Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle.

The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of ß-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.

The Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes.

Tunneling nanotubes (TNTs) are actin-based transient tubular connections that allow direct communication between distant cells. TNTs play an important role in several physiological (development, immunity, and tissue regeneration) and pathological (cancer, neurodegeneration, and pathogens transmission) processes. Here, we report that the Wnt/Ca2+ pathway, an intracellular cascade that is involved in actin cytoskeleton remodeling, has a role in TNT formation and TNT-mediated transfer of cargoes. Specifically, we found that Ca2+ /calmodulin-dependent protein kinase II (CaMKII), a transducer of the Wnt/Ca2+ pathway, regulates TNTs in a neuronal cell line and in primary neurons. We identified the ß isoform of CaMKII as a key molecule in modulating TNT formation and transfer, showing that this depends on the actin-binding activity of the protein. Finally, we found that the transfer of vesicles and aggregated α-synuclein between primary neurons can be regulated by the activation of the Wnt/Ca2+ pathway. Our findings suggest that Wnt/Ca2+ pathway could be a novel promising target for therapies designed to impair TNT-mediated propagation of pathogens.

Combined Therapies for Duchenne Muscular Dystrophy to Optimize Treatment Efficacy.

Duchene Muscular Dystrophy (DMD) is the most frequent muscular dystrophy and one of the most severe due to the absence of the dystrophin protein. Typical pathological features include muscle weakness, muscle wasting, degeneration, and inflammation. At advanced stages DMD muscles present exacerbated extracellular matrix and fat accumulation. Recent progress in therapeutic approaches has allowed new strategies to be investigated, including pharmacological, gene-based and cell-based therapies. Gene and cell-based therapies are still limited by poor targeting and low efficiency in fibrotic dystrophic muscle, therefore it is increasingly evident that future treatments will have to include "combined therapies" to reach maximal efficiency. The scope of this mini-review is to provide an overview of the current literature on such combined therapies for DMD. By "combined therapies" we mean those that include both a therapy to correct the genetic defect and an additional one to address one of the secondary pathological features of the disease. In this mini-review, we will not provide a comprehensive view of the literature on therapies for DMD, since many such reviews already exist, but we will focus on the characteristics, efficiency, and potential of such combined therapeutic strategies that have been described so far for DMD.

Central Role of Transforming Growth Factor Type Beta 1 in Skeletal Muscle Dysfunctions: An Update on Therapeutic Strategies.

Among the soluble factors that regulate skeletal muscle function, Transforming Growth Factor type Beta 1 (TGF-ß1) is one of the most studied. This factor inhibits myogenesis and regeneration by regulating the activity and function of satellite cells (SCs). Indeed, TGF-ß has a central role in muscle pathologies in which there is development of fibrosis and/or atrophy of skeletal muscle. Thus, in this review we present the critical and recent antecedents regarding the mechanisms and cellular targets involved in the effects of TGF-ß1 in the muscle, in pathological processes such as the inhibition of regeneration, fibrosis and atrophy. In addition, an update on the development of new strategies with therapeutic potential to inhibit the deleterious actions of TGF-ß in skeletal muscle is discussed.

Characterizing HSF1 Binding and Post-Translational Modifications of hsp70 Promoter in Cultured Cortical Neurons: Implications in the Heat-Shock Response.

Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1) and histone posttranslational modifications (PTMs) at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression.

SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor ß.

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type ß (TGF-ß) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-ß/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-ß. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-ß in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-ß-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-ß-mediated CTGF expression.

Angiotensin II: role in skeletal muscle atrophy.

Skeletal muscle, the main protein reservoir in the body, is a tissue that exhibits high plasticity when exposed to changes. Muscle proteins can be mobilized into free amino acids when skeletal muscle wasting occurs, a process called skeletal muscle atrophy. This wasting is an important systemic or local manifestation under disuse conditions (e.g., bed rest or immobilization), in starvation, in older adults, and in several diseases. The molecular mechanisms involved in muscle wasting imply the activation of specific signaling pathways which ultimately manage muscle responses to modulate biological events such as increases in protein catabolism, oxidative stress, and cell death by apoptosis. Many factors have been involved in the generation and maintenance of atrophy in skeletal muscle, among them angiotensin II (Ang-II), the main peptide of renin-angiotensin system (RAS). Together with Ang-II, the angiotensin-converting enzyme (ACE) and the Ang-II receptor type 1 (AT-1 receptor) are expressed in skeletal muscle, forming an important local axis that can regulate its function. In many of the conditions that lead to muscle wasting, there is an impairment of RAS in a global or local fashion. At this point, there are several pieces of evidence that suggest the participation of Ang-II, ACE, and AT-1 receptor in the generation of skeletal muscle atrophy. Interestingly, the Ang-II participation in muscle atrophy is strongly ligated to the regulation of hypertrophic activity of factors such as insulin-like growth factor 1 (IGF-1). In this article, we reviewed the current state of Ang-II and RAS function on skeletal muscle wasting and its possible use as a therapeutic target to improve skeletal muscle function under atrophic conditions.

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type ß receptors and the JNK pathway.

