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Umbilical venous cannulation is the favored approach to perinatal central access worldwide but has a failure rate of 25-50% and the insertion technique has not evolved in decades. Improving the success of this procedure would have broad implications, particularly where peripherally inserted central catheters are not easily obtained and in neonates with congenital heart disease, in whom umbilical access facilitates administration of inotropes and blood products while sparing vessels essential for later cardiac interventions. We sought to use real-time, point-of-care ultrasound to achieve central umbilical venous access in patients for whom conventional, blind placement techniques had failed.Multicenter case series, March 2019-May 2021.Cardiac and neonatal ICUs at three tertiary care children's hospitals.We identified 32 neonates with congenital heart disease, who had failed umbilical venous cannulation using traditional, blind techniques.Real-time ultrasound guidance and liver pressure were used to replace malpositioned catheters and achieve successful placement at the inferior cavoatrial junction.In 32 patients with failed prior umbilical venous catheter placement, real-time ultrasound guidance was used to successfully "rescue" the line and achieve central position in 23 (72%). Twenty of 25 attempts (80%) performed in the first 48 hours of life were successful, and three of seven attempts (43%) performed later. Twenty-four patients (75%) were on prostaglandin infusion at the time of the procedure. We did not identify an association between patient weight or gestational age and successful placement.Ultrasound guidance has become standard of care for percutaneous central venous access but is a new and emerging technique for umbilical vessel catheterization. In this early experience, we report that point-of-care ultrasound, together with liver pressure, can be used to markedly improve success of placement. This represents a significant advance in this core neonatal procedure.

OBJECTIVE Single-ventricle congenital heart disease (CHD) in pediatric patients with Glenn and Fontan physiology represents a unique physiology requiring the surgical diversion of the systemic venous return from the superior vena cava (Glenn) and then the inferior vena cava (Fontan) directly to the pulmonary arteries. Because many of these patients are on chronic anticoagulation therapy and may have right-to-left shunts, arrhythmias, or lymphatic disorders that predispose them to bleeding and/or clotting, they are at risk of experiencing neurological injury requiring intubation and positive pressure ventilation, which can significantly hamper pulmonary blood flow and cardiac output. The aim of this study was to describe the complex neurological and cardiopulmonary interactions of these pediatric patients after acute central nervous system (CNS) injury. METHODS The authors retrospectively analyzed the records of pediatric patients who had been admitted to a quaternary children’s hospital with CHD palliated to bidirectional Glenn (BDG) or Fontan circulation and acute CNS injury and who had undergone intubation and mechanical ventilation. Patients who had been admitted from 2005 to 2019 were included in the study. Clinical characteristics, surgical outcomes, cardiovascular and pulmonary data, and intracranial pressure data were collected and analyzed. RESULTS Nine pediatric single-ventricle patients met the study inclusion criteria. All had undergone the BDG procedure, and the majority (78%) were status post Fontan palliation. The mean age was 7.4 years (range 1.3–17.3 years). At the time of acute CNS injury, which included traumatic brain injury, intracranial hemorrhage, and cerebral infarct, the median time interval from the most recent cardiac surgical procedure was 3 years (range 2 weeks–11 years). Maintaining normocarbia to mild hypercarbia for most patients during intubation periods did not cause neurological deterioration, and hemodynamic profiles were more favorable as compared to periods of hypocarbia. Hypocarbia was associated with unfavorable hemodynamics but was necessary to decrease intracranial hypertension. Most patients were managed using low mean airway pressure (MAWP) in order to minimize the impact on preload and cardiac output. CONCLUSIONS The authors highlight the complex neurological and cardiopulmonary interactions with respect to partial pressure of arterial CO2 (PaCO2) and MAWP when pediatric CHD patients with single-ventricle physiology require mechanical ventilation. The study data demonstrated that tight control of PaCO2 and minimizing MAWP with the goal of early extubation may be beneficial in this population. A multidisciplinary team of pediatric critical care intensivists, cardiac intensivists and anesthesiologists, and pediatric neurosurgeons and neurologists are recommended to ensure the best possible outcomes.

