Nitric oxide synthase type II

Wear particles generated with use of total joint replacements incite a chronic macrophage‐mediated inflammatory reaction, which leads to implant failure. Macrophage activation may be polarized into two states, with an M1 proinflammatory state dominating an alternatively activated M2 anti‐inflammatory state. We hypothesized that IL‐4, an activator of M2 macrophages, could modulate polyethylene (PE) particle‐induced osteolysis in an experimental murine model. Four animal groups included (a) calvarial saline injection with harvest at 14 days (b) single calvarial injection of PE particles subcutaneously (SC) without IL‐4 (c) PE particles placed as in (b), then IL‐4 given SC for 14 consecutive days and (d) PE particles as in (b) then IL‐4 beginning 7 days after particle injection for 7 days. The calvarial bone volume to total tissue volume was measured using microCT and histomorphometry. Calvaria were cultured for 24 h to assess release of RANKL, OPG, TNF‐α, and IL‐1ra and isolation and ...

Wear particles generated with use of total joint replacements incite a chronic macrophage‐mediated inflammatory reaction, which leads to implant failure. Macrophage activation may be polarized into two states, with an M1 proinflammatory state dominating an alternatively activated M2 anti‐inflammatory state. We hypothesized that IL‐4, an activator of M2 macrophages, could modulate polyethylene (PE) particle‐induced osteolysis in an experimental murine model. Four animal groups included (a) calvarial saline injection with harvest at 14 days (b) single calvarial injection of PE particles subcutaneously (SC) without IL‐4 (c) PE particles placed as in (b), then IL‐4 given SC for 14 consecutive days and (d) PE particles as in (b) then IL‐4 beginning 7 days after particle injection for 7 days. The calvarial bone volume to total tissue volume was measured using microCT and histomorphometry. Calvaria were cultured for 24 h to assess release of RANKL, OPG, TNF‐α, and IL‐1ra and isolation and ...

Inducible nitric oxide synthase (iNOS) is an enzyme that plays a key role in intracellular immune response against respiratory infections. Since various species of nonhuman primates exhibit different levels of susceptibility to infectious respiratory diseases, and since variation in regulatory regions of genes is thought to play a key role in expression levels of genes, two candidate regulatory regions of iNOS were mapped, sequenced, and compared across five species of nonhuman primates: African green monkeys (Chlorocebus sabaeus), pig-tailed macaques (Macaca nemestrina), cynomolgus macaques (Macaca fascicularis), Indian rhesus macaques (Macaca mulatta), and Chinese rhesus macaques (M. mulatta). In addition, we conducted an in silico analysis of the transcription factor binding sites associated with genetic variation in these two candidate regulatory regions across species. We found that only one of the two candidate regions showed strong evidence of involvement in iNOS regulation. ...

Trypanosoma cruzi, the etiological agent of Chagas' disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1β and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferatoractivated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-β. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas' disease deserves further investigation.

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1+CD11b+ MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr701 in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4+ and CD8+ T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of M...

The objective of this study was to establish the cardioprotective effect of sulfaphenazole (SPZ), a selective inhibitor of cytochrome P450 2C9 enzyme, in an in vivo rat model of acute myocardial infarction (MI). MI was induced by 30 min ligation of left anterior descending coronary artery, followed by 24 h reperfusion (I=R). The study used 6 groups: I=R (control); SPZ; L-NAME; L-NAME þ SPZ; 1400W (an inhibitor of iNOS); 1400W þ SPZ. The agents were administered orally through drinking water for 3 days prior to induction of I=R. Myocardial oxygenation (pO 2 ) at the I=R site was measured using EPR oximetry. The preischemic pO 2 value was 18 AE 2 mm Hg in all groups. At 1 h of reperfusion, the SPZ group showed a significantly higher hyperoxygenation when compared to control (45 AE 1 vs. 34 AE 2 mm Hg). The SPZ group showed a significant improvement in the contractile functions and reduction in infarct size. Histochemical staining of SPZ-treated hearts exhibited significantly lower levels of superoxide and peroxynitrite, and markedly increased levels of iNOS activity and nitric oxide. Western blot analysis indicated upregulation of Akt and attenuation of p38MAPK activities in the reperfused myocardium. The study established that SPZ attenuated myocardial I=R injury through overexpression of iNOS, leading to enhancement of nitric oxide bioavailability and tissue oxygenation. Antioxid. Redox Signal. 11,[725][726][727][728][729][730][731][732][733][734][735][736][737][738]

Background-We reported previously that inducible nitric oxide synthase (iNOS) expression in graft-infiltrating human T cells, which is confined to the bystander population, contributes to T cellmediated rejection of allograft arteries in a humanized mouse model. Here, we examine if CXCL12, a chemokine thought to contribute to recruitment of bystander T cells, induces iNOS in human CD8 T cells. Methods-Human CD8 T cells were treated with CXCL12 and iNOS expression examined. Also, human allograft arteries were immunohistochemically stained for iNOS and CD8, and adjacent sections stained for CXCL12 to determine their localization in human tissues. Results-Resting human CD8 and CD4 T cells expressed the CXCR4, but not the CXCR7, receptor for CXCL12. Treatment with CXCL12 induced expression of both iNOS mRNA and protein in primary human CD8 T cells in a dose-dependent manner, but had no effect on CD4 T cells. Induction of iNOS expression in CD8 T cells was mediated by increased gene transcription. TCR-activated CD8 T cells rapidly down-regulated CXCR4 and this coincided with diminished ability of CXCL12 to induce iNOS in activated T cells. iNOS expression in infiltrating human CD8 T cells was spatially associated with CXCL12 expression both in the humanized mouse model of allograft artery rejection and in clinical specimens of coronary arteries displaying allograft vasculopathy. Conclusions-CXCL12 induces iNOS expression in human CD8 T cells and this response may contribute to allograft rejection.

