Amir F. Kagalwalla

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and γ-glutamyltranspeptidase (γGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

Identification of specific foods responsible for inflammation in children with eosinophilic esophagitis successfully treated with empiric elimination diet.

Objectives: Eosinophilic esophagitis (EoE) is an immune-mediated chronic inflammatory disorder triggered by food antigen(s). A 6-food elimination diet (SFED) excluding cows milk, soy, wheat, egg, peanuts/tree nuts, and seafood has been shown to induce remission in a majority of children with EoE. The goal of the present study was to identify specific food antigens responsible for eosinophilic esophageal inflammation in children with EoE who had achieved histological remission with the SFED. Patients and Methods: In this analysis, we retrospectively analyzed children with EoE who completed subsequent single-food reintroductions that led to identification of foods causing disease recurrence. Repeat upper endoscopy with biopsies was performed after single-food introductions. Recurrence of esophageal eosinophilia following a food reintroduction identified that food antigen as a cause of EoE. Results: A total of 36/46 (25 M/11F) children who were initially successfully treated with SFED completed this trial; the mean age was 7.6 ± 4.3 years. The most common foods identified were 25 to cows milk (74%), 8 to wheat (26%), 4 to eggs (17%), 3 to soy (10%), and 1 to peanut (6%). Milk was 8 times more likely to cause EoE compared with wheat, the next most common food (95% confidence interval 2.41–26.62, P = 0.0007). Conclusions: Serial single-food reintroductions following induction of histological remission with the SFED can lead to the identification of specific causal food antigen(s) in EoE. Cows milk was the most common food identified in subjects with EoE treated with SFED. A subset of children with EoE may develop tolerance to their food sensitivities while on the SFED.

Histopathologic Variability in Children With Eosinophilic Esophagitis

OBJECTIVES:The diagnosis of eosinophilic esophagitis (EoE) is based on histologic findings of eosinophilia in the esophageal squamous epithelium in patients presenting with esophageal symptoms. Variability in the degree of squamous epithelial eosinophil infiltration presents a challenge to diagnostic accuracy. We examined the histopathologic features in a well-defined cohort of children with EoE to derive an optimal number of biopsies needed for diagnosis.METHODS:A total of 30 patients diagnosed with EoE based on>15 eosinophils per high-power field (eos/HPF) were evaluated. The diagnosis of EoE was based on persistent esophageal eosinophilia following acid suppression therapy. All patients had mid and distal biopsy specimens that were analyzed along with 24-h pH probe studies.RESULTS:A total of 221 biopsy specimens were reviewed with a median of 7.8 (range, 5–8) specimens per patient. Eight patients demonstrated abnormal pH studies. The median eos/HPF was 41 (range, 0–288 eos/HPF). Using a criterion of>15 eos/HPF, the diagnostic sensitivity of a single biopsy was 73%, which increased to 84%, 97% and 100% for two, three and six biopsies, respectively. Five patients (17%) would not have met the criteria for diagnosis if biopsies were only taken from the mid esophagus whereas one patient (3%) would have been missed if only distal biopsies were analyzed.CONCLUSIONS:Significant histologic variability exists among biopsy specimens from pediatric patients with EoE. Using the criterion of>15 eos/HPF, three biopsy specimens achieved a diagnosis of EoE in 97% of patients. Histopathologic changes consistent with EoE can persist following proton pump inhibitor therapy in patients with EoE and gastroesophageal reflux disease when the latter is defined by increased distal esophageal acid exposure.

