Evaluación de efectos citotóxicos y genotóxicos con ensayo de micronúcleos en la mucosa bucal de pacientes con aparatología ortodóncica. Revisión de la literatura / Evaluation of cytotoxic and genotoxic effects with micronucleus test in the oral mucosa of patients with orthodontic appliances. Review of the literature

Las maloclusiones representan un problema de salud bucodental, que se resuelven mediante la colocación de aparatología ortodóncica fija (AOF). Esta aparatología provoca corrosión y liberación de iones metálicos por las aleaciones que la constituyen y por el tiempo prolongado del tratamiento. El objetivo de este trabajo fue analizar las alteraciones citotóxicas y genotóxicas de las células de la mucosa oral provocadas por el uso de AOF, reportadas en la literatura y evaluadas con ensayo de micronúcleos (MN); el cual es uno de los ensayos más utilizados para identificar el daño al ácido desoxirribonucleico (ADN). Se realizó una revisión de la literatura de los últimos 10 años, donde se incluye- ron nueve estudios, el 55% de estos mostró evidencia de daño citotóxico y genotóxico posterior a la terapia ortodóncica. El promedio de incremento de MN debido al uso de AOF en estos estudios, fue tres veces mayor con respecto a las células bucales sin trata- miento, este dato es similar a reportes de células orales precancerosas investigadas por otros autores. Además los artículos evaluados, reportaron alteración celular a partir de la primera semana de la colocación de los dispositivos y señalaron que hay una disminu- ción del daño con el tiempo de exposición. En conclusión, el ensayo de MN utilizado en la cavidad bucal demostró ser útil para detectar alteraciones en el ADN debido al uso de AOF. Los datos analizados permiten a los ortodoncistas implementar mejoras en la terapéutica ortodóncica.

Malocclusions represent an oral health problem, which is resolved by the placement of fixed orthodontic appliances (FOA). This orthodontic appliance causes corrosion and release of metal ions due to the alloys they constitute and due to the prolonged time of treatment. The objective of this work was to analyze the cytotoxic and genotoxic altera- tions from oral mucosa cells, caused by the use of FOA, reported in the literature and evaluated with micronucleus (MN) test, which is one of the most widely used tests to identify damage to deoxyribonucleic acid (DNA). A review of the literature of the last 10 years was carried out, where nine studies were included, 55% of these studies showed evidence of cytotoxic and genotoxic damage after orthodontic therapy. The increased average in MN due to the use of FOA in these studies, represent values of approximately three-fold more respect to oral cells without treatment, this data is similar to reports of precancerous oral cells that have been reported by other authors. Besides, the articles analyzed reported cell alterations after the first week of the devices placement and indica- ted a decrease in damage with exposure time. In conclusion, the MN test used in the oral cavity was useful in detecting DNA alterations due to the use of FOA. The data analyzed allow orthodontists to implement improvements in orthodontic therapy.

A biosensor capable of identifying low quantities of breast cancer cells by electrical impedance spectroscopy.

Breast cancer (BC) is a malignant disease with a high prevalence worldwide. The main cause of death is not the primary tumor, but instead the spread of tumor cells to distant sites. The aim of the present study was to examine a new method for the detection of cancer cells in aqueous medium using bioimpedance spectroscopy assisted with magnetic nanoparticles (MNP's) exposure to a constant magnetic field. The spectroscopic patterns were identified for three breast cancer cell lines. Each BC cell line represents a different pathologic stage: the early stage (MCF-7), invasive phase (MDA-MB-231) and metastasis (SK-BR-3). For this purpose, bioimpedance measurements were carried out at a certain frequency range with the aid of nanoprobes, consisting of magnetic nanoparticles (MNPs) coupled to a monoclonal antibody. The antibody was specific for the predominant cell surface protein for each cell line, which was identified by using RT-qPCR and flow cytometry. Accordingly, EpCAM corresponds to MCF-7, MUC-1 to MDA-MB-231, and HER-2 to SK-BR-3. Despite their low concentrations, BC cells could be detected by impedance spectroscopy. Hence, this methodology should permit the monitoring of circulating tumor cells (CTC) and therefore help to prevent recurrences and metastatic processes during BC treatment.

