Ethanol consumption enhances periodontal inflammatory markers in rats

Objective: The aim of this study was to assess the short term effect of ethanol administration on periodontal disease in rats. Design: Rats received either ethanol 2 g/kg or water by gastric gavage twice a day. On the fifth day ligatures were tied around the molars of half of the rats to induce periodontitis. After 7 days gingival tissue was removed and assayed for inflammatory markers. Finally, hemi-mandibles were extracted to evaluate bone loss by histomorphometrical techniques. Results: The experimental periodontitis increased significantly the mRNA expression (p < 0.001) and activity (p < 0.001) of inducible nitric oxide synthase (iNOS) in the gingival tissue, whilst short time ethanol administration increased iNOS activity (p < 0.05) and produced an additive effect on iNOS mRNA expression augmented by periodontitis (p < 0.01). The short time ethanol administration also potentiated the periodontitis stimulatory effect on the mRNA expression of interleukin (IL)-1 beta (p < 0.01 and p < 0.001, in semi-quantitative and real time PCR, respectively) and on the height of periodontal ligament (p < 0.05). However, the ligature-induced periodontitis, but not ethanol administration, increased the prostaglandin E-2 content (p < 0.05) and, diminished the alveolar bone volume (p < 0.05), as compared to sham rats. Conclusion: The present results suggest that ethanol consumption could represent a risk indicator for periodontal disease since augments the expression of inflammatory markers, in healthy rats, and increases them, at short term, during the illness. However, scale longitudinal investigation and more case-control studies are needed to confirm this statement. (C) 2012 Elsevier Ltd. All rights reserved.

Hepatitis B virus genotypes from European origin explains the high endemicity found in some areas from southern Brazil

Southern Brazil is considered an area of low Hepatitis B endemicity, but some areas of higher endemicity have been described in the Southwest of Parana and Santa Catarina states. The aim of this study was to evaluate viral genotypes circulating throughout Parana state. PCR amplification and partial sequencing of the S gene was carried out in 228 samples from HBsAg positive candidate blood donors. Samples have been collected in seven different counties (Cascavel, Curitiba, Foz do Iguacu, Francisco Beltrao, Matinga Londrina and Paranagua). The most common HBV genotype in Parana state was D (82.9%; 189/228), followed by A (14.1%; 32/228). Genotypes F (1.3%; 3/228), C (1.3%; 3/228) and H (0.4%; 1/228) were also found. Distribution of genotypes was different in the studied counties, but genotype D was the most frequent in all of them. In Francisco Beltrao, all studied samples belonged to genotype D. The high prevalence of HBV genotype Din South of Brazil is explained by the intense migration of settlers from Europeans countries. Subgenotypes A1 and A2 were identified circulating in all cities where HBV/A was found. As observed in other areas of Brazil, HBV/A1 is more frequent than the HBV/A2 in Parana state and its presence was significantly larger in black and mulatto individuals. Genotype C was found only in individuals with Asian ancestry from Londrina and Maringa. Most HBV/F sequences identified in this study were classified as subgenotype F2a that was previously described in Brazil. The sole case of subgenotype F4 was from Foz do Iguacu city, near to Northern Argentina, where F4 is highly prevalent. The single genotype H sample was from Curitiba. This is the first case of this genotype described in Brazil. Further studies should be carried out to determine if more genotype H samples can be found in other populations from Brazil. (C) 2012 Elsevier B.V. All rights reserved.

Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats

Martins JM, Longhi-Balbinot DT, Soares DM, Figueiredo MJ, Malvar D do C, de Melo MC, Rae GA, Souza GE. Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats. J Appl Physiol 113: 1456-1465, 2012. First published August 30, 2012; doi:10.1152/japplphysiol.00936.2011.-This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 x 10(10) colony-forming units (CFU)/cavity, and body temperature (T-b) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T-b, whereas the two higher doses promoted similar long-lasting and sustained T-b increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T-b increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 mu g/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

INTERACTION BETWEEN THE CARBON MONOXIDE AND NITRIC OXIDE PATHWAYS IN THE LOCUS COERULEUS DURING FEVER

FAPESP [2010/50882-1]

E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro

Background: In many types of cancer, prostaglandin E-2 (PGE(2)) is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE(1), PGE(2) and Ibuprofen (IBP) observed in the T98G human glioma cell line in vitro. Methods: T98G human glioma cells were treated with IBP, PGE(1) or PGE(2) at varying concentrations for 24-72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively). The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay. Results: A significant decrease was seen in cell number (54%) in the presence of 50 mu M IBP. Mitotic index and bromodeoxyuridine (BrdU) incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167%) and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74%) and the transwell migration assay (36%). In contrast, the presence of exogenous PGE(1) or PGE(2) caused significant increases in cell number (37% PGE(1) and 45% PGE(2)). When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE(1) (62%) and to a greater extent by PGE(2) (100%). The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE(1) and 44% PGE(2)) and the transwell migration assay (28% PGE(1) and 68% PGE(2)). Conclusions: The present study demonstrated that treatments which alter PGE(1) and PGE(2) metabolism influence the proliferative and apoptotic indices of T98G glioma cells. The migratory capacity of the cells was also significantly affected by the change in prostaglandin metabolism. Modifying PG metabolism remains an interesting target for future studies in gliomas.

