Faculty Bibliography

BACKGROUND:Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. METHODS:Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. RESULTS:Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. CONCLUSIONS:Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. TYPE OF STUDY/METHODS:Basic science.

PURPOSE/OBJECTIVE:Large cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system. METHODS:Orthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN. RESULTS:Image analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC >0.90 and 100% accuracy when >30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%. CONCLUSIONS:We recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.

PURPOSE/OBJECTIVE:Limited means exist to assess gastrointestinal activity in pediatric patients postoperatively. Recently, myoelectric gastrointestinal activity recorded by cutaneous patches has been shown in adult patients to be predictive of clinical return of gastrointestinal function postoperatively. The aim of this case series is to demonstrate the feasibility of this system in pediatric patients and to correlate myoelectric signals with return of bowel function clinically. METHODS:Pediatric patients undergoing abdominal surgery were recruited to have wireless patches placed on the abdomen within two hours postoperatively. Myoelectric data were transmitted wirelessly to a mobile device with a user-interface and forwarded to a cloud server where processing algorithms identified episodes of motor activity, quantified their parameters and nominally assigned them to specific gastrointestinal organs based on their frequencies. RESULTS:Three patients (ages 5 months, 4 year, 16 year) were recruited for this study. Multiple patches were placed on the older subjects, while the youngest had a single patch due to space limitations. Rhythmic signals of the stomach, small intestine, and colon could be identified in all three subjects. Patients showed gradual increase in myoelectric intestinal and colonic activity leading up to the first recorded bowel movement. CONCLUSION/CONCLUSIONS:Measuring myoelectric intestinal activity continuously using a wireless patch system is feasible in a wide age range of pediatric patients. The increase in activity over time correlated well with the patients' return of bowel function. More studies are planned to determine if this technology can predict return of bowel function or differentiate between physiologic ileus and pathologic conditions.

BACKGROUND:Corrective surgery for pectus excavatum often relies on the Haller index (HI), derived from chest roentgenograms or computed tomography; however, this exposes children to potentially unnecessary radiation. Our aim was to develop a novel three-dimensional (3D) optical imaging technique to accurately measure chest wall dimensions in a clinically relevant manner. METHODS:Patients with pectus excavatum were imaged using a 3D structured light scanner. Patient characteristics, including height, weight, body mass index, and radiographic HIs (rHI) were recorded. We defined the optical index (OI) as the ratio of the lateral to anterior-posterior measurements obtained from the 3D optical images and compared those to patients' rHIs. Two-thirds of the patients' images were used to develop a predictive model of the rHI, using their OI and biometric data in multilinear regression modeling. The predictive model was applied to the remaining images, and the predicted HIs (pHI) were compared to the rHIs. RESULTS:Optical imaging was performed in 42 patients (ages, 5-35 years) with pectus excavatum; of these, 31 had recent chest roentgenograms, with rHIs ranging from 2.00 to 7.20. The OIs derived from the images correlated closely with rHIs (r = 0.850). Our predictive model, using patients' OI, height, and weight, was able to accurately estimate their rHIs with a median error of 8.11% (interquartile range, 3.5%-17.4%). CONCLUSIONS:3D optical imaging of patients with pectus excavatum is emerging as an alternative method to assess HIs without the use of ionizing radiation. Additional studies will focus on volumetric quantification of chest wall deformities, using the 3D capabilities of this technology.

Neuroblastoma is the most common extracranial childhood tumor, and current treatment requires surgical resection and multidrug chemotherapy. Local, perioperative delivery of chemotherapeutics is a promising treatment method for solid tumors that require surgical removal. In this study, we have aimed to develop a controlled-release implant system to deliver cisplatin in tumor or tumor resection area. Silk fibroin, a biodegradable, nonimmunogenic biopolymer was used to encapsulate different doses of cisplatin in a reservoir system. The physical integrity of the reservoirs was characterized by evaluating the crystalline structure of silk secondary structure using FTIR spectroscopy. The in vitro release of cisplatin was evaluated in phosphate-buffered saline at 37°C, and the reservoirs were able to release the drug up to 30 days. The cytotoxicity of cisplatin and cisplatin reservoirs were tested on KELLY cells. Cytotoxicity data showed 3.2 μg/mL cisplatin was required to kill 50% of the cell population, and the released cisplatin from the silk reservoirs showed significant cytotoxicity up to 21 days. Intratumoral implantation of silk reservoirs into an orthotopic neuroblastoma mouse model decreased tumor growth significantly when compared with control subjects. These results suggest that silk reservoirs are promising carriers for cisplatin delivery to the tumor site.

