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OBJECTIVES: Observational data to support delaying elective pediatric thoracic surgery during peak respiratory viral illness season is lacking. This study evaluated whether lung surgery during peak viral season is associated with differences in postoperative outcomes and resource utilization. METHODS: A retrospective observational cohort study was performed using the Pediatric Health Information System (PHIS). Patients with a congenital lung malformation (CLM) who underwent elective lung resection between 1 January 2016 and 29 February 2020 were included. Respiratory syncytial virus (RSV) incidence was used as a proxy for respiratory viral illness circulation. Monthly hospital-specific RSV incidence was calculated from PHIS data, and peak RSV season was defined by Centers for Disease Control data. Multivariable regression models were built to identify predictors of postoperative mechanical ventilation, which was the main outcome measure, as well as secondary outcomes including 30-day readmission after lung resection, postoperative length of stay (LOS) and hospital billing charges. RESULTS: Of 1542 CLM patients identified, 344 (22.3%) underwent lung resection during peak RSV season. 38% fewer operations were performed per month during peak RSV season than during off-peak months (p < .001). Children who underwent surgery during peak RSV season did not differ from the off-peak group in terms of age at operation, race, or comorbid conditions (i.e., congenital heart disease, newborn respiratory distress, and preoperative pneumonia). There was no association between hospital-specific RSV incidence at the time of surgery and postoperative mechanical ventilation, postoperative LOS, 30-day readmission rate or hospital billing charges. DISCUSSION: Performing elective lung surgery in children with CLMs during peak viral season is not associated with adverse surgical outcomes or increased utilization of healthcare resources.
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Procedimientos Quirúrgicos Electivos , Infecciones por Virus Sincitial Respiratorio , Estaciones del Año , Humanos , Infecciones por Virus Sincitial Respiratorio/epidemiología , Estudios Retrospectivos , Masculino , Femenino , Lactante , Procedimientos Quirúrgicos Electivos/estadística & datos numéricos , Preescolar , Tiempo de Internación/estadística & datos numéricos , Estados Unidos/epidemiología , Incidencia , Niño , Respiración Artificial/estadística & datos numéricos , Complicaciones Posoperatorias/epidemiología , Neumonectomía/estadística & datos numéricos , Readmisión del Paciente/estadística & datos numéricos , Pulmón/cirugía , Recién NacidoRESUMEN
Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development.
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Aminoácidos , Proteoma , Embarazo , Femenino , Ratones , Animales , Aminoácidos/metabolismo , Proteoma/metabolismo , Lisina/metabolismo , Hígado/metabolismo , Desarrollo FetalRESUMEN
Ovarian tumors are rare in children; however, their incidence increases with age. Of these ovarian tumors, Leydig cell tumors are some of the rarest, accounting for less than 0.1% of all ovarian tumors across all ages. Leydig cell tumors predominantly occur in postmenopausal women and are characterized by nodular proliferation of Leydig cells in the ovarian hilum with intracytoplasmic Reinke crystals. These tumors secrete androgens, which can disrupt ovarian function, clinically presenting with abnormal uterine bleeding and virilization. Although they are generally benign, current recommendations are for treatment with a unilateral salpingo-oophorectomy. In adolescents, hyperandrogenism is most commonly caused by polycystic ovarian syndrome (PCOS); however, the differential for hyperandrogenism is broad. We present a case of a 15-year-old girl with a history of primary amenorrhea who presented with a Leydig cell tumor associated with recurrent ovarian torsion and virilization. This case reviews the challenges with diagnosis, management, and future implications of a rare androgen-secreting tumor in young patients.
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Hiperandrogenismo , Tumor de Células de Leydig , Neoplasias Ováricas , Niño , Humanos , Femenino , Adolescente , Tumor de Células de Leydig/complicaciones , Tumor de Células de Leydig/cirugía , Tumor de Células de Leydig/diagnóstico , Hiperandrogenismo/complicaciones , Virilismo/etiología , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/cirugía , AndrógenosRESUMEN
Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novel in utero stable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.
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In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Recién Nacido , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Tolerancia Inmunológica , Feto , Linfocitos T ReguladoresRESUMEN
Gene therapy has traditionally involved the delivery of exogenous genetic material to a cell-most commonly to replace defective genes causing monogenic disorders. This allows cells to produce proteins that are otherwise absent in sufficient quantities, ideally for a therapeutic purpose. Since its inception over 40 years ago, the field of gene therapy has significantly expanded and now includes targeted gene editing strategies, including, but not limited to, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs).
