[68Ga]-NODAGA-RGD Positron Emission Tomography (PET) for Assessment of Post Myocardial Infarction Angiogenesis as a Predictor for Left Ventricular Remodeling in Mice after Cardiac Stem Cell Therapy.

Angiogenesis plays a central role in the healing process following acute myocardial infarction. The PET tracer [68Ga]-NODAGA-RGD, which is a ligand for the αvß3 integrin, has been investigated for imaging angiogenesis in the process of healing myocardium in both animal and clinical studies. It´s value as a prognostic marker of functional outcome remains unclear. Therefore, the aim of this work was to establish [68Ga]-NODAGA-RGD for imaging angiogenesis in the murine infarct model and evaluate the tracer as a predictor for cardiac remodeling in the context of cardiac stem cell therapy. [68Ga]-NODAGA-RGD PET performed seven days after left anterior descending coronary artery (LAD) occlusion in 129S6 mice showed intense tracer accumulation within the infarct region. The specificity was shown in a sub-group of animals by application of the competitive inhibitor cilengitide prior to tracer injection in a subgroup of animals. Myocardial infarction (MI) significantly reduced cardiac function and resulted in pronounced left ventricular remodeling after three weeks, as measured by cardiac MRI in a separate group. Cardiac induced cells (CiC) that were derived from mESC injected intramyocardially in the therapy group significantly improved left ventricular ejection fraction (LVEF). Surprisingly, CiC transplantation resulted in significantly lower tracer accumulation seven days after MI induction. Accordingly, we successfully established the PET tracer [68Ga]-NODAGA-RGD for the assessment of αvß3 integrin expression in the healing process after MI in the mouse model. Yet, our results indicate that the mere extent of angiogenesis following MI does not serve as a sufficient prognostic marker for functional outcome.

Integrative Cluster Analysis of Whole Hearts Reveals Proliferative Cardiomyocytes in Adult Mice.

The recent development and broad application of sequencing techniques at the single-cell level is generating an unprecedented amount of data. The different techniques have their individual limits, but the datasets also offer unexpected possibilities when utilized collectively. Here, we applied snRNA-seq in whole adult murine hearts from an inbred (C57BL/6NRj) and an outbred (Fzt:DU) mouse strain to directly compare the data with the publicly available scRNA-seq data of the tabula muris project. Explicitly choosing a single-nucleus approach allowed us to pin down the typical heart tissue-specific technical bias, coming up with novel insights on the mammalian heart cell composition. For our integrated dataset, cardiomyocytes, fibroblasts, and endothelial cells constituted the three main cell populations accounting for about 75% of all cells. However, their numbers severely differed between the individual datasets, with cardiomyocyte proportions ranging from about 9% in the tabula muris data to around 23% for our BL6 data, representing the prime example for cell capture technique related bias when using a conventional single-cell approach for these large cells. Most strikingly in our comparison was the discovery of a minor population of cardiomyocytes characterized by proliferation markers that could not be identified by analyzing the datasets individually. It is now widely accepted that the heart has an, albeit very restricted, regenerative potential. However there is still an ongoing debate where new cardiomyocytes arise from. Our findings support the idea that the renewal of the cardiomyocyte pool is driven by cytokinesis of resident cardiomyocytes rather than differentiation of progenitor cells. We thus provide data that can contribute to an understanding of heart cell regeneration, which is a prerequisite for future applications to enhance the process of heart repair.

Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition and Velocity.

: Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for the first time an entire adult mammalian heart. Single-nucleus quantification and clustering led to an accurate representation of cell types, revealing 24 distinct clusters with endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) constituting the major cell populations. An additional RNA velocity analysis allowed us to study transcription kinetics and was utilized to visualize the transitions between mature and nascent cellular states of the cell types. We identified subgroups of cardiomyocytes with distinct marker profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting at interrelations.

Nkx2.5 Based Ventricular Programming of Murine ESC-Derived Cardiomyocytes.

