Cincinnati Center for Eosinophilic Disorders Research
Upcoming EGID Research

Upcoming EGID Research

Through the collaborative efforts of the Campaign Urging Research for Eosinophilic Diseases (CURED), Cincinnati Center for Eosinophilic Disorders (CCED) and the awards committee members, we are pleased to present the eight winning abstracts from the 5th CURED EGID Conference and Patient Education Program. Many of the >40 research abstracts were facilitated or generated by the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR), with awardees presenting their findings as late-breaking discoveries. The eight winning abstracts are listed alphabetically by the abstract title.

Authors and Affiliations: Nurit. P. Azouz1, Andrea Klingler1, Mark Rochman1, Misu Paul1, Julie. M. Caldwell1, Michael Brusilovski1, Daniel Miller2, Arthur Lynch2, Carmy Forney2, Leah C. Kottyan2, Matthew T. Weirauch2,3 and Marc E Rothenberg11;Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA., 2Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
3Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

Background: SPINK7 is an epithelial serine protease inhibitor that is highly expressed in the esophagus and markedly decreased in patients with eosinophilic esophagitis (EoE) compared to control individuals. Loss of SPINK7 in esophageal epithelial cells promotes barrier impairment and pro-inflammatory innate immune responses. Despite its important role, the molecular mechanism that regulates SPINK7 expression is unknown.

Methods: We cloned the 4.5 kb region upstream to the transcription start site of SPINK7 into a vector that contains a nano-luciferase reporter. We transfected esophageal epithelial cells (EPC2) with the reporter and analyzed the promoter activity under different conditions. We used a candidate-based approach to survey transcription factors and their effects on SPINK7 promoter activity using a reporter-based assay. We analyzed the expression and cellular localization of Ovo Like Transcriptional Repressor 1 (OVOL1; which was identified by the reporter-based assay as SPINK7 regulator), by western blot and immunofluorescence in esophageal biopsies and EPC2 cells. We manipulated OVOL1 cellular localization by stimulating the cells with cytokines or ligands that activates the aryl hydrocarbon receptor (AHR). We then depleted OVOL1 expression in EPC2 cells by short hairpin RNA and by CRISPR/Cas9 gene deletion. We assessed barrier integrity, gene expression and cytokine production in OVOL1-depleted cells compared to control cells.

Results: A screen of candidate transcription factors revealed that OVOL1 regulates SPINK7 expression. Overexpression of OVOL1 increased SPINK7 expression by 2-fold in a promoter reporter assay (P < 0.0001). Depletion of OVOL1 in epithelial cells decreased SPINK7 expression by 3-fold (p = 0.002), promoted impaired epithelial barrier (> 40 % decrease, p < 0.0001 and increased TSLP production (3-fold, P = 0.001). We uncovered that stimulation with AHR-ligands is required for nuclear translocation and activation of OVOL1 which in turn promotes SPINK7 expression. Blocking AHR with an AHR antagonist, inhibited the AHR-ligands dependent SPINK7 expression. IL-4 and IL-13 abolished OVOL1 nuclear translocation and OVOL1-induced SPINK7 expression after AHR-ligands stimulation in EPC2 cells. The clinical relevance of OVOL1 to EoE pathogenicity was implicated by analysis of esophageal biopsies that demonstrated a 10-fold decrease in OVOL1 protein expression in EoE compared to control individuals (p = 0.02).

Conclusions: We propose that AHR serves as an esophageal sensor, mediates its action via OVOL1-induced SPINK7 transcription, and that IL-4 and IL-13 repress this pathway in EoE.

AUTHORS: Joy W. Chang, Joel H. Rubenstein, Jessica L. Mellinger, Ellyn Kodroff, Mary J. Strobel, Melissa Scott, Denise Mack, Wendy Book, Kathleen Sable, Shay Kyle, Ally Paliana, Evan S. Dellon

BACKGROUND: Given the similar effectiveness but differing philosophies of treatments for eosinophilic esophagitis (EoE), shared decision making (SDM) between patients and providers is important for improving treatment and adherence. Because little is known about this area, we aimed to describe patient-reported SDM and satisfaction in EoE care.

