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Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine observed in the development of many disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis. The anti-IL-17A biological drugs, including Secukinumab, Ixekizumab, and Brodalumab, are monoclonal antibodies approved for several disease treatments. Due to the disadvantages of biological therapies, including their immunogenicity, difficulties in scale generation, and high production costs and time, it is necessary to find new alternative anti- IL-17A agents for these monoclonal antibodies. Our study aimed to identify ssDNA aptamers that block IL-17A activity using the protein-SELEX procedure.The hIL-17A was expressed in codon plus E. coli, and after 14 rounds of the SELEX process, monitoring of aptamer pools was done using the dot blot method. Three families of aptamers were obtained from the selected round 9 aptamer pool, and seven truncates were created. Inhibitory effects of aptamer truncate on IL-17-induced CCL20 expression in HaCaT keratinocytes were evaluated.All aptamer truncates had a significant inhibitory effect compared to the library, but the inhibitory effect of M2 and M7 truncates was more than 80%. Moreover, we evaluated the potential binding site of selected aptamers by ELISA.We introduced a new small 17-nucleotide DNA aptamer that efficiently binds and blocks hIL-17A with a 0.3 nM kd, a potential anti-IL-17A therapeutic agent.

Immunotolerance induction in an antigen-specific manner is the long-term goal of immunotherapy to treat autoimmune diseases. Nanocarriers (NCs) can be designed as a new generation of delivery systems to modulate the immune responses through targeted delivery of antigens and immunomodulators to antigen presenting cells (APCs). In this manuscript, several formulation factors in the preparation of NCs which affect their uptake using APCs and generation of tolerance have been reviewed. The physicochemical properties and composition of NCs have been shown to play essential roles in achieving the desired immunological outcome. Also, targeting of dendritic cells and macrophages as APCs and direct targeting of the autoreactive lymphocytes have been presented as two main ways for induction of antigen-specific tolerance by these tolerogenic nanocarriers (tNCs). These particles herald a promising approach to treat or even prevent unwanted immune reactions in humans specifically.

Background Mesenchymal stem cells-derived adipose tissue (AT-MSCs) are recognized for the treatment of inflammatory diseases including multiple sclerosis (MS). Hypericum perforatum (HP) is an anti-inflammatory pharmaceutical plant with bioactive compounds. Plant tissue culture is a technique to improve desired pharmacological potential. The aim of this study was to compare the anti-inflammatory and proliferative effects of callus with field-growing plant extracts of HP on AT-MSCs derived from MS patients. Materials and methods AT-MSCs were isolated and characterized. HP callus was prepared and exposure to light spectrum (blue, red, blue-red, and control). Total phenols, flavonoids, and hypericin of HP callus and plant extracts were measured. The effects of HP extracts concentrations on proliferation were evaluated by MTT assay. Co-culture of AT-MSCs: PBMCs were challenged by HP plant and callus extracts, and Tregs percentage was assessed by flow cytometry. Results Identification of MSCs was performed. Data showed that blue light could stimulate total phenols, flavonoids, and hypericin. MTT test demonstrated that plant extract in concentrations (0.03, 1.2, 2.5 and 10 mu g/ml) and HP callus extract in 10 mu g/ml significantly increased. Both HP extracts lead to an increase in Tregs percentage in all concentrations. In particular, a comparison between HP plant and callus extracts revealed that Tregs enhanced 3-fold more than control groups in the concentration of 10 mu g/ml callus. Conclusions High concentrations of HP extracts showed effectiveness on AT-MSCs proliferation and immunomodulatory properties with a certain consequence in callus extract. HP extracts may be considered as supplementary treatments for the patients who receiving MSCs transplantation.

Programmed cell death protein-1 (PD-1), as an immune checkpoint molecule, attenuates T-cell activity and induces T-cell exhaustion. Although siRNA has a great potential in cancer immunotherapy, its delivery to target cells is the main limitation of using siRNA. This study aimed to prepare a liposomal formulation as a siRNA carrier to silence PD-1 expression in T cells and investigate it’s in vivo antitumor efficacy. The liposomal siRNA was prepared and characterized by size, zeta potential, and biodistribution. Following that, the uptake assay and mRNA silencing were evaluated in vitro at mRNA and protein levels. siRNA-PD-1 (siPD-1)-loaded liposome nanoparticles were injected into B16F0 tumor-bearing mice to evaluate tumor growth, tumor-infiltrating lymphocytes, and survival rate. Liposomal siPD-1 efficiently silenced PD-1 mRNA expression in T cells (P

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+IFN-γ+, CD8+IFN-γ+, and CD8+granzyme B+T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