Componentes Principales mediante Espectroscopia FTIR como Técnica de Caracterización Innovadora durante la Diferenciación de Células Madre Pluripotentes a Células Pancreáticas / Principal Components by FTIR Spectroscopy as Innovative Characterization Technique during Differentiation of Pluripotent Stem Cells to Pancreatic Cells

Resumen: Dos de los grandes retos en la biología de las Células Madre (CM) y la Medicina Regenerativa, son el control en la diferenciación de estas células y asegurar la pureza de las células diferenciadas, por lo que es necesario contar con técnicas rápidas, eficientes y precisas para la caracterización de CM y su diferenciación a diferentes linajes celulares. El objetivo de este trabajo fue analizar Células Madre Pluripotentes (CMP) y Células Pancreáticas Diferenciadas (CPD) mediante espectroscopía Infrarroja por Transformada de Fourier (FTIR) y Análisis de Componentes Principales (ACP). Para ello se diferenciaron CMP a CPD, caracterizando el proceso de diferenciación a los días 0, 11, 17 y 21 mediante microscopía óptica y espectroscopia vibracional. Los espectros FTIR se analizaron con el método multivariado de ACP, utilizando su segunda derivada en las regiones de proteínas, carbohidratos y ribosas. Los resultados indican que el ACP permite caracterizar y discriminar CMP y CPD en sus diferentes etapas de diferenciación en las regiones espectrales analizadas. Con lo anterior concluimos que el ACP permite caracterizar química y estructuralmente CMP y diferentes etapas de su diferenciación en una forma rápida, precisa y no invasiva.

Abstract: Two of the greatest challenges in Stem Cells (SCs) biology and regenerative medicine, are differentiation control of SCs and ensuring the purity of differentiated cells. In this sense, fast, efficient and accurate techniques for SCs characterization and their differentiation into different cell lineages are needed. The aim of this study was to analyse Pluripotent Stem Cells (PSCs) and Differentiated Pancreatic Cells (DPCs) by Fourier Transform Infrared (FTIR) spectroscopy and Principal Component Analysis (PCA). For this purpose, we differentiated PSCs toward DPCs, characterizing the differentiation process at different stages (0, 11, 17 and 21 days) through light microscopy and vibrational spectroscopy. FTIR spectra were analysed with the multivariate method of PCA, using the second derivatives in the protein, carbohydrate and ribose regions. The results indicate that the PCA allows to characterize and discriminate PSCs and DPCs at different stages of differentiation in the analysed spectral regions. From these results, we concluded that the PCA allows the chemically and structural characterization of PSCs and the different stages of their differentiation in a fast, accurate and non-invasive way.

Effect of Dialyzable Leukocyte Extract on chronic cervicitis in patients with HPV infection.

The objective of the study was to assess the clinical, histopathological and immunochemical changes induced by dialyzable leukocyte extract (DLE) treatment in patients with chronic cervicitis associated to HPV infection. Fifty-four female Mexican patients diagnosed with chronic cervicitis, cervical intra-epithelial neoplasia grade 1 (CIN 1) and HPV infection were divided into two groups: patients treated with placebo and patients treated with DLE. Clinical and colposcopy evaluations were performed before and after treatments. Cervix biopsies were obtained to analyze histopathological features and to determine the local immunological changes by immunohistochemistry analyses. Placebo-treated patients showed no significant changes in the evaluated parameters. Interestingly, in DLE-treated patients, clinical manifestations of cervicitis diminished and 89% of them remitted the colposcopic lesions. Histological analyses of biopsies from DLE-treated patients showed a decreasing leukocyte infiltrate. Immunochemical analyses showed an increased expression of TGF-ß, while expression of IFN-γ, PCNA, and IL-32 decreased. Our results suggest that DLE can stimulate innate immunity of cervical mucosae, diminishing chronic cervicitis in HPV-infected patients. TRIAL REGISTRATION: Register ISRCTN16429164 Abbreviations: HPV = Human Papilloma Virus; DLE = Dialyzable leukocyte extract.

