospective studies on treatments, outcomes, and prenatal diagnoses. This article reviews the diagnostic techniques used to characterize patients with MMA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214677PMC
October 2016

Impact of traffic-related air pollution on the expression of Platanus orientalis pollen allergens.

Int J Biometeorol 2017 Jan 2;61(1):1-9. Epub 2016 Jun 2.

Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Air pollutants and their interaction with environmental allergens have been considered as an important reason for the recent increase in the prevalence of allergic diseases. The aim of this study was to investigate the traffic pollution effect, as a stressor, on Platanus orientalis pollen allergens messenger RNA (mRNA) and protein expression. P. orientalis pollen grains were collected along main streets of heavy traffic and from unpolluted sites in Mashhad city, in northeast Iran. The pollen samples were examined by scanning electron microscopy. To assess the abundance of pollen allergens (Pla or 1, Pla or 2, and Pla or 3) from polluted and unpolluted sites, immunoblotting was performed. Moreover, the sequences encoding P. orientalis allergens were amplified using real-time PCR. Scanning electron microscopy showed a number of particles of 150-550 nm on the surface of pollen from polluted sites. Also, protein and gene expression levels of Pla or 1 and Pla or 3 were considerably greater in pollen samples from highly polluted areas than in pollen from unpolluted areas (p < 0.05). In contrast, no statically significant difference in Pla or 2 protein and mRNA expression level was found between samples from the two areas. We found greater expression of allergens involved in plant defense mechanisms (Pla or 1 and Pla or 3) in polluted sites than in unpolluted ones. The high expression of these proteins can lead to an increase in the prevalence of allergic diseases. These findings suggest the necessity of supporting public policies aimed at controlling traffic pollution to improve air quality and prevent the subsequent clinical outcomes and new cases of asthma.
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http://dx.doi.org/10.1007/s00484-016-1186-zDOI Listing
January 2017

Down-regulation of Th2 immune responses by sublingual administration of poly (lactic-co-glycolic) acid (PLGA)-encapsulated allergen in BALB/c mice.

Int Immunopharmacol 2015 Dec 26;29(2):672-678. Epub 2015 Sep 26.

Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

The goal of this study was to investigate whether poly (lactic-co-glycolic) acid (PLGA) nanoparticles could enhance sublingual immunotherapy (SLIT) efficacy. BALB/c mice sensitized to rChe a 3 were treated sublingually either with soluble rChe a 3 (100μg/dose) or PLGA-encapsulated rChe a 3 (5, 25, or 50μg/dose). SLIT with PLGA-encapsulated rChe a 3 (equivalent to 25 and 50μg rChe a 3 per dose) led to significantly increased antigen-specific IgG2a, along with no effect on allergen-specific IgE and IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in restimulated splenocytes were significantly less, while interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) levels, as well as Foxp3 expression, were significantly greater than in the control groups. Our findings suggest that PLGA nanoparticle-based vaccination may help rational development of sublingual immunotherapy through reduction of the needed allergen doses and also significantly enhanced systemic T regulatory (Treg) and T helper 1 (Th1) immune responses.
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http://dx.doi.org/10.1016/j.intimp.2015.09.011DOI Listing
December 2015

Identification and molecular characterization of the cDNA encoding Cucumis melo allergen, Cuc m 3, a plant pathogenesis-related protein.

Rep Biochem Mol Biol 2014 Apr;2(2):82-7

Allergy Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Melon (Cucumis melo) allergy is one of the most common food allergies, characterized by oral allergy syndrome. To date, two allergen molecules, Cuc m 1 and Cuc m 2, have been fully characterized in melon pulp, but there are few reports about the molecular characteristics of Cuc m 3.

Methods: The Cuc m 3 cDNA has been characterized by rapid amplification of cDNA ends (RACE), which revealed a 456 base-pair (bp) fragment encoding a 151-amino acid polypeptide with a predicted molecular mass of 16.97 kDa, and identified 79 and 178 bp untranslated sequences at the 5' and 3´ ends, respectively.

Results: In silico analysis showed strong similarities between Cuc m 3 and other plant pathogen-related protein 1s from cucumber, grape, bell pepper, and tomato.