This study compares the probiotic Lactobacillus strains isolated from dairy and Iranian traditional food products with those from human sources on intestinal microbiota using BALB/C mice model. First, Lactiplantibacillus plantarum (M11), Limosilactobacillus fermentum (19SH), Lactobacillus acidophilus (AC2), and Lactobacillus gasseri (52b) strains, isolated from either Iranian traditionally fermented products or human (healthy woman vaginal secretions), identified with molecular methods and selected based on the surface hydrophobicity, auto- and co-aggregation, were investigated for their probiotic properties and compared with their standard probiotic strains in vitro. The native strains and their mixtures (MIX) were then orally fed to five groups of female inbred BALB/C mice over the course of 38 days by gavage at 0.5 and 4 McFarland, respectively, equal to 1.5 × 10(8) and 1 × 10(9) cfu/ml. Feeding paused for 6 days to test the bacteria’s adhesion in vivo. According to the findings, the probiotic Lactobacillus strain isolated from human source (52b) exhibited the best in vitro and in vivo adhesion ability. Probiotic Lactobacillus strains isolated from Iranian traditional food products (19SH and AC2) had the most co-aggregation with Listeria monocytogenes (ATTC 7644), Salmonella enterica subsp. enterica (ATCC 13,076), and Escherichia coli (NCTC 12,900 O157:H7) in vitro. These strains produced the most profound decreasing effect on the mice intestinal microbiota and pathogens in vivo. The difference in the strains and their probiotic potential is related to the sources from which they are isolated as well as their cell walls. The results suggest that (19SH and 52b strains) are the best candidates to investigate the cell wall and its effect on the host immune system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00790-6.

Capacity and ability of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus probiotic strains isolated from some human and food sources were evaluated for modulating and regulating the immune system against hypersensitivity type 1 (dependent on IgE). In this study, given the probiotic properties of the strains and the use of mouse models, the strains were orally administered (1×10

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.

Background: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. Objective: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). Methods: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. Results: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. Conclusion: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

Psoriasis is a common inflammatory skin disorder, which is mediated by the immune system and affects 1-4% of the world's population. Psoriasis is caused by a complex interaction between the immune system, autoantigens, psoriasis-associated genetic factors, and various environmental factors. As a chronic disease requiring long-term treatment, psoriasis is associated with follow-up costs and an economic burden on the patients, their families, and healthcare systems. The current treatments for moderate-to-severe plaque psoriasis include topical therapy, phototherapy, and systemic drugs consisting of biological/non-biological drugs. Within the past two decades, recent biological therapies for psoriasis have rapidly advanced. Moreover, new bispecific agents have the potential for better disease control, while small molecule drugs offer a future alternative to biological drugs and the more cost-effective, long-term treatment of the disease. The present study aimed to review updated data regarding the inhibitors used to improve plaque psoriasis that contain biologics, bispecific agents, small molecules, and aptamers (either approved or in the research phase).

Allergen-specific sublingual immunotherapy (SLIT), a safe and efficient route for treating type I hypersensitivity disorders, requires high doses of allergens. SLIT is generally performed without adjuvants and delivery systems. Therefore, allergen formulation with appropriate presentation platforms results in improved allergen availability, targeting the immune cells, inducing regulatory immune responses, and enhancing immunotherapy's efficacy while decreasing the dose of the allergen. In this review, we discuss the adjuvants and delivery systems that have been applied as allergen-presentation platforms for SLIT. These adjuvants include TLRs ligands, 1α, 25-dihydroxy vitamin D3, galectin-9, probiotic and bacterial components that provoke allergen-specific helper type-1 T lymphocytes (TH1), and regulatory T cells (Tregs). Another approach is encapsulation or adsorption of the allergens into a particulate vector system to facilitate allergen capture by tolerogenic dendritic cells. Also, we proposed strategies to increasing the efficacy of SLIT via new immunopotentiators and carrier systems in the future.

Background Early detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets. The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosis mycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. Method M. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. Results The clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosis mycobacterium and other pathogenic bacteria. Conclusions Based on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

Diabetic foot ulcers (DFUs) are chronic severe complications of diabetes disease and remain a worldwide clinical challenge with social and economic consequences. Diabetic wounds can cause infection, amputation of lower extremities, and even death. Several factors including impaired angiogenesis, vascular insufficiency, and bacterial infections result in a delayed process of wound healing in diabetic patients. Treatment of wound infections using traditional antibiotics has become a critical status. Thus, finding new therapeutic strategies to manage diabetic wounds is urgently needed. Nanotechnology has emerged as an efficient approach for this purpose. This review aimed to summarize recent advances using nanotechnology for the treatment of diabetic wounds.