Background— Preconditioning phenomena provide evidence for adaptive responses to ischemia that have important implications for treatment/prevention of myocardial infarction. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia. Methods and Results— Exposure of wild-type mice to intermittent hypoxia resulted in protection of isolated hearts against ischemia-reperfusion injury 24 hours later. Cardiac protection induced by intermittent hypoxia was lost in Hif1a +/ − mice heterozygous for a knockout allele at the locus encoding HIF-1α. Erythropoietin (EPO) mRNA expression was induced in kidneys of wild-type mice subjected to intermittent hypoxia, resulting in increased plasma EPO levels. EPO mRNA expression was not induced in Hif1a +/ − mice. EPO administration to rats increased functional recovery and decreased apoptosis in isolated hearts subjected to ischemia-reperfusion 24 hours later. Conclusions— Hearts isolated from rodents subjected ...

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-κB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-κB p65 and modifies nuclear NF-κBp50. Here, we demonstrated that both nuclear STAT-1 and NF-κB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-κB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-κB prote...

Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β...

Chronic inflammation of the intestinal mucosa is characteristic of inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease. Helminth parasites have developed immunomodulatory strategies that may impact the outcome of several inflammatory diseases. Therefore, we investigated whetherTaenia crassicepsinfection is able to decrease the inflammatory effects of dextran sulfate sodium- (DSS-) induced ulcerative colitis in BALB/c and C57BL/6 mice. Preinfection significantly reduced the manifestations of DSS-induced colitis, as weight loss and shortened colon length, and decreased the disease activity index independently of the genetic background of the mice.Taeniainfection decreased systemic levels of proinflammatory cytokines while increasing levels of IL-4 and IL-10, and the inflammatory infiltrate into the colon was also markedly reduced. RT-PCR assays from colon showed thatT. crassiceps-infected mice displayed increased expression of Arginase-1 but decreased expression...

To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in infl ammatory bowel disease (IBD). We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofl uorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10 -4 mol/ L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18% ± 16% vs 23% ± 15% P <0.01). UC or CD serum caused a signifi cant increase in iNOS compared to control (UC: 300% ± 21%; CD: 275% ± 27% vs 108% ± 14%, P <0.01). Apoptosis/necrosis characteristics did not differ signifi cantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION: IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinfl ammatory protection of endothelial cells.

The molecular mechanism for priapism is not well characterized. Although the nitric oxide (NO) pathway is known to mediate penile erection under normal conditions, we hypothesized that the mechanism of priapism rests in aberrant downstream signaling of this pathway based on our previous findings that mice lacking the gene for endothelial nitric oxide synthase (eNOS —/— ) and mice lacking both neuronal NOS (nNOS) and eNOS (nNOS —/— , eNOS —/— ) have a tendency for priapic activity. We investigated the role of downstream guanylate cyclase and phosphodiesterase type 5 (PDE5A) expression and function in mediating these responses in eNOS —/— and nNOS —/— , eNOS —/— mice. Erectile responses to both cavernous nerve stimulation and intracavernosal injection of the NO donor diethylamine-NONOate were augmented in eNOS —/— and nNOS —/— , eNOS —/— mice but not in WT or nNOS —/— mice. PDE5A protein expression and activity and cGMP levels were significantly lower in eNOS —/— and nNOS —/— , eNOS —...

Transketolaselike (TKTL) 1 indirectly replenishes NADPH preventing damage induced by reactive oxygen species (ROS) formed upon intestinal inflammation. We investigated the function of TKTL1 during murine colitis and ROS detoxification for prevention of tissue damage. Mucosal damage in TKTL1 Ϫ/Ϫ and wild-type (WT) mice was assessed by miniendoscopy and histology during dextran sodium sulfate (DSS) colitis. mRNA levels of interferon (IFN)-␥, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF), transketolase (TKT), and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress nitrosylation, carbonylation and antioxidative enzymes catalase (Cat), superoxide dismutase 1 and 2, as well as glutathione (GSH) were determined. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. TKTL1 knockout or DSS treatment did not influence TKT and TKTL2 mRNA or protein expression. Mucosal damage was significantly increased in TKTL1 Ϫ/Ϫ mice indicated by miniendoscopy as well as a significantly shorter colon and more severe histological scores compared with WT mice during DSS colitis. This was associated with higher mRNA levels of IFN-␥, iNOS, IL-6, and TNF. In addition, iNOS protein expression was significantly enhanced in TKTL1 Ϫ/Ϫ mice as well as MPO activity. Protein modification by nitric oxide (nitrotyrosine) was induced in TKTL1 Ϫ/Ϫ mice. However, introduction of carbonyl groups by ROS was not induced in these mice. The expression of SOD1, SOD2, Cat, as well as GSH content was not significantly changed in TKTL1 Ϫ/Ϫ mice. We conclude that induced colitis in TKTL1 Ϫ/Ϫ mice was more severe compared with WT. This indicates a role of TKTL1 during mucosal repair and restoration.

