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The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we used streptozotocin (S TZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8 mM, 0-24h) ±TNFα (100 ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by the expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBa. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ-/- and WT mice were exposed to STZ and monitored for diabetes. The IκBβ-/- mice developed hyperglycemia and diabetes more frequently than contr ols following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes. [ABSTRACT FROM AUTHOR]

Leprosy is a chronic granulomatous infectious disease caused by the pathogen, Mycobacterium leprae, and the more recently discovered, M. lepromatosis. Described in 1873, M. leprae was among the first microorganisms to be proposed as a cause of a human infectious disease. As an obligate intracellular bacterium, it has still not thus far been reproducibly cultivated in axenic medium or cell cultures. Shepard's mouse footpad assay, therefore, was truly a breakthrough in leprosy research. The generation of immunosuppressed and genetically engineered mice, along with advances in molecular and cellular techniques, has since offered more tools for the study of the M. leprae–induced granuloma. While far from perfect, these new mouse models have provided insights into the immunoregulatory mechanisms responsible for the spectrum of this complex disease. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells that possess multilineage differentiation potential and extensive immunomodulatory properties. In mice and rats, MSCs produce nitric oxide (NO), as immunomodulatory effector molecule that exerts an antiproliferative effect on T cells, while the role of NO in differentiation was less clear. Here, we investigated the role of NO synthase 2 (NOS2) on adipogenic and osteogenic differentiation of rat MSCs. MSCs isolated from NOS2-null (NOS2–/–) and wild type (WT) Sprague–Dawley (SD) rats exhibited homogenous fibroblast-like morphology and characteristic phenotypes. However, after induction, adipogenic differentiation was found significantly promoted in NOS2–/– MSCs compared to WT MSCs, but not in osteogenic differentiation. Accordingly, qRT-PCR revealed that the adipogenesis-related genes PPAR-γ, C/EBP-α, LPL and FABP4 were markedly upregulated in NOS2–/– MSCs, but not for osteogenic transcription factors or marker genes. Further investigations revealed that the significant enhancement of adipogenic differentiation in NOS2–/– MSCs was due to overactivation of the STAT3 signaling pathway. Both AG490 and S3I-201, small molecule inhibitors that selectively inhibit STAT3 activation, reversed this adipogenic effect. Furthermore, after high-fat diet (HFD) feeding, knockout of NOS2 in rat MSCs resulted in significant obesity. In summary, NOS2 is involved in the regulation of rat MSC adipogenic differentiation via the STAT3 signaling pathway.

We investigated Nw-hydroxy l -Arginine (NOHA) predictive response in serous ovarian carcinoma based on estrogen-hormone receptor expression status; and assessed the distinctive NOHA response between estrogen-receptor-negative (ER–) tumor subtypes of ovarian and breast cancer. Three-dimensional (3D) spheroids models of ER– and estrogen-receptor-positive (ER+) from breast and ovarian tumor, cultured for 9 weeks, were assayed for cellular levels of inducible nitric oxide synthase (NOS2), nitric oxide (as total nitrite) and l -Arginine, and compared to NOHA in culture medium. Statistical difference was set at p < 0.01. Nine-week in vitro studies showed a progressive NOHA reduction in culture medium by at least 0.4–0.8 fold, and 0.65–0.92 fold only in the ER– breast tumor and ER– ovarian tumor 3D spheroids, respectively; with increases in cellular NOS2 and nitric-oxide levels, by at least 1.0–2.45 fold in both ER– tumor subtype 3D spheroids (p < 0.01; n = 6). Within ER– subtypes, medium NOHA decreased by ≥ 38.9% in ovarian cancer over breast cancer 3D-spheroids, with cellular increases in NOS2 (by ≥ 17.4%), and nitric oxide (by ≥ 18.8%). Cellular l -Arginine to medium NOHA ratio was higher, and by at least 6.5–22.5 fold in ER– breast tumor 3D-spheroids, and at least 10–70 fold in ER– ovarian tumor 3D spheroids, than in ER+ and control conditions; and was ≥48% higher in ER– ovarian cancer than in ER– breast cancer 3D-spheroids. The present study shows NOHA as a sensitive and selective indicator differentiating and distinguishing ER– subtypes based on the tumor grade. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways.Methods: To determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG)79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry.Results: NCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1β in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase.Conclusions: These studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE. [ABSTRACT FROM AUTHOR]

Background and Objectives: In paediatric population, atopic asthma is associated with increased eosinophil counts in patients, that correlate with the airway inflammation measured by the concentration of nitric oxide in exhaled air (FeNO). As the FeNO level is a biomarker of atopic asthma, we assumed that polymorphisms in nitric synthases genes may represent a risk factor for asthma development. The purpose of this study was to analyse the association of NOS genetic variants with childhood asthma in the Polish population. Materials and methods: In study we included 443 children—220 patients diagnosed with atopic asthma and 223 healthy control subjects. We have genotyped 4 single nucleotide polymorphisms (SNP) from 3 genes involved in the nitric oxide synthesis (NOS1, NOS2 and NOS3). All analyses were performed using polymerase chain reaction with restriction fragments length polymorphism (PCR-RFLP). Results: We observed significant differences between cases and controls in SNP rs10459953 in NOS2 gene, considering both genotypes (p = 0.001) and alleles (p = 0.0006). The other analyzed polymorphisms did not show association with disease. Conclusions: According to our results, 5′UTR variant within NOS2 isoform may have an impact of asthma susceptibility in the population of Polish children. Further functional studies are required to understand the role of iNOS polymorphism in NOS2 translation and to consider it as a novel risk factor in childhood asthma. The next step would be to apply this knowledge to improve diagnosis and develop novel personalized asthma therapies.