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (Bay), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of Bay on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD(2), PGE(2), TXA(2) and LTB4, cytokines TNF-alpha, IL-6, IL-10 and IL-12p70, but not IFN-gamma, and chemokine CCL-2 at different times. The results show that Bay is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2. (c) 2012 Elsevier Ltd. All rights reserved.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement

In a previous study, we reported that the short-term treatment with celecoxib, a nonsteroidal anti-inflammatory drug (NSAID) attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400420 g) were pretreated through oral gavage with celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance: 32.2 +/- 2.49 vs sham: 4.8 +/- 1.79, P<0.05) and celecoxib (appliance: 31.0 +/- 1.45 vs sham: 4.6 +/- 1.82, P<0.05). The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib can indeed be a therapeutic alternative for pain relieve during orthodontic procedures.

Low-level laser therapy in collagenase-induced Achilles tendinitis in rats: Analyses of biochemical and biomechanical aspects

NSAIDs are widely prescribed and used over the years to treat tendon injuries despite its well-known long-term side effects. In the last years several animal and human trials have shown that low-level laser therapy (LLLT) presents modulatory effects on inflammatory markers, however the mechanisms involved are not fully understood. The aim of this study was to evaluate the short-term effects of LLLT or sodium diclofenac treatments on biochemical markers and biomechanical properties of inflamed Achilles tendons. Wistar rats Achilles tendons (n?=?6/group) were injected with saline (control) or collagenase at peritendinous area of Achilles tendons. After 1?h animals were treated with two different doses of LLLT (810?nm, 1 and 3?J) at the sites of the injections, or with intramuscular sodium diclofenac. Regarding biochemical analyses, LLLT significantly decreased (p?<?0.05) COX-2, TNF-a, MMP-3, MMP-9, and MMP-13 gene expression, as well as prostaglandin E2 (PGE2) production when compared to collagenase group. Interestingly, diclofenac treatment only decreased PGE2 levels. Biomechanical properties were preserved in the laser-treated groups when compared to collagenase and diclofenac groups. We conclude that LLLT was able to reduce tendon inflammation and to preserve tendon resistance and elasticity. (c) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:19451951, 2012

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Marine sponges of the order Verongida are a rich source of biologically active bromotyrosine-derived secondary metabolites. However, none of these compounds are known to display anti-inflammatory activity. In the present investigation, we report the anti-inflammatory effects of 11-oxoaerothionin isolated from the Verongida sponge Aplysina fistularis. When RAW264.7 cells and primary macrophages were preincubated with 11-oxoaerothionin and stimulated with LPS (lipopolysaccharide), a concentration-dependent inhibition of iNOS (inducible nitric oxide synthase) protein and NO2- (Nitrite) production were observed. The same effect was observed when proinflammatory cytokines and PGE(2) (Prostaglandin E2) production was evaluated. In summary, we demonstrated that in the presence of LPS, 11-oxoaerothionin suppresses NO2 and iNOS expression as well as inflammatory cytokines and PGE(2).

PTEN Directly Activates the Actin Depolymerization Factor Cofilin-1 During PGE(2)-Mediated Inhibition of Phagocytosis of Fungi

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E-2 (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Temporal relationships of a pulse of prolactin (PRL) to a pulse of a metabolite of PGF2 alpha in mares

Hourly blood samples were collected from 10 mares during 24 h of each of the preluteolytic, luteolytic, and postluteolytic periods. The autocorrelation function of the R program was used to detect pulse rhythmicity, and the intra-assay CV was used to locate and characterize pulses of prolactin (PRL) and a metabolite of prostaglandin F2 alpha (PGFM). Rhythmicity of PRL and PGFM concentrations was detected in 67% and 89% of mares, respectively. Combined for the three periods (no difference among periods), the PRL pulses were 5.2 +/- 0.4 h (mean +/- SEM) at the base, 7.5 +/- 1.5 h between nadirs of adjacent pulses, and 12.3 +/- 1.5 h from peak to peak. The peaks of PRL pulses were greater (P < 0.05) during the luteolytic period (46 +/- 14 ng/mL) and postluteolytic period (52 15 ng/mL) than during the preluteolytic period (17 3 ng/mL). Concentrations of PRL during hours of a PGFM pulse were different (P < 0.003) within the luteolytic period and postluteolytic period and were greatest at the PGFM peak; PRL concentrations during a PGFM pulse were not different during the preluteolytic period. The frequency of the peak of PRL and PGFM pulses occurring at the same hour (synchrony) was greater for the luteolytic period (65%, P < 0.01) and postluteolytic period (50%, P < 0.001) than for the preluteolytic period (17%). This is the first report in mares on characterization and rhythmicity of PRL pulses, synchrony between PRL and PGFM pulses, and greater PRL activity during the luteolytic and postluteolytic periods than during the preluteolytic period. (C) 2012 Elsevier Inc. All rights reserved.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondônia, Brazil

Abstract Background Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondônia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM® 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions These results for the first HBV genotypic characterization in Rondônia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.