Blockade of Proteinase-Activated Receptor 2 (PAR2) Attenuates Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Proteinase-activated receptor-2 (PAR2), which modulates inflammatory responses, is elevated in the central nervous system in multiple sclerosis (MS) and in its murine model, experimental autoimmune encephalomyelitis (EAE). In PAR2-null mice, disease severity of EAE is markedly diminished. We therefore tested whether inhibiting PAR2 activation in vivo might be a viable strategy for the treatment of MS. Using the EAE model, we show that a PAR2 antagonist, the pepducin palmitoyl-RSSAMDENSEKKRKSAIK-amide (P2pal-18S), attenuates EAE progression by affecting immune cell function. P2pal-18S treatment markedly diminishes disease severity and reduces demyelination, as well as the infiltration of T-cells and macrophages into the central nervous system. Moreover, P2pal-18S decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) production and T-cell activation in cultured splenocytes and prevents macrophage polarization in vitro. We conclude that PAR2 plays a key role in regulating neuroinflammation in EAE and that PAR2 antagonists represent promising therapeutic agents for treating MS and other neuroinflammatory diseases. SIGNIFICANCE STATEMENT: Proteinase-activated receptor-2 modulates inflammatory responses and is increased in multiple sclerosis lesions. We show that the proteinase-activated receptor-2 antagonist palmitoyl-RSSAMDENSEKKRKSAIK-amide reduces disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis by inhibiting T-cell and macrophage activation and infiltration into the central nervous system, making it a potential treatment for multiple sclerosis.

rbioapi: user-friendly R interface to biologic web services' API.

SUMMARY: Many packages serve as an interface between R language and the Application Programming Interface (API) of databases and web services. There is usually a 'one-package to one-service' correspondence, which poses challenges such as consistency to the users and scalability to the developers. This, among other issues, has motivated us to develop a package as a framework to facilitate the implementation of API resources in the R language. This R package, rbioapi, is a consistent, user-friendly and scalable interface to biological and medical databases and web services. To date, rbioapi fully supports Enrichr, JASPAR, miEAA, PANTHER, Reactome, STRING and UniProt. We aim to expand this list by collaborations and contributions and gradually make rbioapi as comprehensive as possible. AVAILABILITY AND IMPLEMENTATION: rbioapi is deposited in CRAN under the https://cran.r-project.org/package=rbioapi address. The source code is publicly available in a GitHub repository at https://github.com/moosa-r/rbioapi/. Also, the documentation website is available at https://rbioapi.moosa-r.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation and in vitro Expansion of Regulatory T Cells from ß-Thalassemia Major Kids and in vitro Evaluation of Their Plasticity and Inhibitory Effects in the Presence of Pro-Inflammatory Cytokines.

INTRODUCTION: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential. METHODS: Thirty milliliters of peripheral blood from the ß-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1ß, IL6, TGFß, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant. RESULTS: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055). CONCLUSION: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy.

Neuroinflammatory Response in Reward-Associated Psychostimulants and Opioids: A Review.

Substance abuse is one of the significant problems in social and public health worldwide. Vast numbers of evidence illustrate that motivational and reinforcing impacts of addictive drugs are primarily attributed to their ability to change dopamine signaling in the reward circuit. However, the roles of classic neurotransmitters, especially dopamine and neuromodulators, monoamines, and neuropeptides, in reinforcing characteristics of abused drugs have been extensively investigated. It has recently been revealed that central immune signaling includes cascades of chemokines and proinflammatory cytokines released by neurons and glia via downstream intracellular signaling pathways that play a crucial role in mediating rewarding behavioral effects of drugs. More interestingly, inflammatory responses in the central nervous system modulate the mesolimbic dopamine signaling and glutamate-dependent currents induced by addictive drugs. This review summarized researches in the alterations of inflammatory responses accompanied by rewarding and reinforcing properties of addictive drugs, including cocaine, methamphetamine, and opioids that were evaluated by conditioned place preference and self-administration procedures as highly common behavioral tests to investigate the motivational and reinforcing impacts of addictive drugs. The neuroinflammatory responses affect the rewarding properties of psychostimulants and opioids.