Effect of resveratrol on mitochondrial activity in differentiated mature adipocytes / Efecto del resveratrol sobre la actividad mitocondrial en adipocitos maduros diferenciados

Obesity has reached epidemic proportions, the World Health Organization (WHO) estimates that there are more than 1,000 million overweight adults world-wide. Furthermore, obesity is characterized as an overgrowth of white adipose tissue as a result of adipocyte hypertrophy and hyperplasia. Mitochondria is considered the source of energy within the adipocyte, since it contains the molecular machinery, and it is involved in a large number of metabolic pathways, besides the transformation of chemical energy into adenosine triphosphate. Mitochondria shortage and adipocyte dysfunction result in an excessive accumulation of triacylglycerol in the cytoplasm, which determines an imbalance between energy production and energy expenditure. Resveratrol (RSV) is a polyphenol found in different plants and its effects have been associated with mitochondrial biogenesis. An adipogenesis in vitro model (3T3-L1 preadipocytes) was used, and these cells were differentiated into mature adipocytes. Subsequently the effect of RSV on the adipocytes morphology, the lipid content and mitochondrial activity was evaluated using microscopic and flow cytometry techniques. The effect of RSV on differentiated mature adipocytes, was characterized by the decrease in lipid content and the consequently declination of the mitochondrial activity. 3T3-L1 preadipocytes retained the differentiation ability until passage 18. The RSV at doses of 25 and 50 µM for 48 hours in differentiated mature adipocytes promoted the decreased in lipid content probably due to an increase in mitochondrial activity in the early hours of RSV exposure, causing the consequently declination of mitochondrial activity at the end of 48 hours.

La obesidad ha tomado dimensiones epidémicas globales y la Organización Mundial de la Salud estima que hay más de 1,000 millones de adultos con sobrepeso. Así mismo, la obesidad se ha caracterizado como la expansión del tejido adiposo blanco condicionada por la hipertrofia e/o hiperplasia de los adipocitos. La mitocondria es considerada la fuente de energía dentro del adipocito, debido a que contiene la maquinaria molecular que dirige, a través de diversas vías metabólicas, la transformación de la energía química en adenosíntrifosfato. La escasez de mitocondrias así como su disfunción en el adipocito, resulta en una acumulación excesiva de triacilgliceroles en el citoplasma, lo que condiciona un desequilibrio entre producción de energía y gasto energético. El resveratrol (RSV) es un polifenol que se encuentra en diferentes grupos de plantas y sus efectos se han asociado con la inducción de genes para la biogénesis mitocondrial. Se empleó un modelo de adipogénesis (in vitro) materializado por una línea celular de preadipocitos 3T3-L1, mismos que se diferenciaron a adipocitos maduros. Posteriormente se evaluó el efecto del RSV sobre la morfología, contenido lipídico y actividad mitocondrial en los adipocitos maduros diferenciados a través de las técnicas: microscopía invertida, confocal y citometría de flujo. El efecto del RSV sobre los adipocitos maduros diferenciados, se caracterizó por la disminución del contenido lipídico y consecuentemente de la actividad mitocondrial. Los preadipocitos 3T3-L1 conservaron la capacidad de diferenciación hasta el pase 18. Por otra parte, el resveratrol a dosis de 25 y 50 µM durante 48 horas en adipocitos maduros diferenciados, promueve una disminución en el contenido lipídico probablemente debido a un aumento de la actividad mitocondrial en las primeras horas de exposición al tratamiento, provocando la disminución de la actividad mitocondrial al término de 48 horas.