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

Background The first step of designing any genome-based molecular diagnostic test is to find a specific target sequence. The modified genome comparison method is one of the easiest and most comprehensive ways to achieve this goal. In this study, we aimed to explain this method with the example of Mycobacterium tuberculosis complex and investigate its efficacy in a diagnostic test. Methods A specific target was identified using modified genome comparison method and an in-house PCR test was designed. To determine the analytical sensitivity and specificity, 10 standard specimens were used. Also, 230 specimens were used to determine the clinical sensitivity and specificity. Results The identity and query cover of our new diagnostic target (5KST) were ≥ 90% with M. tuberculosis complex. The 5KST-PCR sensitivity was 100% for smear-positive, culture-positive and 85.7% for smear-negative, culture-positive specimens. All of 100 smear-negative, culture-negative specimens were negative in 5KST-PCR (100% clinical specificity). Analytical sensitivity of 5KST-PCR was approximately 1 copy of genomic DNA per microliter. Conclusions Modified genome comparison method is a confident way to find specific targets for use in diagnostic tests. Accordingly, the 5KST-PCR designed in this study has high sensitivity and specificity and can be replaced for conventional TB PCR tests. Electronic supplementary material The online version of this article (10.1186/s12879-018-3417-x) contains supplementary material, which is available to authorized users.

High levels of CD25, as part of the IL-2 receptor, are expressed on the surface of the activated T lymphocytes and regulatory T cells, indicating that the soluble CD25 (sCD25) could be a clinically valuable tool for treating several diseases. Moreover, progress has been achieved in targeting the IL-2 receptor to treat autoimmune diseases, organ transplantation and certain hematological malignancies. In the current study, generation of an ssDNA aptamer (Apt51) against CD25 is reported. Apt51 bound to CD25 with high affinity (Kd=13.4nM) and specificity. Furthermore, Apt51 was truncated to two shortened variants that almost retained their high affinity for the CD25 protein. Moreover, Apt51 showed good affinity and selectivity for the recognition of CD25 on the cell surface. Importantly, the study showed that Apt51 interfered with the binding of CD25 to its ligand (IL 2) and consequently decreased the IL-2-induced Akt activation.

Background Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Recent reports have shown that probiotics can induce immunomodulatory activity with promising effects in inflammatory diseases. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of Lactobacillus plantarum A7, which comprises human commensal bacteria, and Bifidobacterium animalis, a potential probiotic strain, on alleviation of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Methods To evaluate the therapeutic effects of probiotic strains, female C57BL/6 mice (8–10 wks old) received Lactobacillus plantarum A7, Bifidobacterium animalis PTCC 1631or a mixture of both strains through oral administration daily for 22 days beginning simultaneous with induction of EAE. The clinical parameters were recorded daily. On Day 22, each mouse was bled, and their spinal cord was removed for histology analysis. The effects of the treatments on regulatory T (Treg) cells level were evaluated using flow cytometry, and T-cell proliferation was assessed using a BrdU incorporation assay. The supernatants of spleen and lymph nodes cultured and mononuclear cells were collected for quantification of different panel of pro and anti-inflammatory cytokines by ELISA. The analysis of gene expression was performed at RNA level for transcription factors by real-time PCR. Results The results showed that treatment with a mixture of the two strains caused a more significant delay in the time of disease onset and clinical score compared to when the strains were used alone. The pathological features of the disease, such as mononuclear infiltration into the CNS, were also inhibited more significantly by the combinational approach. The results also revealed that treatment with combination of both strains enhanced the population of CD4+CD25+Foxp3+-expressing T-cells in the lymph nodes and the spleen. Treatment with our probiotic strains markedly inhibited disease associated cytokines while increased anti-inflammatory cytokines. Additionally, L. plantarumA7 and B. animalis ameliorated EAE condition by favoring Th2 and Treg differentiation via up-regulation of Foxp3 and GATA3 in the brain and spleen as well as inhibited the differentiation of Th1 and Th17 cells. Conclusions The current research provided evidence that probiotic therapy with L. plantarum and B. animalis can effectively attenuate EAE progression as well as reinforce the polarization of regulatory T-cells.

Death from infectious diseases has caused concerns about increases in the resistance of pathogens, impelling researchers to create novel therapeutic solutions. The management of intestinal tract problems has been the advance use of probiotics in medicine. The aim of this study was evaluate the physicochemical cell surface and adhesion properties of recombinant Lacotococcus lactis NZ1330 containing Ama r 2 gene, followed by the assessment of the antagonistic activity of this strain against the Escherichia coli causing urinary tract infection (UTI) in humans. For this purpose, cloning and expression of Ama r 2 gene were done. Afterwards, acid and bile resistance, which are the primary characteristics of any probiotic, were evaluated. The r-L. lactis NZ1330 was examined for the physicochemical properties of cell surfaces and the adhesion properties against Escherichia coli. Furthermore, the potential of the recombinant strain to adhere to adenocarcinoma intestinal cell line, Caco-2 cells, as well as the antagonistic properties of r-L. lactis NZ1330 against E. coli was investigated. r-L. lactis NZ1330 was capable of surviving at low pH and different concentrations of bile salts. 40.1% hydrophobicity, 36.5% auto-aggregation and 14.4% co-aggregation were observed for this strain. The adhesion level of r-L. lactis NZ1330 was 5.7% which was also confirmed by scanning electron microscopy (SEM). r-L. lactis NZ1330 was able to compete, inhibit and displace the adhesion of Escherichia coli to Caco-2 cells. r-L. lactis NZ1330 was considered to be a reliable probiotic alternative by showing these desirable properties. Results revealed that Ama r 2 gene expression had no effect on the positive probiotic properties of L. lactis NZ1330, proving this strain could be a suitable probiotic host for the expression of this allergen.

