Imunoexpressão de ING 4, VEGF e NF-κB em lesões periapicais inflamatórias / Immunoexpression of ING 4, VEGF and NF-κB in inflammatory periapical lesions

As lesões periapicais inflamatórias (LPIs) são condições patológicas decorrentes de infecções de origem odontogênica, principalmente representadas pelos granulomas periapicais (GPs) e cistos radiculares (CRs). Sua patogênese está associada a mecanismos imunológicos e angiogênicos. O presente estudo, do tipo retrospectivo, teve como objetivo analisar, de forma semiquantitativa, a expressão imuno-histoquímica de ING-4, VEGF e NF-κB em LPIs, e correlacionar o padrão de expressão dessas proteínas. A amostra consistiu de 26 GPs, 17 CRs e 19 cistos radiculares residuais (CRRs). Foram avaliados espessura epitelial e infiltrado inflamatório, e a associação desses achados com o padrão de expressão das proteínas ING-4, VEGF e NF-κB nas LPIs selecionadas. Para a realização da análise estatística, foram utilizados os testes de qui-quadrado, Kruskal-Wallis, Mann-Whitney e correlação de Spearman (p < 0,05). O infiltrado inflamatório exibiu maior intensidade no GP, seguido pelo CR, e por último, o CRR (p < 0,05). Apesar de não haver associação estatisticamente significativa ao associar a expressão de ING-4 nas células inflamatórias do tecido conjuntivo ou cápsula fibrosa entre os grupos de LPIs, o GP e CR evidenciaram, através da média de postos, maior expressão dessa proteína. Não foi evidenciada associação estatisticamente significativa de ING-4 com a intensidade do infiltrado inflamatório. A imunoexpressão de VEGF no núcleo das células inflamatórias do tecido conjuntivo ou cápsula fibrosa exibem associação significativa com as LPIs, que ocorre maior expressão dessa proteína nos cistos (p= 0,002). A maior expressão de NF-κB foi evidenciada nos casos de GPs, tanto a nível nuclear quanto citoplasmático das células inflamatórias (p = 0,005; p = 0,002). Não houve associação estatisticamente significativa quando comparada a expressão de NF-κB entre os cistos, mas a mediana da expressão dessa proteína foi maior para os CRs. Na cápsula fibrosa, a imunoexpressão nuclear e citoplasmática de NF-κB nas células inflamatórias foi superior nas lesões periapicais com intenso infiltrado inflamatório (p < 0,001). Portanto, sugere-se que ING-4, VEGF e NF-κB participam do desenvolvimento das LPIs, e apesar de ocorrer relação diretamente proporcional entre a expressão dessas proteínas, ING-4 não exerceu atividade reguladora na inflamação associada a essas lesões (AU).

Inflammatory periapical lesions (IPLs) are pathological conditions resulting from infections of odontogenic origin, being mainly represented by periapical granulomas (PGs) and radicular cysts (RCs). Its pathogenesis is associated with immunological and angiogenic mechanisms. This retrospective study, semi-quantitative and comparative aimed to analyze the immunohistochemical expression of ING-4, VEGF and NF-κB in IPLs, and to correlate the pattern of expression of these proteins. The sample consisted of 26 were PGs, 17 RCs and 19 residual radicular cysts (RRCs). Epithelial thickness and inflammatory infiltrate were evaluated, and the correlation of these findings with the expression pattern of ING-4, VEGF and NF-κB proteins in selected IPLs. To perform the statistical analysis, the chi-square, Kruskal-Wallis, Mann-Whitney and Spearman correlation tests were used (p < 0.05). The inflammatory infiltrate exhibited greater intensity in the PG, followed by the RC, and finally, the RRC (p < 0.05). Although there was no statistically significant association when associating the expression of ING-4 in inflammatory cells of the connective tissue or fibrous capsule the groups of IPLs, the PG and RC showed higher expression of this protein. There was no statistically significant association between ING-4 and the intensity of the inflammatory infiltrate. Immunoexpression of VEGF in the nucleus of inflammatory cells in the connective tissue or fibrous capsule shows a significant association with IPLs, in which there is greater expression of this protein occurring in cysts (p= 0,002). The highest expression of NF-κB was evidenced in cases of PGs, both at the nuclear and cytoplasmic level of inflammatory cells (p=0,005; p= 0,002). There was no statistically significant association when comparing the expression of NF-κB between the cysts, but the median expression of this protein was expression was higher for the RCs. In the fibrous capsule, nuclear and cytoplasmic NF-κB immunoexpression in inflammatory cells was higher in periapical lesions with intense inflammatory infiltrate (p<0.001). Therefore, it is suggested that ING-4, VEGF and NF-κB participate in the etiopathogenesis of IPLs, and that there is a directly proportional relationship between the expression of these proteins. ING-4 did not exert regulatory activity in the inflammation associated with these lesions (AU).