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies. The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

Stem Cell Therapy in Heart Diseases - Cell Types, Mechanisms and Improvement Strategies.

A large number of clinical trials have shown stem cell therapy to be a promising therapeutic approach for the treatment of cardiovascular diseases. Since the first transplantation into human patients, several stem cell types have been applied in this field, including bone marrow derived stem cells, cardiac progenitors as well as embryonic stem cells and their derivatives. However, results obtained from clinical studies are inconsistent and stem cell-based improvement of heart performance and cardiac remodeling was found to be quite limited. In order to optimize stem cell efficiency, it is crucial to elucidate the underlying mechanisms mediating the beneficial effects of stem cell transplantation. Based on these mechanisms, researchers have developed different improvement strategies to boost the potency of stem cell repair and to generate the "next generation" of stem cell therapeutics. Moreover, since cardiovascular diseases are complex disorders including several disease patterns and pathologic mechanisms it may be difficult to provide a uniform therapeutic intervention for all subgroups of patients. Therefore, future strategies should aim at more personalized SC therapies in which individual disease parameters influence the selection of optimal cell type, dosage and delivery approach.

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80-90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

Stem cells and heart disease - Brake or accelerator?

After two decades of intensive research and attempts of clinical translation, stem cell based therapies for cardiac diseases are not getting closer to clinical success. This review tries to unravel the obstacles and focuses on underlying mechanisms as the target for regenerative therapies. At present, the principal outcome in clinical therapy does not reflect experimental evidence. It seems that the scientific obstacle is a lack of integration of knowledge from tissue repair and disease mechanisms. Recent insights from clinical trials delineate mechanisms of stem cell dysfunction and gene defects in repair mechanisms as cause of atherosclerosis and heart disease. These findings require a redirection of current practice of stem cell therapy and a reset using more detailed analysis of stem cell function interfering with disease mechanisms. To accelerate scientific development the authors suggest intensifying unified computational data analysis and shared data knowledge by using open-access data platforms.

Cardiac Function Improvement and Bone Marrow Response -: Outcome Analysis of the Randomized PERFECT Phase III Clinical Trial of Intramyocardial CD133+ Application After Myocardial Infarction.

OBJECTIVE: The phase III clinical trial PERFECT was designed to assess clinical safety and efficacy of intramyocardial CD133+ bone marrow stem cell treatment combined with CABG for induction of cardiac repair. DESIGN: Multicentre, double-blinded, randomised placebo controlled trial. SETTING: The study was conducted across six centres in Germany October 2009 through March 2016 and stopped due slow recruitment after positive interim analysis in March 2015. PARTICIPANTS: Post-infarction patients with chronic ischemia and reduced LVEF (25-50%). INTERVENTIONS: Eighty-two patients were randomised to two groups receiving intramyocardial application of 5ml placebo or a suspension of 0.5-5×106 CD133+. OUTCOME: Primary endpoint was delta (∆) LVEF at 180days (d) compared to baseline measured in MRI. FINDINGS (PRESPECIFIED): Safety (n=77): 180d survival was 100%, MACE n=2, SAE n=49, without difference between placebo and CD133+. Efficacy (n=58): The LVEF improved from baseline LVEF 33.5% by +9.6% at 180d, p=0.001 (n=58). Treatment groups were not different in ∆LVEF (ANCOVA: Placebo +8.8% vs. CD133+ +10.4%, ∆CD133+vs placebo +2.6%, p=0.4). FINDINGS (POST HOC): Responders (R) classified by ∆LVEF≥5% after 180d were 60% of the patients (35/58) in both treatment groups. ∆LVEF in ANCOVA was +17.1% in (R) vs. non-responders (NR) (∆LVEF 0%, n=23). NR were characterized by a preoperative response signature in peripheral blood with reduced CD133+ EPC (RvsNR: p=0.005) and thrombocytes (p=0.004) in contrast to increased Erythropoeitin (p=0.02), and SH2B3 mRNA expression (p=0.073). Actuarial computed mean survival time was 76.9±3.32months (R) vs. +72.3±5.0months (NR), HR 0.3 [Cl 0.07-1.2]; p=0.067.Using a machine learning 20 biomarker response parameters were identified allowing preoperative discrimination with an accuracy of 80% (R) and 84% (NR) after 10-fold cross-validation. INTERPRETATION: The PERFECT trial analysis demonstrates that the regulation of induced cardiac repair is linked to the circulating pool of CD133+ EPC and thrombocytes, associated with SH2B3 gene expression. Based on these findings, responders to cardiac functional improvement may be identified by a peripheral blood biomarker signature. TRIAL REGISTRATION: ClinicalTrials.govNCT00950274.

