Jonathan P. Kushner

Coordinate Interaction between IL-13 and Epithelial Differentiation Cluster Genes in Eosinophilic Esophagitis

We have previously proposed that the pathogenesis of eosinophilic esophagitis (EE) is mediated by an IL-13–driven epithelial cell response associated with marked gene dysregulation including eotaxin-3 overproduction. In this study, we compared epithelial responses between healthy patients and those with EE, aiming to uncover molecular explanations for EE pathogenesis. Esophageal epithelial cells could be maintained for up to five passages, with 67% and 62% of cell lines reaching confluence in healthy controls and EE cases, respectively. Both sets of epithelial cells avidly responded to IL-13 at similar levels as assessed by eotaxin-3 production. Acidic pH increased cellular release of eotaxin-3 (4.6 ± 1.98 ng/ml versus 12.46 ± 2.90 ng/ml at pH 7.4 and 4, respectively; p < 0.05). Numerous epidermal differentiation complex (EDC) genes, such as filaggrin and SPRR3, were downregulated both in IL-13–stimulated esophageal epithelial cells and in EE biopsies specimens compared with healthy controls. Whereas the filaggrin loss of function mutation 2282del4 was overrepresented in EE compared with control individuals (6.1% versus 1.3% respectively; p = 0.0172), the decreased filaggrin expression was uniformly seen in all EE cases in vivo. Indeed, expression of the EDC genes filaggrin and involucrin was strongly decreased directly by IL-13. These results establish that the epithelial response in EE involves a cooperative interaction between IL-13 and expression of EDC genes.

Variants of thymic stromal lymphopoietin and its receptor associate with eosinophilic esophagitis

BACKGROUND The genetic cause of eosinophilic esophagitis (EE) has been largely unexplored until a recent genome-wide association study identified a disease susceptibility locus on 5q22, a region that harbors the thymic stromal lymphopoietin (TSLP) gene. However, it is unclear whether the observed genetic associations with EE are disease-specific or confounded by the high rate of allergy in patients with EE. In addition, the genetic contributions of other allergy-associated genes to EE risk have not been explored. OBJECTIVE We aimed to delineate single nucleotide polymorphisms (SNPs) that associated with EE apart from allergy. METHODS We used a custom array containing 738 SNPs in 53 genes implicated in allergic responses, immune responses, or both to genotype 220 allergic or 246 nonallergic control subjects and a discovery cohort of 170 patients with EE. We replicated a statistically significant SNP association in an independent case-control cohort and examined the induction of the candidate gene in primary esophageal epithelial cells. RESULTS A single SNP residing in the TSLP gene reached Bonferroni linkage disequilibrium-adjusted significance but only when patients with EE were compared with allergic control subjects (rs10062929; P = 4.11 x 10(-5); odds ratio, 0.35). A nonsynonymous polymorphism in the thymic stromal lymphopoietin receptor (TSLPR) gene on Xp22.3 and Yp11.3 was significantly associated with disease only in male patients with EE. Primary esophageal epithelial cells expressed TSLP mRNA after Toll-like receptor 3 stimulation. CONCLUSION These data collectively identify TSLP as a candidate gene critically involved in EE susceptibility beyond its role in promoting T(H)2 responses.

A striking local esophageal cytokine expression profile in eosinophilic esophagitis.

BACKGROUND Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. OBJECTIVE Early studies have suggested that esophageal eosinophilia occurs in association with T(H)2 allergic responses, yet the local and systemic expression of relevant cytokines has not been well characterized. METHODS A human inflammatory cytokine and receptor PCR array containing 84 genes followed by PCR validation and multiplex arrays were used to quantify cytokine mRNA in esophageal biopsies and blood samples. RESULTS Esophageal transcripts of numerous chemokines (eg, chemokine [C-C motif] ligand [CCL] 1, CCL1, CCL23, CCL26 [eotaxin-3], chemokine [C-X-C motif] ligand [CXCL] 1, and CXCL2), cytokines (eg, IL13 and ATP-binding cassette, subfamily F, member 1), and cytokine receptors (eg, IL5 receptor, alpha) were induced at least 4-fold in individuals with EE. Analysis of esophageal biopsies (n = 288) revealed that eotaxin-3 mRNA level alone had 89% sensitivity for distinguishing individuals with and without EE. The presence of allergy was associated with significantly increased esophageal expression of IL4 and IL5 mRNA in patients with active EE. We identified 8 cytokines (IL-4, IL-13, IL-5, IL-6, IL-12p70, CD40 ligand, IL-1α, and IL-17) whose blood levels retrospectively distinguished 12 patients without EE from 13 patients with EE with 100% specificity and 100% sensitivity. When applied to a blind, prospectively recruited group of 36 patients, the cytokine panel scoring system had a 79% positive predictive value, 68% negative predictive value, 61% sensitivity, and 83% specificity for identifying EE. CONCLUSION Evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive T(H)2 immunity in regulating eotaxin-3-driven esophageal eosinophilia in the absence of a consistent systemic change in cytokines.

