Inflammatory Mediators Tumor Necrosis Factor-alpha Research Articles

Pomegranate Extract Potentiates the Anti-Demineralizing, Anti-Biofilm, and Anti-Inflammatory Actions of Non-Alcoholic Mouthwash When Associated with Sodium-Fluoride Trimetaphosphate.

This study investigated the anti-caries and anti-inflammatory effects of mouthwash formulations containing Punica granatum (pomegranate) peel extract (PPE), sodium-trimetaphosphate, and low concentrations of fluoride. PPE was characterized using high-performance liquid chromatography (ellagic acid and punicalagin). Total phenolics were quantified among formulations, and their stability was analyzed for 28 days. The formulation effects were evaluated as follows: (1) inorganic component concentration and reduced demineralization on bovine enamel blocks subjected to pH cycling; (2) anti-biofilm effect on dual-biofilms of Streptococcus mutans ATCC 25175 and Candida albicans ATCC 10231 treated for 1 and 10 min, respectively; and (3) cytotoxicity and production of inflammatory mediators (interleukin-6 and tumor necrosis factor-alpha). The formulation containing 3% PPE, 0.3% sodium-trimetaphosphate, and 225 ppm of fluoride resulted in a 34.5% surface hardness loss; a 13% (treated for 1 min) and 36% (treated for 10 min) biofilm reduction in S. mutans; a 26% (1 min) and 36% (10 min) biofilm reduction in C. albicans; absence of cytotoxicity; and anti-inflammatory activity confirmed by decreased interleukin-6 production in mouse macrophages. Thus, our results provide a promising prospect for the development of an alcohol-free commercial dental product with the health benefits of P. granatum that have been recognized for a millennium.

Morin hydrate attenuates chronic stress-induced memory impairment and degeneration of hippocampal subfields in mice: The role of oxidative, nitrergic and neuroinflammatory pathways.

Morin hydrate (MH) is the major flavonoid constituent of Morus alba acclaimed to have antioxidant, anti-inflammatory, anti-stress and neuroprotective properties. However, report on the effect of MH on memory performance and the underlying mechanism following chronic stress exposure is lacking. The current study aimed at investigating the neuroprotective effect of MH on chronic unpredictable stress (CUS)-induced memory impairment in mice using the Y maze test. Mice were subjected to unpredicted stress for 14 days, during which MH (5, 10 and 20 mg/kg i.p) or 25 mg/kg Ginseng was administered to them. On the 14th day, 1 h after treatment, learning and memory deficit was evaluated using the Y maze test and thereafter brains were harvested for the estimation of glutathione (GSH), lipid peroxidation product; malondialdehyde (MDA) and nitrite. Levels of inflammatory mediators tumor necrosis factor-alpha (TNF-α) and interleukin1-beta (IL-1β), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-кB) expressions were also determined. The hippocampus was stained with hematoxylin-eosin (H&E) to examine any morphological changes in the neurons. Mice exposed to CUS showed evidence of impaired memory and increase levels of MDA, nitrite, TNF-α and IL-1β. Furthermore, CUS reduced GSH level, increased the expressions of iNOS and NFкB immune-positive cells and produced loss of neuronal cells in the hippocampus. The MH treatment however improved memory, reduced MDA and nitrite levels, and enhanced brain GSH levels in CUS-mice. Besides, MH reduced brain levels of TNF-α and IL-1β levels, down regulated the expressions of iNOS and NF-кB and rescue neurons in the hippocampal CA3 region of mice exposed to CUS. The results of the study indicate that MH improved CUS-induced memory impairment, which may be related to its ability to boost antioxidant defense system and suppress neuroinflammatory pathways.

Impact of Coenzyme Q10 Administration on Lead Acetate-Induced Testicular Damage in Rats