Data on the fate of MACS® MicroBeads intramyocardially co-injected with stem cell products.

The data presented in this article are related to the research article "Intramyocardial Fate and Effect of Iron Nanoparticles co-injected with MACS® purified Stem Cell Products" (Müller et al., 2017) [1]. This article complements the cellular localization of superparamagnetic iron dextran particles (MACS® MicroBeads) used for magnetic activated cell sorting (MACS®). Data evaluate the time-dependent detachment of these nanoparticles from CD133+ haematopoietic stem cells (HSCs) and CD271+ mesenchymal stem cells (MSCs). Furthermore, the influence of these stem cells as well as of nanoparticles on cardiac remodeling processes after myocardial infarction (MI) was investigated.

Intramyocardial fate and effect of iron nanoparticles co-injected with MACS® purified stem cell products.

BACKGROUND: Magnetic activated cell sorting (MACS®) is routinely used to isolate stem cell subpopulations intended for the treatment of cardiovascular diseases. In strong contrast, studies examining the amount, effect and intramyocardial distribution of iron nanoparticles used for magnetic cell labelling are missing, although iron excess can cause functional disorders in the heart. METHODS AND RESULTS: CD133+ haematopoietic and CD271+ mesenchymal stem cells were purified from bone marrow using automatically and manually MACS® based systems. Flow cytometric measurements demonstrated a rapid loss of MACS® MicroBeads from cells under culture conditions, while storage under hypothermic conditions decelerated their detachment. Moreover, an average loading of ∼11 fg iron/cell caused by magnetic labelling was determined in magnetic particle spectroscopy. Importantly, hemodynamic measurements as well as histological examinations using a myocardial ischemia/reperfusion mouse model showed no influence of MACS® MicroBeads on cardiac regeneration, while the transplantation of stem cells caused a significant improvement. Furthermore, immunostainings demonstrated the clearance of co-injected iron nanoparticles from stem cells and the surrounding heart tissue within 48 h post transplantation. CONCLUSIONS: Our results indicate that iron amounts typically co-injected with MACS® purified stem cells do not harm cardiac functions and are cleared from heart tissue within a few hours. Therefore, we conclude that MACS® MicroBeads exhibit a good compatibility in the cardiac environment.

Cardiac Cell Therapies for the Treatment of Acute Myocardial Infarction: A Meta-Analysis from Mouse Studies.

AIMS: Stem cell-based regenerative therapies for the treatment of ischemic myocardium are currently a subject of intensive investigation. A variety of cell populations have been demonstrated to be safe and to exert some positive effects in human Phase I and II clinical trials, however conclusive evidence of efficacy is still lacking. While the relevance of animal models for appropriate pre-clinical safety and efficacy testing with regard to application in Phase III studies continues to increase, concerns have been expressed regarding the validity of the mouse model to predict clinical results. Against the background that hundreds of preclinical studies have assessed the efficacy of numerous kinds of cell preparations - including pluripotent stem cells - for cardiac repair, we undertook a systematic re-evaluation of data from the mouse model, which initially paved the way for the first clinical trials in this field. METHODS AND RESULTS: A systematic literature screen was performed to identify publications reporting results of cardiac stem cell therapies for the treatment of myocardial ischemia in the mouse model. Only peer-reviewed and placebo-controlled studies using magnet resonance imaging (MRI) for left ventricular ejection fraction (LVEF) assessment were included. Experimental data from 21 studies involving 583 animals demonstrate a significant improvement in LVEF of 8.59%+/- 2.36; p=.012 (95% CI, 3.7-13.8) compared with control animals. CONCLUSION: The mouse is a valid model to evaluate the efficacy of cell-based advanced therapies for the treatment of ischemic myocardial damage. Further studies are required to understand the mechanisms underlying stem cell based improvement of cardiac function after ischemia.

GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration.

BACKGROUND: CD133+ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133+ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics. METHODS: CD133+ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133+ cells was evaluated and compared to manually isolated CD133+ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction. RESULTS: We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 106 viable CD133+ cells with a mean log10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo. CONCLUSIONS: Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133+ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

Cross-cultural adaptation and content and semantic validation of the Difficult Intravenous Access Score for pediatric use in Brazil / Adaptação transcultural e validação de conteúdo e semântica do Difficult Intravenous Access Score para uso pediátrico no Brasil / Adaptación transcultural y validación de contenido y semántica del Difficult Intravenous Access Score para uso pediátrico en Brasil

ABSTRACT Objective: present the cross-cultural adaptation and content and semantic validation of the Difficult Intravenous Access Score for current use in Brazil. Method: cross-cultural adaptation and validation study, structured in six phases: initial translation, synthesis of translations, back-translation, assessment of documents by expert committee of specialized judges, pretest and presentation of the documents to the expert judges and to the author of the original instrument. Twenty health professionals were randomly recruited from a public hospital in the South of Brazil, working in pediatrics, in order to assess the agreement level with the variables in the instrument. In addition, a convenience sample of 30 pediatric patients was selected for the face validation of the same instrument. Cronbach's alpha coefficient, simple and percentage frequencies, the Shapiro-Wilk and Fisher's exact tests were used for the data analysis and reliability measures. Results: the cross-cultural adaptation phases were executed with totally clear translated variables, demonstrating satisfactory results in the content and semantic validation process. Conclusions: the Difficult Intravenous Access Score was adapted and its content and semantics were validated. External clinical validity, measuring equivalence and reproducibility analyses are needed.

RESUMO Objetivo: apresentar a adaptação transcultural e validação de conteúdo e semântica do Difficult Intravenous Access Score para uso corrente no Brasil. Método: pesquisa delineada na adaptação transcultural e validação, estruturada em seis etapas: tradução inicial, síntese das traduções, retrotradução, avaliação da documentação por comitê de juízes especialistas, pré-teste e apresentação da documentação aos juízes especialistas e ao autor do instrumento original. Foram recrutados aleatoriamente 20 profissionais de saúde de uma instituição hospitalar pública do Sul do País, atuantes em pediatria, para avaliar o nível de concordância com as variáveis do instrumento, e, por conveniência, 30 pacientes pediátricos para validação de face do instrumento. Utilizou-se o coeficiente alfa de Cronbach, frequência simples e percentual, testes de Shapiro-Wilk e exato de Fisher, para análise dos dados e mensuração da confiabilidade. Resultados: as etapas da adaptação transcultural foram desenvolvidas com plena clareza das variáveis traduzidas, demonstrando resultados satisfatórios no processo de validação de conteúdo e semântica. Conclusões: o Difficult Intravenous Access Score foi adaptado transculturalmente e validado em seu conteúdo e semântica. Consideram-se necessárias análises de validade clínica externa, da equivalência de mensuração e da reprodutibilidade.