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Edición Génica , Enfermedades Metabólicas , Terapia Genética , Humanos , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/terapia , Nucleasas con Dedos de Zinc/genéticaRESUMEN
BACKGROUND: Total gastrectomy with Roux-en-Y esophagojejunostomy is a life-extending procedure for patients with nonmetastatic proximal gastric and gastroesophageal junction adenocarcinoma, yet it can be a life-altering procedure with negative impact on quality of life.1 Perioperative recovery often involves the need for supplemental nutrition (either enteral or parenteral). Furthermore, long-term effects of early satiety, dysphagia, sustained weight loss, and difficulty in maintaining a healthy weight, dumping syndrome, and intestinal overgrowth are not unusual. Although the alternative of untreated cancer is clearly unacceptable, these lifestyle consequences are not benign. METHODS: A retrospective review of patients who had undergone laparoscopic total and proximal gastrectomy for gastric adenocarcinoma was conducted. Patient demographic data, pathologic parameters, and short-term and long-term clinical data were compared between total gastrectomy and proximal gastrectomy cohorts. RESULTS: Seventeen patients were included in the study: 13 had undergone laparoscopic total gastrectomy (LTG) and 4 had undergone laparoscopic proximal gastrectomy (LPG). Patients who had LPG, given the nature of the procedure, were confined to early stage (up to T2) tumors in the gastric cardia or GE junction. Patients who had LTG tended to be larger, later stage tumors (but not exclusively). The mean operative time was greater for LTG than for LPG (247 ± 54 versus 181 ± 49 min, respectively, P = .036). Length of hospital stay (9.0 ± 3.2 versus 5.0 ± 0.8 days, P < .001) and readmission for postoperative complication (38.5 versus 0%, P = .009) were also higher in the LTG group. There was no significant difference in terms of mean estimated blood loss or blood transfusion rates, overall complications, or anastomotic stricture requiring endoscopic dilation between the patients who underwent LTG and those who underwent LPG. CONCLUSION: In early stage tumors (T1b or T2), proximal gastrectomy (PG) should be considered to mitigate diminished quality of life. PG with esophagogastrostomy, which can easily be performed minimally invasively, can be more tolerable for the patient, with no anatomic basis for dumping syndrome or small intestinal bacterial overgrowth (SIBO), and a greater reservoir for more normal meal habits when compared to total gastrectomy (TG) with Roux-en-Y reconstruction.
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Laparoscopía , Neoplasias Gástricas , Gastrectomía , Humanos , Complicaciones Posoperatorias/epidemiología , Calidad de Vida , Estudios Retrospectivos , Neoplasias Gástricas/cirugía , Resultado del TratamientoRESUMEN
BACKGROUND: Maternal transplacental administration of sildenafil is being considered for a variety of fetal conditions. Clinical translation also requires evaluation of fetal safety in a higher species, such as the fetal lamb. Experiments with the pregnant ewe are curtailed by minimal transplacental transfer as well as limited access to the fetus. The EXTra-uterine Environment for Neonatal Development (EXTEND) model renders the isolated fetal lamb readily accessible and allows for direct fetal administration of sildenafil. METHODS: Five fetal lambs were placed on extracorporeal support in the EXTEND device and received continuous intravenous (IV) sildenafil (0.3-0.5-0.7â¯mg/kg/24hr) for a duration of one to seven days. Plasma sildenafil concentrations were sampled at regular intervals to establish the pharmacokinetic profile using population pharmacokinetic modeling. Serial Doppler ultrasound examination, continuous non-invasive hemodynamic monitoring and blood gas analysis were done to evaluate the pharmacodynamic effects and fetal response. FINDINGS: The target concentration range (47-500â¯ng/mL) was attained with all doses. Sildenafil induced an immediate and temporary reduction of pulmonary vascular resistance, mean arterial pressure and circuit flow, without change in fetal lactate levels and acid-base status. The duration of the systemic effects increased with the dose. INTERPRETATION: Immediate temporary pulmonary vascular and systemic hemodynamic changes induced by sildenafil were biochemically well tolerated by fetal lambs on extracorporeal support, with the 0.5â¯mg/kg/24â¯h dose balancing rapid attainment of target concentrations with short-lived systemic effects. RESEARCH IN CONTEXT: None. SEARCH STRATEGY BEFORE UNDERTAKING THE STUDY: A literature review was conducted searching online databases (Medline, Embase and Cochrane), using search terms: fetal OR prenatal OR antenatal AND sildenafil, without time-limit and excluding human studies. Where relevant, investigators were contacted in order to avoid duplication of work. EVIDENCE BEFORE THIS STUDY: Prenatal therapy with sildenafil, a