Hedieh Saffari

Electron microscopy elucidates eosinophil degranulation patterns in patients with eosinophilic esophagitis

BACKGROUND In patients with eosinophilic esophagitis (EoE), eosinophils accumulate and release granule proteins onto esophageal epithelium. However, little is understood about the mechanism of eosinophil degranulation. OBJECTIVE To determine and quantify eosinophil degranulation patterns, we studied esophageal biopsy specimens from both the proximal and distal esophagi of 9 randomly selected patients with EoE. METHODS The specimens were fixed in glutaraldehyde, embedded, sectioned, and imaged by means of transmission electron microscopy. Eosinophils and their granules were identified by their distinctive morphology, and all eosinophils and granules were imaged. A total of 1672 images from 18 esophageal specimens were evaluated and graded. Eosinophils were categorized based on membrane integrity and by cytoplasmic vesiculation as evidence of piecemeal degranulation. Granules were categorized based on reversal of staining (eosinophil granule core lightening) and localization within and outside the cells. RESULTS The results revealed that greater than 98% of eosinophils infiltrating the esophagus in patients with EoE demonstrate morphologic abnormalities ranging from granule changes with reversal of staining to marked cytoplasmic vesiculation to loss of cellular membrane integrity with cytolytic disruption and release of intact membrane-bound granules into the tissues. Approximately 81% of eosinophils showed membrane disruption. Extracellular granules were abundant in at least 70% of the images, and approximately 50% of these granules showed reversal of staining. On the basis of the prominence of tubulovesicular development, piecemeal degranulation appears closely related to the other morphologic changes seen in patients with EoE. CONCLUSION These findings reveal that eosinophils in esophageal biopsy specimens from patients with EoE are abnormal, with greater than 80% showing cytolysis, and therefore that evaluation by means of light microscopy after hematoxylin and eosin staining might not accurately reflect eosinophil involvement.

Endoscopic appearance and location dictate diagnostic yield of biopsies in eosinophilic oesophagitis

Acknowledging that eosinophilic esophagitis (EoE) is a disease with variable involvement throughout the oesophagus, studies have suggested a minimum of five biopsies to diagnose EoE. Although it is accepted that furrows and exudates appear to represent areas of inflammation, no research to date has looked specifically at EoE endoscopic findings to see if eosinophilic infiltrate correlates with specific endoscopic findings.

99mTechnetium-labeled heparin: A new approach to detection of eosinophilic esophagitis–associated inflammation

FIG 1. A, Two-dimensional planar SPECT image of biopsies from normal, active EoE, and resolved EoE patients. From right, the samples are from patient no. 5, 3, 7, 6, 4, 2, 8, 1, 9, and 10, respectively. Multiple SPECT images were combined to make panel A. B, SPECT count of biopsies as a function of eosinophil counts from the same patient. Technetium-labeledheparin:Anewapproach to detection of eosinophilic esophagitis–associated inflammation

Measurement of Inflammation in Eosinophilic Esophagitis Using an Eosinophil Peroxidase Assay.

OBJECTIVES:We describe a simple, quick method to measure an eosinophil granule protein, eosinophil peroxidase (EPO), as a marker of eosinophil activity, in eosinophilic esophagitis (EoE).METHODS:Esophageal mucosal brushings initially were collected from 36 patients with active EoE (n=13), resolved EoE (n=13), and controls (n=10) before endoscopic biopsy collection; the brushes were frozen at –80 ºC until assayed. EPO on the brush was measured in a colorimetric assay visually and by spectrophotometric absorbance measurements (at 492 nm), and was compared with peak eosinophil counts in esophageal biopsy specimens. The assay was calibrated with known EPO concentrations; as EPO increased in the assay, the color changed from light yellow to dark brown.RESULTS:Mucosal brush specimens from active EoE yielded orange to dark brown colors with absorbance measurements > 1.1 U; in contrast, control and resolved EoE brush specimens yielded a light to dark yellow color with absorbance measurements < 1.1. We then corroborated the results at the bedside (real time) in 16 additional patients. EPO on the brush was measured directly in < 1 h in the assay visually and by absorbance at 492 nm. Absorbance units strongly correlated with peak eosinophil counts both with the frozen brush (rs=0.79, P<0.0001) and with the bedside (rs=0.86, P<0.00017) approaches.CONCLUSIONS:The results support the use of this rapid method to detect and monitor EoE disease activity. Moreover, because eosinophils infiltrate and degranulate in the esophagus in EoE in a patchy manner, this method may be more accurate than current practice by testing for an eosinophil constituent from both intact and degranulated cells, and by sampling large portions of the esophageal lumen rather than small biopsy specimens that may not be representative of eosinophil involvement.

