Jesus Otero

Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors

BackgroundQuantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue.ResultsOur findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated.ConclusionWe propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.

Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation.

Experimental and clinical experiences highlight the need to review some aspects of islet transplantation, especially with regard to site of grafting and control of the immune response. The subcutaneous space could be a good alternative to liver but its sparse vasculature is its main limitation. Induction of graft tolerance by using cells with immunoregulatory properties is a promising approach to avoid graft rejection. Both Fibroblasts and Mesenchymal Stem Cells (MSCs) have shown pro-angiogenic and immunomodulatory properties. Transplantation of islets into the subcutaneous space using plasma as scaffold and supplemented with fibroblasts and/or Bone Marrow-MSCs could be a promising strategy to achieve a functional extra-hepatic islet graft, without using immunosuppressive drugs. Xenogenic rat islets, autologous fibroblasts and/or allogenic BM-MSCs, were mixed with plasma, and coagulation was induced to constitute a Plasma-based Scaffold containing Islets (PSI), which was transplanted subcutaneously both in immunodeficient and immunocompetent diabetic mice. In immunodeficient diabetic mice, PSI itself allowed hyperglycemia reversion temporarily, but the presence of pro-angiogenic cells (fibroblasts or BM-MSCs) within PSI was necessary to improve graft re-vascularization and, thus, consistently maintain normoglycemia. In immunocompetent diabetic mice, only PSI containing BM-MSCs, but not those containing fibroblasts, normalized glycemia lasting up to one week after transplantation. Interestingly, when PSI contained both fibroblasts and BM-MSCs, the normoglycemia period showed an increase of 4-times with a physiological-like response in functional tests. Histology of immunocompetent mice showed an attenuation of the immune response in those grafts with BM-MSCs, which was improved by co-transplantation with fibroblasts, since they increased BM-MSC survival. In summary, fibroblasts and BM-MSCs showed similar pro-angiogenic properties in this model of islet xenotransplantation, whereas only BM-MSCs exerted an immunomodulatory effect, which was improved by the presence of fibroblasts. These results suggest that cooperation of different cell types with islets will be required to achieve a long-term functional graft.

Expression and localization of interleukin 1 beta and interleukin 1 receptor (type I) in the bovine endometrium and embryo

The interleukin-1 (IL1) system likely mediates mammalian embryo-maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium.

Diagnóstico de muerte encefálica mediante tomografia computarizada multicorte : angio-TC y perfusión cérébral

BD was diagnosed by clinical examination, electroencephalogram (EEG), Transcranial Doppler (TCD) and multislice CT of 64 detectors. Initially, a brain perfusion study was performed. This was followed by supra-aortic trunk and brain artery angiography with acquisition of images using 0.5 mm slices, from the origin of the aortic root to the vertex. In all the patients, BD diagnosis was verified by clinical examination, EEG and TCD. Brain perfusion never detected brain blood flow. The angioCT through internal carotid arteries and vertebral arteries demonstrated complete absence of intracranial circulation, observing circulation of the external carotid artery branches. Sensitivity and specificity of the method compared with clinical examination was 100%. These findings demonstrate that the study of brain perfusion and brain angiography by multislice CT scan is a rapid and minimally invasive technique, that is easily available and that shows the absence of brain blood flow through the four vascular trunks. This technique makes it possible to made the diagnosis of BD with high diagnostic safety. Its use has special interest in patients with clinical diagnostic difficulty due to treatment with sedative drugs and serious metabolic alterations.BD was diagnosed by clinical examination, electroencephalogram (EEG), Transcranial Doppler (TCD) and multislice CT of 64 detectors. Initially, a brain perfusion study was performed. This was followed by supra-aortic trunk and brain artery angiography with acquisition of images using 0.5 mm slices, from the origin of the aortic root to the vertex. In all the patients, BD diagnosis was verified by clinical examination, EEG and TCD. Brain perfusion never detected brain blood flow. The angioCT through internal carotid arteries and vertebral arteries demonstrated complete absence of intracranial circulation, observing circulation of the external carotid artery branches. Sensitivity and specificity of the method compared with clinical examination was 100%. These findings demonstrate that the study of brain perfusion and brain angiography by multislice CT scan is a rapid and minimally invasive technique, that is easily available and that shows the absence of brain blood flow through the four vascular trunks. This technique makes it possible to made the diagnosis of BD with high diagnostic safety. Its use has special interest in patients with clinical diagnostic difficulty due to treatment with sedative drugs and serious metabolic alterations.

