ABSTRACT
BACKGROUND: A definitive diagnosis of eosinophilic chronic rhinosinusitis (eCRS) requires invasive surgical tissue sampling and histologic enumeration of intact eosinophils. Eosinophil peroxidase (EPX) is an accurate biomarker of sinonasal tissue eosinophilia in CRS regardless of polyp status. A less invasive and rapid method that accurately identifies tissue eosinophilia would be of great benefit to patients. OBJECTIVE: We sought to evaluate a new clinical tool that uses a nasal swab and colorimetric EPX activity assay to predict a diagnosis of eCRS. METHODS: A prospective, observational cohort study was conducted using nasal swabs and sinonasal tissue biopsies obtained from patients with CRS electing endoscopic sinus surgery. Patients were classified as non-eCRS (n = 19) and eCRS (n = 35) on the basis of pathologically determined eosinophil counts of less than 10 or greater than or equal to 10 eosinophils/HPF, respectively. Swab-deposited EPX activity was measured and compared with tissue eosinophil counts, EPX levels, and CRS-specific disease metrics. RESULTS: EPX activity was significantly increased in patients with eCRS than in patients without eCRS (P < .0001). With a relative absorbance unit cutoff value of greater than or equal to 0.80, the assay demonstrated high sensitivity (85.7%) and moderate specificity (79.0%) for confirming eCRS. Spearman correlations between EPX activity and tissue eosinophil counts (rs = 0.424), EPX levels (rs = 0.503), and Lund-Kennedy endoscopy scores (rs = 0.440) in eCRS were significant (P < .05). CONCLUSIONS: This investigation evaluates a nasal swab sampling method and EPX activity assay that accurately confirms eCRS. This method could potentially address the unmet need to identify sinonasal tissue eosinophilia at the point-of-care, as well as to longitudinally monitor eosinophil activity and treatment response.
Subject(s)
Eosinophilia , Nasal Polyps , Rhinitis , Sinusitis , Humans , Eosinophilia/drug therapy , Eosinophil Peroxidase , Prospective Studies , Rhinitis/drug therapy , Eosinophils/pathology , Sinusitis/drug therapy , Chronic Disease , Nasal Polyps/diagnosis , Nasal Polyps/pathologyABSTRACT
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.
Subject(s)
Enzyme Assays/methods , Eosinophil Peroxidase/analysis , Eosinophilic Esophagitis , Esophageal Mucosa , Inflammation , Adult , Biomarkers/analysis , Biopsy/methods , Comparative Effectiveness Research , Endoscopy/methods , Eosinophilic Esophagitis/diagnosis , Eosinophilic Esophagitis/physiopathology , Esophageal Mucosa/enzymology , Esophageal Mucosa/pathology , Female , Humans , Inflammation/diagnosis , Inflammation/enzymology , Male , Middle Aged , Patient Acuity , Reproducibility of Results , Specimen HandlingABSTRACT
BACKGROUND: Eosinophilic 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. AIM: To 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). METHODS: Esophageal 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. RESULTS: Both 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. CONCLUSIONS: This 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.
Subject(s)
Eosinophil Major Basic Protein/analysis , Eosinophil-Derived Neurotoxin/analysis , Eosinophilic Esophagitis/metabolism , Eosinophilic Esophagitis/pathology , Eosinophils , Esophagus/chemistry , Esophagus/pathology , Adult , Aged , Biopsy , Female , Humans , Leukocyte Count , Male , Middle Aged , Young AdultABSTRACT
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.
Subject(s)
Cell Degranulation/immunology , Eosinophilic Esophagitis/immunology , Eosinophils/immunology , Eosinophils/ultrastructure , Adult , Female , Humans , Male , Microscopy, Electron , Middle Aged , Young AdultABSTRACT
BACKGROUND: Food antigens are clearly implicated in the induction and persistence of eosinophilic oesophagitis. Dietary elimination to identify triggers is tedious and expensive. Alternatives that can mitigate cost and improve patient quality of life during this process are needed. AIMS: To test the hypothesis that antibodies against foods that trigger eosinophilic oesophagitis are secreted into the oesophageal lumen where they can be collected by oesophageal brushings. METHODS: We evaluated food-specific immune responses within brushings in 68 patients undergoing endoscopy (12 controls, 13 resolved eosinophilic oesophagitis and 43 active eosinophilic oesophagitis). Seventeen participants identified their trigger foods via food elimination diets. Immunoglobulin A and immunoglobulin G4 antibodies against the four most common eosinophilic oesophagitis food triggers were measured using the ImmunoCAP assay in the oesophageal brushings. Food-specific antibody values were compared between active eosinophilic oesophagitis, resolved eosinophilic oesophagitis and controls. RESULTS: Patients with active eosinophilic oesophagitis (>15 eosinophils/hpf) demonstrated increased immunoglobulin A and immunoglobulin G4 levels to common eosinophilic oesophagitis triggers compared to controls (327 ± 380 vs 150 ± 130 for immunoglobulin A, and 1534 ± 3346 vs 178 ± 123 for immunoglobulin G4, P < 0.003). Specific trigger foods were associated with elevated immunoglobulin A and immunoglobulin G4 responses compared to foods that did not trigger oesophageal eosinophilia (733 ± 469 vs 142 ± 64, P < 0.001 immunoglobulin A and 2620 ± 3228 vs 526 ± 1050, P < 0.001 immunoglobulin G4). CONCLUSIONS: Food-specific antibodies are easily collected along the oesophageal lumen of eosinophilic oesophagitis patients. Further studies are needed to validate our preliminary findings to determine whether these antibodies can be used to guide elimination diet therapy.
