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The kynurenine pathway (KP) can be involved in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. This study aimed to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line to present more information about the effect of this gene on inflammatory conditions and the KP cycle. The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. To simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24 h before transfection, and transfected by Polyethyleneimine (PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and murine Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed 72 h after transfection, and Acmsd expression level increased 29-fold (P

Objective The main concern about measuring the concentration of rivaroxaban by anti-Xa assay in some laboratories is the lack of a commercial specific calibrator in emergencies. Therefore, this study aimed at providing a homemade anti-Xa calibrator and commercial low molecular weight heparin (LMWH) anti-Xa calibrator. Methods The anti-Xa plasma concentration of rivaroxaban was measured in 70 patients using a commercial specific anti-Xa calibrator, a commercial LMWH anti-Xa calibrator, and a homemade anti-Xa calibrator. Results We demonstrated a significant correlation and agreement (P < .001) between LMWH-calibrated anti-Xa and the commercial specific calibrator. A significant correlation (P < .001) was found between homemade calibrated anti-Xa made by normal pooled plasma and that calibrated with a commercial specific drug. The nonspecific homemade and LMWH calibrators had excellent agreement (P < .001) and can be used interchangeably. Conclusion Our data showed that for estimating rivaroxaban concentrations, the LMWH calibrator could be used as an alternative calibrator in the anti-Xa assay.

p28 is a natural bacterial product, which recently has attracted much attention as an efficient cell penetrating peptide (CPP) and a promising anticancer agent. Considering the interesting biological qualities of p28, maximizing its expression appears to be a prominent priority. The optimization of such bioprocesses might be facilitated by utilizing statistical approaches such as Design of Experiment (DoE). In this study, we aimed to maximize the expression of "biologically active" p28 in Escherichia coli BL21 (DE3) host by harnessing statistical tools and experimental methods. Using Minitab, Plackett-Burman and Box-Behnken Response Surface Methodology (RSM) designs were generated to optimize the conditions for the expression of p28. Each condition was experimentally investigated by assessing the biological activity of the purified p28 in the MCF-7 breast cancer cell line. Seven independent variables were investigated, and three of them including ethanol concentration, OD

Mesenchymal stem cells (MSCs) are mesodermal-origin postnatal stem cells that are able to self-renew and differentiate into several cell lineages. MSCs possess anti-inflammatory and anti-apoptotic activity, immunomodulatory action, as well as regenerative properties. Since MSCs also have antimicrobial properties, it has been suggested that they should be utilized for treating infectious diseases. In this study, the last pre-clinical advances in the efficacy of MSCs' therapy against parasitic diseases were reviewed.Data about the effects of MSCs' therapy on experimental and pre-clinical parasitic infections were collected by searching relevant articles and reviewing them.In the present study, empirical findings on the impacts of MSCs' therapy against parasitic diseases were recapitulated. Studies have reported that the administration of MSCs reduces the burden of the parasite and modulates the levels of inflammatory and anti-inflammatory cytokines in parasitic diseases, including schistosomiasis, malaria, cystic echinococcosis, toxocariasis, leishmaniasis, and trypanosomiasis. Also, the administration of MSCs combined with anti-parasitic drugs enhanced anti-parasitic effects and immunomodulatory actions.Based on this review, empirical studies have revealed the beneficial effects of MSCs against some parasitic infections. This new therapeutic strategy showed both anti-parasitic and immunomodulatory effects. Also, the combination of anti-parasitic drugs with MSCs' therapy promoted anti-parasitic and immunomodulatory activities against parasitic infections.

