Impact of resolvin E1 on murine neutrophil phagocytosis in type 2 diabetes.

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.

LXA4 actions direct fibroblast function and wound closure.

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-ß1, to induce fibroblast activation. The impact of exogenous TGF-ß1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a "scratch" assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-ß1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-ß1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-ß1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Resolvin E1 and chemokine-like receptor 1 mediate bone preservation.

The polyunsaturated ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions.

Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT).

OBJECTIVE: The aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome. DESIGN: Total nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis. RESULTS: The automated extraction method at 63°C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363. CONCLUSIONS: In general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites.

Intermittent therapy with 1,25 vitamin D and calcitonin prevents cyclosporin-induced alveolar bone loss in rats.

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 microg/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 microg/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

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 3(rd), 7(th), 15(th) and 30(th) 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.

The long-term administration of calcineurin inhibitors decreases antioxidant enzyme activity in the rat parotid and submandibular salivary glands.

AIMS: Calcineurin inhibitors are widely used for prevention of graft rejection and treatment of autoimmune disorders, which result in increased longevity and enhanced quality of life for patients. Unfortunately, the toxic side effects of these drugs (mainly renal, hepatic and cardiac) limit their use. In this work, we studied the effects of long-term treatment of rats with the immunosuppressant cyclosporin (CsA) or tacrolimus (Tac) on salivation, saliva composition and on the major salivary glands (parotid and submandibular) in terms of histological alterations and oxidative stress, evaluated as lipoperoxidation (thiobarbituric acid reactive species--TBARS) and antioxidant enzyme activity contents (superoxide dismutase--SOD, catalase--CAT and glutathione peroxidase--GPx). MAIN METHODS: Male adult rats were treated with either CsA (10 mg/kg/day) or Tac (1 mg/kg/day) subcutaneously for 30 or 60 days. At the end of the experimental periods, pilocarpine-stimulated salivary flow rate was measured, saliva samples were collected and the salivary glands were dissected for morphological and biochemical analyses. KEY FINDINGS: After a 60-day treatment with any of the immunosuppressants, the total protein, Ca(2+) and Na(+) saliva concentrations were decreased but salivary flow rates were unaffected. In addition, both parotid and submandibular glands showed decreased SOD, CAT and GPx activities, increased TBARS contents and histomorphological alterations involving the epithelium and acini. SIGNIFICANCE: Based on these results, we suggest that the systemic long-term administration of the calcineurin inhibitor CsA or Tac induces an impairment of the antioxidant enzymatic defense in the rat major salivary glands, which may, in turn, lead to altered saliva composition.

Peripheral blood mononuclear phagocytes from patients with chronic periodontitis are primed for osteoclast formation.

BACKGROUND: During inflammatory periodontal disease, peripheral blood mononuclear cells (PBMCs) are attracted to bone and differentiate into active bone-resorbing osteoclasts (OCs), thus providing evidence that the impact of chronic periodontitis (CP) on the activity of circulating mononuclear cells is of central importance. The authors test the hypothesis that peripheral blood mononuclear phagocytes (PBMPs) from patients with CP are activated and more susceptible to differentiation into OCs, which in turn would lead to more intense bone resorption. METHODS: In vitro cytokine production by both unstimulated and lipopolysaccharide-stimulated PBMCs from individuals with (n = 10) or without (n = 12) periodontitis was determined by cytokine array. OC differentiation from CD14(+) PBMCs was induced by receptor activator of nuclear factor-kappa B ligand (RANKL), either alone or in the presence of macrophage colony-stimulating factor (M-CSF). PBMC differentiation to OCs was confirmed by tartrate-resistant acid phosphatase staining; bone resorbing activity was assessed by using an osteologic plate assay (bone resorption pit formation). RESULTS: PBMCs from patients with CP produced tumor necrosis factor-α and higher amounts of interferon-γ, interleukin (IL)-1α, IL-1ß, IL-1rα, CXC motif chemokine 10, macrophage migration inhibitory factor, macrophage inflammatory protein (MIP)-1α, and MIP-1ß than the control cells. OC differentiation was induced by RANKL alone in PBMCs from patients with CP, but not in PBMCs from the healthy controls, which required the addition of M-CSF. In addition, PBMC-derived OCs from patients with CP showed significantly higher resorption activity than that observed in the healthy controls. Also, the circulating concentrations of M-CSF were significantly higher in patients with CP than in the control participants. CONCLUSIONS: These data indicate that in patients with CP, circulating PBMCs are primed for increased proinflammatory activity and that M-CSF plays a central role in this process by increasing OC formation and the consequent bone resorption activity.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells.

OBJECTIVE: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. DESIGN: Cell line SAOS-2 (1×10(5)cells/mL) were grown in culture medium α-MEM with 10% FBS for 24h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10g/mL) and IL-1ß (1mg/mL) for 24h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO(2)(-) with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. RESULT: There were no significant differences amongst the groups in terms of NO(2)(-), protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p<0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1ß caused up-regulation of this cytokine. CONCLUSIONS: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation.

iNOS-derived nitric oxide stimulates osteoclast activity and alveolar bone loss in ligature-induced periodontitis in rats.

BACKGROUND: Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. METHODS: Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. RESULTS: Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. CONCLUSION: Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.

Influence of antiplatelet drugs in the pathogenesis of experimental periodontitis and periodontal repair in rats.

BACKGROUND: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. METHODS: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. RESULTS: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-α and Il-6 (P <0.05), but only Asp decreased thromboxane A(2) (P <0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P <0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. CONCLUSION: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats.

OBJECTIVE: in this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. MATERIALS AND METHODS: oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. RESULTS: in NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. CONCLUSIONS: in addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney.

O papel da clorexidina no tratamento de pacientes com gengivite no distrito de São Carlos do Jamari - RO / The role of chlorexidine on the treatment of patients with gingivitis in Sao Carlos’ district – RO

A principal causa da gengivite é o acúmulo de placa bacteriana que pode ser evitada com a utilização de meios preventivos mecânicos e químicos. A clorexidina é um agente químico de amplo espectro antibacteriano. O objetivo do estudo foi a avaliação dos efeitos clínicos da clorexidina na saúde gengival, por meio de sua ação antibacteriana no controle da gengivite, em pacientes que apresentam pouca higienização da cavidade bucal no Distrito de São Carlos - Porto Velho – RO. Através do Projeto NAPRA (Núcleo de Apoio a População Ribeirinhada Amazônia) foram selecionados 15 voluntários com gengivite, que foram divididos em grupo controle (cinco voluntários) e grupo clorexidina (10 voluntários). Para o grupo clorexidina foi administrado fármaco a base de clorexidina (Periogard® Colgate) e ao grupo controle foi administrado placebo (água). Os grupos foram monitorados a cada cinco dias, através de exames físicos e Índice Gengival de Lõe, durante um período de 15 dias. Antes do início do estudo, tanto o grupo estudo como o grupo controle encontravam-se equilibrados estatisticamente quanto à média de Índice Gengival de Lõe. Foi observado que o uso de clorexidina por um periodo de 10 dias reduziu e controlou significantemente a gengivite em pacientes que apresentavam higiene bucal deficiente, através de sua ação antibacteriana.