Exposure to lead (Pb) causes multiorgan dysfunction including reproductive impairments. Here, we examined the protective effects of coenzyme Q10 (CoQ10) administration on testicular injury induced by lead acetate (PbAc) exposure in rats. This study employed four experimental groups (n = 7) that underwent seven days of treatment as follows: control group intraperitoneally (i.p.) treated with 0.1 ml of 0.9% NaCl containing 1% Tween 80 (v : v), CoQ10 group that was i.p. injected with 10 mg/kg CoQ10, PbAc group that was i.p. treated with PbAc (20 mg/kg), and PbAc+CoQ10 group that was i.p. injected with CoQ10 2 h after PbAc. PbAc injection resulted in increasing residual Pb levels in the testis and reducing testosterone, luteinizing hormone, and follicle-stimulating hormone levels. Additionally, PbAc exposure resulted in significant oxidative damage to the tissues on the testes. PbAc raised the levels of prooxidants (malondialdehyde and nitric oxide) and reduced the amount of endogenous antioxidative proteins (glutathione and its derivative enzymes, catalase, and superoxide dismutase) available in the cell. Moreover, PbAc induced the inflammatory response as evidenced by the upregulation of inflammatory mediators (tumor necrosis factor-alpha and interleukin-1 beta). Further, PbAc treatment induced apoptosis in the testicular cells, as indicated by an increase in Bax and caspase 3 expression, and reduced Bcl2 expression. CoQ10 supplementation improved testicular function by inhibiting Pb accumulation, oxidative stress, inflammation, cell death, and histopathological changes following PbAc exposure. Our findings suggest that CoQ10 can act as a natural therapeutic agent to protect against the reproductive impairments associated with PbAc exposure.

Mangiferin ameliorates intracerebroventricular-quinolinic acid-induced cognitive deficits, oxidative stress, and neuroinflammation in Wistar rats.

INTRODUCTION:Mangiferin (MGF), a xanthonoid polyphenol, confers neuroprotection via combating oxidative stress and inflammation. The current investigation aimed to assess the neuroprotective potential of MGF on behavioral and neurochemical anomalies evoked by administration of quinolinic acid (QA) through intrastriatal injection in male Wistar rats and to reveal the associated mechanisms.MATERIALS AND METHODS:QA (300 nm/4 μl saline) was administered intracerebroventricular in the striatum (unilaterally) once. Thereafter, MGF 20 and 40 mg/kg (peroral) was administered to the animals for 21 days.RESULTS:QA administration caused marked alteration in motor activity (rotatod), footprint analysis, and cognitive function (Morris water maze test, and novel object recognition test). Furthermore, oxido-nitrosative stress (increased nitrite content, lipid peroxidation, with reduction of GSH), cholinergic dysfunction, and mitochondrial complex (I, II, and IV) dysfunction were observed in hippocampus and striatal region of QA-treated rats in comparison to normal control. Pro inflammatory mediators (tumor necrosis factor-alpha TNF-α and interleukin-1β) were noted to increase in the hippocampus and striatum of QA-treated rats. In addition, we observed BDNF depletion in both the hippocampus and striatum of QA-treated animals. MGF treatment significantly ameliorated memory and motor deficits in QA-administered rats. Moreover, MGF treatment (40 mg/kg) restored the GSH level and reduced the MDA, nitrite level, and pro-inflammatory cytokines in striatum and hippocampus. Furthermore, QA-induced cholinergic dysfunction (AChE), BDNF depletion and mitochondrial impairment were found to be ameliorated by MGF treatment.CONCLUSION:The results suggest that MGF offers the neuroprotective potential that may be a promising pharmacological approach to ameliorate cognitive deficits associated with neurodegeneration.

Evaluation of 4-methyl-2-[(2-methylbenzyl) amino]-1,3-thiazole-5-carboxylic acid against hyperglycemia, insulin sensitivity, and oxidative stress-induced inflammatory responses and β-cell damage in the pancreas of streptozotocin-induced diabetic rats.

4-Methyl-2-[(2-methylbenzyl) amino]-1,3-thiazole-5-carboxylic acid (bioactive compound (BAC)), a novel thiazole derivative, is a xanthine oxidase inhibitor and free radical scavenging agent. Effects of BAC on hyperglycemia, insulin sensitivity, oxidative stress, and inflammatory mediators were evaluated in streptozotocin (STZ)-induced neonatal models of non-insulin-dependent diabetes mellitus (NIDDM) rats where NIDDM was induced in neonatal pups with single intraperitoneal injection of STZ (100 mg/kg). The effect of BAC (10 and 20 mg/kg, p.o.) for 3 weeks was evaluated by the determination of blood glucose, oral glucose tolerance test (OGTT), HbA1c level, insulin level, insulin sensitivity, and insulin resistance (IR). Furthermore, inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) and oxidative stress were estimated in serum and pancreatic tissue, respectively. Significant alteration in the level of blood glucose, OGTT, HbA1c, insulin level, insulin sensitivity, in addition variation in the antioxidant status and inflammatory mediators, and alteration in histoarchitecture of pancreatic tissue confirmed the potential of BAC in STZ-induced neonatal models of NIDDM rats. Pretreatment with BAC restored the level of glucose by decreasing the IR and increasing the insulin sensitivity. Furthermore, BAC balanced the antioxidant status and preserved the inflammatory mediators. Histological studies of pancreatic tissues showed normal architecture after BAC administration to diabetic rats. Altogether, our results suggest that BAC successfully reduces the blood glucose level and possesses antioxidant as well as anti-inflammatory activities. This leads to decreased histological damage in diabetic pancreatic tissues, suggesting the possibility of future diabetes treatments.

