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Subcellular Localization of CAPN14 in Human Esophageal Epithelial Cells
Abstracts AB229
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Half Cow's Milk-Induced Food Protein Induced Enterocolitis Syndrome (FPIES) Require Amino Acid Feeding
Sibylle Blanc1, Delphine Deboissieu, MD2, Nicolas Kalach, MD, Ph D3, Pascale Soulaines2, Florence Campeotto, MD, PhD2, Marie-Pierre Cordier-Collet, MD2, Clara Malka2, Isabelle Montaudie-Dumas, MD1, Carole Piccini-Bailly, MD1, Lisa Giovannini-Chami, MD, PhD1, Thierry Bourrier, MD1, Christophe Dupont, MD, PhD2; 1H^opitaux pediatriques de Nice CHU-Lenval, Nice, France, 2Hopital Necker Enfants Malades, opital Saint Vincent de Paul, Groupement des Hospitaux Paris, France, 3H^ de l’Institut Catholique de Lille (GH-ICL), Lille, France. RATIONALE: FPIES is mainly related to cow’s milk and manifests as a chronic digestive disease or in its acute form with potentially lifethreatening vomiting/diarrhea/dehydration. The objective of this study is to characterize the clinical features of cow’s milk-induced FPIES in children. METHODS: A cohort of patients with FPIES was constituted in French Children’s Hospitals (Necker, Paris – Lenval, Nice). Data were collected from medical records including all patients referred for an acute episode of FPIES, and divided into 2 groups according to their tolerance of extensively hydrolysed formula (eHF) or their need to be fed an amino-acid formula (AAF). RESULTS: 49 children were enrolled. Chronic had occurred in 36 (73%), after a median period of 10 days following introduction of milkbased formula. In the whole group, the acute episode occurred at a median age of 4 months. Allergy testing was rarely positive: patch test 21 (51%), skin prick tests 3 (8%), specific IgE 13 (30%). Recovery was observed in 19 (40%) at a median age of 31 months. The eHF group comprised 24 (49%) infants and the AAF one 25 (51%). They exhibited the following significant or trend towards significance differences : number of hospitalizations before diagnosis per patient 0,9 vs 2,7 (p50,02), age of FA diagnosis 4,5 months vs 2,8 (p50,04), food tolerance acquisition 54% vs 24% (p50,02), associated FA 4% vs 48% (p50,0002). CONCLUSIONS: Half infants with milk-induced FPIES do not tolerate eHF, and need to be fed with an AAF, a condition associated with a delayed diagnosis.
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Investigation of Periostin and TARC Levels in the Search for a Non-Invasive Biomarker in Children and Adults with Eosinophilic Esophagitis
Anubha Tripathi, MD1, Lisa J. Workman, BA2, Kush S. Patel, BS3, Barrett H. Barnes, MD4, Thomas A. E. Platts-Mills, MD, PhD, FAAAAI, FRS5, Scott P. Commins, MD, PhD6; 1Division of Asthma, Allergy & Immunology, Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 2University of Virginia Asthma and Allergic Diseases Center, Charlottesville, VA, 3University of Virginia School of Medicine, Charlottesville, VA, 4Division of Pediatric Gastroenterology, University of Virginia Health System, Charlottesville, VA, 5Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 6Division of Asthma, Allergy and Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: Currently, the only test for pathologic assessment of disease progression and treatment response in Eosinophilic Esophagitis (EoE) is esophagogastroduodenoscopy, which poses the potential for significant procedure-related risk. Therefore, establishing non-invasive biomarkers to monitor disease status is essential. Since periostin and thymus and activation regulated chemokine (TARC) have been implicated in facilitating eosinophil tissue infiltration in allergic esophageal responses, we sought to measure these levels in patients with EoE. METHODS: Sera from children (n520) and adults (n53) with biopsydiagnosed EoE were obtained and analyzed for periostin (Periostin/OSF-2
(human), Phoenix Pharmaceuticals, Inc.) and TARC (human CCL-17 (TARC), BioLegend, Inc.) by ELISA. RESULTS: Mean periostin level (ng/mL) (n523, range510.3-405.5, mean6SEM5 186.7623.2) was significantly higher than reported levels for healthy controls and was higher in patients with signs of fibrosis (esophageal narrowing, mucosal rings, and longitudinal furrows) (n513, mean6SEM5 204.2635.69) versus without signs of fibrosis (n510, mean6SEM5 163.9626.87). Mean TARC level (pg/mL) (n523, range588.4-1159, mean6SEM5 511.8664.06) was significantly higher than reported levels for healthy controls and was higher in patients with signs of fibrosis (n511, mean6SEM5 626.3693.59) versus without signs of fibrosis (n512, mean6SEM5 407.0679.63). In addition, changes in periostin and TARC levels in individual patients’ sera taken at different time points vary with alterations in their clinicopathologic status. CONCLUSIONS: Our results indicate that periostin and TARC levels are elevated in EoE patients and in the subset presenting with fibrosis. Changes in these levels also correlate with changes in clinicopathologic status, suggesting they are reflective of disease activity and, therefore, look promising as biomarkers for EoE.