phosphodiesterase-5 inhibitor with vasodilatory and anti-remodeling effects on vascular smooth muscle cells, has been considered for a variety of fetal conditions. One multicenter clinical trial investigating the benefit of sildenafil in severe intrauterine growth restriction (the STRIDER-trial) was halted early due to excess mortality in the sildenafil-exposed arm at one treatment site. Such findings demonstrate the importance of extensive preclinical safety assessment in relevant animal models. Transplacentally administered sildenafil leads to decreased pulmonary arterial muscularization, preventing or reducing the occurrence of pulmonary hypertension in rat and rabbit fetuses with diaphragmatic hernia (DH). Validation of these results in a higher and relevant animal model, e.g. fetal lambs, is the next step to advance clinical translation. We recently demonstrated that, in contrast to humans, transplacental transfer of sildenafil in sheep is minimal, precluding the in vivo study of fetal effects at target concentrations using the conventional pregnant ewe model. ADDED VALUE OF THIS STUDY: We therefore used the extracorporeal support model for fetal lambs, referred to as the EXTra-uterine Environment for Neonatal Development (EXTEND) system, bypassing placental and maternal metabolism, to investigate at what dose the target concentrations are reached, and what the fetal hemodynamic impact and response are. Fetal hemodynamic and metabolic tolerance to sildenafil are a crucial missing element on the road to clinical translation. This is therefore the first study investigating the pharmacokinetics, hemodynamic and biochemical effects of clinical-range concentrations of sildenafil in fetal lambs, free from placental and maternal interference. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: We demonstrated self-limiting pulmonary vasodilation, a decrease of both systemic arterial pressures and circuit flows, induced by clinical range concentrations of sildenafil, without the development of fetal acidosis. This paves the way for further investigation of prenatal sildenafil in fetal lambs on extracorporeal support. A dose of 0.5â¯mg/kg/24â¯h offered the best trade-off between rapid achievement of target concentrations and shortest duration of systemic effects. This is also the first study using the EXTEND as a model for pharmacotherapy during pregnancy.
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Aorta/efectos de los fármacos , Circulación Extracorporea , Terapias Fetales , Arteria Pulmonar/efectos de los fármacos , Citrato de Sildenafil/farmacocinética , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacocinética , Animales , Aorta/diagnóstico por imagen , Aorta/fisiopatología , Presión Arterial/efectos de los fármacos , Edad Gestacional , Infusiones Intravenosas , Modelos Biológicos , Arteria Pulmonar/diagnóstico por imagen , Arteria Pulmonar/fisiopatología , Oveja Doméstica , Citrato de Sildenafil/administración & dosificación , Citrato de Sildenafil/sangre , Resistencia Vascular/efectos de los fármacos , Vasodilatadores/administración & dosificación , Vasodilatadores/sangreRESUMEN
OBJECTIVES: Contrast-enhanced ultrasound (CEUS) can provide quantitative perfusion metrics and may be useful to detect cerebral pathology in neonates and premature infants, particularly in extrauterine environments. The effect of hemodynamics on cerebral perfusion metrics is unknown, which limits the clinical application of this technology. We aimed to determine associations between systemic hemodynamics and concurrently measured brain perfusion parameters in an animal model of extrauterine support. METHODS: Nine fetal lambs were transferred to an extrauterine support device. Lumason® ultrasound contrast (0.1-0.3 ml) was administered via the umbilical vein and 90-second cine clips were obtained. Time-intensity-curves (TICs) were generated and time-dependent and area-under-curve (AUC) parameters were derived. Associations between brain perfusion metrics and hemodynamics including heart rate (HR) and mean arterial pressure (MAP) were evaluated by multilevel linear mixed-effects models. RESULTS: Eighty-six ultrasound examinations were performed and 72 examinations were quantifiable. Time-dependent measurements were independent of all hemodynamic parameters (all p ≥.05). Oxygen delivery and mean blood flow were correlated with AUC measurements (all p ≤.01). Physiologic HR and MAP were not correlated with any measurements (all p ≥.05). CONCLUSION: Detected aberrations in time-dependent CEUS measurements are not correlated with hemodynamic parameters and are thought to reflect the changes in cerebral blood flow, thus providing a promising tool for evaluation of brain perfusion. CEUS brain perfusion parameters are not correlated with physiologic HR and MAP, but AUC-dependent measurements are correlated with oxygen delivery and blood flow, suggesting that CEUS offers additional value over standard monitoring. Overall, these findings enhance the applicability of this technology.
