Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Updated International Consensus Diagnostic Criteria for Eosinophilic Esophagitis: Proceedings of the AGREE Conference.

BACKGROUND & AIMS: Over the last decade, clinical experiences and research studies raised concerns regarding use of proton pump inhibitors (PPIs) as part of the diagnostic strategy for eosinophilic esophagitis (EoE). We aimed to clarify the use of PPIs in the evaluation and treatment of children and adults with suspected EoE to develop updated international consensus criteria for EoE diagnosis. METHODS: A consensus conference was convened to address the issue of PPI use for esophageal eosinophilia using a process consistent with standards described in the Appraisal of Guidelines for Research and Evaluation II. Pediatric and adult physicians and researchers from gastroenterology, allergy, and pathology subspecialties representing 14 countries used online communications, teleconferences, and a face-to-face meeting to review the literature and clinical experiences. RESULTS: Substantial evidence documented that PPIs reduce esophageal eosinophilia in children, adolescents, and adults, with several mechanisms potentially explaining the treatment effect. Based on these findings, an updated diagnostic algorithm for EoE was developed, with removal of the PPI trial requirement. CONCLUSIONS: EoE should be diagnosed when there are symptoms of esophageal dysfunction and at least 15 eosinophils per high-power field (or approximately 60 eosinophils per mm2) on esophageal biopsy and after a comprehensive assessment of non-EoE disorders that could cause or potentially contribute to esophageal eosinophilia. The evidence suggests that PPIs are better classified as a treatment for esophageal eosinophilia that may be due to EoE than as a diagnostic criterion, and we have developed updated consensus criteria for EoE that reflect this change.

Eosinophilic Esophagitis: Relationship of Subepithelial Eosinophilic Inflammation With Epithelial Histology, Endoscopy, Blood Eosinophils, and Symptoms.

OBJECTIVES: For technical reasons, the histologic characterization of eosinophilic esophagitis (EoE)-specific alterations is almost exclusively based on those found in the esophageal epithelium, whereas little is known about subepithelial abnormalities. In this study, we aimed to systematically assess the nature of subepithelial histologic alterations, and analyze their relationship with epithelial histologic findings, endoscopic features, and symptoms. METHODS: Adult patients with established EoE diagnosis were prospectively included during a yearly follow-up visit. Patients underwent assessment of clinical, endoscopic, and histologic disease activity using EoE-specific scores. RESULTS: We included 200 EoE patients (mean age 43.5±15.7 years, 74% males) with a median peak count of 36 intraepithelial eosinophils/hpf (IQR 14-84). The following histologic features were identified in the subepithelial layer: eosinophilic infiltration (median peak count of 20 eosinophils/hpf (IQR 10-51)), eosinophil degranulation (43%), fibrosis (82%), and lymphoid follicles (56%). Peak intraepithelial eosinophil counts were higher, identical, and lower when compared to the subepithelial layer in 62.5%, 7%, and 30.5% of patients, respectively. Anti-eosinophilic treatment at inclusion did not influence the relation between subepithelial and epithelial peak eosinophil counts. Subepithelial histologic activity correlated with epithelial histologic activity (rho 0.331, P<0.001), endoscopic severity (rho 0.208, P=0.003), and symptom severity (rho 0.179, P=0.011). Forty percent (21/52) of patients with <15 intraepithelial eosinophils/hpf had subepithelial peak counts of ≥15/hpf. CONCLUSIONS: There is a significant but modest correlation between subepithelial histologic activity and epithelial histologic activity, endoscopic severity, and symptom severity. The long-term clinical impact of assessing subepithelial alterations in EoE needs to be further elucidated.

How do gastroenterologists assess overall activity of eosinophilic esophagitis in adult patients?

