Jessica A. Zagory

Characterization of hepatic stellate cells, portal fibroblasts, and mesothelial cells in normal and fibrotic livers

BACKGROUND & AIMS Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs) to myofibroblasts is not fully understood due to insufficient availability of markers and isolation methods. The present study aimed to isolate these cells, characterize their phenotypes, and examine their contribution to myofibroblasts in liver fibrosis. METHODS Liver fibrosis was induced in Collagen1a1-green fluorescent protein (Col1a1(GFP)) mice by bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, or CCl4 injections. Combining vitamin A (VitA) lipid autofluorescence and expression of GFP and glycoprotein M6a (GPM6A), we separated HSCs, PFs, and MCs from normal and fibrotic livers by fluorescence-activated cell sorting (FACS). RESULTS Normal Col1a1(GFP) livers broadly expressed GFP in HSCs, PFs, and MCs. Isolated VitA+ HSCs expressed reelin, whereas VitA-GFP+GPM6A- PFs expressed ectonucleoside triphosphate diphosphohydrolase-2 and elastin. VitA-GFP+GPM6A+ MCs expressed keratin 19, mesothelin, and uroplakin 1b. Transforming growth factor (TGF)-β1 treatment induced the transformation of HSCs, PFs, and MCs into myofibroblasts in culture. TGF-β1 suppressed cyclin D1 mRNA expression in PFs but not in HSCs and MCs. In biliary fibrosis, PFs adjacent to the bile duct expressed α-smooth muscle actin. FACS analysis revealed that HSCs are the major source of GFP+ myofibroblasts in the injured Col1a1(GFP) mice after DDC or CCl4 treatment. Although PFs partly contributed to GFP+ myofibroblasts in the BDL model, HSCs were still dominant source of myofibroblasts. CONCLUSION HSCs, PFs, and MCs have distinct phenotypes, and PFs partly contribute to myofibroblasts in the portal triad in biliary fibrosis.

Recent advances in the pathogenesis and management of biliary atresia.

Purpose of review The purpose of this study is to review advances in both the pathogenesis and clinical management of biliary atresia. Recent findings Immunologic studies have further characterized roles of helper T-cells, B-cells, and natural killer cells in the immune dysregulation following viral replication within and damage of biliary epithelium. Prominin-1-expressing portal fibroblasts may play an integral role in the biliary fibrosis associated with biliary atresia. A number of genetic polymorphisms have been characterized as leading to susceptibility for biliary atresia. Postoperative corticosteroid therapy is not associated with greater transplant-free survival. Newborn screening may improve outcomes of infants with biliary atresia and may also provide a long-term cost benefit. Summary Although recent advances have enhanced our understanding of pathogenesis and clinical management, biliary atresia remains a significant challenge requiring further investigation.

Management of pediatric blunt renal trauma: A systematic review.

BACKGROUND Blunt trauma remains a significant cause of morbidity and mortality in the pediatric population. The use of conservative management for blunt renal trauma is widely accepted in adult trauma literature and is now increasingly accepted for use in the pediatric patient population. This study aimed to review current practices in pediatric blunt renal trauma management and to highlight current practices in conservative protocols, success rates of conservative management strategies, as well as short- and long-term outcomes of blunt renal trauma management. METHODS This is a systematic review of PubMed, Ovid, and the Cochrane Library. The following search was performed in each of the three databases: (Renal or Kidney) AND (Pediatric or Children) AND Trauma AND Management. Publications were limited to publish date after January 1, 2000. Inclusion criteria were (1) original research articles regarding management of pediatric blunt renal trauma, (2) involvement of cases of high-grade renal (Grades IV and V) trauma, and (3) more than one patient presented per study. Literature reviews and meta-analyses were excluded. RESULTS Titles and abstracts (n = 308) were screened to identify scientific articles reporting original research findings. A total of 32 articles met the selection criteria and were included in the review. CONCLUSION The literature supports application of conservative management protocols to high-grade blunt pediatric renal trauma. Criteria for early operative intervention are not well understood. At this time, emergent operative intervention only for hemodynamic instability is recommended. Minimally invasive interventions including angioembolization, stenting, and percutaneous drainage should be used when indicated. Short- and long-term outcomes are favorable when using conservative management approaches to Grade IV and V renal injuries. Further studies including prospective studies and randomized control trials are necessary. Cost analyses of current treatment protocols are also necessary to guide efficient management strategies. LEVEL OF EVIDENCE Systematic review, level III.

Limiting chest computed tomography in the evaluation of pediatric thoracic trauma.