Effects of 1, 25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model

Abstract Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. Objective: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. Methodology: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. Results: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1β (IL-1β) and IL-6 protein expression. Conclusions: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

Expressão imuno-histoquímica das proteínas NF-kB e VEGF165 no líquen plano oral / Immunohistochemical expression of NF-kB and VEGF165 proteins in oral lichen planus

O líquen plano oral (LPO) é uma doença inflamatória crônica relativamente comum cuja etiopatogênese ainda não é totalmente elucidada. Clinicamente o LPO é classificado em seis padrões bem identificados: placa, reticular, bolhoso, atrófico, papular e erosivo. O objetivo dessa pesquisa foi comparar a imunoexpressão do fator nuclear Kappa (NF-κB) e do fator de crescimento endotelial vascular (VEGF165) no LPO. Esta pesquisa consistiu em um estudo transversal retrospectivo de caráter quantitativo e comparativo da expressão imuno-histoquímica da expressão do NF-κB e descritiva de VEGF165 em espécimes de líquen plano oral reticular (30 casos) e erosivo (10 casos) e ainda 15 casos de hiperplasia fibrosa inflamatória (Controle). Em seguida foi realizada a análise imuno-histoquímica para investigar diferenças na expressão do NF-κB (quantitativo de núcleos positivos) em relação ao número de células com núcleos negativos em regiões particulares do espécime, sendo utilizada a metodologia adaptada de Rich et al. (2018). Na análise estatística foram utilizados os testes (teste não-paramétrico de KruskalWallis, ANOVA one-way e o com correções de Bonferroni, com nível de significância de 5% (p < 0,05). Foi observado que para todos os casos de LPO presentes nesse estudo, houve marcação do VEGF165, tanto em tecido epitelial como nas células do infiltrado inflamatório e vasos sanguíneos. Nos casos de HFI, essa marcação teve menor intensidade quando comparada com a das lesões de LPO. Para o NF-κB não foi encontrada diferença estatisticamente significativa no revestimento epitelial em relação a hiperplasia fibrosa inflamatória (controle), já no tecido conjuntivo (infiltrado inflamatório superficial e profundo) a imunoexpressão dessa proteína foi inferior nos casos de LPO em relação a hiperplasia fibrosa inflamatória. Pode-se concluir que a resposta inflamatória no LPO seria coadjuvante na etiopatogenia, e não seria o fator isolado responsável pelo desenvolvimento e perpetuação do LPO, podendo ser crucial a presença de alterações epigenéticas no sistema imunológico para desencadear a doença (AU).

The oral Lichen Planus (LPO) is a relatively common chronic inflammatory disease whose etiopathogenesis is still not fully understood. Clinically the LPO is classified into six well identified standards: plate, Bullous, reticular, papular and atrophic erosive. The objective of this research was to compare the expression of nuclear factor Kappa (NF-κB) and vascular endothelial growth factor (VEGF165) in the LPO. This research consisted of a retrospective cross-sectional study of quantitative character and comparative immunohistochemical expression of expression of NF-κB and descriptive of VEGF165 in specimens of oral Lichen Planus Reticularis (30 cases) and erosion (10 cases) and 15 cases of inflammatory fibrous hyperplasia (control). Then analysis Immunohistochemistry to investigate differences in the expression of NF-κB (number of cores positive) in relation to the number of cells with nuclei in particular regions of the specimen negative, being used the methodology adapted from Rich et al. (2018). In the statistical analysis we used the tests (non-parametric test of Kruskal-Wallis one-way ANOVA with Bonferroni corrections and, with a significance level of 5% (p < 0.05). It was observed that for every case of LPO present in this study, there were marking the VEGF165, both in epithelial tissue as in inflammatory infiltrate cells and blood vessels. In the case of HFI, this markup had lower intensity when compared to that of LPO. For the NF-κB no statistically significant difference was found in the epithelial lining against inflammatory fibrous hyperplasia (control), already in the connective tissue (superficial and deep inflammatory infiltrate) expression in this protein was lower than in the case of LPO about inflammatory fibrous hyperplasia. It can be concluded that the inflammatory response in the LPO would be in a supporting role in pathogenesis, and it would not be the isolated factor responsible for the development and perpetuation of the LPO, and may be crucial to presence of epigenetic changes in the immune system to trigger the disease (AU).

