Esophageal IgG4 levels correlate with histopathologic and transcriptomic features in eosinophilic esophagitis.

BACKGROUND: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses. METHODS: This case-control study included pediatric subjects with EoE (≥15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts. RESULTS: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs 0.2 mg/g protein [0.1-0.9]; P < .0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = .0006); histologic grade (P = .0011) and stage (P = .0112) scores; and IL4, IL10, IL13, but not TGFB1, expression and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression. CONCLUSION: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease; is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines; and is likely produced locally.

Cadherin 26 is an alpha integrin-binding epithelial receptor regulated during allergic inflammation.

Cadherins (CDH) mediate diverse processes critical in inflammation, including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous CDHs by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4ß7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4+ T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional CDH regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

Newly developed and validated eosinophilic esophagitis histology scoring system and evidence that it outperforms peak eosinophil count for disease diagnosis and monitoring.

Eosinophilic esophagitis (EoE) is diagnosed by symptoms, and at least 15 intraepithelial eosinophils per high power field in an esophageal biopsy. Other pathologic features have not been emphasized. We developed a histology scoring system for esophageal biopsies that evaluates eight features: eosinophil density, basal zone hyperplasia, eosinophil abscesses, eosinophil surface layering, dilated intercellular spaces (DIS), surface epithelial alteration, dyskeratotic epithelial cells, and lamina propria fibrosis. Severity (grade) and extent (stage) of abnormalities were scored using a 4-point scale (0 normal; 3 maximum change). Reliability was demonstrated by strong to moderate agreement among three pathologists who scored biopsies independently (P ≤ 0.008). Several features were often abnormal in 201 biopsies (101 distal, 100 proximal) from 104 subjects (34 untreated, 167 treated). Median grade and stage scores were significantly higher in untreated compared with treated subjects (P ≤ 0.0062). Grade scores for features independent of eosinophil counts were significantly higher in biopsies from untreated compared with treated subjects (basal zone hyperplasia P ≤ 0.024 and DIS P ≤ 0.005), and were strongly correlated (R-square >0.67). Principal components analysis identified three principal components that explained 78.2% of the variation in the features. In logistic regression models, two principal components more closely associated with treatment status than log distal peak eosinophil count (PEC) (R-square 17, area under the curve (AUC) 77.8 vs. R-square 9, AUC 69.8). In summary, the EoE histology scoring system provides a method to objectively assess histologic changes in the esophagus beyond eosinophil number. Importantly, it discriminates treated from untreated patients, uses features commonly found in such biopsies, and is utilizable by pathologists after minimal training. These data provide rationales and a method to evaluate esophageal biopsies for features in addition to PEC.

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.

Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis.

The desmosomal cadherin desmoglein-1 (DSG1) is an essential intercellular adhesion molecule that is altered in various human cutaneous disorders; however, its regulation and function in allergic disease remains unexplored. Herein, we demonstrate a specific reduction in DSG1 in esophageal biopsies from patients with eosinophilic esophagitis (EoE), an emerging allergic disorder characterized by chronic inflammation within the esophageal mucosa. Further, we show that DSG1 gene silencing weakens esophageal epithelial integrity, and induces cell separation and impaired barrier function (IBF) despite high levels of desmoglein-3. Moreover, DSG1 deficiency induces transcriptional changes that partially overlap with the transcriptome of inflamed esophageal mucosa; notably, periostin (POSTN), a multipotent pro-inflammatory extracellular matrix molecule, is the top induced overlapping gene. We further demonstrate that IBF is a pathological feature in EoE, which can be partially induced through the downregulation of DSG1 by interleukin-13 (IL-13). Taken together, these data identify a functional role for DSG1 and its dysregulation by IL-13 in the pathophysiology of EoE and suggest that the loss of DSG1 may potentiate allergic inflammation through the induction of pro-inflammatory mediators such as POSTN.

Eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, eosinophil-predominant inflammatory condition that can affect esophageal mucosa at any age. Distinguished from gastroesophageal reflux disease in the mid 1990's, it has seemed to be increasingly prevalent, and is usually a manifestation of food allergy. The endoscopic and histologic features are well described. The clinical manifestations vary considerably by age, with adolescents and adults complaining primarily of dysphagia. Younger children may present with pain, vomiting, other evidence for food allergy, or feeding difficulties. Treatment options include swallowed (non-systemic) steroids and dietary antigen elimination, and must be maintained indefinitely due to the extremely high rate of recurrence off therapy. The complications of untreated disease include fibrosis of the esophageal lamina propria and stricture formation that result in chronic dysphagia, risking food impaction and perforation.

Local B cells and IgE production in the oesophageal mucosa in eosinophilic oesophagitis.

BACKGROUND: Eosinophilic oesophagitis (EO) is an emerging yet increasingly prevalent disorder characterised by a dense and selective eosinophilic infiltration of the oesophageal wall. While EO is considered an atopic disease primarily triggered by food antigens, disparities between standard allergen testing and clinical responses to exclusion diets suggest the participation of distinct antigen-specific immunoglobulin E (IgE) in the pathophysiology of EO. AIM: To find evidence for a local IgE response. METHODS: Endoscopic biopsies of the distal oesophagus of atopic and non-atopic EO and control individuals (CTL) were processed for immunohistochemistry and immunofluorescence to assess the presence of B cells, mast cells, and IgE-bearing cells. Oesophageal RNA was analysed for the expression of genes involved in B cell activation, class switch recombination to IgE and IgE production, including germline transcripts (GLTs), activation-induced cytidine deaminase (AID), IgE heavy chain (Cepsilon) and mature IgE mRNA using polymerase chain reaction and microarray analysis. RESULTS: Regardless of atopy, EO showed increased density of B cells (p<0.05) and of IgE-bounded mast cells compared to CTL. Both EO and CTL expressed muGLT, epsilonGLT, gamma4GLT, AID, Cepsilon and IgE mRNA. However, the frequency of expression of total GLTs (p = 0.002), epsilonGLT (p = 0.024), and Cepsilon (p = 0.0003) was significantly higher in EO than in CTL, independent of the atopic status. CONCLUSION: These results support the heretofore unproven occurrence of both local immunoglobulin class switching to IgE and IgE production in the oesophageal mucosa of EO patients. Sensitisation and activation of mast cells involving local IgE may therefore critically contribute to disease pathogenesis.

Periostin facilitates eosinophil tissue infiltration in allergic lung and esophageal responses.

Periostin is an extracellular matrix protein that has been primarily studied in the context of the heart, where it has been shown to promote cardiac repair and remodeling. In this study, we focused on the role of periostin in an allergic eosinophilic inflammatory disease (eosinophilic esophagitis (EE)) known to involve extensive tissue remodeling. Periostin was indeed markedly overexpressed (35-fold) in the esophagus of EE patients, particularly in the papillae, compared with control individuals. Periostin expression was downstream from transforming growth factor-beta and interleukin-13, as these cytokines were elevated in EE esophageal samples and markedly induced periostin production by primary esophageal fibroblasts (107- and 295-fold, respectively, at 10 ng ml(-1)). A functional role for periostin in eliciting esophageal eosinophilia was demonstrated, as periostin-null mice had a specific defect in allergen-induced eosinophil recruitment to the lungs and esophagus (66 and 72% decrease, respectively). Mechanistic analyses revealed that periostin increased (5.8-fold) eosinophil adhesion to fibronectin. As such, these findings extend the involvement of periostin to esophagitis and uncover a novel role for periostin in directly regulating leukocyte (eosinophil) accumulation in T helper type 2-associated mucosal inflammation in both mice and humans.

The spectrum of pediatric eosinophilic esophagitis beyond infancy: a clinical series of 30 children.