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.

Background Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). This enzyme is encoded by the MUT gene and is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, and threonine, and cholesterol. Method Three unrelated affected patients with isolated MMA and their parents were studied. The MUT gene was analyzed by PCR and sequencing of its entire coding region and the highly conserved exon-intron splice junctions. The homology modeling of the novel mutation found in the MUT gene was performed using the online Swiss-Prot server for automated modeling and then analyzed with special bioinformatics software to better study the structural effects caused by the mutation. Result We found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C > G). The variant is located near the highly conserved acceptor splice site of intron 12. A region at the C-terminus of the protein from ASP709 to GLN748 has been deleted by the alteration of c.2125-3 C > G in intron 12 of the MUT gene. Further studies of the novel mutation in the MUT gene by means of homology modeling revealed abnormalities in the protein's structure, which causes the protein to act malfunctioning and also the mRNA expression analysis of MUT gene confirmed these results. Conclusion We report this novel mutation, including its clinical and biochemical features and genetic defects, in the MUT gene of three patients affected with isolated MMA. Structural analyses of the mutated protein identified changes in the energy and stereochemical features of the protein that unfortunately altered the protein's functionalities. Therefore, we demonstrate that a novel splice site mutation in intron 12 of the MUT gene is a potential highly pathogenic allele via inhibition of alternative splicing.

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.

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.

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.

Aptamers, as a novel class of molecular probes for diagnosis, imaging and targeting therapy, have attracted increasing attention in recent years. Aptamers are generated from libraries of single-stranded nucleic acids against different molecules via the "systematic evolution of ligands by exponential enrichment" (SELEX) method. SELEX is a repetitive process of a sequential selection procedure in which a DNA or RNA library pool is incubated separately with target and control molecules to select specific oligonucleotide aptamers with high affinities and specificities. Cell-SELEX is a modified version of the SELEX process in which whole living cells are used as targets for the aptamers. Dendritic cell (DC) targeting, as a new therapeutic approach, can improve the efficiency of immunotherapy in the treatment of allergies and cancers. DCs use various receptors to continuously induce adaptive immunity via capture and presentation of antigens to naïve T cells. DCs are considered as the best targets in modulating immune responses against cancer, autoimmunity, allergy and transplantation. Aptamers, as a new agent, can be applied in DC targeting. The purpose of this review is to present some general concepts of aptamer production and DC targeting by aptamer molecules.

Background Anthracofibrosis is the black discoloration of the bronchial mucosa with deformity and obstruction. Association of this disease with tuberculosis (TB) was approved. The objective of this study was to find the additional benefit of assessment of TB by the polymerase chain reaction (PCR) method. Methods Bronchoscopy was performed on 103 subjects (54 anthracofibrosis and 49 control subjects) who required bronchoscopy for their pulmonary problems. According to bronchoscopic findings, participants were classified to anthracofibrosis and nonanthracotic groups. They were examined for TB with traditional methods such as direct smear (Ziehl-Neelsen staining), Lowenstein-Jensen culture, and histopathology and the new method "PCR" for Mycobacterium tuberculosis genome (IS6110). Results Age, sex, smoking, and clinical findings were not significantly different in the TB and the non-TB groups. Acid-fast bacilli could be detected by a direct smear in 12 (25%) of the anthracofibrosis subjects, and adding the results of culture and histopathology traditional tests indicated TB in 27 (31%) of the cases. Mycobacterium tuberculosis was diagnosed by PCR in 18 (33%) patients, but the difference was not significant. Detection of acid-fast bacilli in control nonanthracosis subjects was significantly lower (3, 6%), but PCR (20, 40%) and accumulation of results from all traditional methods (22, 44%) showed a nonsignificant difference. Conclusions The PCR method showed a result equal to traditional methods including accumulation of smear, culture, and histopathology.

