A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients.

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

PEG-Interferon-α ribavirin-induced HCV viral clearance: a pharmacogenetic multicenter Spanish study.

OBJECTIVE: Dual PEGylated interferon-α (PEG-IFN) and ribavirin therapy has been the main hepatitis C virus (HCV) treatment of the last decade. Current direct-acting antiviral agents have improved the outcome of therapy but also have increased the cost and management complexity of treatment. The current study analyzes host genetics, viral and clinical predictors of sustained viral response (SVR) to dual PEG-IFN and ribavirin therapy in a representative Spanish population. METHODS: Observational prospective multicentre pharmacogenetic cohort study conducted in 12 different hospitals of 12 different Spanish regions. A total of 98 patients with SVR and 106 with non-SVR in response to PEG-IFN and ribavirin therapy were included. 33 single nucleotide polymorphisms located in 24 different genes related with inflammatory, immune and virus response were selected. Clinical and viral data were also analyzed as candidate of SVR predictors. RESULTS: IL-28B (rs12979860, rs7248668, rs8105790, rs8099917) and TNFRSF1B (rs1061622) genotypes, as well as TNFRSF1B/IL-10/TNFα (-308) non-TTG and TNFRSF1B/IL- 10/IL-4 non-TTC haplotypes together with lower age, lower basal HCV RNA load, higher basal serum LDL cholesterol values, VHC genotypes 2 and 3 and basal low grade fibrosis 0-2 were associated with a SVR in the univariate analysis. Independent predictors of SVR in the multivariate analysis were IL-28B rs12979860 CC, TNFRSF1B/IL-10/IL-4 non-TTC along with low baseline HCV RNA load and HCV genotypes 2 and 3. CONCLUSIONS: IL-28B rs12979860 CC, TNFRSF1B/ IL-10/ IL-4 non-TTC haplotype, low baseline HCV RNA load and HCV genotypes 2 and 3 may help to predict successful outcome to PEG-IFN/ribavirin therapy in Spanish population.

Objetivo: El interferon-pegilado (IFN-PEG) junto a ribavirina ha sido el principal tratamiento de la infeccion por el virus de la hepatitis C (VHC) de la ultima decada. Los agentes antivirales de accion directa actuales han mejorado los resultados de la terapia, pero tambien han aumentado el costo y la gestion de la complejidad del tratamiento. El presente estudio analiza factores geneticos de los pacientes, asi como predictores virales y clinicos de respuesta sostenida viral (RSV) al tratamiento con IFN-PEG y ribavirina en poblacion Espanola. Métodos: Estudio farmacogenetico, multicentrico, prospectivo, observacional de cohortes realizado en 12 hospitales diferentes de 12 comunidades autonomas diferentes. Se incluyeron un total de 98 pacientes con RVS y 106 sin SVR al tratamiento con IFNPEG y ribavirina. Se seleccionaron 33 polimorfismos de nucleotido unico ubicados en 24 genes diferentes relacionados con la respuesta inflamatoria, inmunologica y viral. Los datos clinicos y virales tambien se analizaron como candidatos predictores de RVS. Resultados: Los genotipos IL-28B (rs12979860, rs7248668, rs8105790, rs8099917) y TNFRSF1B (rs1061622), asi como los haplotipos TNFRSF1B / IL-10 / TNF(-308) no-TTG y TNFRSF1B / IL-10 / IL-4 no-TTC junto con la menor edad, menor carga de ARN-VHC basal, valores elevados de colesterol LDL en suero basal, genotipos VHC2 y 3 y bajo grado de fibrosis basal (0-2) se asociaron con una RVS en el analisis univariante. Los predictores independientes de RVS en el analisis multivariante fueron el genotipo IL-28B rs12979860 CC, el haplotipo TNFRSF1B / IL-10 / IL-4 no-TTC junto con los bajos niveles basales de VHCARN y los genotipos virales VHC2 y 3. Conclusiones: El genotipo IL-28B rs12979860 CC, el haplotipo TNFRSF1B / IL-10 / IL-4 haplotipos no-TTC, la carga viral basal baja y los genotipos del VHC2 y 3 pueden ayudar a predecir una buena respuesta a la terapia con IFN-PEG y ribavirina en poblacion espanola.

Impact of administration of vancomycin or linezolid to critically ill patients with impaired renal function.