Objective:A standardised multi-site approach to manage paediatric post-operative chylothorax does not exist and leads to unnecessary practice variation. The Chylothorax Work Group utilised the Pediatric Critical Care Consortium infrastructure to address this gap.Methods:Over 60 multi-disciplinary providers representing 22 centres convened virtually as a quality initiative to develop an algorithm to manage paediatric post-operative chylothorax. Agreement was objectively quantified for each recommendation in the algorithm by utilising an anonymous survey. “Consensus” was defined as ≥ 80% of responses as “agree” or “strongly agree” to a recommendation. In order to determine if the algorithm recommendations would be correctly interpreted in the clinical environment, we developed ex vivo simulations and surveyed patients who developed the algorithm and patients who did not.Results:The algorithm is intended for all children (80%) by the workgroup (range 81–100%). Ex vivo simulations demonstrated good understanding by developers (range 94–100%) and non-developers (73%–100%).Conclusions:The quality improvement effort represents the first multi-site algorithm for the management of paediatric post-operative chylothorax. The algorithm includes transparent and objective measures of agreement and understanding. Agreement to the algorithm recommendations was >80%, and overall understanding was 94%.

Prevention of COVID-19 is widely believed to depend on neutralization of SARS-CoV-2 by vaccine-induced humoral immunity1,2, raising concern that emerging escape variants may perpetuate the pandemic3–6. Here we show that a single intramuscular injection of Adeno-Associated Virus-6 (AAV6) or AAV9 encoding a modified, N-terminal domain deleted (ΔNTD) spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity. The findings may provide an alternative approach to containment of the evolving COVID-19 pandemic and have broader implications for the development of variant-agnostic universal vaccines against a wider range of pathogens.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin

The clinical progression of Duchenne Muscular Dystrophy (DMD) begins with locomotive symptoms in childhood but eventually includes symptoms of severe cardiorespiratory failure. Our studies in mdx mice and GRMD dogs have reavealed surprisingly large early deficits in physiological reserve, attributable to the deficiency of dystrophin. In young GRMD dogs, the findings of diaphragmatic fibrosis and myofiber shortening correlated strongly with non-invasively measured abnormalities in respiratory muscle recruitment. In juvenile GRMD dogs without clinical signs of heart failure, detailed studies of cardiac mechanics revealed profoundly abnormal Frank-Starling and inotropic responses. Thus, submaximal exercise loading has the capactiy to reveal very early, otherwise subclinical deficits in physiological reserve. We have utilized several non-invasive systems to measure integrative cardiopulmonary mechanics in animals trained to undertake submaximal volitional exercise. Our findings demonstrate the possibility of continuous non-invasive monitoring of cardiac output, stroke volume, systemic vascular resistance, and several ventilatory parameters in young dogs and children during treadmill exercise, as well as upright and recumbent bicycling at work outputs appropriate for low risk serial testing in the earliest stages of DMD. As the field contemplates primary endpoints for use in trials of systemic gene therapy, these metrics hold promise as the most sensitive measurements of the earliest functional deficits in organ systems eventually responsible for the lethality of DMD.

Background— The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. Methods and Results— Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (10 13 particles per kilogram) or recombinant adeno-associated virus (10 14 genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher β-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in β-galactosidase levels between 100- or 400-mm Hg groups. The Conclusions— Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.

Powerful masticatory muscles are found in most primates, including chimpanzees and gorillas, and were part of a prominent adaptation of Australopithecus and Paranthropus, extinct genera of the family Hominidae. In contrast, masticatory muscles are considerably smaller in both modern and fossil members of Homo. The evolving hominid masticatory apparatus--traceable to a Late Miocene, chimpanzee-like morphology--shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. Here, we show that the gene encoding the predominant myosin heavy chain (MYH) expressed in these muscles was inactivated by a frameshifting mutation after the lineages leading to humans and chimpanzees diverged. Loss of this protein isoform is associated with marked size reductions in individual muscle fibres and entire masticatory muscles. Using the coding sequence for the myosin rod domains as a molecular clock, we estimate that this mutation appeared approximately 2.4 million years ago, predating the appearance of modern human body size and emigration of Homo from Africa. This represents the first proteomic distinction between humans and chimpanzees that can be correlated with a traceable anatomic imprint in the fossil record.

