Progression of articular and extraarticular damage in oligoarticular juvenile idiopathic arthritis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers

A protocol for DNA damage assessment by the single-cell gel (SCG)/comet assay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcosinate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 mum, to exclude contaminating leukocytes. Distinguishing cell populations is crucial, because significant differential migration was demonstrated for transitional and nontransitional cells, phenomena that may confound the results. When applying the modified protocol to urinary bladder cells from smokers without urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3.23; mean 95% confidence interval; n = 18) when compared with nonsmokers (1.94 +/- 1.41; n = 12). No significant differences were observed between ex-smokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age in nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenesis. Therefore, the present data directly support the classification of individuals with smoking history as patients at high risk for urinary bladder cancer.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of low-level laser therapy on the healing process after tooth replantation: a histomorphometrical and immunohistochemical analysis

Success of tooth replantation is limited because part of the replanted tooth is lost because of progressive root resorption. This study used histomorphometry and immunohistochemistry to evaluate the effect of low-level laser therapy (LLLT) on the healing process of rat teeth replanted after different extra-oral periods, simulating immediate and delayed replantation. Sixty Wistar rats (Rattus norvegicus albinus) had their maxillary right incisors extracted and randomly assigned to six groups (n = 10): C4, C30 and C45, in which the teeth were replanted 4 min (immediate), 30 min (delayed) and 45 min (delayed) after extraction, respectively, and L4, L30 and L45, in which the teeth were replanted after the same extra-alveolar times, but the root surfaces and the alveolar wounds were irradiated with a gallium-aluminum-arsenate (GaAlAs) diode laser before replantation. The animals were sacrificed after 60 days. The anatomic pieces containing the replanted teeth were obtained and processed for either histomorphometrical analysis under optical microscopy or immunohistochemical expression of receptor activator of nuclear factor Kappa-B (RANK), and its ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP) proteins. Areas of external replacement and inflammatory root resorption were observed in all groups, without statistically significant differences (P > 0.05). Ankylosis was more frequent in L30 than in C30 (P < 0.05). RANKL immunostaining predominated over RANK and OPG immunostaining in both groups with immediate tooth replantation (P < 0.05). For the 45-min extra-alveolar time, however, there was greater evidence of RANK immunostaining compared to RANKL for both control and laser-treated groups (P < 0.05). Positive TRAP immunostaining predominated in L4 and L30 (P < 0.05). In conclusion, under the tested conditions, the treatment of the root surface and the alveolar wound with LLLT did not improve the healing process after immediate and delayed tooth replantation in rats.

Nicotine-Induced Damage Affects Gingival Fibroblasts in the Gingival Tissue of Rats

Background: Very limited information is available from in vivo studies about whether smoking and/or nicotine affect gingival tissues in the absence of plaque. The purpose of this study is to evaluate the effect of the systemic administration of nicotine in the proliferation and counting of fibroblast-like cells in the gingival tissue of rats.Methods: Thirty adult male Wistar rats were randomly assigned into two groups to receive subcutaneous injections of a saline solution (control group = group C) or nicotine solution (group N; 3 mg/kg) twice a day. The animals were euthanized 37, 44, or 51 days after the first subcutaneous injection. Specimens were routinely processed for serial histologic sections. Five fields of view in the connective tissue adjacent to the gingival epithelium and above the alveolar bone crest of the maxillary first molar were selected for the counting of fibroblast-like cells. Data were statistically analyzed (P<0.05).Results: The intergroup analysis detected a lower number of fibroblast-like cells in group N compared to group C on days 37 (2.65 +/- 1.41 and 6.67 +/- 3.25, respectively), 44 (2.70 +/- 1.84 and 8.57 +/- 2.37, respectively), and 51(2.09 +/- 1.41 and 7.49 +/- 2.60, respectively) (P<0.05). The quantification of fibroblast-like cells showed no significant difference (P >0.05) in the intragroup analysis of control and nicotine throughout experimental periods. In the intergroup analysis, group N had reduced proliferating cell nuclear antigen positive fibroblasts compared to group C in all periods (P<0.05).Conclusion: The daily systemic administration of nicotine negatively affected, in vivo, the number and proliferation of fibroblast-like cells in the gingival tissue of rats. J Periodontol 2011;82:1206-1211.

