O estudo teve como objetivo avaliar a razão de custo-efetividade, sob a perspectiva do Sistema Único de Saúde SUS, do tratamento da anemia de pacientes em Terapia Renal Substitutiva. Duas alternativas foram comparadas: um novo medicamento recentemente registrado no Brasil, o Ativador Contínuo de Receptor de Eritropoetina (Continuous Erythropoietin Receptor Activator), CERA, e outro, atualmente disponível no sistema de saúde brasileiro, a Eritropoetina Recombinante Humana - Epo-rHu. Métodos: Um modelo de Markov simulou o curso de uma coorte de pacientes em Terapia Renal Substitutiva tratados com CERA e Epo-rHu por quatro anos. A qualidade de vida associada ao uso dos medicamentos foi estimada de forma indireta, por meio de entrevista qualificada com os profissionais cuidadores, previamente submetida e aprovada pelo Comitê de Ética em Pesquisa local. Foi realizada análise de sensibilidade no modelo proposto através da variação dos parâmetros: dose dos medicamentos, custo das estratégias, taxa de desconto e efetividade utilizados para sua construção. Resultados: A média da qualidade de vida atribuída aos pacientes tratados foi 6,3 para a Epo-rHu, 7,8 para o CERA e 9,3 para os pacientes transplantados. O modelo demonstrou que a estratégia mais custo-efetiva é a terapêutica com a Epo-rHu, com um custo por QALY de R$ 21.052,00. O custo incremental por QALY ganho associado ao CERA foi de R$ 72.974,00. Conclusão: A utilização mensal do medicamento CERA está associada à maior qualidade de vida quando comparada a Epo-rHu. No entanto, a terapia com o novo medicamento não se mostrou mais custo-efetiva frente ao tratamento com Epo-rHu / This study sought to determine the cost-effectiveness of anemia treatment in dialysis patients for Brazilian Public Health System. Two alternatives were compared: a new drug, the Continuous Erythropoietin Receptor Activator, CERA, recently registred in Brazil, and another one, provided nowadays by the National Health System, Epo-rHu (Recombinant Human Eythropoietin). Methods: A Markov cohort of dialysis patients treated with CERA and Epo-rHu for four years was used to perform the base case analysis. The model outputs were QALYs and costs. The quality of life associated with each drug was measured by interviews applied to health care professionals. These interviews were previously submitted and approved by the local ethics committee. A sensitivity analysis was applied to the model to test it, varying the values of drugs dosage, costs, discount rate and effectiveness. Results: The average quality of life assigned by health care professionals to the patients treated with Epo-rHu, CERA and to kidney transplant receptors were respectively 6,3, 7,8 and 9,3. The model showed that Epo-rHu treatment was more cost-effective than CERA treatment. The cost-effectiveness ratio of Epo-rHu therapy was R$ 21.052,00. In addition, the cost per QALY gained of CERA therapy was R$ 72.974,00. Conclusion: Anemia treatment with CERA is associated with improvement in quality of life compared to Epo-rHu therapy. However, the new drug is not more cost-effective than the drug provided by the Brazilian Public Health System

The purpose of the study was to elucidate the mechanisms by which polyunsaturated fatty acids (PUFAs) prevent bone loss. MG-63 human osteoblasts and MC3T3-E1 murine osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) as well as oestrogen (E2) and parathyroid hormone (PTH) and the effects thereof tested on a variety of biological parameters characteristic of osteoblasts. These parameters included prostaglandin E2 (PGE2) synthesis, proliferation, differentiation to mature mineralising osteoblasts as well as osteoprotegerin (OPG) and receptor activator of nuclear factor êB ligand (RANKL) secretion. Results showed that AA stimulates PGE2 production significantly in both cell lines. Stimulated PGE2 production by MC3T3-E1 cells however, was significantly higher, which might be attributed to auto-amplification by PGE2 itself in this cell line. Pre-incubation of the MG-63 cells with cyclo-oxygenase (COX)-blockers inhibited PGE2 production significantly, suggesting that both COX enzymes were involved in PGE2 synthesis. The number of functional osteoblasts is important for bone formation therefore in vitro osteoblastic cell proliferation was investigated. In contrast to the hormones E2 and PTH, both AA and DHA inhibited proliferation significantly. The AA-mediated anti-proliferative effect is possibly independent of PGE2 production, as PGE2 per se had little effect on proliferation. DHA