OBJECTIVES: There is no "gold standard" for assessing disease activity in patients with eosinophilic esophagitis (EoE). We aimed to compare physicians' judgment of EoE activity with patients' judgment of symptom severity. We also aimed to examine the relative contribution of symptoms as well as endoscopic and histologic findings in shaping physicians' judgment of EoE activity. METHODS: Six gastroenterologists (all EoE experts) assessed EoE-associated symptoms in adult patients. Patients completed a symptom instrument and provided global assessment of EoE symptom severity (PatGA) (Likert scale: 0 (inactive) to 10 (most active)). Following esophagogastroduodenoscopy with biopsy sampling, gastroenterologists provided a global assessment of EoE activity (PhysGA) (Likert scale from 0 to 10) based on patient history and endoscopic and histologic findings. Linear regression and analysis of variance was used to quantify the extent to which variations in severity of EoE symptoms and endoscopic and histologic findings explain variations in PhysGA. RESULTS: A total of 149 EoE patients were prospectively included (71.8% male, median age at inclusion 38 years, 71.8% with concomitant allergies). A moderate positive correlation between PhysGA and PatGA (rho=0.442, P<0.001) was observed and the mean difference in the Bland-Altman plot was 1.77. Variations in severity of endoscopic findings, symptoms, and histologic findings alone explained 53%, 49%, and 30%, of the variability in PhysGA, respectively. Together, these findings explained 75% of variability in PhysGA. CONCLUSIONS: Gastroenterologists rate EoE activity mainly on the basis of endoscopic findings and symptoms and, to a lesser extent, on histologic findings.

The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia.

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 mAb having been used against B cell leukemia. In this study, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. To confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity, and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 d after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild type control mice, whereas the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the "B cell-specific" inhibitory molecule CD22 on GI eosinophils.

Involvement of mast cells in eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EE) is an emerging disorder with poorly understood pathogenesis. OBJECTIVE: Whereas prior studies have primarily focused on the role of eosinophils in disease diagnosis and pathogenesis, this study investigates the involvement of mast cells. METHODS: Total and degranulated mast cell counts were correlated to microarray and RT-PCR data to generate transcriptome expression profiles related to mast cell number and degranulation in patients with EE and healthy control subjects. RESULTS: Esophageal mastocytosis and mast cell degranulation were readily apparent in patients with EE compared with control subjects (P < .01), as assessed by staining for total mast cells and the presence of extracellular mast cell tryptase (P < .01). Microarray analysis revealed that mast cell levels correlated with the dysregulation of 0.8% (301 genes) of the genome, which was partially distinct from the genes that correlated with tissue eosinophilia. The expression of transcripts for the mast cell proteases carboxypeptidase A3 and tryptase, but not chymase, correlated with mast cell levels and distinguished patients with EE from control subjects. Suprabasilar mast cell counts (P < .01) and degranulation (P < .01) were proportional with KIT ligand mRNA expression. Treatment of patients with EE with swallowed fluticasone propionate normalized levels of mast cells and the mast cell-related transcriptome in responder patients. CONCLUSION: Herein we have identified local mastocytosis and mast cell degranulation in the esophagi of patients with EE; identified an esophageal mast cell-associated transcriptome that is significantly divergent from the eosinophil-associated transcriptome, with carboxypeptidase A3 mRNA levels serving as the best mast cell surrogate marker; and provided evidence for the involvement of KIT ligand in the pathogenesis of EE.

Glucocorticoid-regulated genes in eosinophilic esophagitis: a role for FKBP51.

BACKGROUND: Eosinophilic esophagitis (EE) involves marked accumulation of eosinophils in the esophageal mucosa that responds to swallowed fluticasone propionate (FP) in a subset of patients. OBJECTIVES: We aimed to uncover the mechanism of action of swallowed FP in patients with EE by providing evidence for a topical effect in the esophagus by identifying a molecular signature for FP exposure in vivo. METHODS: Global microarray expression profiles, immunofluorescence microscopy, and cell signaling in esophageal tissue and cell lines were analyzed. RESULTS: Thirty-two transcripts exhibited altered expression in patients who responded to swallowed FP treatment. Esophageal FK506-binding protein 5 (FKBP51) mRNA levels were increased (P < .05) in FP responders compared with those seen in control subjects and patients with untreated active EE. After FP treatment of esophageal epithelial cells, FKBP51 mRNA and protein levels were increased in a dose- and time-dependent manner by FP treatment in vitro. FP-induced FKBP51 was steroid receptor dependent because RU486 completely inhibited gene and protein induction. The half-life of FKBP51 mRNA was 16 to 18 hours independent of FP treatment. FKBP51 overexpression reduced FP action as assessed by FP inhibition of IL-13-induced eotaxin-3 promoter activity. CONCLUSIONS: Our results suggest that swallowed glucocorticoid treatment directly affects esophageal gene expression in patients with EE. In particular, increased FKBP51 transcript levels identify glucocorticoid exposure in vivo and distinguish FP responders from untreated patients with active EE and patients without EE. In addition, FKBP51 reduces glucocorticoid-mediated inhibition of IL-13 signaling in epithelial cells in vitro, suggesting that FKBP51 might influence FP responsiveness. We propose that esophageal FKBP51 levels have diagnostic and prognostic significance in patients with EE.