Conclusion: Here we report the identification and characterization of the Cuc m 3 cDNA, which will be utilized for further analyses of structural and allergenic features of this allergen.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757051PMC
April 2014

Impact of oral 1,25-dihydroxy vitamin D (calcitriol) replacement therapy on coronary artery risk factors in type 2 diabetic patients.

Endocr Metab Immune Disord Drug Targets 2013 Dec;13(4):295-300

Endocrine Research Center, Faculty of Medicine, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Postal Code: 91766, Iran.

Background: Observational data suggest that low 25-hyroxyvitamin D is associated with cardiovascular disease (CVD) and its risk factors include diabetes, metabolic syndrome, insulin resistance, hypertension, microalbuminuria and inflammation. We examined the differences between risk factors of CVD before and after treatment with calcitriol in type 2 diabetic patients with vitamin D deficiency.

Methods: This study was a clinical trial consisting of 119 type 2 diabetic patients. Forty three patients had vitamin D insufficiency (25 OH D less than 30 ng/dl) who underwent calcitriol treatment with 0.5 microgram per day for 8 weeks. Blood pressure, fasting blood sugar (FBS), glycosylated hemoglobin (HbA1C), lipid profile, high sensitive C-reactive protein (HsCRP), Homocysteine and albumin to creatinine ratio were measured, before and after the treatment period. Then the two sets of results were compared with each other.

Results: Following treatment with calcitriol HbA1C, total cholesterol, low density lipoprotein(LDL), high density lipoprotein(HDL) and diastolic blood pressure decreased significantly (p = 0.01, 0.01, 0.04, 0.001 and 0.04 respectively) but the changes in other parameters were not significant.

Conclusion: Replacement of vitamin D may have a beneficial effect on some of the risk factors of CVD in diabetic patients.
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http://dx.doi.org/10.2174/18715303113136660047DOI Listing
December 2013

The role of anti-CCD antibodies in grape allergy diagnosis.

Rep Biochem Mol Biol 2013 Apr;1(2):74-82

Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Allergens are mostly composed of glycoprotein structures. It is believed that glycan-specific antibodies may lead to false-positive reactions in immunoassays. In this study we investigated the glycosylation state of grape allergens as well as the presence of antibodies to cross-reactive carbohydrate determinants (anti-CCDs) in sera from grape-sensitive individuals.

Methods: Grape extract proteins were electrotransferred onto PVDF membranes and their glycosylation states were analyzed by blotting methods. To assess the presence of anti-CCDs, natural and mildly deglycosylated proteins were immunoblotted with grape-allergic subjects' sera. We also measured the IgE reactivity of each subject's sera with other fruit extracts via an indirect ELISA.

Results: Immunoblotting studies showed that mildly deglycosylated grape proteins had lower IgE-binding capacity than their intact natural counterparts, which could be due to the presence of anti-CCDs. Biotinylation studies confirmed that the glycosylation levels of the 24, 32, and 60 kDa IgE-reactive proteins were higher than those of the 38 and 45 kDa ones. Lectin blotting showed that the 24 and 60 kDa bands were highly mannosylated, with the highest level of mannosylation on the 24 kDa allergen.

Conclusion: This study showed that some grape allergens are glycosylated and that anti-CCD antibodies may cause weakly false-positive results during assessment of IgE reactivity to grape allergens.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757059PMC
April 2013

Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo.

Rep Biochem Mol Biol 2013 Apr;1(2):49-63

Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon.

Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli.

Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5'and 3' UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins.

Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757056PMC
April 2013

Type 2 innate lymphoid cells: friends or foes-role in airway allergic inflammation and asthma.

J Allergy (Cairo) 2012 11;2012:130937. Epub 2012 Nov 11.

Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Innate-like lymphocytes (ILLs) and innate lymphoid cells (ILCs) are two newly characterized families of lymphocytes with limited and no rearranged antigen receptors, respectively. These soldiers provide a first line of defense against foreign insults by triggering a prompt innate immune response and bridging the gap of innate and adaptive immunity. Type 2 innate lymphoid cells (ILCs2) are newly identified members of the ILC family that play a key role in type 2 immune responses by prompt production of type 2 cytokines (especially IL-5 and IL-13) in response to antigen-induced IL-25/33 and by recruiting type 2 "immune franchise." Regarding the two different roles of type 2 cytokines, helminth expulsion and type 2-related diseases, here we review the latest advances in ILC2 biology and examine the pivotal role of resident ILCs2 in allergen-specific airway inflammation and asthma.
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http://dx.doi.org/10.1155/2012/130937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503334PMC
December 2012

Decreased levels of soluble Toll-like Receptor 2 in patients with asthma.