inhibited proliferation of MG-63 cells more severely, which might be attributed to the osteosarcoma nature of the MG-63 cells. The anti-proliferative effect of these PUFAs might be attributed to modulation of cell cycle progression or anti-mitotic effects of PUFA peroxidation products. Morphological studies showed apoptotic cells after DHA exposure in MG-63 cells. There is a reciprocal relationship between reduced proliferation and the subsequent induction of cell differentiation in vitro. High basal levels of alkaline phosphatase (ALP) activity, a marker of the mature mineralising osteoblastic phenotype, were detected in MC3T3-E1 cells. Long-term exposure to AA inhibited ALP activity in these cells. This process might be PGE2-mediated. Exposure to PUFAs, however, did not compromise the ability of the MC3T3-E1 cells to differentiate to mature mineralising osteoblasts. In contrast with MC3T3-E1 cells, MG-63 cells demonstrated low basal ALP activity and were unable to differentiate to mature mineralising osteoblasts. In the absence of osteogenic-inducing supplements, PUFAs induced adipocyte-like features that might be due to the expression of high levels of PPARã in this cell line. Lipid-filled vacuoles were absent in the MC3T3-E1 cells suggesting that the MC3T3-E1 cell line may not express PPARã mRNA. The study furthermore demonstrated that PUFAs are able to modulate OPG and RANKL secretion in osteoblasts. AA inhibited OPG secretion dose-dependently in both cell lines, this could be PGE2-mediated. AA dose-dependently stimulated soluble RANKL (sRANKL) secretion in MC3T3-E1 cells thereby affecting the OPG/RANKL ratio in a negative way, supporting various reports that AA and PGE2 do cause bone resorption. No sRANKL could be detected after exposing the MC3T3-E1 cells to DHA suggesting that DHA could be protective to bone. In conclusion, contrary to in vivo evidence, this in vitro study could not indisputably demonstrate protective effects of PUFAs on the osteoblastic cell lines tested. / Thesis (PhD (Physiology))--University of Pretoria, 2006. / Physiology / unrestricted

Embora a grande maioria dos pacientes com asma tenha controle com o padrão de tratamento com corticosteroides, 5 a 10% destes asmáticos ainda desenvolvem algumas formas graves da doença e são considerados pacientes difíceis de controlar. A ativação do NFkB contribui para a manutenção e desenvolvimento de inflamação pulmonar crônica na fisiopatologia da asma. O éster fenetilico do ácido cafeico (CAPE), um componente ativo de própolis das colmeias de abelhas, é conhecido por ter propriedades antiinflamatórias e imunomoduladoras. É conhecido como um potente e um inibidor específico da ativação de NFk B. Nós anteriormente mostramos que Y27632, um inibidor de Rho quinase, atenuou a resposta pulmonar das vias aéreas, inflamação e remodelamento. Para o nosso conhecimento, tratamentos com os inibidores de NFk B ou Rho-quinase em comparação com o uso de corticosteroides não foram previamente investigados em um modelo animal de inflamação pulmonar crônica alérgica. MÉTODOS: Trinta camundongos BALB/C machos (20 a 25g) foram divididos em cinco grupos (n=6 em cada grupo): SAL (instilado com solução salina), OVA (expostos à ovalbumina), OVA-CAPE (expostos à ovalbumina e tratados com CAPE), OVA-RHO (expostos à ovalbumina e tratados com inibidor de Rho), OVA-CORT (expostos à ovalbumina e tratados com corticosteróide). Os animais foram sensibilizados para ovalbumina durante 28 dias e os controles receberam solução salina. A administração de corticosteróide (1mg/Kg/dia) e CAPE (10mg/Kg/dia) foi feita por via intraperitoneal e Y-27632 (10 mg/kg) administrado por via intranasal, todos nos dias 22, 24, 26 e 28 do protocolo experimental. Vinte e quatro horas após a conclusão do protocolo experimental, os animais foram submetidos a uma avaliação da curva de resposta à dose à metacolina, consideramos as respostas máximas da Resistência ao Sistema Respiratório (Rrs) e Elastância (Ers) às 24 horas após o último desafio com ovalbumina (respostas basais e máximas após a metacolina desafio 3, 30 e 300mg/mL). Posteriormente, os pulmões foram removidos e a análise histológica foi realizada usando morfometria. RESULTADOS: não houve diferenças nos valores basais de todos os grupos. expostos à ovalbumina Houve um aumento em Rrs e Ers após o desafio da metacolina em comparação com o grupo SAL (p < 0,05). No grupo exposto ao OVA, os tratamentos com CAPE, CORT e RHO reduziram a resposta máxima de Ers em relação ao OVA (p < 0,05). Em relação ao tratamento com