Eosinophilic esophagitis auxiliary diagnosis based on a peptide ligand to eosinophil cationic protein in esophageal mucus of pediatric patients.

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition of the esophagus characterized by increased number of eosinophils. Currently, EoE diagnosis is based on endoscopic procedures for histopathological examination, eosinophils' counting and, often, in clinical practice, the challenge is the differentiation between EoE and gastroesophageal reflux disease (GERD). Our aim was to develop novel peptide ligand to Eosinophil cationic protein (ECP) present in EoE biopsies of patients with potential to be used for detection. We performed a comparative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of esophageal biopsies from pediatric patients with eosinophilic esophagitis, gastroesophageal reflux disease and control individuals. Then, phage display technology was used to select peptides against specific up-regulated protein from EoE patients. Twelve phage clones were selected after three biopanning rounds, and the best phage clone reactivity was evaluated by phage-ELISA assay using esophageal mucus samples from 94 pediatric patients. Mass spectrometry showed that eosinophil cationic protein (ECP) was one of the most up-regulated proteins in EoE patients, which is an eosinophil granule protein usually deposited on tissues to mediate remodeling, but in excess may cause fibrosis and hypertrophy, especially in allergic responses. A highly reactive ECP-ligand peptide (E5) was able to distinguish reactive mucus of EoE patients from GERD and the control individuals by Phage-ELISA, achieving a sensitivity of 84.62%, and a specificity of 82.72%. This is the first study that successfully demonstrated an antibody-like peptide targeting ECP at the esophagus mucus as a useful auxilliary tool for EoE diagnosis with a significant association with atopic disorders and dysphagia.ClinicalTrials.gov no.: NCT03069573.

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom.

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A(2) activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 degrees C. Assays with metal ions showed that Ca(2+) is an activator, whereas Zn(2+) and Hg(2+) inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.