The underlying reasons for variable clinical outcomes from respiratory viral infections remain uncertain. Several studies suggest that environmental factors contribute to this variation, but limited knowledge of cellular and molecular targets of these agents hampers our ability to quantify or modify their contribution to disease and improve public health. The aryl hydrocarbon receptor (AhR) is an environment sensing transcription factor that binds many anthropogenic and natural chemicals. The immunomodulatory properties of AhR ligands are best characterized with extensive studies of changes in CD4 + T cell responses. Yet, AhR modulates other aspects of immune function. We previously showed that during influenza virus infection, AhR activation modulates neutrophil accumulation in the lung, and this contributes to increased mortality in mice. Enhanced levels of inducible nitric oxide synthase (iNOS) in infected lungs are observed during the same timeframe as AhR-mediated increased pulmonary neutrophilia. In this study, we evaluated whether these two consequences of AhR activation are causally linked. Reciprocal inhibition of AhR-mediated elevations in iNOS and pulmonary neutrophilia reveal that, although they are contemporaneous, they are not causally related. We show using Cre/loxP technology that elevated iNOS levels and neutrophil number in the infected lung result from separate, AhRdependent signaling in endothelial and respiratory epithelial cells, respectively. Studies using mutant mice further reveal that AhR-mediated alterations in these innate responses to infection require a functional nuclear localization signal and DNA binding domain. Thus, gene targets of AhR in non-hematopoietic cells are important new considerations for understanding AhRmediated changes in innate anti-viral immunity.

Riboflavin (vitamin B2) is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is therefore required by all flavoproteins. Riboflavin also works as an antioxidant by scavenging free radicals. The present study was designed to evaluate the effects of riboflavin against acute lungs injury induced by the administration of a single intranasal dose (20 μg/rat) of lipopolysaccharides (LPS) in experimental rats. Administration of LPS resulted in marked increase in malondialdehyde (MDA) level (p &lt; 0.01) and MPO activity (p &lt; 0.001), whereas marked decrease in glutathione (GSH) content (p &lt; 0.001), glutathione reductase (GR) (p &lt; 0.001) and glutathione peroxidase (p &lt; 0.01) activity. These changes were significantly…

The incidence and the mortality of septic acute kidney injury are high, partly because the pathogenesis of sepsis-induced renal dysfunction is not clear. The objective of this study was to investigate the upregulation of renal inducible nitric oxide synthase (iNOS) in human endotoxemia and sepsis and the effect of NO on tubular integrity. Septic patients and endotoxemia that was induced by a bolus injection of 2 ng/kg Escherichia coli LPS in human volunteers were studied. In addition, the effect of co-administration of the selective iNOS inhibitor aminoguanidine was evaluated. The urinary excretion of the cytosolic glutathione-S-transferase-A1 (GSTA1-1) and GSTP1-1, markers for proximal and distal tubule damage, respectively, was determined. In septic patients, an almost 40-fold induction of iNOS mRNA in cells that were isolated from urine was found accompanied by a significant increase in NO metabolites in blood. The mRNA expression of iNOS was induced 34-fold after endotoxin administration. LPS-treated healthy volunteers showed a higher urinary excretion of NO metabolites compared with control subjects. Urinary NO metabolite excretion correlated with urinary GSTA1-1 excretion, indicating proximal tubule damage, whereas no distal tubular damage was observed. Co-administration of aminoguanidine reduced the upregulation of iNOS mRNA, urinary NO metabolite, and GSTA1-1 excretion, indicating that upregulation of iNOS and subsequent NO production may be responsible for renal proximal tubule damage observed.

, for their proliferation and invasion capability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype remains elusive. Here we report that glioma-induced microglia conversion is coupled to a reduction of basal microglial caspase 3 activity, increased Snitrosylation of mitochondria-associated caspase 3 through inhibition of thioredoxin 2 (Trx2) activity, and demonstrate that caspase 3 inhibition regulates microglial tumor-supporting function. Further, we identified nitric oxide synthase 2 (NOS2) activity originating from the glioma cells as a driving stimulus in the control of microglial caspase 3 activity. Repression of glioma NOS2 expression in vivo led to reduction in both microglia recruitment and tumor expansion, whereas depletion of the microglial caspase 3 gene promoted tumor growth. This study provides evidence that the inhibition of Trx2-mediated denitrosylation of SNO-procaspase 3 is part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells. Microglia are necessary for brain development and to maintain normal brain physiology. However, when brain homeostasis is perturbed, microglia react and execute immune functions. In the context of diseases, activation of microglia can contribute to rather contrasting effects; promoting neuronal cell death in the case of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, but promoting cell growth and invasion in the case of glioma 1, 2 . In fact, microglia are attracted toward gliomas in large numbers and microglia density in gliomas positively correlates with malignancy, invasiveness and grading of tumors. Tumor cells shut down the inflammatory properties of microglia and modulate them to exert tumor-trophic functions. Microglia release several factors, including extracellular matrix proteases and cytokines, which in turn directly or indirectly influence tumor invasiveness and growth 1, 2 . As further evidence of

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves&#39; disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression...

Background. Interferon γ (IFN-γ) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-γ downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-γ and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2 -/-mice were used to identify the contribution of signaling molecules during IFN-γ-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-γ -/ , and Nos2 -/-) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-γ repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase ( p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-γ and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-γ and NOS2 enhanced serum amounts of tumor necrosis factor α and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.