Drugs associated with idiosyncratic hepatotoxicity synergize with cytokines to kill hepatocytes in vitro

Idiosyncratic drug‐induced liver injury (IDILI) occurs in a small fraction of patients and often results in removal of otherwise efficacious drugs from the market. The mechanisms of IDILI are unknown; however, in studies using animal models the inflammatory mediators tumor necrosis factor‐alpha (TNFα) and interferon‐gamma (IFNγ) are essential to the pathogenesis of liver injury. The purpose of this study was to test the hypothesis that a drug's ability to synergize with inflammatory cytokines to kill hepatocytes in vitro can classify dugs according to their potential to cause idiosyncratic hepatotoxicity in humans. Human hepatoma (HepG2) cells were treated with drugs associated with IDILI or with drugs lacking IDILI liability and cotreated with TNFα and/or IFNγ.Of 13 drugs associated with IDILI, 12 synergized with TNFα to kill HepG2 cells.IFNγ enhanced the toxicity mediated by 8 IDILI‐positive drugs in the presence of TNFα. Of 10 drugs with little/no IDILI potential, none synergized with inflammatory cytokines to kill HepG2 cells. These findings indicate that drugs associated with IDILI in humans synergize with TNFα and IFNγ in vitro to cause cell death and raise the possibility that drug‐cytokine synergy underlies hepatocellular killing in some IDILI reactions. These results suggest an in vitro approach that could be used during preclinical safety evaluation to identify drug candidates with the potential to cause IDILI.

Effects of different ventilation strategies on exhaled nitric oxide in geriatric abdominal surgery

Exhaled nitric oxide (eNO) has been suggested to be a marker of small airway injury. We investigated the effects of different ventilation strategies on eNO. Sixty-nine patients who received elective open abdominal surgery under general anesthesia with more than 2 h of surgery duration were randomly divided into three groups: high tidal volume of 10–12 ml kg−1 predicted body weight (PBW) with zero end-expiratory pressure (ZEEP) (high VT + ZEEP group); low tidal volume of 6–8 ml kg−1 PBW with 8 cm H2O positive end-expiratory pressure (PEEP) (low VT + PEEP group); and low tidal volume of 6–8 ml kg−1 PBW with 8 cm H2O PEEP and recruitment maneuvers (low VT + PEEP + RMs group). eNO, respiratory system compliance (Crs), oxygenation index, inflammatory mediators tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-8, prostaglandin E2 (PGE2) and PGF2a as well as pulmonary function were measured during the perioperative period. The postoperative eNO decreased in 78.3% of patients in the high VT + ZEEP group and low VT + PEEP group, and increased in 56.5% of patients in the low VT + PEEP + RMs group (P = 0.016). The Crs level in the high VT + ZEEP group significantly decreased with time but significantly increased in the low VT + PEEP + RMs group (P < 0.05). The oxygenation index, inflammatory mediators and pulmonary function did not statistically differ among the three groups. Compared with the low VT + PEEP + RMs group, the decreasing rate of postoperative eNO in the high VT + ZEEP and low VT + PEEP groups was higher, which may imply small airway injury during geriatric abdominal surgery.

Antioxidant and Anti-Inflammatory Activities of Kenyan Leafy Green Vegetables, Wild Fruits, and Medicinal Plants with Potential Relevance for Kwashiorkor.