The orexin 2 receptor plays a central role in maintaining sleep and wakefulness. Recently, it has been shown that sleep and wakefulness orchestrate the proliferation and differentiation of oligodendrocytes. Here, we explored the role of a selective orexin 2 receptor antagonist (JNJ-10397049) in proliferation and differentiation of neural progenitor cells (NPCs). We evaluated the proliferation potential of NPCs after exposure to different concentrations of JNJ-10397049 by using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and neurosphere assays. Moreover, the expression of differentiation markers was assessed by immunocytochemistry and real-time polymerase chain reaction. JNJ-10397049 significantly increased the proliferation of NPCs at lower concentrations. In addition, orexin 2 receptor antagonist facilitated progression of differentiation of NPCs towards oligodendroglial lineage by considerable expression of Olig2 and 2',3'-cyclic-nucleotide 3'-phosphodiesterase as well as decreased expression of nestin marker. The results open a new avenue for future investigations in which the production of more oligodendrocytes from NPCs is needed.

The kynurenine pathway (KP) can be involvement of in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. The aim of this study was to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line learn more about the effect of this gene on inflammatory conditions and the KP cycle. The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. In order to simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24 hours before transfection, and transfected by Polyethyleneimine (PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed after 72 hours, and Acmsd expression level increased 29-fold (P Ido1 gene, and its regulator, Ifn-γ. Consequently, it may be considered as a novel regulatory factor in the KP pathway.

Background: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system characterized by infiltration of inflammatory leukocytes to the CNS followed by oligodendrocyte cell death, myelin sheath destruction, and axonal injury. A logical incidence occurring after demyelination is remyelination. G-protein coupled receptors (GPCRs) activate internal signal transduction cascades through binding to different ligands. This family of receptors are targeted by more than 40% of currently marketed drugs. GPCRs can be successfully targeted for induction of remyelination. GPCRs highly enriched in oligodendrocyte progenitor cells compared to oligodendrocytes are proposed to hamper oligodendrocyte differentiation and therefore their inhibition might induce remyelination. This study aimed to investigate the expression of GPCRs in silico and in vitro. Methods: We performed gene expression analysis using DAVID and Panther websites on a RNA-seq dataset (GSE52564 accession number). Primary embryonic neural stem/progenitor cell isolation and culture were performed and subsequently NSPCs were characterized by Immunocytochemistry with Anti-Nestin antibody. Expression of GPR37L1, EDNRB, PDGFRα, CNPase and GFAP were assessed using real-time PCR. All the experiments were conducted at Shiraz University of Medical Sciences (SUMS), Shiraz, Iran, in the year 2018. Results: The 14 most highly expressed GPCRs in oligodendrocyte progenitor cells (OPCs) compared to Oligodendrocytes were presented in our study. Conclusion: The investigation of the most highly expressed GPCRs in OPCs compared to oligodendrocyte in silico and in vitro presents the significant role of GPCRs in remyelination induction. Among the 14 GPCRs mentioned in this study, GPR37L1 is a potential remyelinating drug target and is suggested for further studies.

Cytochrome P450 3A4 (CYP3A4), the main drug-metabolizing enzyme in the liver, has a functional SNP in intron six named CYP3A4⁎22. Given the CYP3A4⁎22 polymorphism effect on the reduced CYP3A4 mRNA/protein expression, this study aimed to evaluate the prevalence of CYP3A4⁎22 polymorphism in Iranian patients with stroke compared to the control group. A total of 175 patients with stroke and 236 control age-sex matched ones have been examined for CYP3A4⁎22 polymorphism by Tetra ARMS-PCR methods. The results were confirmed by the PCR-RFLP technique and the sequencing of DNA. This study is the first report of CYP3A4⁎22 polymorphism from Iran. The frequency of the CYP3A4⁎22 wild-type (GG genotype) and heterozygous genotypes (GA genotype) was 93.1% and 6.9% in patients, and 92.8% and 7.2% in the controls, respectively. None of the participants was the recessive model for this polymorphism. There were no statistical differences between the patient and control groups (p > 0.05). Also, the GA genotype was not associated with increased risk of stroke compared with the GG genotype (OR = 0.95, 95% CI: 0.44–2.04, P = 0.892). Since CYP3A4⁎22 polymorphism may result in reduced CYP3A4 activity in vivo, due to the large number of drugs that are CYP3A4 substrates, individuals with this polymorphism may experience unusual drug effects. To clarify these impacts, further investigations are, therefore, needed to be done.

