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There remains high morbidity and mortality with mechanical circulatory support (MCS) in failing bidirectional Glenn (BDG) physiology. We performed a retrospective analysis of children with BDG physiology supported with MCS before and after 2018. Fourteen patients met inclusion criteria (median age 1.5 years, weight 9 kg). Prior to 2018 (n = 7), with variable anticoagulation and strategies including pulsatile VAD, continuous flow VAD, and extracorporeal membrane oxygenation (ECMO), 3 (43%) of patients were transplanted with a total of 536 patient-days of support (median 59 days). Major hemocompatability-related adverse event (MHRAE) rate was 63 per 100 patient-months. After 2018 (n = 7), using a staged support strategy (ECMO to pulsatile VAD) and bivalirudin anticoagulation, 5 (71%) patients were transplanted with a total of 1260 patient-days of support (median 188 days) and MHRAE rate of 24 per 100 patient-months. Despite challenging physiology, we have observed improved survival and reduced MHRAE despite longer support duration.
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BACKGROUND: Pediatric heart transplant (HT) candidates experience high waitlist mortality due to a limited donor pool that is constrained in part by anti-HLA sensitization. We evaluated the impact of CDC and Flow donor-specific crossmatch (XM) results on pediatric HT outcomes. METHODS: All pediatric HTs between 1999 and 2019 in the OPTN database were included. Donor-specific XM results were sub-categorized based on CDC and Flow results. Primary outcomes were treated rejection in the first year and time to death or allograft loss. Propensity scores were utilized to adjust for differences in baseline characteristics. RESULTS: A total of 4,695 pediatric HT patients with T-cell XM data were included. After propensity score adjustment, a positive T-cell CDC-XM was associated with 2 times higher odds of treated rejection (OR 2.29 (1.56, 3.37)) and shorter time to death/allograft loss (HR 1.50 (1.19, 1.88)) compared to a negative Flow-XM. HT recipients who were Flow-XM positive with negative/unknown CDC-XM did not have higher odds of rejection or shorter time to death/allograft loss. An isolated positive B-cell XM was also not associated with worse outcomes. Over the study period XM testing shifted from CDC- to Flow-based assays. CONCLUSIONS: A positive donor-specific T-cell CDC-XM was associated with rejection and death/allograft loss following pediatric HT. This association was not observed with a positive T-cell Flow-XM or B-cell XM result alone. The shift away from performing the CDC-XM may result in loss of important prognostic information unless the clinical relevance of quantitative Flow-XM results on heart transplant outcomes is systematically studied.
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Rejeição de Enxerto , Sobrevivência de Enxerto , Transplante de Coração , Humanos , Criança , Masculino , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/epidemiologia , Pré-Escolar , Estudos Retrospectivos , Teste de Histocompatibilidade , Adolescente , Lactente , Doadores de TecidosRESUMO
BACKGROUND: The use of apixaban in the pediatric cardiac population is expanding. We describe our apixaban dosing and monitoring strategy in children and young adults awaiting heart transplantation, along with outcomes related to bleeding and thrombosis during wait-list and early post-transplant periods. METHODS: This study is a retrospective, single-center analysis of all patients receiving apixaban while awaiting cardiac transplantation. Weight-based dosing was monitored with peak drug-specific anti-Xa chromogenic analysis. Significant post-operative bleeding defined by chest tube output or need for surgical intervention. RESULTS: From September 2020 to December 2022, 19 patients, median age 13.5 years (6.1, 15.8 years), weighing 48.9 kg (15.4, 67.6) received apixaban while awaiting transplant. Indication for apixaban was prophylaxis (n = 18, 3 with ventricular assist devices) and treatment of thrombus (n = 1). There were no clinically relevant non-major or major bleeding, nor thrombotic events while awaiting transplant. The median time from last apixaban dose to arrival in the operating room was 23.2 h (15.6-33.8), with median random apixaban level of 37 ng/mL (28.3, 59), 6.3 h (4.8, 8.4) prior to arrival in the operating room. In this study, 32% of patients had significant post-operative bleeding based on chest tube output post-transplant or need for intervention. No patients meeting criteria for significant post-operative bleeding were thought to be attributable to apixaban. CONCLUSIONS: Careful use of apixaban can be safe and effective while awaiting heart transplant. There was no appreciable increase in peri-operative bleeding. The use of apixaban is promising in providing safe, predictable and efficacious anticoagulation while avoiding additional patient stressors.
