Medicina molecular y genética

La hiperplasia suprarrenal congénita (HSC) es un trastorno autosómico recesivo causado por una alteración en el gen CYP21 que, en última instancia, conduce a una deficiencia de 21-hidroxilasa. El presente estudio tuvo como objetivo la evaluación de dos mutaciones comunes, a saber, la mutación de empalme del intrón 2 (INT2S) y las deleciones de 8 pb en el exón 3 del gen CYP21, y establecer sus frecuencias en la población de Cachemira (norte de la India). Las mutaciones se analizaron mediante el sistema de mutación refractaria a la amplificación-PCR (ARMS-PCR) en 50 casos de HSC, comprobados por las características clínicas y los niveles elevados de 17-hidroxiprogesterona (17OHP).

Los resultados revelaron que 15 (30%) casos tenían mutación INT2S mientras que no se detectó una deleción de 8 pb en ningún paciente. En la mutación INT2S, 7 casos fueron homocigotos con genotipo I2-G y 8 casos fueron heterocigotos. La frecuencia de heterocigotos AG se encontró en 5 casos y el genotipo heterocigoto CG se encontró en 3 casos. Se observó que los pacientes con CAH con genitales ambiguos albergaban la mayoría de las mutaciones INT2S con I2-G en 3 casos y heterocigotos CG en 2 casos. En el grupo de pacientes no consanguíneos, se detectaron 4 mutaciones homocigotas I2-G y 4 eran heterocigotos I2-GC en comparación con 3 y 1 en pacientes consanguíneos respectivamente. Nuestro estudio confirma que existen mutaciones INT2S pero no deleciones de 8 pb en el gen CYP21 en pacientes con CAH en la población de Cachemira.

Con el aumento de la conciencia sobre los efectos adversos del uso de productos químicos en los alimentos, existe un esfuerzo continuo para la búsqueda de alternativas naturales. Se están explorando varios extractos de plantas y sus compuestos purificados por su eficacia biológica. En el presente estudio, se ha explorado Docynia indica, que crece como un árbol silvestre en la región noreste de la India, por su posible uso como fuente de conservantes naturales. Aunque existen algunos informes sobre las actividades biológicas de la fruta de D. indica en la literatura, no se sabe mucho sobre su perfil fitoquímico. Se analizaron varios extractos de frutas preparados en diferentes etapas de madurez de frutas de D. indica para determinar su potencial antioxidante y antimicrobiano in vitro, y se determinó el perfil fitoquímico de sus extractos metanólicos mediante cromatografía líquida de alto rendimiento. El extracto de fruta inmadura mostró una alta concentración de fenólicos totales (hasta 196,15 mg de equivalente de ácido gálico/g de extracto) y flavonoides (hasta 100,49 mg de equivalente de rutina/g de extracto) dependiendo del solvente de extracción; y también mostró una mayor actividad antioxidante en ensayos in vitro. Aunque no se observó una tendencia definida para la actividad antibacteriana en función de los estadios de madurez, se encontró que la extracción con la mezcla de metanol, acetona y agua (1:1:1) mostró la menor concentración inhibitoria mínima para todos los estadios de madurez. La catequina y el ácido ferúlico fueron los principales fenólicos presentes en los frutos de D. indica. Los compuestos antioxidantes y antibacterianos presentes en varios extractos de D. indica indican su potencial para su utilización como conservante de alimentos.