somente CAPE atenuou a resposta máxima de Rrs em relação ao OVA (p < 0,05). Houve diminuição nos eosinófilos, conteúdo de fibras de colágeno, células positivas para iNOS, MMP-9, TIMP-1 em grupos OVA-CAPE, OVA-CORT e OVA-RHO em comparação com os animais sensibilizados (grupo OVA) (p < 0,05). CONCLUSÃO: a inibição de NFkB e Rho-quinase contribuiu para o controle da hiperresponsividade, inflamação, processo de remodelamento da matriz extracelular e ativação do estresse oxidativo. Embora os inibidores de NFkB e Rho-quinase sejam uma alternativa ao tratamento da asma, são necessários mais estudos / Although the great majority of asthma patients obtain control with the gold standard treatment using corticosteroids, 5 to 10% of these asthmatics still develop some severe forms of the disease, called difficult to control patients. Activation of NFk B contributes to the maintenance and development of chronic lung inflammation in pathophysiology of asthma. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have anti-inflammatory and immunomodulatory properties. It is known as a potent and a specific inhibitor of NFk B activation. We previously have shown that Y27632, a Rho-quinase inhibitor, attenuated airway lung responsiveness, inflammation and remodeling. To our knowledge, treatments with the NFk B or Rho-kinase inhibitors compared to the use of corticosteroids have not been previously investigated in an animal model of chronic allergic lung inflammation. METHODS: Thirty male BALB/c mice (20 to 25g) were divided into five groups (n=6 in each group): SAL (saline-instilled), OVA (exposed-ovalbumin), OVA-CAPE (exposedovalbumin and treated with CAPE), OVA-RHO (exposed-ovalbumin and treated with Rho inhibitor), OVA-CORT (exposed-ovalbumin and treated with corticosteroid). The BALB/c mice were sensitized to ovalbumin for 28 days and controls received saline. The administration of corticosteroid (1mg/Kg/day) and CAPE (10mg/Kg/day) was made intraperitoneally and Y-27632 (10mg/kg) was intranasally, all on days 22, 24, 26 and 28 of the experimental protocol. Twentyfour hours after completion of the experimental protocol, animals were subject to an evaluation of dose response curve to methacholine. We considered the maximal responses of respiratory system resistance (Rrs) and elastance (Ers) 24 hours after the last challenge with ovalbumin (baseline and maximal responses after metacholine challenge 3, 30 and 300mg/mL). Afterwards, lungs were removed and histological analysis was performed using morphometry. RESULTS: There were no differences on baseline values of all groups. Ovalbumin-exposed mice had an increase in Rrs and Ers after methacholine challenge compared to SAL group (p < 0.05). In OVA-exposed groups, the treatments with CAPE, CORT and RHO reduced maximal response of %Ers compared to OVA (p < 0.05). In relation to %Rrs only CAPE treatment attenuated the maximal response compared to OVA (p < 0.05). There was a decrease in eosinophils, collagen fibers content, iNOS, MMP-9, TIMP-1 positive cells in OVACAPE, OVA-CORT and OVA-RHO groups compared to only sensitized animals (OVA group) (p < 0.05). CONCLUSION: NFkB and Rho-quinase inhibition contributes to the control of hyperresponsiveness, inflammation, extracellular matrix remodeling process and oxidative stress activation. Although NFkB and Rho-quinase inhibitors were an alternative to asthma treatment, more studies are needed

Embora a grande maioria dos pacientes com asma tenha controle com o padrão de tratamento com corticosteroides, 5 a 10% destes asmáticos ainda desenvolvem algumas formas graves da doença e são considerados pacientes difíceis de controlar. A ativação do NFkB contribui para a manutenção e desenvolvimento de inflamação pulmonar crônica na fisiopatologia da asma. O éster fenetilico do ácido cafeico (CAPE), um componente ativo de própolis das colmeias de abelhas, é conhecido por ter propriedades antiinflamatórias e imunomoduladoras. É conhecido como um potente e um inibidor específico da ativação de NFk B. Nós anteriormente mostramos que Y27632, um inibidor de Rho quinase, atenuou a resposta pulmonar das vias aéreas, inflamação e remodelamento. Para o nosso conhecimento, tratamentos com os inibidores de NFk B ou Rho-quinase em comparação com o uso de corticosteroides não foram previamente investigados em um modelo animal de inflamação pulmonar crônica alérgica. MÉTODOS: Trinta camundongos BALB/C machos (20 a 25g) foram divididos em cinco grupos (n=6 em cada grupo): SAL (instilado com solução salina), OVA (expostos à ovalbumina), OVA-CAPE (expostos à ovalbumina e tratados com CAPE), OVA-RHO (expostos à ovalbumina e tratados com inibidor de Rho), OVA-CORT (expostos à ovalbumina e tratados com corticosteróide). Os animais foram sensibilizados para ovalbumina durante 28 dias e os controles receberam solução salina. A administração de corticosteróide (1mg/Kg/dia) e CAPE (10mg/Kg/dia) foi feita por via intraperitoneal e Y-27632 (10 mg/kg) administrado por via intranasal, todos nos dias 22, 24, 26 e 28 do protocolo experimental. Vinte e quatro horas após a conclusão do protocolo experimental, os animais foram submetidos a uma avaliação da curva de resposta à dose à metacolina, consideramos as respostas máximas da Resistência ao Sistema Respiratório (Rrs) e Elastância (Ers) às 24 horas após o último desafio com ovalbumina (respostas basais e máximas após a metacolina desafio 3, 30 e 300mg/mL). Posteriormente, os pulmões foram removidos e a análise histológica foi realizada usando morfometria. RESULTADOS: não houve diferenças nos valores basais de todos os grupos. expostos à ovalbumina Houve um aumento em Rrs e Ers após o desafio da metacolina em comparação com o grupo SAL (p < 0,05). No grupo exposto ao OVA, os tratamentos com CAPE, CORT e RHO reduziram a resposta máxima de Ers em relação ao OVA (p < 0,05). Em relação ao tratamento com somente CAPE atenuou a resposta máxima de Rrs em relação ao OVA (p < 0,05). Houve diminuição nos eosinófilos, conteúdo de fibras de colágeno, células positivas para iNOS, MMP-9, TIMP-1 em grupos OVA-CAPE, OVA-CORT e OVA-RHO em comparação com os animais sensibilizados (grupo OVA) (p < 0,05). CONCLUSÃO: a inibição de NFkB e Rho-quinase contribuiu para o controle da hiperresponsividade, inflamação, processo de remodelamento da matriz extracelular e ativação do estresse oxidativo. Embora os inibidores de NFkB e Rho-quinase sejam uma alternativa ao tratamento da asma, são necessários mais estudos / Although the great majority of asthma patients obtain control with the gold standard treatment using corticosteroids, 5 to 10% of these asthmatics still develop some severe forms of the disease, called difficult to control patients. Activation of NFk B contributes to the maintenance and development of chronic lung inflammation in pathophysiology of asthma. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have anti-inflammatory and immunomodulatory properties. It is known as a potent and a specific inhibitor of NFk B activation. We previously have shown that Y27632, a Rho-quinase inhibitor, attenuated airway lung responsiveness, inflammation and remodeling. To our knowledge, treatments with the NFk B or Rho-kinase inhibitors compared to the use of corticosteroids have not been previously investigated in an animal model of chronic allergic lung inflammation. METHODS: Thirty male BALB/c mice (20 to 25g) were divided into five groups (n=6 in each group): SAL (saline-instilled), OVA (exposed-ovalbumin), OVA-CAPE (exposedovalbumin and treated with CAPE), OVA-RHO (exposed-ovalbumin and treated with Rho inhibitor), OVA-CORT (exposed-ovalbumin and treated with corticosteroid). The BALB/c mice were sensitized to ovalbumin for 28 days and controls received saline. The administration of corticosteroid (1mg/Kg/day) and CAPE (10mg/Kg/day) was made intraperitoneally and Y-27632 (10mg/kg) was intranasally, all on days 22, 24, 26 and 28 of the experimental protocol. Twentyfour hours after completion of the experimental protocol, animals were subject to an evaluation of dose response curve to methacholine. We considered the maximal responses of respiratory system resistance (Rrs) and elastance (Ers) 24 hours after the last challenge with ovalbumin (baseline and maximal responses after metacholine challenge 3, 30 and 300mg/mL). Afterwards, lungs were removed and histological analysis was performed using morphometry. RESULTS: There were no differences on baseline values of all groups. Ovalbumin-exposed mice had an increase in Rrs and Ers after methacholine challenge compared to SAL group (p < 0.05). In OVA-exposed groups, the treatments with CAPE, CORT and RHO reduced maximal response of %Ers compared to OVA (p < 0.05). In relation to %Rrs only CAPE treatment attenuated the maximal response compared to OVA (p < 0.05). There was a decrease in eosinophils, collagen fibers content, iNOS, MMP-9, TIMP-1 positive cells in OVACAPE, OVA-CORT and OVA-RHO groups compared to only sensitized animals (OVA group) (p < 0.05). CONCLUSION: NFkB and Rho-quinase inhibition contributes to the control of hyperresponsiveness, inflammation, extracellular matrix remodeling process and oxidative stress activation. Although NFkB and Rho-quinase inhibitors were an alternative to asthma treatment, more studies are needed

