Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Differential expression of osteoblast and osteoclast chemmoatractants in compression and tension sides during orthodontic movement

Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading, and is supposed to be mediated by several host mediators, such as chemokines. In this study we investigated the pattern of mRNAs expression encoding for osteoblast and osteoclast related chemokines, and further correlated them with the profile of bone remodeling markers in palatal and buccal sides of tooth under orthodontic force, where tensile (T) and compressive (C) forces, respectively, predominate. Real-time PCR was performed with periodontal ligament mRNA from samples of T and C sides of human teeth submitted to rapid maxillary expansion, while periodontal ligament of normal teeth were used as controls. Results showed that both T and C sides exhibited significant higher expression of all targets when compared to controls. Comparing C and T sides, C side exhibited higher expression of MCP-1/CCL2, MIP-1 alpha/CCL3 and RANKL, while T side presented higher expression of OCN. The expression of RANTES/CCL5 and SDF-1/CXCL12 was similar in C and T sides. Our data demonstrate a differential expression of chemokines in compressed and stretched PDL during orthodontic tooth movement, suggesting that chemokines pattern may contribute to the differential bone remodeling in response to orthodontic force through the establishment of distinct microenvironments in compression and tension sides. (C) 2008 Elsevier Ltd. All rights reserved.

PPAR-gamma agonist rosiglitazone prevents inflammatory periodontal bone loss by inhibiting osteoclastogenesis

Rosiglitazone (RGZ), an oral anti-hyperglycemic agent used for non-insulin-dependent diabetes mellitus, is a high-affinity synthetic agonist for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Both in vitro and in vivo experiments have also revealed that RGZ possesses anti-inflammatory properties. Therefore, in the present study, we investigated the anti-inflammatory effects of RGZ in a rat model of periodontal disease induced by ligature placed around the mandible first molars of each animal. Male Wister rats were divided into four groups: 1) animals without ligature placement receiving administration of empty vehicle (control); 2) animals with ligature receiving administration of empty vehicle; 3) animals with ligature receiving administration with oral RGZ (10 mg/kg/day); and 4) animals with ligature receiving administration of subcutaneous RGZ (10 mg/kg/day). Thirty days after induction of periodontal disease, the animals were sacrificed, and mandibles and gingival tissues were removed for further analysis. An in vitro assay was also employed to test the inhibitory effects of RGZ on osteoclastogenesis. Histomorphological and immunohistochemical analyses of periodontal tissue demonstrated that RGZ-treated animals presented decreased bone resorption, along with reduced RANKL expression, compared to those animals with ligature, but treated with empty vehicle. Corresponding to such results obtained from in vivo experiments, RGZ also suppressed in vitro osteoclast differentiation in the presence of RANKL in MOCP-5 osteoclast precursor cells, along with the down-regulation of the expression of RANKL-induced TRAP mRNA. These data indicated that RGZ may suppress the bone resorption by inhibiting RANKL-mediated osteoclastogenesis elicited during the course of experimental periodontitis in rats. (C) 2009 Elsevier B.V. All rights reserved.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease

Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1 beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappa B ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

Clastic cells: Mineralized tissue resorption in health and disease

Clastic cells are responsible for mineralized tissue resorption. Bone resorbing cells are called osteo-clasts; however, they are able to resorb mineralized dental tissues or calcified cartilage and then they are called odontoclasts and chondroclasts, respectively. They derive from mononuclear precursors of the monocyte-macrophage lineage from hemopoietic tissue, reach target mineralized tissues and degrade them under many different physiologic or pathologic stimuli. Clastic cells play a key role in calcium homeostasis, and participate in skeletal growth, tooth movement, and other physiological and pathological events. They interact tightly with forming cells in bone and dental hard tissues; their unbalance may result in disturbed resorptive activity thus, causing local or systemic diseases. (C) 2008 Elsevier Ltd. All rights reserved.

