Anthony P. Olive

PedsQL eosinophilic esophagitis module: feasibility, reliability, and validity.

Objective: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory condition with a paucity of information on health-related quality of life (HRQOL). The objective of the study was to report on the measurement properties of the PedsQL EoE Module. Methods: The PedsQL EoE Module was completed in a multisite study by 196 pediatric patients with EoE and 262 parents of patients with EoE. Results: The PedsQL EoE Module scales evidenced excellent feasibility (0.6%–3.1% missing), excellent group comparison reliability across total scale scores (patient &agr; 0.93; parent proxy &agr; 0.94), good reliability for the 7 individual scales (patient &agr; 0.75–0.87; parent proxy &agr; 0.81–0.92), excellent test–retest reliability (patient intraclass correlation coefficient 0.88; parent intraclass correlation coefficient 0.82), demonstrated no floor effects and low ceiling effects, and demonstrated a high percentage of scaling success for most scales. Intercorrelations with the PedsQL Generic Core Scales were in the medium (0.30) to large (0.50) range. PedsQL EoE Module scores were worse among patients with active histologic disease (≥5 eos/hpf) compared with those in remission (patient self-report: 63.3 vs 69.9 [P < 0.05]; parent proxy report: 65.1 vs 72.3 [P < 0.01]), and those treated with dietary restrictions compared with those with no restrictions (patient self-report: 61.6 vs 74.3 [P < 0.01]; parent proxy report: 65.5 vs 74.7 [P < 0.01]). Conclusions: The results demonstrate excellent measurement properties of the PedsQL EoE Module. Patients with active histologic disease and those treated with dietary restrictions demonstrated worse PedsQL scores. The PedsQL EoE Module may be used in the evaluation of pediatric EoE disease-specific HRQOL in clinical research and practice.

114 A Multicenter Study Assessing the Clinical, Endoscopic and Histologic Response to Four Food Elimination Diet for the Treatment of Eosinophilic Esophagitis

G A A b st ra ct s calculated at baseline and at week 12. Proximal and distal esophageal scores, total scores (summation of proximal and distal), and subscores for individual component of EREFS (edema, rings, exudates, furrows, stricture) were prospectively recorded. Baseline and followup EREFS scores were compared, and post-treatment eosinophil counts and EREFS scores were correlated. Data analysis was performed on the intent-to-treat population. Results: A total of 93 subjects were randomized from 25 centers, and 87 were included in the final analysis. 97% of subjects had endoscopic features identified at baseline. The OBS (n= 49) and placebo (n=38) groups did not differ in baseline demographic and endoscopic characteristics. EREFS scores significantly improved after treatment in both proximal (3.4 to 1.5; p<0.0001) and distal esophagus (4.3 to 2.4; p<0.0001) with OBS but not placebo (proximal 3.3 to 3.4; distal 3.6 to 3.9). Features of edema, rings, exudates and furrows showed significant improvement with OBS but not placebo (Figure). Strictures did not significantly change following OBS or placebo although subjects with high grade strictures were excluded from trial entry. Proximal, distal and total EREFS correlated with peak eosinophil counts after treatment (R: 0.35, p<0.0001). Conclusions: (1) This is the first study to utilize a validated endoscopic scoring instrument in a randomized controlled trial of medical therapy for EoE. (2) Significant benefit was demonstrated in the inflammatory (edema, exudates, furrows), ring, and total EREFS scores. (3) Significant correlation was demonstrated between EREFS and peak eosinophil counts. (4) Endoscopic outcomes may be important endpoints of EoE clinical trials that complement symptom and histologic assessments. Written on behalf of the MPI-101-06 Investigators.

Comparing methods to collect saliva from children to analyze cytokines related to allergic inflammation.