The importance of regulatory pathway mediated by Circ0001955 in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) has become one of the most common cancers in recent years. Given the importance that non-coding RNAs have recently acquired in various diseases including cancers, we decided to design this study to evaluate the expression levels of circ0001955/miR-145-5p/ONECUT2 axis in CRC. METHODS: After bioinformatics analysis of GEO datasets related to CRC, a putative circ0001955/ miR-145-5p/ ONECUT2 pathway was assumed. Then, the expression levels of these genes were measured in 50 CRC samples and adjacent tissues by qRT- PCR. Also, correlation coefficients, receiver operating characteristic (ROC) curves, and correlation between circ0001955 levels with clinicopathological parameters of patients were analyzed. RESULTS: Circ0001955 and ONECUT2 were considerably up-regulated, while the expression level of miR-145-5p was decreased in CRC samples compared with adjacent tissues (p < 0.05). Moreover, statistically significant correlations were observed between expression levels of circ0001955, miR-145-5p, and ONECUT2. We did not find any significant correlation between circ0001955 expression and clinicopathological parameters. CONCLUSION: Our study showed that circ0001955 is dysregulated in CRC. This finding can open a new window for researchers for a better understanding of the potential pathways involved in CRC pathogenesis and, consequently, to find new treatment pathways.

Importance of Circ0009910 in colorectal cancer pathogenesis as a possible regulator of miR-145 and PEAK1.

INTRODUCTION: Colorectal cancer (CRC) is one of the most frequent neoplasms in the world. Based on the emerging role of noncoding RNAs, particularly circular RNAs in pathogenesis of cancers, we designed this study to inspect the expression levels of a circ0009910-mediated regulatory pathway in colorectal cancer. METHODS: After bioinformatics analyses and construction of putative circ0009910/ miR-145-5p/PEAK1 pathway, the expression levels of these components were evaluated in 50 CRC tissues and adjacent specimens by quantitative real-time PCR. Moreover, we appraised the correlation coefficients between these transcripts and calculated the correlation between circ0009910 expression levels with clinicopathological features of patients. RESULTS: Circ0009910 and PEAK1 were significantly upregulated, while miR-145-5p was decreased in CRC samples compared with adjacent tissues (p < 0.05). Moreover, statistically significant correlations were observed between expression levels of circ0009910, miR-145-5p, and PEAK1. We also reported considerable correlations between circ0009910 expression and clinicopathological parameters including sex and perineural invasion. Finally, ROC curve analysis showed circ0009910 level as a discriminative biomarker for CRC. CONCLUSION: For the first time, we could introduce circ0009910 as an important biomarker in CRC. Collectively, this investigation helped us to identify a newly diagnosed pathway in CRC that can be a potential axis for designing effective drugs for treatment of CRC patients.

Inhibiting miR-27a and miR-142-5p attenuate nonalcoholic fatty liver disease by regulating Nrf2 signaling pathway.

The gene Nrf2 (nuclear factor-erythroid 2-related factor 2) is the most important regulator of the cellular antioxidant system and its dysregulation has a role in the etiology of nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the association between Nrf2 targeted miRNAs (miR-27a, miR-142-5p, miR-153, and miR-128) with lipid accumulation in vitro and in vivo models of NAFLD. We used two in vivo and in vitro models of NAFLD. The expression of the genes and miRNAs was assessed by real-time PCR and the protein level was evaluated using western blot. To investigate the potential role of miRNAs in NAFLD, the inhibitors or mimics of the miR-27a and miR-142-5p were transfected into HepG2 cells. The mRNA and protein levels of Nrf2 were significantly decreased in the liver of high fat diet-fed mice as well as in HepG2 cells treated with high glucose (HG). Reduced expression of Nrf2 was associated with increased expression levels of miR-27a and miR-142-5p in both models of NAFLD. HG-induced triglyceride accumulation was attenuated by inhibition of miR-27a or miR-142-5p in HepG2 cells. Overexpression of miR-27a or miR-142-5p suppressed the expression of Nrf2 and its downstream antioxidant genes and increased production of reactive oxygen species, whereas inhibition of miR-27a or miR-142-5p reversed these effects. In conclusion, the data of this study may suggest that miR-27a and miR-142-5p are increased in NAFLD, where they suppress Nrf2 expression and contribute to the accumulation of lipids in the hepatocytes.

Staphylococcal enterotoxin B increased severity of experimental model of multiple sclerosis.