Objective: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5’-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. Materials and Methods: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer. Results: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p

Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20 × 10(6) BM cells and 120 × 10(6)splenocytes, 3 × 10(6) BM cells along with 20 × 10(6)splenocytes were co-cultured in petri dishes for DC generation; 120 × 10(6) splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. BM cells were the control group cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8α, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA. The number of DCs derived from co-culture of BM and splenocytes (DCs(TME)) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2% and 77.2%, respectively; p0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCs(TME) compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p0.05). The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCs(TME) could be more advantageous for DC-based immunotherapy and vaccination techniques.

In the present investigation, polyethylenimine (PEI) was conjugated with succinic anhydride at four substitution degrees and the efficiency of the modified PEI derivatives in transferring the plasmid encoding interleukin-12 gene was evaluated. The results revealed that the conjugated PEI derivatives enhanced the transfection efficiency by up to 3-fold relative to unmodified PEI, with the highest increase occurring at conjugation degrees around 30%. The results demonstrated the ability of the modified PEI derivatives in condensation of the plasmid into the nanoparticles in the size range of approximately 100 nm. Also, the PEI derivatives exhibited substantial decrease in cell-induced toxicity.

Context: There are plenty of evidence that suggest that the potential high doses of radiation result in severe health effects to exposed individuals, although there is no consensus about the health impact of low dose of ionizing radiation (IR). Aims: This study aimed to discuss the effect a range of IR doses on the changes of gene expression and serum protein levels of two immune factors transforming growth factor-β (TGF-β) and interferon gamma (IFN-γ) in rats. Findings from this study can be useful to develop a suitable biomarker for biological dosimetry applications. Subjects and Methods: After 24 h of irradiation of rats with the doses of 1000, 500, 100, 50, and 20 mGy, the gene expression of TGF-β and IFN-γ in lymphocytes was assessed using quantitative polymerase chain reaction. Besides, the protein level of these two factors in blood plasma was determined by enzyme-linked immunosorbent assay (ELISA) kits. Statistical Analysis Used: One-way analysis of variance Tukey–Kramer Multiple Comparisons Test was used. P

In this paper, we investigate the terahertz bistability behavior in a photonic crystal doped with an InSb layer and coupled nonlinear defects by employing the modified transfer matrix method. It is demonstrated that the switch-up threshold for bistability can be strongly controlled by ambient temperature without changing the structure of photonic multilayer. The switch-up threshold increases with increasing temperature to a certain value. No dramatic changes are observed in switch-down threshold with increasing the ambient temperature. Further increase of temperature from 309 K to 311 K leads to the strong reduction of switch-up threshold. Our results reveal that the switch-up threshold for the case T = 311 K is approximately 3000 times lower than that of the case T = 309 K. This sensitivity to temperature can have potential applications in terahertz devices. When temperature increases from 309 K, both switch-up and switch-down threshold increase. However, the switch-down threshold indicates much smaller changes compared to the switch-up threshold. Furthermore, we investigate the effect of incident angle on the bistability behavior. It is shown that the temperature at which the lowest value of switching powers is achieved depends on the incident angle.

Summary Down-regulation of soluble or membrane-bound co-stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow-derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV-DCs) and/or p19 subunit of interleukin (IL)-23 (p19LV-DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In-vitro studies showed that double-transduced BMDCs (CD40+p19LV-DCs) resemble tolerogenic DCs due to profound down-regulation of CD40, lower expression of proinflammatory cytokines (IL-6 and IL-12), increased IL-10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)35–55-specific T cells for production of IL-10 compared with CD40LV-DCs, p19LV-DCs and BMDCs transduced with control lentiviral vector (CoLV-DCs). Moreover, injection of transduced CD40+p19LV- BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL-17 or increased production of IL-10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV-DCs-treated EAE mice. In conclusion, simultaneous knock-down of CD40 and IL-23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL-17-dependent autoimmune diseases, including multiple sclerosis.

