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 mu g/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 mu g/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.

Simvastatin therapy in cyclosporine A-induced alveolar bone loss in rats

Background and Objective: Cyclosporine A treatment is important in the therapy of a number of medical conditions; however, alveolar bone loss is an important negative side-effect of this drug. As such, we evaluated whether concomitant administration of simvastatin would minimize cyclosporine A-associated alveolar bone loss in rats subjected, or not, to experimental periodontal disease. Material and Methods: Groups of 10 rats each were treated with cyclosporine A (10 mg/kg/day), simvastatin (20 mg/kg/day), cyclosporine A and simvastatin concurrently (cyclosporine A/simvastatin) or vehicle for 30 days. Four other groups of 10 rats each received a cotton ligature around the lower first molar and were treated similarly with cyclosporine A, simvastatin, cyclosporine A/simvastatin or vehicle. Calcium (Ca(2+)), phosphorus and alkaline phosphatase levels were evaluated in serum. Expression levels of interleukin-1 beta, prostaglandin E(2) and inducible nitric oxide synthase were evaluated in the gingivomucosal tissues. Bone volume and numbers of osteoblasts and osteoclasts were also analyzed. Results: Treatment with cyclosporine A in rats, with or without ligature, was associated with bone loss, represented by a lower bone volume and an increase in the number of osteoclasts. Treatment with cyclosporine A was associated with bone resorption, whereas simvastatin treatment improved cyclosporine A-associated alveolar bone loss in all parameters studied. In addition, simvastatin, in the presence of inflammation, can act as an anti-inflammatory agent. Conclusion: This study shows that simvastatin therapy leads to a reversal of the cyclosporine A-induced bone loss, which may be mediated by downregulation of interleukin-1 beta and prostaglandin E(2) production.

Hyaluronidase increases the duration of mepivacaine in inferior alveolar nerve blocks

Purpose: To evaluate the duration of the effect of mepivacaine when hyaluronidase is injected immediately prior to the end of pulpal anesthesia. Patients and Methods: Forty bilateral, symmetrical third molar surgeries were performed in 20 healthy patients. Inferior alveolar nerve block was induced using 2.8 mL 2% mepivacaine with epinephrine. Hyaluronidase (75 turbidity-reducing units) or a placebo was injected 40 minutes after the beginning of pulpar anesthesia (randomized and double-blind trial). The duration of effect in the pulpal and gingival tissues was evaluated by response to painful electrical stimuli applied to the Adjacent premolar, and by mechanical stimuli (pin prick) to the vestibular gingiva, respectively. Results: in both tissues, the duration of anesthetic effect with hyaluronidase was longer (P <.01) than with the placebo. Conclusion: Hyaluronidase increases the duration of mepivacaine in inferior alveolar nerve blocks. (c) 2008 American Association of Oral and Maxillofacial Surgeons.

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This `experiment of nature` indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Pivotal Role for Platelet-Activating Factor Receptor in CD36 Expression and oxLDL Uptake by Human Monocytes/Macrophages

The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined. Copyright (C) 2011 S. Karger AG, Basel

Cell activation state influences the modulation of HLA-DR surface expression on human monocytes/macrophages by parenteral fish oil lipid emulsion

Abnormal surface expression of HLA-DR by leukocytes is associated with a poor prognosis in critical care patients. Critical care patients often receive total parenteral nutrition with lipid emulsion (LE). In this study we evaluated the influence of fish oil LE (FO) on human monocyte/macrophage (M phi) expression of surface HLA-DR under distinct activation states. Mononuclear leukocytes from the peripheral blood of healthy volunteers (n = 18) were cultured for 24 hours without LE (control) or with 3 different concentrations (0.1, 0.25, and 0.5%) of the follow LE: a) pure FO b) FO in association (1:1 v/v) with LE composed of 50% medium-chain trygliceride and 50% soybean oil (MCTSO), and c) pure MCTSO. The leukocytes were also submitted to different cell activation states, as determinate by INF-gamma addition time: no INF-gamma addition, 18 hours before, or at the time of LE addition. HLA-DR expression on M phi surface was evaluated by flow cytometry using specific monoclonal antibodies. In relation to controls (for 0.1%, 0.25%, and 0.5%: 100) FO decreased the expression of HLA-DR when added alone [in simultaneously-activated M phi, for 0.1%: 70 (59 +/- 73); for 0.25%: 51 (48 +/- 56); and for 0.5%: 52.5(50 +/- 58)] or in association with MCTSO [in simultaneously-activated M phi, for 0.1%: 50.5 (47 +/- 61); for 25%: 49 (45 +/- 52); and for 05 %: 51 (44 +/- 54) and in previously-activated M phi, for 1.0 % : 63 (44 +/- 88); for 0.25%: 70 (41 +/- 88); and for 0.5%: 59.5 (39 +/- 79)] in culture medium (Friedman p<0.05). In relation to controls (for 0.1%, 0.25%, and 0.5%: 100), FO did not influence the expression of these molecules on non-activated M phi [for 0.1 % : 87.5 (75 +/- 93); for 0.25%: 111 (98 +/- 118); and for 0.5%: 101.5 (84 +/- 113)]. Results show that parenteral FO modulates the expression of HLA-DR on human M phi surface accordingly to leukocyte activation state. Further clinical studies evaluating the ideal moment of fish oil LE infusion to modulate leukocyte functions may contribute to a better understanding of its immune modulatory properties.

TLR4/MYD88-dependent, LPS-induced synthesis of PGE(2) by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells

Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E-2(PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE2 in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.

CCR5 Mediates Pro-osteoclastic and Osteoclastogenic Leukocyte Chemoattraction

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14, CCR2, TNF-alpha, and IL-1 beta, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-gamma, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-alpha+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-alpha, IL-1 beta, IL-6, IFN-gamma, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Influencia do meloxicam sobre a perda ossea alveolar em periodontite experimental: avaliaçao histometrica em ratos

GURGEL, Bruno Cesar de vasconcelos.Influencia do meloxicam sobre a perda ossea alveolar em periodontite experimental: avaliaçao histometrica em ratos. 2003.97f. Dissertaçao (Mestrado) - Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. Piracicaba, 2003. Disponivel em: . Acesso em: 04 out. 2010.

Origem física das curvas sigmoidais respiratórias pressão-volume: recrutamento alveolar e elasticidade não-linear

An important unsolved problem in medical science concerns the physical origin of the sigmoidal shape of pressure volume curves of healthy (and some unhealthy) lungs. Such difficulties are expected because the lung, which is the most important structure in the respiratory system, is extremely complex. Its rheological properties are unknown and seem to depend on phenomena occurring from the alveolar scale up to the thoracic scale. Conventional wisdom holds that linear response, i.e., Hooke s law, together with alveolar overdistention, play a dominant role in respiration, but such assumptions cannot explainthe crucial empirical sigmoidal shape of the curves. In this doctorate thesis, we propose an alternative theory to solve this problem, based on the alveolar recruitment together with the nonlinear elasticity of the alveoli. This theory suggests that recruitment may be the predominant factor shaping these curves in the entire range of pressures normally employed in experiments. The proposed model correctly predicts the observed sigmoidal pressure volume curves, allowing us to discuss adequately the importance of this result, as well as its implications for medical practice