METHODS: We developed and administered a web-based survey about SDM and satisfaction around EoE treatment based on a validated 9-item SDM questionnaire (composite range 9 – 54, low SDM defined as < 32). Adults and caregivers of pediatric patients were recruited via patient advocacy groups and at two tertiary care centers. Regular care was defined by seeing the specialty provider at least annually. Multivariable linear and logistic regression was performed to identify predictors of SDM and satisfaction with treatment.

RESULTS: A total of 243 (47%) adults (mean age 38.7 years, SD 12.1) and 270 (53%) caregivers of pediatric patients (mean age 9.5 years, SD 4.5) completed the survey. A majority (90%) had a diagnosis of EoE alone. Among adults, 31% had regular care with both gastroenterology (GI) and allergy, 30% only with GI, 7% only with allergy, and 32% without specialty care. In contrast, a large majority of the pediatric cohort (70%) had regular follow-up with both GI and allergy, 25% only with GI, 3% only with allergy, and 2% with neither subspecialist. Patients followed by GI providers were more likely to experience greater SDM compared to non-specialists (adjusted β = 6.85; 95% CI 3.31-10.4) (Figure 1, Table 1). Despite this, nearly half of adults (44%) and children (40%) experienced low SDM for EoE management. SDM with allergists alone was similar to non-specialists. There was no difference between adults and children for the effects of provider type. More SDM occurred when patients perceived providers as comfortable with choosing a therapy outside of their recommendation (Table 1). Those receiving more SDM were more satisfied with current treatment, regardless of provider or treatment type (OR 2.77, 95% CI 1.86-4.11; Table 2).

CONCLUSION: SDM around EoE care is practiced most by gastroenterologists and when patients perceived provider comfort with various options, however many patients still do not experience joint decision making. Because patients are more likely to be satisfied with any treatment when SDM is practiced, it is important to improve patient-centered communication and engagement in managing EoE.

Share decision table.

Figure 1: Composite SDM scores by provider type in adult EoE patients. Higher scores denote greater shared decision making around EoE treatment.

Table 1: Associations between shared decision making and provider type.

    Mean, SD Low SDM,
N (%)		 High SDM,
N (%)		 Unadjusted β
(95% CI)		 Mutually Adjusted β
(95% CI)
Provider type
  No specialist
 28.07 (14.62)  46 (51.7%) 43 (48.3%)
 (Reference)
 (Reference)
   GI only
 34.95 (13.45)  52 (36.6%) 90 (63.4%)
  6.88 (3.33, 10.4) 6.85 (3.31, 10.4)
   Allergy only
 27.89 (13.05)
 18 (64.3%)
 10 (35.7%)
  -0.177 (-5.84, 5.48) 0.22 (-5.38, 5.82)
   GI + Allergy
 34.73 (12.74)
 101 (39.8%)
 153 (60.2%)
  6.67 (3.41, 9.92) 6.59 (3.34, 9.84)
Provider comfort
  Comfortable 36.10 (12.63)
 72 (31.4%)
 157 (68.6%)
 (Reference) (Reference)
   Uncomfortable 28.55 (13.48)
 70 (59.3%)
 48 (40.7%)
  -7.55 (-10.5, -4.59) -7.36 (-10.3, -4.45)
   Unsure 32.74 (13.75) 75 (45.5%)
 90 (54.6%)
  -3.37 (-6.03, -0.701) -2.32 (-4.99, 0.367)

Table 2: Associations between satisfaction with current treatment and SDM. 