Objective To study immunophenotype, differential proliferation capacity, invasiveness, adhesion, and cytokine production in ectopic and eutopic endometrial stromal cells (EESCs and EuESCs) from patients with endometriosis. Design In vitro study. Setting Academic research center. Patient(s) Patients with ovarian endometriosis (endometrioma) and nonendometriotic controls. Intervention(s) None. Main Outcome Measure(s) EESCs and EuESCs from 25 patients with endometrioma and ESCs from 20 nonendometriotic controls (CESCs) were isolated, and their immunophenotype, proliferation, invasion, adhesion, and cytokine production were assessed and compared. Result(s) Isolated ESCs from all three sources expressed markers specific for cells of mesenchymal origin but were negative for hematopoietic markers. EESCs exhibited a significantly lower proliferation rate in fibronectin-coated plates and less invasive capacity compared with CESCs or EuESCs. Among all stromal cell groups studied, EuESCs showed the highest invasive behavior. EESCs adhered more firmly to extracellular matrix than EuESCs or CESCs in all time intervals examined. The levels of interleukin (IL) -6 and IL-8 production by EESCs were significantly higher compared with those of EuESCs or CESCs. Conclusion(s) The results of the present study demonstrated that retrograde menstruation alone does not account for the pathogenesis of endometriosis as eutopic and ectopic counterparts of ESCs from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior.

In this study a new multifunctional recombinant gene delivery system (vector) was developed for targeted gene delivery to ZR-75-1 breast cancer cells. The vector backbone contained multiple domains including: (1) two tandem repeating units of truncated histone H1 to condense pDNA, (2) a model cell targeting peptide to target ZR-75-1 cells, (3) a pH-responsive synthetic fusogenic peptide, KALA, to destabilize endosomal membrane, and (4) a nuclear localization signal from human immunodeficiency virus to enhance translocation of pDNA toward the cell nucleus. The vectors were cloned and expressed in Escherichia coli BL21 (DE3) followed by purification with Ni-NTA affinity chromatography. They were then characterized using physicochemical and in vitro biological methods to evaluate the gene transfer efficiency and vector multifunctionality. The results demonstrated that the recombinant vector bearing all four functional domains had the highest rate of gene transfection efficiency as compared to the vectors which lacked one or more functional motifs. Beside the ability to target, the developed multifunctional vector was able to disrupt endosomal membranes, reach cell nucleus by utilizing microtubules and transfect efficiently while showing no detectable toxicity.

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

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 46 kDa 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.

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 14.6 kDa 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 IgEreactivity 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.

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.

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.

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

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.

Background Pollens from the Salsola spp. are an important source of respiratory allergy in tropical countries. Our aim was to characterize the IgE binding proteins of S. incanescens pollen extract and study its cross-reactivity with S. kali pollen allergens. Methods Prick tests with S . kali and S . incanescens pollen extracts were performed on eight respiratory allergy patients from Mashhad, Northeast Iran. The antigenic profiles and IgE-binding patterns of S. kali and S. incanescens pollen extracts were compared by SDS-PAGE and Western blotting, using individual sera from the salsola pollen-sensitive patients. Cross-reactivity of proteins in the two weeds was assessed by IgE- immunoblotting inhibition. Results S. kali and S. incanescens pollen extracts showed similar IgE-binding profiles in Western blotting. The IgE binding components of 39, 45, 66 and 85 kDa were detected in both pollen extracts. Furthermore, inhibition of the immunoblots revealed extensive inhibition of IgE binding to proteins and a close relationship between these two weeds allergens. Conclusions S. incanescens pollen is a potent allergen source with several IgE binding components that shows a close allergenic relationship with S. kali . Our results suggest that in S. incanescens - rich areas, S. kali pollen extracts could be used as a diagnostic reagent for allergic patients to S. incanescens pollen.

Pollen from different tree and grass species represent the greatest inducers of type I allergy worldwide. Oriental plane trees, as Platanus orientalis, are an important source of airborne allergens in cities of the southwest Asia and southeast Europe. This study was aimed to identify relevant allergens of P. orientalis pollen and to ascertain whether P. orientalis allergens have cross-reactivity with related plane trees, such as Platanus acerifolia and Platanus occidentalis pollen components. Nineteen patients with a clinical history of reaction to P. orientalis pollen and a positive skin-prick test (SPT) to P. orientalis pollen extract were included in this study. Identification of IgE-binding proteins in Platanus pollen extracts was elucidated by immunoblotting using sera from P. orientalis pollen-sensitive patients. Cross-reactivity studies among P. orientalis allergens and relevant species was evaluated by immunoblot-inhibition and ELISA inhibition assays. All the patients were polysensitive and exhibited positive SPT to grass and tree pollen allergens. The IgE-binding pattern of P. orientalis pollen extract was well defined. The inhibition experiments showed that the capacity of P. occidentalis was greater than P. acerifolia pollen extract in preventing the reactivity of immobilized P. orientalis pollen extract with the IgE antibodies of patients. The 28-kDa molecule was the most frequent allergen among nine IgE-binding proteins of P. orientalis pollen. The IgE-reactive components of P. orientalis pollen showed a higher level of cross-reactivity with P. occidentalis pollen in comparison with P. acerifolia pollen.