Retinoic acid has been previously proposed in the treatment of Alzheimer's disease (AD). Here, five transgenic mouse models expressing AD and frontotemporal dementia risk genes (i.e., PLB2APP, PLB2TAU, PLB1Double, PLB1Triple, and PLB4) were used to investigate if consistent alterations exist in multiple elements of the retinoic acid signaling pathway in these models. Many steps of the retinoic acid signaling pathway including binding proteins and metabolic enzymes decline, while the previously reported increase in RBP4 was only consistent at late (6 months) but not early (3 month) ages. The retinoic acid receptors were exceptional in their consistent decline in mRNA and protein with transcript decline of retinoic acid receptors β and γ by 3 months, before significant pathology, suggesting involvement in early stages of disease. Decline in RBP1 transcript may also be an early but not late marker of disease. The decline in the retinoic acid signaling system may therefore be a therapeutic target for AD and frontotemporal dementia. Thus, novel stable retinoic acid receptor modulators (RAR-Ms) activating multiple genomic and non-genomic pathways were probed for therapeutic control of gene expression in rat primary hippocampal and cortical cultures. RAR-Ms promoted the non-amyloidogenic pathway, repressed lipopolysaccharide induced inflammatory genes and induced genes with neurotrophic action. RAR-Ms had diverse effects on gene expression allowing particular RAR-Ms to be selected for maximal therapeutic effect. Overall the results demonstrated the early decline of retinoic acid signaling in AD and frontotemporal dementia models and the activity of stable and potent alternatives to retinoic acid as potential therapeutics. [ABSTRACT FROM AUTHOR]

Majority of the human diseases are accounted for by nonsynonymous single nucleotide variations (nsSNVs) that occur in the coding region of genes and alter the amino acid residues at specific positions. The future of genomics research is in identification of nsSNVs that contribute to disease pathophysiology by disrupting protein function. A robust pipeline with several integrated computational prediction tools facilitates prioritization of significant diseaseassociated nsSNVs. In this study, we analysed 30 rare frequency missense variations in NOS2 identified through Whole Exome Sequencing (WES) of six coronary artery disease (CAD) using multiple bioinformatics softwares and tools. We employed a stringent filtering workflow to identify and assess the pathogenic effect of nsSNVs on NOS2 structure and function. We used a combination of deleterious variation detection tools, protein stability changes prediction, post-translational modification site prediction, protein-protein interaction and enrichment analyses to discern disease-associated variations. Our findings implicate four amino acid substitutions - K730N, P769R, P958S and L1012S as candidates in pathological process of NOS2. [ABSTRACT FROM AUTHOR]

Background/Aim: Microbial tetracycline (TC) pastes have been employed to treat oral bacterial infection. In the present study, we investigated the kinetic radicals-cavenging and pro-/anti-inflammatory activity of TC with or without visible light irradiation (VLI). Materials and Methods: The radical-scavenging activity of TC and minocycline (MC) was determined by differential scanning calorimetry (DSC). The stoichiometric factor (n) and the rate constant of inhibition and propagation (kinh/kp) were determined. The levels of cyclooxygenase-2 (Cox2), tumor necrosis factor-α (Tnfα) or nitric oxide synthase 2 (Nos2) mRNA in RAW264.7 cells stimulated with lipopolysaccharide (LPS) were investigated using real-time reverse transcriptasepolymerase chain reaction. Results: The n and kinh/kp values for 1 mM TC in 2,2'-azobisisobutyronitrile and benzoyl peroxide systems were 0.1-0.2 and 119-250, respectively, whereas the corresponding values for quercetin (QU) and resveratrol (RE) were 2-4 and 7-15, respectively. In RAW264.7 cells stimulated with LPS, Cox2 and Tnfα mRNA were over-expressed in the presence of TC. MC downregulated only the expression of Cox2 by about 50% in LPSstimulated cells. The anti-inflammatory activity determined on the basis of Cox2 inhibition declined in the order QU>RE>MC>TC. Upon application of VLI, only TC downregulated the expression of LPS-stimulated Cox2 and Tnfα mRNA. After exposure to VLI, TC, but not MC, markedly up-regulated hemoxygenase-1 (Ho-1) expression. Conclusion: TC is a chain-breaking antioxidant with a large kinh. Upon activation by VLI, TC may undergo degradation and its degradation products affect pleiotropic mediators such as Cox2, Tnfα and Ho-1. TC may be useful as a local photodynamic therapy for periodontal diseases. [ABSTRACT FROM AUTHOR]

Novel brominated flame retardants are widely used in electronics, textiles, furniture, and other products; they can enter the human body through ingestion and respiration and cause harm to the human body, and have been proven to have potential biological toxicity and accumulation effects. 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE) is a widely used novel brominated flame retardant; however, there is a lack of research on its mechanism of toxicity, particularly that of intestinal toxicity. Currently, studies on the functionality of iroquois homeobox 3 (IRX3) are extremely limited. In our study, BTBPE was administered to Sprague-Dawley (SD) rats and rat small intestinal crypt epithelial cells (IEC6 cells) in vivo and in vitro, respectively, and hematoxylin and eosin (HE), immunohistochemical, Alcian blue-periodic acid-Schiff (AB-PAS), CCK8, acridine orange/ethidium bromide (AO/EB), fluorescent probes, qPCR, western blotting, and immunofluorescence analyses were performed. To explore the damage mechanism of BTBPE, we used siRNA to silence IRX3 and iNOs-IN-1 (yeast extract-peptone-wheat; YPW) to inhibit nitric oxide synthase 2 (NOS2). The results showed that BTBPE exposure caused inflammation and necroptosis in the jejunum and ileum, as well as destruction of the tight junctions and mucus layer. Moreover, BTBPE activated the IRX3/NOS2 axis both in vivo and in vitro. Silencing IRX3 or inhibiting NOS2 inhibits necroptosis and restores tight junctions in IEC6 cells. In conclusion, our study found that in the jejunum, ileum, and IEC6 cells, BTBPE exposure caused necroptosis and tight junction destruction by activating the IRX3/NOS2 axis. Blocking the IRX3/NOS2 axis can effectively inhibit necroptosis and restore tight junction. In addition, BTBPE exposure caused inflammation and loss of the mucous layer in the jejunum and ileum. Our study is the first to explore the mechanism of intestinal damage caused by BTBPE exposure and to discover new biological functions regulated by the IRX3/NOS2 axis, providing new research directions for necroptosis and tight junctions. [Display omitted] • The mechanism of BTBPE intestinal toxicity has been explored for the first time. • BTBPE caused inflammation, necroptosis, and destruction of tight junction and mucus layer in rat small intestine. • Activation of the IRX3/NOS2 axis leading to necroptosis was first discovered. • It was first found that the activation of IRX3/NOS2 axis would destroy the tight junction. • NOS2 is a key downstream gene that IRX3 plays roles in inducing necroptosis and destroying tight junction. [ABSTRACT FROM AUTHOR]

