RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Femenino , Ratones , Embarazo , Linfocitos T Reguladores , Quimera por Trasplante , Acondicionamiento PretrasplanteRESUMEN
OBJECTIVE: Neuroimmune cells, particularly microglia and astrocytes, play a critical role in neurodevelopment. Neurocognitive delays are common in children with congenital heart disease, but their etiology is poorly understood. Our objective was to determine whether prenatal hypoxemia, at levels common in congenital heart disease, induced neuroimmune activation to better understand the origins of neurobehavioral disorders in congenital heart disease. METHODS: Eight fetal sheep at gestational age 109 ± 3 days (term â¼145 days) were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid environment for 22 ± 2 days under normoxic (n = 4) or hypoxic (n = 4) conditions. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were stained with ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein antibodies to quantify microglia and astrocytes, respectively, in gray and white matter in frontotemporal and cerebellar sections. Microglia were classified into 4 morphologic types based on cell shape. Data were analyzed with 1-way analysis of variance or Fisher exact test, as appropriate. RESULTS: Oxygen delivery was significantly reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min; P < .01). Rates of apoptosis were similar in hypoxic, normoxic, and intrauterine control animals in all examined areas. There were also no differences between groups in area occupied by glial fibrillary acidic protein-labeled astrocytes or ionized calcium-binding adaptor molecule 1-labeled microglia in all examined areas. However, round microglia were significantly increased in hypoxic animals compared with normoxic animals (33% vs 6%; P < .01) and control animals (33% vs 11%; P < .01). CONCLUSIONS: Prenatal hypoxemia altered microglial morphology without significant gliosis. Additional studies characterizing these mechanisms may provide insight into the origins of neurobehavioral disabilities in children with congenital heart disease.
RESUMEN
OBJECTIVES: To determine which patients with congenital diaphragmatic hernia (CDH) and pulmonary hypertension (PH) benefit from inhaled nitric oxide (iNO) treatment by comparing characteristics and outcomes of iNO responders to nonresponders. STUDY DESIGN: We performed a retrospective chart review of infants with CDH treated at our center between 2011 and 2016. In a subset of patients, iNO was initiated for hypoxemia or echocardiographic evidence of extrapulmonary right to left shunting. Initial post-treatment blood gases were reviewed, and patients were classified as responders (increased PaO2 >20 mm Hg) or nonresponders. Baseline characteristics, echocardiograms and outcomes were compared between groups with Fisher exact tests and Mann-Whitney t tests, as appropriate. RESULTS: During the study period, 95 of 131 patients with CDH (73%) were treated with iNO. All patients with pretreatment echocardiograms (n = 90) had echocardiographic evidence of PH. Thirty-eight (40%) patients met treatment response criteria. Responders had significant improvements in PaO2 (51 ± 3 vs 123 ± 7 mm Hg, P < .01), alveolar-arterial gradient (422 ± 30 vs 327 ± 27 mm Hg, P < .01), and PaO2 to FiO2 ratio (82 ± 10 vs 199 ± 15 mm Hg, P < .01). Nonresponders were more likely to have left ventricular systolic dysfunction (27% vs 8%, P = .03) on echocardiogram. Responders were less likely to require extracorporeal membrane support (50 vs 24%, P = .02). CONCLUSIONS: iNO treatment is associated with improved oxygenation and reduced need for ECMO in a subpopulation of patients with CDH with PH and normal left ventricular systolic function.
Asunto(s)
Hernias Diafragmáticas Congénitas/tratamiento farmacológico , Hipertensión Pulmonar/tratamiento farmacológico , Óxido Nítrico/administración & dosificación , Oxígeno/metabolismo , Administración por Inhalación , Femenino , Hernias Diafragmáticas Congénitas/complicaciones , Humanos , Hipertensión Pulmonar/complicaciones , Lactante , Masculino , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.
Asunto(s)
Comunicación Autocrina , Ingeniería Celular , Inhibidores Enzimáticos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Nanopartículas/química , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: We tested the hypothesis that chronic fetal hypoxia, at a severity present in many types of congenital heart disease, would lead to abnormal neurodevelopment. METHODS: Eight mid-gestation fetal sheep were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid-filled environment for 22 ± 2 days under normoxic or hypoxic conditions. Total parenteral nutrition was provided. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were fixed for histopathology. Neurons were quantified in white matter tracts, and the thickness of the external granular layer of the cerebellum was measured to assess neuronal migration. Capillary density and myelination were quantified in white matter. Data were analyzed with unpaired Student t tests or 1-way analysis of variance, as appropriate. RESULTS: Oxygen delivery was reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min, P < .01), but umbilical blood flow and caloric delivery were not different between the 2 groups. Compared with normoxic and control animals, hypoxic fetuses had reduced neuronal density and increased external granular layer thickness. Compared with normoxic and control animals, hypoxic fetuses had increased capillary density in white matter. Cortical myelin integrity score was lower in the hypoxic group compared with normoxic and control animals. There was a significant negative correlation between myelin integrity and capillary density. CONCLUSIONS: Chronic fetal hypoxia leads to white matter hyper-vascularity, decreased neuronal density, and impaired myelination, similar to the neuropathologic findings observed in children with congenital heart disease. These findings support the hypothesis that fetal hypoxia, even in the setting of normal caloric delivery, impairs neurodevelopment.
