Characterization of NF1 frameshift mutations in pediatric patients with neurofibromatosis type I.

Neurofibromatosis type I is an autosomal dominant disease with complete penetrance and variable age-dependent expressivity. It is caused by heterozygous mutations in neurofibromin 1 (NF1). These occur throughout the length of the gene, with no apparent hotspots. Even though some mutations have been found repeatedly, most have been observed only once. This, along with the variable expressivity, has made it difficult to establish genotype-phenotype correlations. Here, we report the clinical and molecular characteristics of four pediatric patients with neurofibromatosis type I. Patients were clinically examined and DNA was extracted from peripheral blood. The whole coding sequence of NF1, plus flanking intronic regions, was examined by Sanger sequencing, and four frameshift mutations were identified. The mutation c.3810_3820delCATGCAGACTC was observed in a familial case. This mutation occurred within a sequence comprising two 8-bp direct repeats (GCAGACTC) separated by a CAT trinucleotide, with the deletion leading to the loss of the trinucleotide and the 8-bp repeat following it. The deletion might have occurred due to misalignment of the direct repeats during cell division. In the mutation c.5194delG, the deleted G is nested between two separate mononucleotide tracts (AAAGTTT), which could have played a role in creating the deletion. The other two mutations reported here are c.4076_4077insG, and c.3193_3194insA. All four mutations create premature stop codons. In three mutations, the consequence is predicted to be loss of the GAP-related, Sec14 homology, and pleckstrin homology-like domains; while in the fourth, only the latter two domains would be lost.

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.

Characterization of the cytotoxic effects of the combination of cisplatin and flavanol (-)-epicatechin on human lung cancer cell line A549. An isobolographic approach.

BACKGROUND: Among malignancies, lung cancer is a leading cause of death. Platinum-based therapeutic compounds used to treat lung cancer have not been able to increase the survival of patients and such compounds have a high incidence of adverse and toxic effects. It has been proposed that flavonoids such as catechins may significantly reduce the risk of developing cancer, alongside with other health benefits. The aim of this work was to determine the effect of (-)-epicatechin, the main flavanol found in cocoa, on the proliferation of the lung non-small cell adenocarcinoma cancer cell line A549, and to determine its effects when added simultaneously with cisplatin. MATERIALS AND METHODS: Concentration-response curves for cisplatin and epicatechin were obtained, inhibitory concentrations calculated and an isobolographic analysis was then performed. RESULTS: We found that epicatechin has a concentration-dependent inhibitory effect on proliferation of tumor cells and the isobolographic analysis reveals that the effect of its combination with cisplatin is synergistic. It was also observed that epicatechin promotes cell death by apoptosis. CONCLUSIONS: Epicatechin might be considered for future studies to explore its possible use as coadjuvant in cisplatin-based treatments.

Medicina Regenerativa para Úlceras Crónicas: Aplicación Cutánea de Células Troncales Mesenquimales de Gelatina de Wharton / Regenerative Medicine for Chronic Ulcers: Cutaneous Application of Mesenchymal Stem Cells of Wharton's Jelly

Resumen El empleo de células mesenquimales ha emergido como una parte importante en la medicina regenerativa con efectos positivos en la recuperación de tejidos dañados o perdidos. Las úlceras crónicas incluyen lesiones cutáneas que afectan a una gran parte de la población con diabetes mellitus, (por ejemplo 9.14% desarrollan lesiones por pie diabético según ENSANUT 2016) y representan gastos económicos considerables, debido a la pobre calidad de vida que llevan los pacientes. El tratamiento convencional que se lleva a cabo en estos casos es a largo plazo, y los efectos benéficos generados que se presentan son limitados, ya que las lesiones reinciden por no contar con un tratamiento específico y regenerativo, solo paliativo. Es por ello que en este trabajo se obtuvieron y emplearon células troncales mesenquimales derivadas de gelatina de Wharton (CTM-GW) bajo los criterios propuestos por la Sociedad Internacional de la Terapia Celular. El tratamiento fue aplicado en dos pacientes con úlceras crónicas flebostáticas de diferentes condiciones médicas y se monitoreo a corto plazo, por lo que fue un estudio limitado. Los resultados mostraron que las CTM-GW indujeron la reconstrucción cutánea mediante formación de tejido de granulación, además de disminuir el proceso inflamatorio crónico en la zona de daño, lo que favoreció el índice de cierre de la herida. Por lo anterior, se propone la utilización de CTM-GW en úlceras flebostáticas crónicas como tratamiento en la regeneración de la estructura tisular con resultados a corto plazo.