BACKGROUND:Firearm-related injuries are the second leading cause of pediatric deaths in the US. We sought to evaluate the effectiveness of both state child access prevention (CAP) laws and gun regulations on pediatric firearm mortality. We hypothesized that states with more stringent firearm legislation had lower pediatric firearm mortality. STUDY DESIGN:We used 2014-2015 firearm mortality data from the Web-Based Injury Statistics Query and Reporting System, 2014 Brady scores (used to quantify stringency of state gun regulations) and CAP laws. State-level covariates were obtained from government sources, including the Bureau of Labor Statistics and the Department of Education. Spearman rank correlations and linear regression were used to determine the relationship between overall pediatric firearm mortality and gun regulations. We also examined the relationship between gun regulations and firearm-related homicides and suicides. RESULTS:Annually, there were approximately 2,715 pediatric firearm fatalities among children; 62.1% were homicides and 31.4% suicides. There was a moderate negative correlation between states' firearm legislation stringency and overall pediatric firearm mortality (ρ = -0.66; p < 0.001), and between CAP laws and firearm suicide rates (ρ = -0.56; p < 0.001). After controlling for poverty, unemployment, substance abuse, and the number of registered firearms, the association between firearm legislation stringency and overall pediatric firearm mortality remained significant (p = 0.04). The association between CAP laws and firearm suicide rate remained significant after controlling for socioeconomic factors, registered firearms, and other firearm legislation (p = 0.04). CONCLUSIONS:Strict gun legislation and CAP laws are associated with fewer pediatric firearm fatalities and firearm suicides, respectively, though no such association was identified with pediatric firearm homicides. Although more studies are needed to determine causality, state-level legislation could play an important role in reducing pediatric firearm-related deaths.

PURPOSE/OBJECTIVE:MYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461. METHODS:MYCN-amplified (KELLY, IMR5) and nonamplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24-48 h was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed. RESULTS:after 7.8±1.4 days, while those treated with control film reached the same size on 5.1±0.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation. CONCLUSION/CONCLUSIONS:We demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.

BACKGROUND:Pectus excavatum and carinatum are two of the most commonly observed chest wall deformities in pediatrics. The standard diagnostic evaluation for these conditions includes either chest radiograph (CXR) or computed tomography (CT). Our research aims to develop a novel and reliable way of quantifying chest wall deformities in the clinic setting without radiation exposure. METHODS:Using a handheld structured light scanner, we created three-dimensional (3D) models of patients with chest wall deformities through an IRB-approved protocol. Raters from a variety of backgrounds were then asked to take measurements based on the 3D model utilizing commercially available 3D graphical software. The standard deviation of the measurements and intraclass correlation coefficient (ICC) were then calculated to quantify inter-rater reliability. RESULTS:Sixty patients with pectus excavatum (Haller index range 2.0-6.38) and pectus carinatum were enrolled and imaged in our outpatient clinic using a structured light scanner. Five patients were used to verify interuser reliability. The standard deviation of all the measurements was 2.2 mm. The ICC for absolute agreement was 0.99139, with 1.0 being perfect correlation. CONCLUSION/CONCLUSIONS:Structured light scanners provide an alternative approach to quantifying chest wall deformities in pediatric patients without radiation exposure. Our method is highly reliable, even among users with minimal image processing or 3D modeling experience. Our protocol can potentially be used to track treatment progress in children with chest wall deformities.

PURPOSE/OBJECTIVE:Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. METHODS:Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. RESULTS:Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. CONCLUSIONS:Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

BACKGROUND:Advanced-stage neuroblastoma patients require multiagent chemotherapy. Intratumoral implantation of vincristine-loaded silk gel uses local diffusion to decrease orthotopic neuroblastoma tumor growth in mice. We hypothesize that injecting vincristine-loaded silk gel into 8 locations within the tumor, instead of only centrally, decreases the diffusion distance and improves tumor growth suppression. METHODS:and histologic examination. RESULTS:over 3 days. The time to inflection point was 6.6 days for central, 13.3 days for 8-point injection (P < .05). Using the sphere volume equation to approximate tumor volume, splitting the volume into 1/8 decreased the diffusion radius by 1/2. Histologic examination confirmed tumor necrosis adjacent to vincristine-loaded silk gel. CONCLUSION:Injecting vincristine-loaded sustained release silk gel at 8 separate locations halved the diffusion distance and doubled the time for the tumor to reach the growth inflexion point.