Background-Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66 shc protein controls cellular responses to oxidative stress. Mice lacking p66 shc (p66 shcϪ/Ϫ ) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. Methods and Results-Aortic rings from young and old p66 shcϪ/Ϫ or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O 2 Ϫ ) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66 shcϪ/Ϫ mice. Accordingly, an age-related decline of NO release was found in WT but not in p66 shcϪ/Ϫ mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66 shcϪ/Ϫ mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O 2 Ϫ production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66 shcϪ/Ϫ mice. Conclusions-We report that inactivation of the p66 shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66 shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging. (Circulation. 2004;110:2889-2895.) Key Words: aging Ⅲ endothelium Ⅲ free radicals Ⅲ nitric oxide Ⅲ genes S hc proteins are adaptor proteins that exist in 3 different isoforms with relative molecular masses of 46, 52, and 66 kDa. P52 shc /p46 shc is involved in the transmission of mitogenic signals from tyrosine kinases to Ras. 1 p66 shc has the same modular structure of p52 shc /p46 shc (SH2-CH1-PTB) and contains a unique N-terminal region (CH2); however, it is not involved in Ras regulation but rather functions in the intracellular pathway that converts oxidative signals into apoptosis. Indeed, embryo fibroblasts from mice carrying a targeted mutation of p66 shc (p66 shcϪ/Ϫ ) are more resistant to oxidative stress-induced apoptosis. 2 p66 shcϪ/Ϫ mice have an approximately 30% increase in life span and reduced early atherogenesis after long-term consumption of a high-fat diet, 3 suggesting that p66 shc is implicated in aging and in the pathogenesis of aging-associated diseases in mammals. The biochemical function of p66 shc remains, however, largely

Nitric oxide (NO) has been implicated in migraine pathogenesis based on the delayed development of typical migraine headache 4-6 h after infusing the NO donor nitroglycerin [glyceryl trinitrate (GTN)] to migraineurs. Furthermore, inhibiting the synthesis of NO by treatment with a NO synthase (NOS) inhibitor attenuates spontaneous migraine headaches in 67% of subjects. Because NO has been linked to inflammation and cytokine expression, we investigated the delayed consequences of brief GTN infusion (30 min) on the development of meningeal inflammation in a rat model using doses relevant to the human model. We found dose-dependent Type II NOS [inducible NOS (iNOS)] mRNA upregulation in dura mater beginning at 2 h and an increase in the corresponding protein expression at 4, 6 and 10 h after infusion. Type II NOS immunoreactivity was expressed chiefly within resident meningeal macrophages. Consistent with development of a delayed inflammatory response, we detected induction of interleukin 1β in dura mater at 2 and 6 h and increased interleukin 6 in dural

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models (Mol Pharmacol 69: 328 -337, 2006). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH-and time-dependent manner, whereas it acts only as a reversible L-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOSoverexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, This work was supported by ALTANA Pharma AG.

L-Arginine itself and its metabolite-nitric oxide play great roles in intestinal physiology. However, the molecular mechanism underlying nitric oxide pathway regulating L-Arginine transport and cell growth is not yet fully understood. We report that inhibition of nitric oxide synthase (NOS) significantly induced cell apoptosis (p &lt; 0.05), and promoted the rate of Arginine uptake and the expressions of protein for CAT-2 and y+LAT-1 (p &lt; 0.05), while reduced protein expression of CAT-1. And NOS inhibition markedly decreased the activation of mammalian target of rapamycin (mTOR) and PI3K-Akt pathways by Arginine in the IPEC-1 cells (p &lt; 0.05). Taken together, these data suggest that inhibition of NO pathway by L-NAME induces a negative feedback increasing of Arginine uptake and CAT-2 and y+LAT-1 protein expression, but promotes cell apoptosis which involved inhibiting the activation of mTOR and PI3K-Akt pathways.

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to exert anti-inflammatory activities in macrophages by competition for transcriptional coactivators with some transcriptional factors, including NF-κB. In the present study the influence of PPARγ activators on IFN-γ-elicited macrophage stimulation and signaling cascades was investigated. The results show that IFN-γ-induced inducible NO synthase (iNOS) gene transcription, iNOS protein induction, and NO production are more sensitive to inhibition by 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) than by the other two PPARγ agonists, GW1929 and ciglitazone. Delayed addition of 15dPGJ2 for 2 h resulted in reduced inhibition, suggesting action by 15dPGJ2 on the upstream signaling cascades. Immunoblotting, DNA binding, and reporter gene assays consistently revealed the inhibitory ability of 15dPGJ2, but not GW1929 or ciglitazone, on IFN-γ-elicited signaling cascades, including tyrosine phosphorylation of Janus ty...

We investigated the effect of brown ring disease (BRD)(1) development and algal diet on energy reserves and activity of enzymes related to energy metabolism, antioxidant system and immunity in Manila clam, Ruditapes philippinarum. We found that algal diet did not impact the metabolic response of clams exposed to Vibrio tapetis. At two days post-injection (dpi), activities of superoxide dismutase and glutathione peroxidase (GPx) decreased whereas activities of nitric oxide synthase (iNOS) and catalase increased in infected clams, although no clinical signs were visible (BRD-). At 7 dpi, activities of several antioxidant and immune-related enzymes were markedly increased in BRD-likely indicating an efficient reactive oxygen species (ROS) scavenging compared to animals which developed clinical signs of BRD (BRD+). Therefore, resistance to BRD clinical signs appearance was associated with higher detoxification of ROS and enhancement of immune response. This study provides new biochemica...

Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) [Ang-(1-7)] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(1-7)/Mas axis in NO generation and Ca 2+ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca 2+ imaging, and confocal microscopy. Acute Ang-(1-7) treatment (10 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(1-7)–dependent NO raise was abolished by pretreatment with A-779 (1 μmol/L). To confirm that Ang-(1-7) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(1-7) failed to increase NO levels. Moreover, Mas -ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthas...

Background: Carbamazepine, which was developed primarily for the treatment of epilepsy, is now also useful for the treatment of non-epileptic disorders and inflammatory hyperalgesia. However, the mechanism of its anti-neuroinflammatory action remains poorly understood. This study elucidates the anti-neuroinflammatory capacity of carbamazepine on microglial activation and the relative mechanisms involved. The microglial BV-2 cells were pretreated with carbamazepine for 15 min before activation by lipopolysaccharide (LPS). After LPS stimulation, the expression of inducible nitric oxide synthase (iNOS) was analyzed by Western blotting (WB) and reverse transcription-polymerase chain reaction. Signaling proteins and cyclooxygenase (COX)-2 were also evaluated by WB. The levels of nitrate and tumor necrosis factor (TNF)-α were analyzed by the Griess method and enzyme-linked immunosorbant assay, respectively. The formation of intracellular reactive oxygen species (ROS) was examined by fluor...

Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AH...

List of non-standard abbreviations (6)-[ 125 I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl ([ 125 I]DOI; G-protein coupled receptor (GPCR); G-protein coupled receptor kinase (GRK); human herpes virus 8 (HHV8); mitogen-activated protein kinase (MAPK); Multi-PDZ domain containing protein 1 (MUPP1); nuclear factor kappa B (NFkappa B); nitric oxide synthase (NOS); PSD-95, Dlg, ZO-1 (PDZ); serum response element (SRE) MOL#8268

Chlorella possesses various remarkable biological activities. One component, Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe ( Chlorella-11 peptide) was found to be able to suppress LPS-induced NO production and inflammation. However, the molecular mechanism behind these findings and the consistency between in vitro and in vivo data have not been investigated. LPS-activated RAW 264.7 macrophages were used to study in vitro molecular anti-inflammatory effects of Chlorella-11 peptide. After activation, NO production and the expression of iNOS and NF-κB proteins as well as iNOS mRNA were measured using Griess colorimetric assay, Western blotting and RT-PCR, respectively. Alterations in PGE2 and TNF-α contents were also monitored by ELISA. For in vivo studies, thermal injury Wistar rats were used and inflammatory indications e.g. serum malondialdehyde (MDA), TNF-α levels and skin erythema were evaluated 48 h after injury implementation. In vitro results showed that Chlorella-11 peptide prod...

Ischemic diseases such as stroke and proliferative retinopathy are characterized by hypoxia-driven release of angiogenic factors such as vascular endothelial growth factor (VEGF). However, revascularization of the ischemic areas is inadequate resulting in impaired neuro-vascular function. We aim to examine the vascular protective effects of candesartan, an angiotensin receptor blocker, in an ischemic retinopathy mouse model. Vascular density, number of tip cells, and perfusions of capillaries were assessed. Activation of Muller glial cells and levels of peroxynitrite, VEGF, VEGFR2, inducible-nitric oxide synthase, hemoxygenase-1 (HO-1) were assessed. Proangiogenic effects of candesartan were examined in human endothelial cells (EC) that were cultured in normoxia or hypoxia and transduced with siRNA against HO-1. Candesartan (1mg/kg) and (10mg/kg) decreased hypoxia-induced neovascularization by 67% and 70%, respectively. Candesartan (10mg/kg) significantly stimulated the number of tip cells and physiological revascularization of the central retina (45%) compared to untreated pups. The effects of candesartan coincided with reduction of hypoxia-induced Muller glial activation, iNOS expression and restoration of HO-1 expression with no significant change in VEGF levels. In vitro, silencing HO-1 expression blunted the ability of candesartan to induce VEGF expression under normoxia and VEGFR2 activation and angiogenic response under both normoxia and hypoxia. These findings suggest that candesartan improved reparative angiogenesis and hence prevented pathological angiogenesis by modulating HO-1 and iNOS levels in ischemic

Antioxidants have valuable effects on the process of spermatogenesis, particularly with diabetes mellitus (DM). Therefore, the present study investigated the impact and the intracellular mechanisms by which thymoquinone (TQ) works against diabetes-induced testicular deteriorations in rats. Wistar male rats (n = 60) were randomly allocated into four groups; Control, Diabetic (streptozotocin (STZ)-treated rats where diabetes was induced by intraperitoneal injection of STZ, 65 mg/kg), Diabetic + TQ (diabetic rats treated with TQ (50 mg/kg) orally once daily), and TQ (non-diabetic rats treated with TQ) for 12 weeks. Results revealed that TQ significantly improved the sperm parameters with a reduction in nitric oxide (NO) and malondialdehyde (MDA) levels in testicular tissue. Also, it increased testicular reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity. Interestingly, TQ induced downregulation of testicular inducible nitric oxide synthase (iNOS) and nuclear factor kappa-B (NF-κB) and significantly upregulated the aromatase protein expression levels in testicles in comparison with the diabetic rats. In conclusion, TQ treatment exerted a protective effect against reproductive dysfunction induced by diabetes not only through its powerful antioxidant and hypoglycemic effects but also through its downregulation of testicular iNOS and NF-κB along with upregulation of aromatase expression levels in diabetic rats.