Effect of diabetes on orthodontic tooth movement in a mouse model

Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading. Type 1 diabetes results in bone remodeling, suggesting that this disease might affect orthodontic tooth movement. The present study investigated the effects of the diabetic state on orthodontic tooth movement. An orthodontic appliance was placed in normoglycemic (NG), streptozotocin-induced diabetes (DB), and insulin-treated DB (IT) C57BL6/J mice. Histomorphometric analysis and quantitative PCR of periodontium were performed. The DB mice exhibited greater orthodontic tooth movement and had a higher number of tartrate-resistant acid phosphate (TRAP) -positive osteoclasts than NG mice. This was associated with increased expression of factors involved in osteoclast activity and recruitment (Rankl, Csf1, Ccl2, Ccl5, and Tnfa) in DB mice. The expression of osteoblastic markers (Runx2, Ocn, Col1, and Alp) was decreased in DB mice. Reversal of the diabetic state by insulin treatment resulted in morphological findings similar to those of NG mice. These results suggest that the diabetic state up-regulates osteoclast migration and activity and down-regulates osteoblast differentiation, resulting in greater orthodontic tooth movement.

The Potential Role of Suppressors of Cytokine Signaling in the Attenuation of Inflammatory Reaction and Alveolar Bone Loss Associated with Apical Periodontitis

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which down-regulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development. (J Endod 2008;34:1480-1484)

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

O papel do Fator de Necrose Tumoral Alfa (TNF-alfa) no processo de erosão óssea presente no colesteatoma adquirido da orelha média

O colesteatoma adquirido da orelha média causa erosão óssea, com altas taxas de morbidade e mortalidade. O TNF-alfa (TNF-alfa) lambda uma das principais citocinas envolvidas neste processo. OBJETIVO: Avaliar o papel do TNF-alfa na reabsorsão óssea e a ação dele no colesteatoma. MATERIAL E MÉTODOS: Foi realizado um levantamento e uma revisão crítica da literatura. RESULTADOS: Todos os autores estudados concordam com a importância do TNF-alfa no processo de reabsorção óssea presente no colesteatoma e com o grau de destruição observado. Diferentes trabalhos demonstraram que o TNF-alfa é capaz de provocar erosão óssea, através de diferentes vias de ação. Ele pode estimular a diferenciação e a maturação dos osteoclastos ou, ainda, agir na matriz óssea expondo-a à ação dos osteoclastos. Existe a possibilidade de inibir a ação do TNF-alfa, diminuindo seus efeitos e prevenindo a perda óssea em doenças como a artrite reumatóide. Não existe, entretanto, trabalhos específicos em colesteatoma. Não existe consenso sobre a sua localização. Estas diferenças, provavelmente, ocorrem devido à distribuição dos receptores. CONCLUSÃO: O TNF-alfa, presente no colesteatoma promove a reabsorsão óssea, juntamente com outras citocinas (RANKL e IL-1), estando relacionado com a presença de complicações.

Avaliação de técnica de sedimentação em tubos

O rendimento de sedimentação em tubos, aplicada à pesquisa de ovos de Ancylostomidae, Trichuris trichura e Ascaris lumbricoides foi estudado em suspensões fecais diluídas a 2,0%; 1,5% e 1,0% em colunas de 80 mm de altura. De cada um dos helmintos citados, as amostras continham quantidades correspondentes a 12,5; 25,0; 50,0; 100,0; 150,0 e 200,0 ovos por grama de fezes (opg). Avaliou-se o rendimento do processo em termos de freqüências de resultados positivos e de contagens de ovos por lâmina de sedimento (0,078 ml). Ambos aumentaram com os valores de opg. As várias diluições não influíram nos graus de positividade ou nas contagens de ovos por lâmina de sedimento. Os sedimentos de suspensões fecais diluídas a 1,0% (quando o valor de opg era de 150,0). Em trabalho rotineiro é desnecessária a determinação instrumental das quantidades de fezes usadas nas suspensões. Diluições em torno de 1,0% representam um compromisso entre bom rendimento e limpidez dos esfregaços, especialmente quando os valores de opg forem baixos.

