Molecular and immunochemical characterization of Pop n 2: A new allergen of Populus nigra pollen.

BACKGROUND: Pollen is one of the most common allergens that cause respiratory allergies worldwide. Pollen grains from poplars have been reported as important sources of pollinosis in many countries. OBJECTIVE: The aim of the present study was to determine the molecular and immunochemical characterization of Pop n 2, a novel allergen of Populus nigra (P nigra) pollen extract. METHODS: In this study, the pollen extract of P nigra was analysed by SDS-PAGE, and the allergenic profile was determined by IgE immunoblotting and specific ELISA using the sera of twenty allergic patients. The coding sequence of Pop n 2 was cloned and expressed in the Escherichia coli BL21 (DE3) using plasmid the pET-21b (+). Finally, the expressed recombinant Pop n 2 was purified by affinity chromatography. RESULTS: Pop n 2 belongs to the profilin family with a molecular weight of approximately 14 kDa. Pop n 2 is the most IgE-reactive protein (about 65%) in the P nigra pollen extract. The cDNA sequencing results indicated an open reading frame 396 bp that encodes 131 amino acid residues. The results of ELISA and Immunoblotting assays showed that recombinant Pop n 2 could react with the IgE antibody in patients' sera, like its natural counterpart. CONCLUSION: Our data revealed that Pop n 2 is a significant allergen in the P nigra pollen extract. Moreover, we observed that the recombinant Pop n 2 produced by the pET-21b (+) vector in the E colisystem acts as its natural counterpart.

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

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

IL1RL1 single nucleotide polymorphisms are associated with asthma in the Iranian population.

Asthma is a chronic and multifactorial disease which is known to result from environmental and genetic factors. Interleukin 1 receptor-like 1 (IL1RL1) is a receptor, which promotes inflammatory responses after binding to its ligand IL-33. Several studies have shown that IL1RL1 gene polymorphisms are related to susceptibility or protection to asthma. The objective of this study was to evaluate the association between two IL1RL1 single nucleotide polymorphisms (rs10208293 and rs1041973) and the risk of asthma in the Iranian population. We performed genotyping of the IL1RL1 SNPs in 126 adult asthmatics and 300 healthy controls using TaqMan genotyping assay. Moreover, total serum IgE level, eosinophil count, and skin prick test were accomplished. The results indicated that the AA genotype of rs10208293 was positively associated with asthma susceptibility (p=0.028). We did not find any association between rs1041973 and asthma. Overall, our findings indicate that rs10208293 has a positive association with asthma in the Iranian population.

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

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

Safety assays and nutritional values of mycoprotein produced by Fusarium venenatum IR372C from date waste as substrate.

BACKGROUND: Nutritional and environmental benefits of mycoprotein verify its beneficial role on the health of humankind in the next decades. Agro-industrial wastes can be used as cheap substrates to decrease the total cost of product. However, fungi may produce toxins or lead to allergic reactions in consumers. Therefore, the study of the safety and nutritional aspects of this product are very important. RESULTS: Fusarium venenatum IR372C was cultured on date wastes and ammonium salts in submerge fermentation. The safety and nutritional issues of produced mycoprotein were investigated including allergy tests and analyses of toxins, as well as existence of toxin genes, and content of heavy metals, metals, amino acids and fatty acids. The results showed that fumonisin genes in F. venenatum IR372C remain without any gene expression during 1 week fermentation. Zearalenone and deoxynivalenol cannot be detected in the fermentation medium after 3 weeks. Prick tests on 30 volunteers demonstrated no sensitivities to mycoprotein. The content of lead was 658 µg kg-1 as the highest heavy metal followed by arsenic, cadmium and mercury at 161, 30.57 and 0 µg kg-1 , respectively. Produced mycoprotein includes essential amino acids at appropriate contents and the ratio of unsaturated to saturated fatty acid was nearly 2:1. Also, calcium, iron, magnesium and zinc were found in mycoprotein, which could have health beneficial impacts on consumers. CONCLUSION: This study has provided information on safety aspects of mycoprotein production by F. venentaum IR372C from date wastes. However, further studies with focus on long-term clinical benefits of diets containing mycoprotein are necessary. © 2020 Society of Chemical Industry.

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

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

Pro j 2 is mesquite profilin: molecular characteristics and specific IgE binding activity.

BACKGROUND: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries. OBJECTIVE: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants. METHODS: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays. RESULTS: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract. CONCLUSION: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Designing a T-cell epitope-based vaccine using in silico approaches against the Sal k 1 allergen of Salsola kali plant.