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Inibidores do Fator Xa , Transplante de Coração , Pirazóis , Piridonas , Criança , Adulto Jovem , Humanos , Adolescente , Estudos Retrospectivos , Inibidores do Fator Xa/uso terapêutico , Inibidores do Fator Xa/análise , Hemorragia/epidemiologia , Anticoagulantes/uso terapêuticoRESUMO
Despite extracorporeal membrane oxygenation (ECMO) utilization in nearly 20% of cases, there are limited data in children with acute fulminant myocarditis (AFM) requiring ECMO. Herein we identify risk factors for death or heart transplant (HT) in children with AFM supported with ECMO, describe our experience with left atrial (LA) decompression, and depict long-term outcomes of survivors. We performed a retrospective cohort of patients <18 years with AFM (≤14 days of symptoms, rapid cardiogenic shock, and normal left ventricular [LV] size on presentation) supported with ECMO admitted to a single intensive care unit from 1997 to 2021. Among 28 patients (median age 9 years), 21 (75%) survived to discharge without HT. Patients were supported on ECMO for a median of 6 days. Three patients were bridged to HT with durable ventricular assist devices (VAD). Four patients died, two of whom were supported with VAD. At presentation, seven (25%) patients had high grade or complete atrioventricular block and eight (29%) had ventricular tachycardia. Before ECMO cannulation, 21 (75%) patients received CPR. The death/HT group had higher peak troponin levels (12.5 vs . 1.0 ng/ml, p = 0.02) and initial mean LA or pulmonary capillary wedge pressure (27 vs . 18 mm Hg, p = 0.03). Left atrial decompression was performed in 22 patients (79%). Twenty-two (79%) had acute myocarditis on endomyocardial biopsy. Among transplant-free survivors, 18 (86%) had normalization in LV function (median 7 days); the remaining three patients had persistent mild LV dysfunction at last follow-up (median 842 days). Transplant-free survival of pediatric patients with AFM supported on ECMO was 75% and associated with lower initial LA pressure and lower peak troponin. Recovery in ventricular function among survivors was rapid and durable.
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Oxigenação por Membrana Extracorpórea , Miocardite , Humanos , Criança , Miocardite/complicações , Miocardite/terapia , Miocardite/diagnóstico , Oxigenação por Membrana Extracorpórea/efeitos adversos , Estudos Retrospectivos , Choque Cardiogênico/etiologia , Átrios do Coração , Cateterismo Cardíaco , Troponina , Resultado do TratamentoRESUMO
BACKGROUND: A positive crossmatch (+ XM) has traditionally been associated with adverse outcomes following pediatric heart transplantation. However, more recent studies suggest that favorable intermediate-term outcomes may be achieved despite a + XM. This study's hypothesis is that children with a + XM have similar long-term survival, but higher rate of complications such as rejection, coronary allograft vasculopathy (CAV), and infection, compared to patients with a negative (-) XM. METHODS: The Pediatric Heart Transplant Society Registry (PHTS) database was queried from 2010-2021 for all patients <18 years of age with a known XM. Baseline demographics were compared between + XM and - XM groups using appropriate parametric and non-parametric group comparisons. Cox Proportional Hazards Modeling was used to identify risk factors for post-transplant graft loss, rejection, and CAV. RESULTS: Of 4599 pediatric heart transplants during the study period, XM results were available for 3914 (85%), of which 373 (9.5%) had a + XM. Univariate analysis showed lower 10-year survival for patients with + XM (HR = 1.3, p = .04). Multivariate analyses revealed no significant difference in 10-year survival in the 2 groups; however, time to first rejection (p = .0001) remained significantly shorter in the + XM group. CONCLUSIONS: Pediatric patients transplanted across a + XM experience earlier rejection; however, after multivariate adjustment, + XM is not independently associated with intermediate-term graft loss. The risk of heart transplantation against a + XM must be balanced with the ongoing risk of waitlist mortality.