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis-infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat2B and Cat1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr6, Tlr9, Ly96, Tirap, Traf 6, Ticam1, Tollip, Casp8, Map3k1, Mapk8, Nfkbib, Nfkbil1, Ppara, Mapk8ip3, Hspd1, and Ube2n, as well as immunomodulators, such as Ptgs2/Cox2, Csf 2, Csf 3, Ifnb1, Il6ra, and Ilr1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

The high expression of inducible nitric oxide synthase (NOS2) by myeloid-derived suppressor cells (MDSCs) is a key mechanism of immune evasion in cancer. Recently we reported that NOS2 is also expressed by γδ T cells in melanoma, contributing to their polarization towards a pro-tumor phenotype. The molecular mechanisms underlying regulation of NOS2 expression in tumor-induced γδ T cells remain unexplored. By using the model of mice transgenic for the ret oncogene (Ret mice) that develops a spontaneous metastatic melanoma, we evidence that interleukin (IL)-1β and IL-6 drive NOS2 expression in γδ T cells. Indeed, their in vivo neutralization lessens the γδ T cell capacity to produce not only NOS2, but also IL-17 involved in the recruitment of MDSCs at the primary tumor site. The treatment also delayed tumor cell dissemination and induced vitiligo in a significant proportion of Ret mice. Interestingly, Ret mice developing a less aggressive melanoma, characterized by the spontaneous development of a concomitant autoimmune vitiligo, exhibit a weaker concentration of inflammatory cytokines and a reduction of tumor infiltrating γδ T cells expressing NOS2, when compared to Ret mice without any signs of vitiligo. Overall our results support that the level of inflammation at the tumor site regulates NOS2 expression by γδ T cells and the development of vitiligo associated melanoma.

Background Trichotillomania (TTM) is an impulse control disorder characterized by repetitive hair pulling/trimming. Barbering behavior (BB) observed in laboratory animals is proposed as a model of TTM. The neurobiological basis of TTM is unclear, but involves striatal hyperactivity and hypoactivation of the prefrontal cortex. Methods In this study, we evaluated the BB in knockout mice for the inducible isoform of nitric oxide synthase (NOS2KO) and the consequences of silencing this enzyme in PC12 cell differentiation. Results NOS2KO exhibit exacerbated BB, starting four weeks of age, and increased repetitive movements compared to wild-type mice (WT). The expression of BB was attenuated by repeated treatment with clomipramine, a clinically approved drug to treat TTM in humans, or memantine, an antagonist of NMDA receptors, as well as partial rescue of NOS2 expression in haploinsufficient animals. The silencing of NOS2 expression reduced the MAP2 (microtubule-associated protein 2) levels in activity-induced differentiated PC12 cells. Discussion Our data led us to propose that NOS2 is putatively involved in the neuronal maturation of the inhibitory afferent pathways during neurodevelopment, and such inadequate inhibition of motor programs might be associated to the observed phenotype.

Highly specific and efficient drugs have been developed to treat noncommunicable chronic inflammatory skin diseases (ncISD). Due to their specificity, these drugs require precise diagnostics to attribute the most efficient treatment to each patient. Diagnosis is complicated by the complex pathogenesis of ncISD and their clinical and histological overlap. Especially, precise diagnosis of psoriasis and atopic eczema is difficult in special cases, and molecular tools need to be developed to support gold standard diagnosis. Psoriasis and eczema are among the most prevalent skin diseases worldwide with a combined incidence rate of at least 2%. Although more than a dozen of immune-modulating therapies, particularly monoclonal antibodies targeting specific cytokines or their receptors, have become available over the last decade allowing highly effective treatment, the ultimate breakthrough would include accurate diagnostic tools. Diagnosis of both diseases largely depends on a subjective visual exam and histopathological examination. Due to phenotypical overlaps of both diseases, up to 50% of cases with a palmoplantar localization are misdiagnosed. To close this diagnostic gap, a gene expression-based classifier using NOS2 and CCL27 has been proposed and its clinical validity examined and proven in various patient cohorts. This study aims to develop a real-time based molecular classifier (MC) to distinguish psoriasis from atopic eczema in formalin-fixed, paraffin-embedded (FFPE)-fixed skin samples for diagnostic measures and to evaluate the potential of minimally invasive microbiopsies and noninvasive tape strips or sampling disks for molecular diagnosis. The FFPE, micro-(Ø1mm) and macrobiopsies (Ø4–6 mm), and tape strips were collected from psoriasis and eczema lesions and analysed by real-time polymerase chain reaction (PCR) for the expression of NOS2 and CCL27. A molecular classifier (MC) was established using a linear regression model. We transferred the RNAprotect-based molecular classifier to FFPE samples and were able to produce comparable results. Whereas the RNAprotect based MC distinguished psoriasis from eczema with a sensitivity and specificity for psoriasis of 97.7% and 100%, respectively, and an AUC of 0.99, the FFPE-based classifier determined probabilities for psoriasis with a sensitivity and specificity and of 92% and 100%, respectively, and an AUC of 0.97. To test if microbiopsies are also adequate tissue samples for the MC, we analysed gene expression in 83 pairs of macro- and microbiopsies by real-time quantitative-PCR. Delta-CT values showed no significant difference between macro- and microbiopsies. The MC led to reliable results in the vast majority of samples (91.57%) independent of biopsy size, with a small increase of misclassification (4.98%). The FFPE-based molecular classifier (MC) precisely separates eczema from psoriasis and efficiently identifies subtypes of both diseases. The reliability of the two markers NOS2 and CCL27 has been shown in FFPE tissue facilitating a possible implementation of this molecular diagnostic aid in routine clinical pathological practice. We further show that the gene expression profile of RNA later fixed microbiopsies is comparable to standard 4–6 mm biopsies and that microbiopsies are equally suited for the MC. We demonstrate the potential of tape strips of inflamed epidermis for molecular diagnostics of ncISD. Due to the minimally invasive sampling procedure, tape stripping may be particularly useful for the testing of visible and sensitive skin areas and in children as well as for repetitive sampling procedures necessary to monitor treatment responses over time. In summary, the MC discriminates psoriasis from eczema producing meaningful results in a broad range of skin tissue derived from invasive to minimally invasive sampling techniques. Further efforts are required to simplify and shorten the labor- and cost-intensive procedures of RNA isolation and real-time PCR. One possible solution is a fully automated and closed system, which would allow the integration of molecular diagnostics into the routine patient management at the point of care. [ABSTRACT FROM AUTHOR]