RESUMEN Objetivo: presentar la adaptación transcultural y validación de contenido y semántica del Difficult Intravenous Access Score para utilización actual en Brasil. Método: investigación delineada en la adaptación transcultural y validación, estructurada en seis etapas: traducción inicial, síntesis de las traducciones, retrotraducción, evaluación de la documentación por comité de jueces especialistas, pre-prueba y presentación de la documentación a los jueces especialistas y al autor del instrumento original. Fueron reclutados aleatoriamente 20 profesionales de salud de una institución hospitalaria pública del sur del país, activos en pediatría, para evaluar el nivel de concordancia con las variables del instrumento y, por conveniencia, 30 pacientes pediátricos, para validación de faz del instrumento. Fue utilizado el coeficiente alfa de Cronbach, frecuencia simple y porcentaje, pruebas de Shapiro-Wilk y exacto de Fisher, para el análisis de los datos y mensuración de la confiabilidad. Resultados: las etapas de la adaptación transcultural fueron desarrolladas con toda claridad de las variables traducidas, demostrando resultados satisfactorios en el proceso de validación de contenido y semántica. Conclusiones: el Difficult Intravenous Access Score fue adaptado transculturalmente, y validado en su contenido y semántica. Son considerados necesarios análisis de validez clínica externa, de la equivalencia de mensuración y de la reproductibilidad.

Magnet-Bead Based MicroRNA Delivery System to Modify CD133+ Stem Cells.

Aim. CD133+ stem cells bear huge potential for regenerative medicine. However, low retention in the injured tissue and massive cell death reduce beneficial effects. In order to address these issues, we intended to develop a nonviral system for appropriate cell engineering. Materials and Methods. Modification of human CD133+ stem cells with magnetic polyplexes carrying microRNA was studied in terms of efficiency, safety, and targeting potential. Results. High microRNA uptake rates (~80-90%) were achieved without affecting CD133+ stem cell properties. Modified cells can be magnetically guided. Conclusion. We developed a safe and efficient protocol for CD133+ stem cell modification. Our work may become a basis to improve stem cell therapeutical effects as well as their monitoring with magnetic resonance imaging.

Risco de trauma vascular: proposta de intervenções de cuidado pediátrico em enfermagem / Risk of vascular trauma: proposals for nursing intervention in pediatric care

Objetivo: propor intervenções de enfermagem para o cuidado pediátrico relacionadas ao Risco de Trauma Vascular.Método: pesquisa-ação crítica com abordagem qualitativa, realizada na Unidade de Pediatria de hospital públicode ensino, Curitiba (PR). Três ações foram desenvolvidas: levantamento das intervenções já existentes, teorizaçãoe discussão sobre a aplicabilidade de cada uma, e desenvolvimento da intervenção ou ação. Resultados: foramlocalizados, no Sistema Informatizado Hospitalar, 16 registros de intervenções de enfermagem relacionadas ao Riscode Trauma Vascular. Após constatação de que a maioria das intervenções não contemplava especificidades doscuidados pediátricos, desenvolveram-se outras, com base em evidências científicas e correspondências entre a NorthAmerican Nursing Diagnosis Association e a Nursing Interventions Classification. Conclusão: os aspectos propostosindividualizam as intervenções pediátricas e consideram suas especificidades, tendo impacto para prevenção dasiatrogenias, bem como para qualificação da assistência de enfermagem.

Objective: to propose pediatric care nursing interventions related to the Risk of Vascular Trauma. Method: criticalaction research with a qualitative approach; it was performed at the Pediatric Unit of a public teaching hospital,in Curitiba, (PR). Three actions were developed: collection of existing interventions; theorization and discussionon the applicability of each one; and development of the intervention or action. Results: 16 records of nursinginterventions related to the Risk of Vascular Trauma were found in the Hospital Computerized System. After verifyingthat most of the interventions did not include specificities of pediatric care, they were developed others based onscientific evidence and correspondences between the North American Nursing Diagnosis Association and the NursingInterventions Classification. Conclusion: the proposed aspects individualize the pediatric interventions and considertheir specificities; this will have effects on the prevention of iatrogenic and will help to qualify the nursing care.