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Benchmarking , Hemodinámica , Animales , Encéfalo/diagnóstico por imagen , Medios de Contraste , Modelos Animales de Enfermedad , Humanos , Perfusión , Ovinos , UltrasonografíaRESUMEN
BACKGROUND: Minimally invasive fetal surgery, or fetoscopy, is an alternative to open fetal surgery to repair common birth defects like myelomeningocele. Although this hysterotomy-sparing approach reduces maternal morbidity, the effects of in utero insufflation on the fetus are poorly understood. Our purpose was to determine the optimal fetal insufflation conditions. METHODS: Fetal sheep at gestational age 104 to 107 days were studied under insufflation conditions in utero and ex utero. The ex utero fetuses were cannulated via their umbilical vessels into a support device, the EXTra-uterine Environment for Neonatal Development (EXTEND). EXTEND fetuses were exposed to four different insufflation conditions for four hours: untreated carbon dioxide (CO2) (n=5), warm humidified (whCO2) (n=4), whCO2 with the umbilical cord exposed (n=3), and whCO2 without amniotic fluid (skin and cord exposed) (n=3). RESULTS: In utero insufflation led to significant increases in fetal CO2 and reductions in fetal pH. Ex utero insufflation with whCO2 did not lead to changes in fetal blood gas measurements or cerebral perfusion parameters. Insufflation with whCO2 with an exposed umbilical cord led to reduced umbilical blood flow. CONCLUSIONS: Insufflation with warm humidified CO2 with an amniotic fluid covered umbilical cord is well tolerated by the fetus without significant changes in hemodynamics or cerebral perfusion parameters. TYPE OF STUDY: Basic science LEVEL OF EVIDENCE: N/A.
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Enfermedades Fetales , Fetoscopía , Insuflación , Meningomielocele , Animales , Dióxido de Carbono/administración & dosificación , Femenino , Enfermedades Fetales/cirugía , Feto/cirugía , Meningomielocele/cirugía , Embarazo , OvinosRESUMEN
BACKGROUND AND AIMS: The Extra-Uterine Environment for Neonatal Development (EXTEND) aims to avoid the complications of prematurity, such as NEC. Our goal was to determine if bowel development occurs normally in EXTEND-supported lambs, with specific emphasis on markers of immaturity associated with NEC. METHODS: We compared terminal ileum from 17 pre-term lambs supported on EXTEND for 2- 4 weeks to bowel from age-matched fetal lambs that developed in utero. We evaluated morphology, markers of epithelial integrity and maturation, enteric nervous system structure, and bowel motility. RESULTS: EXTEND-supported lamb ileum had normal villus height, crypt depth, density of mucin-containing goblet cells, and enteric neuron density. Expression patterns for I-FABP, activated caspase-3 and EGFR were normal in bowel epithelium. Transmural resistance assessed in Ussing chambers was normal. Bowel motility was also normal as assessed by ex vivo organ bath and video imaging. However, Peyer's patch organization did not occur normally in EXTEND ileum, resulting in fewer circulating B cells in experimental animals. CONCLUSION: EXTEND supports normal ileal epithelial and enteric nervous system maturation in pre-term lambs. The classic morphologic changes and cellular expression profiles associated with NEC are not seen. However, immune development within the EXTEND supported lamb bowel does not progress normally.
