Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

BACKGROUND: The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. METHODS: Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. RESULTS: The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. CONCLUSION: We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

Retrospective Comparison of Fluticasone Propionate and Oral Viscous Budesonide in Children With Eosinophilic Esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is treated with dietary modification and/or pharmacologic management with swallowed topical steroids. Swallowed fluticasone propionate (FP) and oral viscous budesonide (OVB) have proven to be effective in resolving symptoms and reversing histologic changes in children and adults with EoE. There are minimal comparative studies between the 2 agents. OBJECTIVE: The aim of the study was to retrospectively compare endoscopic and histologic outcomes after FP versus OVB therapy in children with EoE in our center. METHODS: We performed a retrospective chart review of subjects diagnosed with EoE at a tertiary care center between 2010 and 2015. Inclusion criteria were FP or OVB therapy for ≥8 weeks along with pre- and post-treatment endoscopic evaluation. Demographic and clinical features and endoscopic and histologic assessment were recorded for comparative analysis. Histologic response was defined as <15 eos/hpf and remission as <5 eos/hpf. RESULTS: The study included 68 EoE patients (20 FP and 48 OVB) with a mean age of 10.6 ±â€Š5.2 years (range 1-20 years); 81% were boys and 68% were Caucasian. No significant demographic or clinical differences were noted between the 2 study groups. Overall histologic response to topical steroids was seen in 44 of 68 (65%) patients. A significantly greater number of patients achieved histologic response with OVB (36/48, 75%) than with FP (8/20, 40%) (P = 0.0059). Mean pretreatment peak eos/hpf was 46 ±â€Š19 in the FP group versus 45 ±â€Š23 in the OVB group. Mean post-treatment peak eos/hpf was 20 ±â€Š29 in the FP group versus 12 ±â€Š16 in the OVB group (P = 0.002). There was also a significantly greater difference in the change of absolute eos/hpf from pre- to post-treatment in the OVB group (-33) versus FP (18) (P = 0.047). A greater number of OVB-treated patients without asthma had a histologic response compared to those with asthma (P = 0.031). The response to OVB was not affected by the delivery vehicle, namely sucralose (Splenda) versus Neocate Duocal. CONCLUSIONS: Our data suggest that treatment with OVB leads to better endoscopic and histologic outcomes than FP. Adherence to treatment and history of asthma are major determining factors in the response to treatments. Using Neocate Duocal as the OVB delivery vehicle is just as effective as sucralose.

Severe Necrotizing Pancreatitis in a Pediatric Patient with COVID-19: A Case Report.

We describe a 15-year-old female diagnosed with necrotizing pancreatitis in the setting of coronavirus disease 2019 with severe complications including splenic vein and portal vein thromboses, pleural effusion requiring chest tube, acute hypoxic respiratory failure requiring noninvasive positive-pressure ventilation, and new-onset insulin-dependent diabetes mellitus, requiring over a month-long hospitalization. Following discharge, the patient experienced a prolonged loss of appetite, nausea, and extreme weight loss., During her prolonged hospitalization, she was diagnosed with necrotizing pancreatitis with walled-off collection which was ultimately treated with transgastric endoscopic ultrasound-guided drainage, multiple endoscopic necrosectomies, lumen-apposing metal stents, and double-pigtail plastic stent. Nine months after her initial presentation, patient's clinical symptoms improved, and her weight stabilized. This case highlights the importance of recognizing acute and necrotizing pancreatitis and its morbidities as complications associated with coronavirus disease 2019.

Efficacy of Dairy Free Diet and 6-Food Elimination Diet as Initial Therapy for Pediatric Eosinophilic Esophagitis: A Retrospective Single-Center Study.

PURPOSE: Management of eosinophilic esophagitis (EoE) varies from center to center. In this study, we evaluated the effectiveness of a dairy-free diet (DFD) and the 6-Food Elimination Diet (SFED) as initial therapies for the treatment of EoE in our practice. METHODS: This was a retrospective study of children who had been treated for EoE at Connecticut Children's Medical Center, Hartford, CT, USA. Pre- and post-treatment endoscopy findings and histology results of patients treated with DFD or SFED were examined. RESULTS: One hundred fifty-two patients (age 9.2±5.2 years, 76.3% male, 69.7% caucasian) met the inclusion criteria for initial treatment with DFD (n=102) or SFED (n=50). Response for DFD was 56.9% and for SFED was 52.0%. Response based on treatment duration (<10, 10-12, and >12 weeks) were 81.8%, 50.0%, and 55.1% for DFD, and 68.8%, 50.0%, and 40.0% for SFED. Response based on age (<6, 6-12, and >12 years) were 59.3%, 42.9%, and 67.5% for DFD, and 36.4%, 58.8%, and 72.7% for SFED. In patients treated with DFD, concomitant proton pump inhibitor (PPI) administration resulted in improved outcomes (p=0.0177). Bivariate regression analysis showed that PPI with diet is the only predictor of response (p=0.0491), however, there were no significant predictors on multiple regression analysis. CONCLUSION: DFD and SFED are effective first line therapies for EoE. DFD should be tried first before extensive elimination diets. Concomitant therapy with PPI's may be helpful.