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt. A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations. The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement. In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

INTRODUÇÃO: Perda óssea é um achado comum em pacientes com Nefrite Lúpica (NL), mesmo naqueles com diagnóstico recente. Algumas evidências indicam um aumento na osteoclastogênese como um dos distúrbios principais no processo de remodelamento ósseo. O objetivo deste estudo foi investigar algumas vias de sinalização (RANKL/OPG, Wnt/Beta-catenin and Th17/IL-17) possivelmente envolvidas na osteoclastogênese anormal detectada em mulheres jovens ao diagnóstico de nefrite lúpica ativa, assim como avaliar a ação da vitamina D (VitD) nesse cenário e sua correlação com fatores inflamatórios. MÉTODOS: Realizamos culturas com a linhagem de células osteoblásticas humanas hFOB 1.19 (ATCC) e as dividimos em um grupo suplementado com soro de pacientes lúpicas (NL) (n=15) e em um grupo com soro de controles saudáveis (CS) (n=15) em vez de soro fetal bovino (SFB). Em seguida, adicionamos 1,25-dihidroxivitamina D3 (1,25(OH)2D3) em dois subgrupos nas concentrações 10-9M e 10-7M, resultando em 6 grupos: CS, CS+vitD 10-9M, CS+vitD 10-7M, NL, NL+vitD 10-9M, NL+vitD 10-7M). Após 48h da adição de 1,25(OH)2D3 ao meio de cultura, células hFOB foram tripsinizadas e separado o lisato celular de cada grupo. Ensaios de citometria de fluxo e multiplex foram realizados para quantificação das seguintes proteínas do lisato cellular: CD166, CD54, fosfatase alcalina, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, Beta-catenina, IL-1-beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22. RT-PCR foi empregado para quantificação de mRNA dos genes RANKL, SOST, OPG e Beta-catenina. RESULTADOS: Pacientes com NL evidenciaram maiores níveis séricos de DKK-1 (2802,04 ± 1380,06 x 696,30 ± 421,22pg/ml, p < 0,001), OPG (560,12 ± 333,56 x 340,24 ± 102,08pg/ml, p=0,0212), TNF-alfa (9,63 ± 14,49 x 1,27 ± 0,35pg/ml, p=0,0337), IL-6 (15,58±39,08 x 8,02±3,49, p= 0,0053) and IL-2 (3,36 ± 3,06 x 1,54 ± 0,9pg/ml, p=0,0353) do que CS. Após exposição ao meio enriquecido com soro de pacientes com NL, células hFOB 1.19 apresentaram maior nível de mRNA de RANKL (p=0,045)) e menor nível de proteína OPG (178,81 ± 66,40 x 298,76 ± 114,94pg/mg, p=0,0016). Suplementação com 1,25(OH)2D3 aumentou a diferença da expressão das proteínas DKK-1 (673,03 ± 171,93 x 456,69 ± 234,53pg/mg, p=0,0215), IL-6 (0,80 ± 0,25pg/mg x 0,66 ± 0,18, p=0,0417) and IL-2 (4,97 ± 2,2 x 3,90 ± 1,66pg/mg, p=0,042) entre hFOB NL comparados com hFOB CS, enquanto diminuiu o nível de mRNA de Beta-catenina em células do grupo NL. DISCUSSÃO: Dentro das limitações deste estudo, os resultados sugerem que os maiores níveis séricos de citocinas pró-inflamatórias, como TNF-alfa, IL-6 e talvez IL-2, detectadas em pacientes com NL pode ter induzido a maior expressão osteoblástica de RANKL, representada pelo maior nível de mRNA RANKL em células do grupo NL, e suprimido OPG, conforme a diminuição observada na quantificação proteica de OPG nos lisatos celulares, o que pode ter contribuído para a aumentada osteoclastogênese evidenciada pela biópsia óssea dessas pacientes. A adição de 1,25(OH)2D3 não preveniu os efeitos inflamatórios do soro de pacientes com NL ativa em células hFOB 1.19 neste estudo / INTRODUCTION: Bone loss is a common finding in Lupus Nephritis (LN) patients even in those recently diagnosed. Some evidences indicate an increased osteoclastogenesis as the main disturb of the bone remodeling process. The aim of this study was to investigate some pathways (RANK-L/OPG, Wnt/?-catenin and Th17/IL-17) possibly involved in the abnormal osteoclastogenesis detected in women at the diagnosis of proliferative LN as well as evaluating the action of vitamin D (vitD) in this scenario and their correlation with inflammatory factors. METHODS: We cultured the human osteoblastic cell line hFOB 1.19 (ATCC), and divided cultures into those supplemented with serum from healthy controls (HC) (n=15) and LN patients (n=15) instead of fetal bovine serum (FBS). Then 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was added in two subgroups at the concentrations of 10-9M e 10-7M while vitD was absent in one subgroup in both HC and LN cultures (HC, HC+vitD 10-9M, HC+vitD 10-7M, LN, LN+vitD 10-9M, LN+vitD 10-7M) . After 48h of vitD addition, hFOB cultures were trypsinized. Flow cytometry and multiplex assays were performed to test CD166, CD54, alkaline phosphatase, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, ?