The aim of this study was to assess the impact of vancomycin (VAN) versus linezolid (LZD) on renal function in patients with renal failure (RF) admitted to intensive care units. This was a multicenter, retrospective, comparative cohort study. Renal failure patients were treated with VAN or LZD for proven or suspected infections by multiresistant Gram-positive cocci. Changes in plasma creatinine levels and creatinine clearance at the start and end of treatment were used as endpoints. A total of 147 patients were treated with VAN (group A, n = 68) or LZD (group B, n = 79). Group B included more patients with diabetes mellitus [9 (13.2%) vs. 25 (31.6%); p = 0.007], septic shock [39 (57.4%) vs. 60 (75.9%); p = 0.013] and greater RF (mean ClCr 42.24 ml/min vs. 37.57 ml/min; p = 0.04). Renal function improved in patients from both groups who did not require renal replacement therapy. A greater improvement was seen in group B [percent decrease in Cr (27.94 vs. 9.48; p = 0.02) and percent increase in ClCr (95.96 vs. 55.06; p = 0.05)]. In group A, nine patients (13.2%) experienced an antibiotic-related increase in RF, and antibiotic was discontinued in five patients due to adverse effects. It is reasonable to avoid use of VAN in critically ill patients with acute renal failure.

[Enteral access and intestinal function assessment in the critically ill patient]. / Acceso enteral y evaluación de la función intestinal en el paciente crítico.

The use of enteral nutrition (EN) in the critically-ill patient makes necessary to evaluate its effectiveness and impact on achieving the target requirements. Gastrically administered EN has a high complication rate, especially increased residue that leads to hyponutrition. The use of the small bowel (jejunum) may achieve greater administered volume, although there are three aspects that directly influence on its use: intestinal access route, motility and absorptive capability, and barrier function. The selection of the access route to the digestive tube has to be done after evaluating the underlying disease and predicted duration of EN. If it is greater than 4-6 weeks a definitive access will be performed through an invasive technique of ostomy (radiologic, endoscopic or surgical jejunostomy) and if it is shorter than 4-6 weeks, an endoscopic, fluoroscopic or ultrasonographic non-invasive or transnasal technique (naso-duodenal, or nasojejunal) will be used. By protocoling procedures and experiences, it has been shown that jejunal nutrition may achieve an increase in the amount of requirements administerd to critically-ill patients with mechanical ventilation as compared to gastric feeding, although the benefits with regards to reducing the number of infectious complications, hospital stay and mortality are not so clear-cut, so that it should be left to those cases in which gastric feeding has been clearly documented. By using the manometrich technique or the acetaminophen absorption tests it has been shown that 50% of critically-ill patients with mechanical ventilation have gastric antral hypomotility with decreased migratory motor complexes and gastric voiding, which considerably hampers nutrition. Under normal circumstances, during fasting, there are regular motor contractions, or an inter-digestive migratory motor complex which pattern prevents nutrient absorption because of being highly propulsive, so that during the nutrient phase, this pattern changes into the postprandial pattern with an irregular and continuous contraction activity, with no activity centers, which is much more adapted to nutrient absorption. In critically-ill patients, this normal propulsive pattern is lost, the postprandial pattern is frequently lost, and the inter-digestive pattern remains, which prevents enteral feeding. There are several factors that have an impact on this change, mainly the underlying disease, sepsis, head trauma, mechanical ventilation, sedation, and muscle relaxation. The use of pro-kinetic agents such as metoclopramide may, at least theoretically, modify motility impainment and facilitate the correct administration of prescribed requirements. Among other functions, the gastrointestinal tract (GIT) has a barrier function between inner and outer media, which prevents bacteria, antigenic agents, and toxicants from entering the blood. Its failure is characterized by decreased nutrient absorption, impaired intestinal immunological response and increased intestinal permeability (IP). Among the hypothesis trying to explain systemic infection and multiorgan failure (MOF), there is precisely anatomical and functional integrity of the intestinal mucosa. Mucosal impairment with increased IP has been shown in burn patients, polytrauma, major surgery, hematopoietic cell transplantation, and sepsis, although its relationship with bacterial translocation has not clearly been established. Before the evidences that link the GIT with MOF, the monitoring methods aimed at early correction of splaenic hypoperfusion focus on the mechanisms implicated in increased IP.

