A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis.
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SUBMITTER: Ogilvie OJ
PROVIDER: S-EPMC8229354 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and aimsThe dietary compliance and its assessment in celiac disease (CD) patients on a strict gluten-free diet (GFD) remain a challenge. Two relatively new, validated methods have been proposed to detect occasional gluten ingestion: standardized dietary questionnaire and determination of urinary gluten immunogenic peptides (GIPs). Our aim was to prospectively assess dietary compliance via these methods and compare their results with those of tissue transglutaminase antibodies (tTGA).MethodsThis was a prospective single-center study. Consecutive CD patients (aged 1-18 years) on a GFD scheduled for regular consultation between March and August 2019 were invited. In addition to standard care, a completed dietary questionnaire and urine sample for GIP were collected. Pearson's chi-square test, Fisher's exact test, and Mann-Whitney U test were performed.ResultsOf the 156 eligible children, 110 provided informed consent. Completed dietary questionnaire, GIP, and tTGA results were available from 86 children (median age 12.8 years, median GFD duration 30 months, 65% female). Adherence to the GFD evaluated by GIP, dietary questionnaire, and anti-tTGA was 94.2%, 75.6% and 94.2%, respectively. No association was found between the tTGA results and the detection of GIP, as well as between the tTGA results and the dietary questionnaires scores (P = .5 and .312, respectively). The participants perceived both the questionnaire and the measurement of GIP as reassuring with regard to correct implementation of the GFD.ConclusionAll the 3 methods have limitations to monitor dietary compliance. The comparison of their performance shows that the best single method is the use of the validated dietary questionnaire, which should therefore be implemented in the regular care for children with CD. The most effective combination of dietary questionnaire and urinary GIP determination should be used in specific clinical situations.
| S-EPMC11307387 | biostudies-literature
Project description:IntroductionThe adherence to a gluten-free diet (GFD) is a trending topic in the management of celiac disease. The aim of our study was to evaluate the diagnostic performance of urinary gluten immunogenic peptides (GIP) determination to detect gluten contamination of the GFD.MethodsIn study A, 25 healthy adults on a standard GFD performed 6 gluten challenges (0, 10, 50, 100, 500, and 1,000 mg) with quantification of urinary GIP before (T0) and during the following 24 hours. In study B, 12 participants on a gluten contamination elimination diet underwent urinary GIP determination at T0 and after challenge with 5 or 10 mg gluten. Urine GIP concentration was determined by an immunochromatographic assay.ResultsIn study A, 51 of 150 baseline urine samples were GIP+ on GFD and 7 of 17 were GIP+ after the zero-gluten challenge, whereas only 55 of 81 were GIP+ after the 10-1,000 mg gluten challenges. There was no significant change in the 24-hour urinary GIP when increasing gluten from 10 to 1,000 mg. In study B, 24 of 24 baseline urine samples were GIP-, whereas 8 of 24 were GIP+ after 5 or 10 mg of gluten.DiscussionTraces of gluten in the standard GFD may cause positivity of urinary GIP determination, whereas a false negativity is common after a gluten intake of 10-1,000 mg. Owing to the impossibility of standardizing the test in normal conditions, it seems unlikely that urinary GIP determination may represent a reliable tool to assess the compliance to the GFD of patients with celiac disease or other gluten-related disorders.
| S-EPMC8500619 | biostudies-literature
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.
| S-EPMC6597064 | biostudies-literature
Project description:Introduction and aimUsually, adherence to the gluten-free diet (GFD) in celiac patients is indirectly assessed through serological analysis, questionnaires, or invasive methods such as intestinal biopsy. The detection of gluten immunogenic peptides in urine (urinary gluten immunogenic peptides-uGIP) is a novel technique that directly evaluates the ingestion of gluten. The aim of this study was to evaluate the clinical efficacy of uGIP in the follow-up of celiac disease (CD).MethodsFrom April 2019 to February 2020, CD patients reporting complete adherence to the GFD were prospectively enrolled but were unaware of the reason for the tests. Urinary GIP, the celiac dietary adherence test (CDAT), symptomatic visual analog scales (VAS), and tissue transglutaminase antibodies (tTGA) titres were evaluated. Duodenal histology and capsule endoscopy (CE) were performed when indicated.ResultsA total of 280 patients were enrolled. Thirty-two (11.4%) had a positive uGIP test (uGIP+). uGIP+ patients did not show significant differences in demographic parameters, CDAT, or VAS scores. The tTGA+ titre was not related to the positivity of uGIP (14.4% vs. 10.9% in patients with tTGA+ and tTGA-). Regarding histology, 66.7% of the GIP+ patients had atrophy compared to 32.7% of the GIP patients (p-value 0.01). However, the presence of atrophy did not correlate with tTGA. Mucosal atrophy was detected in 29 (47.5%) out of 61 patients by CE. With this method, no noticeable dependence on uGIP results (24 GIP- vs. 5 GIP+) was observed.ConclusionsThe single uGIP test was positive in 11% of CD cases referring a correct GFD adherence. Furthermore, uGIP results significantly correlated with the duodenal biopsy, formerly considered the gold standard for assessing CD activity.