Evidence for clock genes circadian rhythms in human full-term placenta

Abstract Biological rhythms are driven by endogenous biological clocks; in mammals, the master clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This master pacemaker can synchronize other peripheral oscillators in several tissues such as some involved in endocrine or reproductive functions. The presence of an endogenous placental clock has received little attention. In fact, there are no studies in human full-term placentas. To test the existence of an endogenous pacemaker in this tissue we have studied the expression of circadian locomoter output cycles kaput (Clock), brain and muscle arnt-like (Bmal)1, period (Per)2, and cryptochrome (Cry)1 mRNAs at 00, 04, 08, 12, 16, and 20 hours by qPCR. The four clock genes studied are expressed in full-term human placenta. The results obtained allow us to suggest that a peripheral oscillator exists in human placenta. Data were analyzed using Fourier series where only the Clock and Bmal1 expression shows a circadian rhythm.

Tratamiento endovascular y trombólisis intraarterial en el ictus isquémico agudo

OBJECTIVE Analysis of the safety and efficacy of intra-arterial thrombolysis therapy and endovascular treatment in acute ischemic stroke. DESIGN AND AREA: An observational prospective study in the Intensive Care Unit. PATIENTS AND METHODS 16 patients had endovascular treatment. Epidemiological data, arterial occlusion site, time between stroke onset and treatment, treatment indication, NIHSS scale at admission and discharge from hospital, complications and functional outcome measured by modified Rankin scale (obtained by telephone survey) were collected. RESULTS Ten male patients with a mean age of 59 years (29-74) were included. The mean stay in the ICU was 6 days (1-33). Seven patients required mechanical ventilation. Treatment indications were: intravenous thrombolysis failure in 4 patients, major vessel occlusion in 5, outside of the therapeutic window in 2, posterior circulation occlusion in 3, outside of the therapeutic window plus major vessel occlusion in 1 and intravenous thrombolysis contraindication in 1. The occlusion site was on posterior circulation in 3 and on carotid territories and branches in 13. Thrombolytic treatment used was Urokinase at a dose of 100,000-600,000IU. Four patients required mechanical embolectomy and 10 stent implantation. Complete recanalization was observed in 11 (69%) and partial in 4 (25%). Three evolved to brain death. Six patients (46%) had a favorable outcome (modified Rankin scale score ≤ 2). Technical complication was 1 femoral artery pseudoaneurysm. CONCLUSIONS With the intra-arterial treatment, high rates of recanalization and favorable outcome are obtained with few complications. It could be indicated in patients with severe neurological injury (NIHSS ≥ 10), evolution time between 3-6h, intravenous thrombolysis contraindication and proximal arterial occlusion.

Doppler transcraneal en el diagnóstico de la muerte encefálica. ¿Es útil o retrasa el diagnóstico?

Transcranial Doppler ultrasound is able to demonstrate cerebral circulatory arrest associated to brain death, being especially useful in sedated patients, or in those in which complete neurological exploration is not possible. Transcranial Doppler ulstrasound is a portable, noninvasive and high-availability technique. Among its limitations, mention must be made of the absence of acoustic windows and false-negative cases. In patients clinically diagnosed with brain death, with open skulls or with anoxia as the cause of death, cerebral blood flow can be observed by ultrasound, since cerebral circulatory arrest is not always synchronized to the clinical diagnosis. The diagnostic rate is therefore time-dependent, and this fact that must be recognized in order to avoid delays in death certification. Despite its limitations, transcranial Doppler ulstrasound helps solve common diagnostic problems, avoids the unnecessary consumption of resources, and can optimize organ harvesting for transplantation.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial.