Molecular diagnosis of eosinophilic esophagitis by gene expression profiling.

BACKGROUND & AIMS Gene expression profiling provides an opportunity for definitive diagnosis but has not yet been well applied to inflammatory diseases. Here we describe an approach for diagnosis of an emerging form of esophagitis, eosinophilic esophagitis (EoE), which is currently diagnosed by histology and clinical symptoms. METHODS We developed an EoE diagnostic panel (EDP) comprising a 96-gene quantitative polymerase chain reaction array and an associated dual-algorithm that uses cluster analysis and dimensionality reduction using a cohort of randomly selected esophageal biopsy samples from pediatric patients with EoE (n = 15) or without EoE (non-EoE controls, n = 14) and subsequently vetted the EDP using a separate cohort of 194 pediatric and adult patient samples derived from both fresh or formalin-fixed, paraffin-embedded tissue: active EoE (n = 91), control (non-EoE and EoE remission, n = 57), histologically ambiguous (n = 34), and reflux (n = 12) samples. RESULTS The EDP identified adult and pediatric patients with EoE with approximately 96% sensitivity and approximately 98% specificity, and distinguished patients with EoE in remission from controls, as well as identified patients exposed to swallowed glucorticoids. The EDP could be used with formalin-fixed, paraffin-embedded tissue RNA and distinguished patients with EoE from those with reflux esophagitis, identified by pH-impedance testing. Preliminary evidence showed that the EDP could identify patients likely to have disease relapse after treatment. CONCLUSIONS We developed a molecular diagnostic test (referred to as the EDP) that identifies patients with esophagitis in a fast, objective, and mechanistic manner, offering an opportunity to improve diagnosis and treatment, and a platform approach for other inflammatory diseases.

Analysis and expansion of the eosinophilic esophagitis transcriptome by RNA sequencing

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)–treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13–induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13–associated responses.

Histologic eosinophilic gastritis is a systemic disorder associated with blood and extragastric eosinophilia, TH2 immunity, and a unique gastric transcriptome

BACKGROUND The definition of eosinophilic gastritis (EG) is currently limited to histologic EG based on the tissue eosinophil count. OBJECTIVE We aimed to provide additional fundamental information about the molecular, histopathologic, and clinical characteristics of EG. METHODS Genome-wide transcript profiles and histologic features of gastric biopsy specimens, as well as blood eosinophil counts, were analyzed in patients with EG and control subjects (n = 15 each). RESULTS The peak gastric antrum eosinophil count was 283 ± 164 eosinophils/×400 high-power field in patients with EG and 11 ± 9 eosinophils/×400 high-power field in control subjects (P = 6.1 × 10(-7)). Patients with EG (87%) had coexisting eosinophilic inflammation in multiple gastrointestinal segments; the esophagus represented the most common secondary site. Increased peripheral blood eosinophil counts (patients with EG: 1.09 ± 0.88 × 10(3)/μL vs control subjects: 0.09 ± 0.08 10(3)/μL, P = .0027) positively correlated with peak gastric eosinophil counts (Pearson r(2) = .8102, P < .0001). MIB-1(+) (proliferating), CD117(+) (mast cells), and FOXP3(+) (regulatory T cells, activated T cells, or both) cell counts were increased in patients with EG. Transcript profiling revealed changes in 8% of the genome in gastric tissue from patients with EG. Only 7% of this EG transcriptome overlapped with the eosinophilic esophagitis transcriptome. Significantly increased IL4, IL5, IL13, IL17, CCL26, and mast cell-specific transcripts and decreased IL33 transcripts were observed. CONCLUSION EG is a systemic disorder involving profound blood and gastrointestinal tract eosinophilia, TH2 immunity, and a conserved gastric transcriptome markedly distinct from the eosinophilic esophagitis transcriptome. The data herein define germane cellular and molecular pathways of EG and provide a basis for improving diagnosis and treatment.

Randomized placebo-controlled trial of pantoprazole for daytime sleepiness in GERD and obstructive sleep disordered breathing:

OBJECTIVE: To determine the efficacy of pantoprazole therapy for daytime somnolence, psychomotor vigilance, and quality of life in patients with mild-moderate obstructive sleep disordered breathing (OSDB) and gastroesophageal reflux disease (GERD). STUDY DESIGN: Randomized, double-blind, placebo-controlled crossover trial. METHODS: Sixty patients with daytime sleepiness, mildmoderate OSDB and GERD were randomly assigned a 2-week treatment with pantoprazole 40 mg or placebo followed by a 2-week washout period and crossover respectively to 2-week treatment with placebo or pantoprazole. Outcomes included Epworth Sleepiness Score (ESS), sleep-related quality-of-life (FOSQ), and reaction time. RESULTS: With pantoprazole, patients reported statistically significantly greater improvement of overall reflux symptoms (P = 0.0003) and in ESS (P = 0.04). A significant improvement was noted in FOSQ for both treatments with a trend toward greater improvement with pantoprazole (P = 0.058). No improvement in reaction times was observed. CONCLUSION: Patients with coexistent GERD and OSDB noted significant improvement in daytime sleepiness after treatment with pantoprazole over placebo likely related to a reduction in nocturnal reflux-related arousals.