748
Subcellular Localization of CAPN14 in Human Esophageal Epithelial Cells
Jeffrey K. Rymer1,2, Jared Travers1, Mark Rochman, PhD1, Benjamin P. Davis, MD, PhD1, Marc E. Rothenberg, MD, PhD, FAAAAI1; 1Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, 2Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. RATIONALE: Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus. An intracellular protease called calpain-14 (CAPN14) has been shown in a previous genome-wide association study (GWAS) to be most highly associated with EoE and is upregulated in EoE esophageal biopsies. In this study, we investigated the localization of CAPN14 in human esophageal epithelial cells. METHODS: Immunofluorescence (IF) and biochemical fractionation were performed on transduced immortalized human esophageal epithelial cells (EPC2) stably overexpressing CAPN14 grown in submerged culture. Fractionation was also performed on untransduced EPC2 cells grown at the air-liquid interface (ALI) and on primary esophageal epithelial cells grown in submerged culture. RESULTS: CAPN14 was readily detectable in whole cell lysates from transduced, but not untransduced, EPC2 cells. Fractionation revealed ;77% of CAPN14 to be in the cytosolic fraction, with ;7% percentage detectable in the membrane and ;16% in the nuclear fractions in human esophageal epithelial cells grown in submerged culture. However, following differentiation into a stratified squamous epithelium, endogenous CAPN14 was mainly localized in the nucleus. IF staining of phorbol12 myristate-13 acetate (PMA) and ionomycin treated esophageal epithelial cells in monolayer culture showed CAPN14 to undergo changes in localization with a shift from the cytoplasmic to nuclear compartments and then to the plasma membrane after 30 and 180 minutes, respectively. CONCLUSIONS: CAPN14 is localized to the cytoplasm, membrane, and nucleus in human esophageal epithelial cells. Following cellular activation (PMA/ionomycin), CAPN14 shows a dynamic distribution, most notable by its presence in the nucleus, consistent with a key cellular function, yet to be described.
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746
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Half Cow's Milk-Induced Food Protein Induced Enterocolitis Syndrome (FPIES) Require Amino Acid Feeding
Sibylle Blanc1, Delphine Deboissieu, MD2, Nicolas Kalach, MD, Ph D3, Pascale Soulaines2, Florence Campeotto, MD, PhD2, Marie-Pierre Cordier-Collet, MD2, Clara Malka2, Isabelle Montaudie-Dumas, MD1, Carole Piccini-Bailly, MD1, Lisa Giovannini-Chami, MD, PhD1, Thierry Bourrier, MD1, Christophe Dupont, MD, PhD2; 1H^opitaux pediatriques de Nice CHU-Lenval, Nice, France, 2Hopital Necker Enfants Malades, opital Saint Vincent de Paul, Groupement des Hospitaux Paris, France, 3H^ de l’Institut Catholique de Lille (GH-ICL), Lille, France. RATIONALE: FPIES is mainly related to cow’s milk and manifests as a chronic digestive disease or in its acute form with potentially lifethreatening vomiting/diarrhea/dehydration. The objective of this study is to characterize the clinical features of cow’s milk-induced FPIES in children. METHODS: A cohort of patients with FPIES was constituted in French Children’s Hospitals (Necker, Paris – Lenval, Nice). Data were collected from medical records including all patients referred for an acute episode of FPIES, and divided into 2 groups according to their tolerance of extensively hydrolysed formula (eHF) or their need to be fed an amino-acid formula (AAF). RESULTS: 49 children were enrolled. Chronic had occurred in 36 (73%), after a median period of 10 days following introduction of milkbased formula. In the whole group, the acute episode occurred at a median age of 4 months. Allergy testing was rarely positive: patch test 21 (51%), skin prick tests 3 (8%), specific IgE 13 (30%). Recovery was observed in 19 (40%) at a median age of 31 months. The eHF group comprised 24 (49%) infants and the AAF one 25 (51%). They exhibited the following significant or trend towards significance differences : number of hospitalizations before diagnosis per patient 0,9 vs 2,7 (p50,02), age of FA diagnosis 4,5 months vs 2,8 (p50,04), food tolerance acquisition 54% vs 24% (p50,02), associated FA 4% vs 48% (p50,0002). CONCLUSIONS: Half infants with milk-induced FPIES do not tolerate eHF, and need to be fed with an AAF, a condition associated with a delayed diagnosis.