Allergens originated from Salsola kali (Russian thistle) pollen grains are one of the most important sources of aeroallergens causing pollinosis in desert and semi-desert regions. T-cell epitope-based vaccines (TEV) are more effective among different therapeutic approaches developed to alleviate allergic diseases. The physicochemical properties, and B as well as T cell epitopes of Sal k 1 (a major allergen of S. kali) were predicted using immunoinformatic tools. A TEV was constructed using the linkers EAAAK, GPGPG and the most suitable CD4+ T cell epitopes. RS04 adjuvant was added as a TLR4 agonist to the amino (N) and carboxyl (C) terminus of the TEV protein. The secondary and tertiary structures, solubility, allergenicity, toxicity, stability, physicochemical properties, docking with immune receptors, BLASTp against the human and microbiota proteomes, and in silico cloning of the designed TEV were assessed using immunoinformatic analyses. Two CD4+ T cell epitopes of Sal k1 that had high affinity with different alleles of MHC-II were selected and used in the TEV. The molecular docking of the TEV with HLADRB1, and TLR4 showed TEV strong interactions and stable binding pose to these receptors. Moreover, the codon optimized TEV sequence was cloned between NcoI and XhoI restriction sites of pET-28a(+) expression plasmid. The designed TEV can be used as a promising candidate in allergen-specific immunotherapy against S. kali. Nonetheless, effectiveness of this vaccine should be validated through immunological bioassays.

Evaluation the frequencies of HLA alleles in moderate and severe COVID-19 patients in Iran: A molecular HLA typing study.

Background: Severe acute respiratory syndrome coronavirus 2 was first reported in December 2019 and it has spread globally ever since. The HLA system is crucial in directing anti-viral immunity and recent studies are investigating the possible involvement of the HLA genes on the severity of immune inflammation in different phases of COVID-19. Methods: In this cross-sectional study, peripheral blood-extracted genomic DNAs of 109 COVID-19 patients and 70 healthy controls were genotyped for different alleles of HLA-A, HLA-B, and HLA-DRB1 loci using sequence-specific primer PCR method. Results: The results indicated that frequencies of HLA-DRB1*11:01 and HLA-DRB1*04:03 were significantly higher in severe patients rather than moderates (p: <0.001 and 0.004, respectively). Also, it was observed that HLA-DRB1*04:01 was more frequent in moderate patients and healthy controls (p:0.002). In addition, HLA-B*07:35, and HLA-DRB1*07:01 showed higher frequencies in patients compared with controls (p: 0.031 and 0.003 respectively). Inversely, due to the higher frequencies of HLA-B*51:01 (p:0.027), HLA-DRB1*11:05 (p:0.003), HLA-DRB1*13:05 (p:0.022), and HLA-DRB1*14:01 (p:0.006) in healthy individuals rather than patients, they may be associated with COVID-19 resistance. Conclusion: The results show that, based on the population differences, the type of alleles related to the severity of COVID-19 is different, which should be clarified by designing large-scale studies in order to develop HLA-based treatments and vaccines.

Insights into Overlappings of Fibrosis and Cancer: Exploring the Tumor-related Cardinal Genes in Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

Investigating PM2.5 toxicity in highly polluted urban and industrial areas in the Middle East: human health risk assessment and spatial distribution.

Exposure to particulate matter (PM) can be considered as a factor affecting human health. The aim of this study was to investigate the concentration of PM2.5 and heavy metals and their influence on survival of A549 human lung cells in exposure to PM2.5 breathing air of Ahvaz city. In order to assess the levels of PM2.5 and heavy metals, air samples were collected from 14 sampling stations positioned across Ahvaz city during both winter and summer seasons. The concentration of heavy metals was determined using ICP OES. Next, the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was employed to ascertain the survival rate of A549 cells. The findings from this research demonstrated that average PM2.5 of the study period was (149.5 µg/m3). Also, the average concentration of PM2.5 in the urban area in winter and summer was (153.3- and 106.9 µg/m3) and in the industrial area this parameter was (191.6 and 158.3 µg/m3). The average concentration of metals (ng/m3) of urban areas against industrial, Al (493 vs. 485), Fe (536 vs. 612), Cu (198 vs. 212), Ni (128 vs. 129), Cr (48.5 vs. 54), Cd (118 vs. 124), Mn (120 vs. 119), As (51 vs. 67), Hg (37 vs. 50), Zn (302 vs. 332) and Pb (266 vs. 351) were obtained. The results of the MTT assay showed that the highest percentage of cell survival according to the exposure concentration was 25 > 50 > 100 > 200. Also, the lowest percentage of survival (58.8%) was observed in the winter season and in industrial areas with a concentration of 200 µg/ml. The carcinogenic risk assessment of heavy metals indicated that except for Cr, whose carcinogenicity was 1.32E-03, other metals were in the safe range (10-4-10-6) for human health. The high concentration of PM2.5 and heavy metals can increase respiratory and cardiovascular diseases and reduce the public health level of Ahvaz citizens.