Evaluación de Termografía Tisular Diferenciada en Mama como Potencial Técnica para Asistir la Detección de Cáncer / Evaluation of Thermography Differentiated in Breast Tissue as Potential Technique to Assist Cancer Detection

Diversos grupos han propuesto el procesamiento de imágenes termográficas para detección de Cáncer de Mama (CaMa). Angiogénesis y vascularización dependientes del ciclo menstrual, edad e Índice de Masa Corporal (IMC) modifican la temperatura absoluta en la superficie tisular sin estar necesariamente asociadas a malignidad, en éste estudio proponemos la Termografía Tisular Diferenciada (TTD) en mama con respecto a su contralateral en espejo con el fin de observar diferencias térmicas características de malignidad. El presente trabajo evalúa la posibilidad de emplear la TTD como potencial técnica para asistir la detección de CaMa. Se muestrearon 110 mujeres voluntarias entre 40 y 60 años de edad segmentadas en dos grupos experimentales: grupo sanas (n=90) y grupo con CaMa (n=20) previamente diagnosticadas por mastografía e histopatología. Imágenes termográficas de ambas mamas fueron adquiridas con una cámara infrarroja y se estimó la TTD en relación a la mama contralateral de la misma paciente, se realizó un análisis de sensibilidad y especificidad y se comparó con el diagnóstico radiológico a través de curvas ROC tomando como referencia el diagnóstico histopatológico. La TTD en mama mostró rangos dinámicos diferenciables entre condiciones de malignidad respecto a benignidad. El análisis ROC mostró valores de sensibilidad y especificidad para el estimado TTD del 70% y 54% mientras que para el diagnóstico radiológico fue del 70% y 96%, respectivamente. La TTD muestra viabilidad técnica para asistir la detección de CaMa.

Several groups have proposed thermographic image processing for Breast Cancer (BC) detection. Angiogenesis and vascularization of menstrual cycle dependent, as well as age and Body Mass Index change the absolute temperature in the tissue surface without necessarily being associated with malignancy. We have proposed the Differentiated Tissue Thermography (DTT) in breast regarding its contralateral mirror in order to observe differences in temperature characteristics of malignancy. This study evaluates the possibility of using breast DTT as a potential technique to assist the detection of BC. We sampled 110 female volunteers between 40 and 60 years old segmented into two experimental groups: healthy group (n=90) and BC group (n=20), which were diagnosed by mammography and histopathology. Thermal images of both breasts were acquired with an infrared camera and the DTT was estimated relative to its contralateral breast in the same patient. A sensitivity and specificity analysis was developed and the DTT was compared with the radiological diagnosis by ROC curves with the histopathological report as reference. The DTT values showed distinguishable dynamic ranges between malignant and healthy conditions. ROC analysis showed sensitivity and specificity values for DTT of 70% and 54% while for the radiological diagnosis was 70% and 96% respectively. DTT showed technical viability to assist BC detection.

c-erbB-2 as a possible target for the use of magnetic nanoparticles in breast cancer cells.

Breast cancer (BC) is the most frequent malignancy among women worldwide and has been associated with high mortality because of the late treatment of the disease. Our group has proposed a selective ablation of breast cancer cells by the use of magnetic fields assisted by magnetic nanoparticles. The principle is to increase the conductivity of tumoral tissue by the use of a bioconjugated "nanoparticle-antibody" that recognizes specific antigens on the surface of the cancer cells. The aim of this study was to evaluate the c-erbB-2 antigen in breast cancer cells of type BT-474, MCF-7, and MDA-MB-231 as a possible target for the use of magnetic nanoparticles coupled to a specific Monoclonal Antibody (Mab). Quantitative real-time polymerase chain reaction and flow cytometry were used to estimate the relative expressions of the c-erbB-2 gene and the c-erbB-2 antigen in the cell lines, respectively. A covalent union of magnetic nanoparticles to anti c-erbB-2 Mab was used to develop the bioconjugate. Fluorescence microscopy was used to determine the cells that were tagged by the bioconjugate. The results show a well-differentiated relative expression of c-erbB-2 in the studied cell lines and are qualitatively in agreement with the fluorescent marking by the magnetic nanoparticles. The selected breast cancer cells appear to be suitable for experimental evaluation of selective targeting by magnetic nanoparticles.

