Fatemeh Vahedi

Diagnosis of Chenopodium album allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.

Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.

Chenopodium album pollen profilin (Che a 2): homology modeling and evaluation of cross-reactivity with allergenic profilins based on predicted potential IgE epitopes and IgE reactivity analysis

The inhalation of Chenopodium album (C. album) pollen has been reported as an important cause of allergic respiratory symptoms. The aim of this study was to produce the recombinant profilin of C. album (rChe a 2) pollen and to investigate its cross-reactivity with other plant-derived profilins based on potential conformational epitopes and IgE reactivity analysis. Che a 2-coding sequence was cloned, expressed, and purified using one step metal affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted IgE potential epitopes among rChe a 2 and other plant-derived profilins. Immunodetection and inhibition assays using sixteen individual sera from C. album allergic patients demonstrated that purified rChe a 2 could be the same as that in the crude extract. The results of inhibition assays among rChe a 2 and other plant-derived profilins were in accordance with those of the homology of predicted conserved conformational regions. In this study, amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins may depend on predicted potential IgE epitopes.

Prevalence of allergic disorders among the population in the city of Mashhad, Northeast Iran

BackgroundAllergic disorders are common ailments, which cause socioeconomic problems. The aim of the study was to determine the prevalence of allergic disorders and symptoms suggestive of asthma in Mashhad city, Northeast Iran.MethodsWe performed a cross-sectional survey in a randomly selected representative sample of the Mashhad population. A questionnaire was used based on the standard questionnaire of the European Community Respiratory Health Survey (ECRHS). The questionnaires asked about allergy-related symptoms including nasal symptoms, eye symptoms, atopic dermatitis symptoms, confirmed asthma, asthma-related symptoms, and nonspecific allergic symptoms. Some of the environmental triggers of allergic reactions such as pollen, dried plant, dust, drugs, food, fruit, condiments, makeup, perfume, jewelry, latex gloves, and keeping a pet were considered in the questionnaire.ResultsThe prevalence of allergic disorders in this study was found to be 27.5% in the city of Mashhad. According to our definitions, the rates detected of allergic rhinitis, atopic dermatitis, conjunctivitis, rhinoconjunctivitis, and asthma were 22.4, 6.6, 13.5, 9.5, and 2.3%, respectively. Half of the allergic group suffered from rhinorrhea and other symptoms were significantly higher than in the non-allergic group. Dust and pollen were found to be the most important triggers of allergic reactions in the allergic group.ConclusionThis report may be useful to understand the factors contributing to asthma and allergic disorders and may help in management and control of these problems.

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

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.

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

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.

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

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.

Characterization of Bacillus anthracis spores isolates from soil by biochemical and multiplex PCR analysis.

Outbreaks of Bacillus anthracis in animals are repeatedly reported in the Islamic Republic of Iran. In this study soil samples were analysed from endemic regions of the country, and B. anthracis isolates were identified by classical bacteriological and biochemical methods. A multiplex polymerase chain reaction (PCR) assay was also developed as an alternative for identification of isolates, and was shown to be a rapid, sensitive and specific diagnostic assay. The results confirmed that 25 samples contained B. anthracis, of which 9 were virulent for mice and guinea pigs. This study suggests that multiplex PCR can be used as a reliable alternative for the detection of B. anthracis spores.

Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

Successful recombinant allergen‐based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility‐enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly‐arginine–lysine: rTAT–Pla or 3 and rPoly‐Arg–Lys–Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high‐level solubility and efficient expression of the fusion proteins (rTAT–Pla or 3 and rPoly‐Arg–Lys–Pla or 3) compared with the wild‐type recombinant. Furthermore, the similar structural characteristics and IgE‐binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment.

Inhibitory effects of cinnamon-water extract on human tumor cell lines

Abstract Objective To question the inhibitory effect of cinnamon-water extract (CWE) on four human tumor cell lines (AGs, HeLa, MCF-7 and MDA-MB234). Naturally, compounds are an important source for clinical proposes. Cinnamon, a plant-derived spice, is widely used as a food additive and has been attracted many researches in recent years to find its pharmaceutical benefits. Methods In order to find the answer to this subject, the water extract of cinnamon was prepared and cell proliferation was evaluated using MTT assay. The effect of apoptosis was investigated by DNA fragmentation analysis. Results The inhibitory effect of CWE on the growth of the cells was significant. DNA fragmentation was found in cultured AGs and MCF-7 cell lines treated by CWE. Conclusions This study showed the anti-neoplastic activity of CWE on tumor cell lines.