Profiling of Pro-Inflammatory Cytokines in Radiation Induced Oral Mucositis (RIOM) among Indian patients

Radiation-induced oral mucositis (RIOM) is aimed at evaluating the expression of NF-κß, IL-1α, IL-6, IL-8 and TNF-α in patients with RIOM so as to validate their role in the pathobiology of the disease. Blood samples were collected and serum of 45 patients isolated with clinical signs and symptoms of mucositis and 10 healthy controls were also included in the study. The expression level of NF-κß, IL-1α, IL-6, IL-8, TNF-α was investigated using ELISA. Mann Whitney U test was applied to find the significance of the expression of these markers in RIOM patients as compared to normal healthy controls and significant expression (P< 0.05) for NF-κß, IL-6, TNF-α and non-significant expression (P > 0.05) IL-1α and IL-8 was found. No significant change in the expression level of the cytokines was observed for patients undergoing chemotherapy and radiation therapy as well as those receiving only the radiation therapy as a part of their treatment. We have also found less expression in grade 1 of mucositis as compared to grade 4. Pro- inflammatory cytokines indeed play a vital role in the pathogenesis as well as progression of RIOM (AU)

Modulation of cell proliferation, survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Objective: The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response. Material and Methods: T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR. Results: RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively. Conclusions: There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types. .

Imunoexpressão de fatores reguladores da osteoclastogênese na doença periodontal em humanos e sua relação com os parâmetros clínicos / Immunoexpression of factors regulating osteoclastogenesis in periodontal disease in humans and its relationship to the parameters of clinical

A doença períodontal é uma infecção oral iniciada por periodonlopatógenos que desencadeiam a resposta imune culminando com a destruição tecidual. Essa destruição é mediada pelo hospedeiro através da indução da produção e ativação de enzimas lííicas, citocinas e da estimulação da osteoclastogênese. O objetivo deste estudo foi comparar a expressão imuno-histoquímica dos fatores envolvidos na reabsorção óssea, RANKL (Ligante do Receptor Ativador do Fator Nuclear kappa B), OPG (Osteoprotegerina) e TNF-a (Fator de Necrose Tmnoral Alfa) entre a gengiva clinicamente saudável, a gengivite e a periodontite crônica, corretacionando-os com os parâmetros clínicos periodontais. A amostra consistiu de 83 casos, sendo 12 de gengivas clinicamente saudáveis. 42 de gengivite e 29 de periodontite, oriundos de 74 pacientes adolescentes e adultos com idade média de 35 anos, sem alterações sistêmicas e não fumantes, predominantemente do sexo feminino e da raça parda. Não houve diferença estatisticamente significativa para expressão do anticorpo anti-RANKL (p=0,58t) e da razão RANK.L/OPG (p=Ü,334) quando se comparou as três condições clínicas, mas o anti-OPG e anti-TNF-a mostraram diferenças estatisticamente significativas entre os tipos de lesão (p=0,001 epO.001, respectivamente), revelando maior imunoexpressão na periodontite. Nos casos de periodontite, a variável perda de inserção clínica (P1C) mostrou diferença estatisticamente significativa e correlação positiva, respectivamente, com a imunomarcação dos anticorpos anti-RANKL (p=0,002; p=0,00! e r=0,642), anti-OPG (p=0,018; p=Ü,014 e 1=0,451), anti-TNF-a (p=0,032; p=0,0t4 e r=0,453) e com a razão percentual de RANKL/OPG {p=0,018; p=0,002 e r=0,544). A mobilidade dentária (MB) apresentou diferença estatisticamente significativa somente com a imunoexpressão do anti-RANKL (p=G,Q26). e a profundidade de sondagem (PS) apresentou correlação positiva com o anti-RANKL (p=0.028 e r=0.4G9), ambos nos casos de periodontite...