OBJECTIVES: Eosinophilic esophagitis, previously confused with esophageal inflammation due to gastroesophageal reflux, has recently begun to be distinguished from it. We undertook this analysis of our large series of children with the condition to clarify its spectrum: its presenting symptoms; its relation to allergy, respiratory disease, and reflux; its endoscopic and histological findings; and its diagnosis and therapy. METHODS: We analyzed the details of our clinical series of 30 children with eosinophilic esophagitis, defining it as > or =5 eosinophils per high power field in the distal esophageal epithelium. Retrospective chart review was supplemented by prospective, blinded, duplicate quantitative evaluation of histology specimens, and by telephone contact with some families to clarify subsequent course. Presentation and analysis of the series as a whole is preceded by a case illustrating a typical presentation with dysphagia and recurrent esophageal food impactions. RESULTS: Presenting symptoms encompass vomiting, pain, and dysphagia (some with impactions or strictures). Allergy, particularly food allergy, is an associated finding in most patients, and many have concomitant asthma or other chronic respiratory disease. A subtle granularity with furrows or rings is newly identified as the endoscopic herald of histological eosinophilic esophagitis. Histological characteristics include peripapillary or juxtaluminal eosinophil clustering in certain cases. Association with eosinophilic gastroenteritis occurs, but is not common. Differentiation from gastroesophageal reflux disease is approached by analyzing eosinophil density and response to therapeutic trials. Therapy encompasses dietary elimination and anti-inflammatory pharmacotherapy. CONCLUSION: Awareness of the spectrum of eosinophilic esophagitis should promote optimal diagnosis and treatment of this elusive entity, both in children and in adults.

Laryngeal cleft and eosinophilic gastroenteritis: report of 2 cases.

Although laryngotracheoesophageal clefts are often found in association with other well-described anomalies, we know of no previous reported association with eosinophilic gastroenteritis, a disorder of unknown etiology characterized by eosinophilic infiltration of the gastrointestinal tract. We treated 2 children who had laryngeal clefts and eosinophilic gastroenteritis. Since the esophageal inflammatory changes found in eosinophilic gastroenteritis may persist despite aggressive therapy, management of the laryngotracheoesophageal clefts is more complicated. The diagnosis of eosinophilic gastroenteritis should not be overlooked in patients with laryngotracheoesophageal clefts and warrants prompt referral to a pediatric gastroenterologist.

Anatomic variability of rejection in intestinal allografts after pediatric intestinal transplantation.

BACKGROUND: Rejection of the allograft is a major contributor to morbidity and mortality in children who undergo a small intestinal transplant. Operational definitions for histologic rejection have been established, but little is known about the anatomic variability of the histologic abnormalities. STUDY DESIGN: Biopsy reports were reviewed retrospectively from more than 1200 endoscopies performed on the 41 children who received intestinal transplantation between 1990 and 1995. RESULTS: Biopsies were performed in the proximal jejunum and distal ileum allograft simultaneously on 248 occasions. In 168 biopsies, neither site was histologically abnormal; in 80, rejection was found. In 42, both regions were abnormal; however, in 17 only the jejunum was involved and in 21 the rejection exclusively involved the ileum. Among children whose allograft included colon, rejection was absent in colon and ileum in 34 biopsies, involved both in 6, involved ileum but not colon in another 6 and involved colon but not ileum in only one. When the allograft included stomach, rejection was absent in the stomach and jejunum 39 times, involved both sites 8 times, involved jejunum and not the stomach 10 times, but involved the stomach and not jejunum only once. Endoscopic appearance correctly predicted histologic rejection 63% of the time. CONCLUSION: Anatomic variability may exist in the rejection process. Sampling the jejunum and ileum seems to have similar sensitivity in detecting rejection, whereas sampling stomach and the colon is less sensitive. When allograft rejection is suspected on clinical grounds, sampling more than one area of the graft may be necessary for histologic confirmation.

Endoscopies in pediatric small intestinal transplant recipients: five years experience.