Treatment with high-dose proton pump inhibitors helps distinguish eosinophilic esophagitis from noneosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EE) is a clinical entity that is recognized increasingly in children. The treatment of EE has been debated since its identification as a clinical entity separate from reflux esophagitis. We hypothesize that the treatment with a high-dose proton pump inhibitor (HDPPI) helps differentiate EE from noneosinophilic esophagitis (NEE). PATIENTS AND METHODS: Retrospective review of 2221 patients who underwent esophagogastroduodenoscopy (EGD) with biopsies was undertaken. Sixty-nine patients had more than or equal to 15 eosinophils/high-power field (eos/HPF) in 1 or more esophageal levels. Of those, 36 were initially treated with HDPPI for 3 months followed by repeat EGD. Patients who demonstrated histologic response were classified as NEE. Patients with no histologic response were diagnosed as having EE and treated with HDPPI+swallowed fluticasone for 3 months followed by repeat EGD. RESULTS: Of the 36 patients, histologic response was seen in 14 (39%) after treatment with HDPPI; 95% confidence interval (0.23-0.54). Swallowed fluticasone was added to the treatment of the 22 patients who did not show histologic response to HDPPI alone. Of those, 15 patients underwent repeat endoscopies. Seven patients were lost to follow-up or did not have repeated EGDs. Histologic response was observed in 9 of 15 (60%) patients. Of the nonresponders (6 of 15), 5 of 6 (83%) self-reported noncompliance with the swallowed fluticasone. Patients with more than or equal to 15 eos/HPF at all 3 levels (25 of 36) were less likely to respond to HDPPI alone and more likely to be categorized as EE (18 of 25), P=or<0.043. Symptomatically, 28 of 36 patients reported resolution of symptoms after HDPPI therapy alone, P=or<0.0001, regardless of histology. Visual endoscopic findings during the first and second EGDs did not show any significance in differentiating EE from NEE, P=0.625 and P=0.2405, respectively. CONCLUSIONS: The study demonstrates that HDPPI can be used to help differentiate EE from NEE histologically. Moreover, patients with more than or equal to 15 eos/HPF at all 3 levels are less likely to respond to HDPPI than patients with more than or equal to 15 eos/HPF at fewer than 3 levels. Therefore, having more than or equal to 15 eos/HPF at 1 or 2 biopsy levels does not necessarily establish the diagnosis of EE. Symptomatic response to HDPPI does not correlate with histologic findings. Clinical management guided by EGD with biopsy helps distinguish patients with EE from those with NEE.

Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model.

BACKGROUND: Pediatric patients suffering from long gap esophageal defects or injuries are in desperate need of innovative treatment options. Our study demonstrates that two different cell sources can adhere to and proliferate on a retrievable synthetic scaffold. In feasibility testing of translational applicability, these cell seeded scaffolds were implanted into piglets and demonstrated esophageal regeneration. METHODS: Either porcine esophageal epithelial cells or porcine amniotic fluid was obtained and cultured in 3 dimensions on a polyurethane scaffold (Biostage). The amniotic fluid was obtained prior to birth of the piglet and was a source of mesenchymal stem cells (AF-MSC). Scaffolds that had been seeded were implanted into their respective Yucatan mini-swine. The cell seeded scaffolds in the bioreactor were evaluated for cell viability, proliferation, genotypic expression, and metabolism. Feasibility studies with implantation evaluated tissue regeneration and functional recovery of the esophagus. RESULTS: Both cell types seeded onto scaffolds in the bioreactor demonstrated viability, adherence and metabolism over time. The seeded scaffolds demonstrated increased expression of VEGF after 6 days in culture. Once implanted, endoscopy 3 weeks after surgery revealed an extruded scaffold with newly regenerated tissue. Both cell seeded scaffolds demonstrated epithelial and muscle regeneration and the piglets were able to eat and grow over time. CONCLUSIONS: Autologous esophageal epithelial cells or maternal AF-MSC can be cultured on a 3D scaffold in a bioreactor. These cells maintain viability, proliferation, and adherence over time. Implantation into piglets demonstrated esophageal regeneration with extrusion of the scaffold. This sets the stage for translational application in a neonatal model of esophageal atresia.

Advanced Glycation End Products Induce Obesity and Hepatosteatosis in CD-1 Wild-Type Mice.