Immunochemical Characterization of Salix alba (S. alba) Pollen Allergens and Evaluation of the Cross-Reactivity Pattern with Common Allergenic Pollen Grains.

BACKGROUND: Pollens, particularly tree and plant pollens, are one of the major causes of allergic respiratory diseases worldwide. Allergy to pollens of different species of Salix trees has been reported in various regions of the world. The most common type of Salix tree in Iran is white willow (Salix alba). OBJECTIVES: This study aimed to identify and determine the immunochemical characteristics of allergenic proteins in S. alba tree pollen extract using SDS-PAGE and IgE- immunoblotting methods. Moreover, the cross-reaction pattern of the specific IgE antibody of S. alba tree pollen proteins with pollen allergens of common allergenic trees, i.e., Populus nigra (P. nigra), Cupressus sempervirens (C. sempervirens), Pinus brutia (P. brutia) and Platanus orientalis (P. orientalis) in the region was investigated. METHODS: The reaction of allergenic proteins in S. alba pollen extract with specific IgE antibodies in patients' sera was investigated using SDS-PAGE and IgE-immunoblotting methods. The cross-reaction of specific IgE antibodies of the proteins present in S. alba pollen extract with pollen allergens of common allergenic trees in the region was investigated using ELISA and immunoblotting inhibition methods. In silico methods such as phylogenetic tree drawing and alignment of amino acid sequences were used to examine the evolutionary relationship and homology structure of common allergenic proteins (Panallergens) responsible for cross reactions. RESULTS: More than 11 protein bands binding to specific IgE antibodies in patients' sera with a molecular weight between 13 and 95 kDa were identified in the S. alba tree pollen extract. ELISA and immunoblotting inhibition results showed that P. nigra extract could inhibit the binding of IgE antibodies to S. alba pollen extract proteins to a greater extent than C. sempervirens, P. brutia, and P. orientalis tree extracts. In silico methods investigated the results of ELISA and immunoblotting inhibition methods. Moreover, a high structural homology and evolutionary relationship were observed between S. alba and P. nigra tree pollen panallergens. CONCLUSION: In this study, it was found that more than 80 % of the sensitive patients who were examined had specific IgE antibodies reacting with the approximately a 15 kDa-protein present in the S. alba pollen extract. Furthermore, the specific IgE-binding proteins found in the pollens of S. alba and P. nigra trees had relative structural homology, and it is likely that if recombinant forms are produced, they can be used for diagnostic and therapeutic purposes for both of the trees.

MiR-27a as a diagnostic biomarker and potential therapeutic target in systemic sclerosis.

Systemic sclerosis (SSc) or scleroderma is a multiorgan rheumatoid disease characterized by skin tightening or organ dysfunction due to fibrosis, vascular damage, and autoimmunity. No specific cause has been discovered for this illness, and hence no effective treatment exists for it. On the other hand, due to the lack of diagnostic biomarkers capable of effectively and specifically differentiating the patients, early diagnosis has not been possible. Due to their potent regulatory roles in molecular pathways, microRNAs are among the novel candidates for the diagnosis and treatment of diseases like SSc. MiR-27a is a microRNA known for its role in the pathogenesis of fibrosis and cancer, both of which employ similar signaling pathways; hence we hypothesized that Mir-27a could be dysregulated in the blood of individuals affected by SSc and it might be useful in the diagnosis or treatment of this disease. Blood was collected from 60 SSc patients (30 limited and 30 diffuse) diagnosed by a rheumatologist according to ACR/AULAR criteria; following RNA isolation and cDNA synthesis; real-time qPCR was performed on the samples using Taq-Man probes and data were analyzed by the ΔΔCT method. Also, potential targets of miR-27a were evaluated using bioinformatics. It was revealed that miR-27a was significantly down-regulated in SSc patients in comparison to healthy individuals, but there was no difference in miR-27 expression between limited and diffused SSc patients. Besides, miR-27a was found to target several contributing factors to SSc. It seems that miR-27a has a protective role in SSc, and its downregulation could result in the disease's onset. Based on bioinformatics analyses, it is speculated that miR-27a likely targets factors contributing to the pathogenesis of SSc, which are elevated upon the downregulation of miR-27a; hence, miR-27a mimics could be considered as potential therapeutic agents for the treatment of SSc in future studies. Since no difference was observed between limited and diffuse patient groups, it is unlikely that this microRNA has a role in disease progression. According to ROC analysis of qPCR data, miR-27a could be employed as a valuable diagnostic biomarker for SSc.

