Search

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.

Background: Hyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early pro-atherosclerotic events., Methods and Findings: In the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1beta (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-kappaB (NF-kappaB) elicited by IL-1beta, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-kappaB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1beta was co-administered., Conclusions: These results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia.

The SIRPa-CD47 axis plays an important role in T cell recruitment to sites of immune reaction and inflammation but its role in T cell antigen priming is incompletely understood. Employing OTII TCR transgenic mice bred to Cd47-/- (Cd47KO) or SKI mice, a knock-in transgenic animal expressing non-signaling cytoplasmic-truncated SIRPa, we investigated how the SIRPa-CD47 axis contributes to antigen priming. Here we show that adoptive transfer of Cd47KO or SKI Ova-specific CD4+ T cells (OTII) into Cd47KO and SKI recipients, followed by Ova immunization, elicited reduced T cell division and proliferation indices, increased apoptosis, and reduced expansion compared to transfer into WT mice. We confirmed prior reports that splenic T cell zone, CD4+ conventional dendritic cells (cDCs) and CD4+ T cell numbers were reduced in Cd47KO and SKI mice. We report that in vitro derived DCs from Cd47KO and SKI mice exhibited impaired migration in vivo and exhibited reduced CD11c+ DC proximity to OTII T cells in T cell zones after Ag immunization, which correlates with reduced TCR activation in transferred OTII T cells. These findings suggest that reduced numbers of CD4+ cDCs and their impaired migration contributes to reduced T cell-DC proximity in splenic T cell zone and reduced T cell TCR activation, cell division and proliferation, and indirectly increased T cell apoptosis. [ABSTRACT FROM AUTHOR]

Resolvin D1, a specialized pro-resolving lipid mediator produced from docosahexaenoic acid by 15- and 5-lipoxygenase, exerts anti-inflammatory effects driving to the resolution of inflammation. The present study aimed to elucidate its role in small intestinal damage induced by nonsteroidal anti-inflammatory drug (NSAID). Indomethacin was administered orally to C57BL/6J male mice, which were sacrificed 24 h later to collect small intestine specimens. Before administration of indomethacin, mice were subjected to intraperitoneal treatment with resolvin D1 or oral administration of baicalein, a 15-lipoxygenase inhibitor. Small intestinal damage induced by indomethacin was attenuated by pretreatment with resolvin D1. Furthermore, resolvin D1 reduced the gene expression levels of interleukin-1β, tumor necrosis factor-α, and CXCL1/keratinocyte chemoattractant. Conversely, the inhibition of 15-lipoxygenase activity by baicalein increased the expression of genes coding for these inflammatory cytokines and chemokine, leading to exacerbated small intestinal damage, and reduced the concentration of resolvin D1 in the small intestinal tissue. Exogenous treatment with resolvin D1 negated the deleterious effect of baicalein. 15-lipoxygenase was mainly expressed in the epithelium and inflammatory cells of the small intestine, and its gene and protein expression was not affected by the administration of indomethacin. Inhibition of the resolvin D1 receptor, lipoxin A4 receptor /formyl peptide receptor 2, by its specific inhibitors Boc-1 and WRW4 aggravated indomethacin-induced small intestinal damage. Collectively, these results indicate that resolvin D1 produced by 15-lipoxygenase contributes to mucoprotection against NSAID-induced small intestinal damage through its anti-inflammatory effect. [ABSTRACT FROM AUTHOR]

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow. [ABSTRACT FROM AUTHOR]

Macrophage cells form part of our first line defense against pathogens. Macrophages become activated by microbial products such as lipopolysaccharide (LPS) to produce inflammatory mediators, such as TNFα and other cytokines, which orchestrate the host defense against the pathogen. Once the pathogen has been eradicated, the activated macrophage must be appropriately deactivated or inflammatory diseases result. Interleukin-10 (IL10) is a key anti-inflammatory cytokine which deactivates the activated macrophage. The IL10 receptor (IL10R) signals through the Jak1/Tyk2 tyrosine kinases, STAT3 transcription factor and the SHIP1 inositol phosphatase. However, IL10 has also been described to induce the activation of the cyclic adenosine monophosphate (cAMP) regulated protein kinase A (PKA). We now report that IL10R signalling leads to STAT3/SHIP1 dependent expression of the EP4 receptor for prostaglandin E2 (PGE2). In macrophages, EP4 is a Gαs-protein coupled receptor that stimulates adenylate cyclase (AC) production of cAMP, leading to downstream activation of protein kinase A (PKA) and phosphorylation of the CREB transcription factor. IL10 induction of phospho-CREB and inhibition of LPS-induced phosphorylation of p85 PI3K and p70 S6 kinase required the presence of EP4. These data suggest that IL10R activation of STAT3/SHIP1 enhances EP4 expression, and that it is EP4 which activates cAMP-dependent signalling. The coordination between IL10R and EP4 signalling also provides an explanation for why cAMP elevating agents synergize with IL10 to elicit anti-inflammatory responses. [ABSTRACT FROM AUTHOR]