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Enterocolitis Necrotizante/prevención & control , Oxigenación por Membrana Extracorpórea/métodos , Madurez de los Órganos Fetales/inmunología , Íleon/embriología , Nacimiento Prematuro/terapia , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/inmunología , Femenino , Feto/inmunología , Humanos , Íleon/inmunología , Recién Nacido , Mucosa Intestinal/embriología , Mucosa Intestinal/inmunología , Nacimiento Prematuro/inmunología , Ovinos , Cordón Umbilical/irrigación sanguíneaAsunto(s)
COVID-19 , Cateterismo , Internado y Residencia , Grupo de Atención al Paciente , Atención al Paciente/métodos , Recursos Humanos/organización & administración , COVID-19/epidemiología , Cateterismo/instrumentación , Cateterismo/métodos , Epidemias , Humanos , Grupo de Atención al Paciente/organización & administración , Diálisis Renal/métodos , SARS-CoV-2RESUMEN
In utero hematopoietic cell transplantation (IUHCT) has the potential to cure congenital hematologic disorders including sickle cell disease. However, the window of opportunity for IUHCT closes with the acquisition of T-cell immunity, beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Here we report that regulatory T cells can promote alloengraftment and preserve allograft tolerance after the acquisition of T-cell immunity in a mouse model of late-gestation IUHCT. We show that allografts enriched with regulatory T cells harvested from either IUHCT-tolerant or naive mice engraft at 20 days post coitum (DPC) with equal frequency to unenriched allografts transplanted at 14 DPC. Long-term, multilineage donor cell chimerism was achieved in the absence of graft-versus-host disease or mortality. Decreased alloreactivity among recipient T cells was observed consistent with donor-specific tolerance. These findings suggest that donor graft enrichment with regulatory T cells could be used to successfully perform IUHCT later in gestation.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Femenino , Ratones , Embarazo , Linfocitos T Reguladores , Quimera por Trasplante , Acondicionamiento PretrasplanteRESUMEN
BACKGROUND: Children with congenital adrenal hyperplasia (CAH) because of 21 hydroxylase deficiency (21OHD) are at risk for early or precocious puberty and a short adult height compared to population means and midparental height. The effect of histrelin in suppressing puberty and improving growth in these children has not been reported. METHODS: Retrospective cohort analysis of all patients (age ≤ 20) at our institution who underwent histrelin implantation between 2008 and 2017. Treated patients with CAH (classic and nonclassic forms of 21OHD) were identified and their growth data analyzed. RESULTS: Fifteen children with CAH were treated with histrelin for a median of 3 years (range 2-5; age at first implantation 7.7 ± 1.5 years). Bone age (BA) to chronologic age (CA) decreased from 1.57 ± 0.4 to 1.25 ± 0.25 (P < .01), while predicted adult height (PAH) increased by 7.1 ± 6.6 cm (P < .01). A subgroup of 10 children reached adult height. Similar changes in BA/CA and PAH were observed with therapy (P = .02). Adult height z improved compared to pretreatment PAH z (-1.42 ± 0.9 vs. -1.96 ± 1.1 respectively, P < .01), but remained lower than midparental height z (P = .01). CONCLUSION: In this retrospective cohort study of children with CAH due to 21OHD and early or precocious puberty, histrelin implantation resulted in a decrease in BA progression compared to CA and an improvement in PAH. In the subgroup who completed growth, adult height remained significantly lower than midparental. These results need to be confirmed with prospective controlled studies.
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Extracorporeal membrane oxygenation is a life-saving intervention, but bleeding complications are frequent. Given that the combination of platelet loss and dysfunction is a major contributor to this acquired bleeding diathesis, efforts to combat these phenomena are of great clinical importance. In this study, we investigated the effects of nitric oxide (NO) added to the sweep gas of an extracorporeal circuit in a neonatal ovine model. Eight lambs (age 9.6 ± 1.9 days) were cannulated via the neck vessels and maintained on a pumpless arteriovenous extracorporeal membrane oxygenation circuit with blood flow restricted to 100 ml/min for 72 hours. All animals were heparinized, and a subset (n = 4) also received NO in the sweep gas at a concentration of 200 ppm. We observed no adverse effects from NO administration, and methemoglobin levels remained unchanged. Platelet counts significantly declined in all animals over the course of the study; however, mean counts were higher in the NO-treated group, and this difference was statistically significant at 24 hours (62 ± 3% vs. 32 ± 7% of baseline, P < 0.01). Likewise, mean plasma levels of beta-thromboglobulin, a marker of platelet activation, were lower in the NO-treated group, and this difference was also significant at the 24 hour time point (9.5 ± 2.2 vs. 19.7 ± 6.5 pg/mL/10 platelets, P < 0.05). We conclude that 200 ppm NO can be safely blended into the oxygenator sweep gas of a low-flow extracorporeal circuit and that it may transiently attenuate platelet consumption and activation.
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Plaquetas/efectos de los fármacos , Oxigenación por Membrana Extracorpórea/métodos , Óxido Nítrico/farmacología , Activación Plaquetaria/efectos de los fármacos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Recuento de Plaquetas , OvinosRESUMEN
Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.