Multiple sclerosis (MS) is a central nervous system disorder mainly characterized by inflammation, demyelination and axonal injury. Anti-inflammatory agents can be used to ameliorate the disease process. Hypericum perforatum L or St. John's wort is widely used as an anti-depressant and anti-inflammatory remedy in traditional and herbal medicine. Based on St. John's wort properties, the therapeutic potentials of an H. perforatum extract (HPE) and a single component, hyperforin were evaluated for effectiveness against MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. Female C57BL/6 mice were immunized with specific antigen MOG35-55 and then administered different doses of hyperforin or HPE post-immunization. Clinical symptoms/other relevant parameters were assessed daily. Histological analysis of the spinal cord was performed. T-cell proliferative activity was also evaluated using a BrdU assay. The effect of hyperforin on regulatory T-cells (Treg cells) was assessed using flow cytometry. The results indicate hyperforin and HPE reduced the incidence and severity of EAE, an outcome that closely correlated with an inhibition of pathological features (leukocyte infiltration and demyelination) and antigen-specific T-cell proliferation. The study also showed that hyperforin caused increased Treg cell levels in the spleen. These results indicated that hyperforin and HPE could attenuate EAE autoimmune responses by inhibiting immune cell infiltration and expansion of Treg cell and could eventually be considered as a potential candidate for use in the treatment of MS.

Aims Cardiac complications are common in carbon monoxide (CO) poisoning and associated with high morbidity and mortality. We have previously shown that erythropoietin (EPO) could reduce CO-induced cardiac ischemia in rat. In the current study, the anti-apoptotic effect of EPO during CO cardiotoxicity was investigated in order to elucidate the mechanism of EPO anti-ischemic action. Main methods Wistar rats were exposed to CO (250, 1000 and 3000 ppm). EPO (5000 IU/kg) was administered to all groups by intraperitoneal injection at the end of CO exposure period. TUNEL and caspase-3 activity levels were assessed to investigate the effects of CO exposure and subsequent EPO administration on myocardial apoptosis. The changes of mitochondrial membrane potential (MMP) were also assessed with sensitive lipophilic dye JC-1 by flow cytometry. The roles of Bcl2 and Bax in EPO protective effect were investigated by Western blotting. Key findings Myocardial apoptosis was observed following CO exposure. Moreover, mitochondrial membrane depolarization and significant reduction in Bcl2/Bax ratio were shown following CO poisoning especially at 3000 ppm. On the other hand, EPO administration could effectively suppress apoptosis in myocardial cells. Also, EPO significantly prevented the CO-induced depolarization of MMP (p Significance EPO reduces myocardial injury due to CO intoxication. Thus EPO could be suggested as a possible candidate for the management of CO cardiotoxicity with clinical applications.

Background The exact pathogenesis of pterygium is still not fully understood. Growth factors are considered to play an important role in the formation of pterygium. Transforming growth factor (TGF)-β1 is considered to be one of the main mediators of fibroblast stimulation and tissue remodeling in allergic conditions. The objective of the present study was to investigate the association between TGF-β1 gene expression and pterygium in atopic and nonatopic participants. Methods We used questionnaires to record demographic and clinical information from patients who underwent pterygium excision surgery. Skin prick examination was done to confirm or rule out atopy in 30 patients with atopy (Case Group) and 30 individuals without atopy (Control Group). Additionally, measurement of serum immunoglobulin E, cytokines, including interleukin-4 and interferon-γ, and peripheral blood eosinophil count was performed to confirm atopy in 30 consecutive patients (Case Group). A semiquantitative reverse transcription polymerase chain reaction was performed to determine TGF-β1 gene expression in all individuals. Results TGF-β1 mRNA gene expression was significantly higher ( p = 0.0001) in atopic patients 2.50 ± 1.11 compared to nonatopic individuals 1.40 ± 0.46. Eosinophil count and serum immunoglobulin E were significantly higher ( p = 0.031 and p = 0.001, respectively) in atopic patients compared to the Control Group. Serum interleukin-4 was also significantly higher ( p = 0.01) in atopic patients compared with nonatopic individuals. Conclusion Excess expression of TGF-β1 gene in pterygium tissue of atopic individuals suggests that growth factors play a role in the pathogenesis of pterygium.