Background Several physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema. Methods We sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro. Results In addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema. Conclusion This is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans.

C oronary artery disease (CAD) is prevalent in candidates for lung volume reduction surgery (LVRS),1 and significant valve dysfunction may also coexist with severe emphysema. The National Emphysema Treatment Trial (NETT) excludes patients with a variety of cardiovascular risk factors from undergoing LVRS,2 as do many surgeons who perform the procedure outside of the trial. Few have performed LVRS in those patients with severe CAD that cannot be managed percutaneously. Thus, although the literature on combining cardiac procedures and pulmonary resections for cancer is extensive, we have identified only 4 published cases3-5 from 3 different groups using diverse operative strategies combining cardiac procedures and LVRS. We report our series of 4 combined, simultaneous cardiac/LVRS procedures to aid in determining whether this is an appropriate approach in selected patients and, if so, to help select the ideal operative strategy.

Top of pageAbstract The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for full complementation of the underlying genetic disorder. As a direct approach to this clinical challenge, somatic gene transfer will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions. We tested the hypothesis that rapid, mechanical distention of the post-capillary venular endothelium by afferent infusion from a distal site would safely facilitate macromolecular transport from the vascular space to the striated muscle interstitium. We show that pressurized infusion through a large-bore catheters in either peripheral, superficial veins or the coronary sinus results in uniform, scale-and vector-independent transduction of myofibers in anatomic domains isolated from the remainder of the circulation. This approach is rapid, minimally invasive as applied to the isolated limb, and avoids pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to virtually 100% of the muscle fibers of an entire extremity in the dog, providing a firm foundation for studies of efficacy in canine models for human diseases. Additional data from a combination of angiographic, tracer dye, and marker gene studies suggests that this approach can be modified to meet the requirements for cardiac-specific or systemic gene delivery as appropriate in a variety of inherited and acquired diseases including hemophilia, muscular dystrophy, and cardiomyopathy. Figure 1 |[ndash]| |[beta]|-galactosidase levels of rat limb, rat cardiac and dog limb muscles after no treatment, vector delivery without afferent transvenular retrograde extravasation (ATVRX) and vector delivery with ATVRX. Figure 2 |[ndash]| LacZ expression after rat quadriceps (left), rat heart (middle), and dog vastus medialis (right) stained with x-galactosidase.

BackgroundPreviously, we used cardiopulmonary bypass with incomplete cardiac isolation and antegrade administration of vector for global cardiac gene delivery. Here we present a translatable cardiac surgical procedure that allows for complete surgical isolation of the heart in situ with retrograde (through the coronary venous circulation) administration of both vector and endothelial permeabilizing agents to increase myocyte transduction efficiency.MethodsIn 6 adult dogs the heart was completely isolated with tourniquets placed around both vena cavae and cannulas and all pulmonary veins. On cardiopulmonary bypass, the aorta and pulmonary artery were crossclamped, and the heart was isolated. Crystalloid cardioplegia at 4°C containing 1013 particles of adenovirus encoding LacZ and 15 μg of vascular endothelial growth factor was infused retrograde into the coronary sinus and recirculated for a total of 30 minutes. The dogs were then weaned from cardiopulmonary bypass and allowed to recover. With a catheter, 3 control dogs underwent retrograde infusion of the same cocktail without cardiac isolation or cardiopulmonary bypass.Resultsβ-Galactosidase activities in the cardiopulmonary bypass group were several orders of magnitude higher in both the right and left ventricles when compared with those in the control group (P < .05). X-gal staining from the cardiopulmonary bypass group showed unequivocal evidence of myocyte gene expression globally in a significant proportion of cardiac myocytes. No myocyte gene expression was observed in the control group.ConclusionA novel cardiac surgical technique has been developed. This approach with cardiac isolation and retrograde delivery of vector through the coronary sinus results in efficient myocyte transduction in an adult large animal in vivo.