The true extent of eosinophil involvement in disease is unrecognized: the secret life of dead eosinophils.

Eosinophil-mediated pathophysiology is tissue destructive and tissue altering with proinflammatory, prothrombotic, and profibrotic effects. The distinctive morphology of an eosinophil reveals a cytoplasm chockfull of unique granules, and the granule proteins have numerous toxic effects on cells, tissues, and organs. Eosinophils are not found in most human tissues, and eosinophil involvement in diseased tissues generally is identified by cell infiltration on histopathologic examination. However, eosinophils characteristically lose their structural integrity and deposit granules and granule proteins at sites of inflammation. Hence, their participation in tissue damage may be underrecognized or entirely overlooked. The eosinophil major basic protein 1 is a toxic granule protein and, when deposited, persists in tissues. Major basic protein 1 deposition can be regarded as a footprint of eosinophil activity. Analyses of numerous eosinophil-related diseases have demonstrated clear-cut evidence of major basic protein 1 deposition in affected tissues where eosinophils were not recognized by hematoxylin and eosin tissue staining and light microscopy. Eosinophil granule protein deposition, as exemplified by localization of major basic protein 1, especially when disproportionately greater than cellular infiltration, emerges as a biomarker of hidden eosinophil-related pathophysiology. Consequently, current assessments of recognized eosinophils may vastly underestimate their role in disease.

Proposed refined diagnostic criteria and classification of eosinophil disorders and related syndromes.

Eosinophilia and eosinophil activation are recurrent features in various reactive states and certain hematologic malignancies. In patients with hypereosinophilia (HE), HE-induced organ damage is often encountered and may lead to the diagnosis of a hypereosinophilic syndrome (HES). A number of known mechanisms and etiologies contribute to the development of HE and HES. Based on these etiologies and the origin of eosinophils, HE and HES are divided into primary forms where eosinophils are clonal cells, reactive forms where an underlying reactive or neoplastic condition is detected and eosinophils are considered to be "non-clonal" cells, and idiopathic HE and HES in which neither a clonal nor a reactive underlying pathology is detected. Since 2012, this classification and the related criteria have been widely accepted and regarded as standard. However, during the past few years, new developments in the field and an increasing number of markers and targets have created a need to update these criteria and the classification of HE and HES. To address this challenge, a Working Conference on eosinophil disorders was organized in 2021. In this conference, a panel of experts representing the relevant fields, including allergy, dermatology, hematology, immunology, laboratory medicine, and pathology, met and discussed new markers and concepts as well as refinements in definitions, criteria and classifications of HE and HES. The outcomes of this conference are presented in this article and should assist in the diagnosis and management of patients with HE and HES in daily practice and in the preparation and conduct of clinical trials.

Diagnostics for Dermatologic Diseases with Autoantibodies.

BACKGROUND: Dermatologic diseases with autoantibodies were recognized early as autoimmunity became accepted as a pathogenic immunologic concept. Laboratory testing to identify disease-defining autoantibodies and investigate their role in pathophysiology has evolved since. CONTENT: Blistering dermatologic diseases, profiled by autoantibody production, target epithelial components critical in cell-cell and cell-matrix adhesion, resulting in epithelial separation and other characteristic features of the disorders. This review covers the clinical indications for dermatologic disease-related autoantibody testing, the specifics of procuring specimens to test, the available diagnostic tests, and information provided by the testing. Atypical, uncharacteristic, and less well-known clinical and autoantibody profiles as well as several of the many future prospects for expansion of the testing applications are elaborated on in the online Data Supplement. SUMMARY: Autoantibody-associated dermatologic diseases are acquired immunologic disorders that have considerable clinical implications affecting essential barrier functions of skin and mucous membranes and causing discomfort, including pain and pruritus. Certain of the diseases can have life-threatening manifestations, and treatments can have significant side-effects. The skin diseases may presage other clinical associations that are important to recognize and treat. Laboratory testing aids in the diagnosis of these diseases through identification of the autoantibodies and is essential for prompt and precise knowledge of the disease type for prognosis, further clinical evaluations, and treatment decisions.

Oral Administration of 99mTechnetium-Labeled Heparin in Eosinophilic Esophagitis.

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.

Eosinophil-Related Disease and the Skin.

Eosinophils are bone marrow-derived cells that infiltrate skin and mucous membrane in a broad spectrum of primary and reactive inflammatory diseases and malignancies. The eosinophil has potent proinflammatory activities, particularly, through the effects of its toxic granule proteins. In addition, eosinophils have prothrombotic and profibrotic activities. Eosinophil participation in the pathogenesis of certain diseases without identifiable intact eosinophil infiltration may not be recognized because eosinophil degranulation is poorly visualized on hematoxylin-and-eosin-stained histopathology sections. Eosinophil-related pathophysiology can involve virtually every component of skin. Commonly recognized dermatoses associated with eosinophils are arthropod bite and sting reactions and drug eruptions, "bugs and drugs." Skin involvement is common in eosinophil-related systemic diseases including the hypereosinophilic syndromes. Eosinophil-related pathophysiology may play a key role in numerous disorders that, therefore, may benefit from therapies targeted to reduce or eliminate eosinophils.

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

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.

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.

Biomarkers of the involvement of mast cells, basophils and eosinophils in asthma and allergic diseases.

Biomarkers of disease activity have come into wide use in the study of mechanisms of human disease and in clinical medicine to both diagnose and predict disease course; as well as to monitor response to therapeutic intervention. Here we review biomarkers of the involvement of mast cells, basophils, and eosinophils in human allergic inflammation. Included are surface markers of cell activation as well as specific products of these inflammatory cells that implicate specific cell types in the inflammatory process and are of possible value in clinical research as well as within decisions made in the practice of allergy-immunology.

Extracellular Eosinophil Granule Protein Deposition in Ringed Esophagus with Sparse Eosinophils.

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.

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.

Pemphigus vulgaris autoantibody profiling by proteomic technique.

Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against the stratified squamous epithelium. Current understanding of PV pathophysiology does not explain the mechanism of acantholysis in patients lacking desmoglein antibodies, which justifies a search for novel targets of pemphigus autoimmunity. We tested 264 pemphigus and 138 normal control sera on the multiplexed protein array platform containing 701 human genes encompassing many known keratinocyte cell-surface molecules and members of protein families targeted by organ-non-specific PV antibodies. The top 10 antigens recognized by the majority of test patients' sera were proteins encoded by the DSC1, DSC3, ATP2C1, PKP3, CHRM3, COL21A1, ANXA8L1, CD88 and CHRNE genes. The most common combinations of target antigens included at least one of the adhesion molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients.

Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD).

BACKGROUND: Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiologic roles that eosinophils may serve in other human "eosinophil-associated" diseases remain obscure. OBJECTIVE: National Institutes of Health (NIH) Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases. METHODS: The taskforce used an organ system approach to identify the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012. RESULTS: The taskforce identified significant research needs. These needs cross disease entities but some are disease specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic, and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communications among the eosinophil-interested communities, for example, governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive. CONCLUSIONS: Significant efforts are required to address our knowledge gaps to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations, and the pharmaceutical industry should consider the taskforce's recommendations in their future research activities.

Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field.

Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.