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PURPOSE: We sought to establish whether nucleated red blood cells (NRBCs) are predictive of disposition, morbidity, and mortality for pediatric patients presenting to the emergency department (ED). METHODS: A single-center retrospective cohort study examining all ED encounters from patients aged younger than 19 years between January 2016 and March 2020, during which a complete blood count was obtained. Univariate analysis and multivariable logistic regression were used to test the presence of NRBCs as an independent predictor of patient-related outcomes. RESULTS: The prevalence of NRBCs was 8.9% (4195/46,991 patient encounters). Patient with NRBCs were younger (median age 4.58 vs 8.23 years; P < 0.001). Those with NRBCs had higher rates of in-hospital mortality (30/2465 [1.22%] vs 65/21,741 [0.30%]; P < 0.001), sepsis (19% vs 12%; P < 0.001), shock (7% vs 4%; P < 0.001), and cardiopulmonary resuscitation (CPR) (0.62% vs 0.09%; P < 0.001). They were more likely to be admitted (59% vs 51%; P < 0.001), have longer median hospital length of stay {1.3 (interquartile range [IQR], 0.22-4.14) vs 0.8 days (IQR, 0.23-2.64); P < 0.001}, and median intensive care unit (ICU) length of stay (3.9 [IQR, 1.87-8.72] vs 2.6 days [IQR, 1.27-5.83]; P < 0.001). Multivariable regression revealed presence of NRBCs as an independent predictor for in-hospital mortality (adjusted odds ratio [aOR], 2.21; 95% confidence interval [CI], 1.38-3.53; P < 0.001), ICU admission (aOR, 1.30; 95% CI, 1.11-1.51; P < 0.001), CPR (aOR, 3.83; 95% CI, 2.33-6.30; P < 0.001), and 30-day return to the ED (aOR, 1.15; 95% CI, 1.15-1.26; P < 0.001). CONCLUSIONS: The presence of NRBCs is an independent predictor for mortality, including in-hospital mortality, ICU admission, CPR, and readmission within 30 days for children presenting to the ED.
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Eritroblastos , Unidades de Terapia Intensiva , Humanos , Criança , Idoso , Pré-Escolar , Estudos Retrospectivos , Mortalidade Hospitalar , Contagem de Células SanguíneasRESUMO
BACKGROUND: Direct oral anticoagulants use in pediatric cardiology is poorly defined. OBJECTIVE: We present the largest experience of apixaban use in children with heart disease, using weight- and level-based dosing. METHODS: Retrospective single-center analysis of cardiac patients ≤19 years treated with apixaban. Patients were evaluated for safety (clinically relevant non-major [CRNM] or major bleeding; thrombotic events) and effectiveness (thrombus improvement by imaging). Peak drug-specific anti-Xa chromogenic assay results ("apixaban levels") were analyzed. RESULTS: Over 3 years (5/2018-9/2021), 219 children, median age 6.8 years (0.3-19), median weight 20.8 kg (4.8-160) received apixaban, totaling 50,916 patient days. Of them, 172 (79%) warranted thromboprophylaxis and 47 (21%) thrombosis treatment (with 10 arterial, 19 venous, 15 intracardiac, and 3 pulmonary). The median initial peak apixaban level was 165 ng/mL (23-474; n = 125) in the prophylaxis subgroup and 153 ng/mL (30-450; n = 33) in the treatment subgroup; dosage was adjusted in response to levels in 25% of the patients. There were 4 bleeding safety events (3 CRNM; 1 major, hemoptysis complicating empyema); the serious bleeding event rate was 2.9 per 100 patient-years of apixaban. Minor bleeding events (42) were noted in 18 patients, with an additional 2 having leukopenia, 1 transaminitis, and 3 rashes. An improvement in thrombosis was seen in 95% of the treated patients with available follow-up imaging (37/39 patients). CONCLUSION: Apixaban use was feasible with a low rate of adverse events across a diverse pediatric cardiac population using commercially available tablets dosed to weight and adjusted based on peak apixaban levels.
