Eraldo L. Batista

Role for periodontitis in the progression of lipid deposition in an animal model.

ABSTRACT Epidemiologic studies have implicated periodontitis as a risk factor for the development of cardiovascular disease. However, no prospective studies investigating this potential relationship have been carried out. Age- and sex-matched New Zealand White rabbits were maintained on a diet consisting of 0.5% fat for 13 weeks to induce the accumulation of lipid deposits in the aorta as a model for atherogenesis. One-half of the animals received silk ligatures around their mandibular premolars followed by an application of a periodontal pathogen, Porphyromonas gingivalis, to induce periodontitis. Animals were sacrificed after 14 weeks. Periodontal disease severity was quantified radiographically, histologically, and by direct visualization of bone loss on defleshed skulls. Lipid deposition was evaluated by computer-assisted morphometry in the aortas en face after lipid deposits were stained with Sudan IV. Animals with experimentally induced periodontitis had more extensive accumulations of lipids in the aorta than did nonperiodontitis animals (P < 0.05), and there was a positive correlation between the severity of periodontal disease and the extent of lipid deposition (r2 = 0.9501). The results provide direct evidence that periodontitis may be a risk factor and may contribute to the pathogenesis of atherosclerosis. The data support the concept that infections at remote locations can modulate atherosclerotic events distantly.

Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase.

Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia‐induced changes was studied by using neutrophil‐like HL‐60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL‐60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL‐60 cells incubated with HG and S100B. HL‐60 cells cultured in HG and S100B exhibited a 1.8‐fold increase in fMLP‐induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE‐ERK1/2 pathway.

Response to Intracanal Medication in Immature Teeth with Pulp Necrosis: An Experimental Model in Rat Molars

INTRODUCTION The present study aimed at developing an experimental model in rat molars for evaluating treatment strategies in necrotic immature teeth. METHODS To define the periods to be adopted in the experimental procedures and to confirm induction of periapical lesions and interruption of root embryogenesis, the left lower first molars of 4-weeks-old Wistar rats underwent pulpectomy and were left open to the oral environment. Comparisons with the right lower first molars (vital teeth) were performed in animals with ages of 7, 10, 13, and 16 weeks. In another group of animals the teeth were left open for 3 weeks, and then interventions for disinfection including the use of an antibiotic paste were carried out. Root formation was then assessed after 3 and 6 weeks on the basis of radiographic and histologic evaluation. RESULTS Vital teeth showed increase of root length and hard tissue thickness throughout the experimental periods. On the other hand, induction of necrosis arrested root formation. Teeth subjected to disinfection with sodium hypochlorite associated with the triple antibiotic paste showed significant reduction of periapical lesions, gain in root length, and increased wall thickness compared with the control (P < .05). CONCLUSIONS The root canal disinfection protocol used was able to reduce periapical lesion size and improve root development. The experimental model presented should contribute to studies that aim at improving therapeutic strategies for necrotic immature teeth by using a rat model.

Differentiation of HL-60 cells to granulocytes involves regulation of select Diacylglycerol Kinases (DGKs)

Diacylglycerol Kinases (DGKs) are a family of enzymes that regulate the levels of different pools of diacylglycerol (DAG), affecting DAG‐mediated signal transduction. Since DAG is known to play several important regulatory roles in granulocyte physiology, we investigated the expression pattern of DGK isoforms throughout differentiation of HL‐60 cells to granulocytes. HL‐60 cells were incubated with 1.25% dimethyl‐sulfoxide (DMSO) to initiate differentiation and total RNA isolated at different time points. DGK expression was assessed through Northern blot, end‐point PCR, and real‐time PCR. The non‐selective inhibitors R59022 and R59949 were used to block DGK at different time points throughout differentiation. CD11b and GPI‐80, reactive oxygen species (ROS) generation, changes in the cell cycle, and apoptosis were used as markers of differentiation. Of the nine isoforms of DGK evaluated (α, δ, ε, γ, ζ, β, θ, ι, η), only five (α, δ, ε, γ, and ζ) were expressed in HL‐60 cells. DGKα was virtually absent in non‐differentiated cells, but was markedly upregulated throughout differentiation. The other isoforms (δ, ε, γ, and ζ) were expressed in undifferentiated HL‐60 cells but were substantially decreased throughout differentiation. Non‐selective blocking of DGK with R59022 and R59949 led to acceleration of differentiation, reducing the time necessary to observe upregulation of CD11b, GPI‐80 and generation of ROS by 50%. Likewise, the cell cycle was disrupted when DGK isoforms were inhibited. These results provide evidence that DGK levels are dynamically regulated throughout differentiation and that expression of DGKs play an important regulatory function during the differentiation of neutrophils.