Abstract Use of mesenchymal cells has emerged as an important part in regenerative medicine with positive effects in the recovery of damaged or lost tissues. Chronic ulcers include skin lesions that affect a large part of the population with diabetes mellitus, (for example, 9.14 develop diabetic foot according to ENSANUT 2016) and represent considerable economic expenses, due to poor quality of life in patients. The conventional treatment that is carried out in these cases is long term, and the generated beneficial effects that are presented are limited, since the injuries recur due to not having a specific and regenerative treatment, only palliative. That is why in this work they were obtained and used mesenchymal stem cells derived from Wharton's jelly (MSC-WJ) under the criteria proposed by the International Society for Cell Therapy. The treatment was applied in two patients with chronic ulcers phlebostatic of different medical conditions and short-term monitoring, so it was a limited study. The results showed that MSC-WJ induced skin reconstruction by tissue formation granulation, in addition to decreasing the chronic inflammatory process in the area of damage, which favored the index of wound healing. Therefore, the use of MSC-WJ in chronic phlebostatic ulcers is proposed as a treatment in the regeneration of the tissue structure with short-term results.

Efecto Regulador de Tibolona sobre la Actividad Antiinflamatoria del Macrófago / Regulatory Effect of Tibolone on Antiinflamatory Activity of Macrophage

Resumen Los macrófagos son células fagocíticas que activan a la sintasa de óxido nítrico (SON) y la NADPH oxidasa con el objetivo de eliminar agentes que reconocen como extraños. Además, participan en el desarrollo y mantenimiento de la respuesta inflamatoria. Tibolona (Tb), a través de sus metabolitos, tiene actividad estrogénica, progestagénica y androgénica. Es utilizada como alternativa de la terapia hormonal de la menopausia, que además disminuye marcadores de inflamación. El objetivo de este estudio fue describir el efecto de Tb sobre la expresión de las interleucinas I, 6, 10 y TNF-α así como la actividad de la NADPH oxidasa del macrófago. Se utilizó la línea celular THP-1, se diferenció a macrófago, y se evaluó la actividad de NADPH oxidasa por el ensayo de NBT y la expresión de IL-1β, IL-6, TNF-α e IL-10 por RT-qPCR. Se encontró que Tb regula la actividad enzimática, así como la expresión de citocinas ante un estímulo proinflamatorio. Por lo que se concluye que Tb favorece la actividad antiinflamatoria del macrófago. Estos resultados contribuyen a la descripción de los mecanismos de acción de este fármaco. Además, se propone al macrófago como un blanco de regulación del proceso inflamatorio por acción de Tb. Se sugiere que se determine la expresión proteica de las citocinas por otras técnicas, tales como western blot, ELISA, o citometría de flujo; así como la actividad de SON.

Abstract Macrophages are phagocytic cells that activate NOS and NADPH oxidase to eliminate agents that recognize like strangers, also participate in development and maintain of inflammatory response. In other hand, tibolone (Tb) has three main metabolites, which have an estrogenic, prostagenic and androgenic activity. This drug is a hormonal therapy to treat symptoms of menopause, with ability to decrease inflammation markers. The aim of this study is to describe the effect of Tb on macrophage activity. This study was carried out on differentiated macrophage THP-1 cell line used. NADPH oxidase activity by NBT assay and expression of IL-1β, IL-6, TNF-α and IL-10 by RT-qPCR were measured. It has been observed that Tb regulates the enzymatic activity, as well as the cytokines expression in the cells that received a proinflammatory stimulus; these results contribute to description of mechanism of action of this drug. The results suggest that macrophage could be a target for antiinflammatory action of Tb. Its remain to investagate the protein expression of cytokines and activity of iNOS.

