Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3'-diaminobenzidine-stained esophageal biopsies

John Plate; Mogens Bove; Helen M Larsson; Elisabeth Norder Grusell; Nabanita Chatterjee; Leif E Johansson; Henrik Bergquist

doi:10.1093/dote/doae034

Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3'-diaminobenzidine-stained esophageal biopsies

Dis Esophagus. 2024 Apr 28:doae034. doi: 10.1093/dote/doae034. Online ahead of print.

Authors

John Plate^{1

2}, Mogens Bove^{1

2}, Helen M Larsson^{1

2}, Elisabeth Norder Grusell³, Nabanita Chatterjee⁴, Leif E Johansson⁵, Henrik Bergquist²

Affiliations

¹ Department of Otorhinolaryngology, Head and Neck Surgery, NU-Hospital Group, Region Västra Götaland, Trollhättan, Sweden.
² Department of Otorhinolaryngology, Head and Neck Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
³ Department of Otorhinolaryngology, Head and Neck Surgery, Kungsbacka Hospital, Region Halland, Kungsbacka, Sweden.
⁴ Core Facilities, Centre for Cellular Imaging (CCI), University of Gothenburg, Gothenburg, Sweden.
⁵ Department of ENT, Head and Neck Surgery, Skövde County Hospital, Region Västra Götaland, Skövde, Sweden.

PMID: 38679488
DOI: 10.1093/dote/doae034

Abstract

Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) share many histopathological features; therefore, markers for differentiation are of diagnostic interest and may add to the understanding of the underlying mechanisms. The nitrergic system is upregulated in GERD and probably also in EoE. Esophageal biopsies of patients with EoE (n = 20), GERD (n = 20), and healthy volunteers (HVs) (n = 15) were exposed to antibodies against inducible nitric oxide synthase (iNOS), nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3. The stained object glasses were randomized, digitized, and blindly analyzed regarding the expression of DAB (3,3'-diaminobenzidine) by a protocol developed in QuPath software. A statistically significant overexpression of iNOS was observed in patients with any of the two inflammatory diseases compared with that in HVs. Eotaxin-3 could differentiate HVs versus inflammatory states. Gastroesophageal reflux patients displayed the highest levels of nitrotyrosine. Neither iNOS nor nitrotyrosine alone were able to differentiate between the two diseases. For that purpose, eosinophil peroxidase was a better candidate, as the mean levels increased stepwise from HVs via GERD to EoE. iNOS and nitrotyrosine are significantly overexpressed in patients with EoE and GERD compared with healthy controls, but only eosinophil peroxidase could differentiate the two types of esophagitis. The implications of the finding of the highest levels of nitrotyrosine among gastroesophageal reflux patients are discussed.

Keywords: eosinophilic esophagitis; gastroesophageal reflux disease; image analysis; immunohistochemistry.