In Vivo Up-Regulation of Kinin B1 Receptors after Treatment with Porphyromonas gingivalis Lipopolysaccharide in Rat Paw

It has been demonstrated that kinin B1 receptors are highly up-regulated under several stressful stimuli, such as infection. However, there is no evidence indicating whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) might lead to B1 receptor up-regulation. In this study, we demonstrate that Pg-LPS injection into the rat paw resulted in a marked functional up-regulation of B1 receptors (as measured by an increase of B1 receptor-induced edema), which was preceded by a rapid rise in B1 receptor mRNA expression. The local administration of Pg-LPS also resulted in a prominent production of the proinflammatory cytokine tumor necrosis factor α (TNF-α), followed by an increase of neutrophil influx; both events were observed at periods before B1 receptor induction. The functional and molecular Pg-LPS-elicited B1 receptor up-regulation was significantly reduced by the glucocorticoid dexamethasone (0.5 mg/kg s.c.), and to a lesser extent by the chimeric anti-TNF-α antibody infliximab (1 mg/kg s.c.). Of high relevance, we show for the first time that a single administration of the proresolution lipid mediator (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (resolvin E1; 300 ng/rat i.p.) was able to markedly down-regulate Pg-LPS-driven B1 receptor expression, probably by inhibiting TNF-α production and neutrophil migration. Collectively, the present findings clearly suggest that Pg-LPS is able to induce the up-regulation of B1 receptors through mechanisms involving TNF-α release and neutrophil influx, which are largely sensitive to resolvin E1. It is tempting to suggest that kinin B1 receptors might well represent a pivotal pathway for the inflammatory responses evoked by P. gingivalis and its virulence factors.

Protective Effects of Resveratrol on Hepatotoxicity Induced by Isoniazid and Rifampicin via SIRT1 Modulation

Acute liver injury was induced in male BALB/c mice by coadministering isoniazid and rifampicin. In this work, the effects of resveratrol (1) were investigated in the hepatotoxicity caused by isoniazid-rifampicin in mice. Compound 1 was administered 30 min prior to isoniazid-rifampicin. Serum biochemical tests, liver histopathological examination, oxidative stress, myeloperoxidase activity, cytokine production (TNF-α, IL-12p70, and IL-10), and mRNA expression of SIRT1-7 and PPAR-γ/PGC1-α were evaluated. The administration of 1 significantly decreased aspartate transaminase and alanine aminotransferase levels, myeloperoxidase activity, and cytokine levels. Furthermore, 1 reverted the decrease of catalase and glutathione activities and ameliorated the histopathological alterations associated with antituberculosis drugs. Modulation of SIRT1 and PPAR-γ/PGC1-α expression is likely involved in the protective effects of 1. The results presented herein show that 1 was able to largely prevent the hepatotoxicity induced by isoniazid and rifampicin in mice, mainly by modulating SIRT1 mRNA expression.

Moesin-induced signaling in response to lipopolysaccharide in macrophages

BACKGROUND AND OBJECTIVE Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane-organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)-mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS-induced moesin signaling pathways in macrophages. MATERIAL AND METHODS Macrophages were stimulated with 500 ng/mL LPS in macrophage serum-free medium. For blocking experiments, cells were pre-incubated with anti-moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real-time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD-2 and Toll-like receptor 4 (TLR4) and co-immunoprecipitation of MyD88-interleukin-1 receptor-associated kinase (IRAK) and IRAK-tumor necrosis factor receptor-activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor kappaB (NF-kappaB) and IkappaBalpha were studied. Tumor necrosis factor alpha, interleukin-1beta and interferon beta were measured by ELISA. RESULTS Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl-terminus. Moesin is temporally associated with TLR4 and MD-2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide-induced signaling is transferred downstream to p38, p44/42 MAPK and NF-kappaB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF-kappaB activation and translocation to the nucleus. CONCLUSION These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.

