Hydrogen peroxide is a novel mediator of inflammatory hyperalgesia, acting via transient receptor potential vanilloid 1-dependent and independent mechanisms

Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H(2)O(2)) in hyperalgesia. In the present study, intraplantar injection of H(2)O(2)-induced a significant dose- and time-dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c-fos activity observed in the dorsal horn of the spinal cord. H(2)O(2) also induced significant nociceptive behavior Such as increased paw licking and decreased body liftings. H(2)O(2) levels were significantly raised in the carrageenan-induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H(2)O(2) (i.pl.) Was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H(2)O(2). In conclusion, we demonstrate a notable effect of H(2)O(2) in mediating inflammatory hyperalgesia, thus highlighting H(2)O(2) removal as a novel therapeutic target for anti-hyperalgesic drugs in the clinic. (C) 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Endothelin A receptor antagonist modulates lymphocyte and eosinophil infiltration, hyperreactivity and mucus in murine asthma

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57B1/6 mice were treated with endothelin ET(A) receptor antagonist (BQ123) or endothelin ET(B) receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD(4)(+), T CD(8)(+), B220(+), T gamma delta(+) and NK1.1(+) lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ET(A) receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus. (C) 2008 Elsevier B.V. All rights reserved.

Menopausal status: A possible predictive factor for recurrence in women with cancer of the uterine cervix without pelvic lymph node metastasis

Objectives: To evaluate risk factors for recurrence of carcinoma of the uterine cervix among women who had undergone radical hysterectomy without pelvic lymph node metastasis, while taking into consideration not only the classical histopathological factors but also sociodemographic, clinical and treatment-related factors. Study design: This was an exploratory analysis on 233 women with carcinoma of the uterine cervix (stages IB and IIA) who were treated by means of radical hysterectomy and pelvic lymphadenectomy, with free surgical margins and without lymph node metastases on conventional histopathological examination. Women with histologically normal lymph nodes but with micrometastases in the immunohistochemical analysis (AE1/AE3) were excluded. Disease-free survival for sociodemographic, clinical and histopathological variables was calculated using the Kaplan-Meier method. The Cox proportional hazards model was used to identify the independent risk factors for recurrence. Results: Twenty-seven recurrences were recorded (11.6%), of which 18 were pelvic, four were distant, four were pelvic + distant and one was of unknown location. The five-year disease-free survival rate among the study population was 88.4%. The independent risk factors for recurrence in the multivariate analysis were: postmenopausal status (HR 14.1; 95% CI: 3.7-53.6; P < 0.001), absence of or slight inflammatory reaction (HR 7.9; 95% CI: 1.7-36.5; P = 0.008) and invasion of the deepest third of the cervix (FIR 6.1; 95% CI: 1.3-29.1; P = 0.021). Postoperative radiotherapy was identified as a protective factor against recurrence (HR 0.02; 95% CI: 0.001-0.25; P = 0.003). Conclusion: Postmenopausal status is a possible independent risk factor for recurrence even when adjusted for classical prognostic factors (such as tumour size, depth of turnout invasion, capillary embolisation) and treatment-related factors (period of treatment and postoperative radiotherapy status). (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Myeloperoxidase as Inflammatory Marker of Periodontal Disease: Experimental Study in Rats

Rationale: Previous studies have used myeloperoxidase (MPO) as an inflammatory marker to estimate the accumulation of neutrophils in inflamed regions. Objective: The aim of this experimental study was to quantify the levels of MPO related to experimental periodontal disease in rats. Methods: Periodontal disease was induced in a group of rats using placement of a ligature around molar teeth. A group of rats without ligature placement served as a control. Measurements were made on the 3rd, 7th, 15th and 30th day from baseline. Gingival tissues were taken for quantification of MPO levels by ELISA. Results: The rats with induced periodontal disease showed statistically higher MPO levels (p 0.05) when compared to control rats. A significant increase in the levels of MPO released on days 7 and 30 was observed, with higher levels in the group with induced periodontitis. Conclusion: The levels of MPO were found to be higher in rats with induced periodontal disease, confirming the hypothesis that MPO may serve as an inflammatory marker for periodontitis.

