Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation.

The rationale for in utero hematopoietic cell transplantation (IUHCT) rests on exploitation of normal events during hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning.  Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine model this can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used a less stringent enrichment protocol of single-step lineage depleted cells alone (lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrow accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity.

Preclinical Canine Model of Graft-versus-Host Disease after In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allogeneic engraftment and associated donor-specific tolerance without the need for toxic conditioning, as we have previously demonstrated in the murine and canine models. This strategy holds great promise in the treatment of many hematopoietic disorders, including the hemoglobinopathies. Graft-versus-host disease (GVHD) represents the greatest theoretical risk of IUHCT and has never been characterized in the context of IUHCT. We recently described a preclinical canine model of IUHCT, allowing further study of the technique and its complications. We aimed to establish a threshold T cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype. Using a range of T cell concentrations within the donor inoculum, we were able to characterize the phenotype of IUHCT-induced GVHD and establish a clear threshold for its induction between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1% to 3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.

Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.

Improved pulmonary function in the nitrofen model of congenital diaphragmatic hernia following prenatal maternal dexamethasone and/or sildenafil.

BACKGROUND: Pulmonary hypoplasia and hypertension is a leading cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). The etiologic insult occurs early in gestation highlighting the potential of prenatal interventions. We evaluated prenatal pharmacologic therapies in the nitrofen CDH model. METHODS: Olive oil or nitrofen were administered alone or with dexamethasone (DM), sildenafil, or DM+sildenafil to pregnant rats. Newborn pups were assessed for lung function, structure and pulmonary artery (PA) flow and resistance. RESULTS: Prenatal DM treatment of CDH pups increased alveolar volume density (Vva), decreased interalveloar septal thickness, increased tidal volumes and improved ventilation without improving oxygenation or PA resistance. Sildenafil decreased PA resistance and improved oxygenation without improving ventilation or resulting in significant histologic changes. DM+sildenafil decreased PA resistance, improved oxygenation and ventilation while increasing Vva and decreasing interalveolar septal and pulmonary arteriole medial wall thickness. Lung and body weights were decreased in pups treated with DM and/or sildenafil. CONCLUSION: Prenatal DM or sildenafil treatment increased pulmonary compliance and decreased pulmonary vascular resistance respectively, and was associated with improved neonatal gas exchange but had a detrimental effect on lung and fetal growth. This study highlights the potential of individual and combined prenatal pharmacologic therapies for CDH management.

Hemolysis-Associated Nitric Oxide Dysregulation during Extracorporeal Membrane Oxygenation.

Acute intravascular hemolysis during extracorporeal membrane oxygenation (ECMO) leads to increased levels of cell-free hemoglobin (FHb). Our aim was to investigate whether FHb levels are associated with nitric oxide (NO) consumption and clinical outcomes. A prospective observational study was performed involving pediatric patients on ECMO. Blood samples were collected before, during, and after the ECMO run, and plasma was evaluated for FHb, oxyhemoglobin, and NO consumption. Clinical data were collected including baseline patient characteristics, indications for ECMO, circuit changes, and mortality. Correlations between laboratory measures and associations between laboratory measures and clinical observations were evaluated. Twenty-three patients (11 male, 17 neonates) were enrolled with a median weight of 3.1 kg (interquartile range, 2.8-14.0 kg) and median ECMO run of 12 days (interquartile range, 5-19 day). There was a significant increase in FHb over time on ECMO (p = .007), and significant correlations were present between NO consumption and both FHb (r = .41, p = .01) and oxyhemoglobin levels (r = .98, p < .0001). Patients on ECMO for sepsis (n = 6) had lower average levels of oxyhemoglobin (mean [standard deviation {SD}] 14.5 [4.4] versus 19.0 [5.0] µM, p = .07) and NO consumption (mean [SD] 15.8 [4.1] versus 19.8 [3.7] µM, p = .04) during ECMO than patients with other indications. In the 3 days leading up to a circuit change, there were increases in mean total cell-free hemoglobin levels (24%/day, p = .08), oxyhemoglobin (37%/day, p = .005), and NO consumption (40%/day, p = .006) (n = 5). There were no significant associations identified between peak or average plasma measures of hemolysis and type of ECMO (venovenous versus venoarterial) or mortality. For children on ECMO, we observed a strong correlation between increased levels of plasma FHb and elevations in oxyhemoglobin and NO consumption; however, these changes were not associated with increased mortality. Increased hemolysis before circuit changes may be both a marker and a contributor to circuit failure.