747
Investigation of Periostin and TARC Levels in the Search for a Non-Invasive Biomarker in Children and Adults with Eosinophilic Esophagitis
Anubha Tripathi, MD1, Lisa J. Workman, BA2, Kush S. Patel, BS3, Barrett H. Barnes, MD4, Thomas A. E. Platts-Mills, MD, PhD, FAAAAI, FRS5, Scott P. Commins, MD, PhD6; 1Division of Asthma, Allergy & Immunology, Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 2University of Virginia Asthma and Allergic Diseases Center, Charlottesville, VA, 3University of Virginia School of Medicine, Charlottesville, VA, 4Division of Pediatric Gastroenterology, University of Virginia Health System, Charlottesville, VA, 5Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA, 6Division of Asthma, Allergy and Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: Currently, the only test for pathologic assessment of disease progression and treatment response in Eosinophilic Esophagitis (EoE) is esophagogastroduodenoscopy, which poses the potential for significant procedure-related risk. Therefore, establishing non-invasive biomarkers to monitor disease status is essential. Since periostin and thymus and activation regulated chemokine (TARC) have been implicated in facilitating eosinophil tissue infiltration in allergic esophageal responses, we sought to measure these levels in patients with EoE. METHODS: Sera from children (n520) and adults (n53) with biopsydiagnosed EoE were obtained and analyzed for periostin (Periostin/OSF-2
(human), Phoenix Pharmaceuticals, Inc.) and TARC (human CCL-17 (TARC), BioLegend, Inc.) by ELISA. RESULTS: Mean periostin level (ng/mL) (n523, range510.3-405.5, mean6SEM5 186.7623.2) was significantly higher than reported levels for healthy controls and was higher in patients with signs of fibrosis (esophageal narrowing, mucosal rings, and longitudinal furrows) (n513, mean6SEM5 204.2635.69) versus without signs of fibrosis (n510, mean6SEM5 163.9626.87). Mean TARC level (pg/mL) (n523, range588.4-1159, mean6SEM5 511.8664.06) was significantly higher than reported levels for healthy controls and was higher in patients with signs of fibrosis (n511, mean6SEM5 626.3693.59) versus without signs of fibrosis (n512, mean6SEM5 407.0679.63). In addition, changes in periostin and TARC levels in individual patients’ sera taken at different time points vary with alterations in their clinicopathologic status. CONCLUSIONS: Our results indicate that periostin and TARC levels are elevated in EoE patients and in the subset presenting with fibrosis. Changes in these levels also correlate with changes in clinicopathologic status, suggesting they are reflective of disease activity and, therefore, look promising as biomarkers for EoE.
748
Subcellular Localization of CAPN14 in Human Esophageal Epithelial Cells
Jeffrey K. Rymer1,2, Jared Travers1, Mark Rochman, PhD1, Benjamin P. Davis, MD, PhD1, Marc E. Rothenberg, MD, PhD, FAAAAI1; 1Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, 2Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. RATIONALE: Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus. An intracellular protease called calpain-14 (CAPN14) has been shown in a previous genome-wide association study (GWAS) to be most highly associated with EoE and is upregulated in EoE esophageal biopsies. In this study, we investigated the localization of CAPN14 in human esophageal epithelial cells. METHODS: Immunofluorescence (IF) and biochemical fractionation were performed on transduced immortalized human esophageal epithelial cells (EPC2) stably overexpressing CAPN14 grown in submerged culture. Fractionation was also performed on untransduced EPC2 cells grown at the air-liquid interface (ALI) and on primary esophageal epithelial cells grown in submerged culture. RESULTS: CAPN14 was readily detectable in whole cell lysates from transduced, but not untransduced, EPC2 cells. Fractionation revealed ;77% of CAPN14 to be in the cytosolic fraction, with ;7% percentage detectable in the membrane and ;16% in the nuclear fractions in human esophageal epithelial cells grown in submerged culture. However, following differentiation into a stratified squamous epithelium, endogenous CAPN14 was mainly localized in the nucleus. IF staining of phorbol12 myristate-13 acetate (PMA) and ionomycin treated esophageal epithelial cells in monolayer culture showed CAPN14 to undergo changes in localization with a shift from the cytoplasmic to nuclear compartments and then to the plasma membrane after 30 and 180 minutes, respectively. CONCLUSIONS: CAPN14 is localized to the cytoplasm, membrane, and nucleus in human esophageal epithelial cells. Following cellular activation (PMA/ionomycin), CAPN14 shows a dynamic distribution, most notable by its presence in the nucleus, consistent with a key cellular function, yet to be described.
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