In Vivo Effects of Amylase Trypsin Inhibitors (ATI)

In Vivo Effects of Amylase Trypsin Inhibitors From Wheat in the Human Gut: Proof of Cause of Non-coeliac Wheat Sensitivity in Irritable Bowel Syndrome?

Wheat is the most important staple food consumed in the Western world and provides beneficial health effects and functional properties. Nevertheless, an increasing proportion of the general population is avoiding or reducing its consumption of wheat products due to self-reported gastrointestinal (GI) symptoms, such as patients with non-coeliac wheat sensitivity (NCWS) and/or irritable bowel syndrome (IBS). There is increasing evidence that the amylase trypsin inhibitors (ATIs), accounting for up to 15% of wheat proteins, play a role in the symptom generation in NCWS and IBS. In vitro studies showed ATIs can induce an innate immune response via direct interaction with the toll-like receptor 4 (TLR4), activating the TLR4-MD2-CD14 complex with subsequent release of pro-inflammatory cytokines. These results were confirmed in mice. Furthermore, in mice ATIs triggered intestinal epithelial lymphocytosis and barrier dysfunction, and modified microbiota composition and metabolism. Thus far, there have been no placebo-controlled studies investigating these effects of isolated ATIs in human subjects. Understanding the role of ATIs in symptom generation in NCWS and IBS patients is important to provide these patients with appropriate dietary advice, improving their quality of life and decreasing their risk of nutritional deficiencies.

The investigators aim to perform a proof-of-concept study to assess the effect of ATIs on the intestinal barrier and immune function in healthy volunteers. The investigators hypothesise that the ATIs either directly affect the intestinal barrier function, or indirectly by activating an immune response via TLR4.

The study conforms a randomized, double-blind, placebo-controlled, cross-over design, using healthy human volunteers (male and female), 18-65 years old. Volunteers will each undergo two test days, separated by a wash-out period of at least 4 weeks. At the test day, volunteers receive either isolated ATIs or placebo (physiological saline), ingested using a nasogastric intraduodenal feeding catheter.

Study Overview

• Status: Not yet recruiting
• Conditions: Irritable Bowel Syndrome; Non-Coeliac Wheat Sensitivity (NCWS)
• Intervention / Treatment: Other: Amylase trypsin inhibitors; Other: Placebo

• Study Type: Interventional
• Enrollment (Anticipated): 8
• Phase: Not Applicable

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 65 years (Adult, Older Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

Description

Inclusion Criteria:

1. Based on medical history and previous self-admitted examination, no gastrointestinal complaints and/or disease can be defined.
2. Age between 18 and 65 years.
3. Body Mass Index (BMI) between 20 and 30 kg/m2.

Exclusion Criteria:

1. History of severe cardiovascular, respiratory, urogenital, gastrointestinal/hepatic, hematological/immunologic, HEENT (head, ears, eyes, nose, throat), dermatological/connective tissue, musculoskeletal, metabolic/nutritional, endocrine, neurological/psychiatric diseases, allergy, major surgery and/or laboratory assessments which might limit participation in or completion of the study protocol. The severity of the disease (major interference with the execution of the experiment or potential influence on the study outcomes) will be decided by the principal investigator.
2. Use of medication, including vitamin supplementation, pre- and probiotic supplementation, except oral contraceptives, within 14 days prior to testing.
3. Administration of investigational drugs or participation in any scientific intervention study, which may interfere with this study (to be decided by the principal investigator), in the 180 days prior to the study.
4. Major abdominal surgery interfering with gastrointestinal function (uncomplicated appendectomy, cholecystectomy and hysterectomy allowed, and other surgery upon judgement of the principal investigator).
5. Pregnancy, lactation.
6. Excessive alcohol consumption (> 14 alcoholic consumptions per week).
7. Smoking.
8. Drug use.
9. Blood donation within 3 months before or after the study period.
10. Self-admitted HIV-positive state.
11. Plan to lose weight or follow a specific diet (e.g. weight loss or gluten-free diet) within the study period.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Crossover Assignment
• Masking: Double

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Amylase trypsin inhibitors (ATIs), then placebo; Test day 1: intraduodenal administration of amylase trypsin inhibitors (ATIs) isolated from Triticum aestivum (bread wheat), dissolved in physiological saline. After a wash-out period of 4-6 weeks, test day 2: intraduodenal administration of placebo (physiological saline).	Other: Amylase trypsin inhibitors; Intraduodenal administration of amylase trypsin inhibitors (ATIs) isolated from Triticum aestivum (wheat), dissolved in physiological saline.; Other: Placebo; Intraduodenal administration of placebo (physiological saline).
Experimental: Placebo, then Amylase trypsin inhibitors; Test day 1: intraduodenal administration of placebo (physiological saline). After a wash-out period of 4-6 weeks, test day 2: intraduodenal administration of amylase trypsin inhibitors (ATIs) isolated from Triticum aestivum (bread wheat), dissolved in physiological saline.	Other: Amylase trypsin inhibitors; Intraduodenal administration of amylase trypsin inhibitors (ATIs) isolated from Triticum aestivum (wheat), dissolved in physiological saline.; Other: Placebo; Intraduodenal administration of placebo (physiological saline).