Prediction of airborne pollen concentrations by artificial neural network and their relationship with meteorological parameters and air pollutants.

After the early rainfall in the autumn of 2013, respiratory syndromes spread in the Khuzestan province of Iran with the most severity in Ahvaz. There have been recurring outbreaks in recent years. Considering that pollen-derived airborne allergens are regarded as key aeroallergens and the main cause of allergic rhinitis and asthma, this work aimed to forecast total pollen concentration in Ahvaz through an artificial neural network (ANN), followed by evaluating the pollen spatial distribution across the city and the association between pollen concentrations and environmental parameters. The utilized ANN in this work included an input layer with 13 parameters, a hidden layer of five neurons, and an output layer. Data were classified into training, validation, and testing sets. The ANN was implemented with 70% and 80% of data for training. The value of the correlation coefficient for the data validation of these two networks was 0.89 and 0.92, respectively. The results also indicated that despite the difference in the mean concentration of the pollens in various areas of Ahvaz, this difference was not statistically significant (P > 0.05). Furthermore, there was a negative correlation between the concentration of total pollen and relative humidity, precipitation, and air pressure. However, it had a positive correlation with temperature. Consequently, considering the logistical challenges of monitoring bioaerosols in the air, the ANN approach could predict total pollen concentrations. Therefore, in addition to measurements, the ANN technique can be a good tool to enable authorities to mitigate the impact of airborne pollen on people.

Immunochemical Characterization of Ligustrum Vulgare (Privet) Pollen Allergens: Study of Common Allergenic Plant in Iran.

Ligustrum vulgare (Privet) pollen proteins are responsible for allergies in susceptible individuals in many regions of the world. This study investigated the immunochemical characterization of Privet pollen extract and the occurrence of skin prick test reactivity to Privet and other allergenic pollen grains in allergic rhinitis patients. All subjects experienced a skin prick test with twenty-two allergen extracts. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separated Privet pollen extract, IgE-immunoblotting, and specific ELISA procedures determined the allergenic profile on forty-five Privet allergic patients. A positive allergic reaction to L. vulgare pollen extract was observed in forty-five (31.4%) out of 145 patients. Ten resolved protein fractions were found on SDS-PAGE, ranging from 10 to 80 kDa. IgE-specific antibodies interacted with several allergenic protein bands from Privet-allergic patients in the immunoblotting assay. The most significant interaction was observed in proteins with molecular weights of approximately 15, 18, 43, and 66 kDa. Privet pollen is regarded as a potent allergen composed of IgE-binding constituents. Considering the high allergenicity of Privet pollen grains and since many countries are rich in this plant, identification and production of recombinant forms of common allergens in this species can be used for developing more efficient diagnostic, therapeutic, and preventive approaches.

Induced Pluripotent Stem-cells Inhibit Experimental Bleomycin-induced Pulmonary Fibrosis through Regulation of the Insulin-like Growth Factor Signaling.

Idiopathic pulmonary fibrosis (IPF) is among the illnesses with a high mortality rate, yet no specific cause has been identified; as a result, successful treatment has not been achieved. Among the novel approaches for treating such hard-to-cure diseases are induced pluripotent stem cells (IPSCs). Some studies have shown these cells' potential in treating IPF. Therefore, we aimed to investigate the impact of IPSCs on insulin-like growth factor (Igf) signaling as a major contributor to IPF pathogenesis.  C57BL/6 mice were intratracheally instilled with Bleomycin (BLM) or phosphate-buffered saline; the next day, half of the bleomycin group received IPSCs through tail vein injection. Hydroxyproline assay and histologic examinations have been performed to assess lung fibrosis. The gene expression was evaluated using specific primers for Igf-1, Igf-2, and insulin receptor substrate 1 (Irs-1) genes and SYBR green qPCR master mix. The data have been analyzed using the 2-ΔΔCT method. The mice that received Bleomycin showed histological characteristics of the fibrotic lung injury, which was significantly ameliorated after treatment with IPSCs comparable to the control group. Furthermore, gene expression analyses revealed that in the BLM group, Igf1, Igf2, and Irs1 genes were significantly upregulated, which were returned to near-normal levels after treatment with IPSCs. IPSCs could modulate the bleomycin-induced upregulation of Igf1, Igf2, and Irs1 genes. This finding reveals a new aspect of the therapeutic impact of the IPSCs on IPF, which could be translated into other fibrotic disorders.