The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis

Objective Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. Design The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot–Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. Results ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. Conclusions The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Eosinophilic Esophagitis: Epithelial Mesenchymal Transition Contributes to Esophageal Remodeling and Reverses with Treatment

BACKGROUND Mechanisms underlying esophageal remodeling with subepithelial fibrosis in subjects with eosinophilic esophagitis (EoE) have not been delineated. OBJECTIVES We sought to explore a role for epithelial mesenchymal transition (EMT) in subjects with EoE and determine whether EMT resolves with treatment. METHODS Esophageal biopsy specimens from 60 children were immunostained for epithelial (cytokeratin) and mesenchymal (vimentin) EMT biomarkers, and EMT was quantified. Subjects studied had EoE (n = 17), indeterminate EoE (n = 15), gastroesophageal reflux disease (n = 7), or normal esophagus (n = 21). EMT was analyzed for relationships to diagnosis, eosinophil counts, and indices of subepithelial fibrosis, eosinophil peroxidase, and TGF-β immunostaining. EMT was assessed in pretreatment and posttreatment biopsy specimens from 18 subjects with EoE treated with an elemental diet, 6-food elimination diet, or topical corticosteroids (n = 6 per group). RESULTS TGF-β1 treatment of esophageal epithelial cells in vitro for 24 hours induced upregulation of mesenchymal genes characteristic of EMT, including N-cadherin (3.3-fold), vimentin (2.1-fold), and fibronectin (7.5-fold). EMT in esophageal biopsy specimens was associated with EoE (or indeterminate EoE) but not gastroesophageal reflux disease or normal esophagus and was correlated to eosinophil counts (r = 0.691), eosinophil peroxidase (r = 0.738), and TGF-β (r = 0.520) immunostaining and fibrosis (r = 0.644) indices. EMT resolved with EoE treatments that induced clinicopathologic remission with reduced eosinophil counts. EMT decreased significantly after treatment by 74.1% overall in the 18 treated subjects with EoE; pretreatment versus posttreatment EMT scores were 3.17 ± 0.82 versus 0.82 ± 0.39 (P < .001), with similar decreases within treatment groups. Pretreatment/posttreatment EMT was strongly correlated with eosinophil counts for combined (r = 0.804, P < .001) and individual treatment groups. CONCLUSIONS EMT likely contributes to subepithelial fibrosis in subjects with EoE and resolves with treatments that decrease esophageal inflammation, and its resolution correlates with decreased numbers of esophageal eosinophils.

Cow's milk elimination: a novel dietary approach to treat eosinophilic esophagitis.

Objectives: Cows-milk protein is one of the food antigens responsible for causing eosinophilic esophageal inflammation in a majority of children. We describe our experience with treating eosinophilic esophagitis (EoE) in children by eliminating only cows milk from their diets. Methods: This retrospective study assessed the short-term clinical and histological response to eliminating cows-milk protein from the diet of children with EoE. Only patients undergoing a subsequent upper endoscopy to assess their histological response were included in this analysis. Results: We identified 17 (12 boys and 5 girls) children with EoE who excluded only cows milk from their diet. Remission was induced in 11 of 17 (65%) patients; within the remission group, 7 (41%) achieved complete histological remission and 4 patients (24%) were in significant histological remission. The mean peak pre- and posttreatment counts for those in remission were 76 ± 40 and 2 ± 4 (P < 0.01), respectively. Conclusions: Elimination of cows milk–induced clinical and histological remission in 65% (95% confidence interval 42%–88%) of children with EoE in whom it was attempted. This approach offers distinct advantages over other dietary treatment approaches for the initial management of children with EoE. The role of eliminating cows milk alone for the treatment of EoE warrants further prospective study.

Symptoms Have Modest Accuracy in Detecting Endoscopic and Histologic Remission in Adults With Eosinophilic Esophagitis