Objective: To investigate the mechanisms that underlie the anti-asthmatic effects of Nepeta bracteata (DBJJ, Dabao Jingjie in Chinese) in rats by integrating metabolomics and network pharmacology., Methods: In this study, the rat model of asthma was induced by ovalbumin (OVA), and the rats were treated with a decoction of N. bracteata . Pathological changes in lung tissue were observed, and the quantification of eosinophils (EOS) and white blood cells (WBC) in bronchoalveolar lavage fluid was performed. Furthermore, the serum levels of asthma-related factors induced by OVA were assessed. 1 H NMR spectroscopy serum metabolomics method was utilized to identify differential metabolites and their associated metabolic pathways. UPLC-QE-MS/MS combined with network pharmacology was employed to predict the core targets and pathways of DBJJ in its action against asthma. The anti-asthmatic properties of DBJJ were investigated using an integrated approach of metabolomics and network pharmacology. The findings were validated through molecular docking and Western blotting analysis of the key targets., Results: The administration of DBJJ effectively alleviated OVA-induced lung histopathological changes and decreased the number of EOS and WBC in BALF. Additionally, DBJJ inhibited the OVA-induced elevation of TNF-α, IL-18, Ig-E, EOS, IL-1β, MDA, VEGF-A, and TGF-β1. A total of 21 biomarkers and 10 pathways were found by metabolomics analysis. A total of 29 compounds were identified by UPLC-QE-MS/MS, in which 13 active components were screened by oral availability and Caco-2 cell permeability, the 120 targets and 173 KEGG pathways were predicted. The integration of metabolomics and network pharmacological analysis revealed that DBJJ's main constituents, including ferulic acid and ursolic acid, exerted their effects on four targets, namely DAO and NOS2, as well as their associated metabolites and pathways. The active constituents of DBJJ demonstrated a high binding affinity towards DAO and NOS2. Furthermore, DBJJ was observed to decrease the protein expression and phosphorylation levels of NOS2, MAPK, and STAT3., Conclusion: The administration of DBJJ demonstrates notable anti-asthma properties in rats with allergic asthma. This effect can be attributed to the modulation of various targets, including NOS2, MAPK, and STAT3, by primary constituents such as ferulic acid and ursolic acid., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V.)

Graphical abstract Highlights • Plasmalogens (Pls) can inhibit NO production in microglial cells. • Pls inhibit NF-kB, p38MAPK and JNK pathways. • PUFA-Pls is more effective than oleic acid (monounsaturated fatty acid)-Pls. Abstract The special lipids plasmalogens (Pls) were reported to be reduced in the neurodegenerative brains such as Alzheimer's disease where a marked increase of glial activation is often observed. We previously found that a reduction of brain Pls can enhance the glial activation in murine brains. However, the detailed role of Pls in the prevention of glial activation was mostly elusive. Here we report that the Pls, extracted from scallop (sPls), significantly inhibited the inducible form of nitric oxide synthase (NOS2) and the production of NO in LPS (lipopolysaccharide)-activated microglial cells. We also observed that the polyunsaturated docosahexaenoic acid (DHA)-containing Pls but not the monounsaturated oleic acid-containing Pls attenuated the NOS2 induction. In addition, sPls blocked the activation of nuclear factor (NF)-kB and mitogen-activated protein kinases (MAPKs) e.g., JNK and p38 MAPK, thereby attenuated the nuclear translocation of NF-kB subunit, p65, and activator protein-1 (AP-1) proteins (c-Fos and c-Jun). Interestingly, LPS treatments suppressed the expression of Pls synthesizing enzymes, glycerone phosphate O-acyltransferase (GNPAT) and alkylglycerone phosphate synthase (AGPS) in the microglial cells by the p38MAPK and JNK pathways. Furthermore, the knockdown of GNPAT and AGPS genes by sh-RNAs accelerated the LPS-induced activation of p38MAPK and JNK, resulting in the increased production of NO. These findings suggested that a decrease of brain Pls can activate the NF-kB, p38MAPK and JNK pathways to induce a prolonged microglial activation which may downplay the neuroprotective events in the brains of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. Methods: In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H2O2 levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. Results: Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H2O2 production. H2O2 metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H2O2. Stat5b silencing finally reduces AKT expression, a prosurvival signal. Conclusion: Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H2O2 accumulation and the ensuing oxidative stress enhances β-cell damage. [ABSTRACT FROM AUTHOR]