Esophageal squamous cell carcinoma (ESCC) is the second-most frequently diagnosed cancer in Northeast Iran, often diagnosed in advanced stages. No standard early diagnostic guideline has been proposed to date and current therapeutic modalities are not effective. Detection of tumor-specific biomarkers, which is the goal of this study, could prove useful in the diagnosis of ESCC. To better understand the gene expression profile of ESCC, we analyzed tumor samples and corresponding adjacent normal tissues from ESCC patients by Chemiluminescent Human Cancer GEArrays. Candidate genes were verified by real-time PCR. Out of 440 cancer-related genes included in the array, 71 were overexpressed compared to normal tissue, with significant differences in 11 genes. There were 108 genes underexpressed, with significant differences in 5 genes. Until now, the AP2M1, FTL, UBE2L6, HLA-C, and HSPA8 overexpressed genes and XRCC5, TP53I3 and RAP1A underexpressed genes were not reported in ESCC. We chose the MMP2, HLA-G, and XRCC5 markers from 58 Iranian ESCC patients to verify the expression validity by real-time PCR. The microarray results were confirmed with two-tailed significance levels of P = 0.003 (MMP2), P = 0.000 (HLA-G) and P = 0.002(XRCC5). Analysis performed for the candidate genes using GNCpro online software highlighted two pathways, an immuno-modulatory response and DNA replication and repair. We successfully performed and validated Chemiluminescent GEArray gene expression profiling in ESCC. Several biomarkers that might be related to tumorigenesis in ESCC were identified.Immuno-modulatory and DNA repair pathways could be used as targets to locate specific diagnostic, prognostic, and therapeutic biomarkers for ESCC.

The exact pathogenesis of pterygium has not been completely elucidated. Growth factors have been considered to play a role in pterygium formation. Vascular endothelial growth factor (VEGF) is one of the principal mediators of angiogenesis, fibroblast stimulation and tissue remodeling in allergic conditions. The aim of this study was to compare the association between pterygium and VEGF gene expression between atopic and non-atopic individuals. At first visit, all patients with pterygium underwent blood tests, serum immunoglobulin E (IgE), serum cytokines including interleukin-4 (IL-4) and interferon-γ (IFN-γ) and peripheral blood eosinophil count. After obtaining informed consents, questionnaires were used to obtain demographic and clinical data from patients who underwent pterygium excision surgery. Skin prick test was performed to confirm or rule out atopy in 30 patients with (case group) and 30 patients without (control group) atopy. Pterygium tissues were then removed by surgery. A semi-quantitative reverse transcriptase polymerase chain reaction was performed to determine VEGF gene expression in all patients. Our results illustrated that VEGF mRNA expression in atopic patients was significantly higher than in the non-atopic group (P = 0.01). Eosinophil count, serum IgE and IL-4 were also significantly higher in atopic patients than in the non-atopic group (P = 0.03, 0.001 and 0.001, respectively). However, no significant difference was noted in serum IFN-γ between the two groups (P = 0.06). The excessive expression of VEGF gene in pterygium tissue of patients with atopy suggests that growth factors may play a role in the pathogenesis of pterygium or accelerate its formation.

Introduction Most of pathogenesis related (PR) proteins possess complicated structures; hence their active recombinant forms are usually produced in eukaryotic systems. In this study, we employed an insect cell line to express a recombinant form of a previously identified grape PR3 allergen categorised as class IV chitinase. Methods Grape chitinase cDNA was amplified by RT-PCR and inserted into pFastBacHTA using restriction enzymes. The recombinant pFastBacHTA was applied for the transformation of Escherichia coli DH10Bac cells. The purified recombinant bacmid was used for transfection of Sf9 cells. Finally, the IgE-immunoreactivity of purified recombinant protein was evaluated using grape allergic patient's sera. Moreover, polyclonal anti-6His-tag and monoclonal anti-chitinase antibodies were used for further assessment of recombinant protein. Results SDS–PAGE analysis of the transfected Sf9 cells showed expression of a monomeric 25 kDa and a dimeric 50 kDa recombinant protein. Western blotting revealed considerable IgE reactivity of the recombinant protein with grape allergic patients’ sera. Furthermore, confirmatory assays showed specific reactivity of the recombinant protein with anti-His tag and anti-chitinase antibodies. Conclusion This study showed that, in contrast to E. coli, insect cells are suitable hosts for the production of a soluble and IgE-reactive recombinant form of grape class IV chitinase. This recombinant allergen could be used for component resolved diagnosis of grape allergy or other immunodiagnostic purposes.