Comparación entre Células Troncales Mesenquimales obtenidas de Médula Ósea, Tejido Adiposo y Gelatina de Wharton en base a los Criterios de la ISCT / Comparison between Mesenchymal Stem Cells obtained from Bone Marrow, Adipose Tissue and Wharton Gelatin according to the ISCT Criteria

RESUMEN: Las células troncales mesenquimales (CTM) representan una población heterogénea con capacidad para auto-renovarse y diferenciarse a distintos tipos celulares. Estas fueron descritas en un inicio en médula ósea (MO) a mediados del siglo pasado, desde entonces este tejido se ha convertido en el estándar de oro para la obtención y caracterización de CTM. Actualmente se sabe que este tipo de células se encuentran alojadas en nichos distribuidos por todo el organismo, donde contribuyen a los procesos de regeneración del tejido donde se localizan. No obstante, encontrar una fuente alterna de CTM con las mismas características que las de MO, pero que su extracción no suponga riesgo para el donador es fundamental para su utilización con fines terapéuticos. En este trabajo se aislaron células troncales de médula ósea, y se compararon con tejido adiposo y gelatina de Wharton y caracterizaron de acuerdo a los criterios de la Sociedad Internacional para la Terapia Celular (ISCT). Los resultados mostraron que la morfología, diferenciación osteogénica y adipogénica, así como la expresión de los antígenos de superficie CD90, CD73 y CD105 cumplen con los estándares, señalando a las provenientes de gelatina de Wharton como mejor opción.

ABSTRACT: Mesenchymal stem cells (MSC) represent a heterogeneous population with the capacity to self-renew and differentiate into different cell types. At the middle of the last century these cells initially were described in bone marrow (BM), thence this tissue has become the gold standard for obtaining and characterization of MSC. It is known that these cells are housed in specific areas called niches distributed throughout all body, where they contribute to tissue regeneration processes of self-tissue were they are located. However, finding an alternative source of CTM with the same characteristics that have showed in MO, but its obtention no represent a risk since the donor is essential to their use for therapeutic purposes. In this study we isolated mesenchymal stem cells from bone marrow, adipose tissue and Wharton's jelly and they were compared in their characteristics in according to the standards of the International Society for Cellular Therapy (ISCT). The results showed that the morphology as well as adipogenic and osteogenic differentiation and also the expression of surface antigens (CD90, CD73, and CD105) from all tissues accomplished the standards, although Wharton's jelly represented the best option.

Efecto de la dehidroepiandroesterona y el acetónido de triamcinolona sobre la línea celular 3T3-L1 / Effect of dehydroepiandroesterone and triamcinolone acetonide on 3T3-L1 cell line

RESUMEN: La piel es el órgano más extenso en el ser humano, su integridad representa protección contra diferentes agentes químicos, biológicos y mecánicos. Las lesiones ocasionadas en este tejido se resuelven mediante la formación de una cicatriz, sin embargo, diferentes alteraciones moleculares pueden sobre estimular este proceso, lo que conlleva a la formación de cicatrices aberrantes (hipertrófica o queloide). El tratamiento más recomendado para este tipo de lesiones es la aplicación intralesional del acetónido de triamcinolona (AT) y por otro lado, la dehidroepiandrosterona (DHEA) es una pro-hormona que posee una gran variedad de efectos biológicos como: regulación de la síntesis de fibras de colágeno, protección celular, propiedades antitumorales, antiinflamatorias y antioxidante. En este trabajo, se estudió la combinación de AT-DHEA sobre la proliferación y muerte celular en la línea celular de fibroblastos 3T3-L1. Los resultados mostraron que la AT a 100 M y la DHEA a 1000 M inhiben la proliferación en un 50 y 40% respectivamente. La combinación de AT-DHEA (10000-10 M) inhibe la proliferación celular e inducen muerte celular programada, entonces esta combinación pudiera utilizarse en cicatrices hipertróficas o queloides para su eliminación.

