Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis.

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFβ1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE

Heat Shock Protein-Derived T-Cell Epitopes Contribute to Autoimmune Inflammation in Pediatric Crohn's Disease

Pediatric Crohn's disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn's disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation

Non-invasive intradermal imaging of cystine crystals in cystinosis.

ImportanceDevelopment of noninvasive methodology to reproducibly measure tissue cystine crystal load to assess disease status and guide clinical care in cystinosis, an inherited lysosomal storage disorder characterized by widespread cystine crystal accumulation.ObjectiveTo develop an unbiased and semi-automated imaging methodology to quantify dermal cystine crystal accumulation in patients to correlate with disease status.Design, setting and participants101 participants, 70 patients and 31 healthy controls, were enrolled at the University of California, San Diego, Cystinosis Clinics, Rady Children's Hospital, San Diego and at the annual Cystinosis Research Foundation family conference for an ongoing prospective longitudinal cohort study of cystinosis patients with potential yearly follow-up.ExposuresIntradermal reflectance confocal microscopy (RCM) imaging, blood collection via standard venipuncture, medical record collection, and occasional skin punch biopsies.Main outcomes and measuresThe primary outcome was to establish an automated measure of normalized confocal crystal volume (nCCV) for each subject. Secondary analysis examined the association of nCCV with various clinical indicators to assess nCCV's possible predictive potential.ResultsOver 2 years, 57 patients diagnosed with cystinosis (median [range] age: 15.1 yrs [0.8, 54]; 41.4% female) were intradermally assessed by RCM to produce 84 image stacks. 27 healthy individuals (38.7 yrs [10, 85]; 53.1% female) were also imaged providing 37 control image stacks. Automated 2D crystal area quantification revealed that patients had significantly elevated crystal accumulation within the superficial dermis. 3D volumetric analysis of this region was significantly higher in patients compared to healthy controls (mean [SD]: 1934.0 μm3 [1169.1] for patients vs. 363.1 μm3 [194.3] for controls, P<0.001). Medical outcome data was collected from 43 patients with infantile cystinosis (media [range] age: 11 yrs [0.8, 54]; 51% female). nCCV was positively associated with hypothyroidism (OR = 19.68, 95% CI: [1.60, 242.46], P = 0.02) and stage of chronic kidney disease (slope estimate = 0.53, 95%CI: [0.05, 1.00], P = 0.03).Conclusions and relevanceThis study used non-invasive RCM imaging to develop an intradermal cystine crystal quantification method. Results showed that cystinosis patients had increased nCCV compared to healthy controls. Level of patient nCCV correlated with several clinical outcomes suggesting nCCV may be used as a potential new biomarker for cystinosis to monitor long-term disease control and medication compliance

Esophageal Compliance Quantifies Epithelial Remodeling in Pediatric Patients With Eosinophilic Esophagitis.

BACKGROUND: The management of eosinophilic esophagitis (EoE) relies on the severity of esophageal eosinophilia, yet there is poor evidence of its prediction of esophageal fibrotic remodeling and subsequent complications such as dysphagia, food impactions, or strictures. Functional luminal imaging planimetry (FLIP) has had limited use in pediatric patients to evaluate esophageal tissue mechanics. We aimed to standardize the FLIP technique and to measure esophageal compliance in children with EoE in comparison to controls. METHODS: Subjects were enrolled into a prospective observational study and had FLIP performed at the time of endoscopy. We calculated esophageal distensibility and compliance for the total and segmental esophagus independently (ie, proximal, middle, and distal esophageal segments). We evaluated esophageal biopsies for eosinophilia and epithelial remodeling, calculated endoscopy scores, and documented patient symptoms. RESULTS: We enrolled 11 EoE and 12 controls subjects, aged 5 to 18 years old. While EoE subjects had lower esophageal compliance (P = 0.004) than controls, the difference in distensibility did not reach significance (P = 0.151). Epithelial remodeling severity was more strongly correlated with compliance than with distensibility. Epithelial remodeling scores ≥2 had a significant association with lower compliance both segmentally and in the entire esophagus (P = 0.029), but not with distensibility. Compliance measures were more sensitive in detecting subjects with remodeling score ≥2 than distensibility (79% vs 64%). CONCLUSIONS: Compliance is a more sensitive measure of esophageal epithelial remodeling in children compared to distensibility, and a more appropriate measure of esophageal tissue mechanics. Standardized placement of the FLIP catheter is important to accurately assess esophageal compliance

Non-invasive intradermal imaging of cystine crystals in cystinosis.

ImportanceDevelopment of noninvasive methodology to reproducibly measure tissue cystine crystal load to assess disease status and guide clinical care in cystinosis, an inherited lysosomal storage disorder characterized by widespread cystine crystal accumulation.ObjectiveTo develop an unbiased and semi-automated imaging methodology to quantify dermal cystine crystal accumulation in patients to correlate with disease status.Design, setting and participants101 participants, 70 patients and 31 healthy controls, were enrolled at the University of California, San Diego, Cystinosis Clinics, Rady Children's Hospital, San Diego and at the annual Cystinosis Research Foundation family conference for an ongoing prospective longitudinal cohort study of cystinosis patients with potential yearly follow-up.ExposuresIntradermal reflectance confocal microscopy (RCM) imaging, blood collection via standard venipuncture, medical record collection, and occasional skin punch biopsies.Main outcomes and measuresThe primary outcome was to establish an automated measure of normalized confocal crystal volume (nCCV) for each subject. Secondary analysis examined the association of nCCV with various clinical indicators to assess nCCV's possible predictive potential.ResultsOver 2 years, 57 patients diagnosed with cystinosis (median [range] age: 15.1 yrs [0.8, 54]; 41.4% female) were intradermally assessed by RCM to produce 84 image stacks. 27 healthy individuals (38.7 yrs [10, 85]; 53.1% female) were also imaged providing 37 control image stacks. Automated 2D crystal area quantification revealed that patients had significantly elevated crystal accumulation within the superficial dermis. 3D volumetric analysis of this region was significantly higher in patients compared to healthy controls (mean [SD]: 1934.0 μm3 [1169.1] for patients vs. 363.1 μm3 [194.3] for controls, PConclusions and relevanceThis study used non-invasive RCM imaging to develop an intradermal cystine crystal quantification method. Results showed that cystinosis patients had increased nCCV compared to healthy controls. Level of patient nCCV correlated with several clinical outcomes suggesting nCCV may be used as a potential new biomarker for cystinosis to monitor long-term disease control and medication compliance