Toll-like receptors (TLRs) play principle roles in recognition of autologous components which have been pointed as the danger-associated molecular patterns (DAMP) and microbial components which are identified as pathogen associated molecular patterns (PAMP).The infiltration of various inflammatory cells such as dendritic cells, lymphocytes (CD4⁺ T, CD8⁺ T as well as B cells), monocytes and macrophages occur into the central nervous sys­tem (CNS) during multiple sclerosis (MS) and its animal model named experimental autoimmune encephalomyelitis (EAE). The infiltrated leukocytes and residential cells of the CNS express several TLRs (especially TLR2) and their expression are elevated in MS and EAE. TLR2 recognizes a large variety DAMP and PAMP molecules due to its ability to create heterodimers with TLR1, TLR6 and probably TLR10. A wide spectrum of  DAMP molecules, including heat shock protein 60 (HSP60), HSP70, high mobility group box 1 (HMGB1), β-defensin 3, surfactant protein A and D, eosinophil-derived neurotoxin, gangliosides, serum amyloid A, hyaluronic acid and biglycan are identified by TLR2, whose their expression is increased in MS patients. TLR2 may contribute in the development of MS and EAE diseases through the reinforcement of Th1/Th17 cell-related responses, downregulation of regulatory T cells, induction of IL-17⁺ γδ T cells, inhibition of oligodendrocyte maturation, induction of poly ADP-ribose polymerase-1 (PARP-1)-dependent pathway in microglia, macrophages and astrocytes and inhibition of type I interferons expression. The contribution of TLR2-related immunopathological responses in the MS and EAE pathogenesis and its possible targeting as promising therapeutic potentials are considered in this review. 

Growing evidence indicated conflicting results that Interleukin-18 (IL-18) promoter polymorphisms rs1946518 (A-607C), rs187238 (G-137C) and rs549908 (A-105C) were associated with asthma risk. The aim of this study is to comprehensively evaluate the IL-18 polymorphisms and asthma by a systematic review and meta-analysis. A total of 12 studies testing the association between these polymorphisms and asthma were examined (8 studies for A-607C, 8 studies for G-137C, and 4 studies for A-105C) in the update meta-analysis, up to Dec 30, 2017. Summary odds ratios (ORs) and 95% confidence intervals (CI) were used to estimate the strength of association between each polymorphism and asthma using fixed- and random-effects models when appropriate. Heterogeneity and publication bias were evaluated. The meta-analysis results indicated that any allele frequencies of the IL-18 polymorphisms (A-607C, G-137C and A-105C) was not associated with asthma risk (p>0.05). And no statistically significant association was observed between genotype frequencies of these polymorphisms and asthma under different genetic models (p>0.05). Subgroup analysis results were similar to the main analysis by ethnicity, sample size, genotyping methods, matching criteria and quality score. There was no evidence of publication bias. The present meta-analysis suggests that IL-18 polymorphisms (A-607C, G-137C and A-105C) were unlikely to be associated with asthma risk.

A growing body of evidence suggests the existence of abnormalities in the immune system of schizophrenic patients. The current study examined serum levels of interleukin (IL) -1β, IL-6, IL-2,interferon(IFN) -γ, and tumor necrosis factor(TNF)-α in schizophrenic patients before and after treatment with risperidone and correlated levels of these cytokines with symptomatology. The study group consisted of 24 schizophrenic patients as defined by Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) criteria and 24 healthy controls. Serum cytokine levels were examined using enzyme-linked immunosorbent assay (ELISA). Schizophrenic symptomatology was assessed with the Positive and Negative Syndrome Scale (PANSS) questionnaire. The serum levels of TNF-α, IL-1β and IL-6 were significantly higher in participants before treatment compared with the healthy controls and after treatment (p<0.001). IFN-γ and IL-2 levels were significantly lower in participants after treatment compared with before treatment and the healthy controls (p<0.001). Except for IL-6 (p<0.05), there was no significant difference in the levels of TNF-α and IL-1β between the patients receiving treatment and the healthy subjects. Moreover, there was no significant difference in levels of IFN-γ and IL-2 between patients before treatment and the healthy subjects. There were no significant correlations between the concentration of cytokines studied and the PANSS. Positive intercorrelations between the production of IFN-γ and IL-2 were detected for sums of all groups(r=0.33, p=0.005). Clinical improvement of treated patients was associated with a reduction in the studied cytokines. It seems that changes in the cytokines level may play a significant role in the psychopathology of these patients.