BACKGROUND: Superantigens can be absorbed trans-mucosal and trans-cutaneous in individuals colonized with superantigen producing Staphylococcus aureus. Ability of superantigens to activate a large numbers of T cells suggests that they may play a role in the course of autoimmune diseases including human multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). In this study we investigated the role of staphylococcal enterotoxin B in immunologic and pathologic changes in experimental animal model of multiple sclerosis. METHODS: C57BL/6 female mice were treatment with SEB protein prior or post immunization with MOG33-35 peptide. Mice were monitored daily and scored for clinical symptoms following EAE induction. Spleen and spinal cord of mice were removed and used for ELISA and histological studies, respectively. RESULTS: Treatment with SEB prior induction of EAE, increased clinical score, the concentration of IL-17A, IFN-γ and histological changes compared to control group. Treatment with SEB after induction of EAE caused these changes, but less severe. DISCUSSION: Since SEB causes demyelination of spinal cord and increases the level of pro-inflammatory cytokine response, infiltration of T-lymphocytes and macrophages to CNS, it may exacerbate the clinical signs of EAE in mice and multiple sclerosis in human.

The Upregulation of hsa-mir-181b-1 and Downregulation of Its Target CYLD in the Late-Stage of Tumor Progression of Breast Cancer.

Some microRNAs are usually dysregulated in the cancers and influencing tumor behavior and progression. Hsa-miR-181b-1 and its target CYLD are involved in regulating the inflammatory pathways. This study aimed to investigate the expression levels of hsa-mir-181b-1 and CYLD in a cohort of breast tumor tissues and normal adjacent tissues to assess their association with breast cancer stages. A total number of 60 breast samples including cancerous and normal adjacent tissue specimens were collected. After pathological study, the expression of hsa-mir-181b-1 and CYLD were measured by qRT-PCR method. The hsa-mir-181b-1 expression level was significantly increased in breast tumor tissues compared to the controls. This increase was associated with the disease progression. Conversely, CYLD expression level was decreased in tumor samples compared to normal samples, significantly. ROC curve data added other prestigious information of hsa-mir-181b-1 and CYLD by defining cancer and healthy tissues with high specificity and sensitivity at a proposed cutoff point. Also, bioinformatic enrichment for the possible targets of mature sequence of "hsa-mir-181b-5p" was performed. Computational analysis showed the five most significant pathways including metabolic, cancer, calcium signaling, PI3K-Akt signaling and focal adhesion pathways which may be influenced by hsa-mir-181b-1. Thus, we suggested hsa-mir-181b-1 and CYLD might be involved in the pathogenesis of breast cancer and could be considered as two biomarkers for prediction, prognosis and diagnosis of the stages of the breast cancer.

Expression level of long noncoding RNA NKILAmiR103-miR107 inflammatory axis and its clinical significance as potential biomarker in patients with colorectal cancer.

BACKGROUND: Inflammatory cytokines have been observed in colorectal cancer (CRC) tissues and can promote the susceptibility to metastasis of CRC cells. Diverse regulatory mechanisms of long ncRNAs (lncRNAs) and microRNAs (miRNAs) involved in the inflammatory responses are associated with tumor progression. The aim of this research was to investigate the expression level of the nuclear factor-kappa B interacting lncRNA (NKILA)-miR103-miR107 regulatory axis and its clinical significance as a potential biomarker in patients with CRC. MATERIALS AND METHODS: In the present study, we investigated the expression levels of miR103, miR107, and NKILA in 21 paired CRC tissues and corresponding adjacent tissues, using real-time polymerase chain reaction technique. Receiver operating characteristic (ROC) curve was used to analyze the prognostic value of biomarkers and to compare their predictive value. RESULTS: It was found that the expression level of miR103 was significantly increased with the development of CRC (cancerous vs. corresponding normal tissues; 2.29 ± 1.65 vs. 1.16 ± 0.64, P = 0.003). Moreover, miR107 was upregulated in CRC tissues compared with paired normal tissues (2.1 ± 1.4 vs. 1.25 ± 0.83, P = 0.005), while NKILA displayed an opposite expression pattern versus miR103/107, but it was not statistically significant (3.69 ± 5.2 vs. 4.35 ± 5.99, P > 0.05). The ROC analysis demonstrated that miR103 had the best diagnostic ability performance with area under curve of 0.723 (0.545-0.901). CONCLUSION: We identified miR103/107 as tumor-promoting miRNAs with diagnostic value in cancer patients and presumptive negative regulators of NKILA, a potential cancer metastatic suppressor. Strategies that disrupt this regulatory axis might block CRC progression.