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis -infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (No s2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos 2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis -infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg 1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat 2B and Cat 1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr 6, Tlr 9, Ly 96, Tirap, Traf 6, Ticam 1, Tollip, Casp 8, Map 3 k 1, Mapk 8, Nfkbib, Nfkbil 1, Ppara, Mapk8i p3, Hspd 1, and Ube2 n, as well as immunomodulators, such as Ptgs 2/ Cox 2, Csf 2, Csf 3, Ifnb 1, Il 6 ra , and Ilr 1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the preventive and curative effects of ethanolic extract of Propolis (EEP) during α-Tropomyosin-induced uveitis in an experimental model using Wistar rats, through the regulation of inducible nitric oxide synthase (NOS2) and arginase-1. In this context, rats received daily injection of EEP (100 mg/kg) for 5 days prior to immunization or for 9 days commencing 5 days post immunization with α-Tropomyosin extract, then were sacrificed at day 14. Histological examination, NOS2, arginase-1, and nuclear factor-κB (NF-κB) expression were evaluated in the retinas. Plasmatic production of nitric oxide (NO), urea, IL-4, and TNF-α was assessed. We have found that treatment with EEP substantially reduced the retinal histological damages induced by α-Tropomyosin. In the same context, a significant decrease of NO and TNF-α levels was noticed. Interestingly, EEP down-modulated NOS2 and NF-κB expression in retina. Also, an increase in urea and IL-4 levels was concomitant to an up-modulation of arginase-1 expression. Hence, it appears that EEP attenuated retinal damages through the induction of Th2 response and the inhibition of NF-κB/NOS2 pathway. [ABSTRACT FROM AUTHOR]

The decline in brain noradrenaline levels is associated with the progression of certain neurodegenerative diseases. This seems to be due, at least in part, to the ability of noradrenaline to limit glial activation and to reduce the damage associated with it. Our previous studies of the mechanisms involved in this process indicate that noradrenaline induces the production of the chemokine CCL2 in astrocytes. While CCL2 can protect neurons against certain injuries, its overproduction has also proven to be harmful and to prevent noradrenaline neuroprotective effects. Therefore, in this study, we analyze if the modifications caused to astrocytes by an excessive production of CCL2 may alter their response to noradrenaline. Using primary cultures of rat cortical astrocytes, we observed that CCL2 enhances the production of beta 2 adrenergic receptors in these cells. While this potentiates noradrenaline signaling through cAMP, the activation of the transcription factor CREB is inhibited by CCL2. Furthermore, although CCL2 potentiates noradrenaline induction of glycogenolysis, this does not translate into an augmented release of lactate, one of the processes through which astrocytes help support neurons. Additionally, other neuroprotective actions of noradrenaline, such as the production of brain derived neurotrophic factor and the inhibition of the inducible nitric oxide synthase in astrocytes were modified by CCL2. These data suggest that some of the central nervous system alterations related to CCL2 could be due to its effects on adrenergic receptors and its interference with noradrenaline signaling. [ABSTRACT FROM AUTHOR]

Summary Phagocyte microbiocidal mechanisms and inflammatory cytokine production are temporally coordinated, although their respective interdependencies remain incompletely understood. Here, we identify a nitric-oxide-mediated antioxidant response as a negative feedback regulator of inflammatory cytokine production in phagocytes. In this context, Keap1 functions as a cellular redox sensor that responds to elevated reactive nitrogen intermediates by eliciting an adaptive transcriptional program controlled by Nrf2 and comprised of antioxidant genes, including Prdx5. We demonstrate that engaging the antioxidant response is sufficient to suppress Toll-like receptor (TLR)-induced cytokine production in dendritic cells and that Prdx5 is required for attenuation of inflammatory cytokine production. Collectively, these findings delineate the reciprocal regulation of inflammation and cellular redox systems in myeloid cells. [ABSTRACT FROM AUTHOR]

Background. Trichotillomania (TTM) is an impulse control disorder characterized by repetitive hair pulling/trimming. Barbering behavior (BB) observed in laboratory animals is proposed as a model of TTM. The neurobiological basis of TTM is unclear, but involves striatal hyperactivity and hypoactivation of the prefrontal cortex. Methods. In this study, we evaluated the BB in knockout mice for the inducible isoform of nitric oxide synthase (NOS2KO) and the consequences of silencing this enzyme in PC12 cell differentiation. Results. NOS2KO exhibit exacerbated BB, starting four weeks of age, and increased repetitive movements compared to wild-type mice (WT). The expression of BB was attenuated by repeated treatment with clomipramine, a clinically approved drug to treat TTM in humans, or memantine, an antagonist of NMDA receptors, as well as partial rescue of NOS2 expression in haploinsufficient animals. The silencing of NOS2 expression reduced the MAP2 (microtubule-associated protein 2) levels in activityinduced differentiated PC12 cells. Discussion. Our data led us to propose that NOS2 is putatively involved in the neuronal maturation of the inhibitory afferent pathways during neurodevelopment, and such inadequate inhibition of motor programs might be associated to the observed phenotype. [ABSTRACT FROM AUTHOR]

Low oxygen environments and accumulation of hypoxia-inducible factors (HIFs) are features of infected and inflamed tissues. Here, we summarize our current knowledge on oxygen levels found in Leishmania-infected tissues and discuss which mechanisms potentially contribute to local tissue oxygenation in leishmanial lesions. Moreover, we review the role of hypoxia and HIF-1 on innate antileishmanial immune responses.

A growing body of evidence supports the use of probiotics in the treatment of several skin conditions, including wounds. Even if in vitro and in vivo studies have highlighted the pro-healing effects of some probiotic bacteria, the underlying mechanisms are still not fully defined. The current investigation aimed to determine the re-epithelialization potential of the soluble fraction from lysate of seven different probiotic strains belonging to different genera (i.e., Streptococcus, Lactobacillus, and Bifidobacterium) on in vitro physically wounded HaCaT monolayer model. The results suggested that the soluble fraction of S. thermophilus, L. plantarum, and L. acidophilus promoted the re-epithelialization of scratched HaCaT monolayers, whereas those from B. longum, B. infantis, and B. breve significantly inhibited the process. On the other hand, L. bulgaricus showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of S. thermophilus, L. plantarum, and L. acidophilus.

