Dental bleaching with ozone: effects on color and enamel microhardness.

The aim of this in vitro study was to evaluate the effects of dental bleaching with ozone (O3) on color change and enamel microhardness. Enamel blocks (3 x 3 x 3mm) were randomly distributed for treatments (n=10). Color change (ΔE) and Knoop microhardness of the enamel blocks were evaluated before and after the following treatments: C - deionized water (control); HP - 37.5% hydrogen peroxide (Pola Office+/ SDI); PLA - placebo gel; O3 - ozone; and O2 - oxygen. Four 8-minute applications were used for HP and PLA, and one 19-minute application for O3 and O2.One-way ANOVA revealed that ΔE was not significantly influenced by the treatment (p = 0.112). For the treatments with HP, PLA, O3 andO2, ΔE was greater than 3.3. The paired t test showed significant decrease in microhardness after treatments (p < 0.001) but no significant difference between treatments (ANOVA; p = 0.313). Dental bleaching treatments with O3, HP, O2 and PLA induced enamel color changes that may be clinically discernible, although enamel microhardeness decreased.

O objetivo deste estudo in vitro foi avaliar os efeitos do clareamento dental com ozônio (O3) quanto à alteração de cor e microdureza do esmalte. Blocos de esmalte (3 x 3 x 3mm) foram aleatoriamente distribuídos entre os tratamentos (n=10). Alteração de cor (ΔE) e microdureza Knoop foram avaliados antes e após cada um dos seguintes tratamentos: C ­ água deionizada (controle); PH ­ peróxido de hidrogênio a 37,5% (Pola Office+/ SDI); PLA ­ gel placebo; O3 ­ ozônio; O2 ­ oxigênio. Quatro aplicações de PH e PLA foram realizadas por 8 minutos cada e uma aplicação de O3 e O2 foram realizados por 19 minutos em cada bloco de esmalte. ANOVA a um critério mostrou que os valores de ΔE não foram significativamente influenciados pelo tratamento (p = 0,112). Para os tratamentos com PH, PLA, O3 e O2, o ΔE foi maior que 3,3. O teste t pareado mostrou diminução significativa dos valores de microdureza no final do tratamento quando comparado com o tempo baseline (p < 0,001), mas não houve diferença significativa entre os tratamentos (ANOVA; p = 0,313). O tratamento com O3, PH, O2 e PLA levou a alteração de cor do esmalte clinicamente perceptível, embora tenha sido observada diminuição da microdureza do esmalte com a realização dos tratamentos.

Dental bleaching with ozone: effects on color and enamel microhardness / Clareamento dental com ozônio: efeitos na cor e na microdureza do esmalte

The aim of this in vitro study was to evaluate the effects of dentalbleaching with ozone (O3) on color change and enamelmicrohardness. Enamel blocks (3 x 3 x 3mm) were randomlydistributed for treatments (n=10). Color change (ΔE) and Knoopmicrohardness of the enamel blocks were evaluated before andafter the following treatments: C – deionized water (control); HP– 37.5% hydrogen peroxide (Pola Office+/ SDI); PLA – placebogel; O3– ozone; and O2– oxygen. Four 8-minute applicationswere used for HP and PLA, and one 19-minute application for O3and O2.One-way ANOVA revealed that ΔE was not significantlyinfluenced by the treatment (p = 0.112). For the treatments withHP, PLA, O3 and O2, ΔE was greater than 3.3. The paired t testshowed significant decrease in microhardness after treatments (p< 0.001) but no significant difference between treatments(ANOVA; p = 0.313). Dental bleaching treatments with O3, HP,O2and PLA induced enamel color changes that may be clinicallydiscernible, although enamel microhardeness decreased.

O objetivo deste estudo in vitro foi avaliar os efeitos doclareamento dental com ozônio (O3) quanto à alteração de core microdureza do esmalte. Blocos de esmalte (3 x 3 x 3mm)foram aleatoriamente distribuídos entre os tratamentos(n=10). Alteração de cor (ΔE) e microdureza Knoop foramavaliados antes e após cada um dos seguintes tratamentos: C– água deionizada (controle); PH – peróxido de hidrogênio a37,5% (Pola Office+/ SDI); PLA – gel placebo; O3– ozônio;O2– oxigênio. Quatro aplicações de PH e PLA foramrealizadas por 8 minutos cada e uma aplicação de O3e O2foram realizados por 19 minutos em cada bloco de esmalte.ANOVA a um critério mostrou que os valores de ΔE não foramsignificativamente influenciados pelo tratamento (p = 0,112).Para os tratamentos com PH, PLA, O3 e O2, o ΔE foi maior que3,3. O teste t pareado mostrou diminução significativa dosvalores de microdureza no final do tratamento quandocomparado com o tempo baseline (p < 0,001), mas não houvediferença significativa entre os tratamentos (ANOVA; p =0,313). O tratamento com O3, PH, O2e PLA levou a alteraçãode cor do esmalte clinicamente perceptível, embora tenha sidoobservada diminuição da microdureza do esmalte com arealização dos tratamentos.

Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial cell in culture.

Angiotensin converting enzyme 2 (ACE2) is a component of the renin-angiotensin system (RAS) which converts Ang II, a potent vasoconstrictor peptide into Ang 1-7, a vasodilator peptide which may act as a negative feedback hormone to the actions of Ang II. The discovery of this enzyme added a new level of complexity to this system. The mesangial cells (MC) have multiple functions in glomerular physiology and pathophysiology and are able to express all components of the RAS. Despite of being localized in these cells, ACE2 has not yet been purified or characterized. In this study ACE2 from mice immortalized MC (IMC) was purified by ion-exchange chromatography. The purified enzyme was identified as a single band around 60-70 kDa on SDS-polyacrylamide gel and by Western blotting using a specific antibody. The optima pH and chloride concentrations were 7.5 and 200 mM, respectively. The N-terminal sequence was homologous with many species ACE2 N-terminal sequences as described in the literature. ACE2 purified from IMC was able to hydrolyze Ang II into Ang 1-7 and the K(m) value for Ang II was determined to be 2.87 ± 0.76 µM. In conclusion, we purified and localized, for the first time, ACE2 in MC, which was able to generate Ang 1-7 from Ang II. Ang 1-7 production associated to Ang II degradation by ACE2 may exert a protective effect in the renal hemodynamic.

Host cell actin remodeling in response to Trypanosoma cruzi: trypomastigote versus amastigote entry.

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a highly prevalent vector-borne disease in Latin America. Chagas' disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host's blood, infected triatomines release in their feces highly motile and infective metacyclic trypomastigotes that may initiate infection. Metacyclic trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellularly, into amastigote-like forms. Unlike their intracellular counterparts, these amastigote-like forms, henceforth called amastigotes, are capable of infecting host cells. Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.

Cell invasion by Trypanosoma cruzi amastigotes of distinct infectivities: studies on signaling pathways.

Trypanosoma cruzi metacyclic trypomastigotes of the major phylogenetic lineages use specific signaling pathways to invade host cells. Using a panel of drugs, we studied if the differences in the ability of extracellular amastigotes (EA) from G (T. cruzi I) and CL (T. cruzi II) strains to invade host cells could be associated to activation of specific signaling routes. Sonicated extracts from G or CL strain EA induced transient raises in HeLa cell intracellular Ca(2+) levels in a dose-dependent manner. Treatment of EA with drugs that affect Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores did not significantly affect the infectivity of either strain, whereas EA of both strains treated with ionomycin plus NH(4)Cl or nigericin that release Ca(2+) from acidocalcisomes had their infectivity reduced. Treatment of parasites with adenylate cyclase activator forskolin increased the infectivity of both strains towards HeLa cells. These data, taken together, suggest that, for host cell invasion, G and CL strain EA engage signaling pathways that lead to an increase of cyclic adenosine monophosphate and Ca(2+) mobilization from acidocalcisomes. Moreover, treatment of EA with genistein reduced by approximately 45% the invasion of HeLa cells by G but not by CL strain, implicating a protein tyrosine kinase in the process. In line with this, HeLa cell extracts contained a protein tyrosine kinase activity that mediated the phosphorylation of 87- and 175-kDa polypeptides of EA from G but not from CL strain. Regarding the target cell response, the activation of host PI3 kinase appears to be required for invasion by either strain as treatment of HeLa cells with wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process involving metacyclic trypomastigotes.

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms.

Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

O agente etiológico da doença de Chagas, Trypanosoma cruzi, ocorre como cepas ou isolados que podem ser agrupados em duas grandes linhagens filogenéticas: T. cruzi I associada ao ciclo silvestre e T. cruzi II ligada à doença humana. No hospedeiro mamífero o parasita tem que invadir células, e vários estudos relacionam as formas flageladas tripomastigotas neste processo. Diferentes componentes de superfície dos parasitas e alguns dos respectivos receptores foram identificados. Em nosso trabalho temos procurado compreender como amastigotas, que normalmente são encontrados crescendo no citoplasma, podem invadir células de mamíferos com infectividade comparável às dos tripomastigotas. Encontramos diferenças nas respostas celulares induzidas por amastigotas e tripomastigotas em relação a componentes de citoesqueleto e projeções de membrana ricas em actina. Amastigotas de cepas de T. cruzi I gerados extracelularmente, podem apresentar infectividade maior que tripomastigotas metacíclicos para linhagens celulares e células com expressão alterada em diferentes classes de componentes celulares. Células albergando a bactéria Coxiella burnetii tem nos permitido obter novos enfoques sobre as propriedades de tráfego intracelular das diferentes formas infectivas do T. cruzi, revelando requerimentos inesperados para o parasita transitar entre seu vacúolo parasitóforo até seu destino final no citoplasma da célula hospedeira.

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages.

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.