Rep Biochem Mol Biol 2012 Oct;1(1):30-6

Immunobiochemistry lab, Immunology Research Center, Bu-Ali Research Institute, Mashhad, Iran.

Background: Recently, reports have indicated a role for the membrane form of Toll-like Receptor 2 (TLR2) in asthma pathogenesis. In this study we examined soluble TLR2 levels in serum and sputum of asthmatic and healthy subjects.

Methods: Serum and sputum samples were obtained from 33 asthmatic and 19 healthy subjects. The asthmatics were classified into four groups according to the Global Initiative for Asthma. A sandwich ELISA was developed to measure soluble TLR2 (sTLR2) in serum and sputum. TLR2 mRNA expression was determined by semi-quantitative RT-PCR of all sputum samples.

Results: The mean sTLR2 levels from serum and sputum of asthmatics were significantly lower than those from healthy subjects. Moreover, sTLR2 concentration decreased concomitantly with asthma severity. The differences observed, however, were not statistically significant. TLR2/GAPDH mRNA of sputum leukocytes was also significantly lower in asthmatics than in healthy subjects.

Conclusion: This study demonstrated for the first time thatsTLR2 levels are lower in serum and sputum samples from asthmatic than from healthy subjects, and this could be an indicator of TLR2 expression. We also found that sTLR2 concentration in serum decreased concomitantly with an increase of asthma severity clinical score .
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757078PMC
October 2012

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli.

Rep Biochem Mol Biol 2012 Oct;1(1):25-9

Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis .

Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method.

Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen.

Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757077PMC
October 2012

Validity of using recombinant melon profilin, Cuc m 2, for diagnosis of melon allergy.

Rep Biochem Mol Biol 2012 Oct;1(1):14-20

Immunobiochemistry Lab, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Allergy is a clinical disorder affecting humans worldwide. Allergenic extracts prepared from natural source materials remain heterogeneous in composition and content, but are regularly used for diagnosis and immunotherapy. Recombinant allergens are suitable candidates to use in place of natural allergens; however, the recombinant allergens should be assessed and compared with the natural ones. Cuc m 2 (profilin), one of the most important allergens of melon (Cucumis melo), has been cloned and was expressed in Escherichia coli (E. coli). We aimed to evaluate the validity of recombinant Cuc m 2 (rCuc m 2) in the diagnosis of melon allergy and investigate whether rCuc m 2 could be used as a replacement for natural Cuc m 2 (nCuc m 2).

Methods: nCuc m 2 was purified by immuno-affinity chromatography and rCuc m 2 was purified by metal-affinity chromatography. SDS-PAGE and western blotting were carried out to evaluate the purification methods. Skin prick tests (SPT), and enzyme immunoassays to determine specific IgE, were performed with the natural and recombinant purified allergens on 53 patients with melon allergy.

Results: rCuc m 2 elicited no significantly different responses in skin compared with nCuc m 2. All patients' sera showed similar ODs in ELISAs with natural and recombinant profilin.

Conclusion: rCuc m 2 evoked strong immuno-reactivity equivalent to nCuc m 2, and has potential for diagnosis of melon allergy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757075PMC
October 2012

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus).

Rep Biochem Mol Biol 2012 Oct;1(1):1-8

INDOOR Biotechnologies, Inc., Charlottesville, Virginia, USA.

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.

Section Title: 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757073PMC
October 2012

Is urinary 5-hydroxyindoleacetic acid helpful for early diagnosis of acute appendicitis?

Am J Emerg Med 2012 May 29;30(4):540-4. Epub 2011 Mar 29.

Surgical Oncology Research Center, Imam Reza Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Objective: Acute appendicitis is the most common abdominal emergency in children and young adults. There are a lot of serotonin-containing cells in the appendix, which release serotonin into the bloodstream in response to inflammation. Consequently, serotonin is converted to 5-hydroxyindoleacetic acid (5-HIAA) and secreted into the urine. On this basis, urinary 5-HIAA could be a marker for acute appendicitis. In this study, we investigated the value of 5-HIAA levels in spot urine in the diagnosis of acute appendicitis.