    Satisfied  Unsatisfied or
indifferent 		 Unadjusted OR
(95% CI) 		 Mutually Adjusted OR
(95% CI) 
SDM composite
 Low SDM
 66 (30.4%)
 151 (69.6%)
 (Reference)
 (Reference)
  High SDM
 169 (57.1%) 127 (42.9%) 3.04 (2.10, 4.41) 2.77 (1.86, 4.11)
Provider type
 No specialist
 26 (29.2%)
 63 (70.8%) (Reference)
 (Reference)
  GI only
74 (52.1%) 68 (47.9%) 2.64 (1.50, 4.63) 1.89 (1.00, 3.57)
  Allergy only
12 (42.9%) 16 (57.1%) 1.82 (0.76, 4.37) 2.56 (0.589, 4.14)
  GI + Allergy
123 (48.4%) 131 (51.6%) 2.28 (1.35, 3.82) 1.60 (0.877, 2.92)
Provider comfort
 Comfortable
118 (51.5%) 111 (48.5%) (Reference)
 (Reference)
  Uncomfortable
54 (45.8%) 64 (54.2%) 0.79 (0.51, 1.24) 1.08 (0.662, 1.75)
  Unsure
 63 (38.2%) 102 (61.8%) 0.58 (0.39, 0.87) 0.712 (0.458, 1.11)
Current treatment
 No therapy
 3 (10.3%) 26 (89.7%) (Reference)
 (Reference)
  PPI only
18 (40.9%) 26 (59.1%) 6.00 (1.57, 22.9) 3.67 (0.917, 14.6)
  Topical corticosteroids
 27 (52.9%) 24 (47.1%) 9.75 (2.62, 36.3) 5.56 (1.40, 22.1)
  Dietary Exclusion
 127 (49.6%) 129 (50.4%) 8.53 (2.52, 28.9) 5.14 (1.44, 18.4)
  Topical corticosteroids + Diet
 55 (45.1%) 67 (54.9%) 7.11 (2.04, 24.8) 4.17 (1.11, 15.7)

Authors and Affiliations: Jeff E. Habel PhD1, Mark Rochman PhD1, Julie M. Caldwell PhD1, Adina Y. Ballaban1, Leah C. Kottyan PhD2, and Marc E. Rothenberg MD, PhD1;1Rothenberg CURED Laboratory, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 2Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH

Background: Calpain-14 is an esophageal specific epithelial protein that is transcriptionally induced in epithelial cells by IL-13, a product of type 2 CD4+ T cells. Genetic variants in the calpain 14 gene (CAPN14) have been linked with susceptibility to eosinophilic esophagitis (EoE), a chronic food allergic disease characterized by the presence of eosinophil accumulation in the esophagus. Dysregulated expression of calpain-14 disrupts epithelial cell barrier function, a process that is impaired in EoE. Yet the specific targets and/or substrates of calpain-14 have not been identified.

Methods: Herein, we performed a proximity-based protein-protein interaction screen, known as BioID, by fusing an active site mutant of the E. coli biotin protein ligase, BirA*, to the C-terminus of human calpain-14. The resulting biotinylated proteins from calpain-14-BirA* overexpressing HEK cells, as well as cells overexpressing negative controls calpain-14 or BirA* alone were purified and digested for mass spectrometry analysis. Potential protein interactors were identified using label-free protein quantitative analysis with SAINT and R Bioconductor packages limma and MSstats by comparing peptide spectral counts of signal and control backgrounds within the datasets.

Results: Identified candidates were filtered through known BioID contaminating proteins, the Crapome database, resulting in an interactome of 176 proteins. Protein-protein interaction using the STRING database produced 237 interactions (p < 1.0e-16). Functional prediction analysis identified 41 proteins involved in cellular junctions (p < 1.0e-13). Out of the 41 cellular junction proteins, the basal polarity module component scribble homolog, scrib, showed the highest spectral counts (SC=46), suggesting stable complex formation with calpain-14. Scrib, a primary component of the basal polarity module involved in cell polarity and cell motility, is known to interact with disks large (DLG1) and lethal giant larval (LLGL1) homologs, and induce epithelial mesenchymal transition. Notably, DLG1 and LLGL1 were part of the calpain-14 interactome and EMT was associated with EoE.

Conclusions: The newly discovered calpain-14 cell junction interactome proteins pose possible direct and regulatory roles of esophageal epithelial calpain-14 in cellular motility and barrier disruption.

Authors and Affiliations: Mats W. Johansson, PhD,a Elizabeth M. McKernan, BS,b Paul S. Fichtinger, BS,b Evelyn L. Angulo, MD,b Jennifer L. Bagley, RN,b Kristine E. Lee, MS,c Michael D. Evans, MS,c* Luis D. Lomeli, MD,b Deane F. Mosher, MD,a,b Shelly M. Cook, MD,d Eric A. Gaumnitz, MD,b and Sameer K. Mathur, MD, PhD, FAAAAIb; aBiomolecular Chemistry, bMedicine, cBiostatistics and Medical Informatics, and dPathology and Laboratory Medicine, University of Wisconsin, Madison, WI.
*Mr. Evans is currently affiliated with the Biostatistical Design and Analysis Center, Clinical and Translational Science Institute, University of Minnesota, Minneapolis, MN.