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.

Chemotactic factors associated with eosinophilic gastrointestinal diseases.

The gastrointestinal (GI) tract is in constant negotiation with the microbial flora present in the lumen. Resident hematopoietic cells (ie, lymphocytes, mast cells, and eosinophils) are part of this ongoing and silent homeostatic battle. Eosinophilic GI diseases are characterized by an increased number of eosinophil infiltrates with no identified cause. This article describes the past and present knowledge regarding the chemotactic factors involved in GI eosinophilia.

Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis.

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.

IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity.

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9-deficient mice fail to develop experimental oral antigen-induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9-mediated mast cell responses have an important role in food allergy.

Clinical, pathologic, and molecular characterization of familial eosinophilic esophagitis compared with sporadic cases.

BACKGROUND & AIMS: Eosinophilic esophagitis (EE) occurs in families. METHODS: Record review confirmed patient kinship and provided clinical information. Slide review confirmed the diagnosis (threshold peak number > or = 24 eosinophils/high-power field). RESULTS: Fifty-nine members (41 males, 18 females) of 26 families were 3 months to 47 years of age (mean age, 10.3 y) at diagnosis. The only recorded race was Caucasian. In 4 families a parent of an affected male had EE. The most common complaint at diagnosis was dysphagia (68% of patients). Endoscopy showed esophageal mucosal furrows (93% of patients) and exudates (44%). Fifty-one percent had asthma. Skin prick tests to food and aeroallergens were positive in 76% and 71%, respectively. Familial EE characteristics (clinical, endoscopic, pathologic, and global esophageal transcript expression profile analysis) were similar to sporadic EE, except among patients with mucosal furrows: familial patients had lower peak eosinophil counts in the distal esophagus (P = .03) compared with sporadic patients. The basic characteristics of EE (eg, eosinophil levels, rate of atopy) did not vary with patient age. By using genome-wide microarray analysis, no significant differences (P < .05, false-discovery rate) were observed between familial and sporadic EE. Among all patients, chest pain was more common in females (P = .02), and thickened mucosa was more common in males (P = .006). CONCLUSIONS: These data support a familial pattern of inheritance of EE and a pathogenesis shared with sporadic EE. EE should be considered in symptomatic family members of patients who have EE.

Esophageal remodeling develops as a consequence of tissue specific IL-5-induced eosinophilia.

BACKGROUND & AIMS: Eosinophilic esophagitis (EE) is an increasingly recognized disease that mimics gastroesophageal reflux disease. Recently, EE has been associated with esophageal remodeling, but the mechanisms involved are poorly understood. We hypothesized that the development of EE in patients and in an experimental murine model would be associated with eosinophil-mediated tissue remodeling. METHODS: Histopathologic analysis of basal layer thickness and collagen accumulation was performed on the biopsy specimens of normal individuals, EE patients, and mouse esophageal tissue sections following experimental induction of EE in wild-type, eosinophil lineage-deficient, interleukin (IL)-5-deficient, and IL-5 transgenic mice, with the latter 2 mice groups having decreased and increased esophageal eosinophilia, respectively. RESULTS: An impressive accumulation of collagen in the epithelial mucosa and lamina propria, as well as basal layer thickening, was observed in the esophagus of patients with EE as well as in mice with experimental EE compared with controls. Significantly reduced lamina propria collagen and basal layer thickness were observed in IL-5-deficient mice and eosinophil lineage-deficient mice compared with wild-type mice following the induction of experimental EE. Furthermore, the esophagus of CD2-IL-5 transgenic mice showed increased basal layer thickness and collagen accumulation compared with nontransgenic mice, yet IL-5 intestine transgenic mice did not have EE-like esophageal changes. Additional analysis revealed increased IL-5 levels in the esophagus of EE patients, allergen-challenged wild-type mice, and CD2-IL-5 transgenic mice but not in IL-5 intestine transgenic mice. CONCLUSIONS: These findings provide evidence that local IL-5-mediated eosinophilia is essential in the induction of esophageal remodeling.

IL-13 involvement in eosinophilic esophagitis: transcriptome analysis and reversibility with glucocorticoids.

