Immune response to co-administration of measles, mumps, and rubella (MMR), and yellow fever vaccines: a randomized non-inferiority trial among one-year-old children in Argentina.

BACKGROUND: In yellow fever (YF) endemic areas, measles, mumps, and rubella (MMR), and YF vaccines are often co-administered in childhood vaccination schedules. Because these are live vaccines, we assessed potential immune interference that could result from co-administration. METHODS: We conducted an open-label, randomized non-inferiority trial among healthy 1-year-olds in Misiones Province, Argentina. Children were randomized to one of three groups (1:1:1): Co-administration of MMR and YF vaccines (MMR1YF1), MMR followed by YF vaccine four weeks later (MMR1YF2), or YF followed by MMR vaccine four weeks later (YF1MMR2). Blood samples obtained pre-vaccination and 28 days post-vaccination were tested for immunoglobulin G antibodies against measles, mumps, and rubella, and for YF virus-specific neutralizing antibodies. Non-inferiority in seroconversion was assessed using a -5% non-inferiority margin. Antibody concentrations were compared with Kruskal-Wallis tests. RESULTS: Of 851 randomized children, 738 were correctly vaccinated, had ≥ 1 follow-up sample, and were included in the intention-to-treat population. Non-inferior seroconversion was observed for all antigens (measles seroconversion: 97.9% in the MMR1YF1 group versus 96.3% in the MMR1YF2 group, a difference of 1.6% [90% CI -1.5, 4.7]; rubella: 97.9% MMR1YF1 versus 94.7% MMR1YF2, a difference of 3.3% [-0.1, 6.7]; mumps: 96.7% MMR1YF1 versus 97.9% MMR1YF2, a difference of -1.3% [-4.1, 1.5]; and YF: 96.3% MMR1YF1 versus 97.5% YF1MMR2, a difference of -1.2% [-4.2, 1.7]). Rubella antibody concentrations and YF titers were significantly lower following co-administration; measles and mumps concentrations were not impacted. CONCLUSION: Effective seroconversion was achieved and was not impacted by the co-administration, although antibody levels for two antigens were lower. The impact of lower antibody levels needs to be weighed against missed opportunities for vaccination to determine optimal timing for MMR and YF vaccine administration. TRIAL REGISTRATION: The study was retrospectively registered in ClinicalTrials.gov (NCT03368495) on 11/12/2017.

TG/HDL index to identify subjects with metabolic syndrome in the Mexican population.

BACKGROUND: The triglyceride/high-density lipoprotein (TG/HDL) index has been proposed as an indicator of cardiovascular risk. In Mexico, there is a study in young adults that relates it to insulin resistance, but no cutoff point that distinguishes subjects with metabolic syndrome has been defined. OBJECTIVE: To determine the cutoff point for the TG/HDL index that identifies subjects with metabolic syndrome in the Mexican population. METHODS: Metabolic syndrome was diagnosed using the criteria established in the Third Report of the Adult Treatment Panel of the National Cholesterol Education Program adapted to the Mexican population. To identify the TG/HDL index cutoff point, ROC curve analysis and the Youden index were used. RESULTS: 1,318 subjects aged 40.9 ± 13.0 years participated in the study; 65.6% were women and 34.4% men; 41.2% had metabolic syndrome. The TG/HDL index obtained an area under the curve of 0.85 and an optimal cutoff point value ≥ 3.46, with a sensitivity of 79.6% and specificity of 76.4%. CONCLUSIONS: TG/HDL index cutoff point ≥ 3.46 is suitable for identifying subjects with metabolic syndrome in the Mexican population.

ANTECEDENTES: El índice triglicéridos/lipoproteína de alta densidad (TG/HDL) ha sido propuesto como un indicador de riesgo cardiovascular. En México, existe un estudio en adultos jóvenes que lo relaciona con resistencia a la insulina, pero no se ha definido un punto de corte que distinga a sujetos con síndrome metabólico. OBJETIVO: Determinar el punto de corte para el índice TG/HDL que identifique a sujetos con síndrome metabólico en población mexicana. MÉTODOS: El síndrome metabólico se diagnosticó mediante los criterios establecidos en el Tercer Reporte del Panel de Tratamiento para Adultos del Programa Nacional de Educación en Colesterol adaptados a la población mexicana. Para identificar el punto de corte del índice TG/HDL se utilizó el análisis de curvas ROC y el índice de Youden. RESULTADOS: En el estudio participaron 1318 sujetos con edad de 40.9 ± 13.0 años; 65.6 % fuerin mujeres y 34.4 % hombres; 41.2% presentó síndrome metabólico. El índice TG/HDL obtuvo un valor del área bajo la curva de 0.85 y un valor óptimo de punto de corte ≥ 3.46, con sensibilidad de 79.6 % y especificidad de 76.4 %. CONCLUSIONES: El punto de corte ≥ 3.46 para el índice TG/HDL es adecuado para identificar a sujetos con síndrome metabólico en población mexicana.

