Análise da influência de proteínas da matriz extracelular e fibrinogênio na formação de biofilme por cepas de Enterococcus faecalis isoladas de infecção endodôntica primária / Analysis of influence of extracellular matrix proteins and fibrinogen in biofilm formation by Enterococcus faecalis strains isolated from primary endodontic infection

Enterococcus faecalis é um patógeno oportunista com peculiar potencial para a manutenção da infecção perirradicular endodôntica após o preparo químico-mecânico do sistema de canais radiculares. Adicionalmente, possui aptidão para desenvolver-se em biofilme e apresenta em sua parede celular adesinas compatíveis com substratos colagênicos, como a composição da matriz extracelular da dentina e dos túbulos dentinários. Esse estudo propôs-se a caracterizar geneticamente 23 amostras de E faecalis isoladas de infecções endodônticas primárias através da técnica da reação em cadeia da polimerase (PCR, do inglês Polymerase Chain Reaction) e investigar a influência de COL I (colágeno tipo I), FN (fibronectina) e fibrinogênio (FBG) na formação de biofilme em superfície abiótica. Assim, após a sensibilização de ¾ dos poços de placas de poliestireno estéreis com 50 µl da solução de proteína de matriz (COL I, FN e FBG) na concentração de 1mg/ml, transferiu-se 50µl de suspensão bacteriana (1,5 x 108 bact/mL) correspondente a cada amostra, de modo a preencher tanto os poços sensibilizados como os não sensibilizados. A quantificação da formação de biofilme foi realizada por meio de leitura por densidade óptica, cujos resultados revelaram que houve formação de biofilme por todas as em superfície abiótica, porém com diferentes graus de intensidade. Todas as cepas foram identificadas geneticamente como Enterococcus faecalis e a presença do gene gelE foi dominante. Contudo, nenhuma apresentou amplificação para os genes esp e agg, e, apesar de 73,9% das amostras amplificarem para o gene ace, apenas 2 cepas (P7 e P75) isoladas de infecções endodônticas primárias tiveram aumento de formação de biofilme na presença de COL I (P<0,05). Embora a presença de FBG não forneça subsídio estatisticamente significante para a formação de biofilme, COL I e FN influenciaram na redução da formação do biofilme para a maior parte das amostras. É possível que a capacidade de formação de biofilme inerente ao E. faecalis e a afinidade para FN e COL I através da expressão gênica de ace contribuam substancialmente para a manutenção desse micro-organismo no ambiente radicular mesmo após o tratamento endodôntico minucioso.

Enterococcus faecalis is an opportunistic pathogen with peculiar potential to maintain the periradicular endodontic infection even after chemical-mechanical preparation of the root canal system. In addition, it has the ability to develop into biofilms and presents in your cell wall adhesins compatible with collagenous substrates, as the composition of the extracellular matrix of the dentine and dentinal tubules. This study aims to characterize genetically 23 samples of E. faecalis isolated from primary endodontic infections by Polymerase Chain Reaction (PCR) technique and investigate the influence of collagen type I (COL I), fibronectin (FN) and fibrinogen (FBG) in biofilm formation on abiotic surface. Thus, after the sensitization of ¾ the wells of sterile microtiter plates with 50 ul of matrix protein solution (COL I and FN FBG) at a concentration of 1mg / ml, was transferred 50mL of bacterial suspension (1.5 x 108 bact / ml) corresponding to each sample in order to fill both wells sensitized and non-sensitized. Quantification of biofilm formation was performed by optical density, so the results showed that there were biofilm formation by all strains on abiotic surface, but with different degrees of intensity. All strains were genetically identified as Enterococcus faecalis and the presence of gelE gene was prevalent. However, none showed amplification for the esp and agg gene, and, while 73.9% of the samples for amplifying ace gene, only 2 strains (P7 and P75) isolated from primary endodontic infections they had increased biofilm formation in the presence of COL I (P <0.05). Although the presence of FBG no provides significant support for the biofilm formation, COL I and FN were relevant influence in the reduction of biofilm formation for most of the samples. It is possible that the biofilm-forming ability inherent in E. faecalis and affinity for FN and COL I through ace gene expression contribute substantially to maintain of this microorganism in the root environment even after thorough endodontic treatment.

Dentin matrix protein 1 (DMP1) expression in developing human teeth

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein that plays an important role in mineralized tissue formation by initiation of nucleation and modulation of mineral phase morphology. The purpose of the present study was to examine the immunoexpression of DMP1 in tooth germs of 7 human fetuses at different gestational ages (14, 16, 19, 20, 21, 23 and 24 weeks) comparing with completed tooth formation erupted teeth. The results showed the presence of DMP1 in the dental lamina, as well as in the cells of the external epithelium, stellate reticulum and stratum intermedium of the enamel organ. However, in the internal dental epithelium, cervical loop region and dental papilla some cells have not labeled for DMP1. In the crown stage, DMP1 was expressed in the ameloblast and odontoblast layer, as well as in the dentinal tubules of coronal dentin near the odontoblast area. Erupted teeth with complete tooth formation exhibited immunolabeling for DMP1 only in the dentinal tubules mainly close to the dental pulp. No staining was observed in the enamel, predentin or dental pulp matrix. DMP1 is present in all developing dental structures (dental lamina, enamel organ, dental papilla) presenting few immunoexpression variations, with no staining in mineralized enamel and dentin.