-catenin, IL-1Beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22 concentrations in the cell lysates. Polymerase reaction chain (RT-PCR) assays analyzed the expression of RANKL, SOST, OPG and Beta-catenin mRNA. RESULTS: LN patients showed higher serum levels of DKK-1 (2802.04 ± 1380.06 x 696.3 ± 421.22pg/ml, p < 0.001), OPG (560.12 ± 333.56 x 340.24 ± 102.08pg/ml, p=0.0212), TNF-alfa (9.63 ± 14.49 x 1.27 ± 0.35pg/ml, p=0.0337), IL-6 (15.58±39.08 x 8.02±3.49, 0.0053) and IL-2 (3.36 ± 3.06 x 1.54 ± 0.9pg/ml, p=0.0353) than HCs. After exposure to medium enriched with LN serum, osteoblasts expressed higher RANKL mRNA (fold change 1.573, p=0.045) and lower OPG protein (178.81 ± 66.40 x 298.76 ± 114.94pg/mg, p=0.0016). 1,25(OH)2D3 supplementation increased the difference between LN and HC expression of DKK-1 (673.03 ± 171.93 x 456.69 ± 234.53pg/mg, p=0.0215), IL-6 (0.80 ± 0.25pg/mg x 0.66 ± 0.18, p=0.0417) and IL-2 (4.97 ± 2.2 x 3.90 ± 1,66pg/mg, p=0.042) proteins and diminished Beta-catenin mRNA in LN cells. DISCUSSION: Within the limitations of this study, the results suggest that the higher serum levels of proinflammatory cytokines, such as TNF-alfa, IL-6 and perhaps IL-2, detected in LN patients would possibly have induced RANKL genes, as demonstrated by an enhanced RANKL mRNA expression in LN osteoblasts, and suppressed OPG protein in cell lysates, which would have contributed to the increased osteoclastogenesis detected in bone biopsies of women with new onset of LN. 1,25(OH)2D3 addition to osteoblast cultures did not prevent the effects of inflammatory LN serum in vitro

INTRODUÇÃO: Perda óssea é um achado comum em pacientes com Nefrite Lúpica (NL), mesmo naqueles com diagnóstico recente. Algumas evidências indicam um aumento na osteoclastogênese como um dos distúrbios principais no processo de remodelamento ósseo. O objetivo deste estudo foi investigar algumas vias de sinalização (RANKL/OPG, Wnt/Beta-catenin and Th17/IL-17) possivelmente envolvidas na osteoclastogênese anormal detectada em mulheres jovens ao diagnóstico de nefrite lúpica ativa, assim como avaliar a ação da vitamina D (VitD) nesse cenário e sua correlação com fatores inflamatórios. MÉTODOS: Realizamos culturas com a linhagem de células osteoblásticas humanas hFOB 1.19 (ATCC) e as dividimos em um grupo suplementado com soro de pacientes lúpicas (NL) (n=15) e em um grupo com soro de controles saudáveis (CS) (n=15) em vez de soro fetal bovino (SFB). Em seguida, adicionamos 1,25-dihidroxivitamina D3 (1,25(OH)2D3) em dois subgrupos nas concentrações 10-9M e 10-7M, resultando em 6 grupos: CS, CS+vitD 10-9M, CS+vitD 10-7M, NL, NL+vitD 10-9M, NL+vitD 10-7M). Após 48h da adição de 1,25(OH)2D3 ao meio de cultura, células hFOB foram tripsinizadas e separado o lisato celular de cada grupo. Ensaios de citometria de fluxo e multiplex foram realizados para quantificação das seguintes proteínas do lisato cellular: CD166, CD54, fosfatase alcalina, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, Beta-catenina, IL-1-beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22. RT-PCR foi empregado para quantificação de mRNA dos genes RANKL, SOST, OPG e Beta-catenina. RESULTADOS: Pacientes com NL evidenciaram maiores níveis séricos de DKK-1 (2802,04 ± 1380,06 x 696,30 ± 421,22pg/ml, p < 0,001), OPG (560,12 ± 333,56 x 340,24 ± 102,08pg/ml, p=0,0212), TNF-alfa (9,63 ± 14,49 x 1,27 ± 0,35pg/ml, p=0,0337), IL-6 (15,58±39,08 x 8,02±3,49, p= 0,0053) and IL-2 (3,36 ± 3,06 x 1,54 ± 0,9pg/ml, p=0,0353) do que CS. Após exposição ao meio enriquecido com soro de pacientes com NL, células hFOB 1.19 apresentaram maior nível de mRNA de RANKL (p=0,045)) e menor nível de proteína OPG (178,81 ± 66,40 x 298,76 ± 114,94pg/mg, p=0,0016). Suplementação com 1,25(OH)2D3 aumentou a diferença da expressão das proteínas DKK-1 (673,03 ± 171,93 x 456,69 ± 234,53pg/mg, p=0,0215), IL-6 (0,80 ± 0,25pg/mg x 0,66 ± 0,18, p=0,0417) and IL-2 (4,97 ± 2,2 x 3,90 ± 1,66pg/mg, p=0,042) entre hFOB NL comparados com hFOB CS, enquanto diminuiu o nível de mRNA de Beta-catenina em células do grupo NL. DISCUSSÃO: Dentro das limitações deste estudo, os resultados sugerem que os maiores níveis séricos de citocinas pró-inflamatórias, como TNF-alfa, IL-6 e talvez IL-2, detectadas em pacientes com NL pode ter induzido a maior expressão osteoblástica