Background. Inflammation, together with related oxidative stress, is linked with the etiology of kwashiorkor, a form of severe acute malnutrition in children. A diet rich in anti-inflammatory and antioxidant phytochemicals may offer potential for the prevention and treatment of kwashiorkor. We selected and assayed five leafy green vegetables, two wild fruits, and six medicinal plants from Kenya for their antioxidant and anti-inflammatory properties. Consensus regarding medicinal plant use was established from ethnobotanical data. Methods. Antioxidant activity and phenolic content were determined using the oxygen radical absorbance capacity (ORAC) assay and Folin-Ciocalteu procedure, respectively. Anti-inflammatory activity was assessed in vitro targeting the inflammatory mediator tumour necrosis factor-alpha (TNF-α). Results. Mangifera indica (leaves used medicinally) showed the greatest antioxidant activity (5940 ± 632 µM TE/µg) and total phenolic content (337 ± 3 mg GAE/g) but Amaranthus dubius (leafy vegetable) showed the greatest inhibition of TNF-α (IC50 = 9 ± 1 μg/mL), followed by Ocimum americanum (medicinal plant) (IC50 = 16 ± 1 μg/mL). Informant consensus was significantly correlated with anti-inflammatory effects among active medicinal plants (r 2 = 0.7639, P = 0.0228). Conclusions. Several plant species commonly consumed by Kenyan children possess activity profiles relevant to the prevention and treatment of kwashiorkor and warrant further investigation.

Diosgenin attenuates inflammatory response induced by myocardial reperfusion injury: role of mitochondrial ATP-sensitive potassium channels

Reperfusion injury is one of the main reasons of cardiac disease morbidity. Phytopharmaceuticals are gaining importance in modern medicine of cardioprotection because of their multiplex capacity. The aim of this study was to investigate the effect of diosgenin on the inflammatory response induced by myocardial ischemia and reperfusion injury and the role of mitochondrial ATP-sensitive potassium (mitoKATP) channels in this regard. Wistar rats (250-300g) were used in this study. The Langendorff-perfused hearts of animals were subjected to a 30-min global ischemia followed by a 90-min reperfusion. The lactate dehydrogenase (LDH) release was measured by spectrophotometry. The levels of inflammatory mediators tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and IL-6 in the supernatant of heart's left ventricle were measured using an enzyme-linked immunosorbent assay rat specific ELISA kit. The LDH release into the coronary effluent during reperfusion was significantly decreased, and cardiac contractility significantly improved by diosgenin preadministration as compared with those of control or Cremophor-EL (solvent of diosgenin) groups (398 ± 48 vs. 665 ± 65 or 650 ± 73ml/min) (P < 0.01). Administration of diosgenin before the main ischemia significantly reduced the levels of IL-6 (P < 0.05), IL-1β, and TNF-α (P < 0.01) in the reperfusion phase of diosgenin-treated hearts as compared with untreated control hearts. Inhibition of mitoKATP channels by 5-hydroxydecanoate significantly reverses the cardioprotective effects of diosgenin (P < 0.05). The findings of the present study indicate that preconditioning with diosgenin may induce cardioprotective effect against reperfusion injury through reducing the production of inflammatory mediators and activating the mitoKATP channels.

Piperine, an active ingredient of black pepper attenuates acetaminophen–induced hepatotoxicity in mice

Objective To explore the hepatoprotective and antioxidant effects of piperine against acetaminophen-induced hepatotoxicity in mice. Methods In mice, hepatotoxicity was induced by a single dose of acetaminophen (900 mg/kg b.w. i.p.). Piperine (25 mg/kg b.w. i.p.) and standard drug silymarin (25 mg/kg b.w. i.p.) were given to mice, 30 min after the single injection of acetaminophen. After 4 h, the mice were decapitated. Activities of liver marker enzymes [(aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP)] and inflammatory mediator tumour necrosis factor-alpha (TNF-α) were estimated in serum, while lipid peroxidation and antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase and glutathione) were determined in liver homogenate of control and experimental mice. Results Acetaminophen induction (900 mg/kg b.w. i.p.) significantly increased the levels of liver marker enzymes, TNF-α, and lipid peroxidation, and caused the depletion of antioxidant status. Piperine and silymarin treatment to acetaminophen challenged mice resulted in decreased liver marker enzymes activity, TNF-α and lipid peroxidation levels with increase in antioxidant status. Conclusions The results clearly demonstrate that piperine shows promising hepatoprotective effect as comparable to standard drug silymarin.