Acceso enteral y evaluación de la función intestinal en el paciente crítico / Enteral access and intestinal function assessment in the critically ill patients

La utilización de la nutrición enteral (NE) en el paciente crítico hace necesario evaluar su eficacia e influencia en la consecución de los requerimientos pautados. La NE administrada a nivel gástrico tiene una tasa alta de complicaciones, particularmente aumento de residuo, que conlleva una infranutrición. La utilización del intestino delgado (yeyuno), puede conseguir un aumento del volumen administrado. Pero para ello hay tres aspectos que influyen directamente en su utilización: la vía de acceso intestinal, la capacidad motora y su capacidad absortiva y función de barrera. La elección de la vía de acceso al tubo digestivo debe realizarse tras valoración de la patología de base y el tiempo previsto de duración de la NE. Si es superior a 4-6 semanas se procederá a acceso definitivo a través de una técnica invasiva y ostomía (yeyunostomía radiológica, endoscópica o quirúrgica) y si es inferior a 4-6 semanas, técnica no invasiva o transnasal (nasoduodenal o nasoyeyunal) con endoscopia, fluoroscopia o ecografía. Se ha constatado que, mediante protocolización de los procedimientos y experiencia, la nutrición yeyunal puede conseguir un aumento de los requerimientos administrados al paciente crítico bajo ventilación mecánica respecto de la nutrición gástrica, aunque no son claros los beneficios en cuanto a la reducción de complicaciones infecciosas, estancia y mortalidad, por lo que su uso debe reservarse para aquellos casos en los que está documentado una clara intolerancia gástrica. Mediante técnica manométrica o con el test de absorción de acetaminofeno, se ha constatado que el 50% de los pacientes críticos con ventilación mecánica tienen, a nivel gástrico, hipomotilidad antral, disminución de los complejos motores migratorios y del vaciamiento gástrico, lo que dificulta considerablemente la nutrición. En condiciones normales, durante el ayuno existen unas contracciones motoras regulares o complejo motor migratorio interdigestivo cuyo patrón no es favorable a la absorción de nutrientes por ser altamente propulsivo, por lo que en la fase nutriente cambia al patrón postprandial, de actividad contráctil irregular y continua, sin frentes de actividad, mucho más adaptado a la absorción de nutrientes. En el paciente crítico se pierde este esquema propulsivo normal, desapareciendo con frecuencia el patrón postprandial y persistiendo el interdigestivo, lo que dificulta o impide la nutrición enteral. Son varios los factores que influyen en ese cambio, principalmente la patología de base, sepsis, TCE, ventilación mecánica, sedación y miorelajación. La utilización de agentes procinéticos, como la metoclopramida, puede, al menos en teoría, modificar la alteración de la motilidad y facilitar la correcta administración de los requerimientos pautados. Entre otras, el tracto gastrointestinal (TGI) desempeña una función de «barrera» entre los medios interno y externo que impide la entrada a sangre de bacterias, agentes antigénicos y tóxicos. Su fracaso se caracteriza por absorción disminuida de nutrientes, alteración de la respuesta inmunológica intestinal y aumento de la permeabilidad intestinal (PI). Entre las hipótesis que pretenden explicar la infección sistémica y el fracaso multiorgánico (FMO) figura precisamente el fallo en la integridad anatómica y funcional de la mucosa intestinal. Se ha constatado una alteración de dicha mucosa con aumento de la PI en patologías como quemados, politraumatismos, cirugía mayor, trasplante de células hemopoyéticas y sepsis, aunque no se ha establecido con claridad su relación con la traslocación bacteriana. Ante las evidencias que implican al TGI en el FMO, los métodos de monitorización dirigidos a corregir precozmente la hipopefusión esplácnica nos orientan sobre los mecanismos implicados en el aumento de la PI

The use of enteral nutrition (EN) in the critically-ill patient makes necessary to evaluate its effectiveness and impact on achieving the target requirements. Gastrically administered EN has a high complication rate, especially increased residue that leads to hyponutrition. The use of the small bowel (jejunum) may achieve greater administered volume, although there are three aspects that directly influence on its use: intestinal access route, motility and absorptive capability, and barrier function. The selection of the access route to the digestive tube has to be done after evaluating the underlying disease and predicted duration of EN. If it is greater than 4-6 weeks a definitive access will be performed through an invasive technique of ostomy (radiologic, endoscopic or surgical jejunostomy) and if it is shorter than 4-6 weeks, an endoscopic, fluoroscopic or ultrasonographic non-invasive or transnasal technique (naso-duodenal, or nasojejunal) will be used. By protocoling procedures and experiences, it has been shown that jejunal nutrition may achieve an increase in the amount of requirements administerd to critically-ill patients with mechanical ventilation as compared to gastric feeding, although the benefits with regards to reducing the number of infectious complications, hospital stay and mortality are not so clear-cut, so that it should be left to those cases in which gastric feeding has been clearly documented. By using the manometrich technique or the acetaminophen absorption tests it has been shown that 50% of critically-ill patients with mechanical ventilation have gastric antral hypomotility with decreased migratory motor complexes and gastric voiding, which considerably hampers nutrition. Under normal circumstances, during fasting, there are regular motor contractions, or an inter-digestive migratory motor complex which pattern prevents nutrient absorption because of being highly propulsive, so that during the nutrient phase, this pattern changes into the postprandial pattern with an irregular and continuous contraction activity, with no activity centers, which is much more adapted to nutrient absorption. In critically-ill patients, this normal propulsive pattern is lost, the postprandial pattern is frequently lost, and the inter-digestive pattern remains, which prevents enteral feeding. There are several factors that have an impact on this change, mainly the underlying disease, sepsis, head trauma, mechanical ventilation, sedation, and muscle relaxation. The use of pro-kinetic agents such as metoclopramide may, at least theoretically, modify motility impainment and facilitate the correct administration of prescribed requirements. Among other functions, the gastrointestinal tract (GIT) has a barrier function between inner and outer media, which prevents bacteria, antigenic agents, and toxicants from entering the blood. Its failure is characterized by decreased nutrient absorption, impaired intestinal immunological response and increased intestinal permeability (IP). Among the hypothesis trying to explain systemic infection and multiorgan failure (MOF), there is precisely anatomical and functional integrity of the intestinal mucosa