| S-EPMC10004805 | biostudies-literature
Project description:The increasing number of persons following a gluten-free (GF) diet and the need for healthy and natural products are forcing researchers and industries to provide gluten-free products with high nutritional value. Here, a biotechnological approach combining the use of teff flour and type-I sourdough has been proposed to produce GF muffins with nutritional benefits. Teff-sourdough was prepared and propagated following the traditional daily refreshment procedure until the biochemical stability was achieved. The sourdough, dominated by Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Saccharomyces cerevisiae strains, was used to produce muffins at three different levels (up to 15%, wt/wt) of fortification, achieving several positive effects on the nutritional properties of the products. The use of teff flour led to high content of fiber (>3 g/100 g) and proteins (>6 g/100 g) in muffins achieving the nutritional requirements for the healthy claims "source of fiber" and "rich in protein". Thanks to their metabolic traits, sourdough lactic acid bacteria caused the increase of the total free amino acids (TFAA, up to 1000 mg/kg, final concentration) and phytic acid decrease (50% lower than control), which positively affect the nutritional properties of the products. Besides, high in vitro protein digestibility (IVPD, 79%) and low starch hydrolysis rate (HI, 52%) characterized the fortified muffins. Muffins also presented high in vitro antioxidant (56%) and mold-inhibitory activities, potentially contributing to an extended shelf-life of the products.
| S-EPMC7466135 | biostudies-literature
Project description:BackgroundCeliac disease is an intestinal chronic disorder with multifactorial etiology resulting in small intestinal mucosal injuries and malabsorption. In genetically predisposed individuals with HLA DQ2/DQ8 molecules, the gluten domains rich in glutamine and proline present gluten domains to gluten reactive CD4+ T cells causing injury to the intestine. In the present experimental design, the indigenous bacteria from wheat samples were studied for their gluten hydrolyzing functionality.ResultsProteolytic activity of Bacillus spp. was confirmed spectrophotometrically and studied extensively on gliadin-derived synthetic enzymatic substrates, natural gliadin mixture, and synthetic highly immunogenic 33-mer peptide. The degradation of 33-mer peptide and the cleavage specificities of the selected isolates were analyzed by tandem mass spectrometry. The gluten content of the sourdough fermented by the chosen bacterial isolates was determined by R5 antibody based competitive ELISA. All the tested isolates efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA, and Z-PFP-pNA and also cleaved 33-mer immunogenic peptide extensively. The gluten content of wheat sourdough was found to be below 110 mg/kg.ConclusionIt has been inferred that four Bacillus spp especially GS 188 could be useful in developing gluten-reduced wheat food product for celiac disease prone individuals.
| S-EPMC7291523 | biostudies-literature
Project description:Gluten content labels inform food choice and people practicing a gluten-free diet rely upon them to avoid illness. The regulations differ between jurisdictions, especially concerning fermented foodstuffs such as beer. Gluten abundance is typically measured using ELISAs, which have come into question when testing fermented or hydrolysed foodstuffs such as beer. Mass spectrometry can be used to directly identify gluten peptides and reveal false negatives recorded by ELISA. In this survey of gluten in control and gluten-free beers, gluten protein fragments that contain known immunogenic epitopes were detected using liquid chromatography-mass spectrometry in multiple beers that claim to be gluten-free and have sufficiently low gluten content, as measured by ELISA, to qualify as being gluten-free in some jurisdictions. In fact, several purportedly gluten-free beers showed equivalent or higher hordein content than some of the untreated, control beers. The shortcomings of ELISAs for beer gluten testing are summarised, the mismatch between ELISA and mass spectrometry results are explored, and the suitability of existing regulations as they pertain to the gluten content in fermented foods in different jurisdictions are discussed.
| S-EPMC10486350 | biostudies-literature
Project description:A lifelong gluten-free diet (GFD) is the only current treatment for celiac disease (CD), but strict compliance is complicated. Duodenal biopsies are the "gold standard" method for diagnosing CD, but they are not generally recommended for disease monitoring. We evaluated the sensitivity and specificity of fecal gluten immunogenic peptides (GIPs) to detect duodenal lesions in CD patients on a GFD and compared them with serum anti-tissue transglutaminase (tTG) IgA antibodies. A prospective study was conducted at two tertiary centers in Spain on a consecutive series of adolescents and adults with CD who maintained a long-lasting GFD. Adherence to a GFD and health-related quality of life were scored with validated questionnaires. Mucosal damage graded according to the Marsh-Oberhüber classification (Marsh 1/2/3) was used as the reference standard. Of the 97 patients included, 27 presented duodenal mucosal damage and 70 had normal biopsies (Marsh 0). The sensitivity (33%) and specificity (81%) of GIPs were similar to those provided by the two assays used to measure anti-tTG antibodies. Scores in questionnaires showed no association with GIP, but an association between GIPs and patients' self-reported gluten consumption was found (p = 0.003). GIP displayed low sensitivity but acceptable specificity for the detection of mucosal damage in CD.
| S-EPMC7824460 | biostudies-literature
Project description:Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces matrix metalloproteinase (MMP)-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial 65 kDa heat shock protein (HSP65) chaperone is the major target for the immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs that cleave HSP65, we used MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, membrane-associated MMP-14, MMP-15, MMP-16 and MMP-24, and glycosylphosphatidylinositol-linked MMP-17 and MMP-25. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follow. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection.
| S-EPMC3197701 | biostudies-literature
Project description:BackgroundMatrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.Methodology/principal findingsTo elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context.Conclusions/significanceIn sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.
| S-EPMC2654159 | biostudies-literature