T helper 1 and T helper 17 cells play important roles in immunity against foreign invaders. Differentiation of these Th subsets is affected by state of maturation and cytokines that are produced by dendritic cells (DCs). Curdlan is a linear (1→3)-β-glucan and has shown activity against tumors and infectious agents.This study aims to investigate whether curdlan plays its role through affecting the maturation and cytokine production by DCs.DCs were isolated from the spleen of BALB/c mice by MACS method. After an overnight culture of DCs in the presence of curdlan, the expression levels of CD40, CD86, and MHC-II molecules were determined by flow cytometry. The production of cytokines involved in Th1 and Th17 cell differentiation (IL-12 and IL-6, respectively) was also evaluated by ELISA. Lipopolysaccharide (LPS) treated and untreated cells were considered as positive and negative controls, respectively.The results of this study did not show a significant difference in the levels of surface expression of CD40 (p=0.82), CD86 (p=0.79), and MHC class II (p=0.84) molecules upon exposure to curdlan. However, LPS increased the intensity of CD40 expression on dendritic cells (p=0.04). In addition, it was revealed that curdlan-exposed DCs are not able to produce a significant amount of IL-6 and IL-12 cytokines. Conversely, LPS-treated DCs were able to make a significant amount of IL-12 (p=0.005).The results of the present study suggest that curdlan has no effect on Th1 or Th17 differentiation while LPS may induce Th1 deviation by induction of CD40 expression and IL-12 production.

Immunotherapy with dendritic cells (DCs), which have been manipulated ex vivo to become immunogenic or tolerogenic, has been tested in clinical trials for disease therapy. DCs are sentinels of the immune system, which after exposure to antigenic or inflammatory signals and crosstalk with effector CD4(+) T cells express high levels of costimulatory molecules and cytokines. Upregulation of either costimulatory molecules or cytokines promotes immunologic DCs, whereas their downregulation generates tolerogenic DCs (TDCs), which induce T regulatory cells (Tregs) and a state of tolerance. Immunogenic DCs are used for the therapy of infectious diseases such as HIV-1 and cancer, whereas tolerogenic DCs are used in treating various autoimmune diseases and in transplantation. DC vaccination is still at an early stage, and improvements are mainly needed in quality control of monitoring assays to generate clinical-grade DC products and to assess the effect of DC vaccination in future clinical trials. Here, we review the recent work in DC generation and monitoring approaches for DC-based trials with immunogenic or tolerogenic DCs.

Background and Aim: Ulcerative colitis (UC) is a multifactorial disease associated with dysregulated immunity. Recently, cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms have been reported in association with several autoimmune diseases in several populations. In the present study, the possible implication of the CTLA-4 gene as a risk factor for UC in the Iranian population was investigated. Methods: One hundred UC patients and 100 healthy subjects were studied. CTLA-4 exon 1 position 49 (A/G: codon 17: Thr/Ala) polymorphisms were investigated by polymerase chain reaction single strand confirmation polymorphism method. Four of the patients and one of the healthy controls were excluded from the study because of incomplete DNA extraction. Results: The allele frequencies of A and G in 96 patients (A: 66.1%; G: 33.9%) were not significantly different from the 99 control subjects (A: 63.1%; G: 36.9%, P > 0.05). No significant differences in the distribution of genotype frequencies were observed between A + 49G gene polymorphisms and UC in the Iranian population (P > 0.05). Conclusion: CTLA-4 polymorphism is not associated with UC in the Iranian population.

Although, the cytotoxic T lymphocyte antigen-4 gene polymorphism at position 49 of exon-1 has been strongly elucidated in different autoimmune diseases, but its role in predisposition to systemic sclerosis (SSc) is yet controversial. This study intends to analyze the genetic correlation of the ctla-4 gene locus with diffuse systemic sclerosis (dSSc), as well as to understand the influence of these genotypes in disease expression. Seventy known cases of SSc, and 151 age-matched healthy controls, were participated in this investigation. The frequencies of AA, GG and AG genotypes were found to be 26 (37.1%), 5 (7.2%) and 39 (55.7%) in patients, and 60 (39.7%), 19 (12.6%) and 72 (47.7%) in controls, respectively. As indicated, the differences in genotype and allele frequencies between patients and controls were insignificant (P>0.05). Moreover, the distribution of CTLA-4 polymorphism between patients did not differ significantly according to clinical and serologic features. In Iranian patients, susceptibility to SSc is not influenced by a bi-allelic ctla-4 gene (A49G) polymorphism.