Cada vez hay más pruebas de que los estrógenos pueden promover la progresión tumoral, incluidos los tumores ováricos. Los estrógenos ejercen sus acciones en los tejidos a través de dos subtipos de receptores diferentes (ESR1 y ESR2). Los estudios han demostrado que las gallinas desarrollan cáncer de ovario de forma espontánea, por lo que proporcionan un modelo animal adecuado para la enfermedad. Nuestro objetivo fue determinar la expresión de ARNm y proteína de los subtipos de receptores de estrógeno en ovarios de gallinas normales y ovarios de gallinas con cáncer de ovario. Se recolectó tejido ovárico de gallinas normales y gallinas con cáncer de ovario para análisis de PCR cuantitativa en tiempo real e inmunofluorescencia. Los resultados de PCR cuantitativa en tiempo real mostraron que la expresión relativa de ARNm de ESR1 y la relación de ESR1/ESR2 son significativamente mayores en gallinas con cáncer de ovario en comparación con el tejido ovárico normal. El análisis de inmunofluorescencia mostró una expresión diferencial de las proteínas ESR1 y ESR2 en secciones de tejido ovárico de gallinas normales y gallinas con cáncer de ovario, con resultados paralelos a los datos de ARNm. No hubo una diferencia significativa en los niveles plasmáticos de estradiol entre las gallinas normales y las gallinas con cáncer de ovario. Estos datos sugieren un aumento en las acciones mediadas por estrógenos en los tumores ováricos de pollo y, de hecho, el análisis de microarrays revela una vía de señalización de estradiol funcionalmente significativa en los tumores ováricos de pollo. Curiosamente, la expresión de un supuesto supresor de tumores ováricos, EPB41L3, está regulada a la baja en esta vía. En conjunto, estos resultados sugieren que, en la gallina, ESR1 puede estar mediando una respuesta proliferativa en las células de cáncer de ovario. Aunque se requieren estudios adicionales para definir el papel de ESR1 en la formación de tumores en la gallina, estos resultados respaldan la utilidad de la gallina para probar posibles terapias endocrinas.

Autoimmune diseases cause numerous health and social problems throughout the world. The common spectrum of autoimmune diseases affect the majority of tissues within the body, including pancreatic beta cells in type 1 diabetes (T1DM), myelin surrounding nerve axons in Multiple sclerosis (MS) and synovial joint antigens in Rheumatoid Arthritis (RA). The diseases are likely caused by a complex interaction between multiple HLA- and non- HLA related genes and environmental factors. The well documented co-clustering of autoimmune diseases within families and individuals, together with apparent sharing of number risk genes between the diseases suggests at least some common mechanisms of autoimmune development.

Diabetic Retinopathy (DR) is the most common microvascular complication of diabetes and is one of the leading causes of visual impairment worldwide. DR is a chronic eye disease that eventually can result in legal blindness due to the evolution towards the major vision-threatening disorders diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR). Current treatments with steroids, anti-VEGF compounds or retinal laser photocoagulation have shown a significant improvement in visual acuity in the advanced stage of the disease. However, main concerns are possible side effects and/or the relatively large number of clinical non-responders. A better understanding of the biological and molecular pathways not only in DR patients but also in the preclinical in vivo models would aid the development of novel and more efficient (personalized) therapeutic approaches for DR. This review aims to describe the pathways in a selection of diabetic, non-diabetic and surrogate rodent models of pathogenic neovascularization, vascular permeability, inflammation and neurodegeneration. None of these models can mimic the entire human pathophysiological progression but they all exhibit joint pathophysiological features with human DR pathogenesis. These combined features make these models very relevant in gaining a better understanding of the disease etiology and eventually improved strategies for drug screening. Through highlighting the most important biochemical and molecular pathways that these animal models have in common with DR patients, selection of the most suitable model for mechanistic studies and drug screening can be facilitated.

Aim: Ovarian cancer prognosis is influenced by factors such as intratumoral T-lymphocyte presence and the amount of stroma formation relative to the epithelial tumor compartment. The regulation of these factors is unknown. Matrix metalloproteinases are involved in stromal remodeling and may be involved in T-cell trafficking as well. This study investigates the quantitative relationships between the matrix metalloproteinases MMP-2 and MMP-14 and both relative stroma amounts (‘stroma percentage’) and the presence of T lymphocytes in the stromal and epithelial compartments of ovarian cancer.

Patients and methods: In 86 patients with ovarian cancer, MMP-2 and MMP-14 expression and T-lymphocyte presence was determined using semi quantitative immunohistochemistry. Stroma percentage was determined by area calculation in haematoxylin eosin slides. Per slide, 3 random images at the tumor-stroma interface were investigated.

Results: Epithelial MMP-14 scoring correlated negatively with T-lymphocytes in tumor epithelium: correlation coefficient rho −0.36 (p < 0.01) for CD3, −0.30 (p < 0.01) for CD8 and −0.24 (p < 0.05) for CD45Ro. Stromal MMP-2 was positively related to stroma percentage (rho 0.29; p < 0.01). No significant correlations were found for MMP-14 with stromal amounts, or MMP-2 with T-cell presence in stroma or tumor epithelium.