Roux-en-Y drainage of a pancreatic fistula for disconnected pancreatic duct syndrome after acute necrotizing pancreatitis.

BACKGROUND: After acute necrotizing pancreatitis (ANP), a pancreatic fistula may occur from disconnected pancreatic duct syndrome (DPDS) where a segment of the pancreas is no longer in continuity with the main pancreatic duct. AIM: To study the outcome of patients treated using Roux-Y pancreatic fistula tract-jejunostomy for DPDS after ANP. METHODS: Between 2002 and 2011, patients treated for DPDS in the setting of endoscopic retrograde cholangiopancreatography (ERCP) or magnetic resonance cholangiopanreatography (MRCP) documented main pancreatic duct disruption with Roux-Y pancreatic fistula tract-jejunostomy. RESULTS: In all, seven patients with DPDS were treated. The median age was 62 years (range 49-78) and five were men. The cause of ANP was gallstones (2), alcohol (1), ERCP (1) and idiopathic (3). Pancreatic necrosectomy was done in six patients. Time from onset of pancreatitis to fistula drainage was 270 days (164-365). Pancreatic fistulae arose from DPDS in the head/neck (4) and body/tail (3). Patients had a median fistula output of 140 ml (100-200) per day before surgery. The median operative time was 142 min (75-367) and estimated blood loss was 150 ml (25 to 500). Patients began an oral diet on post-operative day 4 (3-6) and were hospitalized for a median of 7 days (5-12). The median follow-up was 264 days (29-740). Subsequently, one patient required a distal pancreatectomy. After surgery, three patients required oral hypoglycaemics. No patient developed pancreatic exocrine insufficiency. CONCLUSION: Internal surgical drainage using Roux-en-Y pancreatic fistula tract-jejunostomy is a safe and definitive treatment for patients with DPDS.

Enhanced recovery after surgery in children.

Enhanced recovery after surgery (ERAS) is a systematic approach to optimize a patient's health and improve clinical outcomes, increase patient satisfaction and decrease healthcare costs. Enhanced recovery protocols have been used across a variety of surgical disciplines and patient groups to improve patient safety and reduce hospital length of stay without increasing return visits to the system. ERAS involves the application of clinical decision making throughout the patient experience with interventions in the preoperative, perioperative and post operative phases. In addition, ERAS is multidisciplinary and the success of an ERAS program is dependent on the effort and integration of stakeholders across the healthcare system. Utilization of ERAS systems have grown across the global adult surgical community over the last three decades and adoption in pediatric surgery has only occurred recently. Hospitals in both adult and pediatric surgery have found that implementation of ERAS systems lead to a shortened length of stay and reduced complications without increasing patient returns to the system. Importantly patients who have surgery within an ERAS program experience less pain, less opioid utilization, a quicker recovery and increased satisfaction. In pediatric surgery ERAS has successfully been employed across most all disciplines from congenital cardiac surgery to colorectal surgery. The evolution of ERAS continues as a paradigm of quality and safety.

Nonoperative management of appendicitis in children.

Appendicitis is a common condition in childhood and adolescence that frequently requires urgent surgical intervention. For almost two centuries appendicitis has been recognized as a medical problem with a surgical solution. Currently the appendix can be removed with a minimally invasive approach, low anesthetic and surgical risk, and swift hospital discharge. Despite these advances, surgery and anesthesia have associated risks including postoperative infection, bleeding, hernia and organ injury among others. In addition, surgery requires time off of school and work to recover and associated healthcare costs can be significant. In both adult and pediatric populations, quality data suggesting a nonoperative approach is suggesting a change to the traditional surgical paradigm. Adults studies have demonstrated both safety and efficacy in the nonoperative management of acute appendicitis. In selected children with uncomplicated appendicitis, initial nonoperative management has been shown to be safe with fewer complications, fewer disability days and less healthcare costs while avoiding the risks inherent to surgery. Ongoing randomized controlled clinical trials in both the United States and Europe seek to further demonstrate the safety of nonoperative management and assist physicians with educating patients about the risk profile of their treatment decision. In complicated appendicitis presenting with abscess or acute appendiceal phlegmon, an initial nonoperative strategy with or without abscess drainage followed by interval appendectomy is the current state of the art though the utility of interval appendectomy is questioned.