Índice TG/HDL para identificar a sujetos con síndrome metabólico en población mexicana / TG/HDL index to identify subjects with metabolic syndrome in the Mexican population

Resumen Antecedentes: El índice triglicéridos/lipoproteína de alta densidad (TG/HDL) ha sido propuesto como un indicador de riesgo cardiovascular. En México, existe un estudio en adultos jóvenes que lo relaciona con resistencia a la insulina, pero no se ha definido un punto de corte que distinga a sujetos con síndrome metabólico. Objetivo: Determinar el punto de corte para el índice TG/HDL que identifique a sujetos con síndrome metabólico en población mexicana. Métodos: El síndrome metabólico se diagnosticó mediante los criterios establecidos en el Tercer Reporte del Panel de Tratamiento para Adultos del Programa Nacional de Educación en Colesterol adaptados a la población mexicana. Para identificar el punto de corte del índice TG/HDL se utilizó el análisis de curvas ROC y el índice de Youden. Resultados: En el estudio participaron 1318 sujetos con edad de 40.9 ± 13.0 años; 65.6 % fuerin mujeres y 34.4 % hombres; 41.2% presentó síndrome metabólico. El índice TG/HDL obtuvo un valor del área bajo la curva de 0.85 y un valor óptimo de punto de corte ≥ 3.46, con sensibilidad de 79.6 % y especificidad de 76.4 %. Conclusiones: El punto de corte ≥ 3.46 para el índice TG/HDL es adecuado para identificar a sujetos con síndrome metabólico en población mexicana.

Abstract Background: The triglyceride/high-density lipoprotein (TG/HDL) index has been proposed as an indicator of cardiovascular risk. In Mexico, there is a study in young adults that relates it to insulin resistance, but no cutoff point that identifies subjects with metabolic syndrome has been defined. Objective: To determine the cutoff point for the TG/HDL index that identifies subjects with metabolic syndrome in the Mexican population. Methods: Metabolic syndrome was diagnosed using the criteria established by the Third Report of the Adult Treatment Panel of the National Cholesterol Education Program adapted to the Mexican population. To identify the TG/HDL index cutoff point, ROC curve analysis and the Youden index were used. Results: 1,318 subjects aged 40.9 ± 13.0 years participated in the study; 65.6% were women and 34.4% men; 41.2% had metabolic syndrome. The TG/HDL index obtained an area under the curve of 0.85 and an optimal cutoff point value ≥ 3.46, with a sensitivity of 79.6% and specificity of 76.4%. Conclusions: TG/HDL index cutoff point ≥ 3.46 is suitable for identifying subjects with metabolic syndrome in the Mexican population.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Evaluation of Systemic Gentamicin as Translational Readthrough Therapy for a Patient With Epidermolysis Bullosa Simplex With Muscular Dystrophy Owing to PLEC1 Pathogenic Nonsense Variants.

IMPORTANCE: Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) is an autosomal recessive disorder caused by pathogenic variants in PLEC1, which encodes plectin. It is characterized by mild mucocutaneous fragility and blistering and muscle weakness. Translational readthrough-inducing drugs, such as repurposed aminoglycoside antibiotics, may represent a valuable therapeutic alternative for untreatable rare diseases caused by nonsense variants. OBJECTIVE: To evaluate whether systemic gentamicin, at a dose of 7.5 mg/kg/d for 14 consecutive days, is clinically beneficial in a patient with EBS-MD. DESIGN, SETTING, AND PARTICIPANTS: A single patient in Madrid, Spain, received 2 treatment courses with gentamicin on July 2019 and February 2020 with a follow-up period of 120 and 150 days, respectively. RESULTS: In this case report of a woman in her 30s with EBS-MD, before gentamicin treatment, the patient had mucocutaneous involvement, skeletal and respiratory muscle weakness, and myalgia that negatively affected her quality of life. Outcomes were evaluated with extensive laboratory tests and clinical scales. No nephrotoxic or ototoxic effects were detected after intravenous gentamicin administration. Gentamicin treatment was followed by plectin expression in the skin for at least 5 months. Although minimal changes were noted in skeletal muscle function (as measured by the Hammersmith functional motor scale and its expanded version: 6/40 to 7/40 and from 10/66 to 11/66, respectively) and respiratory musculature (maximal inspiratory and expiratory pressures D0 vs D16, MIP: 2.86 vs 3.63 KPa and MEP: 2.93 vs 4.63 KPa), myalgia disappeared (VAS dropped from 6 to 0), and quality of life improved (EuroQoL-5D-3L pain and anxiety dropped from 2 to 1). CONCLUSIONS AND RELEVANCE: The findings of this single case report suggest that gentamicin treatment may help suppress PLEC1 premature termination codons and induce plectin expression in EBS-MD primary keratinocytes and skin. Our study suggests that gentamicin may play an important role in treating EBS-MD owing to nonsense variants.

Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing.

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

Dry Eye Disease Association with Computer Exposure Time Among Subjects with Computer Vision Syndrome.

PURPOSE: To assess the time of exposure to the computer and dry eye disease (DED) in subjects with computer vision syndrome (CVS). METHODS: A cross-sectional study was conducted in office workers, computer users of both sexes, with an age range of 18-45 years without comorbidities; we included 108 subjects divided into 3 groups according to the time of computer exposure in hours per day (H/D): <4 (n = 23), 4 -7.9 (n = 49), >8 (n = 39). A specific questionnaire was applied to them on the exposure time and the type of visual display terminal (VDT) used, as well as the computer vision symptoms scale (CVSS17). DED was diagnosed with the Ocular Surface Disease Index (OSDI). Ocular surface damage and signs of DED were evaluated with the tear rupture time test (TBUT), the integrity of the ocular surface by ocular surface staining (OSS) and the production of the aqueous basal tear film using the Schirmer test. RESULTS: Average computer exposure time, measured differently, was positively correlated with DED development. The computer exposure time measured in hours per year and TBUT showed a significant negative correlation (p <0.001) (rho -0.463). Years of computer exposure and staining of the ocular surface showed a significant positive correlation (p <0 0.001; rho 0.404). The accumulated exposure time was negatively correlated with TBUT (p <0.001; rho -0.376) and positively with OSS (p <0.001; rho 0.433). Schirmer test did not correlate with computer exposure time. CONCLUSION: The prolonged time of exposure to the computer in subjects with CVS was significantly correlated with the DED tests, in the different ways of measuring it; but not with the Schirmer test.

Relación entre el índice lipoproteínas de baja densidad (LDL)/lipoproteínas de alta densidad (HDL) con enzimas antioxidantes y el índice oxLDL/HDL.

INTRODUCTION: The low-density lipoprotein (LDL)/high-density lipoprotein (HDL) index is a predictive factor for atherosclerosis, which is associated with oxidative modifications. OBJECTIVE: To assess the association of the index with oxidative stress markers. METHODS: 444 subjects were included and were clinically, anthropometrically and biochemically characterized; superoxide dismutase, glutathione peroxidase 3 (GPx3), magnesium and oxidized LDL (oxLDL) index (oxLDL/HDL) were quantified. RESULTS: A decrease of 1.014 units in the LDL/HDL index was associated with a superoxide dismutase increase of 1 unit/mL (p = 0.030), while a decrease of 0.023 units was associated with a GPx3 increase of 1 nmol/min/mL (p < 0.0005). An increase of one unit in the index was associated with an increase of 0.831 in the oxLDL/HDL index (p < 0.05). After controlling for the effect of gender, age, smoking, obesity and insulin resistance, a reduction of 0.001 per index unit was associated with an increase of 1 µg/g of magnesium in the nails (p = 0.020). CONCLUSIONS: The LDL/HDL index shows an inverse relationship with the antioxidant status and a direct relationship with oxidation status, regardless of other cardiovascular and oxidative stress risk factors.

INTRODUCCIÓN: El índice de lipoproteínas de baja densidad (LDL)/lipoproteínas de alta densidad (HDL) es un factor predictivo de aterosclerosis, la cual está asociada con modificaciones oxidativas. OBJETIVO: Evaluar la asociación del índice con marcadores de estrés oxidativo. MÉTODO: Se incluyeron 444 sujetos, caracterizados clínica, antropométrica y bioquímicamente; se cuantificó superóxido dismutasa, glutation peroxidasa 3 (GPx3), magnesio e índice LDL oxidadas (oxLDL/HDL). RESULTADOS: La disminución en 1.014 unidades del índice LDL/HDL se asoció con aumento de 1 unidad/mL de superóxido dismutasa (p = 0.030) y la de 0.023 unidades con aumento de 1 nmol/minuto/mL de GPx3 (p < 0.0005). El aumento en 1 unidad del índice se asoció con aumento de 0.831 unidades en el índice oxLDL/HDL (p < 0.05). Después de controlar el efecto del sexo, edad, fumar, obesidad y resistencia a la insulina, la reducción de 0.001 por unidad del índice se asoció con aumento de 1 µg/g de magnesio en uñas (p = 0.020). CONCLUSIONES: El índice LDL/HDL presenta relación inversa con el estado antioxidante y relación directa con el estado de oxidación, independientemente de otros factores de riesgo cardiovascular y de estrés oxidativo.

