Wheat Flour Treatment With Microbial Transglutaminase and Lysine Ethyl Ester: New Frontiers in Celiac Disease Treatment.

Study Overview
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Brief Summary
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Celiac disease is one of the most common forms of food intolerance (prevalence 1/200). The disease occurs in genetically predisposed individuals after ingestion of foods containing gluten. Celiac patients can suffer from severe malabsorption syndrome, mainly characterized by diarrhea and weight loss. The only therapeutic approach currently recognized is a life-long gluten-free diet. Specific regions of gluten molecule become recognizable by lymphocytes and activate them, due to changes made by tissue transglutaminase. These changes consist in the conversion of specific residues of glutamine into glutamic acid. The consequence is an increased binding affinity between gluten and histocompatibility molecule (HLA-DQ2), localized on the surface of the antigen presenting cells (APC); the exposure of the fragments of modified gluten on the surface of APC is a phenomenon that eventually activates T lymphocytes. Recent studies on modified gluten confirmed the hypothesis that it is possible to block the presentation of gluten to lymphocytes by means of lysine ethyl ester binding exclusively to those gluten regions responsible for lymphocyte activation. The enzymatic treatment is performed directly on flour instead of extracted gluten, maintaining the same anti-inflammatory effectiveness. The procedure uses a food-grade enzyme, the microbial transglutaminase (mTGasi) isolated from Streptoverticillium mobarensis, able to catalyze the formation of intermolecular cross-links that modify the functional properties of the products. Objective of the study is to validate the ability of the enzyme treatment of wheat flour with mTGasi and lysine ethyl ester to block the toxic effect of gluten in celiac patients.

Detailed Description
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Celiac disease is one of the most common forms of food intolerance (prevalence 1/200). The disease occurs in genetically predisposed individuals after ingestion of foods containing wheat gluten and similar proteins found in other common cereals such as barley and rye. Celiac patients can suffer from severe malabsorption syndrome, mainly characterized by diarrhea, weight loss and growth retardation. The only therapeutic approach currently recognized is a life-long gluten-free diet. Specific regions of gluten molecule become recognizable by lymphocytes and activate them, due to changes made by tissue transglutaminase. These changes consist in the conversion of specific residues of glutamine (Q) into glutamic acid (E). The consequence is an increased binding affinity between gluten and histocompatibility molecule (HLA-DQ2), localized on the surface of the antigen presenting cells (APC); the exposure of the fragments of modified gluten on the surface of APC is a phenomenon that eventually activates T lymphocytes. Recent studies on modified gluten confirmed the hypothesis that it is possible to block the presentation of gluten to lymphocytes by means of lysine ethyl ester binding exclusively to those gluten regions responsible for lymphocyte activation. Specifically, it is possible to perform the enzymatic treatment directly on flour instead of extracted gluten, maintaining the same anti-inflammatory effectiveness. The final procedure consists in dissolving the flour in water in the presence of appropriate concentrations of enzyme and lysine ethyl ester, maintaining the suspension in constant motion for two hours at room temperature. The procedure uses a food-grade enzyme, the microbial transglutaminase (mTGasi) isolated from Streptoverticillium mobarensis, already used for the preparation of food. The mTgasi is able to catalyze the formation of intermolecular cross-links modifying the functional properties of the products through the aggregation and polymerization of proteins. The peculiar method identified by our laboratory reduces the possibility of cross-links between proteins: consequently, minimal changes involve the gluten structure and, consequently, the visco-elastic properties of the dough. Objective of the study is to validate the ability of the enzyme treatment of wheat flour with mTGasi and lysine ethyl ester to block the toxic effect of gluten in celiac patients.

Official Title
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Wheat Flour Subjected to Microbial Transglutaminase Enzymatic Treatment in the Presence of Lysine Ethyl Ester for Alimentary Use in the Treatment of Celiac Disease.

Conditions
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Celiac Disease

Intervention / Treatment
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* Dietary Supplement: rusks made of wheat flour enzymatically treated
* Dietary Supplement: rusks made of wheat flour not enzimatically modified
* Dietary Supplement: Gluten-free diet