Asunto(s)
Encefalopatías/fisiopatología , Encéfalo/crecimiento & desarrollo , Capilares/fisiopatología , Hipoxia Fetal/fisiopatología , Neovascularización Fisiológica , Neurogénesis , Neuronas , Animales , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Encefalopatías/sangre , Encefalopatías/patología , Capilares/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Sangre Fetal/metabolismo , Desarrollo Fetal , Hipoxia Fetal/sangre , Hipoxia Fetal/patología , Edad Gestacional , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Neuronas/patología , Oxígeno/sangre , Embarazo , Oveja DomésticaRESUMEN
OBJECTIVE: To evaluate the effect of continuous treprostinil in infants with severe pulmonary hypertension associated with congenital diaphragmatic hernia (CDH) on specific markers of pulmonary hypertension severity and to report the safety and tolerability of treprostinil. STUDY DESIGN: We conducted a retrospective cohort study of infants with CDH-associated pulmonary hypertension treated with treprostinil from January 2011 to September 2016. Severity of pulmonary hypertension was assessed by echocardiogram and serum B-type natriuretic peptide (BNP) by using time points before initiation and 24 hours, 1 week, and 1 month after treprostinil initiation. Fisher exact tests, Wilcoxon-rank sum tests, and mixed-effects models were used for analysis. RESULTS: Seventeen patients were treated with treprostinil for a median of 54.5 days (IQR 44.3-110 days). Compared with the concurrent CDH population (n = 147), infants treated with treprostinil were more likely to require extracorporeal support (76.5% vs 25.2%, P < .0001), to have a longer hospital stay (144 vs 60 days, P < .0001), and to need longer mechanical ventilator support (76.5 vs 30.9 days, P < .0001). Following treprostinil initiation, there was a significant reduction in BNP at 1 week (1439 vs 393 pg/mL, P < .01) and 1 month (1439 vs 242 pg/mL, P = .01). Severity of pulmonary hypertension by echocardiogram improved at 1 month (OR 0.14, CI 95% 0.04-0.48, P = .002). Despite these improvements, overall mortality remained high (35%). There were no adverse events related to treprostinil, including no hypotension, hypoxia, or thrombocytopenia. CONCLUSIONS: In this cohort, treprostinil use was associated with improved severity of pulmonary hypertension assessed by echocardiogram and decreased BNP, with no significant side effects.
Asunto(s)
Epoprostenol/análogos & derivados , Hernias Diafragmáticas Congénitas/complicaciones , Hipertensión Pulmonar/tratamiento farmacológico , Presión Esfenoidal Pulmonar/efectos de los fármacos , Sistema de Registros , Antihipertensivos/administración & dosificación , Relación Dosis-Respuesta a Droga , Epoprostenol/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Lactante , Recién Nacido , Masculino , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
OBJECTIVES: The purpose of this study was to assess the need and timing of extracorporeal membrane oxygenation in relation to congenital diaphragmatic hernia repair as modifiers of short-term neurodevelopmental outcomes. DESIGN: Retrospective study. SETTING: A specialized tertiary care center. PATIENTS: Between June 2004 and February 2016, a total of 212 congenital diaphragmatic hernia survivors enrolled in our follow-up program. Neurodevelopmental outcome was assessed at a median age of 22 months (range, 5-37) using the Bayley Scales of Infant Development, third edition. Fifty patients (24%) required extracorporeal membrane oxygenation support. Four patients (8%) were repaired prior to cannulation, 25 (50%) were repaired on extracorporeal membrane oxygenation, and 21 (42%) were repaired after decannulation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Children with congenital diaphragmatic hernia, who required extracorporeal membrane oxygenation scored on average 4.6 points lower on cognitive composite (p = 0.031) and 9.2 points lower on the motor composite (p < 0.001). Language scores were similar between groups. Mean scores for children with congenital diaphragmatic hernia repaired on extracorporeal membrane oxygenation were significantly lower for cognition (p = 0.021) and motor (p = 0.0005) outcome. Language scores were also lower, but did not reach significance. A total of 40% of children repaired on extracorporeal membrane oxygenation scored below average in all composites, whereas only 9% of the non-extracorporeal membrane oxygenation, 4% of the repaired post-extracorporeal membrane oxygenation, and 25% of the repaired pre-extracorporeal membrane oxygenation patients scored below average across all domains. Only 20% of congenital diaphragmatic hernia survivors repaired on extracorporeal membrane oxygenation support scored within the average range for all composite domains. Duration of extracorporeal membrane oxygenation support was not associated with a higher likelihood of adverse cognitive (p = 0.641), language (p = 0.147), or motor (p = 0.720) outcome. CONCLUSIONS: Need for extracorporeal membrane oxygenation in congenital diaphragmatic hernia survivors is associated with worse neurocognitive and neuromotor outcome. Need for congenital diaphragmatic hernia repair while on extracorporeal membrane oxygenation is associated with deficits in multiple domains. Overall time on extracorporeal membrane oxygenation did not impact neurodevelopmental outcome.