Expression Pattern of Inflammatory and Anti-Inflammatory Cytokines and Key Differential Transcription Factors in Peripheral Blood Mononuclear Cells of Iranian Coronavirus Disease 2019 Patients with Different Disease Severity.

The purpose of this research was to investigate the gene expression levels of inflammatory cytokines interferon (IFN)γ, tumor necrosis factor (TNF)α, interleukin (IL)1ß, IL2, IL6, IL8, and IL17, and anti-inflammatory cytokines IL4, IL10, IFNα, and IFNß, as well as relevant key transcription factors (TFs), including GATA3, PU1, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), IRF3 (interferon regulatory factor 3), BCL6 (B cell lymphoma 6 protein), FOXP3 (forkhead box P3), RORγt, and T-bet (T-box expressed in T cell) in Iranian patients with moderate and severe coronavirus disease 2019 (COVID-19). Fifty-six patients with COVID-19, and 25 healthy controls (HCs) age and sex matched were investigated. Based on the interim guidance of COVID-19 from the World Health Organization, the patients were classified into 33 moderate and 23 severe patients with COVID-19. The gene expression levels of cytokines and relevant TFs were quantified in peripheral blood mononuclear cells by quantitative real-time polymerase chain reaction (qRT-PCR). The gene expression levels of TFs RoRγ (RAR-related orphan nuclear receptor γ), NF-κB, and T-bet were significantly higher in patients with COVID-19 compared with HCs. Furthermore, the gene expression levels of cytokines, including IL2, IFNγ, IL6, TNFα, IL1ß, IL8, and IL17, were significantly higher in patients with COVID-19 than HCs. However, there was a significant increase for IL6, TNFα, and IL17 in severe compared with moderate patients with COVID-19. Finally, The Spearman correlation analysis revealed a significantly positive correlation for IL6 and TNFα, IL6 and IL2, IL6, IFNγ, and IL2 and IFNγ. These data suggest that expression of IL6, TNFα, and IL17 as well as the synergic effect of elevated values of IL2 and IFNγ should be considered in the treatment and management of patients with severe COVID-19.

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.

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

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

Identification of a new allergen from Amaranthus retroflexus pollen, Ama r 2.

BACKGROUND: Pollinosis from Amaranthus retroflexus pollen is a common cause of respiratory allergy in Iran with a high positive rate (68.8%) among Iranian allergic patients. The aim of the present study was to evaluate the allergenicity of the A. retroflexus pollen profilin. METHODS: Using sera from twelve patients allergic to A. retroflexus pollen, IgE-binding proteins from the A. retroflexus pollen extract was identified by immunoblotting. The cDNA of A. retroflexus pollen profilin was amplified, then cloned into the pET-21b (+) vector, expressed in Escherichia coli, and finally purified by metal affinity chromatography. The IgE-binding capacity of the recombinant protein was then analyzed by the ELISA, immunoblotting, and inhibition assays, as well as by the skin prick test (SPT). RESULTS: Immunoblotting results indicated a 14.6kDa protein with IgE-reactivity to 33% (4/12) among A. retroflexus pollen-allergic patients. Nucleotide sequencing of the cDNA revealed an open reading frame of 399 bp encoding for 133 amino acid residues which was belonged to the profilin family and designated as Ama r 2. A recombinant Ama r 2 (rAma r 2) was then produced in E. coli as a soluble protein which showed a strong IgE-reactivity via ELISA confirmed by the SPT. Inhibition experiments revealed high IgE cross-reactivities with the profilins from other plants. CONCLUSIONS: The profilin from the A. retroflexus pollen, Ama r 2, was firstly identified as an allergen. Moreover, rAma r 2 was produced in E. coli as a soluble immunoreactive protein with an IgE-reactivity similar to that of its natural counterpart.