Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

BACKGROUND: The human eosinophil Charcot-Leyden Crystal (CLC) protein is a member of the Galectin superfamily and is also known as Galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE: We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS: Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by co-immunoprecipitation and co-affinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by employing enzyme activity assays, confocal microscopy, and shRNA knock-out of CLC/Gal-10 expression in human CD34(+) cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS: CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases, eosinophil-derived neurotoxin (EDN, RNS2) and eosinophil cationic protein (ECP, RNS3), and with murine eosinophil-associated ribonucleases. The interaction is independent of glycosylation and is not inhibitory toward endoribonuclease activity. Activation of eosinophils with INF-γ induces the rapid co-localization of CLC/Gal-10 with EDN/RNS2 and CD63. ShRNA knock-down of CLC/Gal-10 in human cord blood-derived CD34(+) progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS: CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic ribonucleases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 mu M CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules

Novel Peptide Nanoparticle Biased Antagonist of CCR3 Blocks Eosinophil Recruitment and Airway Hyperresponsiveness

Background—Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason may be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. Objective—We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G-protein signaling, but enable or promote receptor internalization. Methods—Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by dynamic light scattering and NMR. Inhibitory activity on CCR3 signaling was assessed in vitro using flow cytometry, confocal microscopy, and western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed using a triple allergen mouse asthma model. Results—R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. IC50 values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of ERK1/2 activation and not the late phase generally associated with β-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo, R321 effectively blocks eosinophil recruitment into the lungs and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. Conclusions—R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism may hold significant therapeutic promise by eluding the formation of drug tolerance

Design and cloning strategies of recombinant allergens for diagnosis and specific immunotherapy

Persons suffering from allergy (Type I hypersensitivity) produce immunoglobulin E against innocuous environmental antigens such as pollen, house dust, animal dander, food proteins. Diagnosis of allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens in skin prick test. Diagnostic reagents based on allergen extracts obtained from natural biological material often reveal unbalanced allergen content, a presence of nonallergenic components, and are difficult to standardize. Replacement of allergen extracts with a set of individual allergens in component-resolved diagnostics is regarded as a tool for patient selection for specific immunotherapy. The concept of using single recombinant allergens to determine the patient's sensitization profile was coined "component-resolved diagnosis" and is regarded as a precondition for patient-tailored immunotherapy, i.e.,"component resolved immunotherapy"To provide reliable, more specific reagents for allergy diagnosis and therapy recombinant DNA technology has been widely applied. The majority of recombinant allergens by far, have been produced in the prokaryotic expression system; however eukaryotic cells (yeast, plant, insect and mammalian cells) were also exploited. To avoid side effects in the course of immunotherapy various approaches in design of hypoallergenic molecules have been performed. This chapter will give an overview of the concepts and approaches in producing recombinant allergens for component resolved diagnosis and component resolved immunotherapy. © 2014 by Nova Science Publishers, Inc. All rights reserved.Advances in Genetics Researc

Design and cloning strategies of recombinant allergens for diagnosis and specific immunotherapy

Persons suffering from allergy (Type I hypersensitivity) produce immunoglobulin E against innocuous environmental antigens such as pollen, house dust, animal dander, food proteins. Diagnosis of allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens in skin prick test. Diagnostic reagents based on allergen extracts obtained from natural biological material often reveal unbalanced allergen content, a presence of nonallergenic components, and are difficult to standardize. Replacement of allergen extracts with a set of individual allergens in component-resolved diagnostics is regarded as a tool for patient selection for specific immunotherapy. The concept of using single recombinant allergens to determine the patient's sensitization profile was coined "component-resolved diagnosis" and is regarded as a precondition for patient-tailored immunotherapy, i.e.,"component resolved immunotherapy"To provide reliable, more specific reagents for allergy diagnosis and therapy recombinant DNA technology has been widely applied. The majority of recombinant allergens by far, have been produced in the prokaryotic expression system; however eukaryotic cells (yeast, plant, insect and mammalian cells) were also exploited. To avoid side effects in the course of immunotherapy various approaches in design of hypoallergenic molecules have been performed. This chapter will give an overview of the concepts and approaches in producing recombinant allergens for component resolved diagnosis and component resolved immunotherapy. © 2014 by Nova Science Publishers, Inc. All rights reserved

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions

BACKGROUND: The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). METHODS: Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. RESULTS: Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. CONCLUSION: Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. GENERAL SIGNIFICANCE: In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy

One-Hour Esophageal String Test: A Nonendoscopic Minimally Invasive Test That Accurately Detects Disease Activity in Eosinophilic Esophagitis

OBJECTIVES: Eosinophilic esophagitis (EoE), a chronic food allergic disease, lacks sensitive and specific peripheral biomarkers. We hypothesized that levels of EoE-related biomarkers captured using a 1-hour minimally invasive Esophageal String Test (EST) would correlate with mucosal eosinophil counts and tissue concentrations of these same biomarkers. We aimed to determine whether a 1-hour EST accurately distinguishes active from inactive EoE or a normal esophagus. METHODS: In a prospective, multisite study, children and adults (ages 7–55 years) undergoing a clinically indicated esophagogastroduodenoscopy performed an EST with an esophageal dwell time of 1 hour. Subjects were divided into 3 groups: active EoE, inactive EoE, and normal esophageal mucosa. Eosinophil-associated protein levels were compared between EST effluents and esophageal biopsy extracts. Statistical modeling was performed to select biomarkers that best correlated with and predicted eosinophilic inflammation. RESULTS: One hundred thirty-four subjects (74 children, 60 adults) with active EoE (n 5 62), inactive EoE (n 5 37), and patient controls with a normal esophagus (n 5 35) completed the study. EST-captured eosinophil-associated biomarkers correlated significantly with peak eosinophils/high-power field, endoscopic visual scoring, and the same proteins extracted from mucosal biopsies. Statistical modeling, using combined eotaxin-3 and major basic protein-1 concentrations, led to the development of EoE scores that distinguished subjects with active EoE from inactive EoE or normal esophagi. Eightyseven percent of children, 95% of parents, and 92% of adults preferred the EST over endoscopy if it provided similar information. DISCUSSION: The 1-hour EST accurately distinguishes active from inactive EoE in children and adults and may facilitate monitoring of disease activity in a safe and minimally invasive fashion

Novel peptide nanoparticle–biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness

Background—Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason may be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. Objective—We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G-protein signaling, but enable or promote receptor internalization. Methods—Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by dynamic light scattering and NMR. Inhibitory activity on CCR3 signaling was assessed in vitro using flow cytometry, confocal microscopy, and western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed using a triple allergen mouse asthma model. Results—R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. IC50 values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of ERK1/2 activation and not the late phase generally associated with β-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo, R321 effectively blocks eosinophil recruitment into the lungs and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. Conclusions—R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism may hold significant therapeutic promise by eluding the formation of drug tolerance