910 resultados para Avascular necrosis
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Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.
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The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.
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Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.
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Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70° C for later determination of plasma tumor necrosis factor-a and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.
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OBJECTIVE: To investigate the role of recombinant bactericidal/permeability-increasing protein (rBPI21) in the attenuation of the sepsis syndrome and acute lung injury associated with lower limb ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Gut-derived endotoxin has been implicated in the conversion of the sterile inflammatory response to a lethal sepsis syndrome after lower torso I/R injury. rBPI21 is a novel antiendotoxin therapy with proven benefit in sepsis. METHODS: Anesthetized ventilated swine underwent midline laparotomy and bilateral external iliac artery occlusion for 2 hours followed by 2.5 hours of reperfusion. Two groups (n = 6 per group) were randomized to receive, by intravenous infusion over 30 minutes, at the start of reperfusion, either thaumatin, a control-protein preparation, at 2 mg/kg body weight, or rBPI21 at 2 mg/kg body weight. A control group (n = 6) underwent laparotomy without further treatment and was administered thaumatin at 2 mg/kg body weight after 2 hours of anesthesia. Blood from a carotid artery cannula was taken every half-hour for arterial blood gas analysis. Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 by bioassay, and IL-8 by enzyme-linked immunosorbent assay (ELISA), as a markers of systemic inflammation. Plasma endotoxin concentration was measured using ELISA. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Bronchoalveolar lavage protein concentration was measured by the bicinclinoic acid method as a measure of capillary-alveolar protein leak. The alveolar-arterial gradient was measured; a large gradient indicated impaired oxygen transport and hence lung injury. RESULTS: Bilateral hind limb I/R injury increased significantly intestinal mucosal acidosis, intestinal permeability, portal endotoxemia, plasma IL-6 concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, edema, capillary-alveolar protein leak, and impaired gas exchange. Conversely, pigs treated with rBPI21 2 mg/kg at the onset of reperfusion had significantly reduced intestinal mucosal acidosis, portal endotoxin concentrations, and circulating phagocytic cell priming and had significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSIONS: Endotoxin transmigration across a hyperpermeable gut barrier, phagocytic cell priming, and cytokinemia are key events of I/R injury, sepsis, and pulmonary dysfunction. This study shows that rBPI21 ameliorates these adverse effects and may provide a novel therapeutic approach for prevention of I/R-associated sepsis syndrome.
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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL-induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.
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Fas (CD95/Apo-1) is a member of the tumor necrosis factor receptor family. Receptor binding results in activation of caspase 8, leading to activation of proapoptotic downstream molecules. We found that expression of Fas was up-regulated >10-fold in MCF-7 breast and HCT116 and RKO colon cancer cell lines after treatment with IC(60) doses of 5-fluorouracil (5-FU) and raltitrexed (RTX). Combined treatment with the agonistic Fas antibody CH-11 and either 5-FU or RTX resulted in a highly synergistic induction of apoptosis in these cell lines. Similar results were obtained for another antifolate, Alimta. Induction of thymidylate synthase expression inhibited Fas induction in response to RTX and Alimta, but not in response to 5-FU. Furthermore, thymidylate synthase induction abrogated the synergy between CH-11 and both antifolates but had no effect on the synergistic interaction between 5-FU and CH-11. Inactivation of p53 in MCF-7 and HCT116 cell lines blocked 5-FU- and antifolate-mediated up-regulation of Fas. Furthermore, Fas was not up-regulated in response to 5-FU or antifolates in the p53-mutant H630 colon cancer cell line. Lack of Fas up-regulation in the p53-null and -mutant lines abolished the synergistic interaction between 5-FU and CH-11. Interestingly, synergy was still observed between the antifolates and CH-11 in the p53-null HCT116 and p53-mutant H630 cell lines, although this was significantly reduced compared with the p53 wild-type cell lines. Our results indicate that Fas is an important mediator of apoptosis in response to both 5-FU and antifolates.
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Neutrophil elastase (NE) is thought to be the most important protease which damages the cystic fibrosis (CF) lung. Attempts have been made to suppress this activity using the plasma-derived inhibitor, alpha1-antitrypsin (AAT). In this pilot study, the safety and efficacy of inhaled recombinant human AAT (rAAT) as a treatment for CF were investigated. Thirty-nine patients participated in a prospective, double-blinded, randomized, placebo-controlled phase II trial to examine the effect of rAAT (500, 250, and 125 mg) on sputum NE activity. Sputum myeloperoxidase (MPO), interleukin-8, tumor necrosis factor receptors, sputum and plasma NE/AAT complexes, and safety parameters were also measured. Subjects were randomized to receive nebulized treatment once a day for 4 weeks, followed by 2–4 weeks with no study treatment, and then a 2-week rechallenge phase. Trends toward a reduction in NE activity were observed in patients treated with 500 mg and 250 mg of rAAT compared to placebo. Sputum NE/AAT complex and MPO levels were lower on rAAT compared to placebo. No major adverse events and, in particular, no allergic reactions to rAAT were observed. Although significant differences between rAAT and placebo for sputum NE activity were not observed, some improvements were found for secondary efficacy variables. This study demonstrated that nebulized rAAT is safe and well-tolerated, but has a limited effect on NE activity and other markers of inflammation. Pediatr Pulmonol.