M1152 Integrity of Esophageal Emptying in Spasm Patients: A High-Resolution Impedance Manometry Study

Chest pain and dysphagia are the most common symptoms reported by patients diagnosed with esophageal spasm. The role of esophageal emptying in the etiology of symptoms in these patients is unclear. Our AIM was to determine the integrity of esophageal emptying in patients with manometrically defined esophageal spasm using high-resolution impedance manometry (HRIM). METHODS: Esophageal HRIM data were retrospectively reviewed from 12 patients (7 male) classified as esophageal spasm (Pandolfino et al, AJG, 2008). Data were collected using a solid state HRIM system with 36 manometry and 8 impedance sensors (MMS Inc, Dover, NH). All patients underwent 10 5-ml water swallows and 10 5-ml viscous swallows. Patients were classified based on symptoms as either chest pain dominant (n=5) or dysphagia dominant (n=7). Esophageal motility and lower sphincter relaxation were quantified using the distal contractile integral (DCI), propagation front velocity (PFV) and the 4-second integrated relaxation pressure (IRP). Manometric parameters reflecting bolus transit, i.e. flow permissive time (FPT) and bolus domain pressure (BDP) were quantified using MATLAB programs (The Mathworks Inc, Natick, MA). Complete esophageal emptying was defined as a recovery back to 50% of baseline impedance on all intraesophageal sensors. All results are average ± std dev. RESULTS: Two manometric patterns were noted in spasm patients, (i) spastic contractions with DCI >5000 mmHg.cm.s and contractions restricted to the mid-esophagus (Type I), and (ii) spastic contractions with DCI > 7000 mmHg.cm.s and contractions spanning the mid and distal esophagus (Type II). 1/5 chest pain dominant patients was classified in type I and the remaining 4 chest pain patients were classified in Type II. Of the 7 dysphagia dominant patients, 3 were type I and 4 type II. Type I patients had complete bolus transit in all 4 patients, while 5 of the 8 type II patients had incomplete bolus emptying on at least two consecutive impedance sensors. FPT was longer in type I vs type II patients (3.2 ± 2.7 s vs. 1.9 ± 1.0 s; p 0.05). All patients had complete LES relaxation, 4-s IRP = 12.8±4.1 mmHg). Viscous swallows showed higher frequency of bolus retention compared to water swallows. CONCLUSIONS: Two manometric variations are seen in spasm patients. Type II patients have a stronger association with incomplete bolus transit and are more likely to have symptoms of dysphagia than chest pain. This suggests that impaired bolus transit is likely an important factor in the etiology of dysphagia but not of chest pain in the symptomatology of spasm patients.

584 An Optimal Dysphagia-Sensitive Algorithm for Assessing Deglutitive Intrabolus Pressure Using Esophageal High-Resolution Manometry

esophageal motility (IEM), 9 with nutcracker esophagus (NE) and 1 with diffuse esophageal spasm (DES). Abnormal manometry in VS was demonstrated in 31 patients (67.4%, 21 IEM, 9 NE, 1 DES). Abnormal transit in LS and VS was demonstrated in 14 (25.9 %) and 18 (33.3%), respectively. The manometric and transit abnormalities were more common in patients with dysphagia (78.5% vs. 21.4%, p=0.04) and in patients with posture instability gait disturbance assessed by UPDRS (p=0.02). Abnormal esophageal function was not correlated to age or Hoehn & Yahr stage. Repetitive deglutition during single swallow was observed in 143 of 540 (26.5%) swallows (mean 1.4±0.4/LS, 1.4±0.5/VS). Repetitive deglutition was significantly correlated with failed peristalsis and incomplete bolus transit (p<0.0001). Abnormal inhibition of motor activity in the oesophageal body during MRS was observed in 30 out of 49 (61.2%) patients; 26 with incomplete inhibition (repetitive contraction), and 4 with failed peristalsis. Abnormal MRS response correlated with abnormal manometry (p=0.043). Conclusion: Repetitive deglutition with abnormal inhibition of motor activity in the oesophageal body is frequently observed in patients with early PD. MRS provides further evidence of selective involvement of either the brain stem or the esophageal myenteric plexus in the early PD.