Stromal-derived factor-1 alpha (CXCL12) levels increase in periodontal disease

Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

Comparison of biological behavior between early-stage adenocarcinoma and squamous cell carcinoma of the uterine cervix

Objectives: (1) To compare the anatomopathological variables and recurrence rates in patients with early-stage adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix; (2) to identify the independent risk factors for recurrence. Study design: This historical cohort study assessed 238 patients with carcinoma of the uterine cervix (113 and IIA), who underwent radical hysterectomy with pelvic lymph node dissection between 1980 and 1999. Comparison of category variables between the two histological types was carried out using the Pearson`s X-2 test or Fisher exact test. Disease-free survival rates for AC and SCC were calculated using the Kaplan-Meier method and the curves were compared using the log-rank test. The Cox proportional hazards model was used to identify the independent risk factors for recurrence. Results: There were 35 cases of AC (14.7%) and 203 of SCC (85.3%). AC presented lower histological grade than did SCC (grade 1: 68.6% versus 9.4%; p < 0.001), lower rate of lymphovascular space involvement (25.7% versus 53.7%; p = 0.002), lower rate of invasion into the middle or deep thirds of the uterine cervix (40.0% versus 80.8%; p < 0.001) and lower rate of lymph node metastasis (2.9% versus 16.3%; p = 0.036). Although the recurrence rate was lower for AC than for SCC (11.4% versus 15.8%), this difference was not statistically significant (p = 0.509). Multivariate analysis identified three independent risk factors for recurrence: presence of metastases in the pelvic lymph nodes, invasion of the deep third of the uterine cervix and absence of or slight inflammatory reaction in the cervix. When these variables were adjusted for the histological type and radiotherapy status, they remained in the model as independent risk factors. Conclusion: The AC group showed less aggressive histological behavior than did the SCC group, but no difference in the disease-free survival rates was noted. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Objective We investigated the effects of high-fat diet-induced obesity on vascular proinflammatory factors and oxidative stress on endothelium-dependent relaxation of the aorta. Methods Female Swiss mice were submitted to a high-fat diet for 16 weeks. At the end of the experimental period, we evaluated blood pressure, relaxation in response to acetylcholine in aortic rings in the absence and the presence of the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), and the nuclear factor (NF)-kappa B inhibitor, sodium salicylate (5 mmol/l). Aortic protein expression of endothelial nitric oxide synthase, Cu/Zn-SOD, NF-kappa B, I kappa B-alpha, and proinflammatory cytokines were also evaluated. Results Obese mice presented higher systolic and diastolic blood pressure than control mice (P<0.05). The relaxation of aortas to acetylcholine, but not to sodium nitroprusside, was significantly decreased in obese mice and was corrected by both SOD and sodium salicylate (P<0.05). The protein expression of endothelial nitric oxide synthase and Cu/Zn-SOD was significantly decreased in aorta from obese mice (P<0.05). Total p65 NF-kappa B subunit protein expression was not affected by obesity, but the protein expression of NF-kappa B inhibitor I kappa B-alpha was lower in aorta from obese mice (P<0.05). There were no significant differences in the interleukin (IL)-1 beta and IL-6 protein expression between groups. In contrast, the expression of TNF-alpha was significantly increased in aortas from obese mice. Conclusion Our resultssuggest that the reducedantioxidant defense and the local NF-kappa B pathway play an important role in the impairment of endothelium-dependent relaxation in aorta from obese mice. J Hypertens 28: 2111-2119 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and beta 2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2 alpha beta (cytokine-induced neutrophil chemoattractant-2 alpha beta), IL-1 beta (interleukin-1 beta), MIP-1 alpha (macrophage inflammatory protein-1 alpha) and TNF-alpha (tumour necrosis factor-alpha)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2 alpha beta into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of beta 2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2 alpha beta release.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

The aim of the present study was to evaluate the effect of overstimulation of beta-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the beta-adrenoceptor agonist isoproterenol (0.3 mg(.)kg(-1.)day(-1) sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1 beta (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-alpha expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-beta B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of I kappa B-alpha (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-kappa B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg(.)kg(-1.)day(-1) for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of beta-adrenoceptors increased proinflammatory cytokines and upregulated NF-kappa B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic beta-adrenergic stimulation.