Objective One of the major complications of pregnancy, preeclampsia makes pregnancy termination inevitable in most cases. Similarities exist between the mechanisms that maintain normal pregnancy, allograft transplants, and, it is postulated, peripheral self-tolerance. In addition, the critical role of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) molecule in maintaining self-tolerance has been established. Therefore, the frequency of CTL A-4 A49G polymorphism was investigated in severe preeclampsia. Patients and Methods Genomic DNA extracted from mononuclear cells of the peripheral blood of 36 pregnant women with severe preeclampsia and 151 healthy women was analyzed. A49G polymorphism in position 49 of exon-1 of the CTL A-4 gene was studied by the polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) method. Results The frequency of the GG genotype was 2 (5.6%) in patients and 19 (12.6%) in controls, while the frequency of the AA genotype was 4 (11.1%) and 60 (39.7%). Interestingly, the frequency of the AG genotype was significantly higher in preeclamptic than in healthy women from the general population (83.3% vs. 47.7%; P =0.0005). Conclusion These data suggest that heterozygosity in the CTL A-4 A49G allele might be a predisposing factor for severe preeclampsia. Whether the observed association results from linkage imbalance with other loci on chromosome 2 or other polymorphisms of the CTL A-4 gene or even from a preferential transfer and/or expression of one allele from a heterozygous mother to the fetus will be the subject of future investigations.

The exon 1 polymorphism (49A/G) of ctla-4 gene corresponds to an amino acid exchange (threonine to alanine) in the leader peptide of the expressed protein. There are reports concerning the higher level of G allele in subjects with various autoimmune diseases, which has resulted in the hypothesis that CTLA-4 may play a role in regulating self-tolerance by the immune system and in the pathogenesis of autoimmune disorders. This study was undertaken to investigate the correlation of exon 1 (49A/G) polymorphism in the ctla-4 gene and breast cancer. The ctla-4 49A/G polymorphism was studied in 197 women with primary breast cancer and 151 age/sex matched normal individuals. The results indicated a significant difference between frequency of ctla-4 genotypes in patients and controls. The frequency of GG genotype was significantly decreased in breast cancer patients compared to controls (4.6% v.s. 12.6%, P = 0.012). There was also a significant positive correlation between tumor size and the existence of AA genotype in patients (P = 0.016). In addition, a positive correlation between AA genotype and lymph node involvement was observed (P = 0.042). The observed decrease in the frequency of GG genotype in the breast cancer patients is contrary to the frequently reported increase of GG genotype in autoimmune diseases. In addition, the data implies that polymorphism of ctla-4 exon 1 contributes in tumor progression.

Polymorphisms in ctla-4 gene have been shown to be associated with the Graves' disease (GD) susceptibility in different populations in the world. This study was undertaken to disclose the probable association of exon-1 polymorphism of ctla-4 with GD in Iranian patients. A49G polymorphism was investigated in 90 patients and 90 age/sex matched normal healthy controls, using PCR-SSCP and PCR-RFLP methods. Frequencies of AA, AG and GG genotypes among patients were found to be 21 (23.3%), 49 (54.5%) and 20 (22.2%) while these frequencies among healthy controls were 30 (33.3%), 53 (58.9%) and 7(7.8%), respectively. A significant increase of GG genotype and G allele was observed in patients (p = 0.012 and p = 0.025). In conclusion, consistent with the results of most other studies, the presence of a G allele in position 49 of ctla-4 exon-1 is associated with susceptibility to GD in Iranian population.