Low oxygen environments and accumulation of hypoxia-inducible factors (HIFs) are features of infected and inflamed tissues. Here, we summarize our current knowledge on oxygen levels found in Leishmania-infected tissues and discuss which mechanisms potentially contribute to local tissue oxygenation in leishmanial lesions. Moreover, we review the role of hypoxia and HIF-1 on innate antileishmanial immune responses. [ABSTRACT FROM AUTHOR]

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triplenegative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER- patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feedforward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFa-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in themore aggressiveMDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDAMB- 231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER- and TNBC disease. [ABSTRACT FROM AUTHOR]

Both mesenchymal stromal cells (MSCs) and myeloid-derived suppressor cells (MDSCs) have immunosuppressive properties, and their presence may confer a worse prognosis upon cancer patients. However, whether MSCs can enhance the immunosuppressive effects of MDSCs in MM remains unknown. We evaluated the influence of MSCs on MDSCs growth, apoptosis, and functions. Our results show that MSCs promote proliferation and inhibit apoptosis in MDSCs. Additionally, MSCs enhance the ability of MDSCs by inhibiting T-cell proliferation and IFN-γ production. Furthermore, both the mRNA and protein levels of Arg1 and NOS2 were upregulated in MDSCs. These results suggest that MSCs may exert immunomodulatory effects on MDSCs by upregulating Arg1 and NOS2. [ABSTRACT FROM AUTHOR]

Leptin, following its discovery, has developed a formidable interest in the scientific community to delineate its contribution towards overall metabolic homeostasis. Contradictory reports have been published on leptin administration effects on whole body insulin sensitivity. Following late reports, we surveyed human serum leptin levels along with other metabolic parameters including BMI and HOMA-IR. We found a positive correlation between leptin levels and insulin resistance parameters. Considering the presence of the long form of leptin receptor on adipocytes, we explored the effects of chronic physiological hyper-leptinemic exposure on adipocyte insulin sensitivity. Chronic leptin (50 ng/ml) treatment in 3T3-L1 adipocytes decreased insulin-induced phosphorylation of nodal insulin signaling proteins along with reduced glucose uptake. Metabolic flux studies indicated mitochondrial dysfunction and reduced oxygen consumption rate. Leptin treatment also increased both cellular and mitochondrial superoxide levels concomitant to increased expression of nitric oxide synthase-2 (NOS2). Further, pharmacological depletion of NOS2 reversed leptin mediated effects on insulin signaling. In-vivo implantation of leptin osmotic pumps in C57BL/6 mice also decreased insulin responsiveness. Interestingly, these effects were lacking in NOS2 knockout strain. In conclusion, our studies put forward a potential link between leptin and adipocyte insulin responsiveness in an NOS2 dependent manner. [ABSTRACT FROM AUTHOR]

The pathogen Mycobacterium leprae causes leprosy that affects mainly skin and nerves. Polymorphisms of certain genes are substantiated to be associated with the susceptibility/resistance to leprosy. The present investigation addressed the association of Nitric Oxide Synthase2 gene polymorphisms and leprosy in a population from northern part of India. A total of 323 leprosy cases and 288 healthy controls were genotyped for four NOS2 promoter variants (rs1800482, rs2779249, rs8078340 and rs2301369) using FRET technology in Real Time PCR. None of these SNPs in promoter sites was associated with susceptibility/resistance to leprosy. NOS2 rs1800482 was found to be monomorphic with GG genotype. However, NOS2 - 1026T allele was observed to be in higher frequency with leprosy cases (BL and LL) who were not suffering from any reactional episodes compared to cases with ENL reaction {OR = 0.30, 95% CI (0.10–0.86), p = 0.024}. NOS2 - 1026GT genotype was more prevalent in cases without reaction (BT, BB and BL) compared to RR reactional patients {OR = 0.38, 95% CI (0.17–0.86), p = 0.02}. Although haplotype analysis revealed that no haplotype was associated with leprosy susceptibility/resistance with statistical significance, GTG haplotype was noted to be more frequent in healthy controls. These SNPs are observed to be in linkage disequilibrium. Although, these SNPs are not likely to influence leprosy vulnerability, -1026G > T SNP was indicated to have noteworthy role in leprosy reactions. [ABSTRACT FROM AUTHOR]

Significance: Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Development and progression of malignancy are associated with diverse cell signaling pathways that control cell proliferation, survival, motility, invasion, and metastasis. Recent Advances: An increasing number of clinical studies have implicated a strong relationship between elevated tumor nitric oxide synthase-2 (NOS2) expression and poor patient survival. Critical Issues: Herein, we review what we believe to be key mechanisms in the role(s) of NOS2-derived nitric oxide (NO) as a driver of breast cancer disease progression. High NO increases cyclooxygenase-2 activity, hypoxia inducible factor-1 alpha protein stabilization, and activation of important cell signaling pathways, including phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, epidermal growth factor receptor, and Ras, through post-translational protein modifications. Moreover, dysregulated NO flux within the tumor microenvironment has other important roles, including the promotion of angiogenesis and modulation of matrix metalloproteinase/tissue inhibitor matrix metalloproteinase associated with tumor progression. Future Directions: The elucidation of these and other NO-driven pathways implicates NOS2 as a key driver of breast cancer disease progression and provides a new perspective in the identification of novel targets that may be therapeutically beneficial in the treatment of estrogen receptor-negative disease. Antioxid. Redox Signal. 26, 1044-1058. [ABSTRACT FROM AUTHOR]

Studies of cognitive behavior in rodent models of Alzheimer’s disease (AD) are the mainstay of academic and industrial efforts to find effective treatments for this disorder. However, in the majority of such studies, the nature of rodent behavioral tests is considerably different from the setting associated with cognitive assessments of individuals with AD. The recently developed touchscreen technique provides a more translational way of rodent cognitive testing because the stimulus (images in different locations on the screen) and reaction (touch) are similar to those employed in human test routines, such as the Cambridge Neuropsychological Test Automated Battery. Here, we used Visual Discrimination and Reversal of Visual Discrimination touchscreen tasks to assess cognitive performance of APPSwDI/Nos2 −/− (CVN) mice, which express mutated human APP and have a homozygous deletion of the Nos2 gene. We revealed that CVN mice made more first-time errors and received more correction trials than WT mice across both discrimination and reversal phases, although mutation effect size was larger during the latter phase. These results indicate sensitivity of touchscreen-based measurements to AD-relevant mutations in CVN mice and warrant future touchscreen experiments aimed at evaluating other cognitive and motivational phenotypes in this AD mouse model. [ABSTRACT FROM AUTHOR]