The aim of this in vitro study was to examine the possible effect of [2-methyl-5-(4methylsulphonyl)phenyl-1-phenyl-3-(2-n-propyloxyethyl)]-1H-pyrrole (VA441), a new selective cyclooxygenase (COX)-2 inhibitor, on human osteoarthritic (OA) chondrocyte cultivated in the presence or absence of interleukin-1β (IL-1β). In particular, we assessed the effects of 1 and 10 μM of VA441, celecoxib, and indomethacin on cell viability, COX-2 and inducible nitric oxide synthase (iNOS) gene expression, prostaglandin E 2 (PGE 2 ) production, and nitric oxide (NO) and metalloproteinase-3 (MMP-3) release. Furthermore, we carried out morphological assessment by transmission electron microscopy (TEM). The presence of IL-1β led to a significant increase in PGE 2 , MMP-3, and NO production, as well as a significant increase in gene expression of COX-2 and iNOS. All the drugs tested had a statistically significant inhibitory effect on PGE 2 production and gene expression of COX-2 stimulated by IL-1β. VA441 and celecoxib significantly suppressed IL-1β-stimulated MMP-3 and NO and iNOS gene expression in a dose-dependent manner, while indomethacin did not show any significant effect on MMP-3 and NO production or on iNOS gene expression. TEM demonstrated that IL-1β severely alters the structure of chondrocytes; after coincubation with VA441 or celecoxib, the cells recovered their ultrastructure. Our data suggest that VA441 and celecoxib may have a beneficial effect on chondrocyte metabolism.

Purpose: To determine the influence of tanshinone on the levels of nitric oxide synthase (NOS) and acetylcholinesterase (AChE) in the brain of an Altzheimer Disease (AD) rat model and on its potential therapeutic mechanism. Methods: 100 Male Sprague Dawley rats were divided into three groups: control group, model group and tanshinone treatment group. 10µg A?1-42 was injected bilaterally into the dorsal lateral region of the dentate gyrus in the hippocampus of rats in the model and tanshinone treatment groups to prepare the AD models. 24h after modeling, tanshinone, 50mg/kg, was administered by gastric perfusion to rats in the tanshinone treatment group. Later, immunohistochemical assay and Western blot analysis were used to detect expression of neuronal NOS (nNOS) and inducible NOS (iNOS) in the rat hippocampus. Activity of AChE in each subregion (CA1~CA4) of rats’ hippocampus was determined by a histochemical technique. Results: Expression of nNOS in the model group was down-regula...

The importance of protein-small molecule interaction in drug discovery, medicinal chemistry and biology has driven the development of new analytical methods to disclose the whole interactome of bioactive compounds. To accelerate targets discovery of N-formyl-7-amino-11cycloamphilectene (CALe), a marine bioactive diterpene isolated from the Vanuatu sponge Axinella sp., a chemoproteomic-based approach has been successfully developed. CALe is a potent anti-inflammatory agent, modulating NO and prostaglandin E2 overproduction by dual inhibition of the enhanced inducible NO synthase expression and cyclo-oxygenase-2 activity, without any evidence of cytotoxic effects. In this paper, several isoforms of tubulin have been identified as CALe off-targets by chemical proteomics combined with bio-physical orthogonal approaches. In the following biological analysis of its cellular effect, CALe was found to protect microtubules against the colcemid depolymerizing effect. † Electronic supplementary information (ESI) available: HPLC run of CALe-NH 2 bearing linker, MS and MSMS analysis of the covalent adduct between CALe-NH 2 and dithiobis-succinimidylpropionate, HPLC runs of CALe-NH 2 bearing linker immobilization on amine beads, electrophoretic profile of proteins eluted from CALe and control beads, proteins identified from control experiments, proteins sequence coverage of the selected CALe interactors, Mascot report and peak lists for Hsp90β and tubulin, analysis of CALe and Hsp90β affinity by surface plasmon resonance and cell viability assay. See

The present study has demonstrated the translocation of zinc ferrite nanoparticles (ZnFe 2 O 4 -NPs) into the cytoplasm of human amnion epithelial (WISH) cells, and the ensuing cytotoxicity and genetic damage. The results suggested that in situ NPs induced oxidative stress, alterations in cellular membrane and DNA strand breaks. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and neutral red uptake (NRU) cytotoxicity assays indicated 64.48 ± 1.6% and 50.73 ± 2.1% reduction in cell viability with 100 μg/ml of ZnFe 2 O 4 -NPs exposure. The treated WISH cells exhibited 1.2-fold higher ROS level with 0.9-fold decline in membrane potential (ΔΨm) and 7.4-fold higher DNA damage after 48 h of ZnFe 2 O 4 -NPs treatment. Real-time PCR (qPCR) analysis of p53, CASP 3 (caspase-3), and bax genes revealed 5.3, 1.6, and 14.9-fold upregulation, and 0.18-fold down regulation of bcl 2 gene vis-à-vis untreated control. RT 2 Profiler™ PCR array data elucidated differential up-regulation of mRNA transcripts of IL-1b, NFKB1, NOS2 and CCL21 genes in the range of 1.5 to 3.7folds. The flow cytometry based cell cycle analysis suggested the transfer of 15.2 ± 2.1% (p b 0.01) population of ZnFe 2 O 4 -NPs (100 μg/ml) treated cells into apoptotic phase through intrinsic pathway. Over all, the data revealed the potential of ZnFe 2 O 4 -NPs to induce cellular and genetic toxicity in cells of placental origin. Thus, the significant ROS production, reduction in ΔΨm, DNA damage, and activation of genes linked to inflammation, oxidative stress, proliferation, DNA damage and repair could serve as the predictive toxicity and stress markers for ecotoxicological assessment of ZnFe 2 O 4 -NPs induced cellular and genetic damage.