A proteína da matriz dentinária 1 (DMP1) é uma fosfoproteína ácida que tem sido relacionada diretamente ao processo de mineralização dos tecidos em formação sendo iniciadora do processo de nucleação e modulação da fase mineral. O objetivo desse trabalho foi avaliar a imunoexpressão da DMP1 em germes dentários em diferentes fases da odontogênese, obtidos de 7 fetos humanos em diversos estágios gestacionais (14, 16, 19, 20, 21, 23 e 24 semanas), comparando-se com dentes com rizogênese completa. Os resultados mostraram que a DMP1 esteve expressa na lâmina dentária, bem como, nas células do epitélio externo, retículo estrelado e estrato intermediário do órgão do esmalte. Diferentemente, no epitélio interno do órgão do esmalte, alça cervical e papila dentária algumas células não apresentaram a DMP1. Nas fases de coroa, os ameloblastos e odontoblastos apresentaram marcação positiva para a DMP1, bem como os túbulos dentinários da dentina coronária próximos à região odontoblástica. Os dentes com rizogênese completa exibiram marcação para a DMP1 apenas nos túbulos dentinários principalmente próximos à polpa dentária. Nenhuma marcação foi observada na matriz de esmalte ou pré-dentina, nem na polpa dentária. Concluímos que a DMP1 está presente em todas as fases da odontogênese, tanto na lâmina dentária, órgão do esmalte, bem como na papila dentária, com pequenas variações de nuances de expressão, estando ausente na dentina e esmalte mineralizados.

Chronic liver injury, TGF-beta, and cancer

Cells termed myofibroblasts are prominent in the injury response of all epithelial tissues. They exhibit proliferation, migration, production of collagen and other extracellular matrix (ECM) molecules, and contraction, all for containing the injury and closing the wound. When the injury is limited in time, the final stage of the repair involves a dismantling of the cellular apparatus and restoration of normal tissue structure. With multiple cycles of repair, however, there is net accumulation of ECM, to the detriment of tissue structure and function. Repair-related ECM coalesces into fibrous bundles and, over time, undergoes changes that render it resistant to degradation. The result is a scar. In the skin, a scar may have cosmetic importance only. In the liver, however, extensive scarring is the setting for unregulated growth and neoplasia; also, fibrous bands disrupt normal blood flow, leading to portal hypertension and its complications. With regard to therapy for fibrosis, the first consideration is elimination of the injury factor. However, given that many liver diseases do not have effective therapies at present, strategies targeting fibrogenesis per se are under development. The main source of myofibroblast-like cells and ECM production in the liver is the perisinusoidal stellate cell, which responds to injury with a pleiotypic change termed activation. Activation is orchestrated by cytokines and the ECM itself. Among the cytokines involved in this process, transforming growth factor-beta (TGF-beta) is particularly prominent. The early changes in ECM include de novo production of a specific "fetal" isoform of fibronectin, which arises from sinusoidal endothelial cells. It is stimulated by TGF-beta and acts directly on stellate cells to promote their activation. Based on these and other advances in understanding the fundamentals of the injury response, several strategies now exist for altering fibrogenesis, ranging from agents that block TGF-beta to traditional Chinese herbal extracts. Arrest of fibrogenesis, even with underlying cirrhosis, is likely to extend life or prolong the time to transplant. Whether it reduces the risk of hepatocellular carcinoma remains to be proven. Although TGF-beta antagonists are effective anti-fibrogenic agents, they will require detailed safety testing because of the finding that several forms of epithelial neoplasia are associated with altered regulation of TGF-beta.

Effect of PMA and IL-1 on matrix proteins with special reference to kidney mesangial cells.

Cytokines have different effects on the extracellular matrix proteins which were produced by a number of different cell lines. The extracellular matrix proteins fibronectin, collagen IV, vitronectin, laminin, proteoglycan, fetal proteoglycan and actin; and the cells human mesangial, BeWo, Hep G2 and BHK using APAAP and dot-blot assay were used. It was shown that IL-1 and PMA increased the staining intensity of proteoglycan, fibronectin and collagen IV and vitronectin and did not alter the intensity of staining. While PMA and IL-1 decreased but did not alter laminin, PMA increased the staining intensity of proteoglycan in mesangial cells but not altered in mesangial cells stimulated with IL-1. These results show that PMA and IL-1 altered the production and secretion of extracellular matrix proteins, these in turn could lead to the alteration of pore size, matrix properties and filtration of mesangial cells, and thus influence pathological conditions of renal diseases.