Hepatoprotective and antioxidant effects of gallic acid in paracetamol-induced liver damage in mice

The aim of this research paper was to investigate the hepatoprotective and antioxidant effects of gallic acid in paracetamol-induced liver damage in mice. In the present study, the hepatoprotective and antioxidant effects of gallic acid were evaluated against paracetamol-induced hepatotoxicity in mice and compared with the silymarin, a standard hepatoprotective drug. The mice received a single dose of paracetamol (900 mg/kg body weight i.p.). Gallic acid (100 mg/kg body weight i.p.) and silymarin (25 mg/kg body weight i.p.) were administered 30 min after the injection of paracetamol. After 4 h, liver marker enzymes (aspartate transaminase, alanine transaminase and alkaline phosphatase) and inflammatory mediator tumour necrosis factor-alpha (TNF-alpha) were estimated in serum, while the lipid peroxidation and antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glutathione) were determined in liver homogenate of the control and experimental mice. Increased activities of liver marker enzymes and elevated TNF-alpha and lipid peroxidation levels were observed in mice exposed to paracetamol (P < 0.05), whereas the antioxidant status was found to be depleted (P < 0.05) when compared with the control group. However gallic acid treatment (100 mg/kg body weight i.p.) significantly reverses (P < 0.05) the above changes by its antioxidant action compared to the control group as observed in the paracetamol-challenged mice. The results clearly demonstrate that gallic acid possesses promising hepatoprotective effects.

Low levels of soluble receptor for advanced glycation end products in non-ST elevation myocardial infarction patients

Interaction of the receptors for advanced glycation end products (RAGEs) with advanced glycation end products (AGEs) results in expression of inflammatory mediators (tumor necrosis factor-alpha [TNF-α] and soluble vascular cell adhesion molecule-1 [sVCAM-1]), activation of nuclear factor-kappa B and induction of oxidative stress - all of which have been implicated in atherosclerosis. Soluble RAGE (sRAGE) acts as a decoy for the RAGE ligand and is protective against atherosclerosis. To determine whether levels of serum sRAGE are lower, and whether levels of serum AGEs, TNF-α and sVCAM-1 are higher in non-ST elevation myocardial infarction (NSTEMI) patients than in healthy control subjects; and whether sRAGE or the ratio of AGEs to sRAGE (AGEs/sRAGE) is a predictor/biomarker of NSTEMI. Serum levels of sRAGE, AGEs, TNF-α and sVCAM-1 were measured in 46 men with NSTEMI and 28 age- and sex-matched control subjects. Angiography was performed in the NSTEMI patients. sRAGE levels were lower, and levels of AGEs, TNF-α, sVCAM-1 and AGEs/sRAGE were higher in NSTEMI patients than in control subjects. sRAGE levels were negatively correlated with the number of diseased coronary vessels, serum AGEs, AGEs/sRAGE, TNF-α and sVCAM-1. The sensitivity of the AGEs/sRAGE test is greater than that of the sRAGE test, while the specificity and predictive values of the sRAGE test are greater than those of the AGEs/sRAGE test for identifying NSTEMI patients. Serum levels of sRAGE were low in NSTEMI patients, and were negatively correlated with extent of lesion, inflammatory mediators, AGEs and AGEs/sRAGE. Both sRAGE and AGEs/sRAGE may serve as biomarkers/predictors for identifying NSTEMI patients.

Surgery-Induced Inflammation in Relation to Age, Muscle Endurance, and Self-Perceived Fatigue

Elective abdominal surgery can be considered as a model for an important acute inflammatory trigger in human participants. The aim of the study was to explore the effect of surgery-induced inflammation on muscle strength, endurance, and self-perceived fatigue and its relation with age. Sixty-six elective abdominal surgery patients aged 24-91 years were assessed before and at the second and fourth day after surgical intervention. Outcome parameters were grip strength, muscle endurance, fatigue subscale of the Profile of Mood State and visual analog scale for pain, and the circulating inflammatory mediators C-reactive protein, interleukin (IL)-6, and tumor necrosis factor-alpha (TNF-alpha). All parameters worsened postoperatively (p < .01) and remained significantly (p < .05) worse until the fourth day postsurgery, except for TNF-alpha (no significant change). Older age was related to higher surgery-induced IL-6 levels at the second (p < .05) and fourth postoperative (p < .01) day and to worse self-perceived fatigue and muscle endurance (both p < .05) at the fourth postoperative day. Higher pain levels at the second day following surgery was related to more self-perceived fatigue (p < .05). Worsening muscle endurance following surgery was significantly related to higher IL-6 release following surgery (p < .01) and self-perceived fatigue (p < .05) at the fourth day following the intervention. Age and surgery-induced increase in circulating IL-6 at Day 4 postsurgery was highest in patients showing both worsened muscle endurance and self-perceived fatigue (p < .05). Surgery-induced inflammation is related to reduced muscle endurance and the sensation of fatigue. Elderly patients suffer from a higher impact of surgery on muscle endurance.