Asunto(s)
Desarrollo Infantil , Oxigenación por Membrana Extracorpórea/efectos adversos , Hernias Diafragmáticas Congénitas/complicaciones , Herniorrafia/efectos adversos , Trastornos del Neurodesarrollo/etiología , Preescolar , Oxigenación por Membrana Extracorpórea/métodos , Femenino , Estudios de Seguimiento , Hernias Diafragmáticas Congénitas/terapia , Humanos , Lactante , Recién Nacido , Masculino , Trastornos del Neurodesarrollo/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Centros de Atención Terciaria , Factores de TiempoRESUMEN
PURPOSE: Right-sided congenital diaphragmatic hernia (R-CDH) occurs in up to 25% of all CDH cases, but has been less widely studied compared to left-sided defects. We sought to compare characteristics and outcomes of left- versus right-sided defects in a large cohort of CDH patients who underwent standardized treatment and follow-up at a single institution. METHODS: We retrospectively reviewed charts of all CDH patients in our pulmonary hypoplasia program from January 2002 through December 2014. Categorical variables were analyzed by Fisher's exact test and continuous variables by Mann-Whitney t-test (p≤0.05). RESULTS: A total of 330 CDH patients were treated more than the 12-year study period, with 274 (83%) left-sided and 56 (17%) right-sided cases identified. Specific pulmonary morbidities were associated with R-CDH, with increased duration of nitric oxide therapy, increased requirement for tracheostomy, increased requirement for supplemental oxygen at the time of NICU discharge, and increased chronic pulmonary hypertension with requirement for long-term Sildenafil therapy. CONCLUSIONS: In this series, R-CDH was not associated with increased mortality, but was associated with increased requirement for pulmonary vasodilatory therapy and requirement for tracheostomy. The high incidence of pulmonary complications indicates increased severity of pulmonary hypoplasia in R-CDH, supporting a role for delivery in tertiary centers with expertise in CDH management.
Asunto(s)
Predicción , Hernias Diafragmáticas Congénitas/complicaciones , Herniorrafia/métodos , Hipertensión Pulmonar/complicaciones , Femenino , Hernias Diafragmáticas Congénitas/diagnóstico , Hernias Diafragmáticas Congénitas/cirugía , Humanos , Hipertensión Pulmonar/epidemiología , Incidencia , Recién Nacido , Masculino , Estudios Retrospectivos , Estados Unidos/epidemiologíaRESUMEN
PURPOSE: Successful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery. METHODS: Six different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR). RESULTS: Fluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification. CONCLUSION: Adeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.
Asunto(s)
Dependovirus/clasificación , Terapia Genética/métodos , Vectores Genéticos/clasificación , Enfermedades Pulmonares/terapia , Pulmón , Serogrupo , Transducción Genética , Animales , Biomarcadores/metabolismo , Dependovirus/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/metabolismo , Pulmón/virología , Enfermedades Pulmonares/congénito , Enfermedades Pulmonares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Serotipificación , Ovinos , Oveja Doméstica , TransgenesAsunto(s)
Suministros de Energía Eléctrica/efectos adversos , Esófago/lesiones , Cuerpos Extraños/etiología , Mediastinitis/etiología , Traumatismos Torácicos/etiología , Antibacterianos/uso terapéutico , Preescolar , Esofagoscopía , Esófago/patología , Femenino , Cuerpos Extraños/diagnóstico , Cuerpos Extraños/cirugía , Humanos , Imagen por Resonancia Magnética , Mediastinitis/diagnóstico por imagenRESUMEN
PURPOSE: Hydrops and pulmonary hypoplasia are associated with significant morbidity and mortality in the setting of a congenital lung lesion or pleural effusion (PE). We reviewed our experience using in utero thoracoamniotic shunts (TA) to manage fetuses with these diagnoses. METHODS: A retrospective review of fetuses diagnosed with a congenital lung lesion or pleural effusion who underwent TA shunt placement from 1998-2013 was performed. RESULTS: Ninety-seven shunts were placed in 75 fetuses. Average gestational age (±SD) at shunt placement and birth was 25±3 and 34±5 weeks. Shunt placement resulted in a 55±21% decrease in macrocystic lung lesion volume and complete or partial drainage of the PE in 29% and 71% of fetuses. 69% of fetuses presented with hydrops, which resolved following shunt placement in 83%. Survival was 68%, which correlated with GA at birth, % reduction in lesion size, unilateral pleural effusions, and hydrops resolution. Surviving infants had prolonged NICU courses and often required either surgical resection or tube thoracostomy in the perinatal period. CONCLUSION: TA shunts provide a therapeutic option for select fetuses with large macrocystic lung lesions or PEs at risk for hydrops and/or pulmonary hypoplasia. Survival following shunting depends on GA at birth, reduction in mass size, and hydrops resolution.