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

Cystic echinococcosis (hydatidosis) is a zoonotic helminthic disease of human and other intermediated hosts wherein infection is caused by the larval stages of tapeworm Echinococcus granulosus. Growth of the larval stage is formed throughout the internal organs, the liver and lung, causing their destruction. Important pathways are unknown about suppression and survival of cysts in human body. In this study, apoptotic bifunctional effects are evaluated in relationship between host and parasite in cystic echinococcosis. Human lymphocytes were treated with hydatid fluid (HF). After 6 h of exposure, caspase-3 activity was measured by fluorometric assay in the HF-treated lymphocytes and control cells. Also, the expression of Bax (as pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) mRNA was assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after 12 h of exposure. For surveying of apoptosis-inducing ligands TNF-related apoptosis-inducing ligand and Fas-L, germinal layer and accompaniment peripheral tissues as healthy control were separated by scalpel from each cyst in sterile condition, then were assess by semiquantitative RT-PCR method in mRNA expression. Both the ratio of Bax/Bcl-2 mRNA expression and caspase-3 activity were higher in the fertile fluid-treated lymphocytes relative to infertile fluid-treated lymphocytes and control group versus the expression level of apoptosis-inducing ligands having a relatively high level in germinal layer of infertile cyst in comparison to fertile cyst and healthy tissue. Apoptosis of germinal layer of fertile cysts is possibly one of the suppression mechanisms in hydatidosis patients, in contrast to lymphocytes apoptosis by modulator of hydatid fluid, one of the hydatid cyst survival mechanisms.

The cyclooxygenase-2 (COX-2) inhibitors including celecoxib inhibit cell growth and induce apoptosis in cancer cells. As COX-2 is over expressed in solid tumors such as colorectal cancer, it can be a suitable target for cancer treatment studies. In this study we designed and synthesized 4,5-bisaryl imidazolyl imidazoles as novel COX-2 inhibitors and evaluated their apoptosis inducing activities. The ability of our synthetic compounds to inhibit ovine COX-1 and COX-2 was determined using a colorimetric method. The effects of these COX-2 inhibitors and celecoxib on the proliferation of Caco-2 cells were evaluated by MTT assay. Cell apoptosis was determined by flow cytometry and DNA fragmentation assay. cDNA microarray technique was used to evaluate the effects of these synthetic compounds on 112 genes involved in apoptosis pathways. The expression of five apoptosis-related genes Bak-1, Bcl-x, BIRC (Survivin), TNFSF10 and CASP3 were evaluated by quantitative real-time PCR. Among our synthetic compounds (3a-c), 4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl derivative (compound 3c) exhibited the highest COX-1/COX-2 selectivity index (SI=262.9) and lowest growth inhibitory concentration (IC(50)=21.20μM). In addition, compounds 3a-c could up-regulate pro-apoptotic genes and down-regulate anti-apoptotic genes. So, these synthetic compounds seem to be inducers of apoptosis in Caco-2 cell line. This study indicates that 4,5-bisaryl imidazolyl imidazole is a suitable scaffold to design COX-2 inhibitors and 4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl derivative exhibited highly COX-2 inhibitory potency and selectivity even more than celecoxib. It seems that it could induce apoptosis in Caco-2 colorectal carcinoma cell line.

The goal of study was first to design multi-epitope peptides from the rat HER2/neu (rHER2/neu) oncogene and then to evaluate the effectiveness of these peptides encapsulated in liposome-polycation-DNA(LPD) nanoparticles (NPs) for the induction of immune response in BALB/c mice. Four multi-epitope peptides derived from the rHER2/neu were designed and different groups of mice were vaccinated with free peptides or peptides encapsulated in NPs. Two of the four tested peptides (p5 and p435), as well as their combinations with the LPD NPs induced a significantly higher IFN-γ and CTLresponses in comparison with the control groups. Consequently, these responses led to lower tumor sizes and longer survival time in TUBO tumor mice model. Our results demonstrate that rHER2/neu-peptides (p5 and p435) and their encapsulation can induce an antigen-specific immunity. This study also presents the first attempt to evaluate the effectiveness of natural rHER2/neu-peptides containing CTL multi-epitope and encapsulated in LPD NPs. From the Clinical Editor This study represents the first attempt to evaluate the effectiveness of natural rHER2/neu-peptides containing CTL multi-epitope encapsulated in LPD NPs, demonstrating that rHER2/neu-peptides (p5 and p435) and their encapsulation can induce tumor antigen-specific immunity.

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.

The aim of this study was to investigate a new allergen of Salsola kali, Sal k 4, and to investigate the predictive value of the conserved conformational regions in cross-reactivity with other plant-derived profilins. The Sal k 4-coding sequence was cloned, expressed, and purified by one-step Ni2+ affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted conserved conformational regions among rSal k 4 and other plant-derived profilins. Immunodetection and inhibition assays using 30 individual sera from S. kali allergic patients indicated that purified rSal k 4 might be the same as that in the crude extract. The results of inhibition assays among rSal k 4 and other plant-derived profilins were in accordance with the homology of the predicted conserved conformational regions. Amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins might depend on the predicted conserved conformational regions.