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Cardiopatias , Trombose , Tromboembolia Venosa , Humanos , Criança , Anticoagulantes/efeitos adversos , Varfarina/efeitos adversos , Estudos Retrospectivos , Tromboembolia Venosa/tratamento farmacológico , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Piridonas/efeitos adversos , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Trombose/induzido quimicamente , Cardiopatias/complicações , Cardiopatias/diagnóstico , Inibidores do Fator Xa/efeitos adversosRESUMO
There is a growing population of pediatric and adult patients supported with the HeartMate 3 ventricular assist device (HM3 VAD) all of whom require anticoagulation. Apixaban is an anticoagulant requiring less testing than warfarin which has been shown to be effective in other indications. We report five pediatric and young adult patients managed on HM3 VAD with apixaban anticoagulation for 1589 days of VAD support between January 6, 2019 and January 7, 2022. The median age was 17 years and the weight was 69 kg. Four patients had congenital heart disease (2 single-ventricle Fontan circulation, and 2 biventricular circulations) and one had dilated cardiomyopathy. Apixaban was initiated at a median of 7 days postoperatively and doses were titrated based on peak apixaban-specific anti-Xa chromogenic analysis levels (goal 150-250 ng/ml). All patients received aspirin 81 mg daily. There was one major hemocompatibility-related event observed (outflow graft thrombus in the setting of medication nonadherence and chronic VAD infection); there was no major bleeding, death, or stroke. Three patients underwent heart transplantation and two remain on VAD support. In this limited series, apixaban paired with a level-based dosing regimen and low-dose aspirin provided safe and effective antithrombosis with only one hemocompatibility-related event related to medication non-adherence.
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Insuficiência Cardíaca , Coração Auxiliar , Adolescente , Criança , Humanos , Adulto Jovem , Anticoagulantes/efeitos adversos , Aspirina , Insuficiência Cardíaca/cirurgia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
PURPOSE: Cardiac disease results in significant morbidity and mortality in patients with muscular dystrophy (MD). Single centers have reported their ventricular assist device (VAD) experience in specific MDs and in limited numbers. This study sought to describe the outcomes associated with VAD therapy in an unselected population across multiple centers. METHODS: We examined outcomes of patients with MD and dilated cardiomyopathy implanted with a VAD at Advanced Cardiac Therapies Improving Outcomes Network (ACTION) centers from 9/2012 to 9/2020. RESULTS: A total of 19 VADs were implanted in 18 patients across 12 sites. The majority of patients had dystrophinopathy (66%) and the median age at implant was 17.2 years (range 11.7-29.5). Eleven patients were non-ambulatory (61%) and 6 (33%) were on respiratory support pre-VAD. Five (28%) patients were implanted as a bridge to transplant, 4 of whom survived to transplant. Of 13 patients implanted as bridge to decision or destination therapy, 77% were alive at 1 year and 69% at 2 years. The overall frequencies of positive outcome (transplanted or alive on device) at 1 year and 2 years were 84% and 78%, respectively. Two patients suffered a stroke, 2 developed sepsis, 1 required tracheostomy, and 1 experienced severe right heart failure requiring right-sided VAD. CONCLUSIONS: This study demonstrates the potential utility of VAD therapies in patients with muscular dystrophy. Further research is needed to further improve outcomes and better determine which patients may benefit most from VAD therapy in terms of survival and quality of life.
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Insuficiência Cardíaca , Coração Auxiliar , Distrofias Musculares , Humanos , Criança , Adulto Jovem , Adolescente , Adulto , Resultado do Tratamento , Qualidade de Vida , Insuficiência Cardíaca/cirurgia , Distrofias Musculares/terapia , Sistema de Registros , Estudos RetrospectivosRESUMO
There is significant uncertainty in describing prognosis and a lack of reliable entry criteria for palliative care studies in children with advanced heart disease (AHD). This study evaluates the utility of the surprise question-"Would you be surprised if this child died within the next year?"-to predict one-year mortality in children with AHD and assess its utility as entry criteria for future trials. This is a prospective cohort study of physicians and nurses caring for children (1 month-19 years) with AHD hospitalized ≥ 7 days. AHD was defined as single ventricle physiology, pulmonary vein stenosis or pulmonary hypertension, or any cardiac diagnosis with signs of advanced disease. Primary physicians were asked the surprise question and medical record review was performed. Forty-nine physicians responded to the surprise question for 152 patients. Physicians responded "No, I would not be surprised if this patient died" for 54 (36%) patients, 20 (37%) of whom died within 1 year, predicting one-year mortality with 77% sensitivity, 73% specificity, 37% positive predictive value, and 94% negative predictive value. Patients who received a "No" response had an increased 1-year risk of death (hazard ratio 7.25, p < 0.001). Physician years of experience, subspecialty, and self-rated competency were not associated with the accuracy of the surprise question. The surprise question offers promise as a bedside screening tool to identify children with AHD at high risk for mortality and help physicians identify patients who may benefit from palliative care and advance care planning discussions.