AGEs are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and/or nucleic acids. AGEs have been shown to play a role in various conditions including cardiovascular disease and diabetes. In this study, we hypothesized that AGEs play a role in the "multiple hit hypothesis" of nonalcoholic fatty liver disease (NAFLD) and contribute to the pathogenesis of hepatosteatosis. We measured the effects of various mouse chows containing high or low AGE in the presence of high or low fat content on mouse weight and epididymal fat pads. We also measured the effects of these chows on the inflammatory response by measuring cytokine levels and myeloperoxidase activity levels on liver supernatants. We observed significant differences in weight gain and epididymal fat pad weights in the high AGE-high fat (HAGE-HF) versus the other groups. Leptin, TNF-α, IL-6, and myeloperoxidase (MPO) levels were significantly higher in the HAGE-HF group. We conclude that a diet containing high AGEs in the presence of high fat induces weight gain and hepatosteatosis in CD-1 mice. This may represent a model to study the role of AGEs in the pathogenesis of hepatosteatosis and steatohepatitis.

Characterizing the inflammatory response in esophageal mucosal biopsies in children with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an emerging allergic, IgE- and non-IgE (Th2 cell)-mediated disease. There are major gaps in the understanding of the basic mechanisms that drive the persistence of EoE. We investigated whether esophageal biopsies from children with EoE demonstrate an inflammatory response that is distinct from normal controls. We prospectively enrolled 84 patients, of whom 77 were included in our analysis, aged 4-17 years (12.8±3.8 years; 81% males). Five esophageal biopsies were collected from each patient at the time of endoscopy. Intramucosal lymphocytes were isolated, phenotyped and stimulated with phorbol 12-myristate 13-acetate/ionomycin to measure their potential to produce cytokines via flow cytometry. We also performed cytokine arrays on 72-h biopsy culture supernatants. CD8(+) T cells, compared with CD4(+) T cells, synthesized more TNF-α and interferon (IFN)-γ after mitogen stimulation in the EoE-New/Active vs EoE-Remission group (P=0.0098; P=0.02) and controls (P=0.0008; P=0.03). Culture supernatants taken from explant esophageal tissue contained 13 analytes that distinguished EoE-New/Active from EoE-Remission and Controls. Principal component analysis and cluster analysis based on these analytes distinctly separated EoE-New/Active from EoE-Remission and Controls. In summary, we have identified a previously unappreciated role for CD8(+) T lymphocytes with potential to produce TNF-α and IFN-γ in EoE. Our results suggest that CD8(+) T cells have a role in the persistence or progression of EoE. We have also identified a panel of analytes produced by intact esophageal biopsies that differentiates EoE-New/Active from EoE-Remission and controls. Our results suggest that esophageal epithelial cells may have specific immune effector functions in EoE that control the type and amplitude of inflammation.

Biomimetic and synthetic esophageal tissue engineering.

BACKGROUND/PURPOSE: A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. METHODS: Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. RESULTS: Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. CONCLUSIONS: We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue.

Esophagitis in children with celiac disease.

Objectives. To our knowledge, the occurrence of esophagitis in children with celiac disease (CD) has never been evaluated. The aim of this study is to determine the prevalence of esophagitis in children with CD. Patients and Methods. Between 2003 and 2007, children with biopsy confirmed CD were retrospectively identified. Biopsy reports were reviewed for esophageal inflammation. Biopsy reports of 2218 endoscopies performed during the same period were also evaluated for inflammation. Results. Forty-nine children diagnosed with CD (47% boys). Nineteen of 49 (39%) patients with CD had esophagitis (95% CI 0.23-0.5). Thirty percent of boys and 46% of girls with CD had esophagitis (95% CI 0.12-0.40). Overall, 45% of patients who underwent upper endoscopy had esophagitis. The prevalence of esophagitis in CD (39%) compared to the prevalence of esophagitis (45%) in our practice was not significantly different, P = 0.2526. Conclusion. There was no difference in the prevalence of esophagitis between children diagnosed with CD at the time of their diagnostic EGD and the prevalence of esophagitis in children without CD. A prospective study to determine whether the esophagitis should be treated with acid suppression or whether the esophagitis heals with the gluten-free diet is warranted.

Two Cases of Helicobacter pylori-Negative Gastric Outlet Obstruction in Children.

Gastric outlet obstruction (GOO) in children is most commonly caused by idiopathic hypertrophic pyloric stenosis. Prior to proton pump inhibitors and H2 blockers, peptic ulcer disease (PUD) secondary to H. pylori was a cause of GOO. Both patients presented with a history of weight loss, vomiting, and abdominal pain. Their diagnosis of PUD and GOO was made by EGD and UGI. H. pylori testing was negative for both on multiple occasions but still received H. pylori eradication therapy. Patient 1 after failing pharmaceutical management underwent surgery for definitive treatment. Patient 2 underwent six therapeutic pyloric dilations before undergoing surgery as definitive treatment. These cases suggest that GOO secondary to PUD occurs in the absence of H. pylori infection and surgical management can provide definitive therapy.