de RANKL, representada pelo maior nível de mRNA RANKL em células do grupo NL, e suprimido OPG, conforme a diminuição observada na quantificação proteica de OPG nos lisatos celulares, o que pode ter contribuído para a aumentada osteoclastogênese evidenciada pela biópsia óssea dessas pacientes. A adição de 1,25(OH)2D3 não preveniu os efeitos inflamatórios do soro de pacientes com NL ativa em células hFOB 1.19 neste estudo / INTRODUCTION: Bone loss is a common finding in Lupus Nephritis (LN) patients even in those recently diagnosed. Some evidences indicate an increased osteoclastogenesis as the main disturb of the bone remodeling process. The aim of this study was to investigate some pathways (RANK-L/OPG, Wnt/?-catenin and Th17/IL-17) possibly involved in the abnormal osteoclastogenesis detected in women at the diagnosis of proliferative LN as well as evaluating the action of vitamin D (vitD) in this scenario and their correlation with inflammatory factors. METHODS: We cultured the human osteoblastic cell line hFOB 1.19 (ATCC), and divided cultures into those supplemented with serum from healthy controls (HC) (n=15) and LN patients (n=15) instead of fetal bovine serum (FBS). Then 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was added in two subgroups at the concentrations of 10-9M e 10-7M while vitD was absent in one subgroup in both HC and LN cultures (HC, HC+vitD 10-9M, HC+vitD 10-7M, LN, LN+vitD 10-9M, LN+vitD 10-7M) . After 48h of vitD addition, hFOB cultures were trypsinized. Flow cytometry and multiplex assays were performed to test CD166, CD54, alkaline phosphatase, RANKL, OPG, CD14, TLR4, NF-KappaB, SOST, DKK-1, ?-catenin, IL-1Beta, IL-2, IL-6, TNF-alfa, IL-17A, IL-17F, IL-21 and IL-22 concentrations in the cell lysates. Polymerase reaction chain (RT-PCR) assays analyzed the expression of RANKL, SOST, OPG and Beta-catenin mRNA. RESULTS: LN patients showed higher serum levels of DKK-1 (2802.04 ± 1380.06 x 696.3 ± 421.22pg/ml, p < 0.001), OPG (560.12 ± 333.56 x 340.24 ± 102.08pg/ml, p=0.0212), TNF-alfa (9.63 ± 14.49 x 1.27 ± 0.35pg/ml, p=0.0337), IL-6 (15.58±39.08 x 8.02±3.49, 0.0053) and IL-2 (3.36 ± 3.06 x 1.54 ± 0.9pg/ml, p=0.0353) than HCs. After exposure to medium enriched with LN serum, osteoblasts expressed higher RANKL mRNA (fold change 1.573, p=0.045) and lower OPG protein (178.81 ± 66.40 x 298.76 ± 114.94pg/mg, p=0.0016). 1,25(OH)2D3 supplementation increased the difference between LN and HC expression of DKK-1 (673.03 ± 171.93 x 456.69 ± 234.53pg/mg, p=0.0215), IL-6 (0.80 ± 0.25pg/mg x 0.66 ± 0.18, p=0.0417) and IL-2 (4.97 ± 2.2 x 3.90 ± 1,66pg/mg, p=0.042) proteins and diminished Beta-catenin mRNA in LN cells. DISCUSSION: Within the limitations of this study, the results suggest that the higher serum levels of proinflammatory cytokines, such as TNF-alfa, IL-6 and perhaps IL-2, detected in LN patients would possibly have induced RANKL genes, as demonstrated by an enhanced RANKL mRNA expression in LN osteoblasts, and suppressed OPG protein in cell lysates, which would have contributed to the increased osteoclastogenesis detected in bone biopsies of women with new onset of LN. 1,25(OH)2D3 addition to osteoblast cultures did not prevent the effects of inflammatory LN serum in vitro

Made available in DSpace on 2014-12-17T15:32:29Z (GMT). No. of bitstreams: 1 CassianoFWN_TESE.pdf: 3914655 bytes, checksum: c6fb9bd86bba433eb6a5a047a9337861 (MD5) Previous issue date: 2010-09-23 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The odontogenic keratocysts are distinguished from other odontogenic cystic lesions by their potentially aggressive clinical behavior and association, in some cases, with Gorlin syndrome. Studies have suggested that syndrome keratocysts, in comparison with sporadic lesions, have higher growth and infiltration capacity and higher recurrence tendency. The aim of this study was to analyze, by means of immunohistochemistry, the expressions of receptor activator of nuclear factor ?B ligand (RANKL) and osteoprotegerin (OPG), the angiogenic index (CD34) and the presence of myofibroblasts (?-SMA) in primary and recurrent sporadic keratocysts and in keratocysts associated with Gorlin syndrome. The sample was composed by 30 sporadic keratocysts (22 primary and 8 recurrent) and 22 syndrome keratocysts. In the epithelium and in the fibrous capsule of the lesions, the immunoexpression of RANKL and OPG was evaluated by determination of the percentage of positive cells, according to the following scores: 0 (less than 10% of positive cells), 1 (11% - 50% of positive cells), 2 (51% - 75% of positive cells) and 3 (more than 76% of positive cells). In addition, cases were classified according to the RANKL score/ OPG score ratio, as follows: RANKL > OPG, RANKL < OPG, and RANKL = OPG. The angiogenic index was analyzed by counting the microvessels immunoreactive to anti-CD34 antibody in 5 fields (200?). The analysis of myofibroblasts was performed by counting the cells immunoreactive to anti-?