Evaluación in Vitro de la Biocompatibilidad de Cuatro Cementos Selladores con Osteoblastos Humanos / In vitro Evaluation of Four Sealer Cements Biocompatibility with Human Osteoblasts

RESUMEN: El correcto sellado apical es un paso importante durante el tratamiento de conductos, para esto, se utilizan puntas de gutapercha y cemento sellador, de este último existen diversas formulaciones químicas en el mercado, por lo cual es importante tomar en cuenta los efectos que estas pueden tener en el proceso de cicatrización periapical. El propósito de este estudio fue evaluar la biocompatibilidad de cuatro cementos selladores con diferente composición química con osteoblastos humanos. Se prepararon extractos de cementos selladores a con dos concentraciones (10 mg/mL y 40 mg/mL) y dos tiempos de exposición (10 min y 8 h), estos fueron colocados en contacto con osteoblastos humanos para evaluar la proliferación y citotoxicidad a 24, 72 y 96 h con sus respectivos controles y blancos. Se realizó un análisis estadístico con ANOVA de un factor y la prueba de comparaciones múltiple de Bonferroni. Los resultados obtenidos, tanto en el ensayo de citotoxicidad como en el de proliferación, indicaron que el cemento a base de resina no es biocompatible con osteoblastos. El cemento a base de poli-dimetilxilosano fue el único que no mostró citotoxicidad a ningún de tiempo de exposición y concentración examinadas en este estudio.

ABSTRACT: Correct apical sealing is an important step during root canal treatment, hence, gutta-percha points and sealant are used. There are several chemical compositions on the market, so it is important to evaluate the effects of these in the periapical healing process. The aim of this study was to evaluate the biocompatibility of four sealer cements with different chemical composition placed in contact with human osteoblast. Different extracts were prepared at two concentrations (10 mg/mL and 40 mg/mL) and two exposure times (10 min and 8 h) these were placed in contact with human osteoblast to evaluate cytotoxicity and proliferation at 24, 48 and 72 h with their respective controls and blanks. A statistical analysis was performed with ANOVA of one factor and Bonferroni post hoc. Results obtained in cytotoxicity and proliferation assays, indicated that the resinbased cement is not biocompatible with osteoblast. The poly-dimethylxilosanbased cement was the only that did not show cytotoxicity at any time of exposure and concentration examined in this study.

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation.

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A(-202A/376G), G6PDA(-376G/968C) and G6PD Santamaria(376G/542T). Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF(193A>G) (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.

Parasitaemia levels in Plasmodium chabaudi infected-mice modify IFN-gamma and IL-10 expression after a homologous or heterologous challenge.

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti-malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF-alpha, IL-2 and IFN-gamma response which is followed by an IL-4 and IL-10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN-gamma and a weaker IL-10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.

The effect of an anti-inflammatory pentapeptide produced by Entamoeba histolytica on gene expression in the U-937 monocytic cell line.

OBJECTIVE: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression of genes encoding human cytokines, receptors and related factors in the human cell line U-937. MLIF (Met-Gln-Cys-Asn-Ser) is an anti-inflammatory pentapeptide produced by Entamoeba histolytica that inhibits many human monocyte functions. MATERIAL AND METHODS: U-937 cell line cultured (24 hrs/RPMI). RNA extracted by Trizol method. 385 genes were analyzed on microarray membranes, complement by real-time RT-PCR and protein expression of some affected genes. RESULTS: MLIF had a preferentially inhibitory effect on gene expression; four genes were over-expressed and 13 underexpressed in MILF vs. simple medium - constitutive expression. Three genes are over-expressed and 19 under-expressed in MLIF/PMA vs. PMA - induced expression. CONCLUSIONS: Many modified genes are products regulated by the Nuclear Factor-kappaB and Mitogen Activated Protein Kinase pathways, suggesting MLIF involvement with these two major pathways for the modulation of the inflammation and immune responses.

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes.

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.