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells.

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells-thereby reducing the risk of tumor relapse-but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19+ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.

Cell injury and receptor expression in the epileptic human amygdala.

Neuropathological findings in the amygdala obtained from patients with mesial temporal lobe epilepsy (MTLE) indicate varying degrees of histopathological alterations, such as neuronal loss and gliosis. The mechanisms underlying cellular damage in the amygdala of patients with MTLE have not been fully elucidated. In the present study, we assess cellular damage, determine the receptor expression of major inhibitory and excitatory neurotransmitters, and evaluate the correlation between the expression of various receptors and cell damage in the basolateral complex and the centromedial areas in the amygdala specimens resected during brain surgery on 30 patients with medically intractable MTLE. Our data reveal an increased rate of cell damage and apoptosis as well as decreased expression levels of several GABAergic receptor subunits (GABAARα1, GABAARß3, and GABABR1) and GAD65 in the amygdalae obtained during epilepsy surgery compared to autopsy specimens. Analyses of the expression of glutamate excitatory receptor subunits (NR1, NR2B, mGluR1α, GluR1, and GluR2) reveal no significant differences between the epileptic amygdalae and autopsy control tissues. Furthermore, the increased occurrence of apoptotic cells in the amygdala is negatively correlated with the reduced expression of the studied GABAergic receptor subunits and GAD65 but is not correlated with the expression of excitatory receptors. The present data point to the importance of GABAergic neurotransmission in seizure-induced cell injury in the amygdala of patients with MTLE and suggest several GABA receptor subunits as potential druggable target structures to control epilepsy and its comorbid disorders, such as anxiety.

Switching from high-fat diet to foods containing resveratrol as a calorie restriction mimetic changes the architecture of arcuate nucleus to produce more newborn anorexigenic neurons.

PURPOSE: These days, obesity threatens the health for which one of the main interventions is calorie restriction (CR). Due to the difficulty of compliance with this treatment, CR mimetics such as resveratrol (RSV) have been considered. The present study compared the effects of RSV and CR on hypothalamic remodeling in a diet-switching experiment. METHODS: C57BL/6 male mice received high-fat diet (HFD) for 4 weeks, subsequently their diet switched to chow diet, HFD + RSV, chow diet + RSV or CR diet for a further 6 weeks. Body weight, fat accumulation, hypothalamic apoptosis and expression of trophic factors as well as generation and fate specification of newborn cells in arcuate nucleus (ARC) were evaluated. RESULTS: Switching diet to RSV-containing foods leading to weight and fat loss after 6 weeks. In addition, not only a significant reduction in apoptosis but also a considerable increase in production of newborn cells in ARC occurred following consumption of RSV-enriched diets. These were in line with augmentation of hypothalamic ciliary neurotrophic factor and leukemia inhibitory factor expression. Interestingly, RSV-containing diets changed the fate of newborn neurons toward generation of more proopiomelanocortin than neuropeptide Y neurons. The CR had effects similar to those of RSV-containing diets in the all-evaluated aspects besides neurogenesis in ARC. CONCLUSIONS: Although both RSV-containing and CR diets changed the fate of newborn neurons to create an anorexigenic architecture for ARC, newborn neurons were more available after switching to RSV-enriched diets. It can be consider as a promising mechanism for future investigations.

The imbalance of circulating T helper subsets and regulatory T cells in patients with LRBA deficiency: Correlation with disease severity.

Patients with lipopolysaccharides responsive beige-like anchor protein (LRBA) deficiency suffer from a variety of immunological abnormalities. In the current study, we investigated the role of T helper (Th) cell subsets and regulatory T (Treg) cells and their related cytokines and transcription factors in the immune dysregulation of LRBA deficiency. The study population comprised of 13 LRBA-deficient patients and 13 age- and sex-matched healthy controls (HCs). Th subsets and Treg were examined by flow cytometry. The expression of determinant cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-22, and IL-10), and cell subset-specific transcription factors were evaluated before and after proliferation and activation stimuli. The frequencies of Th1, Th1-like Th17 and Th22 cells along with the expression of T-box transcription factor (TBET) and runt-related transcription factor 1 (RUNX1) were significantly increased in patients with LRBA. Moreover, IFN-γ and IL-22 production in LRBA-deficient CD4+ T cells were elevated after lymphocyte stimulation, particularly in patients with enteropathy. However, CD4+ CD25+ FoxP3+ CD127- cells were significantly decreased in LRBA-deficient patients compared with those of HCs, particularly in patients with autoimmunity. There was a negative correlation between the frequencies of CD4+ CD25+ FoxP3+ CD127- cells and Th1-like Th17 cells in LRBA-deficient patients, and an overlapping phenotype of autoimmunity and enteropathy were observed in ~70% of patients. The frequency of Th17 cells was lower in patients with enteropathy, while Th1-like Th17 cells were higher than in those without enteropathy. Our findings demonstrated an imbalance in Th subsets, mainly in Th1-like Th17 and Treg cells and their corresponding cytokines in LRBA deficiency, which might be important in the immunopathogenesis of autoimmunity and enteropathy.

Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Microenvironment proteinases, proteinase-activated receptor regulation, cancer and inflammation.

We propose that in the microenvironment of inflammatory tissues, including tumours, extracellular proteinases can modulate cell signalling in part by regulating proteinase-activated receptors (PARs). We have been exploring this mechanism in a variety of inflammation and tumour-related settings that include tumour-derived cultured cells from prostate and bladder cancer, as well as immune inflammatory cells that are involved in the pathology of inflammatory diseases including multiple sclerosis. Our work showed that proteinase signalling via the PARs affects prostate and bladder cancer-derived tumour cell behaviour and can regulate calcium signalling in human T-cell and macrophage-related inflammatory cells as well as in murine splenocytes. Further, we found that the tumour-derived prostate cancer cells and immune-related cells (Jurkat, THP1, mouse splenocytes) can produce PAR-regulating proteinases (including kallikreins: kallikrein-related peptidases), that can control tissue function by both a paracrine and autocrine mechanism. We suggest that this PAR-driven signalling process involving secreted microenvironment proteinases can play a key role in cancer and inflammatory diseases including multiple sclerosis.

Suppressed oligodendrocyte steroidogenesis in multiple sclerosis: Implications for regulation of neuroinflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Neurosteroids are reported to exert anti-inflammatory effects in several neurological disorders. We investigated the expression and actions of the neurosteroid, dehydroepiandrosterone (DHEA), and its more stable 3ß-sulphated ester, DHEA-S, in MS and associated experimental models. CNS tissues from patients with MS and animals with experimental autoimmune encephalomyelitis (EAE) displayed reduced DHEA concentrations, accompanied by diminished expression of the DHEA-synthesizing enzyme CYP17A1 in oligodendrocytes (ODCs), in association with increased expression of inflammatory genes including interferon (IFN)-γ and interleukin (IL)-1ß. CYP17A1 was expressed variably in different human neural cell types but IFN-γ exposure selectively reduced CYP17A1 detection in ODCs. DHEA-S treatment reduced IL-1ß and -6 release from activated human myeloid cells with minimal effect on lymphocyte viability. Animals with EAE receiving DHEA-S treatment showed reduced Il1b and Ifng transcript levels in spinal cord compared to vehicle-treated animals with EAE. DHEA-S treatment also preserved myelin basic protein immunoreactivity and reduced axonal loss in animals with EAE, relative to vehicle-treated EAE animals. Neurobehavioral deficits were reduced in DHEA-S-treated EAE animals compared with vehicle-treated animals with EAE. Thus, CYP17A1 expression in ODCs and its product DHEA were downregulated in the CNS during inflammatory demyelination while DHEA-S provision suppressed neuroinflammation, demyelination, and axonal injury that was evident as improved neurobehavioral performance. These findings indicate that DHEA production is an immunoregulatory pathway within the CNS and its restoration represents a novel treatment approach for neuroinflammatory diseases.

MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis.

BACKGROUND: MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation. METHODS: The expression levels of two mature isoforms of miR-142 were measured in the brains of patients with multiple sclerosis (MS) and the CNS tissues from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression analyses were also performed in mitogen and antigen-stimulated splenocytes, as well as macrophages and astrocytes using real-time RT-PCR. The role of the mature miRNAs was then investigated in T cell differentiation by transfection of CD4+ T cells, followed by flow cytometric analysis of intracellular cytokines. Luciferase assays using vectors containing the 3'UTR of predicted targets were performed to confirm the interaction of miRNA sequences with transcripts. Expression of targets were then analyzed in activated splenocytes and MS/EAE tissues. RESULTS: Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords. CONCLUSIONS: Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.