BDNF gene transfer to the mammalian brain using CNS-derived neural precursors.

Neural stem cell lines represent a homogeneous source of cells for genetic, developmental, and gene transfer and repair studies in the nervous system. Since both gene transfer of neurotrophic factors and cell replacement strategies are of immediate interest for therapeutical purposes, we have generated BDNF-secreting neural stem cell lines and investigated to what extent different endogenous levels of BDNF expression affect in vitro survival, proliferation and differentiation of these cells. Also, we have investigated the in vivo effects of such BDNF gene transfer procedure in the rat neostriatum. Hippocampus- and cerebellum-derived cell lines reacted differently to manipulations aimed at varying their levels of BDNF production. Over-expression of BDNF enhanced survival of both cell types, in a serum-deprivation assay. Conversely, and ruling out unspecific effects, expression of an antisense version of BDNF resulted in compromised survival of cerebellum-derived cells, and in a lethal phenotype in hippocampal progenitors. These data indicate that endogenous BDNF level strongly influences the in vitro survival of these cells. These effects are more pronounced for hippocampus- than for cerebellum-derived progenitors. Hippocampus-derived BDNF overproducers showed no major change in their capacity to differentiate towards a neuronal phenotype in vitro. In contrast, cerebellar progenitors overproducing BDNF did not differentiate into neurons, whereas cells expressing the antisense BDNF construct generated cells with morphological features of neurons and expressing immunological neuronal markers. Taken together, these results provide evidence that BDNF controls both the in vitro survival and differentiation of neural stem cells. After in vivo transplantation of BDNF-overproducing cells to the rat neostriatum, these survived better than the control ones, and induced the expected neurotrophic effects on cholinergic neurons. However, long-term (3 months) administration of BDNF resulted in detrimental effects, at this location. These findings may be of importance for the understanding of brain development, for the design of therapeutic neuro-regenerative strategies, and for cell replacement and gene therapy studies.

Affinity chromatography purification of mitochondrial inner membrane proteins with calcium transport activity.

Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into preformed asolectin liposomes which contained 120 mM KCl as internal high K+ medium. 45Ca2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H+i/45Ca2+o or Na+i/45Ca2+o exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca2+ uptake in liposomes was studied in parallel. Na+i/45Ca2+o exchange activity was not detectable. H+i/45Ca2+o exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100-200 nmoles 45Ca2+ per mg protein in 30 s. 45Ca2+ uptake in voltage-dependent assays (a K+ diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca2+/H+ countertransport. H+i/45Ca2+o uptake showed a saturable component at around 80 microM Ca and was coupled to an increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K+ medium. The results are consistent with the presence of a functional nH+/Ca2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H+/45Ca2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H+/Ca2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.

[Bilateral malignant Brenner's tumor. Apropos of a case]. / Tumor de Brenner maligno bilateral. A propósito de un caso.

This is the case of a 51 year old woman presented with sensation of pelvic fullness, hypogastric pain, anorexia, asthenia and weight loss of one year duration. The gynecological exam revealed a normal uterus and a hard oval mass in the posterior aspect of the vagina, and another spherical mass in right vaginal cul de sac and adnexial area. After surgery malignant a Brenner tumor was diagnosed. A course of chemotherapy was started postoperatively. The "second-look" was negative macroscopically but positive cytologically. A second course of chemotherapy was done. The second "second-look" was negative macroscopically and cytologically. The patient at present is in good health.