Objetivo: proponer intervenciones de enfermería para el cuidado pediátrico relacionadas al Riesgo de Trauma Vascular.Método: investigación-acción crítica con abordaje cualitativo, realizada en la Unidad de Pediatría de un hospitalpúblico de enseñanza, en Curitiba (PR). Tres acciones fueron desarrolladas: levantamiento de las intervencionesya existentes; teorización y discusión sobre la aplicabilidad de cada una; y desarrollo de la intervención o acción.Resultados: en el Sistema Informatizado Hospitalario fueron localizados 16 registros de intervenciones de enfermeríarelacionados con el Riesgo de Trauma Vascular. Después de constatar que la mayoría de las intervenciones nocontemplaba especificidades de los cuidados pediátricos, se desarrollaron otras con base en evidencias científicas ycorrespondencias entre la North American Nursing Diagnosis Association y la Nursing Interventions Classification.Conclusión: los aspectos propuestos individualizan las intervenciones pediátricas y consideran sus especificidades;lo que tendrá impacto en la prevención de las iatrogenias, así como ayudará a calificar la asistencia de enfermería.

Adaptação transcultural e validação clínica do Difficult Intravenous Access Score - Diva Score - para uso no Brasil / Cross-cultural adaptation and clinical validation of the Difficult Intravenous Access Score - Diva Score - for use in Brazil

A Punção Intravenosa Periférica é considerada um dos procedimentos mais desgastante para crianças hospitalizadas, familiares e equipe de saúde. Para minimizar possíveis traumas, o uso de score de predição ao insucesso à primeira tentativa, no estabelecimento de PIP, já é uma realidade internacional. A tradução informal de instrumentos, desenvolvidos em nacionalidades diferentes compromete a qualidade dos resultados esperados com seu emprego. Portanto, procedeu-se a adaptação transcultural e validação clínica de instrumento aplicável à pediatria, o Difficult Intravenous Access score - DIVA score. Os seguintes passos foram implementados: 1. Tradução inicial; 2. Síntese das traduções; 3. Retro tradução; 4. Avaliação da documentação por comitê de juízes especialistas; 5. Pré-teste; 6. Apresentação da documentação aos juízes especialistas e autor do instrumento original. Com o emprego da metodologia proposta, obteve-se instrumento adaptado transculturalmente, que foi validado para uso confiável no Brasil, como recurso adjuvante à punção intravenosa de crianças. Os principais atributos que cercam a adaptação transcultural e validação de instrumento de saúde internacional, é sua aplicabilidade coerente, para que possam surtir efeitos positivos à nossa realidade. Destarte, tem-se um instrumento útil para diagnóstico do insucesso na primeira tentativa de punção e de risco de trauma vascular, que poderá, apontar pela adoção de recursos tecnológicos adjuvantes à venopunção, disponível para profissionais de saúde, sobretudo para a enfermagem, no território brasileiro.

The Peripheral Venipuncture is considered one of the most tiring processes for hospitalized children, family members and health team. In order to minimize possible traumas, the use of a failure prediction score to the first attempt, at establishing the Peripheral Venipuncture, is already an international reality. The informal translation of instruments, developed in different nationalities, compromises the quality of the expected results with its employment. Therefore, a transcultural adaptation and clinical validation of instrument applied to pediatrics, the Difficult Intravenous Access score - DIVA score. The following steps were implemented: 1. Initial translation; 2. Translations synthesis; 3. Back-translation; 4. Documentation evaluation by specialized judges committee. 5. Pretest. 6. Presentation of the documentation to the specialized judges and author of the original instrument. By employing the proposed methodology, it was obtained a transculturally-translated instrument that was validated for reliable use in Brazil as an adjuvant resource to the peripheral venipuncture of children. The main attribute that concerns the transcultural adaptation and the international health instrument validation is its consistent application so that they may result in positive effects to our reality. This way, we have an useful instrument for the diagnosis of failure on the first attempt of puncture and of vascular trauma risk, that may point to the adoption of adjuvant technological resources to the venipuncture, available to health professionals, above all to nursing, in Brazilian territory.

Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts.

BACKGROUND: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. METHODS: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins. RESULTS: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. CONCLUSION: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105(+) hMSCs.

Genetic modifications of bone marrow derived human mesenchymal stem cells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag). We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allowfor initial cell modulation in vitro.