HIV-1 infection is characterized by generalized deregulation of the immune system, resulting in increased chronic immune activation. However, some individuals called HIV controllers (HICs) present spontaneous control of viral replication and have a more preserved immune system. Among HICs, discordant results have been observed regarding immune activation and the frequency of different T cell subsets, including Treg and Th17 cells. We evaluated T cell immune activation, differentiation and regulatory profiles in two groups of HICs—elite controllers (ECs) and viremic controllers (VCs)—and compared them to those of cART-treated individuals (cART) and HIV-1-negative (HIV-neg) individuals. ECs demonstrated similar levels of activated CD4+ and CD8+ T cells in comparison to HIV-neg, while cART and VCs showed elevated T cell activation. CD4+ T cell subset analyses showed differences only for transitional memory T cell frequency between the EC and HIV-neg groups. However, VC individuals showed higher frequencies of terminally differentiated, naïve, and stem cell memory T cells and lower frequencies of transitional memory and central memory T cells compared to the HIV-neg group. Among CD8+ T cell subsets, ECs presented higher frequencies of stem cell memory T cells, while VCs presented higher frequencies of terminally differentiated T cells compared to the HIV-neg group. HICs showed lower frequencies of total Treg cells compared to the HIV-neg and cART groups. ECs also presented higher frequencies of activated and a lower frequency of resting Treg cells than the HIV-neg and cART groups. Furthermore, we observed a high frequency of Th17 cells in ECs and high Th17/Treg ratios in both HIC groups. Our data showed that ECs had low levels of activated T cells and a high frequency of activated Treg and Th17 cells, which could restrict chronic immune activation and be indicative of a preserved mucosal response in these individuals. [ABSTRACT FROM AUTHOR]

Paneth cells (PCs) are specialized epithelial cells of the small bowel that contain multiple secretory granules filled with antimicrobial peptides and trophic factors, which are essential for the control of the microorganisms growth and maintaining intestinal integrity. Alterations in their function are associated with an imbalance of the normal microbiota, gastrointestinal infections and inflammatory processes, such as Crohn’s disease (CD). One of the most common murine models for studying CD is IL-10-/- mouse. IL-10-/- mice when housed in conventional conditions and take contact with commensal microorganisms develop an acute enterocolitis mediated by a Th1 immune response. Even though, alterations in PCs function are related to CD, they had not been characterized yet in this mouse model. Here we show that in specific pathogen free conditions IL-10-/- mice have aberrant granules and a large number of immature PCs at the bottom of the crypt in the ileum of IL-10-/- mice before developing intestinal inflammation, along with a reduced expression of Indian Hedgehog. In addition, IL-10-/- Paneth cells presented a reduced expression of cryptidin-4, and a heterogeneous distribution of lysozyme+ granules. The alterations in the maturation of the PCs at the bottom of the crypt were not modified after the colonization by the conventional microbiota. On the other hand, depletion of microbiota altered the phenotype, but did not normalize PCs. Our results suggest that IL-10 could be necessary for the integrity of PCs. Moreover, our results help to explain why IL-10-/- mice develop enterocolitis in response to microorganisms. [ABSTRACT FROM AUTHOR]