Apical periodontium response to enamel matrix derivative as an intracanal medication in rat immature teeth with pulp necrosis: radiographic and histologic findings.

INTRODUCTION The aim of this study was to evaluate the enamel matrix derivative (EMD) biomaterial in nonvital immature teeth. METHODS To arrest root development, pulpectomies were performed in the lower first molars of 36 4-week-old rats; the cavities were left exposed to the oral environment for 3 weeks. Then, chemical disinfection was performed, and triple antibiotic paste (TAP) or EMD was applied in the root canals. A control group did not receive any treatment. Radiographic and histological data were evaluated after 3 and 6 weeks. RESULTS At 3 weeks, TAP promoted a milder inflammatory response and increased root lengths compared with the control group. At 6 weeks, root development and reduced periapical lesions could be observed in both test groups, mainly because of the deposition of a cementum-like tissue. EMD promoted narrower canals compared with TAP (P < .05). CONCLUSIONS EMD deserves attention as a potential tool in the treatment of nonvital immature teeth. The ingrowth of cementum-like tissues into canal spaces favored dental wall thickness and may contribute to tooth resistance and support.

Increased Tissue Factor Expression and Poor Nephroblastoma Prognosis

PURPOSE There is potential interaction between malignant cell growth and the coagulation pathway. Recent studies suggest that tissue factor, a primary initiator of the extrinsic coagulation pathway, is expressed in various solid tumors in association with increased angiogenesis. To our knowledge we report for the first time the detection of tissue factor expression by immunohistochemistry in Wilms tumors and its correlation with clinical outcomes. MATERIAL AND METHODS Tissue factor expression detected by immunohistochemistry was assessed in 41 formalin fixed, paraffin embedded Wilms tumor cases treated at university hospitals. We correlated findings with tumor recurrence and cancer specific survival. RESULTS Positive immunohistochemistry detection of tissue factor was observed in 88.3% of the tumors analyzed. Tissue factor on immunohistochemistry was associated with tumor recurrence and survival (p = 0.01 and 0.02, respectively). Increased immunohistochemical detection of tissue factor was the most important risk factor for recurrence and mortality in our population on bivariate and multivariate analysis. CONCLUSIONS Tissue factor is a promising research subject as a prognostic factor for Wilms tumor. More studies are needed to clarify the mechanisms by which tissue factor affects cancer progression and outcome, and its potential role as a therapeutic target.

Analysis of select members of the E26 (ETS) transcription factors family in colorectal cancer

The E-twenty-six (ETS) family of transcription factors is known to act as positive or negative regulators of the expression of genes that are involved in diverse biological processes, including those that control cellular proliferation, differentiation, hematopoiesis, apoptosis, metastasis, tissue remodeling, and angiogenesis. Identification of target gene promoters of normal and oncogenic transcription factors provides new insights into the regulation of genes that are involved in the control of normal cell growth and differentiation. The aim of the present investigation was to analyze the differential expression of 11 ETS (ELF-3, ESE3, ETS1, ETV3, ETV4, ETV6, NERF, PDEF, PU1, Spi-B, and Spi-C) as potential markers for prognostic of colorectal cancer. A series of paired tissue biopsies consisting of a tumor and a non-affected control sample were harvested from 28 individuals suffering from diagnosed colorectal lesions. Total RNA was isolated from the samples, and after reverse transcription, differential expression of the select ETS was carried out through real-time polymerase chain reaction. Tumor staging as determined by histopathology was carried out to correlate the degree of tumor invasiveness with the expression of the ETS genes. The results demonstrated a different quantitative profile of expression in tumors and normal tissues. ETV4 was significantly upregulated with further increase in the event of lymph node involvement. PDEF and Spi-B presented downregulation, which was more significant when lymph node involvement was present. These findings were supported by immunohistochemistry of tumoral tissues. The results suggest that select ETS may serve as potential markers of colorectal cancer invasiveness and metastasis.