Dickkopf (DKK) family of proteins are known as antagonists for the Wnt-β-catenin signaling pathway. It is suggested that the Dickkopf-1 (DKK-1) has a role in several diseases such as hepatocellular carcinomas, hepatoblastomas, Wilms’ tumors, lung cancer and Myeloma bone disease. The aim of the present study was to produce a chimeric-recombinant DKK-1 protein in order to induce immune response against the antigen. The recombinant Dickkopf-1 (rDKK-1) protein was designed using bioinformatics analysis. The standard methods were used for cloning, expression and purification. The structure of recombinant protein was analyzed by spectroscopy methods. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were performed to confirm the recombinant protein using a commercial anti-DKK-1 (whole protein) polyclonal antibody. The immunogenicity of the recombinant DKK-1 was assessed by immunizing, intraperitoneally, BALB/C mice four times with the 31-kDa and 45-kDa purified rDKK-1 cloned in pET28a and pET32a vectors respectively. The antibody titer was measured in due course of time. Stronger immunogenic parts of the protein were selected based on in-silico predictions and recombinant protein was successfully designed. The chimeric gene was sub-cloned, expressed, purified and refolded. The purified protein was confirmed by Western blotting and ELISA. The three dimensional structural was confirmed by CD spectrum and predicted structures by bioinformatics tools, revealed the stability of helix structures. rDKK-1 protein was capable of inducing immune response with high titer antibody and  excessive humoral immune response. No significant difference was observed between immunization by 31-kDa and 45-kDa antigen.

Angiogenesis has essential role in growth and metastasis of tumors. Development of therapies aimed to suppress angiogenesis using medicinal plants is one of the effective approaches for prevention/treatment of cancer. The current study was performed to investigate in vitro anti-angiogenic effect of Teucrium Polium (TP) extract and its fractions. The aerial part of Teucrium Polium was powdered and extracted with 50% ethanol. The extract was fractionated in to aqueous (AQ), n-butanol (BU), ethyl acetate (EA) and n-hexane (HE) fractions. Anti-angiogenic effect of TP was examined on human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen matrix. The endothelial cells form capillary-like branches that can be visualized using phase contrast microscope and the number of tube-like structures can be quantified as a measure of in vitro angiogenesis. Furthermore, anti-proliferative and vascular endothelial growth factor(VEGF )suppressive effect of TP as important factors in the process of angiogenesis were assessed using3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)and quantitative ELISA, respectively. Based on our findings, among the TP fractions, EA fraction showed the highest inhibitory activity on angiogenesis. This fraction with IC50: 68 µg/mL, inhibited angiogenesis at 60 µg/mL. The crude extract and other fractions of TP inhibited angiogenesis in a dose-dependent manner at doses higher concentrations than EA fraction, significantly.TP extract and EA fraction were able to inhibit proliferation of HUVEC and inhibited VEGF secretion in a dose dependent manner. The ethyl acetate fraction at 60 µg/ml inhibited VEGF secretion perfectly. Our data indicated that ethyl acetate fraction of Teucrium Polium could be a potential candidate for the prevention of angiogenesis in cancer and other related disorders. However, this suggestion needs more quantitative and in vivo investigations for confirmation.

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system.MS creates a wide range of symptoms with lifelong debilitating consequences. The hallmark of the disease is the inflammation of the nervous system, which can lead to damage to the nerve tissue and loss of function of the neurons. IL-17 has a prominent role in the beginning of inflammatory reactions. Here, we analyzed a mouse model developed using anti-myelin antibodies. This mouse model mimics many symptoms of MS in humans. C57BL/6 mice were randomly divided into five groups. Mice were immunized subcutaneously with 50 μg, 100 μg, 150 μg and 200 μg myelin oligodendrocyte glycoprotein in complete Freund’s adjuvant containing 4 mg/Ml Mycobacterium tuberculosis and two injections of 800 ng of pertussis toxin intraperitoneally, on day 0 and 2 post immunization. Serum level of IL-17 was measured, inflammation and demyelination of brain tissue were also evaluated. Mice with experimental autoimmune encephalomyelitis demonstrated inflammatory cell accumulation, different degrees of demyelination in the brain, and rising levels of serum IL-17 depending on the dose of the anti-myelin antibody. Our study demonstrates that level of IL-17 production is directly associated with inflammation and demyelination. In addition, different degrees of experimental autoimmune encephalomyelitis in mice can be utilized to test a wide range of therapeutic interventions for MS treatment.

