Marina L. Fernandez

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.

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 ± 5.2 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 ± 19 in the FP group versus 45 ± 23 in the OVB group. Mean post-treatment peak eos/hpf was 20 ± 29 in the FP group versus 12 ± 16 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.

Distinctive colonic mucosal cytokine signature in new-onset, untreated pediatric Crohn disease

Objective: The aim of the study was to compare the colonic mucosal immune response in children with new, untreated Crohn disease (CD-New), CD in remission (CD-Remission), and unaffected children (CTRL [controls]). Methods: We performed flow cytometry of mitogen-stimulated colonic lamina propria mononuclear cells isolated from colonic biopsies and 72-hour biopsy explant cultures, and analyzed the supernatant by an unbiased multiplex cytokine array of 45 analytes. Results: Thirty-six children were studied (mean age 14 ± 3 years, 14 girls): 12 CD-New, 11 CD-Remission, and 13 CTRL. We found that stimulation of lamina propria mononuclear cells isolated from colonic biopsies induced comparable intracellular cytokine levels of interferon (IFN-&ggr;), interleukin (IL)-17, and tumor necrosis factor (TNF)-&agr; in T cells from CD-New, CD-Remission, and CTRL, suggesting that mucosal innate inflammation plays a larger role than activated T cells in CD-New. To measure factors released during the ongoing inflammatory response in CD-New, we cultured colonic biopsy explants and uncovered 13/45 factors that were significantly higher in CD-New versus CD-Remission, whereas 10 were increased in CD-New over CTRL. Ingenuity Pathway Analysis software revealed the anticipated interconnectivity of TNF-&agr;, IL-6, and CSF-2 in CD-New of the colon. A novel subnetwork of chemokines was, however, evident, whereas IL-17a appeared as a peripheral factor. Principal component analysis and hierarchal clustering showed that CD-New and CD-Remission separated into distinct subgroups based on the 13 factors. Conclusions: At diagnosis of inflammatory bowel disease, the colonic cytokine response contains a predominance of innate immune factors, with chemoattractants and vascular adhesion molecules playing a central role.

Programmed cell death-1, PD-1, is dysregulated in T cells from children with new onset type 1 diabetes

Background Programmed death cell 1 (PD-1) is an inhibitor of T cell activation and is also functionally linked to glycolysis. We hypothesized that PD-1 expression is defective in activated T cells from children with type 1 diabetes (T1D), resulting in abnormal T cell glucose metabolism. Methods In this pilot study, we enrolled children with new onset T1D within 2 weeks of diagnosis (T1D), unaffected siblings of T1D (SIBS), unaffected, unrelated children (CTRL), children with new onset, and untreated Crohn disease (CD). We repeated the assays 4–6 months post-diagnosis in T1D (T1D follow up). We analyzed anti-CD3/-CD28-stimulated peripheral blood mononuclear cells (PBMC) subsets for PD-1 expression by flow cytometry at baseline and after 24 h in culture. We measured cytokines in the culture medium by multiplex ELISA and glycolytic capacity with a flux analyzer. Results We enrolled 37 children. T cells derived from subjects with T1D had decreased PD-1 expression compared to the other study groups. However, in T1D follow-up T cells expressed PD-1 similarly to controls, but had no differences in PBMC cytokine production. Nonetheless, T1D follow up PBMCs had enhanced glycolytic capacity compared to T1D. Conclusions Activated T cells from T1D fail to upregulate PD-1 upon T-cell receptor stimulation, which may contribute to the pathogenesis of T1D. T1D follow up PBMC expression of PD-1 normalizes, together with a significant increase in glycolysis compared to T1D. Thus, insulin therapy in T1D children is associated with normal PD1 expression and heightened glycolytic capacity in PBMC.