Background: Management of eosinophilic esophagitis (EoE) would benefit from non-invasive biomarkers for response to therapy and resolution of inflammation to replace endoscopic biopsy.
Our objective was to identify blood eosinophil surface antigens that correlate with esophageal eosinophil count, with a focus on the N29 epitope on activated beta1-integrins, which predicts disease activity in non-severe asthma; P-selectin (CD62P), which is exposed on activated platelets and activates beta1-integrin; and alphaIIb (CD41), the major platelet alpha integrin subunit, which reports platelet-eosinophil complexes.

Methods: Twenty-five EoE patients were studied following two-month proton-pump-inhibitor therapy and diagnostic endoscopy, with visit 1 (V1) one week after endoscopy. Patients received standard of care treatment for eight weeks followed by V2 and repeat endoscopy. N29, CD62P, CD41, and 13 other markers were assayed by whole blood flow cytometry. Peak eosinophil count (PEC) per high power field (HPF) was assessed on esophageal biopsy.

Results: N29, CD62P, and CD41 correlated among each other. Receiver operating characteristic curve analysis demonstrated that percentage CD41-positive blood eosinophils was the best predictor of PEC < 6/HPF at V2 with an area under curve = 0.84 (P < 0.001). An optimal cutoff of < 23% CD41-positive eosinophils predicted PEC < 6/HPF with a specificity of 93% and sensitivity of 82%. Dividing the patients according to median CD41, CD41-low patients had median PEC = 0/HPF and CD41-high patients median 31/HPF (P < 0.001).

Conclusions: The results indicate that a pathway in which activated platelets associate with and activate eosinophils operates in EoE, such that CD41 associated with circulating eosinophils is a potential non-invasive biomarker for disease activity.

Authors and affiliations: Hemanth Kumar Kandikattu and Anil Mishra; Department of Medicine, Section of Pulmonary Diseases, Tulane Eosinophilic Disorder Center, Tulane University School of Medicine, New Orleans, LA 70112

Background: Eosinophilic Gastrointestinal Disease (EGID) is a chronic disease, and the most compelling clinical feature of EGID is its responsiveness to elemental and highly restricted diets. Systemic steroids are also used for acute exacerbations, while topical glucocorticoids are used to provide long-term control; however, their long-term use may have some health issues in patients. Similarly, anti-IL-5 and anti-IL-13 treatment approaches have also demonstrated some promise; but the early clinical results have not been strikingly positive as hoped. We recently observed that calcineurin inhibitor “Tacrolimus” has the property to reduce the allergen, IL-5, and IL-13 induced blood, and tissue eosinophilia in the IL-5 overexpressed murine model of EGID.

Methods: We used intranasal Aspergillus fumigatus sensitized mice, CD2-IL-5 transgenic and DOX regulated IL-13 transgenic mice to examine blood and tissue eosinophilia following tacrolimus treatment using flow cytometer, anti-major basic protein (MBP) immunostaining ELISA and western blot analyses for the regulator of calcineurin 1 (RCAN1) protein.

Results: Tacrolimus administration in allergen challenge, CD2-IL-5 transgenic and DOX expose-IL-13 mice showed highly significantly reduced CCR3/SigleF+ eosinophils in the blood and anti- MBP+ eosinophils in esophageal, lung and intestinal tissues. Additionally, we observed reduced IL-5, IL-13 TGF-β, and RCAN1 levels in the blood and tissues of tacrolimus treated mice compared to vehicle-treated mice. Notably, RCAN1 reduction following Tacrolimus treatment indicates that tacrolimus inhibits eosinophils generation by directly targeting its progenitors in the bone marrow compared to anti-IL-5 and anti-IL-13 therapy that target eosinophils maturation, and development.

Conclusions: Taken together, we provide direct in vivo evidence that Tacrolimus ameliorates eosinophilic Gastrointestinal Disease; however, considering it reactivity to several other drugs and some side effects, tropical use of low dose tacrolimus may be a novel approach for the steroid-refractory adult EGID patients.

