Assessment and comparison of nuclear changes seen in gingivitis and periodontitis using fluorescent microscopy / Valoración y comparación de los cambios nucleares observados en gingivitis y periodontitis, mediante microscopio fluorescente

INTRODUCTION AND OBJECTIVES: The objective of this study was to observe the evolution of genotoxicity (micronucleation, binucleation and multinucleation) from normal to periodontally compromised gingival epithelium (gingivitis and periodontitis) and to compare the severity of damage. METHODS AND MATERIAL: 45 participants formed 3 different categories; a control group of 15 healthy subjects, 15 subjects with gingivitis and 15 with chronic periodontitis. Smears were collected from all the gingiva and stained with acridine orange stain. A total of 500 cells were evaluated under fluorescent microscopy for nuclear abnormalities such as micronuclei, binucleation and multinucleation. The statistical analysis used was one way ANOVA and posthoc Tukey test. RESULTS AND CONCLUSIONS: A statistically significant difference was observed when the age of the 3 groups were compared (p = 0.002); the control group were younger than those with chronic periodontitis or gingivitis. With respect to genotoxic changes, the differences for binucleation (p = 0.002) and multinucleation (p < 0.001) were statistically significant thus suggesting advanced damage in the nucleus. Such changes in genotoxicity could be of help to a clinician in determining prognosis

INTRODUCCIÓN Y OBJETIVOS: El objetivo de este estudio fue observar las tendencias en cuanto a genotoxicidad (micronucleación, binucleación y multinucleación) a partir de epitelio gingival de normal a periodoncialmente-comprometido (gingivitis y periodontitis), y comparar la gravedad del daño entre ambas situaciones. MÉTODOS Y MATERIAL: Seleccionamos a cuarenta y cinco sujetos, que agrupamos en 3 categorías (15 sujetos sanos, 15 con gingivitis y 15 con periodontitis crónica). Se recogieron frotis del tejido gingival de dichos sujetos, que se tiñeron con acridina naranja. Evaluamos un total de 500 células bajo el microscopio fluorescente en busca de anomalías nucleares de tipo micronúcleos, binucleación y multinucleación. El análisis estadístico utilizado fue ANOVA unidireccional y Tukey posthoc. RESULTADOS Y CONCLUSIÓN: Observamos una diferencia estadísticamente significativa al comparar los 3 grupos de edad (p = 0,002), donde el grupo control (sujetos sanos) pertenecía a un rango de edad inferior a los sujetos con periodontitis o gingivitis crónicas. Con respecto a los cambios genotóxicos, las diferencias para binucleación (p = 0,002) y multinucleación (p < 0,001) fueron estadísticamente significativas, lo cual sugiere un daño avanzado en el núcleo. Las tendencias en cuanto a genotoxicidad podrían ayudar a los clínicos a determinar el pronóstico de la enfermedad

Candidate predisposing germline copy number variants in early onset colorectal cancer patients

Purpose. A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called ‘missing heritability’. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. Methods/patients. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. Results. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. Conclusions. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC (AU)

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Flow cytometry assay for counting micronucleated erythrocytes: development process.

Development of any new assay proceeds in several phases. When an assay is intended for regular use to support regulatory decision-making, there are significant additional stages in the development process beyond the initial description of the method. In this paper we discuss some of the studies related to the development of a flow cytometric method for counting micronuclei in rodent erythrocytes. Studies related to fixation methods and conditions, standardization of DNA staining, and antibody staining are discussed. These studies, while not part of the formal description of the method, are needed as part of the preparation for the formal validation of the method. In addition, the lessons learned in transferring the method to other laboratories are briefly discussed in relation to defining the final protocol.