Eficiência do diagnóstico coproscópico de Schistosoma mansoni em fezes prensadas

A eficácia do controle da esquistossomose depende em grande parte da sensibilidade da coproscopia. Passamos a utilizar, além da técnica de Kato-Katz, a de prensagem de fezes entre lâmina e lamínula de polipropileno, segundo Ferreira. De 1.282 amostras fecais colhidas entre 1998 e 2000 no Bairro da Palha, Município de Bananal, São Paulo, Brasil, 105 (8,2%) resultaram positivas. A primeira técnica revelou 90 (7%) e a segunda, 88 (6,9%) amostras positivas. Os resultados concordaram, segundo a estatística kappa. Valores baixos de percentagens de positivos e de opg (ovos por grama) de fezes, em Bananal, demandaram aumento do volume de material a examinar. Além de permitir a observação da viabilidade dos ovos de Schistosoma mansoni, a segunda técnica dispensa o uso de glicerina, de placa medidora e de tamisação; esta última, responsável por ulterior perda de precisão nas determinações de opg.

Estudo comparativo de técnicas parasitológicas: Kato-Katz e coprotest®

O diagnóstico parasitológico deve ser realizado de maneira apropriada, com maior sensibilidade e especificidade para a detecção dos parasitas intestinais, uma vez que dele dependerá o tratamento específico. Foi desenvolvido um estudo comparativo para avaliar a concordância entre os métodos Kato-Katz e coprotest® na detecção de helmintos em 332 indivíduos do município de Pedro de Toledo. Destacou-se uma diferença significativa para Trichuris trichiura, 16,2% no Kato-Katz e 7,5% no coprotest®. Devido a essa diferença compararam-se amostras positivas e negativas do método de coprotest® com número de ovos por grama de fezes (opg) obtido pelo método de Kato-Katz. Quando o método de coprotest® era negativo, contaram-se 65 opg de Trichuris trichiura pelo Kato-Katz e quando o coprotest® era positivo, esse número foi maior, 199 opg. O coprotest® mostrou-se inferior ao Kato-Katz nas infecções de baixa carga parasitária.

Identification and differentiation of Candida species from pediatric patients by random amplified polymorphic DNA

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Denosumab for prevention of fractures in postmenopausal women with osteoporosis.

BACKGROUND: Denosumab is a fully human monoclonal antibody to the receptor activator of nuclear factor-kappaB ligand (RANKL) that blocks its binding to RANK, inhibiting the development and activity of osteoclasts, decreasing bone resorption, and increasing bone density. Given its unique actions, denosumab may be useful in the treatment of osteoporosis. METHODS: We enrolled 7868 women between the ages of 60 and 90 years who had a bone mineral density T score of less than -2.5 but not less than -4.0 at the lumbar spine or total hip. Subjects were randomly assigned to receive either 60 mg of denosumab or placebo subcutaneously every 6 months for 36 months. The primary end point was new vertebral fracture. Secondary end points included nonvertebral and hip fractures. RESULTS: As compared with placebo, denosumab reduced the risk of new radiographic vertebral fracture, with a cumulative incidence of 2.3% in the denosumab group, versus 7.2% in the placebo group (risk ratio, 0.32; 95% confidence interval [CI], 0.26 to 0.41; P<0.001)--a relative decrease of 68%. Denosumab reduced the risk of hip fracture, with a cumulative incidence of 0.7% in the denosumab group, versus 1.2% in the placebo group (hazard ratio, 0.60; 95% CI, 0.37 to 0.97; P=0.04)--a relative decrease of 40%. Denosumab also reduced the risk of nonvertebral fracture, with a cumulative incidence of 6.5% in the denosumab group, versus 8.0% in the placebo group (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01)--a relative decrease of 20%. There was no increase in the risk of cancer, infection, cardiovascular disease, delayed fracture healing, or hypocalcemia, and there were no cases of osteonecrosis of the jaw and no adverse reactions to the injection of denosumab. CONCLUSIONS: Denosumab given subcutaneously twice yearly for 36 months was associated with a reduction in the risk of vertebral, nonvertebral, and hip fractures in women with osteoporosis. (ClinicalTrials.gov number, NCT00089791.)