Multiple immunosuppressive mechanisms exist in the tumor microenvironment that drive poor outcomes and decrease treatment efficacy. The co-expression of NOS2 and COX2 is a strong predictor of poor prognosis in ER- breast cancer and other malignancies. Together, they generate pro-oncogenic signals that drive metastasis, drug resistance, cancer stemness, and immune suppression. Using an ER- breast cancer patient cohort, we found that the spatial expression patterns of NOS2 and COX2 with CD3+CD8+PD1- T effector (Teff) cells formed a tumor immune landscape that correlated with poor outcome. NOS2 was primarily associated with the tumor-immune interface, whereas COX2 was associated with immune desert regions of the tumor lacking Teff cells. A higher ratio of NOS2 or COX2 to Teff was highly correlated with poor outcomes. Spatial analysis revealed that regional clustering of NOS2 and COX2 was associated with stromal-restricted Teff, while only COX2 was predominant in immune deserts. Examination of other immunosuppressive elements, such as PDL1/PD1, Treg, B7H4, and IDO1, revealed that PDL1/PD1, Treg, and IDO1 were primarily associated with restricted Teff, whereas B7H4 and COX2 were found in tumor immune deserts. Regardless of the survival outcome, other leukocytes, such as CD4 T cells and macrophages, were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to a massive cell infiltration, thus validating the hypothesis that COX2 is an essential component of the Teff exclusion process and, thus, tumor evasion. Our study indicates that NOS2/COX2 expression plays a central role in tumor immunosuppression. Our findings indicate that new strategies combining clinically available NOS2/COX2 inhibitors with various forms of immune therapy may open a new avenue for the treatment of aggressive ER-breast cancers.

Spermatogenesis is the developmental process that produces spermatozoa. The aim of this study was to investigate the single nucleotide polymorphisms (SNPs) within C7H15orf39 and NOS2 genes and to determine the correlations between two SNPs and semen quality in Duroc boars ( n  = 604). The polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method was used for genotyping the selected two nonsynonymous SNPs. The significant correlation was observed between two SNPs (rs80969873: g.58385473 G > A within C7H15orf39 ; rs325865291: g.44175445 G > A within NOS2 ) and semen traits in Duroc boars. This study indicates the SNPs in C7H15orf39 and NOS2 may be the potential molecular marker for improving the semen quality traits in Duroc boars.

Prostaglandin E2 (PGE2) plays crucial roles in managing microglial activation through the prostanoid EP2 receptor, a PGE2 receptor subtype. In this study, we report that PGE2 enhances interferon-γ (IFN-γ)-induced nitric oxide production in microglia. IFN-γ increased the release of nitrite, a metabolite of nitric oxide, which was augmented by PGE2, although PGE2 by itself slightly affects nitrite release. The potentiating effect of PGE2 was positively associated with increased expression of inducible nitric oxide synthase. In contrast to nitrite release induced by IFN-γ, lipopolysaccharide-induced nitrite release was not affected by PGE2. An EP2 agonist, ONO-AE1-259-01 also augmented IFN-γ-induced nitrite release, while an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, or an EP4 agonist, ONO-AE1-329, did not. In addition, the potentiating effect of PGE2 was inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10−6 M. Among the EP agonists, ONO-AE1-259-01 alone was able to accumulate cyclic adenosine monophosphate (AMP), and among the EP antagonists, PF-04418948 was the only one able to inhibit PGE2-increased intracellular cyclic AMP accumulation. On the other hand, IFN-γ promoted phosphorylation of signal transducer and activator of transcription 1, which was not affected by PGE2. Furthermore, other prostanoid receptor agonists, PGD2, PGF2α, iloprost, and U-46119, slightly affected IFN-γ-induced nitrite release. These results indicate that PGE2 potentiates IFN-γ-induced nitric oxide production in microglia through the EP2 receptor, which may shed light on one of the pro-inflammatory aspects of PGE2. [ABSTRACT FROM AUTHOR]

Objective: To investigate the spatial memory and the expression of hippocampal NOS2 in rat model of acute alcohol intoxication combined with TBI. Methods: After practice in water maze,96 male SD rats were randomly divided into four groups: N group（saline injection）,A group（alcohol injection）,T group（traumatic brain injury and saline injection） and AT group（traumatic brain injury and alcohol injection）. The animal model of acute alcohol intoxication with TBI was established by hitting rat forehead vertically 2hours following injecting of 25% alcohol 2.5 g/kg intraperitoneally. Animals of each group sacrificed at 1,3,5,7,14 days after behavior tests respectively. The spatial memory was tested by Morris-maze. The expression of NOS2 in hippocampus neurons was detected by immunohistochemistry method. Results: All experimental groups exhibit higher expression of NOS2 than that of N control group. NOS2 expression in T group was higher than in AT group at the 1stday after surgery（P<0.01）. NOS2 expression in AT group was higher than that of T group from 5thday to14thday after surgery（P<0.05）. In water maze tests,latency in T group was shorter than that in AT group（P<0.05） in the1stand the 14thday. However,in the 3rdday,latency in NT group was longer than in AT group（P<0.05）. Conclusions: Acute alcohol intoxication with advanced TBI had long latency in finding the platform,suggesting that capacity of spatial learning and memory was impaired. The increased expression of NOS2 in rat CA1 neurons area of hippocampus may contribute to secondary brain injury,and may be one of the reasons for unfavourable prognosis in later stage. [ABSTRACT FROM AUTHOR]

