Diagnostic potential of circulating cell-free DNA in patients needing mechanical ventilation: Promises and challenges

Circulating cell-free DNA (cf-DNA) mainly comes from apoptotic cells and can refl ect the extent of cellular damage. Increased plasma levels of cf-DNA have been found in many acute disorders, including septic and clinically ill patients, and usually correlate well with clinical outcome. Acute respiratory failure, the most frequent organ failure in ICU patients, can be related to various acute diseases that may cause cell death and release of DNA into the bloodstream. In a recent issue of Critical Care, Okkonen and colleagues evaluate levels of cf-DNA in plasma as a prognostic marker in patients needing mechanical ventilation. They report that plasma cf-DNA was higher than normal in patients with mechanical ventilation, and even higher in patients who eventually died compared to survivors. However, its usefulness as a death predictor may be limited in the heterogeneous group of mechanically ventilated patients, probably due to confounding eff ects of comorbidities, among other factors

Efecto precoz del B-bloqueante Esmolol sobre la regresión del remodelado coroonario en ratas espontáneamente hipertensas y estudio observacional de la influencia del tratamiento crónico B-bloqueantes en la aparición de complicaciones perioperatorias en cirugía no cardiaca

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Cirugía. Fecha de lectura: 29-04-2016Esta tesis tiene embargado el acceso al texto completo hasta el 29-10-2017La hipertensión arterial produce remodelado de cavidades cardiacas y pared vascular, que conduce a la aparición de complicaciones cardíacas (insuficiencia cardiaca, infarto de miocardio, arritmias graves) con aumento secundario de la morbimortalidad. Varios fármacos antihipertensivos (ARA-II, IECAS, calcioantagonistas y β-bloqueantes) han mostrado su capacidad para revertir el anterior proceso de remodelado en un periodo de tratamiento de meses-años, mejorando así el pronóstico de estos pacientes. El esmolol es un fármaco bloqueante 1-adrenérgico (cardioselectivo) que posee ciertas ventajas farmacocinéticas respecto a otros hipotensores, junto con un efecto bradicardizante e hipotensor rápido y potente. En un estudio experimental previo, realizado en ratas espontáneamente hipertensas (SHR), nuestro grupo demostró que el esmolol reducía significativamente, en tan solo 48 horas de perfusión intravenosa, la hipertrofia ventricular izquierda, siendo el primer fármaco capaz de producir dicho efecto de manera precoz (15). En base a este hallazgo, diseñamos el presente trabajo experimental con el objetivo de investigar si el anterior tratamiento con esmolol revertía el remodelado de la arteria coronaria en la SHR. Nuestros resultados muestran que el fármaco produce dicho efecto y que lo hace a través de los siguientes mecanismos: 1º) reducción del grosor de la pared y de la capa media arterial; 2º) normalización de la reactividad vascular, favorecida por una mayor biodisponibilidad de óxido nítrico; 3º) reducción del estrés oxidativo con un aumento de actividad de la superóxido dismutasa y catalasa; y 4º) disminución de la concentración de dimetilarginina asimétrica que conduce a una mayor producción de óxido nítrico. Adicionalmente, durante una estancia de 3 meses en el hospital St George´s de Londres, hemos diseñado y realizado un estudio clínico observacional con el objetivo de analizar la eficacia del tratamiento crónico -bloqueante en la profilaxis de las complicaciones de la cirugía no cardíaca. Para ello, hemos evaluado retrospectivamente una cohorte de 80 pacientes sometidos a cirugía mayor no cardíaca, e ingresados en la Unidad de Cuidados Intensivos de este hospital. Tras un periodo de seguimiento de 2-4 semanas desde el postoperatorio inmediato hasta el alta hospitalaria/fallecimiento, los pacientes incluidos en el brazo de tratamiento -bloqueante presentaron un mayor número de factores de riesgo cardiovascular y mayor incidencia de arritmias en el postoperatorio. Sin embargo, no se encontraron diferencias en la incidencia de otros eventos cardiacos, complicaciones no cardiacas, duración de la estancia hospitalaria o tasa de mortalidad entre los pacientes tratados o sin tratar con -bloqueantes. Los resultados de este estudio sugieren que el tratamiento crónico con -bloqueantes no induce la aparición de complicaciones perioperatorias (salvo arritmias postoperatorias) en cirugía no cardíaca. No obstante, se necesitan estudios prospectivos y aleatorizados adicionales, con un tamaño muestral mayor, para dilucidar la eficacia real del tratamiento crónico con -bloqueantes en la profilaxis de complicaciones perioperatorias tras cirugía no cardíaca.Hypertension induces remodeling of the cardiac cavities and vascular wall, leading to the onset of cardiac complications (heart failure, myocardial ischemia, serious arrhythmias) and the resulting increase in morbidity and mortality. Several antihypertensive drugs (ARA-II, ACE inhibitors, calcium channel and β- adrenergic receptor blockers) have shown their ability to reverse the above remodeling process within months or years of treatment, thus improving the patients’ prognosis. Esmolol is a -adenergic blocking agent (cardioselective) that possesses certain pharmacokinetic advantages over other antihypertensive drugs, and also has fast and powerful bradycardic and hypotensive effects. In a previous experimental study in spontaneously hypertensive rats (SHRs), our group had shown that esmolol significantly reduced, after just 48 hours of intravenous infusion, left ventricular hypertrophy, being the first drug known to produce this effect in such a short period of time (15). Based on this finding, we designed the present experimental study with the objective of investigating whether the above esmolol treatment revert coronary artery remodeling in SHR. Our results show that the drug produces the above effect, and that it does so through the following mechanisms: 1st) reducing the thickness of the arterial wall and the middle layer; 2nd) normalization of vascular reactivity by increasing nitric oxide bioavailability; 3rd) reducing oxidative stress by increasing the activity of superoxide dismutase and catalase; and 4) decreasing the concentration of the asymmetric dimethylarginine, which leads to increased nitric oxide production. Additionally, during a 3 month stay at St George's Hospital in London, an observational clinical study was designed and performed with the aim of analyzing the effectiveness of chronic therapy with β- blockers in the prophylaxis of perioperative complications of major noncardiac surgery. This was done by retrospectively evaluating a cohort of 80 patients who underwent major noncardiac surgery and were transferred to the Intensive Care Unit of the hospital. After a follow-up period of 2-4 weeks after surgery until discharge/death, the patients who were included in the beta-blocker arm had a greater number of cardiovascular risk factors and a higher incidence of postoperative arrhythmias. However, no differences in relation to the incidence of other cardiac events, noncardiac complications, length of hospital stay or mortality rate were found between patients treated or untreated with β-blockers. The results of this study suggest that chronic treatment with β-blockers does not induce perioperative complications (except postoperative arrhythmias) after noncardiac surgery. However, additional prospective and randomized studies, with a larger sample size, are needed to elucidate the actual effectiveness of chronic treatment with β-blockers as prophylaxis for perioperative complications after noncardiac surgery

