Formaldehyde inhalation reduces respiratory mechanics in a rat model with allergic lung inflammation by altering the nitric oxide/cyclooxygenase-derived products relationship

Bronchial hyperresponsiveness is a hallmark of asthma and many factors modulate bronchoconstriction episodes. A potential correlation of formaldehyde (FA) inhalation and asthma has been observed; however, the exact role of FA remains controversial. We investigated the effects of FA inhalation on Ovalbumin (OVA) sensitisation using a parameter of respiratory mechanics. The involvement of nitric oxide (NO) and cyclooxygenase-derived products were also evaluated. The rats were submitted, or not, to FA inhalation (1%, 90 min/day, 3 days) and were OVA-sensitised and challenged 14 days later. Our data showed that previous FA exposure in allergic rats reduced bronchial responsiveness, respiratory resistance (Rrs) and elastance (Ers) to methacholine. FA exposure in allergic rats also increased the iNOS gene expression and reduced COX-1. L-NAME treatment exacerbated the bronchial hyporesponsiveness and did not modify the Ers and Rrs, while Indomethacin partially reversed all of the parameters studied. The L-NAME and Indomethacin treatments reduced leukotriene B4 levels while they increased thromboxane B2 and prostaglandin E2. In conclusion, FA exposure prior to OVA sensitisation reduces the respiratory mechanics and the interaction of NO and PGE2 may be representing a compensatory mechanism in order to protect the lung from bronchoconstriction effects.

Endotoxin exposure during sensitization to Blomia tropicalis allergens shifts TH2 immunity towards a TH17-mediated airway neutrophilic inflammation: role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.

Periodontal treatment downregulates protease-activated receptor 2 in human gingival crevicular fluid cells

Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

Role of an indole-thiazolidine molecule PPAR pan-agonist and COX inhibitor on inflammation and microcirculatory damage in acute gastric lesions

The present study aimed to show the in vivo mechanisms of action of an indole-thiazolidine molecule peroxisome-proliferator activated receptor pan-agonist (PPAR pan) and cyclooxygenase (COX) inhibitor, LYSO-7, in an ethanol/HCl-induced (Et/HCl) gastric lesion model. Swiss male mice were treated with vehicle, LYSO-7 or Bezafibrate (p.o.) 1 hour before oral administration of Et/HCl (60%/0.03M). In another set of assays, animals were injected i.p. with an anti-granulocyte antibody, GW9962 or L-NG-nitroarginine methyl ester (L-NAME) before treatment. One hour after Et/HCl administration, neutrophils were quantified in the blood and bone marrow and the gastric microcirculatory network was studied in situ. The gastric tissue was used to quantify the percentage of damaged area, as well as myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) protein and PPARγ protein and gene expression. Acid secretion was evaluated by the pylorus ligation model. LYSO-7 or Bezafibrate treatment reduced the necrotic area. LYSO-7 treatment enhanced PPARγ gene and protein expression in the stomach, and impaired local neutrophil influx and stasis of the microcirculatory network caused by Et/HCl administration. The effect seemed to be due to PPARγ agonist activity, as the LYSO-7 effect was abolished in GW9962 pre-treated mice. The reversal of microcirculatory stasis, but not neutrophil influx, was mediated by nitric oxide (NO), as L-NAME pre-treatment abolished the LYSO-7-mediated reestablishment of microcirculatory blood flow. This effect may depend on enhanced eNOS protein expression in injured gastric tissue. The pH and concentration of H(+) in the stomach were not modified by LYSO-7 treatment. In addition, LYSO-7 may induce less toxicity, as 28 days of oral treatment did not induce weight loss, as detected in pioglitazone treated mice. Thus, we show that LYSO-7 may be an effective treatment for gastric lesions by controlling neutrophil influx and microcirculatory blood flow mediated by NO

Role of M2 muscarinic receptor in the airway response to methacholine of mice selected for minimal or maximal acute inflammatory response

Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation

Elevated tissue omega-3 fatty acid status prevents age-related glucose intolerance in fat-1 transgenic mice