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Novel specific therapies for psoriasis and eczema have been developed, and they mark a new era in the treatment of these complex inflammatory skin diseases. However, within their broad clinical spectrum, psoriasis and eczema phenotypes overlap making an accurate diagnosis impossible in special cases, not to speak about predicting the clinical outcome of an individual patient. Here, we present a novel robust molecular classifier ( MC) consisting of NOS2 and CCL27 gene that diagnosed psoriasis and eczema with a sensitivity and specificity of >95% in a cohort of 129 patients suffering from (i) classical forms; (ii) subtypes; and (iii) clinically and histologically indistinct variants of psoriasis and eczema. NOS2 and CCL27 correlated with clinical and histological hallmarks of psoriasis and eczema in a mutually antagonistic way, thus highlighting their biological relevance. In line with this, the MC could be transferred to the level of immunofluorescence stainings for iNOS and CCL27 protein on paraffin-embedded sections, where patients were diagnosed with sensitivity and specificity >88%. Our MC proved superiority over current gold standard methods to distinguish psoriasis and eczema and may therefore build the basis for molecular diagnosis of chronic inflammatory skin diseases required to establish personalized medicine in the field. [ABSTRACT FROM AUTHOR]

Background: Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca(2+)-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca(2+) influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2.Methods: We studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8-/- and Trpm4-/- mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation.Results: In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca(2+)]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca(2+)]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca(2+)/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation.Conclusions: Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca(2+)-sensitive gene expression, including Nos2. [ABSTRACT FROM AUTHOR]

Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy. [ABSTRACT FROM AUTHOR]

Many studies suggest significant genetic variation in the resistance of cattle and humans to infection with Mycobacterium bovis, the causative agent of zoonotic tuberculosis. The inducible nitric oxide synthase (iNOS which is encoded by the NOS2 gene) plays a key role in the immunological control of a broad spectrum of infectious agents. This study aimed to investigate the influence of genetic variations in the promoter of the NOS2 gene on bovine tuberculosis (bTB) susceptibility. In this study, the NOS2 genes of 74 bTB-infected Holstein cows and 90 healthy controls were genotyped using PCR followed by nucleotide sequencing. Polymorphisms at rs207692718, rs109279434, rs209895548, rs385993919, rs433717754, rs383366213, rs466730386, rs715225976, rs525673647, rs720757654 and g.19958101T>G in the promoter region of the NOS2 gene were detected. The g.19958101T>G SNP produced two different conformation patterns (TT and TG) and the TG genotype was over-represented in the bTB group (20.27%) compared with the control group (2.22%). The TG genotype frequency of the g.19958101T>G variant was significantly higher in bTB cattle than in healthy controls (OR, 11.19; 95% CI, 2.47-50.73; P=0.0002). The G allele of the g.19958101T>G polymorphism was more frequent in bTB group when compared to control group (10.14% versus 1.11%). Furthermore, the G allele was a risk factor for bTB susceptibility (OR, 10.04; 95% CI, 2.26-44.65; P=0.0002). In conclusion, the g.19958101T>G polymorphism of the NOS2 gene may contribute to the susceptibility of Holstein cattle to bTB. [ABSTRACT FROM AUTHOR]

The role of nitric oxide (NO) in host defense against Echinococcus granulosus larvae was previously reported. However, NO production by NOS2 (inducible NO synthase) is counteracted by the expression of Arginase. In the present study, our aim is to evaluate the involvement of the laminated layer (external layer of parasitic cyst) in Arginase induction and the protoscoleces (living and infective part of the cyst) survival. Our in vitro results indicate that this cystic compound increases the Arginase activity in macrophages. Moreover, C-type lectin receptors (CLRs) with specificity for mannan and the TGF-β are implicated in this effect as shown after adding Mannan and Anti-TGFβ. Interestingly, the laminated layer increases protoscoleces survival in macrophages–parasite co-cultures. Our results indicate that the laminated layer protects E. granulosus against the NOS2 protective response through Arginase pathway, a hallmark of M2 macrophages. [ABSTRACT FROM AUTHOR]

Objectives: Caspase-1 (casp1), a key protease involved in the systemic inflammatory response syndrome (SIRS), controls the brain expression of a set of eight genes: Nos2 and Ptgs2 (nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, two inducible enzymes), Cxcl1 and Cxcl10 (C-X-C motif chemokine ligand 1 and ligand 10), Tgtp and Gbp2 (T cell-specific GTPase 1 and guanylate-binding protein 2, two GTPases), Adamts1 (a disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 1, a metalloprotease) and Il1rn (interleukin-1 receptor antagonist). Our objective was to ascertain whether casp1 also controlled the peripheral expression of these genes and, if so, to compare their central versus peripheral patterns of gene expression in immune and endocrine tissues during SIRS. Methods: Wild-type (wt) and casp1 knockout (casp1-/-) mice were injected with either saline or a high dose of endotoxin/lipopolysaccharide (LPS; 800 μg/mice i.p.). Saline-injected mice were immediately euthanized after injection, whereas LPS-injected mice were sacrificed 6 and 12 h after LPS administration. Hippocampal, splenic and adrenal gene expressions were determined by real-time PCR. Results: Overall, casp1-/- mice showed a lower inflammatory response than wt mice. The expression levels of powerful proinflammatory factors such as Nos2 and Ptgs2 was reduced in casp1-/- mice. Moreover, a hierarchical clustering analysis aimed at studying patterns of gene coexpression revealed large alterations in the hippocampal pattern of casp1-/- mice. Surprisingly, the expression of Adamts1 was increased in the hippocampus and adrenals of casp1-/- mice. Conclusions: The resilience of casp1-/- mice to SIRS lethality is associated with a lower inflammatory response, loss of hippocampal gene coexpression patterns, and increased hippocampal Adamts1 gene expression. The latter might be beneficial for casp1-/- mice, since ADAMTS1 is likely to play a role in neuronal plasticity. The mechanisms described here may help the development of either novel biomarkers or therapeutic targets against SIRS/sepsis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]