Celiac sprue is an inheritable inflammatory disease of the upper small intestine caused by proline- and glutamine-rich gluten proteins contained in wheat, rye, and barley. In contrast to virtually all other dietary proteins, gluten is incompletely digested by gastric and pancreatic enzymes, resulting in the release of metastable peptides that persist in the intestinal lumen until they are translocated across the epithelial barrier by unknown mechanisms. Translocated peptides are selectively deamidated at key glutamine residues by the endogenous enzyme transglutaminase 2, thereby increasing their affinity for the disease-associated major histocompatibility complex molecule, HLA-DQ2. Presentation of DQ2-bound gluten peptides to disease-specific intestinal T cells initiates a localized inflammatory immune response as well as a systemic humoral immune response. Left untreated, the inflammatory response causes gross disruption of epithelial architecture and barrier function, which in many individuals results in nutrient malabsorption, chronic diarrhea, and wasting. Conversely, dietary abstention from gluten constitutes an effective treatment in most patients. Due to the ubiquity of gluten in most human diets, however, a gluten-free diet is extraordinarily difficult to maintain. Consequently, non-dietary therapies have the potential to substantially improve quality of life for the 0.5-1% of human beings afflicted with this life-long disease.
This thesis focuses on two potential therapeutic interventions: oral administration of proteases capable of digesting gluten (i.e. glutenases) and modulation of intestinal permeability toward gluten peptides. To investigate the potential of oral glutenase therapy, a naturally evolved barley glutenase, EP-B2, was heterologously expressed in E. coli and characterized biochemically and structurally, via X-ray crystallography. Recombinant EP-B2 enhanced digestion of gluten epitopes in vitro and in vivo (in rats), thereby mitigating their immunogenicity toward disease-specific T cells. Complementation of EP-B2 with a microbial prolyl endopeptidase resulted in complete gluten detoxification, indicating the clinical promise of combination glutenase therapy. The mechanisms controlling gluten peptide translocation are not well understood, hampering the development of therapeutics aimed at modulating intestinal permeability. Therefore, basic biochemical and cell culture studies were undertaken that identified interferon-γ as the primary modulator of epithelial permeability secreted by gluten-stimulated, celiac patient-derived intestinal T cells. In furtherance of these studies, and in collaboration with others, mass spectrometric and immunological assays were developed to detect and quantify gluten peptides and glutenase activity in complex biological samples.
At the time of these studies, there was no animal model for celiac sprue. Therefore, to further investigate enzyme therapeutics and peptide transport in vivo, a condition of gluten sensitivity in captive rhesus macaques was identified and characterized. Gluten-sensitive macaques exhibited many of the hallmark signs and symptoms of celiac sprue, including gluten-dependent clinical, serological, histological, and dermatological manifestations. In this model, a prophylactic effect of oral EP-B2 against gluten-induced clinical relapse was demonstrated, as well as the intact transepithelial transport of a disease-relevant gluten peptide. The immunotoxicity of gluten in celiac sprue precludes similar studies in human patients. Therefore, synthetic gluten peptide analogues were designed that retained resistance to gastrointestinal proteases, susceptibility to therapeutic glutenases, and similar transepithelial transportability, but that exhibited greatly reduced deamidation by transglutaminase 2, binding to HLA-DQ2, and immunogenicity toward celiac patient-derived, gluten-specific intestinal T cells. Thus, these non-inflammatory biomarkers may be used to safely evaluate the efficacy of glutenases and gut permeability-modulating therapeutics in celiac sprue patients.
|School Location:||United States -- California|
|Source:||DAI-B 70/03, Dissertation Abstracts International|
|Subjects:||Pharmacology, Biochemistry, Immunology|
|Keywords:||Biomarkers, Celiac sprue, Gluten peptides, Interferon-gamma, Peptide transport|
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