Relationship of the low-density lipoprotein (LDL)/high-density lipoprotein (HDL) index with antioxidant enzymes and with the oxLDL/HDL index

Introduction: The low-density lipoprotein (LDL)/high-density lipoprotein (HDL) index is a predictive factor for atherosclerosis, which is associated with oxidative modifications. Objective: To assess the association of the index with oxidative stress markers. Methods: 444 subjects were included and were clinically, anthropometrically and biochemically characterized; superoxide dismutase, glutathione peroxidase 3 (GPx3), magnesium and oxidized LDL (oxLDL) index (oxLDL/HDL) were quantified. Results: A decrease of 1.014 units in the LDL/HDL index was associated with a superoxide dismutase increase of 1 unit/mL (p = 0.030), while a decrease of 0.023 units was associated with a GPx3 increase of 1 nmol/min/mL (p < 0.0005). An increase of one unit in the index was associated with an increase of 0.831 in the oxLDL/HDL index (p < 0.05). After controlling for the effect of gender, age, smoking, obesity and insulin resistance, a reduction of 0.001 per index unit was associated with an increase of 1 µg/g of magnesium in the nails (p = 0.020). Conclusions: The LDL/HDL index shows an inverse relationship with the antioxidant status and a direct relationship with oxidation status, regardless of other cardiovascular and oxidative stress risk factors.

Relationship of the low-density lipoprotein (LDL)/high-density lipoprotein (HDL) index with antioxidant enzymes and with the oxLDL/HDL index.

INTRODUCTION: The low-density lipoprotein (LDL)/high-density lipoprotein (HDL) index is a predictive factor for atherosclerosis, which is associated with oxidative modifications. OBJECTIVE: To assess the association of the index with oxidative stress markers. METHODS: 444 subjects were included and were clinically, anthropometrically and biochemically characterized; superoxide dismutase, glutathione peroxidase 3 (GPx3), magnesium and oxidized LDL (oxLDL) index (oxLDL/HDL) were quantified. RESULTS: A decrease of 1.014 units in the LDL/HDL index was associated with a superoxide dismutase increase of 1 unit/mL (p = 0.030), while a decrease of 0.023 units was associated with a GPx3 increase of 1 nmol/min/mL (p < 0.0005). An increase of one unit in the index was associated with an increase of 0.831 in the oxLDL/HDL index (p < 0.05). After controlling for the effect of gender, age, smoking, obesity and insulin resistance, a reduction of 0.001 per index unit was associated with an increase of 1 µg/g of magnesium in the nails (p = 0.020). CONCLUSIONS: The LDL/HDL index shows an inverse relationship with the antioxidant status and a direct relationship with oxidation status, regardless of other cardiovascular and oxidative stress risk factors.

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene-editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

An RNA-targeted therapy for dystrophic epidermolysis bullosa.

Functional impairment or complete loss of type VII collagen, caused by mutations within COL7A1, lead to the severe recessive form of the skin blistering disease dystrophic epidermolysis bullosa (RDEB). Here, we successfully demonstrate RNA trans-splicing as an auspicious repair option for mutations located in a wide range of exons by fully converting an RDEB phenotype in an ex vivo pre-clinical mouse model based on xenotransplantation. Via a self-inactivating (SIN) lentiviral vector a 3' RNA trans-splicing molecule, capable of replacing COL7A1 exons 65-118, was delivered into type VII collagen deficient patient keratinocytes, carrying a homozygous mutation in exon 80 (c.6527insC). Following vector integration, protein analysis of an isolated corrected single cell clone showed secretion of the corrected type VII collagen at similar levels compared to normal keratinocytes. To confirm full phenotypic and long-term correction in vivo, patches of skin equivalents expanded from the corrected cell clone were grafted onto immunodeficient mice. Immunolabelling of 12 weeks old skin specimens showed strong expression of human type VII collagen restricted to the basement membrane zone. We demonstrate that the RNA trans-splicing technology combined with a SIN lentiviral vector is suitable for an ex vivo molecular therapy approach and thus adaptable for clinical application.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Classification of skin phenotypes caused by diabetes mellitus using complex scattering parameters in the millimeter-wave frequency range.