Papel das proteínas intracelulares nod e da proteína adaptadora myD88 na regulação de espressão de RANKL e modulação da resposta inflamatória induzidos por antígenos bacterianos in vitro: estudo em células relevantes de periodonto / Role of nod an myD88 proteins on the regulation of bacterial antigen-induced expression of RANKL and on the modulation of inflammation: a study on relevant cells of the periodontium

A reabsorcao do osso alveolar e uma das principais caracteristicas associadas a progressao da doenca periodontal. Apesar da enorme complexidade da microbiota envolvida, considera-se que bacterias Gram-negativas tenham um papel relevante em sua etiopatogenese. Um dos fatores de virulencia destes microrganismos e representado por um componente de sua parede externa denominado lipopolissacarideo (LPS). A presenca de LPS na proximidade dos tecidos periodontais e capaz de induzir a producao de diversos mediadores inflamatorios que levam a degradacao tanto do tecido conjuntivo quanto osseo. Atualmente acredita-se que a interacao do ligante do receptor-ativador do fator nuclear kappa-B (RANKL) com seu receptor (RANK) presente em precursores hematopoieticos e necessaria e suficiente para a inducao da diferenciacao de osteoclastos. Por outro lado, a ligacao de RANKL com seu falso-receptor, denominado osteoprotegerina (OPG), reduz sua biodisponibilidade e inibe, desta forma, a osteoclastogenese. Assim, a razao da expressao de RANKL e OPG e considerada como o principal determinante do “turnover” do tecido osseo. A producao de RANKL e OPG depende das vias de sinalizacao ativadas, as quais sao influenciadas pela natureza do estimulo extracelular. Atualmente, a familia de receptores NLRs (nod-like receptors) foi identificada como receptor intracelular para componentes bacterianos e agentes moduladores de diferentes vias de sinalizacao. Considerando a relevancia do LPS bacteriano na patogenese da doenca periodontal, o papel do RANKL no processo de reabsorcao ossea e a possivel implicacao das proteinas Nod na transducao de sinais regulando a expressao de RANKL, o objetivo geral deste projeto foi estudar os mecanismos de regulacao da expressao de RANKL induzido por LPS bacteriano em celulas relevantes do periodonto (macrofagos, osteoblastos e fibroblastos). Os objetivos especificos propostos...

Bone resorption is one of the major characteristics of destructive periodontal disease. Despite the great number of different bacterial species in the dental biofilm, Gramnegative microorganisms were demonstrated to have a very important role on periodontal disease pathogenesis. Lipopolysaccharide (LPS) is a bacterial cell wall component, which is acknowledged as one of the main virulence factors of these microorganisms. The mere presence of LPS in proximity with the periodontal tissues initiates the expression and production of inflammatory mediators and other cytokines which can culminate in degradation of both soft and hard tissues. It is currently accepted that the interaction between receptor-activator of nuclear factor kappa-B ligand (RANKL) and its receptor (RANK) is both necessary and sufficient to induce osteoclast differentiation and activation. However, RANKL can interact with its soluble decoy receptor osteoprotegerin (OPG) inhibiting osteoclastogenesis by decreasing the bioavailability of RANKL. Production of RANKL/OPG is the result of the signaling pathways activated by external stimuli. Recently, the NLR (nod-like receptors) family was identified as cytosolic receptors for bacterial components and also, as capable of modulating different signaling pathways. Considering the relevance of LPS and RANKL in bone resorption and the possible implication of Nod proteins in signal transduction regulating RANKL expression, the aim of this study was to evaluate the influence of different intracellular signaling pathways on the regulation of RANKL expression induced by LPS in relevant cells of the periodontium (macrophages, osteoblasts and fibroblasts). The specific objectives proposed were to determine after LPS and interleukin-1 beta stimulation the role of MyD88-dependent and independent signaling pathways, Nod1 and Nod2 on the expression of RANKL, OPG, IL-10 and IFN-beta...