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: INCLUSION CRITERIA diagnosis of celiac disease according to ESPGHAN criteria: class III according to Marsh-Oberhuber classification, clear response to gluten-free diet, serological antiendomysium and antitransglutaminase antibodies positive results before GFD; HLA DQ2-DQ8 positive results; gluten-free diet from at least one year; negative serology from at least 1 year; EXCLUSION CRITERIA inflammatory bowel diseases; tumors; infectious liver disease; renal impairment.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: rusks made with treated flour<br>Gluten-free diet adding rusks (100g / day) produced with commercial wheat flour enzymatically treated with mTGasi and lysine ethyl ester. | Dietary Supplement: rusks made of wheat flour enzymatically treated<br>* 100 g of rusks obtained from commercial wheat flour enzymatically treated with mTGasi and lysine ethyl ester, every day for 3 months<br>Dietary Supplement: Gluten-free diet<br>* gluten-free diet<br>|
| Active Comparator: rusks made with not-treated flour<br>Gluten-free diet adding rusks (100g / day) produced with untreated wheat flour. | Dietary Supplement: rusks made of wheat flour not enzimatically modified<br>* 100 g of rusks obtained from commercial wheat flour NOT enzymatically treated with mTGasi and lysine ethyl ester, every day for 3 months<br>Dietary Supplement: Gluten-free diet<br>* gluten-free diet<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change from baseline in IgA anti-tissue transglutaminase (anti-tTG) antibodies at 30 days | Proof of any positivization of specific serological antibodies for celiac disease. Results quantified by an ELISA reader at 450 nm (A450nm) is expressed in U/mL and the antibody level 10 U/mL was used as a cutoff value to identify anti-tTG positive results. | After 1 month | 
| Change from baseline in IgA anti-tissue transglutaminase (anti-tTG) antibodies at 60 days | Proof of any positivization of specific serological antibodies for celiac disease. Results quantified by an ELISA reader at 450 nm (A450nm) is expressed in U/mL and the antibody level 10 U/mL was used as a cutoff value to identify anti-tTG positive results. | After 2 months | 
| Change from baseline in IgA anti-tissue transglutaminase (anti-tTG) antibodies at 90 days | Proof of any positivization of specific serological antibodies for celiac disease. Results quantified by an ELISA reader at 450 nm (A450nm) is expressed in U/mL and the antibody level 10 U/mL was used as a cutoff value to identify anti-tTG positive results. | After 3 months | 
| Change from baseline in IgA anti-endomysium antibodies (EMA) at 30 days | Test used to confirm serological activation of celiac disease. IgA EMA were searched in sera diluted 1:5 by indirect immunofluorescence analysis on cryostat sections of monkey esophagus. The results are expressed as ''positive/negative''. | After 1 month | 
| Change from baseline in IgA anti-endomysium antibodies (EMA) at 60 days | Test used to confirm serological activation of celiac disease. IgA EMA were searched in sera diluted 1:5 by indirect immunofluorescence analysis on cryostat sections of monkey esophagus. The results are expressed as ''positive/negative''. | After 2 months | 
| Change from baseline in IgA anti-endomysium antibodies (EMA) at 90 days | Test used to confirm serological activation of celiac disease. IgA EMA were searched in sera diluted 1:5 by indirect immunofluorescence analysis on cryostat sections of monkey esophagus. The results are expressed as ''positive/negative''. | After 3 months | 
| Pathologic variations in histologic analysis from baseline | Proof of duodenal mucosa histological alterations induced by celiac disease, consisting in altered (<3:1) villous height/crypt depth ratio and increased (>25) intraepithelial lymphocytes per 100 intestinal epithelial cells, according to Marsh-Oberhuber classification. | After 3 months, or in case of serum anti-tTG IgA/EMA IgA positive results | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Presence of bloating at baseline | Evaluation of bloating intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | At baseline | 
| Presence of abdominal pain at baseline | Evaluation of abdominal pain intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | At baseline | 
| Presence of diarrhea at baseline | Evaluation of diarrhea intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | At baseline | 
| Presence of asthenia at baseline | Evaluation of asthenia intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | At baseline | 
| Variation of bloating from baseline at 30 days | Evaluation of bloating intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 1 month | 
| Variation of abdominal pain from baseline at 30 days | Evaluation of abdominal pain intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 1 month | 
| Variation of diarrhea from baseline at 30 days | Evaluation of diarrhea intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 1 month | 
| Variation of asthenia from baseline at 30 days | Evaluation of asthenia intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 1 month | 
| Variation of bloating from baseline at 60 days | Evaluation of bloating intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 2 months | 
| Variation of abdominal pain from baseline at 60 days | Evaluation of abdominal pain intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 2 months | 
| Variation of diarrhea from baseline at 60 days | Evaluation of diarrhea intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 2 months | 
| Variation of asthenia from baseline at 60 days | Evaluation of asthenia intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 2 months | 
| Variation of bloating from baseline at 90 days | Evaluation of bloating intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 3 months | 
| Variation of abdominal pain from baseline at 90 days | Evaluation of abdominal pain intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 3 months | 
| Variation of diarrhea from baseline at 90 days | Evaluation of diarrhea intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 3 months | 
| Variation of asthenia from baseline at 90 days | Evaluation of asthenia intensity is reported according to the Visual Analog or Analogue Scale (VAS), with a range of 0-10. | After 3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Celiac disease, Gluten-free diet, Wheat flour treatment, Microbial transglutaminase, Lysine Ethyl Ester, Celiac Disease Treatment