Subject(s)
Allergens/immunology , Endoscopy , Eosinophilic Esophagitis/immunology , Food/adverse effects , Adult , Aged , Bodily Secretions/immunology , Eosinophilic Esophagitis/therapy , Eosinophils/metabolism , Female , Humans , Immunoglobulin G/immunology , Male , Middle Aged , Quality of Life , Young AdultABSTRACT
OBJECTIVE: To determine if heparin labeled with 99mTechnetium (99mTc) could be an imaging probe to detect eosinophil-related inflammation in eosinophilic esophagitis and to determine the biodistribution and radiation dosimetry of 99mTc-heparin oral administration using image-based dosimetry models with esophageal modeling. METHODS: Freshly prepared 99mTc-heparin was administered orally to 5 research subjects. Radioactivity was measured by whole-body scintigraphy and single-photon emission computed tomography during the 24 hours postadministration. Following imaging, endoscopic examination was performed. The biodistribution of esophageal radioactivity was compared with endoscopic findings, eosinophil counts in biopsy tissues, and immunostaining for eosinophil granule major basic protein-1 (eMBP1). These studies were conducted from July 1, 2013, until April 22, 2017. RESULTS: Oral administration of 99mTc-heparin was well tolerated in all 5 subjects. The entire esophagus could be visualized dynamically during oral administration. Bound esophageal radioactivity marked areas of inflammation as judged by endoscopy scores, by eosinophils per high power field and by localization of eMBP1 using immunostaining. Ninety percent of the radioactivity did not bind to the esophagus and passed through the gastrointestinal tract. CONCLUSION: The biodistribution of ingested 99mTc-heparin is almost exclusively localized to the gastrointestinal tract. Radiation exposure was highest in the lower gastrointestinal tract and was comparable with other orally administered diagnostic radiopharmaceuticals. The use of swallowed 99mTc-heparin may aid in assessing eosinophil-related inflammation in the esophagus.
Subject(s)
Eosinophilic Esophagitis/diagnostic imaging , Heparin/administration & dosage , Organotechnetium Compounds/administration & dosage , Radiopharmaceuticals/administration & dosage , Tomography, Emission-Computed, Single-Photon , Administration, Oral , Adult , Esophagoscopy , Humans , Male , Middle Aged , Tissue Distribution , Whole Body ImagingSubject(s)
Eosinophilic Esophagitis/diagnostic imaging , Eosinophils/immunology , Esophagus/diagnostic imaging , Heparin , Organotechnetium Compounds , Technetium , Cell Degranulation , Eosinophil Cationic Protein/analysis , Eosinophil Cationic Protein/metabolism , Esophagus/pathology , Humans , Protein Binding , Radioligand Assay , Tomography, Emission-Computed, Single-PhotonABSTRACT
BACKGROUND: Pemphigoid (herpes) gestationis (PG) is an uncommon, self-limited disease with other autoimmune associations; however, celiac disease (CD) is not recognized as one. METHODS: From 71 patients' sera submitted for herpes gestationis factor (HGF) testing over a 5-year period, 12 were consistent with PG demonstrating HGF and increased IgG BP180 antibody levels; these sera were tested for IgA and IgG endomysial antibodies (EMA), epithelial basement membrane zone and cell surface antibodies by indirect immunofluorescence, and for IgA and IgG tissue transglutaminase (transglutaminase 2 or TG2) antibodies, IgA epidermal transglutaminase (transglutaminase 3 or TG3) antibodies, IgG BP230, and IgG desmoglein 1 and desmoglein 3 antibodies by enzyme-linked immunosorbent assays (ELISAs). RESULTS: Three of 12 patients' sera with PG (25%) had CD antibodies with positive IgA EMA and increased IgA TG2 antibody levels; two of these had positive IgG EMA, and one other had an increased IgA TG3 antibody level. CONCLUSIONS: A subset of patients with serological findings of PG also has serological evidence of CD, which may have implications in the etiopathogenesis of PG and which reveals important information about the mother's, and possibly her infant's, health.
Subject(s)
Autoantibodies/blood , Celiac Disease/blood , Pemphigoid Gestationis/blood , Pemphigoid, Bullous/blood , Serologic Tests/methods , Adult , Celiac Disease/immunology , Celiac Disease/physiopathology , Cohort Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Fluorescent Antibody Technique, Indirect , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Pemphigoid Gestationis/immunology , Pemphigoid Gestationis/physiopathology , Pemphigoid, Bullous/immunology , Pemphigoid, Bullous/physiopathology , Pregnancy , Prognosis , Remission, Spontaneous , Retrospective Studies , Risk Assessment , Young AdultABSTRACT
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.