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Cardiopatias , Médicos , Criança , Humanos , Cuidados Paliativos , Estudos Prospectivos , PrognósticoRESUMO
Pneumatosis intestinalis (PI) is a rare complication after thoracic organ transplantation. There are several theories for explaining the pathophysiology of this disease. In this paper, we highlight three cases of PI in a single pediatric center, one after lung transplantation and two after heart transplantation. Although the presentations differed, all cases improved with non-surgical therapies. There are not many articles in the pediatric literature about post-transplantation PI, and there are still many questions regarding the incidence, etiology, and treatment for this disease.
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Transplante de Coração , Transplante de Pulmão , Pneumatose Cistoide Intestinal , Criança , Transplante de Coração/efeitos adversos , Humanos , Incidência , Transplante de Pulmão/efeitos adversos , Pneumatose Cistoide Intestinal/diagnóstico , Pneumatose Cistoide Intestinal/etiologia , Pneumatose Cistoide Intestinal/terapiaRESUMO
Cardiac disease has emerged as a leading cause of mortality in Duchenne muscular dystrophy in the current era. This survey sought to identify the diagnostic and therapeutic approach to DMD among pediatric cardiologists in Advanced Cardiac Therapies Improving Outcomes Network. Pediatric cardiology providers within ACTION (a multi-center pediatric heart failure learning network) were surveyed regarding their approaches to cardiac care in DMD. Thirty-one providers from 23 centers responded. Cardiac MRI and Holter monitoring are routinely obtained, but the frequency of use and indications for ordering these tests varied widely. Angiotensin converting enzyme inhibitor and aldosterone antagonist are generally initiated prior to onset of systolic dysfunction, while the indications for initiating beta-blocker therapy vary more widely. Seventeen (55%) providers report their center has placed an implantable cardioverter defibrillator in at least 1 DMD patient, while 11 providers (35%) would not place an ICD for primary prevention in a DMD patient. Twenty-three providers (74%) would consider placement of a ventricular assist device (VAD) as destination therapy (n = 23, 74%) and three providers (10%) would consider a VAD only as bridge to transplant. Five providers (16%) would not consider VAD at their institution. Cardiac diagnostic and therapeutic approaches vary among ACTION centers, with notable variation present regarding the use of advanced therapies (ICD and VAD). The network is currently working to harmonize medical practices and optimize clinical care in an era of rapidly evolving outcomes and cardiac/skeletal muscle therapies.
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Cardiomiopatias , Insuficiência Cardíaca , Distrofia Muscular de Duchenne , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Cardiomiopatias/etiologia , Criança , Coração , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/terapia , Humanos , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/terapiaRESUMO
We describe waiting times for pediatric heart transplant (HT) candidates after the 2016 revision to the US allocation policy. The OPTN database was queried for pediatric HT candidates listed between 7/2016 and 4/2019. Of the 1789 included candidates, 65% underwent HT, 14% died/deteriorated, 8% were removed for improvement, and 13% were still waiting at the end of follow-up. Most candidates were status 1A at HT (81%). Median wait times differ substantially by listing status, blood type, and recipient weight. The likelihood of HT was lower in candidates <25 kg and in those with blood type O; The <25 kg, blood type O subgroup experiences longer wait times and higher wait list mortality. For status 1A candidates, median wait times were 108 days (≤25 kg, blood type O), 80 days (≤25 kg, non-O), 47 days (>25 kg, O), and 24 days (>25 kg, non-O). We found that centers with more selective organ acceptance practices, based on a lower median Pediatric Heart Donor Assessment Tool (PH-DAT) score for completed transplants, experience longer status 1A wait times for their listed patients. These data can be used to counsel families and to select appropriate advanced heart failure therapies to support patients to transplant.
