Introducing a Stabilizer Formulation for Allergenic Mold Extracts.

BACKGROUND: Sensitization to common mold allergens is one of the major causes of allergic rhinitis and asthma. Therefore, there is a critical need for standard sensitivity tests including skin prick tests to improve the stability of fungi extracts in traditional allergenic formulations. To address this concern, the present study aimed to develop a formulation to preserve allergenic activity of mold extracts. METHODS: 48 stabilizer formulations were designed and monitored for allergenic activity during a 40-days incubation period at 37 °C using an ELISA. Specifically, the IgE reactivity of allergenic A. alternata extracts were examined. After establishing the most effective stabilizer formulation, we evaluated whether it could protect the allergenic activity of Alt a1, A. fumigatus, and C. herbarum using an IgE inhibition ELISA after 40 days at 37 °C. RESULTS: We demonstrated that the most effective stabilizer formulation was a glycerol-based extract containing Arg and Glu. This formulation had an equal ratio of sucrose, sorbitol and protein and was able to preserve more than 95% of allergenic A. alternata extract activity during a 40-days incubation period at 37 °C. CONCLUSION: The present study reveals a novel formulation that is an efficient stabilizer of allergenic mold extract activity and has practical applications in mold skin prick tests, ELISAs, immunotherapies, and RAST.

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

OBJECTIVES: Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogen, causes 1.5 million deaths globally. Bacilli Calmette-Guérin (BCG) is commonly administered to protect people against M. tuberculosis 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-É£ (IFN-É£) production and higher INF-É£/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.

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

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 Erwinia amylovora 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 E. amylovora, 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 (Mal d 1, 2, 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 Mal d 2 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 Mal d 1, 2, and 3 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 E. amylovora-resistant than in E. amylovora-sensitive cultivars.

Determining the Effect of Amino Acids on the Allergenic Activity of Pollen Extracts.

BACKGROUND: The diagnosis and treatment of allergic diseases require high quality pollen allergen extracts for reliable test results and effective treatments. The quality of the pollen allergen extracts is influenced by pharmacologically inert ingredients, such as stabilizers which are added to prevent the degradation of the allergenic activity. This study was conducted to develop a stabilizer formulation in order to protect the allergenic activity of the pollen's extracts. METHODS: Pine and orchard grass pollen allergen extracts were incubated for 40 days at 37 °C. The effects of chemicals were examined via inhibition ELISA on days 7, 14, 21, 28, and 40 to evaluate the ability of the pollen allergen extracts to inhibit specific IgE in the sera of sensitized patients. RESULTS: Our findings showed that the pine pollen and orchard grass allergen extracts treated with Lys/Glu had the best stabilizing effect resulting in a 97% IgE inhibition following the 40 days of incubation. In the non-treatment group, the IgE inhibition decreased to 23% at the end of the 40 days. The orchard grass pollen allergen extracts receiving no treatment decreased to 12% IgE inhibition following the 40-day incubation. CONCLUSION: Amino acids are able to act as an effective stabilizer for pollen allergen extracts and prevent the degradation of their activity over time. Particularly applying Lys/ Glu in pollen allergenic extracts can protect allergenic activity and potency of the pollen extracts to inhibit specific IgE in human sera.

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

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-ß 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- ß, 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.

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

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-ß, 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-ß, 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.

The Most Common Allergenic Tree Pollen Grains in the Middle East: A Narrative Review.

Allergy is becoming a major disease burden globally. Pollens are considered as the main component of aeroallergens that lead to rhinitis and asthma. Due to the lack of a comprehensive investigation on most allergic pollens of trees in the Middle East, the present study aimed to conduct a comprehensive literature review on this topic. The main goal of the study was to provide a checklist for allergists and patients to easily identify the commonest allergic pollens in their locality. The present review provides a broad range of information on the types and geographic locations of the most common allergic pollens of trees in each studied country. In general, among the 23 studied countries, palm and mesquite trees were the common producers of pollen allergen in the Persian Gulf region. Olive tree is common in Turkey, Palestine, and Israel, whereas sycamore tree is the common allergen pollen in Iran. Considering the uneven geographical distribution of these trees in the world, allergists are unable to accurately select the appropriate extracts for the skin prick test based on the information from the neighboring countries. This scenario becomes more complicated if one adds the imported ornamental trees in the picture.

Mutation analysis of genes related to methylmalonic acidemia: identification of eight novel mutations.

Methylmalonic acidemia (MMA), an inherited metabolic disease, results from genetic defects in methylmalonyl-CoA mutase or any of the proteins involved in adenosylcobalamin synthesis. This enzyme is classified into several complementation groups and genotypic classes. In this work we explain the biochemical, structural and genetic analysis of 25 MMA patients, from Iran. The diagnosis was established by the measurement of propionylcarnitine in blood using tandem mass spectrometry and confirmed using a gas chromatography-flame ionization detector. Using clinical, biochemical, structural and molecular analyses we identified 15 mut MMA, three cblA, one cblB, and four cblC-deficient patients. Among mutations identified in the MUT gene (MUT) only one, the c.1874A>C (p.D625A) variant, is likely a mut- mutation. The remaining mutations are probably mut0. Here, we present the first molecular analysis of MMA in Iranian patients and have identified eight novel mutations. Four novel mutations (p.D625A, p.R326G, p.V157F, p.F379L) were seen exclusively in patients from northern Iran. One novel splice site mutation (c.2125-3C>G) in MUT and two novel mutation (p.N225M and p.A99P) in the MMAA gene were associated with patients from eastern Iran. The rs184829210 SNP was recognized only in patients with the novel c.958G>A (p.A320T) mutation. This study confirms pathogenesis of deficient enzyme activity in MUT, MMAA, MMAB, and MMACHC as previous observations. These results could act as a basis for the performance of pharmacological therapies for increasing the activity of proteins derived from these mutations.