ABSTRACT: The skin in the human is the largest organ, his integrity represents protection against various chemical, biological and mechanical agents. The injuries in this tissue are solved by forming a scar, however, different molecular alterations may overstimulate this process, leading to the formation of aberrant scars (hypertrophic or keloid). The most recommended treatment for such injuries is the intralesional application of triamcinolone acetonide (TA) and on the other hand, dehydroepiandrosterone (DHEA) is a pro-hormone that has a wide variety of biological effects such as regulation of the synthesis of collagen fibers, cell protection, anti-tumor properties, anti-inflammatory and antioxidant. In this paper, the combination of AT-DHEA on proliferation and cell death in fibroblast cell line 3T3-L1 was studied. The results showed that the AT 100 and 1000 M DHEA to inhibit proliferation by 50 and 40% respectively. The combination of AT-DHEA (10000-10 M) inhibits cell proliferation and induce programmed cell death, so this combination could be used in hypertrophic or keloid scars for disposal.

Efecto del Medio Condicionado de la Línea Celular H9C2 Tratada con Dehidroepiandrosterona y Expuesta a Daño sobre la Motilidad de Células Troncales Mesenquimales / Effect of the Conditioned Medium from H9C2 Cell Line Treated with Dehydroepiandrosterone and Exposed to Damage on the Motility of Mesenchymal Stem Cells

RESUMEN: Las enfermedades cardiovasculares (ECV) son la principal causa de muerte a nivel mundial, donde la terapia con Células Troncales Mesenquimales (CTM) representa una alternativa para los pacientes que no logran recuperarse con los tratamientos actuales. El lograr que las CTM residentes se movilicen al órgano afectado representaría una ventaja para el manejo terapéutico de las ECV. La dehidroepiandrosterona (DHEA) es un precursor hormonal cuyos niveles disminuyen a lo largo de la vida, lo que se ha asociado al desarrollo de ECV. Diversos estudios han demostrado que el consumo de DHEA previene y mejora la condición cardiaca, aunque no se sabe si esto ocurre porque se ejerce un efecto en los cardiomiocitos y estos, a su vez, hacia las CTM. El objetivo del presente estudio fue determinar el efecto del medio condicionado procedente de la línea H9C2 pretratada con DHEA y sometida a daño, sobre la motilidad de CTM, llevando a cabo un ensayo de cierre de herida. El pretratamiento con DHEA y el daño en la línea H9C2, promueve la motilidad de CTM. El estímulo de la motilidad de CTM por un efecto indirecto de DHEA podría ser una estrategia terapéutica para el daño cardiaco.

ABSTRACT: Cardiovascular diseases (CVD) are the leading cause of death worldwide. Mesenchymal Stem Cell (MSC) therapy is an alternative for patients who cannot recover with current treatments. Ensure movilization of MSC to the affected organs would represent an advantage for therapeutic management of CVD. Dehydroepiandrosterone (DHEA) is a hormone precursor whose levels decrease throughout life, which has been associated with the onset of CVD. Several studies have shown that DHEA consumption, prevents and improves heart condition, although it is not known if this is because an effect on cardiomyocytes is exercised on these cells and this, in turn, to CTM. The aim of this study was to determine the effect of conditioned medium from H9C2 cell line pretreated with DHEA and subjected to damage, on the motility of CTM, performing a wound healing assay. Pretreatment with DHEA and damage to H9C2 cell line, promotes motility of CTM. Stimulation of CTM motility by an indirect effect of DHEA could be a therapeutic strategy for heart damage.