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Cardiopatias Congênitas , Transplante de Coração , Obtenção de Tecidos e Órgãos , Criança , Humanos , Políticas , Doadores de Tecidos , Estados Unidos , Listas de EsperaRESUMO
OBJECTIVE: To characterize viscoelastic testing profiles of children with multisystem inflammatory syndrome in children (MIS-C). METHODS: This single-center retrospective review included 30 patients diagnosed with MIS-C from March 1 to September 1, 2020. Thromboelastography (TEG) with platelet mapping was performed in 19 (63%) patients and compared to age- and sex-matched controls prior to cardiac surgery. Relationships between TEG parameters and inflammatory markers were assessed using correlation. RESULTS: Patients with MIS-C had abnormal TEG results compared to controls, including decreased kinetic (K) time (1.1 vs. 1.7 minutes, p < .01), increased alpha angle (75.0° vs. 65.7°, p < .01), increased maximum amplitude (70.8 vs. 58.3 mm, p < .01), and decreased lysis in 30 minutes (Ly30) (1.1% vs. 3.7%, p = .03); consistent with increased clot formation rate and strength, and reduced fibrinolysis. TEG maximum amplitude was moderately correlated with erythrocyte sedimentation rate (ESR) (r = 0.60, p = .02), initial platelet count (r = 0.67, p < .01), and peak platelet count (r = 0.51, p = .03). TEG alpha angle was moderately correlated with peak platelet count (r = 0.54, p = .02). Seventeen (57%) patients received aspirin (ASA) and anticoagulation, five (17%) received only ASA, and three (10%) received only anticoagulation. No patients had a symptomatic thrombotic event. Six (20%) patients had a bleeding event, none of which was major. CONCLUSIONS: Patients with MIS-C had evidence of hypercoagulability on TEG. Increased ESR and platelets were associated with higher clot strength. Patients were prophylactically treated with ASA or anticoagulation with no symptomatic thrombosis or major bleeding. Further multicenter study is required to characterize the rate of thrombosis and optimal thromboprophylaxis algorithm in this patient population.
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Coagulação Sanguínea , COVID-19/complicações , Síndrome de Resposta Inflamatória Sistêmica/sangue , Trombofilia/sangue , Adolescente , Anticoagulantes/uso terapêutico , Aspirina/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , COVID-19/sangue , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Estudos Retrospectivos , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Tromboelastografia , Trombofilia/tratamento farmacológico , Tratamento Farmacológico da COVID-19RESUMO
Survival of pediatric patients with heart failure has improved due to medical and surgical advances over the past decades. The complexity of pediatric heart transplant patients has increased as medical and surgical management for patients with congenital heart disease continues to improve. Quality of life in patients with heart failure and transplant might be affected by the impact on functional status that heart failure, heart failure complications or treatment might have. Functional areas affected might be motor, exercise capacity, feeding, speech and/or cognition. The goal of rehabilitation is to enhance and restore functional ability and quality of life to those with physical impairments or disabilities. Some of these rehabilitation interventions such as exercise training have been extensively evaluated in adults with heart failure. Literature in the pediatric population is limited yet promising. The use of additional rehabilitation interventions geared toward specific complications experienced by patients with heart failure or heart transplant are potentially helpful. The use of individualized multidisciplinary rehabilitation program that includes medical management, rehabilitation equipment and the use of physical, occupational, speech and feeding therapies can help improve the quality of life of patients with heart failure and transplant.
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Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs, and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs.
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Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endopeptidases/metabolismo , Epigênese Genética , Fatores de Transcrição SOXB1/genética , Transcrição Gênica , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Endopeptidases/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos/genética , Histonas/metabolismo , Camundongos , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Sirtuína 1/metabolismo , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genéticaRESUMO
Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.
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Cálcio/metabolismo , Diferenciação Celular , Fibroblastos/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/biossíntese , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Humanos , Camundongos , Contração Miocárdica/genética , Miócitos Cardíacos/química , Fatores de Transcrição/genéticaRESUMO
Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.