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Sistemas CRISPR-Cas/genética , Enfermedades Pulmonares/genética , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Edición Génica/métodos , Humanos , Ratones , Mutación/genética , Proteína C Asociada a Surfactante Pulmonar/genéticaRESUMEN
BACKGROUND: In an effort to mitigate the major morbidities and mortality associated with extreme prematurity, we have developed an EXTrauterine Environment for Neonatal Development (EXTEND) designed to provide physiologic support of extremely premature infants. OBJECTIVES: We have previously shown that long-term, physiologic support of premature fetal lambs is possible with EXTEND, but in this study, we sought to demonstrate bioenergetic equipoise at the tissue level. METHODS: Four premature fetal lambs were delivered by hysterotomy at gestational ages (GA) of 105-107 days (term â¼145 days), cannulated via the umbilical vessels, and transitioned to support on EXTEND for 3-4 weeks. Five control fetuses were age-matched to the GA of experimental fetuses at the time of study end (128-134 days GA) and immediately sacrificed after hysterotomy. Mitochondria were isolated from the heart, liver, kidney, and skeletal muscle of fetuses at the time of sacrifice, and oxygen consumption rates (OCRs) were measured. RESULTS: There were no differences in basal mitochondrial OCR between EXTEND and control fetuses for heart, kidney, or skeletal muscle. For liver, the basal OCR was higher in EXTEND fetuses compared to controls. There were no differences in physiologic maximal OCR or reserve capacity for any tissue analyzed. CONCLUSIONS: Fetal lambs supported by EXTEND demonstrate physiologic mitochondrial function as evidenced by adequate basal and physiologic maximal cellular respiration as well as preserved reserve capacity.
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Órganos Artificiales , Metabolismo Energético , Oxigenación por Membrana Extracorpórea , Mitocondrias/metabolismo , Nacimiento Prematuro/terapia , 8-Hidroxi-2'-Desoxicoguanosina/sangre , Animales , Animales Recién Nacidos , Bilirrubina/sangre , Biomarcadores/sangre , Respiración de la Célula , Oxigenación por Membrana Extracorpórea/instrumentación , Femenino , Monitoreo Fetal , Edad Gestacional , Consumo de Oxígeno , Oxigenadores de Membrana , Embarazo , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/fisiopatología , Oveja Doméstica , Factores de TiempoRESUMEN
In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.
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Terapias Fetales/métodos , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Animales , Femenino , Feto , Ratones , Modelos Animales , EmbarazoRESUMEN
In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.
Asunto(s)
Terapia Genética , Oxidorreductasas/genética , Proproteína Convertasa 9/genética , Tirosinemias/terapia , Animales , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Edición Génica , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Oxidorreductasas/uso terapéutico , Proproteína Convertasa 9/uso terapéutico , Tirosinemias/genética , Tirosinemias/patologíaRESUMEN
BACKGROUND: Aggressive fluid resuscitative strategies have been the cornerstone of early trauma management for decades. However, recent prospective adult studies have challenged this practice, underlining the detrimental effect of positive fluid balance on cardiopulmonary function. Fluid overload has been associated with impaired oxygenation and morbidity in critically ill adults, but data is lacking in pediatric trauma patients. METHODS: We completed a retrospective chart review of all pediatric trauma patients 0-18â¯years old admitted to a level 1 trauma center from January 2013 to December 2015. Four patient cohorts were established based on volume of fluid administered: <20â¯ml/kg/day, 20-40â¯ml/kg/day, 40-60â¯ml/kg/day, andâ¯>â¯60â¯ml/kg/day. The primary outcome was death. Secondary outcomes included the number of days on the ventilator, intensive care unit length of stay (ICU LOS), overall length of stay (LOS), number of days nil per os (NPO) as an indicator of ileus, and incidence of bloodstream infection and/or surgical site infection. RESULTS: The mean volume of fluid administered over the first 24â¯h was 41â¯ml/kg/day, and 28â¯ml/kg/day over the first 48â¯h. ICU length of stay and overall length of stay were increased in patients who received more than 60â¯ml/kg/day in the first 24â¯h of their hospitalization. Furthermore, ventilator use, ICU length of stay, overall length of stay, and time to resumption of a regular diet were all increased in patients who received >60â¯ml/kg/day over 48â¯h. CONCLUSIONS: Early administration of high volumes of crystalloid fluid greater than 60â¯ml/kg/day significantly correlates with pulmonary complications, days NPO, and hospital length of stay. These results span the first 48â¯h of a patient's hospital stay and should encourage surgical care providers to exercise judicious use of crystalloid fluid administration in the trauma bay, ICU, and floor. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: Level III.