Human saliva is a complex fluid, rich in immunological components, which reflects real time systemic concentrations. Advances in biotechnology have enabled us to measure minute concentrations of immunological components such as cytokines in saliva samples with precision and accuracy 1. There are few data evaluating how the collection method may influence the results in addition to other methodological challenges, resulting in lack of consensus on a universally accepted saliva collection technique2, especially in children. Specifically, the impact of saliva collection methods on detection and analysis of salivary concentrations of allergic inflammatory cytokines such as the interleukin (IL)-4, IL-5, IL-13, eotaxin 3 (Eo3) and thymic stromal lymphopoietin (TSLP), that mediate allergic inflammation3 has not been reported. These cytokines play an important role in the development of allergic inflammatory states in increasingly prevalent conditions such as allergic rhinitis (AR), food allergy (FA), asthma (AS), atopic dermatitis (AD), and eosinophilic esophagitis (EoE)4. The overall goal of this study was to compare two methods of saliva collection in the detection and analysis of salivary concentrations of IL-4, IL-5, IL-13, Eo3 and TSLP in children. Our study was approved by the Baylor Institutional Review Board and informed consent was obtained from the parent and assent, where appropriate, from the child. We studied 20 children; median age: 14 years (9–17, IQR), female: 14 (64%), Caucasian: 13 (59%). Three (15%) children did not report any allergies and the remaining 17 (85%) children reported one or more of following commonly prevalent allergic conditions: AD (n=1), oral allergy syndrome (n=1), AS (n=2), FA (n=2), AR (n=6) and EoE (n=9). No attempt was made to distinguish salivary cytokine profiles between children with and without allergies in this study. Participants were nil per os for at least 4 hours. They rinsed their mouth with water, and following a 10 min. period, passive drool (PD) was collected followed by saliva collection via an oral swab (OS), in order to minimize the effect of saliva on the flow rate and concentration of cytokines in PD. PD samples were obtained with the head tilted forward while drooling down a 5.5 cm polypropylene Salimetrics Collection Aid® (Salimetrics LLC, State College, PA). Next, the participant placed a Salimetrics Oral Swab® (Salimetrics LLC, State College, PA) in their mouth and gently rolled it side-to-side for approximately 60–90 sec. to saturate the swab, which was then placed in a storage tube that allows saliva to be centrifuged. All samples were collected between 7:30 am and 12:30 pm. Two (9%) participants aged 11 and 14 years were unable to provide PD because they were not able to direct their drool into the collection device. All participants were able to use the OS. After providing paired saliva samples, all children responded to a short questionnaire assessing their knowledge of salivary diagnostics, asking them to rate their experience in providing each saliva sample in terms of perceived ease of use, acceptability and safety. Only 23% were aware of saliva testing in the surveillance of health or disease. A significantly higher proportion of children preferred the OS over PD sampling (82 % vs. 18 %; P < 0.05). Ease (68%) and speed (14%) of providing samples were the most common reasons cited by those who preferred the OS. Saliva samples were maintained at 4°C and transport ed to the laboratory within 2 hr of collection and were centrifuged at 3,000 rpm for 15 minutes. Supernatants were stored in aliquots and frozen at −80°C. On the day of anal ysis, samples were brought to room temperature, vortexed, and centrifuged for 15 minutes at 3,000 rpm. A magnetic, high sensitivity human multi-analytes profiling bead-based assay kit was used (EMD Millipore, Billerica, MA; lower limit of detection (LLD) for IL-4, IL-5, and IL-13: 0.13 pg/mL, and coefficient of variation (CV) < 10%, <6% and <8% respectively; Eo3 and TSLP: LLD: 3.2 pg/mL, and CV < 9% and <12% respectively). All cytokines were measured on 96-well plates (50 µL saliva/well). The standards and controls were plated on every run using a saliva based sample matrix. Plates were read on the Luminex® 200™ platform (Luminex, Austin, TX) according to the manufacturer’s recommendations. Intraassay precision was performed using 10 samples in duplicates and CV was calculated. IL-4, -5 and -13 were detected in all PD and OS samples. Eo3 and TSLP were undetectable in 3 (17%) PD samples but were detectable in all OS samples. The Wilcoxon-rank sum test revealed that the median concentrations were similar between PD and OS samples (Table); however greater precision was noted in OS compared to PD concentrations. Bland-Altman analysis5 showed no systematic bias and revealed insufficient agreement between PD and OS, suggesting that PD and OS may not be used interchangeably. Table Salivary concentrations (pg/mL) cytokines associated with allergic inflammation in passive drool (PD) and saliva collected in oral swab (OS). To our knowledge, this is the first study to describe the presence of cytokines related to allergic inflammation in saliva in a pediatric population. Our results suggest that OS provides more precise concentrations and is more acceptable than PD for detection and analysis of cytokines related to allergic inflammation. Furthermore, our data suggest that PD and OS may not be used interchangeably. Our data extend previous findings showing that saliva collection method can affect the salivary concentrations of stress and non-allergic inflammatory markers 6–9. Strengths to our study included the ability to minimize the effects of external factors such as tooth brushing and recent meals by collecting saliva samples from children who were nil per os for an acceptable period of time prior to providing saliva samples. We were successful in collecting saliva samples (both PD and OS) from children as young as 6 years of age. Limitations to our study include our use of a small convenience sample. In addition, our focus was on cytokines relevant to allergic inflammation. It is possible that either PD or OS may be acceptable for other biomarkers. Presence of eosinophils, eosinophil degranulation products, and/or epithelial cells and their influence on the concentrations of specific cytokines in PD or OS remains unclear. In summary, in pediatric population, saliva collected by OS appears to offer methodological advantages over PD in analyzing cytokines related to allergic inflammation. Adequately designed studies to evaluate the potential of salivary cytokines as non-invasive, point of care markers to diagnose and/or manage common allergic conditions affecting children are warranted.