Introduction: Studies on metabolomics and CKD have primarily addressed CKD incidence defined as a decline on eGFR or appearance of albuminuria in the general population, with very few evaluating hard outcomes. In the present study, we investigated the association between metabolites and mortality and ESRD in a CKD cohort. Setting and methods: Data on 454 participants of the Progredir Cohort Study, Sao Paulo, Brazil were used. Metabolomics was performed by GC-MS (Agilent MassHunter) and metabolites were identified using Agilent Fiehn GC/MS and NIST libraries. After excluding metabolites present in <50% of participants, 293 metabolites were analyzed. An FDR q value <0.05 criteria was applied in Cox models on the composite outcome (mortality or incident renal replacement therapy) adjusted for batch effect, resulting in 34 metabolites associated with the outcome. Multivariable-adjusted Cox models were then built for the composite outcome, death, and ESRD incident events. Competing risk analysis was also performed for ESRD. Results: Mean age was 68±12y, mean eGFR-CKDEPI was 38.4±14.6 ml/min/1.73m2 and 57% were diabetic. After adjustments (GC-MS batch, sex, age, DM and eGFR), 18 metabolites remained significantly associated with the composite outcome. Nine metabolites were independently associated with death: D-malic acid (HR 1.84, 95%CI 1.32–2.56, p = 0.0003), acetohydroxamic acid (HR 1.90, 95%CI 1.30–2.78, p = 0.0008), butanoic acid (HR 1.59, 95%CI 1.17–2.15, p = 0.003), and docosahexaenoic acid (HR 0.58, 95%CI 0.39–0.88, p = 0.009), among the top associations. Lactose (SHR 1.49, 95%CI 1.04–2.12, p = 0.03), 2-O-glycerol-α-D-galactopyranoside (SHR 1.76, 95%CI 1.06–2.92, p = 0.03), and tyrosine (SHR 0.52, 95%CI 0.31–0.88, p = 0.02) were associated to ESRD risk, while D-threitol, mannitol and myo-inositol presented strong borderline associations. Conclusion: Our results identify specific metabolites related to hard outcomes in a CKD population. These findings point to the need of further exploration of these metabolites as biomarkers in CKD and the understanding of the underlying biological mechanisms related to the observed associations. [ABSTRACT FROM AUTHOR]

Chronic inflammation results when the immune system responds to trauma, injury or infection and the response is not resolved. It can lead to tissue damage and dysfunction and in some cases predispose to cancer. Some viruses (including Epstein-Barr virus (EBV)) can induce inflammation, which may persist even after the infection has been controlled or cleared. The damage caused by inflammation, can itself act to perpetuate the inflammatory response. The latent membrane protein 1 (LMP1) of EBV is a pro-inflammatory factor and in the skin of transgenic mice causes a phenotype of hyperplasia with chronic inflammation of increasing severity, which can progress to pre-malignant and malignant lesions. LMP1 signalling leads to persistent deregulated expression of multiple proteins throughout the mouse life span, including TGFα S100A9 and chitinase-like proteins. Additionally, as the inflammation increases, numerous chemokines and cytokines are produced which promulgate the inflammation. Deposition of IgM, IgG, IgA and IgE and complement activation form part of this process and through genetic deletion of CD40, we show that this contributes to the more tissue-destructive aspects of the phenotype. Treatment of the mice with N-acetylcysteine (NAC), an antioxidant which feeds into the body’s natural redox regulatory system through glutathione synthesis, resulted in a significantly reduced leukocyte infiltrate in the inflamed tissue, amelioration of the pathological features and delay in the inflammatory signature measured by in vivo imaging. Reducing the degree of inflammation achieved through NAC treatment, had the knock on effect of reducing leukocyte recruitment to the inflamed site, thereby slowing the progression of the pathology. These data support the idea that NAC could be considered as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. Additionally, the model described can be used to effectively monitor and accurately measure therapies for chronic inflammation. [ABSTRACT FROM AUTHOR]

Diabetes mellitus accelerates atherosclerosis that causes most cardiovascular events. Several metabolic pathways are considered to contribute to the development of atherosclerosis, but comprehensive metabolic alterations to atherosclerotic arterial cells remain unknown. The present study investigated metabolic changes and their relationship to vascular histopathological changes in the atherosclerotic arteries of rabbits with alloxan-induced diabetes. Diabetic atherosclerosis was induced in rabbit ilio-femoral arteries by injecting alloxan (100 mg/kg), injuring the arteries using a balloon, and feeding with a 0.5% cholesterol diet. We histologically assessed the atherosclerotic lesion development, cellular content, pimonidazole positive-hypoxic area, the nuclear localization of hypoxia-inducible factor-1α, and apoptosis. We evaluated comprehensive arterial metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose and pimonidazole. Plaque burden, macrophage content, and hypoxic areas were more prevalent in arteries with diabetic, than non-diabetic atherosclerosis. Metabolomic analyses highlighted 12 metabolites that were significantly altered between diabetic and non-diabetic atherosclerosis. A half of them were associated with glycolysis metabolites, and their levels were decreased in diabetic atherosclerosis. The uptake of glucose evaluated as 18F-fluorodeoxyglucose in atherosclerotic lesions increased according to increased macrophage content or hypoxic areas in non-diabetic, but not diabetic rabbits. Despite profound hypoxic areas, the nuclear localization of hypoxia-inducible factor-1α decreased and the number of apoptotic cells increased in diabetic atherosclerotic lesions. Altered glycolysis metabolism and an impaired response to hypoxia in atherosclerotic lesions under conditions of insulin-dependent diabetes might be involved in the development of diabetic atherosclerosis. [ABSTRACT FROM AUTHOR]