Complement activation and plasma levels of C4b-binding protein in critical limb ischemia patients

Critical limb ischemia (CLI) is a peripheral arterial disease manifested by drastically diminished blood flow to the legs, pain at rest, nonhealing wounds, and gangrene caused by atherosclerosis. Significant tissue necrosis is associated with late stage CLI and the patients have a poor prognosis. Necrotic and apoptotic cells activate complement and bind complement inhibitor C4b-binding protein (C4BP). The major isoform of C4BP is composed of seven identical alpha-chains and one beta-chain, here termed C4BP(beta), whereas upon inflammation a normally less abundant isoform is upregulated that is exclusively composed of alpha-chains. Measuring the alpha-chains of C4BP includes both isoforms and is termed total C4BP (C4BP(tot)). The hypothesis of this study was that levels of complement activation and C4BP are predictive for the severity of the disease and that their measurement might be of clinical advantage. This was a prospective, single-center study of 259 consecutive patients with CLI admitted to a secondary referral center for vascular diseases. Interventions included evaluation of soluble terminal complement complexes (C5b-9), C4BP(tot) and C4BP(beta), lipid levels, the inflammatory mediators tumor necrosis factor-alpha, interleukin-6, 8-iso-prostaglandin F(2alpha), high-sensitivity C-reactive protein, neopterin, plasma homocysteine, and plasma endothelin-1 in plasma as well as resistance to activated protein C and ankle blood pressure. All data were compared with an age-matched population based control group of 219 currently healthy individuals. The data are presented as mean +/- SEM/median. CLI patients showed systemic complement activation (1.17 +/- 0.06/1.13 AU/mL vs 0.69 +/- 0.07/0.59 AU/mL in healthy controls, P < .0001), which was even higher in patients with gangrene (1.33 +/- 0.11/1.28 AU/mL vs 1.1 +/- 0.08/1.0 AU/mL, P = .0264), who also showed increased C4BP levels (421 +/- 28.6/386 microg/mL vs 341 +/- 10.8/318 microg/mL for C4BP(tot), P = .0248; 374 +/- 25.4/332 microg/mL vs 305 +/- 9.5/285 microg/mL for C4BP(beta), P = .0581). C4BP plasma levels were significantly elevated in CLI patients in comparison to healthy controls (351 +/- 8.1/322 microg/mL vs 297 +/- 8.0/288 microg/mL for C4BP(tot), P = .0001; 314 +/- 7.0/287 microg/mL vs 265 +/- 7.0/263 microg/mL for C4BP(beta), P = .0004) and correlated to levels of interleukin-6 (P(tot/beta) = .0048/.0019), high-sensitivity C-reactive protein (P < .0001), leukocyte (P(tot/beta) = .0086/.0043) and platelet count (P = .0001), LDL/HDL ratio (P(tot) = .0151) and HDL (P(tot/beta) = .0047/.0177), but not to tumor necrosis factor-alpha. Increased complement activation and C4BP plasma levels are related to the degree of tissue necrosis and disease severity of critical limb ischemia. This knowledge in combination with the found correlations to other biomarkers is useful for understanding the pathophysiology of the disease.

The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.

The Toll-like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immune-response-domain-containing adaptor inducing interferon (IFN)-beta. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol-induced liver injury in wild-type, TLR2-deficient, TLR4-deficient, or MyD88-deficient (knockout [KO]) mice after administration of the Lieber-De-Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum alanine aminotransferase levels, liver steatosis and triglyceride levels suggesting liver damage in WT, TLR2-KO, and MyD88-KO but not in TLR4-KO mice. Expression of inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) and TLR4 coreceptors (CD14 and MD2) was significantly higher in livers of alcohol-fed WT, TLR2-KO, or MyD88-KO, but not in TLR4-KO mice, compared to controls. Reactive oxygen radicals produced by cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complexes contribute to alcoholic liver damage. Alcohol feeding-induced expression and activation of cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complex were prevented by TLR4-deficiency but not by MyD88-deficiency. Liver expression of interferon regulatory factor 3 (IRF3), a MyD88-independent signaling molecule, was not affected by chronic alcohol treatment in whole livers of WT mice or in any of the KO mice. However, the induction of IRF7, an IRF3-inducible gene, was found in Kupffer cells of alcohol-fed WT mice. Alcohol feeding also induced nuclear factor-kappaB activation in a TLR4-dependent MyD88-independent manner. While TLR4 deficiency was protective, MyD88 deficiency failed to prevent alcohol-induced liver damage and inflammation. These results suggest that the common TLR adapter, MyD88, is dispensable in TLR4-mediated liver injury in ALD.

