Investigación para el diagnóstico situacional local de la epidemia de ETM: Intervenciones preventivas para su contención: Área Programática del Hospital General de Agudos Juan A. Fernández 2015-2016 / Research for the local situational diagnosis of ETM epidemic: Preventive interventions for its containment: Acute General Hospital Juan A. Fernández Programmatic Area 2015-2016

El plan de Prevención y Control de Enfermedades Transmitidas por Mosquito (ETM): Dengue, Fiebre Chikungunya, Amarilla, y Zika de la CABA establece cuatro escenarios teóricos de riesgo que orientan la implementación de las acciones de prevención y control. En el presente informe se presentan los estudios de foco investigados en nuestra Área Programática (AP) y su georreferencia, a fin de obtener un diagnóstico situacional local que permita la ejecución de intervenciones oportunas y eficaces a fin de limitar la aparición de nuevos casos en el contexto epidémico 2015-2016. El objetivo general fue estudiar la epidemia de ETM para su comprensión y abordaje en el Área Programática del Hospital General de Agudos Juan A. Fernández, y el objetivo específico, prevenir y limitar la aparición de nuevos casos de ETM. (AU)

The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity.

Hedgehog proteins mediate many of the inductive interactions that determine cell fate during embryonic development. Hedgehog signaling has been shown to regulate slow muscle fiber type development. We report here that mutations in the zebrafish slow-muscle-omitted (smu) gene disrupt many developmental processes involving Hedgehog signaling. smu(-/-) embryos have a 99% reduction in the number of slow muscle fibers and a complete loss of Engrailed-expressing muscle pioneers. In addition, mutant embryos have partial cyclopia, and defects in jaw cartilage, circulation and fin growth. The smu(-/-) phenotype is phenocopied by treatment of wild-type embryos with forskolin, which inhibits the response of cells to Hedgehog signaling by indirect activation of cAMP-dependent protein kinase (PKA). Overexpression of Sonic hedgehog (Shh) or dominant negative PKA (dnPKA) in wild-type embryos causes all somitic cells to develop into slow muscle fibers. Overexpression of Shh does not rescue slow muscle fiber development in smu(-/-) embryos, whereas overexpression of dnPKA does. Cell transplantation experiments confirm that smu function is required cell-autonomously within the muscle precursors: wild-type muscle cells rescue slow muscle fiber development in smu(-/-) embryos, whereas mutant muscle cells cannot develop into slow muscle fibers in wild-type embryos. Slow muscle fiber development in smu mutant embryos is also rescued by expression of rat Smoothened. Therefore, Hedgehog signaling through Slow-muscle-omitted is necessary for slow muscle fiber type development. We propose that smu encodes a vital component in the Hedgehog response pathway.

Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families.

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

Tumor necrosis factor concentrations in hemolytic uremic syndrome patients and children with bloody diarrhea in Argentina.

Hemolytic uremic syndrome (HUS) is thought to be a vascular endothelial injury disease. The mechanism of injury is unknown although verocytotoxins (Shiga-like toxins (SLTs)) are known to be associated with it. Recent evidence suggests that in vitro treatment of some endothelial cells with tumor necrosis factor alpha (TNF-alpha) dramatically increases their susceptibility to SLTs. We studied 25 children with HUS, 63 children with SLT-positive bloody diarrhea, 62 children with bloody diarrhea not associated with SLTs and 39 children admitted for elective surgery, included as an age- and season-matched control group. The TNF-alpha concentrations were found to be significantly elevated in children with HUS (range, 1 to 95 pg/ml; geometric mean, 32.2 pg/ml) compared with the healthy controls (range, 0 to 53 pg/ml; mean, 12.5 pg/ml; P < 0.001). Because it is hypothesized that TNF-alpha elevation might precede development of HUS, we also studied children with blood diarrhea. The TNF-alpha serum concentrations were significantly higher during the first 10 days after onset of bloody diarrhea than after the first 10 days (P < 0.02). Such elevation could be associated with vascular endothelial glycolipid receptor up-regulation and increased susceptibility to the effects of SLTs.

Interactions of the p107 and Rb proteins with E2F during the cell proliferation response.

The E2F transcription factor is found in complexes with a variety of cellular proteins including the retinoblastoma tumor suppressor protein. Various assays have demonstrated a tight correlation between the functional capacity of Rb as a growth suppressor and its ability to bind to E2F. Moreover, only the underphosphorylated form of Rb, which appears to be the active species, interacts with E2F. Despite the fact that the majority of Rb becomes hyperphosphorylated at the end of G1, we now show that the E2F-Rb interaction persists through the G1/S transition and into S phase. A distinct E2F complex does appear to be regulated in relation to the transition from G1 to S phase. We now demonstrate that this complex contains the Rb-related p107 protein. Moreover, like the Rb protein, p107 inhibits E2F-dependent transcription in a co-transfection assay. This result, together with the observation that free, uncomplexed E2F accumulates as cells leave G1 and enter S phase, suggests that the p107 protein may regulate E2F-dependent transcription during G1. In contrast, although Rb does regulate the transcriptional activity of E2F, this association does not coincide with the G1 to S phase transition.