Assuntos
Células-Tronco Embrionárias/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Feminino , Estimativa de Kaplan-Meier , Camundongos , Infarto do Miocárdio/mortalidade , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco/métodosRESUMO
OBJECTIVES: To evaluate different refractive cutoffs for spectacle provision with regards to their impact on visual improvement and spectacle compliance. DESIGN: Prospective study of visual improvement and spectacle compliance. PARTICIPANTS: South African school children aged 6-19 years receiving free spectacles in a programme supported by Helen Keller International. METHODS: Refractive error, age, gender, urban versus rural residence, presenting and best-corrected vision were recorded for participants. Spectacle wear was observed directly at an unannounced follow-up examination 4-11 months after initial provision of spectacles. The association between five proposed refractive cutoff protocols and visual improvement and spectacle compliance were examined in separate multivariate models. MAIN OUTCOMES: Refractive cutoffs for spectacle distribution which would effectively identify children with improved vision, and those more likely to comply with spectacle wear. RESULTS: Among 8520 children screened, 810 (9.5%) received spectacles, of whom 636 (79%) were aged 10-14 years, 530 (65%) were girls, 324 (40%) had vision improvement > or = 3 lines, and 483 (60%) were examined 6.4+/-1.5 (range 4.6 to 10.9) months after spectacle dispensing. Among examined children, 149 (31%) were wearing or carrying their glasses. Children meeting cutoffs < or = -0.75 D of myopia, > or = +1.00 D of hyperopia and > or = +0.75 D of astigmatism had significantly greater improvement in vision than children failing to meet these criteria, when adjusting for age, gender and urban versus rural residence. None of the proposed refractive protocols discriminated between children wearing and not wearing spectacles. Presenting vision and improvement in vision were unassociated with subsequent spectacle wear, but girls (p < or = 0.0006 for all models) were more likely to be wearing glasses than were boys. CONCLUSIONS: To the best of our knowledge, this is the first suggested refractive cutoff for glasses dispensing validated with respect to key programme outcomes. The lack of association between spectacle retention and either refractive error or vision may have been due to the relatively modest degree of refractive error in this African population.
Assuntos
Óculos/estatística & dados numéricos , Refração Ocular , Erros de Refração/fisiopatologia , Erros de Refração/terapia , Acuidade Visual , Adolescente , Adulto , Criança , Protocolos Clínicos , Feminino , Humanos , Masculino , Cooperação do Paciente/estatística & dados numéricos , Avaliação de Programas e Projetos de Saúde , Estudos Prospectivos , Saúde da População Rural/estatística & dados numéricos , Saúde da População Urbana/estatística & dados numéricos , Seleção Visual/métodosRESUMO
PURPOSE: To quantify the impact on self-reported visual functioning of spectacle provision for school-aged children in Oaxaca, Mexico. METHODS: The Refractive Status Vision Profile (RSVP), a previously validated tool to measure the impact of refractive correction on visual functioning, was adapted for use in rural children and administered at baseline and 4 weeks (27.3 +/- 4.4 days) after the provision of free spectacles. Visual acuity with and without correction, age, sex, and spherical equivalent refraction were recorded at the time of follow-up. RESULTS: Among 88 children (mean age, 12 years; 55.7% girls), the median presenting acuity (uncorrected or with original spectacles), tested 4 weeks after the provision of free spectacles, was 6/9 (range, 6/6-6/120). Significant improvements in the following subscales of the RSVP were seen for the group as a whole after the provision of free spectacles: function, 11.2 points (P = 0.0001); symptoms, 14.3 points (P < 0.0001); total score, 10.3 points (P = 0.0001). After stratification by presenting vision in the better-seeing eye, children with 6/6 acuity (n = 22) did not have significant improvement in any subscale; those with acuity of 6/7.5 to 6/9 (n = 34) improved only on function (P = 0.02), symptoms (P = 0.005), and total score (P = 0.003); and those with acuity of 6/12 or worse improved on total score (P < 0.0001) and all subscales. Subjects (n = 31) with uncorrected myopia of -1.25 D or more had a mean improvement in total score of 15.9 points (P < 0.0001), whereas those with uncorrected myopia between -0.50 and -1.00 D inclusive (n = 53) had a mean improvement of 8 points (P = 0.01). CONCLUSIONS: Provision of spectacles to children in this setting had a significant impact on self-reported function, even at modest levels of baseline visual disability. The correlation between presenting vision/refraction and improvement and the failure of children 6/6 at baseline to improve offer evidence for a real effect.