PULMONARY INFLAMMATORY RESPONSES AFTER ETHANOL EXPOSURE AND BURN INJURY

Previous studies suggest that ethanol use imposes a greater risk of trauma-associated mortality due to the development of sepsis and multiple organ dysfunction syndromes (MODS). Burn patients with prior ethanol exposure have a worse prognosis than those who sustain injury but had not been drinking. Evidence suggests that pulmonary infection may be responsible, in part, for the difference in mortality and mortality between the two groups. We previously reported that mice given a 15% total body surface area scald injury with a blood ethanol content of 100 mg/dl had an elevated pulmonary inflammatory response. In the present study, we report that at 2 hours after injury the levels of the inflammatory mediators macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNFα) were decreased in bronchoalveolar lavage (BAL) fluid obtained from mice exposed to ethanol and burn injury when compared to BAL fluid from mice given burn alone (P < 0.05). At this time point, there were no differences in the expression of these mediators in lung homogenates from mice from burn vs. burn ethanol groups. In contrast, we found elevated levels of the anti-inflammatory cytokine interleukin-10 (IL-10) at 6 hours post injury in the lungs of mice given both ethanol and burn injury relative to burn alone. Taken together, these observations could help explain the aberrant inflammatory response seen in burn patients who had alcohol in their system at the time of injury (NIH AA12034, T32 AA135527, the Illinois Excellence in Academic Medicine Grant and the Dr. Ralph and Marion C. Falk Medical Research Trust).

Inflammatory mediators are associated with 1-year mortality in critical limb ischemia

The atherosclerotic process has inflammatory features. Patients with peripheral atherosclerosis and critical limb ischemia have a poor prognosis. This study evaluated the hypothesis that inflammatory markers are associated with mortality among patients admitted to the hospital because of critical limb ischemia. This was a prospective, single-center, 1-year, follow-up study of 259 consecutive patients with critical limb ischemia who were admitted to a secondary referral center of vascular diseases. Interventions included evaluation of intercurrent disease, ankle and arm blood pressures, plasma glucose and lipid levels, plasma homocysteine, cardiolipin antibodies, resistance to activated protein C, plasma endothelin-1, and the inflammatory mediators tumor necrosis factor-alpha, interleukin-6, neopterin, high-sensitivity C-reactive protein, CD40 ligand, and 8-iso-prostaglandin F alpha in plasma. The main outcome measure was total mortality and causes of death assessed 1 year after admission. During the first year after admission, 61 patients (24%) died. These patients were older (P < .0001), showed a higher leukocyte count (P = .0011) and levels of serum creatinine (P < .0001), lower levels of high-density lipoprotein (HDL) cholesterol (P = .003) and frequency of active treatment (P = .014) than the 198 (76%) survivors. More nonsurvivors had gangrene (P < .0001), and fewer (P = .004) had lipid-lowering treatment. The plasma levels of interleukin-6 (P < .0001), tumor necrosis factor-alpha (P < .0001), neopterin (P < .0001), and high-sensitivity C-reactive protein (P = .002) at admission for critical limb ischemia were all significantly lower in the survivors, whereas there was no difference concerning CD40 ligand. In logistic regression adjusted for age, sex, lipid-lowering therapy, active treatment, gangrene, leukocyte count, creatinine, and serum HDL cholesterol, the inflammatory mediators tumor necrosis factor-alpha (P = .0084), neopterin (P = .0035), but not interleukin-6 (P = .585) or high-sensitivity C-reactive protein (P = .314) were independent risk variables of death within 1 year. Increased age, leukocyte count, creatinine, and inflammatory mediators, together with gangrene, were associated with 1-year mortality despite intervention in critical limb ischemia. For tumor necrosis factor-alpha and neopterin in plasma, this association was independent of the other parameters.

Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9).