BACKGROUND: Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. Early studies have established that esophageal eosinophilia occurs in association with T(H)2 allergic responses, and we recently identified an EE-specific esophageal transcriptome that included eotaxin-3. OBJECTIVE: We sought to determine the mechanism by which this T(H)2 response leads to EE. METHODS: Real-time PCR and microarray analysis were performed on RNA extracted from esophageal biopsy specimens and primary esophageal epithelial cell cultures stimulated with IL-13 (0-100 ng/mL). Transient transfections in esophageal cell lines were performed with plasmids containing the luciferase gene driven by eotaxin-3 promoter fragments and modified forms of signal transducer and activator of transcription 6. RESULTS: The IL-13 mRNA level was markedly increased (16-fold) in esophageal biopsy specimens from patients with EE compared with those from healthy individuals. Furthermore, IL-13 treatment of primary esophageal epithelial cells was sufficient to induce a global-expression transcript profile that remarkably overlapped with the EE-specific esophageal transcriptome. In addition, esophageal epithelial cells markedly produce eotaxin-3 after IL-13 stimulation through a transcriptional mechanism dependent on signal transducer and activator of transcription 6. Lastly, increased IL-13 mRNA levels and the EE transcriptome were largely reversible with glucocorticoid treatment in vivo. CONCLUSIONS: Taken together, we propose that the pathogenesis of EE is mediated by an IL-13-stimulated keratinocyte-derived transcriptome that is largely reversible with corticosteroid treatment. Furthermore, we identify an in vivo IL-13-induced transcriptome that has potential utility for target assessment after anti-IL-13 therapeutics. CLINICAL IMPLICATIONS: IL-13-induced pathways and genes are fundamental processes in the cause and manifestations of EE; as such, therapeutic agents that interfere with IL-13 might be particularly useful for disease treatment.

Interplay of adaptive th2 immunity with eotaxin-3/c-C chemokine receptor 3 in eosinophilic esophagitis.

BACKGROUND: Pediatric eosinophilic esophagitis (EE) is a recently described disorder associated with atopy. Although studies of esophageal tissue suggest that Th2 cytokines and eotaxin-3 may be crucial in disease pathogenesis, little is known about the systemic immunological phenotypes of children with EE. OBJECTIVES: To define the phenotypes of peripheral blood eosinophils and lymphocytes in EE and to examine for correlations between these parameters and tissue eosinophil numbers and disease severity. PATIENTS AND METHODS: Blood was collected from children with EE, atopic control children without EE, and nonatopic control children without EE. Flow cytometry was used to measure eosinophil expression of chemokine receptor 3 (CCR3) and interleukin-5 receptor-alpha (IL-5Ralpha), and intracellular lymphocyte expression of IL-4, IL-5, IL-13, interferon-gamma, and tumor necrosis factor-alpha. Eosinophil numbers and eotaxin-3 mRNA levels were quantitated in esophageal biopsy specimens. RESULTS: Compared with nonatopic control children, EE patients with active disease had increased peripheral blood eosinophil percentages, mean channel of fluorescence (MCF) of CCR3 on eosinophils, and percentage of CD4+ T cells expressing IL-5. Notably, these parameters positively correlated with esophageal eosinophil numbers. Eotaxin-3 tissue expression positively correlated with esophageal eosinophil numbers and peripheral blood eosinophil CCR3 MCF. The percentage of peripheral blood eosinophils, eosinophil CCR3 MCF, and CD4+ T cell expression of IL-5 were lower in EE patients in disease remission than in patients with active disease. CONCLUSIONS: Collectively, these studies demonstrate cooperation between systemic CD4+ Th2-cell-mediated immunity and an enhanced eosinophil-CCR3/eotaxin-3 pathway in EE pathogenesis. Furthermore, the imbalanced Th2 immunity and increased CCR3 expression are reversible with disease remission.

Potential of blood eosinophils, eosinophil-derived neurotoxin, and eotaxin-3 as biomarkers of eosinophilic esophagitis.