Analysis of failed commercially pure titanium dental implants: A scanning electron microscopy and energy-dispersive spectrometer X-ray study

Background: the failure of osseointegration in oral rehabilitation has gained importance in current literature and in clinical practice. The integration of titanium dental implants in alveolar bone has been partly ascribed to the biocompatibility of the implant surface oxide layer. The aim of this investigation was to analyze the surface topography and composition of failed titanium dental implants in order to determine possible causes of failure.Methods: Twenty-one commercially pure titanium (cpTi) implants were retrieved from 16 patients (mean age of 50.33 +/- 11.81 years). Fourteen implants were retrieved before loading (early failures), six after loading (late failures), and one because of mandibular canal damage. The failure criterion was lack of osseointegration characterized as dental implant mobility. Two unused implants were used as a control group. All implant surfaces were examined by scanning electron microscopy (SEM) and energy-dispersive spectrometer x-ray (EDS) to element analysis. Evaluations were performed on several locations of the same implant.Results: SEM showed that the surface of all retrieved implants consisted of different degrees of organic residues, appearing mainly as dark stains. The surface topography presented as grooves and ridges along the machined surface similar to control group. Overall, foreign elements such as carbon, oxygen, sodium, calcium, silicon, and aluminum were detected in failed implants. The implants from control group presented no macroscopic contamination and clear signs of titanium.Conclusion: These preliminary results do not suggest any material-related cause for implant failures, although different element composition was assessed between failed implants and control implants.

Diffusion analysis of one photosensitizer in bovine teeth using fluorescence optical imaging

Some photosensitizers (PSs) used for PACT (Antimicrobial Photodynamic Therapy) show an affinity for bacterial walls and can be photo-activated to cause the desired damage. However, on dentine bacterias may be less susceptible to PACT as a result of limited penetration of the PS. The aim of this study was to evaluate the diffusion of one PS based on hematoporphyrin on dentine structures. Twelve bovine incisors were used. Class III cavities (3 x 3 x 1 mm) were prepared on the mesial or distal surfaces using a diamond bur. Photogem (R) solution at 1 mg/mL (10 uL for each cavity) was used. The experimental Groups were divided according to thickness of dentine remaining and etched or no-etched before the PS application. The fluorescence excitation source was a VelScope (R) system. For image capture a scientific CCD color camera PixelFly (R) was coupled to VelScope. For image acquisition and processing, a computational routine was developed at Matlab (R). Fick's Law was used to obtain the average diffusion coefficient of PS. Differences were found between all Groups. The longitudinal temporal diffusion was influenced by the different times, thickness and acid etching.

Antioxidant activity of indigo and its preventive effect against ethanol-induced DNA damage in rat gastric mucosa

Ethanol-induced oxidative damage is commonly associated with the generation of reactive oxygen molecules, leading to oxidative stress. Considering that antioxidant activity is an important mechanism of action involved in cytoprotection, the aim of this work was to evaluate the antioxidant properties of the alkaloid indigo (1) (2 mg/kg, p. o.), obtained from the leaves of Indigofera truxillensis Kunth (Fabaceae), on rat gastric mucosa submitted to ethanol-induced (100%, 1 mL, p.o.) gastric ulcer. Enzymatic assays and DNA fragmentation analysis were performed. When ethanol was administered to the control group, the sulfhydryl content (SH) and the glutathione peroxidase (GPx) activity decreased by 41% and 50%, respectively; in contrast, superoxide dismutase (SOD) and glutathione reductase (GR) activities increased by 56% and 67%, respectively. Additionally, myeloperoxidase (MPO) activity, a marker for free radical generation caused by polymorphonuclear neutrophil (PMN) tissue infiltration, also increased 4.5-fold after ethanol treatment. Rat gastric mucosa exposed to ethanol showed DNA fragmentation. Indigo alkaloid pretreatment protected rats from ethanol-induced gastric lesions. This effect was determined by the ulcerative lesion area (ULA), indicating an inhibition of around 80% at 2 mg/kg. This alkaloid also diminished GPx activity, which was higher than that observed with ethanol alone. However, this effect was counterbalanced by increased GR activity. Indigo was unable to restore alterations in SOD activity promoted by ethanol. After indigo pretreatment, SH levels and MPO activity remained normal and gastric mucosa DNA damage caused by ethanol was also partially prevented by indigo. These results suggest that the gastroprotective mechanisms of indigo include non-enzymatic antioxidant effects and the inhibition of PMN infiltration which, in combination, partially protect the gastric mucosa against ethanol-induced DNA damage.