Conclusion: Opposite to our hypothesis that MMPs reduce stromal amounts and permit T cell trafficking into both stromal and epithelial tumor parts, MMP expression was associated with higher relative stromal amounts and lower presence of T lymphocytes. In particular, we found that epithelial MMP-14 expression was negatively associated with T-lymphocyte presence in the tumor suggesting a role for MMP-14 in negatively regulating T-cell trafficking by other means. Stromal MMP-2 expression was associated with an increase in stromal amounts suggesting an ineffective feedback on tumor stroma volume growth of the expression of this gelatinase. More research is necessary to determine the specific actions of MMPs in ovarian cancer.

The purpose of our study was to develop new delivery systems for drugs effective against breast cancer by using biodegradable and biocompatible capsules. Redox-active microcapsules based on thiolated polymethacrylic acid (PMA) were employed. The interaction of these PMASH capsules with breast cancer cells and the mechanism of their internalization was investigated. PMASH biocompatibility was evaluated by MTT assay. To analyze their potential as drug carrier, we incorporated doxorubicin into the capsules. Confocal microscopy observations showed the presence of capsules inside the cells. Although some drug molecules still appeared co-localized with PMASH capsules, strong doxorubicin fluorescence was observed both in the cytoplasm and nucleus, indicating the disassembling of the capsule into PMASH-drug conjugate after internalization. These results were confirmed by both flow cytometry (time course of capsule uptake) and scanning electron microscopy. PMASH capsules were also internalized in 3D cell structures (spheroids) suggesting their potential use as drug delivery system for treatment of human diseases.

Objectives: Several candidate genes are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM), one of which is interleukin (IL-10). In this investigation, we aimed to study the association between IL-10 (-592A/C) gene polymorphism with its level in T2DM with and without nephropathy.

Methods: IL-10 (-592A/C) gene polymorphism was genotyped using restriction fragment length polymorphism (RFLP-PCR) technique and IL-10 levels were measured in two groups of T2DM, one group complicated with nephropathy and the second non-complicated.

Results: Our results revealed no significant differences in IL-10 (−592A/C) genotypes distribution between the two groups of T2DM patients. IL-10 levels were found significantly elevated in diabetic nephropathy patients compared to T2DM without nephropathy (P<0.05) and were positively correlated to the degree of albuminuria (r=0.61, P<0.01). IL-10 levels increased in IL-10- (−592C/C) genotype compared to IL-10-(−592A/A) and IL-10- (−592A/C) genotypes in diabetic nephropathy patients while such difference was not found in T2DM patients without nephropathy.

Conclusion: IL-10 (-592A/C) gene polymorphism and IL-10 level play a role in the pathogenesis of nephropathy in T2DM.

Epigenetic mechanisms are known to be involved in tissue-specific differentiation. DNA methylation patterns have been shown to be largely conserved across tissues but with variation for specific genes. However, it is unclear whether the variability observed in the methylation profile of a metabolically active tissue is reflected in other sources such as hematopoietic tissue. This study aimed to test blood genome-wide CpG site methylation levels as a surrogate model for visceral adipose tissue (VAT) methylation and to verify whether it appropriately reflects differences in methylation levels found in VAT between men discordant for the metabolic syndrome (MetS). Tissue specimens (VAT and blood samples) were obtained from 16 severely obese individuals discordant for the MetS. CpG sites methylation levels were measured with the Infinium HumanMethylation450 BeadChip and correlations of methylation levels between VAT and blood were computed. Differences in methylation levels between individuals with and without MetS were tested in both tissues. Pathway analysis was conducted for differentially methylated CpG sites common to both tissues. High cross-tissue correlations were observed for VAT and blood (0.952±0.014) while some CpG sites had significantly different methylation levels in VAT versus blood. Differential methylation analysis between individuals with and without MetS demonstrated a higher number of differentially methylated CpG sites in VAT than in blood (11,778 vs. 881, respectively) with nearly 4% of differentially methylated sites found in VAT being also represented in blood. Common differentially methylated sites were involved in inflammatory-, lipid- and diabetes-related pathways. These results suggest that blood methylation levels of specific CpG sites may adequately reflect VAT methylation levels for some of the MetS-related genes, specifically for inflammatory, lipid and glucose metabolism genes.