Celiac disease, induced by dietary gluten, is characterized by mucosal atrophy and local inflammation associated with cell infiltration and activation. Unlike other food proteins, gluten and its proteolytic fragments, besides inducing a specific immune response, were shown to activate components of innate immunity and cause, e.g., direct stimulation of TNF-α and IL-10 and a significant rise in NO production by peritoneal macrophages. The identity of the active fragments was established by separating the peptic digest of gliadin by RP-HPLC chromatography. The purest fraction with the highest activity was analyzed by mass spectrometry, and the gliadin peptide sequence was identified as VSFQQPQQQYPSSQ. This peptide (T) and its N- and C-terminally shortened forms (A, B, C and D, E, F) were synthesized. Peptide B (FQQPQQQYPSSQ) elicited the highest TNF-α, IL-10, and RANTES secretion and increase in IFN-γ-primed NO production by mouse macrophages. In contrast, C-terminally shortened pepti...

The aim of this prospective study was to investigate the diurnal change in serum nitric oxide (NO) levels in active and remission phases of patients with panic disorder. This study included 15 patients fulfilling the criteria for panic disorder of Diagnostic and Statistical Manual of Mental Disorders -Fourth Edition and 15 healthy controls matched for age and sex. All patients were receiving a selective serotonin reuptake inhibitor at therapeutic doses. The serum nitrite and nitrate levels of subjects were determined at 10 : 00 a.m. after overnight fasting and at 3 : 00 p.m. 2 hours after lunch. NO levels of all patients measured in the morning were significantly higher than those of controls. The patients were also divided into active and remission groups according to clinical status and Panic Agoraphobia Scale's cut-off point. There were no statistically significant differences in serum nitrite and nitrate levels of the active group between the 10 : 00 a.m. and 3 : 00 p.m. measurements. In contrast, statistically significant differences were found in the serum levels of nitrite (p<0.05) and nitrate (p<0.05) in the remission group. Notably, the afternoon nitrite and nitrate levels of the remission group were higher than those of the morning levels as seen in control subjects. Thus, diurnal variation of NO production is altered in patients with panic disorder but is resumed in the remission phase. The present study suggests that serum NO levels are a good marker for evaluation of panic disorder. ──── oxidative stress; nitric oxide; diurnality; panic disorder; SSRI

Endothelial nitric oxide synthase (eNOS) plays an important role in control of vascular tone and angiogenesis among other functions. Its regulation is complex and has not been fully established. Several studies have emphasized the importance of phosphorylation in the regulation of eNOS activity. Although it is commonly accepted that protein kinase C (PKC) signaling inhibits eNOS activity by phosphorylating Thr497and dephosphorylating Ser1179, the distinct role of different PKC isoforms has not been studied so far. The PKC family comprises roughly 12 different isozymes that activate distinct downstream pathways. The present study was designed to investigate the role of PKCα isoform in regulation of eNOS activity. Overexpression of PKCα in primary endothelial cells was associated with increased eNOS-Ser1179phosphorylation and increased NO production. Inhibition of PKCα activity either by siRNA transfection or by overexpression of a dominant negative mutant resulted in a marked decreas...

Background/aim-Age-related metabolic diseases are often associated with low-grade inflammation. The aim of the present study was to investigate the role of the transcriptional coactivator PGC-1α in the potential beneficial effects of exercise training and/or resveratrol in the prevention of age-associated low-grade inflammation. To address this, a long-term voluntary exercise training and resveratrol supplementation study was conducted. Experimental setup-Three month old whole body PGC-1α KO and WT mice were randomly assigned to four groups: untrained chow-fed, untrained chow-fed supplemented with resveratrol, chow-fed voluntarily exercise trained and chow-fed supplemented with resveratrol and voluntarily exercise trained. The intervention lasted 12 months and three month old untrained chow-fed mice served as young controls. Results-Voluntary exercise training prevented an age-associated increase (p<0.05) in systemic IL-6 and adiposity in WT mice. PGC-1α expression was required for a training-induced prevention of an age-associated increase (p<0.05) in skeletal muscle TNFα protein. Independently of PGC-1α, both exercise training and resveratrol prevented an age-associated increase (p<0.05) in skeletal muscle protein carbonylation. The present findings highlight that exercise training is a more effective intervention than resveratrol supplementation in reducing age-associated inflammation and that PGC-1α in part is required for the exercise training-induced anti-inflammatory effects.

Although nuclear factor (NF)-κB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-κB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-κB responsive genes. By sequence comparison with the IFN-β and IL-8 promoters, we identified an NRE in the hiNOS promoter located at −6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the −6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an...

Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3′-untranslated region (3′-UTR) revealed five putative miR-939 binding sites. The hiNOS 3′-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3′-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutag...

Two different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (TGF)-b and interleukin (IL)-10. Finally, in-vitro treatment of macrophages with anti-LPG and/or anti-TLR-2 antibodies before infection reduces the number of amastigotes in macrophages and co-treatment of mice with anti-TLR-2 antibodies and cytosinephosphate-guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)-gpredominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR-2 reduce anti-leishmanial responses via cytokine-mediated decrease of TLR-9 expression.