OBJECTIVE: Intestinal transplantation has become an option as a treatment for permanent intestinal failure. Endoscopy is an essential tool in assessing the intestinal allograft after intestinal transplantation. The aim of this study was to analyze our experience using endoscopy in intestinal transplant recipients. METHODS: This was a retrospective review of endoscopic and histological reports in 41 children who received an intestinal transplant between 1990 and 1995 at Children's Hospital of Pittsburgh. RESULTS: A total of 1273 endoscopies was performed of which 760 were ileoscopies via allograft ileostomy, 273 were upper endoscopies, and 240 were colonoscopies. One hundred four rejection episodes were documented histologically in 32 patients, 6 days to >4 yr after transplantation. Most episodes were mild and easily treated with increased immunosuppression; however, severe rejection with mucosal exfoliation was seen in nine patients. Rejection sometimes involved only part of the allograft. Endoscopic appearance alone without biopsies was sensitive enough to diagnose only 63% of the rejection episodes. Epstein-Barr and cytomegalovirus infections occurred in 11 and eight patients, respectively, and involved both native bowel and allograft in some. Complications of endoscopy were few: one perforation, three episodes of bleeding, and three episodes of transient respiratory compromise. CONCLUSIONS: Endoscopy is an essential tool in the postoperative assessment of intestinal transplant recipients. Frequent surveillance ileoscopies with biopsies should be performed after transplantation. If patients clinically deteriorate with fever, diarrhea, bacteremia, or gastrointestinal bleeding and a clear cause is not elucidated by ileoscopy, an upper endoscopy with biopsies is indicated.

Postviral gastroparesis: presentation, treatment, and outcome.

We describe the clinical features and long-term outcome of 11 children who had persistent gastroparesis after an acute viral illness, eight of whom tested positive for rotavirus. Gastric emptying was delayed in the 10 children evaluated with scintigraphy. Antroduodenal manometry confirmed postprandial antral hypomotility in 10 subjects. All children recovered within 6 to 24 months.

Gastroesophageal reflux disease and dysphagia in children.

Gastroesophageal reflux disease (GERD) is a common problem in children that is sometimes associated with dysphagia. Choking, food refusal, and food "getting stuck" are non-specific symptoms that may arise consequent to reflux and esophagitis. Swallowing plays a role in reflux physiology, functioning as a major clearance mechanism after reflux episodes. Therefore, failure of swallowing to effectively perform that function contributes to reflux pathophysiology. The diagnosis and treatment of GERD in children must be carried out systematically and thoroughly. Multiple interacting factors are common, thus complicating the process.

Rectal bleeding in Prader-Willi syndrome.

Individuals with Prader-Willi syndrome manifest severe skin picking behavior. We report three patients with this syndrome in whom an extension of this behavior to rectal picking resulted in significant lower gastrointestinal bleeding and anorectal disease. The recognition of this behavior is important to avoid misdiagnosing inflammatory bowel disease in this group of patients.

Imaging of post-transplant spindle cell tumors.

Spindle/smooth muscle cell proliferation is an additional neoplastic process related to immunosuppression and EBV infection. We describe four post transplant children with this diagnosis. Multiple organ systems may be involved, particularly the liver, gastrointestinal tract, and lungs. Lesions are radiographically, clinically, and endoscopically indistinguishable from those of post-transplant lymphoproliferative disease (PTLD).

Chronic pancreatitis in a patient with cystic fibrosis and clinical pancreatic insufficiency.

Pancreatitis occurs in up to 15% of patients with cystic fibrosis and pancreatic sufficiency, but the possibility of its occurrence in patients with pancreatic insufficiency has not been recognized. We describe a patient with homozygous delta F508 cystic fibrosis and typical symptoms of pancreatic insufficiency (greasy, fatty stools) in whom pancreatitis developed.

Gastrointestinal bleeding due to delayed perianastomotic ulceration in children.

Delayed perianastomotic ulcers are a poorly recognized complication of intestinal surgery. We report two patients with this complication of their remote intestinal surgery who developed significant iron deficiency anemia. Patient 1 had intestinal resection for perforated necrotizing enterocolitis as a newborn and presented at 16 yr of age with an ulcer at the ileocolonic anastomosis. Patient 2 had intestinal resection for strangulated internal hernia at 9 yr and was diagnosed with two ulcers at the ileoileal anastomosis at 14 yr of age. Fifteen patients with delayed anastomotic ulcers have so far been reported in the literature. We add two more cases and also emphasize the difficulty in establishing the diagnosis and importance of performing a retrograde ileoscopy.