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.

Post-operative Outcomes in Children Undergoing Fontan Palliation in a Regionalized Surgical System.

Evidence suggests that outcomes in pediatric cardiac surgery are improved by consolidating care into centers of excellence. Our objective was to determine if outcomes are equivalent in patients across a large regional referral base, or if patients from centers without on-site surgery are at a disadvantage. Since 1996, all pediatric cardiac surgery has been offered at one of two centers within the region assessed, with the majority being performed at Stollery Children's Hospital. All patients who underwent a Fontan between 1996 and 2016 were included. Follow-up data including length of stay (LOS), repeat surgical interventions, and transplant-free survival were acquired for each patient. The association between post-operative outcomes and home center was assessed using Kaplan-Meier survival analysis and Cox proportional Hazards models. 320 children (median age 3.3 years, IQR 2.8-4.0) were included; 120 (37.5%) had the surgical center as their home center. Cardiac anatomy was hypoplastic left heart syndrome in 107 (33.4%) subjects. Median LOS was 11 days (IQR, 8-17), and there were 8 in-hospital deaths. There were 17 deaths and 11 transplants over the course of follow-up. Five-year transplant-free survival was 92.5%. There was no difference in hospital re-intervention, late re-intervention, or survival by referral center (all p > 0.05). In multivariable analysis, home center was not predictive of either LOS (R 2 = -0.40, p = 0.87) or transplant-free survival (1.52, 95%CI 0.66, 3.54). In children with complex congenital heart disease, a regionalized surgical care model achieves good outcomes, which do not differ according to a patient's home base.

Persistent methicillin-resistant Staphylococcus aureus bacteremia from an infected superior vena cava thrombus.

Infection is among the leading causes of mortality for patients with end-stage renal disease. The placement of catheters for hemodialysis are common culprits of infection and have been associated with the development of complications such as venous thrombosis, bacteremia and thromboembolism. Calcification of a venous thrombus is a rare complication and infection of a right-sided thrombus can result in life-threatening septicemia and embolic complications. Herein, we describe the case of a 46-year-old patient found to have a calcified superior vena cava thrombus and bacteremia refractory to antibiotic therapy requiring surgical intervention under circulatory arrest to remove the infected thrombus gaining infectious source control and preventing future complications.

Infection is among the leading causes of mortality for patients with end-stage renal disease. The placement of catheters for dialysis are common culprits of infection and have been associated with the development of complications such as blood clotting in the veins and bacteria in the blood. Calcification of a venous blood clot is a rare complication and infection of a venous blood clot can result in life-threatening blood stream infection and dislodgement of clots. Herein, we describe the case of a 46-year-old patient found to have a calcified superior vena cava clot and a blood stream infection resistant to antibiotic therapy requiring surgical intervention to remove the infected clot gaining infectious source control and preventing future complications.

Neonatal Tracheal and Intracardiac Repair in a High-Risk Premature Infant Requiring Preoperative ECMO Transport.

Congenital tracheal stenosis is an uncommon malformation that portends a poor outcome in children who are symptomatic in the neonatal period. Over time, the management of significant tracheal disease has been consolidated at high-volume centers, and increasingly complex patients have undergone surgical repair. We present a premature newborn boy who was diagnosed with critical multi-level airway and cardiac disease who decompensated at a remote site, requiring extracorporeal membrane oxygenation support for transport. He underwent a complete repair including a slide tracheoplasty and was successfully discharged home, with no residual stenosis at follow-up.

Carotid Artery Dissection Following Neck Cannulation for Extracorporeal Life Support.

A term neonate was cannulated for venoarterial extracorporeal life support (ECLS) via the right neck for non-postoperative junctional ectopic tachycardia. Initial echocardiogram demonstrated an echogenic strand in the transverse arch. Computed tomography angiogram confirmed arterial dissection of the right common carotid artery that extended into the proximal transverse arch. Dissection flap was repaired at the time of ECLS decannulation without cardiopulmonary bypass. Follow-up computed tomography angiogram revealed a segment of narrowing of approximately 50% of the right common carotid artery without false lumen or aneurysm.

Tricuspid Valve Repair in Infancy Using Neochordae: Three-Dimensional Echocardiographic Imaging.

Tricuspid regurgitation (TR) in infancy poses a surgical challenge. Both two- and three-dimensional echocardiography (3DE) can provide detailed information about the mechanism(s) of valve failure and insights into valve adaptation during follow-up. We report two patients who underwent tricuspid valve repair using Gore-Tex neochordae, repairs which were facilitated by and assessed with 3DE. Both infants had less than mild residual TR and no valve tethering at hospital discharge. Furthermore, follow-up 3DEs have helped to confirm valve competence, lack of tethering, and growth of the valve and valve apparatus.

ß(3)-Adrenoceptor Antagonist SR59230A Attenuates the Imbalance of Systemic and Myocardial Oxygen Transport Induced by Dopamine in Newborn Lambs.

BACKGROUND: In neonates, the increase in O(2)-delivery (DO(2)) by dopamine is offset by a greater increase in O(2)-consumption (VO(2)). This has been attributed to ß(3)-adrenergic receptors in neonatal brown fat tissue. ß(3) receptors in the heart have negative inotropic properties. We evaluated the effects of SR59230A, a ß(3)-antagonist, on the balance of systemic and myocardial O(2)-transport in newborn lambs treated with dopamine. METHODS: Lambs (2-5 days old, n = 12) were anesthetized and mechanically ventilated. Heart rate (HR) and rectal temperature were monitored. VO(2) was measured by respiratory mass spectrometry and cardiac output (CO) by a pulmonary artery transonic flowmeter. Arterial, jugular bulb venous and coronary sinus blood gases and lactate were measured to calculate DO(2), O(2) extraction ratio (ERO(2)), myocardial O(2) and lactate extraction ratios (mERO(2), mERlac). After baseline measurements, lambs were randomized to receive SR59230A at 5 mg/kg iv (SRG) or placebo. Both groups received incremental doses of a dopamine infusion (0-5-10-15-20 mcg/kg/min) every 15 min. Measurements were repeated at the end of each dose. RESULTS: After SR59230A infusion, CO and HR trended to decrease (P = 0.06), but no significant changes occurred in other parameters. Over the incremental doses of dopamine, temperature increased in both groups (P < 0.0001) but to a lesser degree in SRG (P = 0.004). CO and HR increased (P = 0.005 and 0.04) and similarly in both groups (P > 0.1). DO(2) trended to a small increase (P = 0.08). VO(2) increased in both groups (P < 0.0001) but to a lesser degree in SRG (P < 0.0001). As a result, ERO(2) increased in both groups (P < 0.0001), but to a lesser degree in SRG (P < 0.0001). mERO(2) was lower in SRG (P = 0.01) with a faster increase (P < 0.0001). mERlac was higher in SRG (P = 0.06) with a faster decrease (P = 0.04). CONCLUSION: Although SR59230A tends to induce an initial drop in CO, it significantly attenuates the rise in VO(2) and hence the imbalance of systemic and myocardial O(2) transport induced by dopamine at higher doses. Studies are warranted to examine the effect of SR59230A in cases of cardiac dysfunction and increased VO(2), observed after cardiac surgery.