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Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.
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Abnormal maternal inflammation during pregnancy is linked to complications such as preeclampsia and fetal growth restriction. There is growing evidence that insulin resistance is also associated with a heightened inflammatory state, and is linked to pregnancy complications such as gestational diabetes. This study tested the hypothesis that abnormal inflammation during pregnancy is causally linked to elevations in blood glucose and insulin resistance. To induce a state of abnormal systemic inflammation, bacterial lipopolysaccharide (LPS) was administered to pregnant rats on gestational days (GD) 13.5-16.5. Dams treated with LPS exhibited an abnormal immune response characterized by an elevation in white blood cells, which was linked to reduced fetal weight and increased glucose levels over pregnancy. Abnormal inflammation is characterized by increased levels of circulating pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) and interleukin-6, which contribute to insulin resistance by inhibiting the insulin signalling pathway. TNF in particular induces a serine phosphorylation (pSer307) of insulin receptor substrate 1 (IRS-1). In our model, insulin resistance was assessed by measuring the extent of pSer307 of IRS-1 and total IRS-1 expression in skeletal muscle, as well as changes in metabolic parameters and pancreas tissue morphology associated with insulin resistance. LPS-treated dams exhibited a significant reduction in IRS-1 expression, elevation in fasting glucose levels, and reduction in insulin sensitivity indices. There were also biologically relevant increases in fasting plasma insulin levels and insulin resistance indices, but not pSer307 of IRS-1 and pancreatic islet size. To determine whether inflammation plays a role in reducing insulin signalling and the other changes associated with LPS administration, etanercept, a TNF antagonist, was administered on GDs 13.5 and 15.5 prior to LPS injections. With the exception of IRS-1 expression, in rats treated with etanercept all of the measured parameters remained at the levels observed in saline controls, indicating a link between abnormal inflammation and insulin resistance. The results of this study support the practice of monitoring the inflammatory conditions of the mother prior to and during pregnancy, and support further investigation into the potential use of anti-inflammatory agents during pregnancy in women at risk of insulin resistance and gestational diabetes.
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Abstract
Thiazolidinediones (TZDs) have been used for the treatment of hyperglycaemia in type 2 diabetes for the past 10 years. They may delay the development of type 2 diabetes in individuals at high risk of developing the condition, and have been shown to have potentially beneficial effects on cardiovascular risk factors. TZDs act as agonists of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) primarily in adipose tissue. PPAR-gamma receptor activation by TZDs improves insulin sensitivity by promoting fatty acid uptake into adipose tissue, increasing production of adiponectin and reducing levels of inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor-1(PAI-1) and interleukin-6 (IL-6). Clinically, TZDs have been shown to reduce measures of atherosclerosis such as carotid intima-media thickness (CIMT). However, in spite of beneficial effects on markers of cardiovascular risk, TZDs have not been definitively shown to reduce cardiovascular events in patients, and the safety of rosiglitazone in this respect has recently been called into question. Dual PPAR-alpha/gamma agonists may offer superior treatment of insulin resistance and cardioprotection, but their safety has not yet been assured
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Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c+FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear factor-kappa B-mediated transcription attenuated IL-8-induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy.
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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]
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Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.
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Polymethyl methacrylate (PMMA) bone cement–multiwalled carbon nanotube (MWCNT) nanocomposites with a weight loading of 0.1% were prepared using 3 different methods of MWCNT incorporation. The mechanical and thermal properties of the resultant nanocomposite cements were characterised in accordance with the international standard for acrylic resin cements. The mechanical properties of the resultant nanocomposite cements were influenced by the type of MWCNT and method of incorporation used. The exothermic polymerisation reaction for the PMMA bone cement was significantly reduced when thermally conductive functionalised MWCNTs were added. This reduction in exotherm translated in a decrease in thermal necrosis index value of the respective nanocomposite cements, which potentially could reduce the hyperthermia experienced in vivo. The morphology and degree of dispersion of the MWCNTs in the PMMA matrix at different scales were analysed using scanning electron microscopy. Improvements in mechanical properties were attributed to the MWCNTs arresting/retarding crack propagation through the cement by providing a bridging effect into the wake of the crack, normal to the direction of crack growth. MWCNT agglomerations were evident within the cement microstructure, the degree of these agglomerations was dependent on the method used to incorporate the MWCNTs into the cement.