BACKGROUND & AIMS It is not clear whether symptoms alone can be used to estimate the biologic activity of eosinophilic esophagitis (EoE). We aimed to evaluate whether symptoms can be used to identify patients with endoscopic and histologic features of remission. METHODS Between April 2011 and June 2014, we performed a prospective, observational study and recruited 269 consecutive adults with EoE (67% male; median age, 39 years old) in Switzerland and the United States. Patients first completed the validated symptom-based EoE activity index patient-reported outcome instrument and then underwent esophagogastroduodenoscopy with esophageal biopsy collection. Endoscopic and histologic findings were evaluated with a validated grading system and standardized instrument, respectively. Clinical remission was defined as symptom score <20 (range, 0-100); histologic remission was defined as a peak count of <20 eosinophils/mm(2) in a high-power field (corresponds to approximately <5 eosinophils/median high-power field); and endoscopic remission as absence of white exudates, moderate or severe rings, strictures, or combination of furrows and edema. We used receiver operating characteristic analysis to determine the best symptom score cutoff values for detection of remission. RESULTS Of the study subjects, 111 were in clinical remission (41.3%), 79 were in endoscopic remission (29.7%), and 75 were in histologic remission (27.9%). When the symptom score was used as a continuous variable, patients in endoscopic, histologic, and combined (endoscopic and histologic remission) remission were detected with area under the curve values of 0.67, 0.60, and 0.67, respectively. A symptom score of 20 identified patients in endoscopic remission with 65.1% accuracy and histologic remission with 62.1% accuracy; a symptom score of 15 identified patients with both types of remission with 67.7% accuracy. CONCLUSIONS In patients with EoE, endoscopic or histologic remission can be identified with only modest accuracy based on symptoms alone. At any given time, physicians cannot rely on lack of symptoms to make assumptions about lack of biologic disease activity in adults with EoE. ClinicalTrials.gov, Number: NCT00939263.

Esophageal microbiome in eosinophilic esophagitis

Objective The microbiome has been implicated in the pathogenesis of a number of allergic and inflammatory diseases. The mucosa affected by eosinophilic esophagitis (EoE) is composed of a stratified squamous epithelia and contains intraepithelial eosinophils. To date, no studies have identified the esophageal microbiome in patients with EoE or the impact of treatment on these organisms. The aim of this study was to identify the esophageal microbiome in EoE and determine whether treatments change this profile. We hypothesized that clinically relevant alterations in bacterial populations are present in different forms of esophagitis. Design In this prospective study, secretions from the esophageal mucosa were collected from children and adults with EoE, Gastroesophageal Reflux Disease (GERD) and normal mucosa using the Esophageal String Test (EST). Bacterial load was determined using quantitative PCR. Bacterial communities, determined by 16S rRNA gene amplification and 454 pyrosequencing, were compared between health and disease. Results Samples from a total of 70 children and adult subjects were examined. Bacterial load was increased in both EoE and GERD relative to normal subjects. In subjects with EoE, load was increased regardless of treatment status or degree of mucosal eosinophilia compared with normal. Haemophilus was significantly increased in untreated EoE subjects as compared with normal subjects. Streptococcus was decreased in GERD subjects on proton pump inhibition as compared with normal subjects. Conclusions Diseases associated with mucosal eosinophilia are characterized by a different microbiome from that found in the normal mucosa. Microbiota may contribute to esophageal inflammation in EoE and GERD.

Locus heterogeneity in progressive familial intrahepatic cholestasis.

Progressive familial intrahepatic cholestasis (PFIC or Byler disease) is a rare autosomal recessive form of severe and fatal cholestatic liver disease. A locus for PFIC has recently been mapped to chromosome 18q21-q22 in the original Byler pedigree. This region harbours the locus for a related phenotype, benign recurrent intrahepatic cholestasis (BRIC), suggesting that these traits are allelic. Linkage analysis was undertaken in five consanguineous PFIC pedigrees from Saudi Arabia using marker loci (D18S69, D18S41, D18S64, D18S38, D18S42, D18S55, D18S68, and D18S61) which span the Byler disease/BRIC region on 18q21-q22. In this family set the disease locus was excluded from this region, showing that locus heterogeneity exists for the PFIC phenotype.

Comparison of radionuclear scintigraphy and liver biopsy in the evaluation of neonatal cholestasis.

Summary Twenty-eight patients with neonatal cholestasis were given appropriate tests to arrive at a definitive diagnosis. Comparison of the results showing total obstruction on scintigraphy but not on liver biopsy is presented. It is suggested that liver biopsy be performed to confirm the diagnosis prior to undertaking exploratory laparotomy when radionuclear scan suggests extrahepatic biliary atresia.