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Difference in gene expression associated with intestinal barrier function, measured in duodenal biopsies collected after intervention with ATIs or placebo	Gene expression associated with intestinal barrier function, such as gene expression of tight junction and adherens junction proteins: e.g. peri-junctional Factin, myosin, ZO-1, claudin-3, occluding, myosin light chain kinase, phosphorylated myosin light chain, E-cadherin. Duodenal biopsies will be obtained using upper gastrointestinal endoscopy.	Biopsies will be collected 2 hours after administration of ATIs or placebo, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Difference in protein levels associated with intestinal barrier function, measured in duodenal biopsies collected after intervention with ATIs or placebo.	Expression of proteins associated with intestinal barrier function, such as tight junction and adherens junction proteins by Western blot and immunofluorescent staining. Duodenal biopsies will be obtained using upper gastrointestinal endoscopy.	Biopsies will be collected 2 hours after administration of ATIs or placebo, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.
Differences in gene expression associated with immune function, measured in duodenal biopsies collected after intervention with ATIs or placebo.	Gene expression associated with immune activation, such as TLR4, and pro-inflammatory cytokines and antimicrobiota peptides. Duodenal biopsies will be obtained using upper gastrointestinal endoscopy.	Biopsies will be collected 2 hours after administration of ATIs or placebo, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.
Differences in infiltration of immune cells in duodenal tissue, measured in duodenal biopsies collected after intervention with ATIs or placebo.	Infiltration of immune cells (neutrophils, mast cells, dendritic cells) into duodenal tissue by immunofluorescent staining. Duodenal biopsies will be obtained using upper gastrointestinal endoscopy.	Biopsies will be collected 2 hours after administration of ATIs or placebo, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.
Difference in change in gastrointestinal symptoms from baseline after intervention with ATIs or placebo.	Measured on the Visual Analogue Scale ranged from 0-100, in which 0 is absence of symptoms and 100 is severe symptoms; ATIs vs placebo	Various time points throughout each test day: 1 hour before and 5 minutes before intervention with ATIs or placebo, and 1 hour, 2 hours and 12 hours after intervention with ATIs or placebo
Difference in blood immune cell populations after intervention with ATIs or placebo	Quantification of immune cell populations (T helper cells, cytotoxic T cells, CD25+ effector T cells, CD69+ effector T cells, regulatory T cells, and NK-cells) in whole blood.	Blood samples will be collected directly after upper gastrointestinal endoscopy, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.
Difference in blood biomarkers of immune stimulation after intervention with ATIs or placebo	Measurement of biomarkers of immune stimulation (such as the human cytokines IL-10, TGF-β, IL-12p40 subunit, IL-12p70 subunit, IL-1β, IL-2, IL-6, IL-4, IL-17, IL-22 and IFN-γ, TNFα) in whole blood.	Blood samples will be collected directly after upper gastrointestinal endoscopy, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.
Blood markers for intestinal barrier function after intervention with ATIs or placebo	Markers for intestinal barrier function (plasma citrulin, metabolites) and small intestinal mucosal tissue injury (intestinal fatty acid binding protein (I-FABP)), measured in whole blood.	Blood samples will be collected directly after upper gastrointestinal endoscopy, on two separate test days. The test days are separated by a wash-out period of 4-6 weeks.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Participant characteristics	To assess suitability for participation of to characterise the study population, data will be collected on demographics and medical history using screening questionnaires.	Data on demographic factors and medical history will be collected during the screening visit
One-day food record to ensure dietary intake is similar during the 24 hours before testday 1 and testday 2.	Participants will be asked to repeat intake prior to testday 1 on the day prior to testday 2. Thus, the intake is written down during the 24 hours prior to test day 1, and repeated (and again written down) during the 24 hours prior to testday 1, which is 4-6 weeks after test day 1.	24 hours prior to testday 1, and after a wash-out period of 4-6 weeks, also during the 24 hours prior to testday 2

Collaborators and Investigators

• Sponsor: Maastricht University
• Investigators: Principal Investigator: Daisy MAE Jonkers, Prof, PhD, Maastricht University

Study record dates

Study Major Dates

• Study Start (Anticipated): September 1, 2023
• Primary Completion (Anticipated): September 1, 2024
• Study Completion (Anticipated): September 1, 2024

Study Registration Dates

• First Submitted: October 6, 2021
• First Submitted That Met QC Criteria: December 1, 2021
• First Posted (Actual): December 15, 2021

Study Record Updates

• Last Update Posted (Actual): March 27, 2023
• Last Update Submitted That Met QC Criteria: March 24, 2023
• Last Verified: March 1, 2023

More Information

Terms related to this study

• Keywords: Irritable Bowel Syndrome; Non-Coeliac Wheat Sensitivity; Amylase Trypsin Inhibitors
• Additional Relevant MeSH Terms: Digestive System Diseases; Pathologic Processes; Immune System Diseases; Disease; Gastrointestinal Diseases; Colonic Diseases, Functional; Colonic Diseases; Intestinal Diseases; Hypersensitivity; Syndrome; Irritable Bowel Syndrome; Molecular Mechanisms of Pharmacological Action; Enzyme Inhibitors; Protease Inhibitors; Serine Proteinase Inhibitors; Trypsin Inhibitors
• Other Study ID Numbers: NL77666.608.21

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No