Extracellular Eosinophil Granule Protein Deposition in Ringed Esophagus with Sparse Eosinophils

BackgroundEosinophilic esophagitis (EoE) remains difficult to classify because of varying presentations. Not uncommonly, patients present with symptoms of esophageal dysfunction and have esophageal changes on endoscopy resembling EoE but without >15 eosinophils/HPF. Patients with low numbers of eosinophils in esophageal biopsy specimens may have esophageal changes and symptomatic disease brought about by eosinophil granule protein deposition without recognizable intact cells.AimTo determine whether extracellular eosinophil granule protein deposition is present in the esophagi of patients with low eosinophil numbers who have clinical symptoms and characteristic endoscopic esophageal changes of EoE including ringed esophagus (RE).MethodsEsophageal biopsy specimens were studied from eight EoE patients with >15 eosinophils per high power field (HPF) and nine patients with RE (<15 eosinophils/HPF). The specimens were analyzed for eosinophil granule proteins, major basic protein 1 (eMBP1) and eosinophil-derived neurotoxin (EDN), by indirect immunofluorescence.ResultsBoth EoE and RE showed positive EDN and eMBP1 extracellular deposition; control esophagus showed minimal or none. Comparing EoE and RE, extracellular EDN and eMBP1 were similar except that EDN in EoE was greater in the distal esophagus.ConclusionsThis study highlights the importance of assessing eosinophil granule protein deposition in esophageal disease with potential eosinophil involvement. Persistent/progressive esophageal changes may be brought about by eosinophil granule proteins despite low numbers of intact cells. The meaning of “resolution” in EoE may need to be redefined based on numbers of esophageal eosinophils, extracellular eosinophil granule protein deposition, and subsequent clinical course of patients.

Non-invasive ultrasound to identify eosinophil granule proteins in eosinophilic esophagitis.

Although traditional microbubble contrast agents are bright, the high contrast of gas bubbles and air-water interfaces in the upper gastrointestinal tract renders these agents less useful for diagnosing diseases such as eosinophilic esophagitis, a disease characterized by patchy infiltration of eosinophils into the esophagus. Here we report a first-in-class ultrasound contrast enhancement agent composed of echogenic insulin particles, which are labeled with molecular recognition elements to diagnose eosinophil-associated diseases. We prepared solid echogenic insulin particles, tethered antibodies to eosinophil granule major basic protein 1 (MBP-1) to their surfaces and experimentally evaluated binding of these agents to MBP-1 on ex vivo non-human primate esophagi. We found that insulin particles can be readily observed by ultrasound and bind to MBP-1-coated esophagi within minutes. Our results suggest the potential of this new class of solid contrast agents to image, diagnose and improve management of eosinophilic esophagitis.

A subset of patients with pemphigoid (herpes) gestationis has serological evidence of celiac disease

Pemphigoid (herpes) gestationis (PG) is an uncommon, self‐limited disease with other autoimmune associations; however, celiac disease (CD) is not recognized as one.

Su1850 Eosinophil Degranulation Patterns in Eosinophilic Esophagitis

with EoE had a lower BMI at the time of diagnosis than non-EoE controls, and PPI-REE subjects had an intermediate BMI. This finding may be a proxy for dietary modification to minimize symptoms in untreated EoE patients or increased metabolism associated with a chronic inflammation. In conjunction with other markers, BMI might serve as a clinical variable to differentiate EoE from other conditions.

Sa1185 Biodistribution of Orally Administered 99mtc-Heparin: A Radiolabeled Contrast Agent for Eosinophilic Esophagitis Associated Inflammation

Background: 99mTc-Heparin provides contrast for localized inflammation in eosinophilic esophagitis (EoE), because themarkedly basic eosinophil granule proteins deposit in inflamed regions of the esophagus and eosinophil granule proteins, such as major basic protein 1 (MBP-1), bind strongly to heparin (Swaminathan GJ, et al., Biochemistry 44 (2005) 141528). In prior experiments, we tested the ability of 99mTc-heparin to bind to cationic eosinophil granule proteins, and we demonstrated that the 99mTc-heparin binds to Macaca monkey tissues coated with MBP-1. We also demonstrated that radiolabeled heparin binds in vitro to esophageal biopsies from patients with EoE. We are now poised to determine whether radiolabeled heparin localizes specifically to the esophagus of patients with eosinophilic esophagitis by having patients swallow the reagent and image its binding by single-photon emission computed tomography (SPECT). Aim: To evaluate the radiation risk from 99mTcheparin by studying the tissue distribution and calculating the radiation dose from orally administered 99mTc-heparin to mice. Method: 99mTc-Heparin (500 μCi/mouse, 15 mg) was administered to mice (C57-B16) by oral gavage. At each time point (0.75, 1.5, 3, 6, 18, and 30 hours), mice were euthanized with CO2 and positioned prone on the scanner bed. SPECT/CT images of mice were acquired by using an Inveon trimodality PET/SPECT/ CT scanner (Siemens Preclinical Solutions, Knoxville, TN). The activity of the harvested organs was measured in a well counter. Results: 99mTc-Heparin was not absorbed significantly through theGI tract into other bodily tissue. The highest radioactivities occurredmainly in stomach, small and large intestine. Discussion: These proof-of-principle experiments clearly show that 99mTc-heparin stays in GI tract and is not strongly absorbed. Furthermore, because heparin is a physiological substance that has long been used in clinical medicine, we believe that it will be well tolerated in our patients. This finding is important because it initiates a new avenue for clinical detection of EoE associated inflammation.