-SMA antibody in 10 fields (400?). The analysis of the expressions of RANKL and OPG in the epithelial lining and in the fibrous capsule did not reveal significant differences between groups (p > 0.05). Regarding the RANKL/ OPG ratio in the epithelial lining, most sporadic primary (54.5%) and syndrome lesions (59.1%) showed RANKL < OPG ratio and RANKL = OPG ratio, respectively (p > 0.05). With respect to the RANKL/ OPG ratio in the fibrous capsule, the majority of sporadic primary (81.8%) and sporadic recurrent lesions (75.0%) and most syndrome lesions (45.5%) showed RANKL = OPG ratio (p > 0.05). The mean number of microvessels was 69.2 in sporadic primary lesions, 67.6 in recurrent lesions, and 71.6 in syndrome lesions, with no significant differences between groups (p > 0.05). The mean number of myofibroblasts was 34.4 in sporadic primary lesions, 29.3 in recurrent lesions, and 33.7 in syndrome lesions, with no significant differences between groups (p > 0.05). In conclusion, the results of the present study suggest that the differences in the biological behavior between sporadic keratocysts and keratocysts associated with Gorlin syndrome may not be related to the expressions of RANKL and OPG, the RANKL/ OPG ratio, the angiogenic index or the number of myofibroblasts in these lesions / Os ceratocistos odontog?nicos se destacam em rela??o a outras les?es c?sticas odontog?nicas pelo comportamento cl?nico potencialmente agressivo e por se apresentarem associados, em alguns casos, ? s?ndrome de Gorlin. Estudos t?m sugerido que os ceratocistos sindr?micos, em compara??o ?s les?es isoladas, possuem maior capacidade de crescimento e infiltra??o e maior tend?ncia ? recorr?ncia. O objetivo do presente trabalho consistiu em analisar, por meio de imuno-histoqu?mica, as express?es do ligante do receptor ativador do fator nuclear ?B (RANKL) e da osteoprotegerina (OPG), o ?ndice angiog?nico (CD34) e a presen?a de miofibroblastos (?-SMA), em ceratocistos isolados prim?rios e recorrentes e ceratocistos associados ? s?ndrome de Gorlin. A amostra foi composta por 30 ceratocistos isolados (22 prim?rios e 8 recorrentes) e 22 ceratocistos sindr?micos. A express?o de RANKL e OPG foi avaliada no epit?lio e na c?psula fibrosa das les?es, estabelecendo-se o percentual de c?lulas imunopositivas, de acordo com os escores: 0 (? 10% das c?lulas positivas), 1 (11% - 50% das c?lulas positivas), 2 (51% - 75% das c?lulas positivas) e 3 (? 76% das c?lulas positivas). Al?m disso, os casos foram categorizados, segundo a propor??o RANKL/ OPG, em: RANKL > OPG, RANKL < OPG e RANKL = OPG. O ?ndice angiog?nico foi analisado por meio da contagem dos microvasos imunomarcados pelo anticorpo anti-CD34, em 5 campos (200?). Para a avalia??o dos miofibroblastos, foram quantificadas as c?lulas imunorreativas ao anticorpo anti-?-SMA, em 10 campos (400?). A an?lise das express?es de RANKL e OPG, no revestimento epitelial e na c?psula fibrosa, n?o revelou diferen?as significativas entre os grupos (p > 0,05). Em rela??o ? propor??o RANKL/ OPG no revestimento epitelial, grande parte das les?es isoladas prim?rias (54,5%) e sindr?micas (59,1%) exibiu propor??o RANKL < OPG e propor??o RANKL = OPG, respectivamente (p > 0,05). Em rela??o ? propor??o RANKL/ OPG na c?psula fibrosa, a maioria das les?es isoladas prim?rias (81,8%) e isoladas recorrentes (75,0%) e grande parte das les?es associadas ? s?ndrome de Gorlin (45,5%) revelaram propor??o RANKL = OPG (p > 0,05). O n?mero m?dio de microvasos foi de 69,2 nas les?es isoladas prim?rias, 67,6 nas les?es recorrentes e 71,6 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). A an?lise dos miofibroblastos revelou valores m?dios de 34,4 nas les?es isoladas prim?rias, 29,3 nas les?es recorrentes e 33,7 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). Em conclus?o, os resultados do presente estudo sugerem que as diferen?as no comportamento biol?gico entre ceratocistos isolados e associados ? s?ndrome de Gorlin podem n?o estar relacionadas ?s express?es de RANKL e OPG, ? propor??o RANKL/ OPG, ao ?ndice angiog?nico ou ? quantidade de miofibroblastos presentes nas les?es