Objectives: Our aim was to investigate if genetic variations in the visfatin gene (SNPs rs7789066/ rs11977021/rs4730153) could modify the cardiovascular-risk (CV-risk) despite the metabolic phenotype (obesity and glucose tolerance). In addition, we investigated the relationship between insulin sensitivity and variations in visfatin gene. Material and Methods: A population-based study in rural and urban areas of the Province of Segovia, Spain, was carried out in the period of 2001–2003 years. A total of 587 individuals were included, 25.4% subjects were defined as obese (BMI ≥30 Kg/m2). Results: Plasma visfatin levels were significantly higher in obese subjects with DM2 than in other categories of glucose tolerance. The genotype AA of the rs4730153 SNP was significantly associated with fasting glucose, fasting insulin and HOMA-IR (Homeostasis model assessment-insulin resistance) after adjustment for gender, age, BMI and waist circumference. The obese individuals carrying the CC genotype of the rs11977021 SNP showed higher circulating levels of fasting proinsulin after adjustment for the same variables. The genotype AA of the rs4730153 SNP seems to be protective from CV-risk either estimated by Framingham or SCORE charts in general population; and in obese and non-obese individuals. No associations with CV-risk were observed for other studied SNPs (rs11977021/rs7789066). Conclusions: In summary, this is the first study which concludes that the genotype AA of the rs4730153 SNP appear to protect against CV-risk in obese and non–obese individuals, estimated by Framingham and SCORE charts. Our results confirm that the different polymorphisms in the visfatin gene might be influencing the glucose homeostasis in obese individuals. [ABSTRACT FROM AUTHOR]

Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development. [ABSTRACT FROM AUTHOR]

We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. [ABSTRACT FROM AUTHOR]

Objectives: Chronic renal disease (CRD) accelerates atherosclerosis and cardiovascular calcification. Statins reduce low-density lipoprotein-cholesterol levels in patients with CRD, however, the benefits of statins on cardiovascular disease in CRD remain unclear. This study has determined the effects of pitavastatin, the newest statin, on arterial inflammation and calcification in atherogenic mice with CRD. Methods and Results: CRD was induced by 5/6 nephrectomy in cholesterol-fed apolipoprotein E-deficient mice. Mice were randomized into three groups: control mice, CRD mice, and CRD mice treated with pitavastatin. Ultrasonography showed that pitavastatin treatment significantly attenuated luminal stenosis in brachiocephalic arteries of CRD mice. Near-infrared molecular imaging and correlative Mac3 immunostaining demonstrated a significant reduction in macrophage accumulation in pitavastatin-treated CRD mice. Pitavastatin treatment reduced levels of osteopontin in plasma and atherosclerotic lesions in CRD mice, but did not produce a significant reduction in calcification in atherosclerotic plaques as assesses by histology. CRD mice had significantly higher levels of phosphate in plasma than did control mice, which did not change by pitavastatin. In vitro, pitavastatin suppressed the expression of osteopontin in peritoneal macrophages stimulated with phosphate or calcium/phosphate in concentrations similar to those found in human patients with CRD. Conclusion: Our study provides in vivo evidence that pitavastatin reduces inflammation within atherosclerotic lesions in CRD mice. [ABSTRACT FROM AUTHOR]

Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction. [ABSTRACT FROM AUTHOR]

