Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells.

BACKGROUND: Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents. METHODS: Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality. RESULTS: The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures. CONCLUSION: These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.

Coronary artery augmentation with the right subclavian artery for single coronary artery variants of dextro transposition of the great arteries treated by an arterial switch operation.

Objective: Single coronary artery variants in dextro transposition of the great arteries increase the technical demands of performing coronary translocations during the arterial switch operation (ASO). Coronary augmentation using the right subclavian artery as an interposition graft is a viable option in certain cases. The aim of this study is to describe this operative technique and review outcomes. Methods: Of 59 patients who underwent an ASO, from July 2015 to May 2021, 6 had single coronary variants in which the right subclavian artery was used as an interposition graft. Mean follow-up was 21.5 months. Results: Patients mean age and weight at the time of surgery were 7.1 ± 3.8 days and 2.84 ± 0.76 kg, respectively. Four patients had the left anterior descending coronary artery and right coronary artery coming from the left sinus and the circumflex coronary artery coming from the right sinus. One patient had a single ostium arising from the left sinus and another patient had a single ostium coming from the right sinus. All patients are alive and free of cardiac symptoms at follow-up. Conclusions: Single coronary artery variants in dextro transposition of the great arteries can pose technical challenges for coronary translocation during ASO. The augmentation of coronary buttons using a segment of the right subclavian artery is an option which should be considered in selective cases.

Preoperative Evaluation of Refugee Children With Heart Disease.

The objective of this report is to identify nutritional deficiencies and health concerns in Canadian paediatric refugee patients with heart disease before cardiac surgery. A retrospective case study was conducted with 4 refugee children, aged 0-18 years, with heart disease referred to Cardiac Surgery. A total of 75% of patients had low vitamin D levels, 50% had selenium deficiency, 50% had iron deficiency anaemia, 75% were below the third percentile for height and weight for age, and 75% had dental caries. Early laboratory, nutritional, and dental screening of paediatric refugee patients at the time of heart disease diagnosis can optimize general and cardiovascular health before surgical intervention.

Notre étude visait à dépister des carences nutritionnelles et des problèmes de santé présents chez les enfants réfugiés au Canada atteints de cardiopathie, avant une intervention chirurgicale cardiaque. Une étude de cas rétrospective a été menée auprès de quatre enfants réfugiés, âgés de 0 à 18 ans, souffrant de cardiopathie et orientés vers la chirurgie cardiaque. Au total, 75 % des patients présentaient un faible taux de vitamine D, 50 % avaient une carence en sélénium, 50 % souffraient d'anémie ferriprive, 75 % avaient une taille et un poids inférieurs au troisième percentile pour leur âge et 75 % présentaient des caries dentaires. Un dépistage précoce comprenant des analyses de laboratoire, une évaluation nutritionnelle et un examen dentaire, au moment d'un diagnostic de cardiopathie chez les enfants réfugiés, pourrait permettre d'améliorer leur état de santé général et cardiovasculaire avant une intervention chirurgicale.

Hybridizing the hybrid: a "serendipitous bridge" to heart transplantation in a child with hypoplastic left heart syndrome.

We report a unique experience with stage 1 palliation of hypoplastic left heart syndrome (HLHS) using the hybrid approach. The tortuous anatomy of the ductus arteriosus precluded stenting. Persistent ductal patency and ability to support the systemic circulation adequately after prolonged prostaglandin E1 (PGE1) infusion and its subsequent discontinuation presented us with a novel approach to hybrid palliation of hypoplastic left heart syndrome. Our experience offers a tempting approach, but it should not be considered a standard alternative management option to stage I hybrid palliation based on a single case report. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12055-020-01088-8.

Intraoperative Extubation Post Arterial Switch Operation for Transposition of the Great Arteries With Intact Ventricular Septum: A One-Year, Single Center Experience.