BACKGROUND Computed tomography (CT) of the chest (chest CT) is overused in blunt pediatric thoracic trauma. Chest CT adds to the diagnosis of thoracic injury but rarely changes patient management. We sought to identify a subset of blunt pediatric trauma patients who would benefit from a screening chest CT based on their admission chest x-ray (CXR) findings. We hypothesize that limiting chest CT to patients with an abnormal mediastinal silhouette identifies intrathoracic vascular injuries not otherwise seen on CXR. METHODS All blunt trauma activations that underwent an admission CXR at our Level 1 pediatric trauma center from 2005 to 2013 were retrospectively reviewed. Patients who had a chest CT were evaluated for added diagnoses and change in management after CT. RESULTS An admission CXR was performed in 1,035 patients. One hundred thirty-nine patients had a CT, and the diagnosis of intra-thoracic injury was added in 42% of patients. Chest CT significantly increased the diagnosis of contusion or atelectasis (30.3% vs 60.4%; p < 0.05), pneumothorax (7.2% vs 18.7%; p < 0.05), and other fractures (4.3% vs 10.8%; p < 0.05) on CXR compared to chest CT. Chest CT changed the management of only 4 patients (2.9%). Two patients underwent further radiologic evaluation that was negative for injury, one had a chest tube placed for an occult pneumothorax before exploratory laparotomy, and one patient had a thoracotomy for repair of aortic injury. Chest CT for select patients with an abnormal mediastinal silhouette on CXR would have decreased CT scans by 80% yet still identified patients with an intrathoracic vascular injury. CONCLUSIONS The use of chest CT should be limited to the identification of intrathoracic vascular injuries in the setting of an abnormal mediastinal silhouette on CXR. LEVEL OF EVIDENCE Therapeutic study, level IV; diagnostic study, level III.

Heller Myotomy Is Superior to Balloon Dilatation or Botulinum Injection in Children with Achalasia: A Two-Center Review

INTRODUCTION Achalasia is an uncommon disorder in children. Currently, there is no consensus regarding the optimal treatment for achalasia. We investigate the effectiveness of symptom relief in patients who underwent endoscopic treatments versus Heller myotomy (HM). METHODS We conducted a retrospective review of all children (age 0-18 years) treated for achalasia at two pediatric hospitals from 2004 to 2014. Demographics, presenting symptoms, outcomes, and complications were analyzed. RESULTS Twenty-three patients (61% male) were identified with a mean age at diagnosis of 11.6 ± 5.0 years. About 47.8% of the cohort had no comorbidities. Common presenting symptoms included weight loss/failure to thrive (87.0%), emesis (69.6%), and dysphagia (69.6%). Mean time from symptom onset to diagnosis was 18 ± 18.9 months. Nine patients underwent laparoscopic HM as their primary treatment, whereas 14 received esophageal dilatation (ED) as their first-line therapy. Patients who underwent ED as their initial treatment were younger (9.92 versus 15.6 years, P = .047). Patients who underwent HM were more likely to attain symptom resolution compared to those managed with ED alone (P = .004). Of the 14 patients who underwent ED initially, 10 subsequently required HM due to persistent symptoms. None of the 4 patients who underwent ED alone achieved long-term symptom relief and, on the average, required an increased number of procedures compared to their HM counterparts (5.25 versus 2.47, P = .010). There was a trend toward increased intraoperative mucosal perforation in patients who underwent preoperative ED and botulinum injections. CONCLUSION Our data suggest that HM is superior to balloon dilatation or botulinum injection in children with achalasia. We conclude that HM should be recommended for newly diagnosed children with achalasia as a first-line therapy.

Hepatic Prominin-1 expression is associated with biliary fibrosis

Background. Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin‐1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin‐1 expression is biliary fibrosis‐specific. Methods. Gene expression of Prominin‐1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin‐1‐expressing cells and Collagen‐1&agr;‐expressing cells was performed after bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl and Collagen‐1&agr;Gfp transgenic mice, respectively. Results. Prominin‐1 expression increased significantly after bile duct ligation compared with sham (6.6 ± 0.9‐fold change at 2 weeks, P < .05) but not with carbon tetrachloride (−0.7 ± 0.5‐fold change, not significant). Upregulation of Prominin‐1 was observed histologically throughout the liver as early as 5 days after bile duct ligation in Prominin‐1cre‐ert2−lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin‐1‐expressing cells labeled prior to bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN‐19 within ductular reactions up to 5 weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker &agr;‐SMOOTH MUSCLE ACTIN in Prominin‐1cre‐ert2−lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ‐1 in Collagen‐1&agr;Gfp mice, indicate minimal contribution of Prominin‐1 progenitor cells to the pool of collagen‐producing myofibroblasts. Conclusion. During biliary fibrosis Prominin‐1‐expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Focused assessment with sonography for trauma in children after blunt abdominal trauma: A multi-institutional analysis