Transforming growth factor ß (TGF-ß) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-ß on CTGF expression. We found that myoblasts treated with LPA and TGF-ß1 produced an additive effect on CTGF expression. In the absence of TGF-ß, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-ß receptor type II (TGF-ßRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-ßRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-ßRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-ßRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-ßRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-ß are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.

Pex3p-dependent peroxisomal biogenesis initiates in the endoplasmic reticulum of human fibroblasts.

The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p-GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N-glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross-expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER-to-peroxisomal route in mammalian cells and provides new clues to understand its function.

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells.

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.

Evaluation of the role of the Endoplasmic Reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.

Factores de Riesgo para muerte perinatal en el hospital Los Andes, El Alto, Bolivia / Risk factors for prerinatal deaths at the Los Andes Hospital, el Alto, Bolivia

Objetivo: Identificar los factores de riesgo asociados a muerte perinatal en el Hospital Los Andes de la ciduad de El Alto, Bolivia. Diseño. Caoss y controles incidentes. Lugar: Hospital Los Andes , centro materno infantil institucional de sugundo nivel. Participantes. Se enrolaron al estudio 70 madres de mortinatos in útero y/o fallecidos en los primeros 7 días de vida (casos) y 140 madres de recién nacidos vivos como controles (Controles). Investigación. Ninguna. Mediciónes principales Usando un instrumento previamente probado, 6 enfermeras antes capacitadas en su manejo, entrevistaron alas madres motivo del estudio. El instrumento evaluó el estado socioeconómico, la historia obstétrica. La calidad de atención del parto y la atención del recién nacido fueron solicitados a los médicos tratantes. Resultados. el 25 porciento sucedieron fuera del servicio y el 47.1 porciento en servicio(au)

El espermograma en la valoración de la pareja infértil / The sperm count rating scale of the infertility partners

La infertilidad es un problema que afecta a millones de parejas en el mundo. En Costa Rica no conocemos con exactitud la magnitud del problema. De acuerdo con estudios realizados en otros países el 15 por ciento de las parejas tiene problemas de infertilidad y en la mitad de éstas existe un factor masculino. El espermograma es un estudio pivote en la valoración inicial en la praeja infértil. Es un examen sencillo, rápido, de bajo costo y no invasivo. Es de suma utilidad ya que permite estudiar el factor masculino y valorar la respuesta a la terapia, ya sea ésta médica o quirúrgica.

Estudio de la hematuria en niños: enfoque urológico / Study of hematuria in children: urological criteria

Con el fin de hacer una valoración ordenada y sistemática de la patología urológica de nuestros niños y de uniformar criterios en el personal médico de nuestra institución, el Servicio de Urología del Hospital Nacional de Niños presenta su protocolo de estudio de la hematuria

Esfinter urinario artificial: primeros ocho casos en Costa Rica / Artificial urinary sphincter: first eight cases in Costa Rica

La incontingencia urinaria es uno de los problemas urológicos de más díficil manejo. No sólo el paciente se siente incapacitado para realizar una serie de actividades, sino que el urólogo no siempre cuenta con armas tan efectivas como quisiera. El esfinter urinario artificial es tal vez la única excepción a lo anterior, y hoy día, se considera el tratamiento de elección la incontinencia urinaria debida a falla del mecanismo esfinteriano. Este dispositivo proporciona continencia aceptable a más del 85 por ciento de los pacientes a quienes se les implanta. El presente trabajo describe la experiencia con los primeros ocho casos de esfínter urinario artificial implatados en Costa Rica

Tacto rectal y antígeno prostático específico en el diagnóstico del cáncer de próstata / Rectal tact and prostatic especific antigen in the prostate cancer diagnosis

Con el descubrimiento del antígeno prostático específico (APE) el diagnóstico del cáncer de próstata, en los últimos 15 años, tomó una nueva dimensión. Los esfuerzos actualmente se centran en detectar esta enfermedad en estadíos tempranos para así ofrecer al paciente las mayores oportunidades de sobrevida y una mejor calidad de vida. En este estudio se revisan los resultados de un período de 5 años, con el uso del tacto rectal como único método de detección de cáncer de próstata. Se analizan aspectos como el diagnóstco de ingreso, edad y estadío al momento del diagnóstico así como hallazgos al tacto rectal. Posteriormente se revisa los resultados luego de un año de inclusión del APE al armamento diagnóstico y se comparan con el período anterior

Cáncer de próstata. Diagnóstico temprano / Prostate cancer. Early diagnosis

Conforme aumenta la expectativa de vida en nuestro país, aumenta la prevalencia de algunas enfermedades propias de la edad. En Costa Rica, el cáncer de próstata es la segunda causa de muerte por cáncer en hombres. La mayoría de estos tumores, al igual que en el resto del mundo, se diagnostican en estadío avanzado, cuando ya no es posible ofrecer cura. La presente revisión tiene por objeto dar una visión clara acerca de la epidemiología, la historia natural y los métodos con que contamos hoy en día para el diagnóstico del cáncer de próstata y de como utilizar esta información para procurar un diagnóstico temprano.