The Role of Minimally Invasive Surgery in Pediatric Trauma.

Minimally invasive surgery (MIS) in the management of blunt and penetrating pediatric trauma has evolved in the past 30 years. Laparoscopy and thoracoscopy possess high levels of diagnostic accuracy with low associated missed injury rates. Currently available data advocate limiting the use of MIS to blunt or penetrating injuries in the hemodynamically stable child. In the pediatric trauma population, MIS offers both diagnostic and therapeutic potential, as well as reduced postoperative pain, a decreased rate of postoperative complications, shortened hospital stay, and potentially reduced cost.

Pediatric thoracic trauma: Current trends.

Pediatric thoracic trauma is relatively uncommon but results in disproportionately high levels of morbidity and mortality when compared with other traumatic injuries. These injuries are often more devastating due to differences in children׳s anatomy and physiology relative to adult patients. A high index of suspicion is of utmost importance at the time of presentation because many significant thoracic injuries will have no external signs of injury. With proper recognition and management of these injuries, there is an associated improved long-term outcome. This article reviews the current literature and discusses the initial evaluation, current management practices, and future directions in pediatric thoracic trauma.

Caspase-3 cleavage and nuclear localization of caspase-activated DNase in human temporal lobe epilepsy.

Programmed cell death (apoptosis) signaling pathways have been implicated in seizure-induced neuronal death and the pathogenesis of human temporal lobe epilepsy (TLE). End-stage DNA fragmentation during cell death may be mediated by nucleases including caspase-activated DNase (CAD), apoptosis-inducing factor (AIF) and endonuclease G. In the present study, we investigated the subcellular localization of these nucleases in resected hippocampus from TLE patients and autopsy controls. Subcellular fractionation determined levels of CAD were significantly higher in the nuclear fraction of TLE samples compared with controls, and semiquantitative immunohistochemistry revealed cleaved caspase-3 positive cells in TLE sections but not controls. While mitochondrial levels of AIF and endonuclease G were higher in TLE samples than controls, nuclear localization of AIF was limited and restricted to cells that were negative for cleaved caspase-3. Nuclear accumulation of endonuclease G was not found in TLE samples. These data support ongoing caspase-dependent apoptosis signaling in human TLE and suggest that interventions targeting such pathways may have potential as adjunctive neuroprotective therapy in epilepsy.

The first 100 infant thoracoscopic lobectomies: Observations through the learning curve and comparison to open lobectomy.

OBJECTIVE: The objective of the study is to describe our initial 100 attempted infant thoracoscopic lobectomies for asymptomatic, prenatally diagnosed lung lesions, and compare the results to contemporaneous age-matched patients undergoing open lobectomy. BACKGROUND: Infant thoracoscopic lobectomy is a technically challenging procedure, which has only gained acceptance worldwide in recent years. METHODS: This is a retrospective review of all patients undergoing thoracoscopic or open lung lobectomy between March 2005 and January 2014. Included were all asymptomatic infants younger than 4months. Excluded were patients undergoing emergent lobectomy and patients with isolated extralobar bronchopulmonary sequestrations. RESULTS: A total of 100 attempted thoracoscopic lobectomies were compared with 188 open lobectomies. In the thoracoscopic group, mean age and weight at surgery were 7.3weeks and 4.8kg, mean operative time was 185minutes, and mean hospital stay was 3days. Twelve cases were converted to open (12%). Ten conversions occurred within the first third of the series and none in the last third. There were no mortalities. There were no differences between the thoracoscopic and open groups in perioperative complications or hospital stay. There was a significant difference in the operative time: 111minutes vs. 185minutes (open vs. thoracoscopic; p<0.001). There was a higher mean end-tidal carbon dioxide (ETCO2) and lower mean peripheral capillary oxygen saturation (SpO2) in the thoracoscopic group versus the open group (51.7 versus 38.6mmHg and 97.5 versus 99.1%, respectively). CONCLUSION: In high volume centers, the learning curve of thoracoscopic lobectomy can be overcome and the procedure can be performed with equivalent outcomes and, in our opinion, superior cosmetic results to open lobectomy.