1149 Osteoprotegerin Expression is Upregulated in the Colon of Children With Active Inflammatory Bowel Disease

Background: Inflammatory Bowel Diseases (IBD) are chronic, relapsing conditions. Infections and other environmental exposures that vary by season have been postulated to trigger relapses; however, little information has been published on the seasonality of pediatric-onset IBD. Further understanding of this topic may provide valuable data on the epidemiology of Crohns Disease (CD) and Ulcerative Colitis (UC). Aim: To examine whether season is associated with disease activity in pediatric-onset IBD. Methods: ImproveCareNow (ICN) is a multicenter network of health care providers formed in 2007 to improve the quality of care of children with IBD. The ICN registry contains disease and treatment data, including physician global assessment (PGA), prospectively collected during outpatient encounters. This study analyzed data collected from December 2008-November 2010 at 12 centers across the USA. The percentage of visits during each season in which patients were classified as being in remission based on PGA was calculated. For patients with multiple visits per season, only the visit with the most severe PGA score was included. The distribution of sick visits (PGA of mild, moderate, or severe) and well visits (PGA of remission) across each season was also calculated. Seasons were defined as summer (Jun-Aug), fall (Sep-Nov), winter (Dec-Feb), and spring (Mar-May). Data was further divided into northern and southern regions (defined by 37° latitude). A generalized linear mixed model and the multinomial chi-square test were used for statistical analysis with p<0.05 considered significant. Results: The percentage of UC visits (n=1129)in remission was highest in summer and lowest in winter (summer 51.6%, fall 48.8%, spring 45.0%, winter 41.9%) with significant seasonal variation (p=0.01). A similar pattern was seen in CD (summer 51.4%, fall 47.5%, winter 46.9%, spring 45.8%, n=2937), though not statistically significant (p=0.09). The results were consistent across the northern and southern regions (UC p=0.58, CD p=0.08). The percentage of all sick visits was evenly distributed across seasons for UC (n=954, p=0.81) and CD (n=2414, p=0.07). However, the percentage of well visits was highest in the summer and lowest in the winter in both UC (28.7% vs. 20.5%, n=1440, p<0.001) and CD (28.1% vs. 22.6%, n=3688, p<0.001). Conclusion: In this large multicenter study, seasonal variation of disease activity was not found, nor was there seasonal variation when examined by region. Although the percentage of visits of UC patients in remission increased during the summer, this was due to a higher percentage of all well visits during this season. One potential explanation is that well visits may be intentionally scheduled during summer months to limit the amount of school missed by children.

Su1830 T-Lymphocyte Isolation and Phenotyping From Esophageal Biopsies in Children With Eosinophilic Esophagitis: Pilot Study

Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). Currently the diagnosis of EoE necessitates endoscopy with biopsy that characterizes less than 2% of the esophagus. Furthermore, the inflammation is patchy, necessitating numerous biopsies to ensure that the involved area has been adequately sampled. Improved methods are needed to characterize the eosinophil density throughout the esophagus and to thoroughly understand this disease. Aim: To prepare the first MBP-1 specific ultrasound contrast agents to facilitate the detection of EoE associated inflammation by tethering EoE specific antibodies to their surfaces and demonstrating the binding of these agents to eosinophil proteins on ex vivo monkey esophagi. Method: The contrast agent was synthesized by preparing 2 mg/ mL of insulin in HCl (pH 1.6) at 65C for 12 hours. The resulting particles were incubated withmaleimide activated streptavidin. Purified antibodies specific to MBP-1 were biotinylated and reacted with the streptavidin functionalized fibers to form an insulin aggregate-steptavidin-biotin antibody complex. This complex comprises the MBP-1 specific ultrasound contrast agent. A Macaca monkey esophagus, incubated overnight in MBP-1, was filled with this solution. Ultrasound images were taken before and after washing at 15 MHz. Results: Insulin aggregates were observed using ultrasound. Incubating the insulin complex in monkey esophagi coated with MBP-1 yields an extra contrast layer at the inner wall of the esophagus, demonstrating the specific binding of these agents to eosinophil granule proteins. No such layer is visualized in control samples (not treated with MBP-1). Discussion: This is the first reported use of insulin particles as contrast agents for ultrasound. These findings indicate that insulin aggregates provide clear ultrasound contrast in liquid environments. When coupled with disease specific antibodies, these novel agents have the potential to be used for detection, minimizing the need for anesthesia or radiation associated with other diagnostic or imaging modalities. This finding is important because it provides a new avenue for clinical detection of EoE.