The pathological skin phenotype caused by hyperglycemia is an important indicator for the progress of diabetes mellitus. An early detection of diabetes assures an early intervention to regulate the carbohydrate metabolism. In this publication a non-invasive detection principle based on the measurement of complex scattering parameters in the millimeter-wave frequency range is presented. The measurement principle provides evidence of the applicability for the identification of different glycemic states in animal models. The method proposed here can be used to predict diabetes status in animal models and is interesting for application on humans in view of safeness of millimeter-wave radiation. Furthermore the complex scattering parameters give important information about the anatomic varieties between the analyzed skin samples of the different mice strains. In contrast to other methods, our approach is less sensitive to skin variations between animals.

Triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) index as a reference criterion of risk for metabolic syndrome (MetS) and low insulin sensitivity in apparently healthy subjects.

AIM: To evaluate if the TG/HDL-C index can be considered as a reference criterion of MetS and low insulin sensitivity in apparently healthy subjects. METHODS: The subjects were Mexican mestizos who resided in Puebla City, Mexico, who were anthropometrically, biochemically, and clinically characterized. The TG/HDL-C index was calculated by dividing triglyceride (TG) levels by HDL-C levels. MetS was diagnosed by the Third Report from the Adult Treatment Panel-National Cholesterol Education Program (ATP-III NCEP) criteria, while insulin sensitivity was evaluated by the Quantitative Insulin sensitivity Check Index (QUICKI). RESULTS: The study included 813 subjects, with an average age of 38.6 ± 12.1 years, of which 564 were women and 249 men. An association was found between high TG/HDL-C index and low insulin sensitivity (Odds ratio [OR]: 4.09; p < 0.01) and with MetS (OR: 15.29; p < 0.01). A correlation was found between the TG/HDL-C index and QUICKI (rho: -0.4989; p < 0.01) and with MetS (rho: 0.6581; p < 0.01). CONCLUSION: The results indicate that the TG/HDL-C index is associated with low insulin sensitivity and MetS in apparently healthy subjects, suggesting this index as a reference criterion of risk for low insulin sensitivity and MetS.

Analysis of cytokine gene polymorphisms in Mestizo and native populations from Mexico.

OBJECTIVES: To determine whether the well-known genetic structure of the Mexican population observed with other multiallelic markers can be detected by analyzing functional polymorphisms of cytokine and other inflammatory-response-related genes. METHODS: A total of 834 Mestizo individuals from five Mexican cities and 92 Lacandonians - an Amerindian group from southeastern Mexico - were genotyped for 14 polymorphisms in the CRP, IL10, IL6, TGFB1, TNFA, LTA, ICAM1 IFNG, and IL1RN genes. Allele and haplotype frequencies were used for genetic structure analysis using F-statistics pairwise distances and multidimensional scaling plot. Ancestry analysis was performed, as well. RESULTS: Significant interpopulational differences at the allele and haplotype frequency level were observed, mainly between Northern (Guadalajara, Monterrey, and Culiacan) and Southern (Tierra Blanca and Puebla) Mexican populations. Also, low but significant substructure was detected between some populations from these two broad regions. Interestingly, both Lacandonian populations were highly differentiated from each other and with respect to Mestizos. Consistent with previous data, Amerindian ancestry in the Southern Mexican groups was higher compared to Northern ones. CONCLUSIONS: The Mexican population exhibits regional differences in functional polymorphisms of inflammatory-response genes, as observed for other genetic markers. This information constitutes a reference for epidemiological studies that include these genetic markers to assess the susceptibility of the Mexican population to several immune-response-related diseases, such as diabetes, obesity, and renal disease, which have been shown to be common in the Mexican population but with prevalence differences within this country.

In-vivo, non-invasive detection of hyperglycemic states in animal models using mm-wave spectroscopy.

Chronic or sustained hyperglycemia associated to diabetes mellitus leads to many medical complications, thus, it is necessary to track the evolution of patients for providing the adequate management of the disease that is required for the restoration of the carbohydrate metabolism to a normal state. In this paper, a novel monitoring approach based on mm-wave spectroscopy is comprehensively described and experimentally validated using living animal models as target. The measurement method has proved the possibility of non-invasive, in-vivo, detection of hyperglycemia-associated conditions in different mouse models, making possible to clearly differentiate between several hyperglycemic states.

The transition from linear to highly branched poly(ß-amino ester)s: Branching matters for gene delivery.

Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(ß-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(ß-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(ß-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(ß-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their translation to a clinical environment.

Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.