Methods: The urinary 5-HIAA was measured by an enzyme-linked immunosorbent assay in the spot urine of 70 patients who presented to the emergency department with a clinical picture of acute appendicitis. Urine concentration results were correlated to final histopathologic reports, and the diagnostic value of this factor was measured.

Results: Diagnosis of appendicitis was confirmed by histopathologic reports in 59 of 70 patients with presumptive diagnosis of appendicitis. Considering 5.25 mg/L as the cutoff point for urinary 5-HIAA, 28 patients had high urinary 5-HIAA levels, whereas 42 patients had values within reference range. The sensitivity and specificity of this test was 44% and 81%, respectively.

Conclusions: The measurement of urinary 5-HIAA levels is not an ideal diagnostic tool for ruling out or determination of acute appendicitis.
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http://dx.doi.org/10.1016/j.ajem.2011.01.027DOI Listing
May 2012

Cloning and expression of Che a 1, the major allergen of Chenopodium album in Escherichia coli.

Appl Biochem Biotechnol 2011 Apr 25;163(7):895-905. Epub 2010 Sep 25.

Biotechnology Department, Razi Vaccine and Serum Research Institute, Mashhad, Iran.

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b+ vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni-NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.
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http://dx.doi.org/10.1007/s12010-010-9093-yDOI Listing
April 2011

Metabolic factors associated with urinary calculi in children.

Iran J Kidney Dis 2010 Jan;4(1):32-8

Department of Pediatrics, Dr Sheikh Children Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.

Introduction: We aimed to identify metabolic and anatomical abnormalities present in children with urinary calculi.

Materials And Methods: Metabolic evaluation was done in 142 pediatric calculus formers. Evaluation included serum biochemistry; measurement of daily excretion of urinary calcium, uric acid, oxalate, citrate, and magnesium (in older children); and measurement of calcium, uric acid, oxalate, and creatinine in random urine samples in nontoilet-trained patients. Urinary tests for cystinuria were also performed. All of the patients underwent renal ultrasonography.

Results: Sixty-one patients (42.7%) had metabolic abnormalities. Anatomical abnormalities were found in 12 patients (8.4%). Three children (2.1%) had infectious calculi, and 3(2.1%) had a combination of metabolic and anatomic abnormalities. In 66 children (46.2 %) we did not find any reasons for calculus formation (idiopathic). Urinalysis revealed hypercalciuria in 25 (17.6%), hyperuricosuria in 23 (16.1%), hyperoxaluria in 17 (11.9%), cystinuria in 9 (6.3%), hypocitraturia in 3 (2.1%), and low urinary magnesium level in 1 (0.7%) patients. Sixteen patients (11.2%) had mixed metabolic abnormalities.

Conclusions: Metabolic abnormalities are common in pediatric patients with urinary calculi. In our study, calcium and uric acid abnormalities were the most common, and vesicoureteral reflux seemed to be the most common urological abnormality which led to urinary stasis and calculus formation.
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January 2010

Cloning and expression of the allergen Cro s 2 profilin from saffron (Crocus sativus).

Allergol Int 2009 Sep 25;58(3):429-35. Epub 2009 Jul 25.

Immunobiochemistry Lab, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Profilin is a panallergen that is recognized by IgE in allergic patients. Allergy to saffron (Crocus sativus) pollen has been described in people exposed to its pollen. Saffron contains a profilin that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression and purification of saffron profilin from pollen.

Methods: Cloning of saffron profilin was performed by polymerase chain reaction using specific primers from saffron pollen RNA. Expression was carried out in Escherichia coli BL21 (DE3) using a vector pET-102- TOPO. A recombinant fusion protein was expressed and the recombinant profilin was purified by metal precipitation. Immunological characterization was performed by immunoblotting experiments.

Results: The 34kDa- recombinant saffron profilin, Cro s 2, as a fusion protein was purified. Immunoblotting tested with the sera of allergic patients showed a specific reaction with the recombinant Cro s 2 band.

Conclusions: The sequence of Cro s 2 showed a high degree of identity and similarity to other plant profilins and the recombinant saffron profilin, Cro s 2, may be used for target-specific diagnosis and structural analyses and investigation of cross reactivity of Cro s 2 with other plant profilins.
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http://dx.doi.org/10.2332/allergolint.09-OA-0088DOI Listing
September 2009