Diagnostic methods for Lysosomal Storage Disease.

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.

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

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.

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

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 Platanus (P.) orientalis 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 P. orientalis extract. CONCLUSION: The P. orientalis extract stability was increased by a mixture of lysine (20 mM), trehalose (20 mM), and D-mannitol.

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

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.

Preparation allergenic pollen extracts; the points should be considered to make high-quality products.

Atopic diseases have an increasing trend worldwide during the last two decades. Determining the main cause of allergic diseases, allergens, is the first step in managing and improving the issue, usually is done by Skin Prick tests (SPTs). Having allergenic extract in high quality is desired to perform a reliable SPT. Several parameters of extracts are considered including composition, stability, potency, preservation conditions, and unit definition. In this review, these factors have been explained pointing to factors might have profitable points or harmful drawback in the quality of allergen extracts.

The immunomodulatory role of probiotics in allergy therapy.

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.

Frequency of Inborn Errors of Metabolism in a Northeastern Iranian Sample with High Consanguinity Rates.

OBJECTIVE: Inborn errors of metabolism (IEMs) are disorders with various manifestations that occur mainly in the pediatric population. In countries where consanguineous marriage is common, the association between consanguinity and IEMs is highly important. No studies have been conducted in Iran examining the impact of consanguinity on IEMs. METHODS: In this retrospective study, the incidences of metabolic disorders were evaluated for the years 2006 through 2016 in the North East Iran Regional Diagnostic Laboratory (Pardis Clinical and Genetic Laboratory). A total of 13,327 infants with clinical symptoms were referred and investigated for IEMs. Newborn screening was performed on samples from all patients suspected of having IEMs. RESULTS: Of 13,327 infants examined, 60 different IEMs were diagnosed in 1,118. The most frequent disorders among our patients were glucose-6-phosphate dehydrogenase deficiency (G6PDD) (14.04%), methylmalonic and propionic acidurias (MMA/PA) (9.12%), phenylketonuria (PKU) (8%), and isovaleric acidemia (IVA) (6.98%). A significant difference was found in the prevalence of amino acid disorders between the offspring of consanguineous and those of non-consanguineous parents. No statistically significant differences were found between the 2 groups for organic or fatty acids, carnitine or urine cycles, or lysosomal storage disorders. A total of 707 of the 1,118 infants with metabolic diseases (63.24%) were children of consanguineous parents. These findings show that consanguinity can be an important factor in the inheritance of recessive mutations in a homozygous state. CONCLUSION: This study found a greater frequency of metabolic diseases in offspring of consanguineous parents than in those of non-consanguineous parents in a population with a high rate of consanguinity.

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

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 TGFß 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 TGFß 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.

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

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.

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

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.

A Description of Reference Ranges for Organic Acids in Urine Samples from A Pediatric Population in Iran.

BACKGROUND: Organic acids refer to a family of compounds that are intermediates in a variety of metabolic pathways. Many organic acids are present in urine from clinically normal individuals. Elevated levels of urine organic acids cause to the organic acidurias, disorders in which some metabolic pathways in organic acid metabolism are blocked. The present work identified major and minor urinary acidic metabolites in normal subjects, and their quantitative ranges in a pediatric population of Iran. METHODS: Two hundred and fifty-one healthy subjects, including 132 males and 119 females, from 2 days to 15 years of age were enrolled. Urinary organic acids were extracted from urine with organic solvents and identified and quantified by gas chromatography-mass spectrometry. RESULTS: The results provide a foundation on which to check results for patients with potentially abnormal organic acidurias. By this method 98 organic acids were identified in a pediatric population of Iran. CONCLUSION: The present work identifies and quantifies major and minor urinary metabolites excreted by normal subjects. We also analyzed urine from 30 patients with organic acid metabolism abnormalities and compared the concentrations of specific organic acids with those from urines of normal individuals.

Common solvents for making extraction of allergenic proteins from plants' pollens for prick tests and related factors: a technical review.

Collecting information on influencing factors in developing consistent and high-quality extracts results in accurate diagnosis and effective treatment of type I allergy (IgE mediated). Furthermore, considering that a large number of allergens are currently in practice, any attempt to develop a more effective procedure for preparing extract may be useful. Nowadays, different saline solvents, temperature, incubation time, and PH are being incorporated for preparing allergen extracts. The objective of the current study was to clear and address the commonest of solvent buffers and allied conditions for making extracts of pollens of grasses, trees, and weeds. The literature review was done in Jan 2016 on PubMed and Google Scholar medical search engines without any time limitation. After reading abstracts of 87 articles, finally 37 relevant papers were selected and their full texts were retrieved. In conclusion, 24 full-text papers were recognized appropriate and chosen. The extracted information for papers has been described fully in the text. On the basis of these data, PBS buffer with PH 7.4, temperature of 4 °C and with overnight incubation time, may be the optimized condition in order to have a proper extract for carrying out skin prick tests.