The objective of this study was to investigate the impact of elevated tissue omega-3 (n-3) polyunsaturated fatty acids (PUFA) status on age-related glucose intolerance utilizing the fat-1 transgenic mouse model, which can endogenously synthesize n-3 PUFA from omega-6 (n-6) PUFA. Fat-1 and wild-type mice, maintained on the same dietary regime of a 10% corn oil diet, were tested at two different ages (2months old and 8months old) for various glucose homeostasis parameters and related gene expression. The older wild-type mice exhibited significantly increased levels of blood insulin, fasting blood glucose, liver triglycerides, and glucose intolerance, compared to the younger mice, indicating an age-related impairment of glucose homeostasis. In contrast, these age-related changes in glucose metabolism were largely prevented in the older fat-1 mice. Compared to the older wild-type mice, the older fat-1 mice also displayed a lower capacity for gluconeogenesis, as measured by pyruvate tolerance testing (PTT) and hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). Furthermore, the older fat-1 mice showed a significant decrease in body weight, epididymal fat mass, inflammatory activity (NFκ-B and p-IκB expression), and hepatic lipogenesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression), as well as increased peroxisomal activity (70-kDa peroxisomal membrane protein (PMP70) and acyl-CoA oxidase1 (ACOX1) expression). Altogether, the older fat-1 mice exhibit improved glucose homeostasis in comparison to the older wild-type mice. These findings support the beneficial effects of elevated tissue n-3 fatty acid status in the prevention and treatment of age-related chronic metabolic diseases

Ovariectomized OVA-sensitized mice display increased frequency of CD4+Foxp3+ T regulatory cells in the periphery

It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs.

Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury

The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.

Low temperatures reduce skin healing in the Jacaré do Pantanal (Caiman yacare, Daudin 1802)

Studies of skin wound healing in crocodilians are necessary given the frequent occurrence of cannibalism in intensive farming systems. Air temperature affects tissue recovery because crocodilians are ectothermic. Therefore, the kinetics of skin wound healing in Caiman yacare were examined at temperatures of 33°C and 23°C. Sixteen caiman were selected and divided into two groups of eight maintained at 23°C or 33°C. The studied individuals' scars were photographed after 1, 2, 3, 7, 15 and 30 days of the experimental conditions, and samples were collected for histological processing after 3, 7, 15 and 30 days. Macroscopically, the blood clot (heterophilic granuloma) noticeably remained in place covering the wound longer for the caiman kept at 23°C. Microscopically, the temperature of 23°C slowed epidermal migration and skin repair. Comparatively, new blood vessels, labeled using von Willebrand factor (vWF) antibody staining, were more frequently found in the scars of the 33°C group. The collagen fibers in the dermis were denser in the 33°C treatment. Considering the delayed healing at 23°C, producers are recommended to keep wounded animals at 33°C, especially when tanks are cold, to enable rapid wound closure and better repair of collagen fibers because such lesions tend to compromise the use of their skin as leather.

Effects of MK-801 and amphetamine treatments on allergic lung inflammatory response in mice

Glutamate acts as a neurotransmitter within the Central Nervous System (CNS) and modifies immune cell activity. In lymphocytes, NMDA glutamate receptors regulate intracellular calcium, the production of reactive oxygen species and cytokine synthesis. MK-801, a NMDA receptor open-channel blocker, inhibits calcium entry into mast cells, thereby preventing mast cell degranulation. Several lines of evidence have shown the involvement of NMDA glutamate receptors in amphetamine (AMPH)-induced effects. AMPH treatment has been reported to modify allergic lung inflammation. This study evaluated the effects of MK-801 (0.25mg/kg) and AMPH (2.0mg/kg), given alone or in combination, on allergic lung inflammation in mice and the possible involvement of NMDA receptors in this process. In OVA-sensitized and challenged mice, AMPH and MK-801 given alone decreased cellular migration into the lung, reduced IL-13 and IL10 levels in BAL supernatant, reduced ICAM-1 and L-selectin expression in granulocytes in the BAL and decreased mast cell degranulation. AMPH treatment also decreased IL-5 levels. When both drugs were administered, treatment with MK-801 reversed the decrease in the number of eosinophils and neutrophils induced by AMPH in the BAL of OVA-sensitized and challenged mice as well as the effects on the expression of L-selectin and ICAM-1 in granulocytes, the IL-10, IL-5 and IL-13 levels in BAL supernatants and increased mast cell degranulation. At the same time, treatment with MK-801, AMPH or with MK-801+AMPH increased corticosterone serum levels in allergic mice. These results are discussed in light of possible indirect effects of AMPH and MK-801 via endocrine outflow from the CNS (i.e., HPA-axis activity) to the periphery and/or as a consequence of the direct action of these drugs on immune cell activity, with emphasis given to mast cell participation in the allergic lung response of mice.

Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function

The role of Nox2-derived ROS in the development of cognitive impairment after sepsis

BACKGROUND: Sepsis- associated encephalopathy (SAE) is an early and common feature of severe infections. Oxidative stress is one of the mechanisms associated with the pathophysiology of SAE. The goal of this study was to investigate the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. METHODS: Sepsis was induced in WT and gp91phox knockout mice (gp91phox-/-) by cecal ligation and puncture (CLP) to induce fecal peritonitis. We measured oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in the hippocampus at six hours, twenty-four hours and five days post-sepsis. Mice were also treated with apocynin, a NADPH oxidase inhibitor. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance test and the Morris water maze in control and apocynin-treated WT mice. RESULTS: Acute oxidative damage to the hippocampus was identified by increased 4-HNE expression in parallel with an increase in Nox2 gene expression after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibited hippocampal oxidative stress in septic animals. Pharmacologic inhibition or the absence of Nox2 in gp91phox-/- mice prevented glial cell activation, one of the central mechanisms associated with SAE. Finally, treatment with apocynin and inhibition of hippocampal oxidative stress in the acute phase of sepsis prevented the development of long-term cognitive impairment. CONCLUSIONS: Our results demonstrate that Nox2 is the main source of reactive oxygen species (ROS) involved in the oxidative damage to the hippocampus in SAE and that Nox2-derived ROS are determining factors for cognitive impairments after sepsis. These findings highlight the importance of Nox2-derived ROS as a central mechanism in the development of neuroinflammation associated with SAE.

The putative role of ovary removal and progesterone when considering the effect of formaldehyde exposure on lung inflammation induced by ovalbumin

OBJECTIVE: Formaldehyde exposure during the menstrual cycle is known to affect the course of allergic lung inflammation. Because our previous data demonstrated that formaldehyde combined with an ovariectomy reduced allergic lung inflammation, we investigated the putative role of ovary removal and progesterone treatment when considering the effect of formaldehyde on allergic lung inflammation. METHOD: Ovariectomized rats and their matched controls were exposed to formaldehyde (1%, 3 days, 90 min/day) or vehicle, and immediately after exposure, the rats were sensitized to ovalbumin by a subcutaneous route. After 1 week, the rats received a booster by the same route, and after an additional week, the rats were challenged with ovalbumin (1%) by an aerosol route. The leukocyte numbers, interleukin-10 (IL-10) release, myeloperoxidase activity, vascular permeability, ex vivo tracheal reactivity to methacholine and mast cell degranulation were determined 24 h later. RESULTS: Our results showed that previous exposure to formaldehyde in allergic rats decreased lung cell recruitment, tracheal reactivity, myeloperoxidase activity, vascular permeability and mast cell degranulation while increasing IL-10 levels. Ovariectomy only caused an additional reduction in tracheal reactivity without changing the other parameters studied. Progesterone treatment reversed the effects of formaldehyde exposure on ex vivo tracheal reactivity, cell influx into the lungs and mast cell degranulation. CONCLUSION: In conclusion, our study revealed that formaldehyde and ovariectomy downregulated allergic lung inflammation by IL-10 release and mast cell degranulation. Progesterone treatment increased eosinophil recruitment and mast cell degranulation, which in turn may be responsible for tracheal hyperreactivity and allergic lung inflammation

José sarney é internado em São Paulo

Quadro de inflamação da vesícula biliar indica cirurgia de emergência.