Background Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. Methods In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patients lymphocytes with CB-SCs in vitro and returns the “educated” lymphocytes (but not the CB-SCs) into the patients blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Findings Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C–C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Interpretation Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Funding Obra Social “La Caixa”, Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

Platelet‐Derived Mitochondria Display Embryonic Stem Cell Markers and Improve Pancreatic Islet β‐cell Function in Humans

Diabetes is a major global health issue and the number of individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D) increases annually across multiple populations. Research to develop a cure must overcome multiple immune dysfunctions and the shortage of pancreatic islet β cells, but these challenges have proven intractable despite intensive research effort more than the past decades. Stem Cell Educator (SCE) therapy—which uses only autologous blood immune cells that are externally exposed to cord blood stem cells adhering to the SCE device, has previously been proven safe and effective in Chinese and Spanish subjects for the improvement of T1D, T2D, and other autoimmune diseases. Here, 4‐year follow‐up studies demonstrated the long‐term safety and clinical efficacy of SCE therapy for the treatment of T1D and T2D. Mechanistic studies found that the nature of platelets was modulated in diabetic subjects after receiving SCE therapy. Platelets and their released mitochondria display immune tolerance‐associated markers that can modulate the proliferation and function of immune cells. Notably, platelets also expressed embryonic stem cell‐ and pancreatic islet β‐cell‐associated markers that are encoded by mitochondrial DNA. Using freshly‐isolated human pancreatic islets, ex vivo studies established that platelet‐releasing mitochondria can migrate to pancreatic islets and be taken up by islet β cells, leading to the proliferation and enhancement of islet β‐cell functions. These findings reveal new mechanisms underlying SCE therapy and open up new avenues to improve the treatment of diabetes in clinics. Stem Cells Translational Medicine 2017;6:1684–1697

OriginalTratamiento endovascular y trombólisis intraarterial en el ictus isquémico agudoEndovascular treatment and intra-arterial thrombolysis in acute ischemic stroke

OBJECTIVE Analysis of the safety and efficacy of intra-arterial thrombolysis therapy and endovascular treatment in acute ischemic stroke. DESIGN AND AREA: An observational prospective study in the Intensive Care Unit. PATIENTS AND METHODS 16 patients had endovascular treatment. Epidemiological data, arterial occlusion site, time between stroke onset and treatment, treatment indication, NIHSS scale at admission and discharge from hospital, complications and functional outcome measured by modified Rankin scale (obtained by telephone survey) were collected. RESULTS Ten male patients with a mean age of 59 years (29-74) were included. The mean stay in the ICU was 6 days (1-33). Seven patients required mechanical ventilation. Treatment indications were: intravenous thrombolysis failure in 4 patients, major vessel occlusion in 5, outside of the therapeutic window in 2, posterior circulation occlusion in 3, outside of the therapeutic window plus major vessel occlusion in 1 and intravenous thrombolysis contraindication in 1. The occlusion site was on posterior circulation in 3 and on carotid territories and branches in 13. Thrombolytic treatment used was Urokinase at a dose of 100,000-600,000IU. Four patients required mechanical embolectomy and 10 stent implantation. Complete recanalization was observed in 11 (69%) and partial in 4 (25%). Three evolved to brain death. Six patients (46%) had a favorable outcome (modified Rankin scale score ≤ 2). Technical complication was 1 femoral artery pseudoaneurysm. CONCLUSIONS With the intra-arterial treatment, high rates of recanalization and favorable outcome are obtained with few complications. It could be indicated in patients with severe neurological injury (NIHSS ≥ 10), evolution time between 3-6h, intravenous thrombolysis contraindication and proximal arterial occlusion.