Single-stage versus multi-stage pull-through for Hirschsprung's disease: practice trends and outcomes in infants.

PURPOSE: The aim of this study was to evaluate surgical treatments and outcomes in a multi-institutional cohort of neonates with Hirschsprung's disease (HD). METHODS: Using the Pediatric Health Information System (PHIS) from 1999 to 2009, neonates diagnosed with HD were identified and classified as having a single stage pull-through (SSPT) or multi-stage pull-through (MSPT). Diagnosis and classification algorithms and clinical variables and outcomes were validated by multi-institutional chart review. Groups were compared using logistic regression modeling and propensity-score matched analysis to account for baseline differences between groups. RESULTS: 1555 neonates with HD were identified; 77.2% underwent SSPT and 22.8% underwent MSPT. Misclassification of disease or surgical treatment was <2%. Rates of SSPT increased over time (p=0.03). Compared to SSPT, patients undergoing MSPT had significantly lower birth weights and higher rates of prematurity, non-HD gastrointestinal anomalies, enterocolitis, and preoperative mechanical ventilation. Patients undergoing MSPT had significantly higher rates of readmissions (58.5 vs. 37.9%) and additional operations (38.7 vs. 26%). Results were consistent in the propensity-score matched analysis. CONCLUSION: Most neonates with HD undergo SSPT. In patients with similar observed baseline characteristics, MSPT was associated with worse outcomes suggesting that some infants currently selected to undergo MSPT may have better outcomes with SSPT. However, there remains a subgroup of MSPT patients who were too ill to be adequately compared to SSPT patients; for this subgroup of severely ill infants with HD, MSPT may be the best option.

Stem cell and genetic therapies for the fetus.

The prenatal diagnosis and management of congenital disease has made significant progress over the previous decade. Currently, fetal therapy (including open surgery and fetoscopic intervention) provides therapeutic options for a range of congenital anomalies; however, it is restricted to the treatment of fetal pathophysiology. Improvements in prenatal screening and the early diagnosis of genetic disease allow for preemptive treatment of anticipated postnatal disease by stem cell or genetic therapy. While currently awaiting clinical application, in utero stem cell therapy has made significant advances in overcoming the engraftment and immunologic barriers in both murine and pre-clinical large animal models. Likewise, proof in principle for fetal gene therapy has been demonstrated in rodent and large animal systems as a method to prevent the onset of inherited genetic disease; however, safety and ethical risks still need to be addressed prior to human application. In this review, we examine the current status and future direction of stem cell and genetic therapy for the fetus.

Decompressive laparotomy for abdominal compartment syndrome in children: before it is too late.

PURPOSE: Abdominal compartment syndrome (ACS) in children is an infrequently reported, rapidly progressive, and often lethal condition underappreciated in the pediatric population. This underrecognition can result in a critical delay in diagnosis causing increased morbidity and mortality. This study examines the clinical course of patients treated for ACS at our institution. METHODS: A review of children requiring an emergency laparotomy (n = 264) identified 26 patients with a diagnosis of ACS. ACS was defined as sustained intraabdominal hypertension (bladder pressure >12 mm Hg) that was associated with new onset organ dysfunction or failure. RESULTS: Patients ranged in age from 3 months to 17 years old and were cared for in the pediatric intensive care unit (PICU). Twenty-seven percent (n = 7) were transferred from referring hospitals, 50% (n = 13) were admitted directly from the emergency department, and 23% (n = 6) were inpatients before being transferred to PICU. Admission diagnoses included infectious enterocolitis (n = 12), postsurgical procedure (n = 10), and others (n = 4). Patients progressed to ACS rapidly, with most requiring decompressive laparotomy within 8 hours of PICU admission (range, <1-96 hours). Preoperatively, all patients had maximum ventilatory support and oliguria, 85% (n = 22) required vasopressors/inotropes, and 31% (n = 8) required hemodialysis. Mean bladder pressure was 25 mm Hg (range, 12-44 mm Hg). In 42% (n = 11), cardiac arrest preceeded decompressive laparotomy. All patients showed evidence of tissue ischemia before decompressive laparotomy with an average preoperative lactate of 8 (range, 1.2-20). Decompressive laparotomy was done at the bedside in the PICU in 13 patients and in the operating room in 14 patients. Abdominal wounds were managed with open vacuum pack or silastic silo dressings. Physiologic data including fluid resuscitation, oxygen index, mean airway pressure, vasopressor score, and urine output were recorded at 6-hour intervals beginning 12 hours before decompressive laparotomy and extending 12 hours after operation. The data demonstrate improvement of all physiologic parameters after decompressive laparotomy except for urine output, which continued to be minimal 12 hours post intervention. Mortality was 58% (n = 15) overall. The only significant factor related to increased mortality was bladder pressure (P = .046; odds ratio, 1.258). Cardiac arrest before decompressive laparotomy, need for hemodialysis, and transfer from referring hospital also trended toward increased mortality but did not reach significance. CONCLUSION: Abdominal compartment syndrome in children carries a high mortality and may be a consequence of common childhood diseases such as enterocolitis. The diagnosis of ACS and the potential need for emergent decompressive laparotomy may be infrequently discussed in the pediatric literature. Increased awareness of ACS may promote earlier diagnosis, treatment, and possibly improve outcomes.

