Eric J. Rellinger

Natural history of grade I-II blunt traumatic aortic injury.

BACKGROUND Endovascular aortic repair has revolutionized the management of traumatic blunt aortic injury (BAI). However, debate continues about the extent of injury requiring endovascular repair, particularly with regard to minimal aortic injury. Therefore, we conducted a retrospective observational analysis of our experience with these patients. METHODS We retrospectively reviewed all BAI presenting to an academic level I trauma center over a 10-year period (2000-2010). Images were reviewed by a radiologist and graded according to Society for Vascular Surgery guidelines (grade I-IV). Demographics, injury severity, and outcomes were recorded. RESULTS We identified 204 patients with BAI of the thoracic or abdominal aorta. Of these, 155 were deemed operative injuries at presentation, had grade III-IV injuries or aortic dissection, and were excluded from this analysis. The remaining 49 patients had 50 grade I-II injuries. We managed 46 grade I injuries (intimal tear or flap, 95%), and four grade II injuries (intramural hematoma, 5%) nonoperatively. Of these, 41 patients had follow-up imaging at a mean of 86 days postinjury and constitute our study cohort. Mean age was 41 years, and mean length of stay was 14 days. The majority (48 of 50, 96%) were thoracic aortic injuries and the remaining two (4%) were abdominal. On follow-up imaging, 23 of 43 (55%) had complete resolution of injury, 17 (40%) had no change in aortic injury, and two (5%) had progression of injury. Of the two patients with progression, one progressed from grade I to grade II and the other progressed from grade I to grade III (pseudoaneurysm). Mean time to progression was 16 days. Neither of the patients with injury progression required operative intervention or died during follow-up. CONCLUSIONS Injury progression in grade I-II BAI is rare (~5%) and did not cause death in our study cohort. Given that progression to grade III injury is possible, follow-up with repeat aortic imaging is reasonable.

Surgical necrotizing enterocolitis

Although currently available data are variable, it appears that the incidence of surgical necrotizing enterocolitis (NEC) has not decreased significantly over the past decade. Pneumoperitoneum and clinical deterioration despite maximal medical therapy remain the most common indications for operative treatment. Robust studies linking outcomes with specific indications for operation are lacking. Promising biomarkers for severe NEC include fecal calprotectin and S100A12; serum fatty acid-binding protein; and urine biomarkers. Recent advances in ultrasonography make this imaging modality more useful in identifying surgical NEC and near-infrared spectroscopy (NIRS) is being actively studied. Another fairly recent finding is that regionalization of care for infants with NEC likely improves outcomes. The neurodevelopmental outcomes after surgical treatment are known to be poor. A randomized trial near completion will provide robust data regarding neurodevelopmental outcomes after laparotomy versus drainage as the initial operative treatment for severe NEC.

Promoter analysis of ventricular myosin heavy chain (vmhc) in zebrafish embryos

In zebrafish, ventricular myosin heavy chain (vmhc) gene is initially expressed at the anterior lateral mesoderm and thereafter its expression is restricted to the cardiac ventricle. The transcriptional control mechanisms in regulating chamber‐specific expression of myosin heavy chains are not well defined. We isolated and analyzed zebrafish vmhc upstream region to examine the spatial and temporal regulation of vmhc using transgenic and transient expression techniques. Promoter deletion analyses defined a basal promoter region sufficient to drive vmhc expression in the ventricle and an upstream fragment necessary for repressing ectopic vmhc expression in the atrium. The transcriptional mechanism that prevents vmhc expression in the atrium is mediated through Nkx2.5 binding elements (NKE). We have further discovered that paired‐related homeobox transcriptional factor 2 (Prx2/S8)‐like binding elements are required for promoting vmhc expression, and Prrx1b, a Prx‐related homeobox protein, participates in the regulation of vmhc expression with other transcriptional factors. Developmental Dynamics 238:1760–1767, 2009.

Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells.

Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia‐inducible factor‐1 alpha (HIF‐1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin‐releasing peptide receptor (GRP‐R) signaling regulates glucose metabolism.

FX11 inhibits aerobic glycolysis and growth of neuroblastoma cells

Background. The MYC family of proteins promotes neuroblastoma tumorigenesis at least in part through the induction of aerobic glycolysis by promoting the transcription of key glycolytic enzymes, such as LDHA. FX11 is a selective inhibitor of LDHA that has demonstrated preclinical efficacy in adult cancers. Herein, we hypothesized that FX11 would inhibit aerobic glycolysis and block growth of neuroblastoma cells. Methods. We surveyed 3 MYCN–single copy and 5 MYCN‐amplified neuroblastoma cell lines to correlate C‐MYC/N‐MYC protein levels with LDHA expression. Cell viability was measured with FX11 using a tetrazolium‐based assay. Cell cycle analysis using propidium iodide with flow cytometry was performed to evaluate for growth arrest. Immunoblotting demonstrated PARP and Caspase 3 cleavage as evidence of apoptosis. Results. LDHA is frequently expressed in both MYCN‐‐amplified and MYCN‐single copy cell lines. N‐MYC and C‐MYC protein levels did not correlate with LDHA protein expression. FX11 inhibits aerobic glycolysis and growth in three MYCN‐amplified and one MYCN–single copy neuroblastoma cell lines. FX11 induces modest G1 cell cycle arrest with selective induction of apoptosis. Conclusion. Small molecule LDHA inhibition is capable of blocking aerobic glycolysis and growth of neuroblastoma cell lines in vitro and merits further in vivo evaluation of its preclinical efficacy in neuroblastomas.