A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex.

The E2F transcription factor has been found in association with the cyclin A protein, and this complex accumulates during the S phase of the cell cycle, suggesting that E2F may play a role in cell cycle control. In independent studies, cyclin A has been shown to be associated with two other proteins, the Rb-related p107 protein and the cdc2-related p33 cdk2 protein kinase. Through an analysis of the E2F-cyclin A complex, we now find that both the p107 protein and the cdc2-related p33cdk2 kinase are components of the previously described complex. Moreover, the complex possesses H1 kinase activity. These results thus define a cyclin A-cdk2 kinase complex that possesses sequence-specific DNA binding activity. This suggests that the cdk2 kinase may phosphorylate other DNA-bound substrates, and that one role of the E2F factor may be to localize this protein kinase to the DNA.

Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes.

Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.

Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A.

We have examined E2F binding activity in extracts of synchronized NIH 3T3 cells. During the G0 to G1 transition, there is a marked increase in the level of active E2F. Subsequently, there are changes in the nature of E2F-containing complexes. A G1-specific complex increases in abundance, disappears, and is then replaced by another complex as S phase begins. Analysis of extracts of thymidine-blocked cells confirms that the complexes are cell cycle regulated. We also show that the cyclin A protein is a component of the S phase complex. Each complex can be dissociated by the adenovirus E1A 12S product, releasing free E2F. The release of E2F from the cyclin A complex coincides with the stimulation of an E2F-dependent promoter. We suggest that these interactions control the activity of E2F and that disruption of the complexes by E1A contributes to a loss of cellular proliferation control.

Adenovirus 12S E1A gene represses differentiation of F9 teratocarcinoma cells.

The F9 teratocarcinoma cell line differentiates in vitro after treatment with retinoic acid and cAMP and has been a widely used model system for the study of the molecular events that are responsible for cellular commitment and differentiation during early development. Previous experiments have suggested intriguing parallels between the control of gene expression during F9 cell differentiation and the regulation of gene expression by adenovirus E1A. Transfection of a 12S E1A-expressing plasmid into terminally differentiated, nonproliferating F9 cells generates, at high frequency, colonies of dividing cells, each of which expresses E1A. Cell lines established from these colonies proliferate in the presence of retinoic acid and have lost the fully differentiated phenotype as characterized by the absence of expression of a series of differentiation-specific genes. We conclude that expression of the viral 12S E1A gene product interferes with retinoic acid-induced F9 cell differentiation. Moreover, the results suggest that the differentiation process, as defined by markers of terminal differentiation, may not be a permanent event but can be reversed by E1A expression.

Infecciones extraintestinales por Salmonella enterica en pacientes padiátricos / Enteric salmonella, extraintestinal infections in pediatric patients

Las infecciones por Salmonellas no tificas se presentan habitualmente como gastroenteritis agudas de curso autolimitado sin alto riesgo de bacteriemia e infecciones extraintestinales (IEI). En el período comprendico entre enero de 1987 y julio de 1989 se diagnosticaron en el Hospital de Niños "Dr. Ricardo Gutiérrez" 12 casos de IEI por Salmonella enterica. Las edades oscilaron entre 1 y 62 meses (X = 16 meses) con ocho (66 por ciento) pacientes menores de 12 meses. Cuatro enfermos (33 por ciento) eran huéspedes inmunocomprometidos y uno presentaba mielomeningocele y era portador de una válvula de derivación ventrículoperitoneal. Seis pacientes (50 por ciento) presentaron diarrea pero todos los coprocultivos realizados fueron negativos. Se registró fiebre mayor de 38,5§C en diez niños (83 por ciento) con una duración media de 6,8 días (1-17 días). Se aisló Salmonella enterica en 13 muestras de hemocultivos de nueve pacientes, en siete cultivos de líquido cefalorraquídeo de dos niños, en un líquido peritoneal, en dos líquidos articulares de una lactante, en un líquido pleural y en un urocultivo. Los diagnósticos de egreso fueron: sepsis en cinco pacientes, dos de ellas asociadas a meningitis, bacteriemia en cuatro pacientes, dos de ellas asociadas a neumonía y las otras dos, una lactante de 51 días de vida, desnutrida de II grado y una niña con leucemia linfoblástica aguda con neutropenia. Otros diagnósticos de egreso fueron: supuración pleuropulmonar; artritis séptica y peritonitis. La evolución fue favorable en diez pacientes; un paciente falleció por septicemia y otro adquirió una infección intrahospitalaria