Innate immune cells express toll-like receptor-9 (TLR9) and respond to unmethylated, CG dinucleotide motif-rich DNA released from bacteria during infection or endogenous cells during autoimmune tissue injury. Oligonucleotides containing CG dinucleotide (CpG-DNA) mimic the effect of unmethylated DNA and stimulate TLR9. CpG-DNA was cytotoxic to neurons in organotypic brain cultures. Neurotoxicity of CpG-DNA was mediated via microglial cells and started primarily from neurites as determined by time-lapse imaging of enhanced green fluorescent protein (EGFP)-transfected neurons. Cultured brain microglial cells expressed TLR9 and responded to CpG-DNA by production of the inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Blockade of NO synthase and TNF-alpha prevented damage of neurites and neurotoxicity of CpG-DNA. The data suggest that stimulation of microglia via TLR9 and subsequent release of NO and TNF-alpha is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury.

Regulation of the Hypoxia-inducible Factor 1α by the Inflammatory Mediators Nitric Oxide and Tumor Necrosis Factor-α in Contrast to Desferroxamine and Phenylarsine Oxide

Hypoxic/ischemic conditions provoke activation of the hypoxia-inducible factor-1 (HIF-1), which functions as a transcription factor. HIF-1 is composed of the HIF-1alpha and -beta subunits, and stability regulation occurs via accumulation/degradation of HIF-1alpha with the notion that a prolyl hydroxylase accounts for changes in protein level. In addition, there is evidence that HIF-1 is up-regulated by diverse agonists during normoxia. We investigated the impact of inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) on HIF-1alpha regulation. For comparison, LLC-PK(1) cells were exposed to hypoxia, stimulated with desferroxamine (DFX, known to mimic hypoxia), and the thiol-cross-linking agent phenylarsine oxide (PAO). Although all stimuli elicited HIF-1alpha stabilization with differences in the time-dependent accumulation pattern, significant variations appeared with regard to signaling. With the use of a superoxide anion (O(2-)) generator, we established an O(2-)-sensitive pathway that blocked HIF-1alpha stabilization in response to NO and TNF-alpha while DFX- and PAO-evoked HIF-1alpha stabilization appeared O(2-)-insensitive. NO and TNF-alpha signaling required phosphorylation events, especially activation of the phosphatidylinositol 3-kinase/Akt, which is in contrast to DFX and PAO. Based on HIF-1-dependent luciferase reporter gene analysis, it was found that, in contrast to NO and TNF-alpha, PAO resembled a stimulus that induced a dysfunctional HIF-1 complex. These data indicate that diverse agonists activate HIF-1alpha under normoxic conditions by employing different signaling pathways.

Proapoptotic Fas ligand is expressed by normal kidney tubular epithelium and injured glomeruli.

Fas ligand (FasL) is a cell membrane cytokine that can promote apoptosis through activation of Fas receptors. Fas receptor activation induces glomerular cell apoptosis in vivo and participates in tubular cell death during acute renal failure. However, there is little information on the expression of FasL in the kidney. This study reports that FasL mRNA and protein are present in normal mouse and rat kidney. In situ hybridization and immunohistochemistry showed that proximal tubular epithelium is the main site of FasL expression in the normal kidney. In addition, increased total kidney FasL mRNA and de novo FasL protein expression by glomerular cells were observed in two different models of glomerular injury : rat immune-complex proliferative glumerulonephritis and murine lupus nephritis. Both full-length and soluble FasL were increased in the kidneys of the mice with nephritis. Cultured murine proximal tubular epithelial MCT cells and primary cultures of murine tubular epithelial cells expressed FasL mRNA and protein. Tubular epithelium-derived FasL induced apoptosis in Fassensitive lymphoid cell lines but not in Fas-resistant lymphoid cell lines. By contrast, MCT cells grown in the presence of the survival factors of serum were resistant to FasL, and only became partially sensitive to apoptosis induced by high concentrations (100 ng/ml) of FasL upon serum deprivation. However, MCT cells stimulated with inflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide) increased cell surface Fas expression and were sensitized to apoptosis induced by FasL (FasL 55 +/- 5% versus control 8.3 +/- 4.1% apoptotic cells at 24 h, P < 0.05). Cytokine-primed primary cultures of tubular epithelial cells also acquired sensitivity to FasL-induced apoptosis. These results suggest that FasL expression by intrinsic renal cells may play a role in cell homeostasis in the normal kidney and during renal injury.