Caracterización morfológica, génica y protéica en la diferenciación de células madre embrionarias de ratón a células pancreáticas tempranas / Morphological, genetic and protein characterization in the differentiation of embryonic stem cells to early pancreatic cells

Debido al auge de la medicina regenerativa, las Células Madre (SC) representan una fuente de reemplazo celular para cualquier tejido, decidiendo emprender este trabajo de investigación con el objetivo de diferenciar células madre embrionarias de ratón (mESC) a células pancreáticas tempranas, realizando su caracterización génica y morfológica. Primeramente se cultivaron y arrestaron en su ciclo celular fibroblastos embrionarios de ratón (MEF) con mitomicina, posteriormente se expandieron las mESC y se sometieron a un protocolo de diferenciación de 21 días hacía células pancreáticas tempranas, evaluándose durante la diferenciación su morfología y expresión relativa de los genes sox-17, pdx-1, ins-1 e ins-2, determinando además la producción de las proteínas insulina y glucagón mediante inmunocitoquímica y citometría de flujo. Se obtuvieron cuerpos embrionarios (EBs) a partir de mESC, con características morfológicas diferentes de acuerdo a su diferenciación, los cuales expresaron genes de la línea germinal endodérmica (sox-17 y pdx-1) a los días 0, 11 y 17 de diferenciación, gen inductor del desarrollo embrionario pancreático (pdx-1) al día 11 de diferenciación y, genes de expresión pancreática (ins-1 e ins-2) a los días 17 y 21 de diferenciación. Finalmente se detectó la producción de proteínas insulina y glucagón en los EBs al día 21 de diferenciación. Se logró diferenciar mESC. El análisis morfológico evidenció cúmulos celulares tridimensionales correspondientes a EBs. Con el análisis de los patrones de expresión génica, se distinguieron inicialmente células con características genéticas de endodermo y posteriormente a partir del día 17 células pancreáticas tempranas, las cuales al día 21 de diferenciación expresaron las proteínas insulina y glucagón...

Due to the boom in regenerative medicine, Stem Cells (SC) represent a source of cell replacement to any tissue, we decided to undertake this research with the objective of differentiating mouse embryonic stem cells (mESC) to early pancreatic cells, developing their genetic and morphological characterization. Initially Mouse embryonic fibroblasts (MEF) were grown and arrested in their cell cycle with mitomycin, subsequently mouse embryonic SC (mESC) were expanded and subjected in to a pancreatic cell differentiation protocol of 21 days. During differentiation, morphology and the relative expression of sox-17, pdx-1, Ins-1 and Ins-2 genes were assessed, also the production of insulin and glucagon proteins was determinated by fluorescence microscopy and flow cytometry. Embryoid bodies (EBs) were obtained from mESC, with different morphological characteristics according to their differentiation, which expressed endodermal germ line genes (sox-17 y pdx-1) at days 0, 11 and 17 of differentiation, an inductor gene of embryonic pancreas development (pdx-1) was detected at day 11 of differentiation. Pancreas genes (ins-1 e ins-2) were expressed at day 17 and 21 of differentiation. Finally the production of insulin and glucagon proteins was detected on the EBS at day 21 of differentiation. In conclusion, the mESC differentiation was achieved. The morphological analysis evidenced three-dimensional cell clusters corresponding to EBs. Analysis of the gene expression patterns in the differentiation process, cells initially showed genetic characteristics of endoderm and thereafter from day 17 of differentiation characteristics of early pancreatic cells which by day 21 of differentiation expressed insulin and glucagon proteins...