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Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques.

ABSTRACT: Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement-but not remission-of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm-by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-? and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea-all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

SUBMITTER: Sestak K

PROVIDER: S-EPMC4963877 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques.

Sestak Karol K Thwin Hazel H Dufour Jason J Liu David X DX Alvarez Xavier X Laine David D Clarke Adam A Doyle Anthony A Aye Pyone P PP Blanchard James J Moehs Charles P CP Moehs Charles P CP

Nutrients 20160628 7

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are n ...[more]

PMID: 27367722

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Project description:Gluten is a complex of proteins present in barley, wheat, rye and several varieties of oats that triggers celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and therefore, proline-rich digestion-resistant peptides contain multiple immunogenic epitopes. Prolyl endopeptidases (PEP) hydrolyse internal proline residues on the carboxyl side of peptides and have been proposed for food gluten detoxification and as oral enzyme supplementation for celiacs. The aim of this study was to identify new gluten-degrading microbial enzymes with the potential to reduce gluten immunogenicity by neutralizing its antigenic epitopes. Using a gluten-degrading colony screening approach, a bacterial isolate (2RA3) displaying the highest glutenase activity was selected, characterized and its genome completely sequenced. The identification through 16S rDNA gene sequencing showed a 99,1% similarity to Chryseobacterium taeanense. Hydrolysis of gluten immunogenic peptides (GIP) was further monitored, over a 48-hour period, by colony encapsulation in gliadin-containing microspheres, followed by detection with the G12 anti-GIP monoclonal antibody. Glutenase activity was detected in the extracellular medium of 2RA3 cultures, where gel electrophoresis and gliadin zymography revealed the presence of a ~50 kDa gluten-degrading enzyme. Nano-ESI-Q-TOF of the excised active band identified 7 peptides contained in the protein product predicted for an open reading frame (ORF) in the 2RA3 genome. Based on sequence similarity to the PEP family, the new enzyme was named PEP 2RA3. The PEP 2RA3 coding sequence was PCR-amplified from C. taeanense 2RA3, cloned and expressed in Escherichia coli as a C-terminally His-tagged recombinant protein and purified by Ni-NTA affinity chromatography. The recombinant protein, with predicted molecular mass and isoelectric point of 78.95 kDa and 6.8, respectively, shows PEP activity with standard chromogenic substrates, works optimally at pH 8.0 and 30°C and remains stable at pH 6.0 and 50°C, indicating a potential use in gluten-containing food process applications. The ability of the recombinant enzyme to degrade GIP in beer into smaller peptides was confirmed.

Project description:Primary immunodeficiencies are heritable disorders of immune function. CD19 is a B cell co-receptor important for B cell development, and CD19 deficiency is a known genetic risk factor for a rare form of primary immunodeficiency known as "common variable immunodeficiency" (CVID); an antibody deficiency resulting in low levels of serum IgG and IgA. Enteropathies are commonly observed in CVID patients but the underlying reason for this is undefined. Here, we utilize CD19-/- mice as a model of CVID to test the hypothesis that antibody deficiency negatively impacts gut physiology under steady-state conditions. As anticipated, immune phenotyping experiments demonstrate that CD19-/- mice develop a severe B cell deficiency in gut-associated lymphoid tissues that result in significant reductions to antibody concentrations in the gut lumen. Antibody deficiency was associated with defective anti-commensal IgA responses and the outgrowth of anaerobic bacteria in the gut. Expansion of anaerobic bacteria coincides with the development of a chronic inflammatory condition in the gut of CD19-/- mice that results in an intestinal malabsorption characterized by defects in lipid metabolism and transport. Administration of the antibiotic metronidazole to target anaerobic members of the microbiota rescues mice from disease indicating that intestinal malabsorption is a microbiota-dependent phenomenon. Finally, intestinal malabsorption in CD19-/- mice is a gluten-sensitive enteropathy as exposure to a gluten-free diet also significantly reduces disease severity in CD19-/- mice. Collectively, these results support an effect of antibody deficiency on steady-state gut physiology that compliment emerging data from human studies linking IgA deficiency with non-infectious complications associated with CVID. They also demonstrate that CD19-/- mice are a useful model for studying the role of B cell deficiency and gut dysbiosis on gluten-sensitive enteropathies; a rapidly emerging group of diseases in humans with an unknown etiology.

Dataset Information

Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques.

Publications

Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques.

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