Induced differentiation inhibits sphere formation in neuroblastoma

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

Isoxazole compound ML327 blocks MYC expression and tumor formation in neuroblastoma

Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ∼30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ~30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.

cFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition

Epithelial cancers (carcinomas) comprise the top four causes of cancer-related deaths in the United States. While overall survival has been steadily improving, therapy-resistant disease continues to present a major therapeutic challenge. Carcinomas often exploit the normal developmental program, epithelial-to-mesenchymal transition (EMT), to gain a mesenchymal phenotype associated with increased invasiveness and resistance to apoptosis. We have previously shown that an isoxazole-based small molecule, ML327, partially reverses TGF-β-induced EMT in an immortalized mouse mammary epithelial cell line. Herein, we demonstrate that ML327 reverses much of the EMT gene expression program in cultured carcinoma cell lines. The reversal of EMT sensitizes these cancer cells to the apoptosis-inducing ligand TRAIL. This sensitization is independent of E-cadherin expression and rather relies on the downregulation of a major anti-apoptotic protein, cFLIPS. Loss of cFLIPS is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIPS restores resistance to TRAIL-induced apoptosis despite EMT reversal with ML327. In summary, we have utilized an isoxazole-based small molecule that partially reverses EMT in carcinoma cells to demonstrate that cFLIPS critically regulates the apoptosis resistance phenotype associated with EMT.

Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma

Approximately two-thirds of patients with neuroblastoma are found to have metastatic disease at time of diagnosis with frequent skeletal, lymph node, central nervous system, and liver involvement. Using a serial in vivo splenic injection model, we have isolated an aggressive subclone (BE(2)-C/LM2) from MYCN-amplified neuroblastomas that demonstrate an enhanced propensity to develop metastatic liver lesions. BE(2)-C/LM2 subclone cells demonstrate increased adherent, soft agar colony and tumorsphere growth in vitro. Components of the tumor microenvironment regulate cancer progression, via networks of cytokines and growth factors. Cytokine array analysis identified increased TIMP-1 in the plasma of mice injected with BE(2)-C/LM2 subclone cells, leading us to hypothesize that TIMP-1 may play a role in our observed prometastatic phenotype. Immunoblotting and ELISA demonstrated enhanced endogenous TIMP-1 expression in our isolated neuroblastoma subclone. Silencing endogenous TIMP-1 successfully blocked in vitro proliferation, soft agar colony formation and tumorsphere formation by BE(2)-C/LM2 cells. Stable RNA interference of endogenous TIMP-1 failed to reverse the prometastatic phenotype of our BE(2)-C/LM2 subclone in our liver metastasis model, suggesting that endogenous TIMP-1 levels may not be an essential component of this in vivo behavior. Notably, tissue microarray analysis and Kaplan-Meier by gene expression demonstrates that elevated TIMP-1 expression is correlated with increased disease relapse and mortality in patients with neuroblastoma. Taken together, our study identifies TIMP-1 as a novel soluble factor that is associated with a prometastatic phenotype in our in vivo model and adverse outcomes in patients with neuroblastoma.

Acute Right Lower Quadrant Abdominal Pain

A previously healthy 4-year-old girl with a history of prematurity (31 weeks’ gestational age) presented as a transfer from an outside facility with abdominal pain. She had presented to an outside emergency department 2 days prior with lower abdominal pain that subsequently localized to the right lower quadrant. Her pain was accompanied by anorexia and nonbloody, nonbilious emesis. She denied fevers or chills. She was noted to have voluntary guarding and focal right lower quadrant tenderness to palpation. Laboratory analyses demonstrated a mild leukocytosis (white blood cell count = 14 × 10/μL [to convert to ×10/L, multiply by 0.001]) and normal urinalysis results. Abdominal ultrasonography images were obtained that failed to show her appendix and demonstrated no secondary signs of inflammation. Because of her notable tenderness, she was admitted for observation at the referring facility. No antibiotics were initiated, and her activity improved with intravenous hydration. However, her right lower abdominal pain persisted, prompting further evaluation. Computed tomography images of the abdomen and pelvis were obtained, and the representative images are shown in Figure 1. She was transferred to our facility for surgical evaluation. In our emergency department, pelvic ultrasonography images were obtained that failed to show both the appendix and right adnexal structures. Axial section A Coronal section B