We sought to examine the clinical impact of intraoperative extubation (IE) in neonates undergoing the arterial switch operation (ASO) for D-transposition of the great arteries with intact ventricular septum (dTGA/IVS). This was a single center retrospective study of patients who underwent ASO for dTGA/IVS in the 12 months after an institutional change in practice favoring IE when clinically feasible. A control group was obtained by identifying the same number of consecutive patients with dTGA/IVS who underwent ASO immediately prior to this institutional change in practice, none of whom were extubated intraoperatively. Primary outcome measures included morbidity, mortality, length of hospital and intensive care unit stay and reintubation rates. There were no significant differences in the preoperative and operative characteristics between the 2 groups. Of the 10 patients who underwent ASO for dTGA/IVS in the 12 months post institutional change in practice, all (100%) were extubated intraoperatively and none (0%) required reintubation. The median length of intensive care unit stay was 2 days for both the intraoperative and non-IE groups (mean 2.2 and 3 days respectively). The median length of stay in hospital was 4 days in the IE group and 5.5 days in the non-IE group (mean 4.5 and 6 days respectively). No patients died and there was no significant difference in morbidity between the 2 groups. Our data suggests IE post ASO for dTGA/IVS is safe and displays a statistically insignificant trend toward earlier discharge from hospital.

Tricuspid Intervention Following Pulmonary Valve Replacement in Adults With Congenital Heart Disease.

BACKGROUND: Tricuspid regurgitation (TR) is common among adults with corrected tetralogy of Fallot (TOF) or pulmonary stenosis (PS) referred for pulmonary valve replacement (PVR). Yet, combined valve surgery remains controversial. OBJECTIVES: This study sought to evaluate the impact of concomitant tricuspid valve intervention (TVI) on post-operative TR, length of hospital stay, and on a composite endpoint consisting of 7 early adverse events (death, reintervention, cardiac electronic device implantation, infection, thromboembolic event, hemodialysis, and readmission). METHODS: The national Canadian cohort enrolled 542 patients with TOF or PS and mild to severe TR who underwent isolated PVR (66.8%) or PVR+TVI (33.2%). Outcomes were abstracted from charts and compared between groups using multivariable logistic and negative binomial regression. RESULTS: Median age at reintervention was 35.3 years. Regardless of surgery type, TR decreased by at least 1 echocardiographic grade in 35.4%, 66.9%, and 92.8% of patients with pre-operative mild, moderate, and severe insufficiency. In multivariable analyses, PVR+TVI was associated with an additional 2.3-fold reduction in TR grade (odds ratio [OR]: 0.44; 95% confidence interval [CI]: 0.25 to 0.77) without an increase in early adverse events (OR: 0.85; 95% CI: 0.46 to 1.57) or hospitalization time (incidence rate ratio: 1.17; 95% CI: 0.93 to 1.46). Pre-operative TR severity and presence of transvalvular leads independently predicted post-operative TR. In contrast, early adverse events were strongly associated with atrial tachyarrhythmia, extracardiac arteriopathy, and a high body mass index. CONCLUSIONS: In patients with TOF or PS and significant TR, concomitant TVI is safe and results in better early tricuspid valve competence than isolated PVR.

Are Blood Products Routinely Required in Pediatric Heart Surgery?

A restrictive blood transfusion strategy has emerged in adult cardiac surgery. However, the feasibility in children is poorly investigated. 352 consecutive patients undergoing open-heart surgery were retrospectively reviewed, excluding patients requiring extracorporeal membrane oxygenation. Patient demographics, perioperative blood product usage, and clinical outcome parameters were investigated. Variables predicting the need for blood products were delineated. Of the 352 study patients, 148 patients (42%) underwent bloodless surgery and 204 (58%) were transfused. Of the 204 transfused patients, 170 (83.4%) patients received one blood transfusion and 34 (16.6%) received two or more blood transfusions. Patient's weight and preoperative hematocrit (Hct) were statistically significant in predicting the need for blood priming the CPB circuit (AUC 0.99, p < 0.001, sensitivity 96.6%, specificity 95.2%). A body weight of 8.5 kg carried a sensitivity of 100% and specificity of 94.5% (p < 0.001) for a blood prime. Among patients with a weight less than 8.5 kg (n = 171), only 27 patients (15.8%, p < 0.001) required additional transfusion of PRBCs. Factors impacting the need for a blood transfusion during CPB included redo surgery [odds ratio (OR) 4.61, p = 0.001] and the highest lactate level on CPB (OR 1.65, p = 0.006). Redo surgery had the highest impact (OR 7.27, p = 0.012) for requiring a postoperative PRBC transfusion. A restrictive transfusion strategy can be safely implemented in pediatric cardiac surgery. The majority of children with a BW > 8.5 kg required no blood products and those with a BW ≤ 8.5 kg required only 1 unit of blood, to prime the cardiopulmonary bypass circuit.

Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1-3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Aortic Arch Homograft Reconstruction of Nonconfluent Pulmonary Arteries During Extracardiac Fontan.

Reconstruction of nonconfluent pulmonary arteries during Fontan completion is a challenging technical issue. In this case report, we describe the use of an aortic homograft, including the aortic arch, to complete a Fontan and reconstruct the pulmonary artery confluence in a child with discontinuous pulmonary arteries and bilateral superior caval veins who had undergone bilateral unidirectional Glenn palliation. The configuration of the aortic homograft was ideal to ensure laminar flow from the inferior vena cava to both pulmonary arteries and in maintaining durable elastance posterior to the native aorta.

Jellyfish-Like Accessory Mitral Valve Tissue Causing Near-Collapse in a Young Child.

Accessory mitral valve tissue (AMVT) causing left ventricular outflow tract obstruction (LVOTO) is rare. We report a case of AMVT causing severe LVOTO resulting in acutely progressive symptoms of near-collapse. Urgent surgical resection eliminated the patient's life-threatening symptoms. AMVT should be considered among potential LVOTO diagnoses, and early surgical intervention may be required.

Hybrid stenting for left ventricular outflow tract obstruction in congenitally corrected transposition of the great arteries.

Congenitally corrected transposition of the great arteries is commonly associated with left ventricular outflow tract obstruction. We describe a case of congenitally corrected transposition of the great arteries and previous surgical ventricular septal defect repair with recurrent left ventricular outflow tract obstruction. The patient underwent a hybrid procedure to stent the left ventricular outflow tract, which was successful with no re-intervention through 3 years of follow-up.

Valvar aortico-ventricular tunnel: an insight into the development of the great arteries.

Aorto-left ventricular tunnel is a rare congenital heart lesion, with an incidence of <0.1% of all CHD. We present a unique case of a valvar aorto-left ventricular tunnel in a neonate, in belief that our findings may shed some light on the developmental origins of this lesion.

Levoatriocardinal vein in D-transposition of the great arteries.

The levoatriocardinal vein is the embryological remnant of the connection between the pulmonary and systemic venous systems. It is a rare lesion that usually occurs in the presence of left-sided obstruction, developing as a pathway for decompression of the pulmonary veins. We report the first case of a levoatriocardinal vein in a patient with D-transposition of the great arteries.

Minimizing the risk of preoperative brain injury in neonates with aortic arch obstruction.

OBJECTIVE: To determine whether prenatal diagnosis lowers the risk of preoperative brain injury by assessing differences in the incidence of preoperative brain injury across centers. STUDY DESIGN: From 2 prospective cohorts of newborns with complex congenital heart disease studied by preoperative cerebral magnetic resonance imaging, one cohort from the University Medical Center Utrecht (UMCU) and a combined cohort from the University of California San Francisco (UCSF) and University of British Columbia (UBC), patients with aortic arch obstruction were selected and their imaging and clinical course reviewed. RESULTS: Birth characteristics were comparable between UMCU (n = 33) and UCSF/UBC (n = 54). Patients had a hypoplastic aortic arch with either coarctation/interruption or hypoplastic left heart syndrome. In subjects with prenatal diagnosis, there was a significant difference in the prevalence of white matter injury (WMI) between centers (11 of 22 [50%] at UMCU vs 4 of 30 [13%] at UCSF/UBC; P < .01). Prenatal diagnosis was protective for WMI at UCSF/UBC (13% prenatal diagnoses vs 50% postnatal diagnoses; P < .01), but not at UMCU (50% vs 46%, respectively; P > .99). Differences in clinical practice between prenatally diagnosed subjects at UMCU vs UCSF/UBC included older age at surgery, less time spent in the intensive care unit, greater use of diuretics, less use of total parenteral nutrition (P < .01), and a greater incidence of infections (P = .01). In patients diagnosed postnatally, the prevalence of WMI was similar in the 2 centers (46% at UMCU vs 50% at UCSF/UBC; P > .99). Stroke prevalence was similar in the 2 centers regardless of prenatal diagnosis (prenatal diagnosis: 4.5% at Utrecht vs 6.7% at UCSF/UBC, P = .75; postnatal diagnosis: 9.1% vs 13%, respectively, P > .99). CONCLUSION: Prenatal diagnosis can be protective for WMI, but this protection may be dependent on specific clinical management practices that differ across centers.