Prolonged Physical Training Decreases mRNA Levels of Glucocorticoid Receptor and Inflammatory Genes

Background/Aims: Prolonged physical exercise induces adaptive alterations in the hypothalamic-pituitary axis, increasing cortisol metabolism, and reducing cortisol synthesis and glucocorticoid sensitivity. The mechanisms responsible for this relative glucocorticoid resistance remain unknown but may involve expression of genes encoding glucocorticoid receptor (GR) and/or inflammatory molecules of nuclear factor kappa B1 (NFkB1) signaling pathway and cytokines. This study aimed to determine the impact of prolonged physical training on the expression of genes involved in glucocorticoid action and inflammatory response. Methods: Normal sedentary male cadets of the Brazilian Air Force Academy were submitted to 6 weeks of standardized physical training. Eighteen of 29 initially selected cadets were able to fully complete the training program. Fasting glucose, insulin and cortisol levels, cytokine concentration and the expression of genes encoding GR, NFkB1, inhibitor of NFkB1 and IkB kinase A were determined before and after the training period. Results: Prolonged physical exercise reduced the basal cortisol levels and the percent cortisol reduction after dexamethasone. These findings were associated with a significant reduction in the mRNA levels of GR (6.3%), NFkB1 (63%), inhibitor of NFkB1 (25%) and IkB kinase A (46%) with concomitant reduction in cytokine concentrations (ELISA). Conclusions: Prolonged physical training decreases the glucocorticoid sensitivity and the mRNA levels of the GR gene combined with decreased mRNA of genes related to the NFkB pathway. Copyright (C) 2010 S. Karger AG, Basel

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response

Trypanosoma cruzi trypomastigotes continuously shed into the medium plasma membrane fragments sealed as vesicles enriched in glycoproteins of the gp85 and trans-sialidase (TS) superfamily and alpha-galactosyl-containing glycoconjugates. Injection of a vesicle fraction into BALB/c mice prior to T. cruzi infection led to 40% of deaths on the 16th day post-infection and 100% on day 20th whereas 20% of untreated animals survived for more than 30 clays. The vesicle-treated animals developed severe heart pathology, with intense inflammatory reaction and higher number of amastigote nests. Analysis of the inflammatory infiltrates 15 days after infection showed predominance of TCD4(+) lymphocytes and macrophages, but not of TCD8(+) cells, as well as a decrease of areas labeled with anti-iNOS antibodies as compared to the control. Higher levels of IL-4 and IL-10 mRNAs were found in the hearts and higher IL-10 and lower NO levels in splenocytes of vesicles pretreated animals. Treatment of mice with neutralizing anti-IL-10 or anti-IL-4 antibodies precluded the effects of pre-inoculation of membrane vesicles on infection. These results indicate that T. cruzi shed membrane components increase tissue parasitism and inflammation by stimulation of IL-4 and IL-10 synthesis and thus may play a central role in the pathogenesis of Chagas` disease acute phase. (c) 2008 Elsevier Masson SAS. All rights reserved.

Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis

Background Epidemiological and experimental data suggest that bacteria] lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll-like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro-Type 1 T helper cells (Th 1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways, we used TLR4-, MyD88-, TRIF-, or IL-12/IFN-gamma-deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co-adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co-adsorbed onto alum impaired in dose-dependent manner OVA-induced Th2-mediated allergic responses such as airway eosinophilia, type-2 cytokines secretion, airway hyper-reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1 -affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL-12/IFN-gamma axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll-like receptor 4 agonists co-adsorbed with allergen onto alum down-modulate allergic lung disease and prevent the development of polarized T cell-mediated airway inflammation.