C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the “Loss of a primer binding site” technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The “Loss of a primer binding site” technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.

In this study we aimed to examine the relaxant effect of berberine, a compound extracted from a variety of herbs, on rat tracheal smooth muscle (TSM) and its possible mechanism(s). Cumulative concentrations of berberine (20, 65, 200 and 600 μg/mL) were added on pre-contracted TSM by methacholine or KCl in non-incubated or incubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, L-NG-nitro arginine methyl ester (L-NAME) and papaverine. The relaxant effects of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 mL) as negative control were also examined in non-incubated tissues. Berberine showed significant and concentration-dependent relaxant effects in non-incubated tissues contracted by KCl and methacholine (p<0.01 to p<0.001). There was no significant difference in the relaxant effects of berberine between non-incubated and incubated tissues with atropine, propranolol, diltiazem, glibenclamide, and papaverine. The relaxant effects of second concentrations of berberine in incubated tissues with L-NAME, its three lower concentration in incubated tissues with chlorpheniramine and its all concentrations in incubated tissues with indomethacin were significantly lower than non-incubated tissues (p<0.05 to p<0.001). The EC50 values of berberine in incubated tissues with chlorpheniramine was significantly higher than the non-incubated condition (p<0.05). Our findings reveal a relatively potent relaxant effect of berberine that is lower than the effect of theophylline. Proposed mechanisms for the relaxant effect of berberine are histamine (H₁) receptor blockade, inhibition of cyclooxygenase pathways and/or nitric oxide formation.

Sulfur mustard (SM)-exposed individuals develop late pulmonary complications, which are associated with chronic inflammation and fibrotic changes in the lung tissue. MicroRNAs are known to act as important regulators of inflammatory responses, including inflammation and fibrosis-related cytokine signaling. In this study, we investigated the expression miR-15b-5p and miR-21-5p, two regulators of TGF-β signaling, as well as their target molecule, SMAD7, in lung tissues from SM-exposed and control individuals. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue biopsies obtained during surgery from SM-exposed (n=20) or control (n=20) cases. Quality of the extracted RNA was evaluated by an Agilent Bioanalyzer and RNA was quantified using a NanoDrop. MiR-21-5p, miR-15b-5p and SMAD7 expression levels were measured by real-time RT-PCR. miR-21-5p expression levels were significantly decreased (2.7 fold) in the lung tissues from SM-exposed individuals compared with tissues obtained from the control group (p=0.02). There were no significant differences in miR-15b-5p expression levels between the two groups (p=0.94). Interestingly, SMAD7 expression levels were significantly higher (5.8 fold) in SM-exposed individuals’ lung tissues compared with the control group (p=0.045). Our data indicate that exposure to sulfur mustard affects the expression of miR-21-5p as well as its target, SMAD7, in lung tissues many years after exposure. Considering the role of SMAD7 in the regulation of TGF-β signaling, these changes might point to a potential mechanism by which SM-exposure regulates inflammatory/fibrotic alterations in lung tissue.

Asthma is a common respiratory disease with huge economic burden leading to activity limitations, morbidity, and mortality. In this study, we aim to investigate the prevalence of Oppositional Defiant Disorder (ODD), Attention Deficit Hyperactivity Disorder (ADHD) and Conduct Disorder (CD) among children with asthma. This case-control study was performed in a pediatric referral health care center(Children's Medical CenterinTehran University of Medical Sciences) in 2017.With random selection, the 80 children with asthma and 92 controls with age range of 5 to 11 years were enrolled in this study. In addition to the demographic information and family history of allergy, asthma symptoms, and control quality evaluated with a validated Childhood Asthma Control Test (C-ACT). The mode of measurement for ADHD, ODD and CD was based on Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) psychiatric scales from clinical interviews conducted by child psychiatrists. Totally, 42.5% and 25% in the case and control groups had ADHD respectively withsignificant difference (p=0.01). Also, 25% and 5.4% in thecase and control groups had ODD respectively with significant difference (p=0.001). But conduct disorder was 10% and 10.9% in case and control groups respectively without significant difference (p=0.8). Children with asthma were associated with exhibiting ADHD and ODD but not CD. Therefore, appropriate evaluation and treatment are needed for asthmatic children with attention-deficit and ODD symptoms. Besides, further research is needed to determine the etiological approach towards ADHD, ODD and asthma.

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