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD

Plasma levels of mitochondrial and nuclear DNA in patients with massive pulmonary embolism in the emergency department: A prospective cohort study

Introduction: Cell-free plasma mitochondrial DNA (mt-DNA) and nuclear DNA (n-DNA) are biomarkers with prognostic utility in conditions associated with a high rate of cell death. This exploratory study aimed to determine the plasma levels of both nucleic acids in patients with massive and submassive pulmonary embolism (PE) and to compare them with other biomarkers, such as heart-type fatty acid-binding protein (H-FABP) and troponin I (Tn-I) Methods: This was a prospective observational study of 37 consecutive patients with massive PE, 37 patients with submassive PE, and 37 healthy subjects. Quantifications of plasma mt-DNA and n-DNA with real-time quantitative polymerase chain reaction (PCR), and plasma H-FABP and Tn-I by commercial assays, were done on blood samples drawn within 4 hours after presentation at the emergency department. Results: Plasma mt-DNA and n-DNA concentrations were much higher in patients with massive PE (median, 2,970 GE/ml; interquartile range (IQR), 1,050 to 5,485; and 3,325 GE/ml, IQR: 1,080 to 5,790, respectively) than in patients with submassive PE (870 GE/ml and 1,245 GE/ml, respectively; P < 0.01) or controls (185 GE/ml and 520 GE/ml, respectively). Eighteen patients with massive PE died of a PE-related cause by day 15 of observation. Plasma mt- DNA and n-DNA values were 2.3-fold and 1.9-fold higher in the subgroup of nonsurviving patients than in survivors. H-FABP and Tn-I values were also higher in patients with massive PE who died (7.3 ng/ml and 0.023 ng/ml, respectively) than in those who survived (6.4 ng/ml, and 0.016 ng/ml, respectively). By receiver operating curve (ROC) analysis, the best cutoff values for predicting 15-day mortality were 3,380 GE/ml for mt-DNA, 6.8 ng/ml for H-FABP, 3,625 GE/ml for n-DNA, and 0.020 ng/ml for Tn-I, based on the calculated areas under the curve (AUCs) of 0.89 (95% confidence interval (CI), 0.78 to 0.99), 0.76 (95% CI, 0.69 to 093), 0.73 (95% CI, 0.58 to 0.91), and 0.59 (95% CI, 0.41 to 0.79), respectively. By stepwise logistic regression, a plasma mt-DNA concentration greater than 3,380 GE/ml (adjusted odds ratio (OR), 8.22; 95% CI, 1.72 to 39.18; P 6.8 ng/ml (OR, 5.36; 95% CI, 1.06 to 27.08; P < 0.01) were the only independent predictors of mortality. Conclusions: mt-DNA and H-FBAP might be promising markers for predicting 15-day mortality in massive PE, with mt-DNA having better prognostic accuracy.This work was supported partially by grants from Plan Nacional I+D+I (SAF 2008-05347 and SAF2011-23575) and from Fundación Mutua Madrileña de Investigación Biomédica (2008 and 2011) to Francisco Arnalich and Carmen Montie

Expression patterns for nicotinic acetylcholine receptor subunit genes in smoking-related lung cancers

Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and underrepresentation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer typeThis study was supported by grants to C. Montiel and F. Arnalich from the Ministry of Economy, Industry and Competitiveness, Government of Spain (SAF2014-56623-R) and Foundation “Mutua Madrileña Investigación Biomédica” (FMM2011), Spain. A.B. is recipient of a fellowship (Beca FPI, Universidad Autónoma Madrid). J.L.C. and C.M.S. are recipients of fellowships (Beca FPU from Ministerio de Educación, Cultura y Deporte and Beca FPI from Ministry of Economy, Industry and Competitiveness, Government of Spain, respectively

The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.Ministerio de Economía Españ