Combination Steroid and Test-based Food Elimination for Eosinophilic Esophagitis: A Retrospective Analysis

Objectives: Eosinophilic esophagitis (EoE) is a clinicopathologic disorder characterized by infiltration of eosinophils into the esophagus. Primary treatment approaches include topical corticosteroids and/or food elimination. The aim of the present study was to compare the effectiveness of combination therapy (topical corticosteroid plus test-based food elimination [FS]) with single therapy (topical corticosteroid [S] or test-based food elimination [F]). Methods: Chart review of patients with EoE at Texas Childrens Hospital (age <21 years) was performed. Clinical and histological statuses were evaluated after a 3-month treatment with either single or combination therapy. Comparisons were analyzed using Fisher exact test, Kruskal-Wallis tests, and multiple logistic regression models. Results: Among 670 charts, 63 patients (1–21 years, median 10.3 years) with clinicopathologic diagnoses of EoE were identified. Combination FS therapy was provided to 51% (n = 32) and single treatment (S, F) to 27% (n = 17) or 22% (n = 14) of patients, respectively. Clinical responses were noted in 91% (n = 29), 71% (n = 12), and 64% (n = 14) of patients in the FS, S, and F groups, respectively. The odds of clinically improving were 4.6 times greater (95% confidence interval: 1.1–18.8) with combination versus single therapy. The median peak number of eosinophils per high-power field after 3-month therapy was not significantly different in the S, F, and FS groups. Conclusions: The combination of topical corticosteroids with specific food elimination is as effective in achieving clinical and histological remissions as the single-treatment approaches. Responses were achieved with the combination in patients who had previously failed single-agent therapy. Prospective research of this combination approach in young patients with EoE is needed.

Fish oil supplementation does not impair the gut immune response to Trichinella spiralis infection in rats.

BACKGROUND Fish oil has been recommended as a source of omega-3 fatty acids for preterm infants and for therapy of some inflammatory diseases. METHODS Because fish oil supplementation could downregulate the hosts immune response, we studied the gut inflammatory response to an enteric infection in 72 rats assigned to three dietary groups with differing fatty acid profile: 1) fish oil, rich in eicosapentaenoic and docosahexaenoic acid; 2) olive oil, containing 71% monounsaturated fat; and 3) rat chow, containing 57% saturated fat. One half (n = 36) of the rats were infected with Trichinella spiralis larvae; the other half served as controls. The inflammatory response to initial infection (study 1), and type I hypersensitivity response to a subsequent parasite-derived antigenic challenge (study 2) were assessed. Jejunal inflammatory cell infiltrate, mean villus height, disaccharidase levels, changes in short-circuit current in response to glucose absorption, and chloride secretagogues (study 1) were measured 9 days after infection. Short-circuit current changes induced by chloride secretion were measured when the proximal jejunum was challenged with T. spiralis-derived antigen 40 days after infection (study 2). RESULTS In study 1, jejunal tissue from infected animals had more eosinophilic infiltrate, lower disaccharidase levels, and less glucose absorptive and chloride secretory capacity than tissue from noninfected animals. In study 2, the jejunum of infected animals showed an antigen-induced chloride secretory response, whereas no response was obtained from jejunal tissue from noninfected animals. Type of diet did not affect the response in either study. CONCLUSION Under the conditions of this experiment, fish oil supplementation did not interfere with the local intestinal inflammatory response after T. spiralis infection.