Momordica charantia is used to treat various diseases, including inflammatory conditions. Previous reports indicated that the extract of this plant inhibits activation of nuclear transcription factor-jB (NF-jB) but activates peroxisome proliferator-activated receptor (PPAR). Additionally, cucurbitane-type triterpene glycosides are the main bioactive components of the fruit of M. charantia. Therefore, we investigated the anti-inflammatory activity of 17 cucurbitane-type triterpene glycosides (1-17) isolated from this plant. Their inhibition of NF-jB and activation of PPAR activities in HepG2 cells were measured using luciferase reporter and PPAR subtype transactivation assays. Compounds 6 and 8 were found to inhibit NF-jB activation stimulated by tumor necrosis factor-a (TNFa) in a dose-dependent manner. With 50% inhibition concentration (IC50) values of 0.4 lM, compounds 6 and 8 were more potent inhibitors than the positive control, sulfasalazine (IC50 = 0.9 lM). Compounds 4, 6, and 8 also inhibited TNFa-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA. However, only compound 13 significantly increased PPARc transactivation.

ABSTRACT. This study demonstrates that intraperitoneal injections of DHA (all cis 4,7,10,13,16,19 docosahexaenoic acid C22: n-3) bound to bovine serum albumin ameliorate murine acute renal failure (ARF) induced by temporary occlusion of the renal artery. Three micromoles of DHA decreased serum creatinine (Scr) from 2.3 mg/dl to 1.1 mg/dl 24 h after reperfusion (n = 15; P &lt; 0.05). Scr of the treated animals were significantly lower than controls throughout a 7-d time course. Although lower doses of DHA were less effective, higher doses were not more effective. Ribonuclease (RNase) protection assays showed that ischemia increased mRNA abundance for TNF-α and inducible nitric oxide synthase (iNOS) at 24 h. This increase was prevented by DHA administration. Because TNF-α and iNOS contribute to renal ischemic injury, their inhibition may contribute to DHA’s salutary effect. In addition, the data may have therapeutic implications, because the DHA improves ARF even when administered at ...

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days. a Scoring for cytokine staining was as follows: 0, negative; 1, Ͻ10% of cells staining positive; 2, 10 to 30% of cells staining positive; 3, 50 to 80% of cells staining positive; and 4, 80 to 100% of cells staining positive. Scoring for cellular infiltrate was as follows: 0, no cellular infiltrate; 1, small granulomas; 2, medium-sized granulomas; and 3, large granulomas. b BL-RR, borderline lepromatous leprosy in reversal reaction; BT-RR, borderline tuberculoid leprosy in reversal reaction; MDT, multidrug therapy.

The activity and protein expression of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) were investigated during the development of hypertension in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto rats (WKY) were studied at three different ages: 4, 14 to 17, and 63 weeks of age. After treatment with saline or lipopolysaccharide (LPS, 10 mg/kg IV) for 3 hours, the aortas were removed for measurement of NOS activity and protein expression assay by [ 3 H]-L-citrulline formation method and Western blot analysis, respectively. Plasma levels of nitrite/nitrate (NO 2 Ϫ /NO 3 Ϫ ) and tumor necrosis factor-␣ (TNF-␣) were also determined. At 14 to 17 weeks and 63 weeks, the basal activity and protein expression of eNOS in the aortas were significantly lower in SHR than in WKY. In addition, the aged WKY exhibited lower eNOS activity than that of adult WKY, but this change was not seen in SHR. By comparison, the basal activity and protein expression of iNOS were only observed in SHR of the 14-to-17-week group and in the 63-week group; SHR still exhibited higher activities, and these differences were further exaggerated by treatment with LPS. The basal and LPS-induced NO 2 Ϫ /NO 3 Ϫ and TNF-␣ levels in the plasma were also higher in the SHR except the 4-week group. After treatment with quinapril, the basal and LPS-induced expressions of iNOS in SHR were significantly attenuated. Our results demonstrated that alterations of activity and protein expression of eNOS and iNOS occurred in SHR. In addition, aging may reduce the activity of eNOS in WKY but not in SHR. The decline of eNOS activity and/or expression may contribute to the development of hypertension, whereas the increase of iNOS expression may be a consequence of the pathological state of vessels associated with hypertension in SHR. However, the augmented expression of iNOS in SHR was attenuated by antihypertensive therapy, suggesting that the abnormal expression of iNOS is associated with hypertension.

The appearance of excessive inflammatory activity is associated with onset of many disease states. Such non-productive responses are often the basis of the mortality consequent to incurring numerous disorders. The current outbreak of coronavirus disease 2019 caused by the virus "severe acute respiratory syndrome coronavirus 2" is a striking reflection of the inadequacy of current medical science to adequately address this issue. The usefulness of a range of materials of botanical origin in the attenuation of both chronic and acute inflammatory responses to various disease stressors is described. The properties of preparations of plant-based origin often parallel those of synthesized pharmacologics, but differ from them in some key respects. These differences can lead to more traditional preparations having distinct therapeutic advantages but also a number of specific shortcomings. The strengths and weaknesses of these materials are objectively contrasted with that of a more orthodox pharmacological approach. Each of these emphases in style has specific advantages and they should not be considered as competitors, but rather as accomplices in combating adverse states involving derangement of immune function.