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Effects of ginger (Zingiber officinale Roscoe) on DNA damage and development of urothelial tumors in a mouse bladder carcinogenesis model

Extracts of the spice ginger (Zingiber officinale Roscoe) are rich in gingerols and shogaols, which exhibit antioxidant, anti-inflammatory, antifungal, anti mycobacterial, and anticarcinogenic proprieties. The present study evaluated the chemoprotective effects of a ginger extract on the DNA damage and the development of bladder cancer induced by N-butyl-N-(4-hydroxibutyl) nitrosamine (BBN)/N-methyl-N-nitrosourea (MNU) in male Swiss mice. Groups G1-G3 were given 0.05% BBN in drinking water for 18 weeks and four i.p. injections of 30 mg/kg body weight MNU at 1, 3, 10, and 18 weeks. Group G4 and G5 received only the BBN or MNU treatments, respectively, and groups G6 and G7 were not treated with BBN or MNU. Additionally, Groups G2, G3, and G6 were fed diets containing 1, 2, and 2% ginger extract, respectively, while Groups G1, G4, G5, and G7 were fed basal diet. Samples of peripheral blood were collected during the experiment for genotoxicity analysis; blood collected 4 hr after each MNU dose was used for the analysis of DNA damage with the Comet assay (assay performed on leukocytes from all groups), while reficulocytes collected 24 hr after the last MNU treatment of Groups G5-G7 were used for the micronucleus assay. At the end of the experiment, the urinary bladder was removed, fixed, and prepared for histopathological, cell proliferation, and apoptosis evaluations. Ginger by itself was not genotoxic, and it did not alter the DNA damage levels induced by the BBN/MNU treatment during the course of the exposure. The incidence and multiplicity of simple and nodular hyperplasia and transitional cell carcinoma (TCC) were increased by the BBN/MNU treatment, but dietary ginger had no significant effect on these responses. However, in Group G2 (BBN/MNU/2% ginger-treated group), there was an increased incidence of Grade 2 TCC. The results suggest that ginger extract does not inhibit the development of BBN-induced mouse bladder tumors.

Analysis of Mortality in Africanized Honey Bee Colonies with High Levels of Infestation by Varroa destructor

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultrastructural Analysis of the Oocytes of Female Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) Ticks Subjected to the Action of Azadirachta indica A. Juss (Neem)

The present study provides ultrastructural information about the acaricidal effects of neem (Azadirachta indica A. Juss) on the ovaries of R. sanguineus engorged females. In general, the main damage caused in the oocytes was alteration in the shape of the cell and of the germinal vesicle, ring-shaped nucleolus, cytoplasmic vacuolation, and disorganization of the organelles and of the cell membranes (including the chorion), all of which indicate that these cells could be in the process of death. The results showed that azadirachtin would be an efficient acaricide agent for inhibiting and/or neutralizing the reproduction process of R. sanguineus females, impairing the reproductive ability of this species.

Brown Recluse Spider Venom: Proteomic Analysis and Proposal of a Putative Mechanism of Action

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteomic analysis of kidney in rats chronically exposed to fluoride

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1 beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.