Introduction The utility of focused assessment with sonography for trauma (FAST) in children is poorly defined with considerable practice variation. Our purpose was to investigate the role of FAST for intra-abdominal injury (IAI) and IAI requiring acute intervention (IAI-I) in children after blunt abdominal trauma (BAT). Methods We prospectively enrolled children younger than 16 years after BAT at 14 Level I pediatric trauma centers over a 1-year period. Patients who underwent FAST were compared with those that did not, using descriptive statistics and univariate analysis; p value less than 0.05 was considered significant. FAST test characteristics were performed using computed tomography (CT) and/or intraoperative findings as the gold standard. Results Two thousand one hundred eighty-eight children (age, 7.8 ± 4.6 years) were included. Eight hundred twenty-nine (37.9%) received a FAST, 340 of whom underwent an abdominal CT. Ninety-seven (29%) of these 340 patients had an IAI and 27 (7.9%) received an acute intervention. CT scan utilization after FAST was 41% versus 46% among those who did not receive FAST. The frequency of FAST among centers ranged from 0.84% to 94.1%. There was low correlation between FAST and CT utilization (r = −0.050, p < 0.001). Centers that performed FAST at a higher frequency did not have improved accuracy. The test performance of FAST for IAI was sensitivity, 27.8%; specificity, 91.4%; positive predictive value, 56.2%; negative predictive value, 76.0%; and accuracy, 73.2%. There were 81 injuries among the 70 false-negative FAST. The test performance of FAST for IAI-I was sensitivity, 44.4%; specificity, 88.5%; positive predictive value, 25.0%; negative predictive value, 94.9%; and accuracy, 85.0%. Fifteen children with a negative FAST received acute interventions. Among the 27 patients with true positive FAST examinations, 12 received intervention. All had an abnormal abdominal physical examination. No patient underwent intervention before CT scan. Conclusion As currently used, FAST has a low sensitivity for IAI, misses IAI-I and rarely impacts management in pediatric BAT. LEVEL OF EVIDENCE Prognostic and epidemiologic study, level II; diagnostic tests or criteria study, level II; therapeutic/care management study, level III.

Toll-like receptor 3 mediates PROMININ-1 expressing cell expansion in biliary atresia via Transforming Growth Factor-Beta

BACKGROUND In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-β (TGFβ) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFβ pathway activation in vitro. METHODS PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), ± small molecule inhibitors nafamostat, or SB431542. RESULTS Poly I:C induced myofibroblastic-like morphologic changes, degradation of IκB-α consistent with TLR3-NFκB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1α staining following Poly I:C treatment. Degradation of IκBα was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. CONCLUSIONS TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFβ pathway activation to promote BA-associated biliary fibrosis.

Liquid chromatography–mass spectroscopy as a tool in the rapid diagnosis of biliary atresia: a pilot study

INTRODUCTION Biliary atresia (BA) is a neonatal obstructive cholangiopathy requiring rapid intervention to prevent end-stage liver failure and death. Low bile acid levels in stool, detectable with high-performance liquid chromatography-mass spectroscopy, may reflect extrahepatic biliary obstruction in cholestasis. HYPOTHESIS Stool bile acid content can differentiate BA from non-BA forms of cholestasis. METHODS Stool samples from four healthy and nine cholestatic patients were collected following internal review board approval. Bile acids were extracted and separated on a 4000-Q-Trap HPLC-MS system. RESULTS Total bile acid content was highest in samples from healthy relative to cholestatic patients: 3354.01 ± 2102.56, 1476.27 ± 1361.07, and 34.29 ± 10.30 μM/mg of stool in healthy, total parenteral nutrition-associated cholestasis, and BA samples, respectively. Mean cholic acid and chenodeoxycholic acid concentrations in healthy samples (2017.5 ± 1413.6 and 876.83 ± 660.60 μM/mg) were higher than in TPN cholestatic samples (93.99±131.55 and 232.34 ± 293.41 μM/mg). The most dramatic reduction in cholic acid and chenodeoxycholic acid was observed in BA samples (0.65 ± 0.47 and 1.22 ± 0.80 μM/mg). CONCLUSION Bile acid content in stool is reduced in cholestatic patients relative to healthy patients with the most dramatic reduction observed in BA-patients.

Planning for the Pediatric Patient During a Disaster

Purpose of ReviewDisaster and mass casualty events are unpredictable and quickly overwhelm response systems. Children are affected by disasters; however, pediatric-specific disaster preparedness is lacking. Preparedness is required at the individual, physician, and hospitals/community levels and constitutes material allocation, education, and communication. Specifically, pediatricians, pediatric subspecialists, and pediatric surgical subspecialists have important roles during a disaster, mass casualty, or pandemic response; thus, integration of both community and subspecialty physicians is important. Disaster response is not limited to planning and response; major efforts in recovery, reunification, community health safety, and follow-up are required.Recent FindingsEvents have highlighted the need for improved preparation and execution of mass casualty response. There is a need for training in the event of a mass casualty disaster, and pediatric professionals have indicated that they do not feel adequately prepared to care for children during such events. Incorporation of pediatric-specific simulation scenarios can help address this deficiency.SummaryThus, efforts to incorporate pediatric-specific education, disaster simulation, and preparation including a multidisciplinary approach integrating families, physicians, and hospital and community organizations are required.