Authors and Affiliations: Tetsuo Shodaa, Ting Wena, Julie M. Caldwella, Margaret H. Collinsb, John A. Bessea, Garrett A. Osswalda, J. Pablo Aboniaa, Nicoleta C. Arvac, Dan Atkinsd, Kelley E. Capocellie, Evan S. Dellonf, Gary W. Falkg, Nirmala Gonsalvesh, Sandeep K. Guptai, Ikuo Hiranoh, Vincent A. Mukkadaj, Philip E. Putnamj, Rachel M. Sheridanb, Rudman Spergel AKk, Jonathan M. Spergell, Joshua B. Wechslerm, Guang-Yu Yangn, Seema S. Aceveso, Glenn T. Furutap and Marc E. Rothenberga on behalf of the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR)*; aDivision of Allergy and Immunology, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, bDivision of Pathology, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, cDepartment of Pathology, Ann & Robert H. Lurie Children’s Hospital of Chicago, Feinberg School of Medicine, Northwestern University, dSection of Pediatric Allergy and Immunology, Children’s Hospital Colorado, eDepartment of Pathology, Children’s Hospital Colorado, fDivision of Gastroenterology and Hepatology, University of North Carolina School of Medicine, gDivision of Gastroenterology, University of Pennsylvania Perelman School of Medicine, hDivision of Gastroenterology & Hepatology, Northwestern University, iDivision of Pediatric Gastroenterology, Hepatology and Nutrition, University of Illinois College of Medicine/Children's Hospital of Illinois, jDivision of Gastroenterology, Hepatology, and Nutrition, University of Cincinnati College of Medicine and Cincinnati Children's Hospital Medical Center, kAllergy, Asthma and Airway Biology Branch Division of Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases, NIH, lDivision of Allergy and Immunology, University of Pennsylvania Perelman School of Medicine/Children's Hospital of Philadelphia, mGastroenterology, Hepatology & Nutrition, Ann & Robert H. Lurie Children’s Hospital of Chicago, nDepartment of Pathology and Laboratory Medicine, Northwestern University, oDivision of Allergy Immunology, University of California-San Diego, Rady Children’s Hospital, San Diego, pSection of Pediatric Gastroenterology, Hepatology and Nutrition, Digestive Health Institute, Children's Hospital Colorado.

Background: Eosinophilic gastritis (EG) is a clinicopathologic disorder with marked gastric eosinophilia and clinical symptoms. There is an unmet need in EG for more precise diagnostic tools. We aimed to develop tissue- and blood-based diagnostic platforms for EG.

Methods: Patients with EG and non-EG controls were enrolled across 9 Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR)-associated sites. An EG Diagnostic Panel (EGDP; gastric transcript subset) and EG blood biomarker panel (protein multiplex array) were analyzed. The EGDP18 scores were derived from the expression of 18 highly dysregulated genes and Blood EG scores from dysregulated cytokines/chemokine levels.

Results: Gastric biopsies and blood samples from 185 subjects (EG 74, non-EG 111) were analyzed. The EGDP a) identified patients with active EG (P < .01, AUC ≥0.95); b) effectively monitored disease activity in longitudinal samples (P < .01); c) highly correlated in same-patient samples (antrum vs. body, r = 0.85, P < .01); and d) correlated with gastric peak eosinophil levels (r = -0.83, P < .01), periglandular circumferential collars (r = -0.73, P < .01), and endoscopic nodularity (r = -0.45, P < .01). For blood-based platforms, eotaxin-3, TARC, IL-5, and TSLP levels were significantly increased. Blood EG scores a) distinguished patients with EG from non-EG controls (P < .01, AUC ≥0.91); b) correlated with gastric eosinophil levels (P < .01); and c) correlated with EGDP18 scores (P < .01). Plasma eotaxin-3 strongly associated with EGDP18 score and gastric CCL26 expression (P < .01).

Conclusion: We developed tissue- and blood-based platforms for assessment of EG and uncovered robust associations between specific gastric molecular profiles and histologic and endoscopic features, providing insight and tools for this emerging rare disease.

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The Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR) is dedicated to improving the lives of individuals with eosinophilic gastrointestinal disorders through innovative research, clinical expertise and education via collaborations between scientists, health care providers, patients, and professional organizations. CEGIR is part of the Rare Diseases Clinical Research Network (RDCRN) and has a contact registry. Consider joining today. Read more
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