Objectives: Community-acquired Staphylococcus aureus necrotizing pneumonia is a life-threatening disease. Panton Valentine Leukocidin (PVL) has been associated with necrotizing pneumonia. PVL triggers inflammasome activation in human macrophages leading to IL-1β release. IL-1β activates lung epithelial cells to release IL-8. This study aimed to assess the relevance of this inflammatory cascade in vivo and to test the potential of an IL-1 receptor antagonist (IL-1Ra/Kineret) to decrease inflammation-mediated lung injury. Methods: We used the sequential instillation of Heat-killed S. aureus and PVL or S. aureus infection to trigger necrotizing pneumonia in rabbits. In these models, we investigated inflammation in the presence or absence of IL-1Ra/Kineret. Results: We demonstrated that the presence of PVL was associated with IL-1β and IL-8 release in the lung. During PVL-mediated sterile pneumonia, Kineret/IL-1Ra reduced IL-8 production indicating the relevance of the PVL/IL-1/IL-8 cascade in vivo and the potential of Kineret/IL-1Ra to reduce lung inflammation. However, Kineret/IL-1Ra was ineffective in blocking IL-8 production during infection with S. aureus. Furthermore, treatment with Kineret increased the bacterial burden in the lung. Conclusions: Our data demonstrate PVL-dependent inflammasome activation during S.aureus pneumonia, indicate that IL-1 signaling controls bacterial burden in the lung and suggest that therapy aimed at targeting this pathway might be deleterious during pneumonia. [ABSTRACT FROM AUTHOR]

Chronic inflammation closely associates with obesity, metabolic syndrome, diabetes mellitus, and atherosclerosis. Evidence indicates that the immunomodulator pentraxin 3 (PTX3) may serve as a biomarker of these cardiometabolic disorders, but whether PTX3 predicts cardiovascular complications is unknown. We examined the association of plasma PTX3 levels with recurrent coronary events via a prospective, nested, case-control design in the CARE trial. Among 4159 patients who had a prior myocardial infarction 3 to 20 months before enrollment and also had total cholesterol levels <240 mg/dL and LDL cholesterol levels between 115 and 175 mg/dL, we measured plasma PTX3 levels at baseline by high-sensitivity ELISA in 413 cases with recurrent myocardial infarction or coronary death during a 5-year follow-up period, and in 366 sex- and age-matched controls. Cases with recurrent coronary events and controls had similar PTX3 levels, and PTX3 did not predict recurrent coronary events — a finding that contrasts with that of C-reactive protein (CRP) and serum amyloid A (SAA) in this cohort. We then associated PTX3 levels with metabolic disorders. Low plasma PTX3 levels correlated with high body-mass index, waist circumference, and triglycerides; and with low HDL cholesterol. Overall, PTX3 levels correlated inversely with the number of metabolic syndrome components. PTX3 levels also correlated inversely with apoCIII and tissue plasminogen activator, but did not associate with CRP. Although the study further links low PTX3 levels with various features associated with metabolic syndrome, the results do not indicate that PTX3 can predict recurrent coronary events among MI survivors. [ABSTRACT FROM AUTHOR]

Aims/Hypothesis: Obesity is associated with a dysregulation of beta-cell and adipocyte function. The molecular interactions between adipose tissue and beta-cells are not yet fully elucidated. We investigated, whether or not the adipocytokine Nicotinamide phosphoribosyltransferase (Nampt) and its enzymatic product Nicotinamide mononucleotide (NMN), which has been associated with obesity and type 2 diabetes mellitus (T2DM) directly influence beta-cell survival and function. Methods: The effect of Nampt and NMN on viability of INS-1E cells was assessed by WST-1 assay. Apoptosis was measured by Annexin V/PI and TUNEL assay. Activation of apoptosis signaling pathways was evaluated. Adenylate kinase release was determined to assess cytotoxicity. Chronic and acute effects of the adipocytokine Nampt and its enzymatic product NMN on insulin secretion were assessed by glucose stimulated insulin secretion in human islets. Results: While stimulation of beta-cells with the cytokines IL-1β, TNFα and IFN-γ or palmitate significantly decreased viability, Nampt and NMN showed no direct effect on viability in INS-1E cells or in human islets, neither alone nor in the presence of pro-diabetic conditions (elevated glucose concentrations and palmitate or cytokines). At chronic conditions over 3 days of culture, Nampt and its product NMN had no effects on insulin secretion. In contrast, both Nampt and NMN potentiated glucose stimulated insulin secretion acutely during 1 h incubation of human islets. Conclusion/Interpretation: Nampt and NMN neither influenced beta-cell viability nor apoptosis but acutely potentiated glucose stimulated insulin secretion. [ABSTRACT FROM AUTHOR]

Visfatin, also known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague- Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 mmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 μmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 mmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 μmol/L). Additionally, the Nampt inhibitor APO866 (10 μmol/L to 10 μmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 mmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis." Our study would advance current understanding of visfatin physiology. [ABSTRACT FROM AUTHOR]

Background:Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. Methods and Results:Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1β (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 μmol/L) markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. Conclusions:Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation. [ABSTRACT FROM AUTHOR]