Should early extubation be the goal for children after congenital cardiac surgery?

OBJECTIVE: We sought to determine the feasibility and assess the clinical outcomes associated with an early extubation strategy for all children undergoing congenital heart surgery, including neonates (age, <30 days). METHODS: We performed a linked database analysis of all patients undergoing congenital heart surgery from July 1, 2010 to December 31, 2012. We collected data on the cardiac diagnoses, preoperative status, procedure, and postoperative course, including the duration of invasive and noninvasive ventilation, failure of extubation, hemodynamic data, length of stay, complications, and mortality. A multivariable model was used to assess the independent factors associated with an inability to extubate within the operating room and with delayed extubation (>24 hours). RESULTS: We operated on 613 children, including 97 neonates. Intraoperative extubation was achieved in 71% of the cases and early extubation (≤ 24 hours) was achieved in 89% of the cases. The overall mortality was 1.5% (9 of 613 patients). Early extubation was associated with lower mortality (1% vs 9%, P < .001) and a lower rate of reintubation (4% vs 23%, P < .001) compared with delayed extubation. Notably, 63% of the neonates were extubated within 24 hours, including 67% of arterial switch operations and 54% of total anomalous pulmonary venous return repairs. Norwood operations were the only procedure in which no patient was extubated within the first 24 hours. Multivariable logistic regression demonstrated that the predictors of delayed extubation included preoperative mechanical ventilation, weight < 5 kg, a longer procedure time, and the need for postoperative inotrope support. Implementation of an early extubation strategy was associated with low rates of complications (5.1 per 10 procedures), short lengths of intensive care unit stay (median, 1 day; interquartile range, 1-3), and short hospital stays (median, 4 days; interquartile range, 3-6). CONCLUSIONS: Most children undergoing congenital heart surgery can be extubated in the operating room. Most neonates, including many undergoing complex procedures, can be extubated within the first 24 hours after surgery. Early extubation was associated with low morbidity rates and short lengths of intensive care unit and hospital stays.

Nonuniform and variable arrangements of ryanodine receptors within mammalian ventricular couplons.

RATIONALE: Single-tilt tomograms of the dyads in rat ventricular myocytes indicated that type 2 ryanodine receptors (RYR2s) were not positioned in a well-ordered array. Furthermore, the orientation and packing strategy of purified type 1 ryanodine receptors in lipid bilayers is determined by the free Mg2+ concentration. These observations led us to test the hypothesis that RYR2s within the mammalian dyad have multiple and complex arrangements. OBJECTIVES: To determine the arrangement of RYR2 tetramers in the dyads of mammalian cardiomyocytes and the effects of physiologically and pathologically relevant factors on this arrangement. METHODS AND RESULTS: We used dual-tilt electron tomography to produce en-face views of dyads, enabling a direct examination of RYR2 distribution and arrangement. Rat hearts fixed in situ; isolated rat cardiomyocytes permeabilized, incubated with 1 mmol/L Mg2+, and then fixed; and sections of human ventricle, all showed that the tetramer packing within a dyad was nonuniform containing a mix of checkerboard and side-by-side arrangements, as well as isolated tetramers. Both phosphorylation and 0.1 mmol/L Mg2+ moved the tetramers into a predominantly checkerboard configuration, whereas the 4 mmol/L Mg2+ induced a dense side-by-side arrangement. These changes occurred within 10 minutes of application of the stimuli. CONCLUSIONS: The arrangement of RYR2 tetramers within the mammalian dyad is neither uniform nor static. We hypothesize that this is characteristic of the dyad in vivo and may provide a mechanism for modulating the open probabilities of the individual tetramers.