BACKGROUND & AIMS: Eosinophilic esophagitis (EE) is an increasingly recognized disorder characterized by eosinophilic inflammation of the esophageal mucosa, and typically requires serial invasive endoscopic biopsy examinations to document the characteristic histologic features of the disorder. The aim of this study was to identify noninvasive biomarkers that correlated with disease activity and response to treatment as measured by esophageal eosinophilia. METHODS: A prospective, cross-sectional analysis was performed on 47 pediatric patients undergoing endoscopic evaluation of possible EE. Blood samples were collected for measurement of peripheral blood absolute eosinophil count (AEC) and levels of eosinophil-derived neurotoxin (EDN), eotaxin-1, -2, and -3, and interleukin-5. Stool samples were collected for measurement of EDN. Biomarker levels were correlated with esophageal eosinophil density, and differences in biomarker levels based on disease activity and treatment were determined. RESULTS: AEC, plasma EDN levels, and eotaxin-3 levels significantly correlated with esophageal eosinophil density (AEC: r = 0.56, P < .0001; EDN: r = 0.54, P < .0001; eotaxin-3: r = 0.32, P = .04), and were increased in patients with active EE vs controls (AEC: 440 vs 140 eosinophils/muL, P < .05; EDN: 50.3 vs 31.1 ng/mL, P = .01; eotaxin-3: 37.7 vs 11.5 pg/mL, P = .01). Cut-off values were established to maximize the sensitivity, specificity, and predictive values of these biomarkers alone and in combination. Eotaxin-1, eotaxin-2, interleukin-5, and fecal EDN levels did not correlate with esophageal eosinophil density, and were not increased in active EE vs controls or those with inactive EE. CONCLUSIONS: These data show that blood levels of AEC, EDN, and eotaxin-3 may have value as noninvasive biomarkers for monitoring EE.

Resistin-like molecule beta regulates innate colonic function: barrier integrity and inflammation susceptibility.

BACKGROUND: Resistin-like molecule (RELM) beta is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function primarily remains an enigma. OBJECTIVE: We sought to elucidate the function of RELM-beta in the gastrointestinal tract. METHODS: We generated RELM-beta gene-targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation. RESULTS: We show that RELM-beta is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-beta. Using acute colonic inflammatory models, we demonstrate that RELM-beta has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-beta regulates expression of type III regenerating gene (REG) (REG3beta and gamma), molecules known to influence nuclear factor kappaB signaling. CONCLUSIONS: These data define a critical role for RELM-beta in the maintenance of colonic barrier function and gastrointestinal innate immunity. CLINICAL IMPLICATIONS: These findings identify RELM-beta as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies.

Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.

Eosinophilic esophagitis (EE) is an emerging disorder with a poorly understood pathogenesis. In order to define disease mechanisms, we took an empirical approach analyzing esophageal tissue by a genome-wide microarray expression analysis. EE patients had a striking transcript signature involving 1% of the human genome that was remarkably conserved across sex, age, and allergic status and was distinct from that associated with non-EE chronic esophagitis. Notably, the gene encoding the eosinophil-specific chemoattractant eotaxin-3 (also known as CCL26) was the most highly induced gene in EE patients compared with its expression level in healthy individuals. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia and mastocytosis. Furthermore, a single-nucleotide polymorphism in the human eotaxin-3 gene was associated with disease susceptibility. Finally, mice deficient in the eotaxin receptor (also known as CCR3) were protected from experimental EE. These results implicate eotaxin-3 as a critical effector molecule for EE and provide insight into disease pathogenesis.

Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription 6.

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at sites of inflammation. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. Eotaxin-3/CCL-26 belongs to a family of CC chemokines, which are potent chemoattractants for eosinophils. In this study, we hypothesized that intestinal epithelial cells could release eotaxin-3. We demonstrate that the T helper 2 type cytokines interleukin-4 or interleukin-13 increase eotaxin-3 mRNA levels and eotaxin-3 protein expression in the human intestinal epithelial cell lines HT-29 CL.19A and T84 in a dose-dependent manner. Addition of actinomycin-D prior to interleukin-4/-13 stimulation led to decreases in eotaxin-3 mRNA levels similar to those observed in controls without interleukin-4/-13. Interleukin-4 and interleukin-13 activated signal transducer and activator of transcription 6 which was found to bind the two canonical signal transducer and activator of transcription 6 binding sites located in the eotaxin-3 promoter. Experiments with the eotaxin-3 promoter luciferase constructs revealed that the most proximal signal transducer and activator of transcription 6 binding site located between positions -62 and -71 relative to the transcriptional start was necessary for full eotaxin-3 promoter activity. Importantly, we present evidence that the signal transducer and activator of transcription 6 is necessary and sufficient for interleukin-4 or interleukin-13 mediated eotaxin-3 gene up-regulation using HT-29 CL.19A cells expressing a dominant-negative signal transducer and activator of transcription 6. Overall, these results demonstrate that epithelial eotaxin-3 is up-regulated in the context of a T helper 2 mediated inflammatory bowel disease via the signal transducer and activator of transcription 6, thus suggesting that the intestinal epithelium actively participates in the recruitment of eosinophils at the site of inflammation.

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6.

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1-4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.