Reflux esophageal stricture--a review of 30 years' experience in children.

PURPOSE: Strictures of the esophagus in children may have multiple etiologies including congenital, inflammatory, infectious, caustic ingestion, and gastroesophageal reflux (peptic stricture [PS]). Current literature lacks good data documenting long-term outcomes in children. This makes it difficult to counsel some patients about realistic treatment expectations. The objective of this study is to evaluate our institutional experience and define the natural history and treatment outcomes. METHODS: A retrospective review of clinical data obtained from children who underwent dilation for PS was performed. RESULTS: Over the past 30 years, 114 children and adolescents received 486 dilations. The most common indications for stricture dilation were PS (42%) and esophageal atresia (38%). Other lesser indications included congenital, foreign body, corrosive, cancer, radiation, allergic, and infectious. This review focuses on the 48 children with PS. Of the children with PS, a congenital anomaly was identified in 23 children; and 12 had neurologic impairment. Average age at presentation was 10.2 years (range, 0.5-18.3 years). Most patients had had symptoms for many months before diagnosis. Peptic stricture was most common in the lower esophagus (n = 39). However, middle (n = 8) and upper (n = 1) strictures were occasionally identified. Noncompliance with medical therapy was a challenge in 12% (n = 5) of children. Children with a PS received a median of 3 dilations, but a subset of 5 patients with severe strictures underwent up to 48 dilations (range, 1-48). Repeated dilations were required for a median of 20 months (range, 1-242 months). Among patients receiving esophageal dilation for PS, 94% required an antireflux procedure (19% required a second antireflux surgery). A subgroup of patients (n = 10) was identified who required extended dilations, multiple surgeries, and esophageal resection. This subgroup had a significantly longer period of symptomatic disease and increased risk of esophageal resection compared with those patients requiring fewer dilations. Surgical resection of the esophageal stricture was ultimately required in 3 children with PS after failure of more conservative measures. CONCLUSION: Children and adolescents presenting with reflux esophageal stricture (PS) frequently require antireflux surgery, redo antireflux surgery, and multiple dilations for recurrent symptoms. We hope that these data will be of use to the clinician attempting to counsel patients and parents about treatment expectations in this challenging patient population.

Laparoscopic treatment of celiac artery compression syndrome: case series and review of current treatment modalities.

INTRODUCTION: Compression of the celiac artery by the diaphragmatic crura, the median arcuate ligament, or the fibrous periaortic ganglionic tissue results in a rare constellation of symptoms known as celiac artery compression syndrome (CACS). ANATOMY: First described in 1963 by Harjola in a patient with symptoms of mesenteric ischemia, it remains an elusive diagnosis. CLINICAL PRESENTATION: Patients commonly present with a wide variety of symptoms resulting in multiple diagnostic tests. DIAGNOSIS: A firm diagnosis is difficult to establish, and treatment is equally challenging. These challenges are illustrated by the following case series, and evidence supporting current treatment modalities is reviewed. TREATMENT: We describe a laparoscopic approach to decompression of the celiac artery facilitated by intraoperative ultrasound.