Phylicia Dupree

Interdisciplinary approach to esophageal replacement and major airway reconstruction

PURPOSE Severe esophageal disease warranting replacement often presents with additional airway anomalies in children. Colon interposition and airway reconstruction have separately proven successful in attaining satisfactory outcomes. The aim of this study was to determine outcomes associated with an interdisciplinary approach to care of the patient with complex esophageal and airway disease. METHODS After IRB approval, a retrospective cohort study was performed spanning 2011 through 2015. Eleven patients underwent colon interposition and airway surgery. Review of medical records was performed, extracting patient demographics, clinical and operative courses and outcomes. RESULTS The mean age of patients was 44months (range 2-108). 91% (n=10) were transferred to our institution with primary diagnoses of caustic ingestion (45%, n=5), long gap esophageal atresia (27% n=3), tracheoesophageal fistula (18%, n=2) and necrotizing pharyngitis (9% n=1). All patients had associated airway anomalies. Pulmonology, gastroenterology and speech therapy were involved in preoperative evaluation and postoperative care of all patients. Intraoperatively, a multi-team approach was utilized. The most common postoperative complication was esophageal stricture (54%, n=6). All patients are capable of taking some to full nutrition per orum. CONCLUSION Colonic interposition with major airway reconstruction at our institution attains satisfactory functional results through utilization of a multidisciplinary approach.

RHESUS ROTAVIRUS VP4 SEQUENCE-SPECIFIC ACTIVATION OF MONONUCLEAR CELLS IS ASSOCIATED WITH CHOLANGIOPATHY IN MURINE BILIARY ATRESIA

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRVs VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

The SRL peptide of Rhesus Rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia.

Biliary atresia (BA) is a neonatal obstructive cholangiopathy that progresses to end‐stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that the RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Using rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains that cause obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4‐derived peptide (TRTRVSRLY) significantly reduced the ability of RRV to bind and infect cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains that do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by small interfering RNAs reduced RRVs ability to infect cholangiocytes. This virus‐cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV‐injected mice. Conclusion: The tripeptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70, defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response to this interaction to understand how it contributes to the pathogenesis of BA. (Hepatology 2017;65:1278‐1292)

Cystic Biliary Atresia and Choledochal Cysts Are Distinct Histopathologic Entities

Cystic biliary atresia (CBA), a rare cystic expansion of atretic extrahepatic bile ducts in young infants, overlaps in age at presentation and imaging features with early choledochal cysts (CC). Treatment and prognosis differ; histologic differences are unsettled. We compared 10 patients with CBA, 1975 to 2015, to an age-similar cohort of 13 infants, and to older patients who had surgery for CC. Operative details, imaging, and clinical courses were correlated to pathologic specimens. Immunostains for smooth muscle actin and myosin heavy chain were used to evaluate cyst walls and atretic segments. CBA cysts typically lacked epithelium and inflammation; cyst walls had an inner, dense cicatricial layer associated with myofibroblastic (MF) hyperplasia that often delaminated producing a grossly visible inner cyst wall. Seven proximal biliary remnants in CBA featured circumferential peribiliary MF hyperplasia/fibrosis with little or no inflammation, similar to isolated BA. Extrahepatic atresia was usually both proximal and distal to the cyst. Features in 10/13 CC from infants and 8/8 CC in older patients had mostly preserved uninjured epithelium and no subepithelial cicatrix. Mural smooth muscle (absent in CBA) was present to some extent in CC at all ages. Unexpectedly, focal MF hyperplasia and laminar sclerosis was present in a few CC in infants, resembling CBA. CBA and infant CC are distinct histologic entities that occasionally overlap. CBA bile duct injury mimics non-CBA. Cystification is an aberrant manifestation of stromal proliferation in BA. The current management approach assuming CBA and CC in infants are 2 separate disease processes is supported but caution is advised.

Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes.

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.

Quality assessment of lymph node sampling in rhabdomyosarcoma: A surveillance, epidemiology, and end results (SEER) program study

BACKGROUND Lymph node sampling is integral in the management of extremity and paratesticular rhabdomyosarcoma (RMS). The aim of this study was to determine overall surgical compliance with treatment protocols and impact of nodal sampling outcomes in these tumors. METHODS A query of the surveillance, epidemiology, and end results program (SEER) database was performed from 2003 to 2008 for patients <19years of age with RMS. Data obtained included demographics, five-year survival and rate of nodal sampling. Analysis was performed utilizing chi-squared, Kaplan-Meier and hazard ratio modeling. RESULTS Of 537 patients with extremity RMS, nodal sampling was performed in 25.7% (n=138). This lack of nodal sampling had a negative outcome on survival (p=0.004). Sixty five patients with paratesticular RMS aged greater than 10 were identified and also displayed low rates of lymph node sampling (47.7%, n=31). For paratesticular patients, a similar increase in survival was seen in patients who underwent nodal evaluation (p=0.024). CONCLUSION Lymph node sampling is the standard of care in RMS. However, surgical compliance with treatment protocols is poor. Nodal evaluation correlated significantly with overall survival. These findings suggest a need for improved education among surgeons and oncologists regarding the need lymph node assessment in pediatric oncology patients. Evidence rating/classification: Prognosis study, Level III.

A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

The management of pediatric renovascular hypertension: a single center experience and review of the literature

INTRODUCTION Renal artery occlusive disease is poorly characterized in children; treatments include medications, endovascular techniques, and surgery. We aimed to describe the course of renovascular hypertension (RVH), its treatments and outcomes. METHODS We performed literature review and retrospective review (1993-2014) of children with renovascular hypertension at our institution. Response to treatment was defined by National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents at most-recent follow-up. RESULTS We identified 39 patients with RVH. 54% (n=21) were male, with mean age of 6.93 ± 5.27 years. Most underwent endovascular treatment (n=17), with medication alone (n=12) and surgery (n=10) less commonly utilized. Endovascular treatment resulted in 18% cure, 65% improvement and 18% failure; surgery resulted in 30% cure, 50% improvement and 20% failure. Medication alone resulted in 0% cure, 75% improvement and 25% failure. 24% with endovascular treatment required secondary endovascular intervention; 18% required secondary surgery. 20% of patients who underwent initial surgery required reoperation for re-stenosis. Mean follow-up was 52.2 ± 58.4 months. CONCLUSIONS RVH treatment in children includes medications, surgical or endovascular approaches, with all resulting in combined 79% improvement in or cure rates. A multidisciplinary approach and individualized patient management are critical to optimize outcomes. TYPE OF STUDY Retrospective comparative study LEVEL OF EVIDENCE: Level III.