Mucosal mast cell counts in pediatric eosinophilic gastrointestinal disease

To the Editor, Primary eosinophilic gastrointestinal disorders (EGID) include a spectrum of conditions where increased collection of eosinophil leukocytes occurs within the intestinal tract in the setting of symptomatic gastrointestinal (GI) disease. Disorders falling under this broad category include eosinophilic esophagitis (EoE), eosinophilic gastroenteritis, eosinophilic colitis, and eosinophilic proctitis.(1). With the emergence of EoE, mutual efforts of allergists and gastroenterologists continue to identify candidate biomarkers of EGID, including mast cell-associated ones.(2) These markers may help to understand the pathophysiology of the disorders, guide their diagnosis and treatments, as well as aid the monitoring of treatment response. Increased numbers of mast cells have been described in adults and pediatrics patients with EoE (3), but there is limited knowledge of these cells in other EGID conditions. We conducted a retrospective study to investigate correlation between eosinophil and mast cell numbers in pediatric EGID (excluding EoE) and controls. Patients with EGID were identified from the EGID clinic at Texas Children’s Hospital (TCH). The diagnosis was based on increased number of eosinophils above empirically set internal reference ranges at TCH in stomach, duodenum, terminal ileum, right, or left colon. Control patients included those that underwent endoscopic evaluation for constipation and suspected polyp. Patients with any other conditions where mucosal eosinophilia has been observed (e.g., inflammatory bowel disease, irritable bowel syndrome, diarrhea, abdominal pain, and celiac disease) were excluded from the control group. Age, gender, and allergies were recorded for the purpose of this study. Percent of peripheral eosinophils within 2 months of the endoscopy was documented. The biopsy specimens were examined by a pediatric gastrointestinal pathologist, who was blinded to the clinical diagnosis, and the previously reported histology reports. The biopsy specimens were fixed in 10% neutral-buffered formalin immediately following endoscopy. Paraffin sections (3 micron) were cut and stained with hematoxylin and eosin (H&E) with 8 tissue sections on 1 slide. They were reviewed for eosinophil count at 9400 magnification. Duplicated 3-micron sections were stained for mast cell tryptase (MCT) via immunohistochemistry using a monoclonal mouse antihuman MCT antibody (clone AA1, code number 7052, DAKO, Glostrup, Denmark). After pre-treatment with trypsin for 5 min at room temperature, the specimens were incubated with the primary antibody dilution 1:200 at room temperature for 15 minutes. Novocastra Bond Polymer Refine Detection System (biotinfree, peroxide conjugated) was used (from LEICA, Newcastle upon Tyne, United Kingdom) with diaminobenzidine tetrahydrochloride (DAB) as the chromogen and hematoxylin as the counter stain. Nonparametric Mann–Whitney U-test (Prism software version 2.0 [Graph Pad Inc., San Diego, California, USA]) was employed for group comparisons. Pearson correlation coefficient was calculated in Excel (Microsoft Office Excel 2007). Significance for correlation was calculated with public Statistics Calculator version 3.0 (http://www.danielsoper.com/ statcalc3/calc.aspx?id=44). Statistical significance was determined at p < 0.05. The study was approved by the IRB of Baylor College of Medicine (protocol number H-32539). Twenty-six patients were in the study; 14 controls and 12 with EGID. Intestinal biopsies from stomach, duodenum,

Endoscopic pneumatic reduction of a pediatric ileo-ileocolic intussusception during diagnostic colonoscopy.

y An 11-year-old girl presented with acute abdominal pain after a 5-month history of intermittent colicky abdominal pain associated with nonbilious vomiting and weight loss. An ileo-ileocolic intussusception was noted on ultrasound, but the barium enema was negative. During diagnostic colonoscopy, a spontaneous ileo-ileocolic intussusception occurred at the level of the hepatic flexure. Endoscopic air insufflation was used to (transverse colon).

SMAD4 haploinsufficiency associates with augmented colonic inflammation in select humans and mice: O-22.

SMAD4 is a common mediator of the TGF-beta signaling pathway. One of the members of this pathway, TGF-beta 1, has an important role in controlling gut inflammation in relation to the continuous stimulation of the intestinal microbiota. SMAD4 haploinsufficiency in humans has been linked to juvenile polyposis hereditary hemorrhagic telangiectasia syndrome (JP/HHT; OMIM#17505). Hematochezia and colonic mucosal inflammation suggestive of inflammatory bowel diseases (IBD) have been reported in JP/HHT. Stimulated by recent experience with two affected pediatric patients presented here, we explored the potential role of Smad4 haploinsufficiency in a murine model of colonic inflammation. Smad4(+/-) mice were maintained on a mixed C57/129SvEv background. Chronic colitis was induced with repeated administration of dextran sulfate sodium (DSS) in drinking water. The colonic mucosal microbiota was interrogated by massively parallel pyrosequencing of the bacterial 16S rRNA gene. 66.7% of Smad4(+/-) mice were sensitive to DSS colitis compared to 14.3% of wild type (Chi-Square p=0.036). The augmented colitis was associated with microbiota separation in the Smad4(+/-) mice. Enterococcus and Enterococcus faecalis specifically was increased in abundance in the colitis-prone animals. Smad4 haploinsufficiency can associate with increased susceptibility to large bowel inflammation in mammals with variable penetrance in association with the colonic mucosal microbiota. These findings may reveal implications not only towards colonic inflammation in the setting of SMAD4 haploinsufficiency, but for colorectal cancer as well.