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Celiac.com 12/26/2016 - Could gluten-degrading enzymes offer a better future for celiac patients? Rothia mucilaginosa is an oral microbial colonizer that can break down proline- and glutamine-rich proteins present in wheat, barley, and rye that contain the immunogenic sequences that drive celiac disease. A team of researchers recently set out to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for celiac disease. The research team included G Wei, N Tian, R Siezen, D Schuppan, and EJ Helmerhorst. They are variously affiliated with the Department of Molecular and Cell Biology at the Henry M. Goldman School of Dental Medicine in Boston, Massachusetts; the Bacterial Genomics Group, Center for Molecular and Biomolecular Informatics at Radboud University Medical Centre, Nijmegen, the Netherlands; the Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; and with the Institute of Translational Immunology and Research Center for Immunology, University Medical Center, Johannes-Gutenberg-University, Mainz, Germany. They first extracted and separated membrane-associated R. mucilaginosa proteins using DEAE chromatography. They tracked enzyme activities using paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. They determined epitope elimination in R5 and G12 ELISAs. They identified gliadin-degrading Rothia enzymes by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in celiac disease. To do this, the team cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. They found that Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. Rothia and food-grade Bacillus subtilisins show promise for development as enzyme therapies for celiac disease. Source: Am J Physiol Gastrointest Liver Physiol. 2016 Sep 1;311(3):G571-80. doi: 10.1152/ajpgi.00185.2016. Epub 2016 Jul 28
Celiac.com 02/27/2014 - For many people with celiac disease, one of the numerous downsides of the condition is the constant threat of an adverse reaction triggered by accidental gluten consumption. Because reactions to gluten ingestion can be severe for some celiac patients, many clinicians are looking to see if anything can be done to lessen the effects gluten reactions in celiac patients once they have started. A team of researchers sought to provide at least one possible answer by looking into the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to lessen effects gluten reactions in celiac patients. The researchers included G.J. Tack, J.M. van de Water, M.J. Bruins, E.M Kooy-Winkelaar, J. van Bergen, P. Bonnet, A.C. Vreugdenhil, I. Korponay-Szabo, L. Edens, B.M. von Blomberg, M.W. Schreurs, C.J. Mulder, and F. Koning. They are all affiliated with the Department of Gastroenterology and Hepatology, VU University Medical Centre, 1007 MB Amsterdam, The Netherlands. For their study, the team enrolled 16 adults with celiac disease as confirmed by positive blood test and biopsy-confirmed subtotal or total villous atrophy. All patients were following a strict gluten-free diet, and showed normalized antibodies and mucosal healing classified as Marsh 0 or I. In their randomized double-blind placebo-controlled pilot study, the team had patients consume wheat toast, totaling about 7 grams of gluten per day, with AN-PEP for a two-week safety phase. After a two-week washout period with adherence of the usual gluten-free diet, 14 patients were randomized to receive gluten with either AN-PEP or placebo for there two-week efficacy phase. Baseline measurements included complaints, quality-of-life, serum antibodies, immuno-phenotyping of T-cells and duodenal mucosa immuno-histology. The team collected both serum samples and quality of life questionnaires during and after the safety, washout and efficacy phase. They conducted duodenal biopsies after both safety and efficacy phases. The primary endpoint was a change in histological evaluation according to the modified Marsh classification. None of the sixteen adults in the study suffered serious adverse events, and no patients withdrew during the trial. Overall scores for the gastrointestinal subcategory of the celiac disease quality (CDQ) remains fairly high throughout the study, indicating that AN-PEP was well tolerated. Through the efficacy phase, CDQ scores for patients consuming gluten with placebo or gluten with AN-PEP remained largely unchanged, and researchers observed no differences between the groups. Moreover, neither the placebo group nor the AN-PEP group developed significant antibody titers, and IgA-EM concentrations remained negative for both groups. The team excluded two patients from entering the efficacy phase because their mucosa showed an increase of two Marsh steps after the safety phase, even though their serum antibodies remained undetectable. A total of 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, the team saw no significant deterioration in immunohistological and flow cytometric values between the group consuming placebo compared to the group receiving AN-PEP. Furthermore, compared to baseline, after two weeks of gluten four out of seven patients on placebo showed increased IgA-tTG deposit staining. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP. Source: World J Gastroenterol. 2013 Sep 21;19(35):5837-47. doi: 10.3748/wjg.v19.i35.5837.
Celiac.com 03/24/2014 - Two new studies have confirmed colonization of gluten-degrading bacteria in the human mouth and in the upper gastrointestinal tracts respectively. Both studies come out of the Department of Periodontology and Oral Biology, Boston University Henry M. Goldman School of Dental Medicine in Boston, Massachusetts. The research teams included Maram Zamakhchari, Guoxian Wei, Floyd Dewhirst, Jaeseop Lee, Detlef Schuppan, Frank G. Oppenheim, and Eva J. Helmerhorst. Gluten is notoriously hard for mammals to digest, because gliadin proteins resist mammalian proteolytic enzymes in the gut, so researchers wanted to find sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract. These microbial enzymes have the potential to neutralize the gluten peptides that act as celiac disease triggers. In the first study the researchers assessed proteolytic activity in suspended dental plaque towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of α2-gliadin and 26-mer of γ-gliadin). In addition, they conducted gliadin zymography to establish the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and performed liquid iso-electric focusing to determine overall enzyme iso-electric points. Their results provide the first known evidence of gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk. In the second study, the team employed a selective plating strategy using gluten agar to obtain oral microorganisms with gluten-degrading capacity. They then used16S rDNA gene sequencing to carry out microbial speciations. To determine enzyme activity, they used gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer a-gliadin and 26-mer c-gliadin immunogenic peptides. They separated fragments of the gliadin peptides by RP-HPLC, and structurally characterized them using mass spectrometry. They found that strains Rothia mucilaginosa and Rothia aeria showed high gluten-degrading activity. For example, gliadins (250 mg/ml) added to Rothia cell suspensions (OD620 1.2) degraded by 50% after 30 minutes of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were 70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10). While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. Taken together, these studies suggest a potential for these bacteria to fuel the development of compounds that can degrade of harmful gluten peptides that trigger celiac disease in susceptible individuals. Source: PLoS One. 2011;6(9):e24455. doi: 10.1371/journal.pone.0024455. http://www.ncbi.nlm.nih.gov/pubmed/20948997
Celiac.com 10/14/2013 - A team of researchers recently set out to assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN–PEP) to mitigate the effects of gluten in celiac patients. For their study, the researchers included celiac patients with positive serology and subtotal or total villous atrophy on duodenal biopsies, who follow a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. Prior to this randomized double-blind placebo-controlled pilot study, the team measured complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. They then had patients consume approximately 7 grams of gluten per day as toast, along with AN-PEP for a two week safety phase. The team put subjects through a two week washout phase where they followed their usual gluten-free diets. The team then randomly assigned 14 patients to receive gluten, with either AN-PEP or placebo, for a two week efficacy phase. They also collected patient questionnaires on serum and quality of life during and after the safety, washout and efficacy phase. They conducted duodenal biopsies after both the safety phase and the efficacy phase. Change in histological evaluation according to the modified Marsh classification served as the primary endpoint. In all, 16 adults participated in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The average score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the team saw no significant deterioration in the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP, nor did they observe any other differences between the groups. During the efficacy phase, neither the placebo nor the AN-PEP group showed significant antibody titers. IgA-EM concentrations remained negative in both groups. The team excluded two patients from entering the efficacy phase because their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies. A total of 14 patients were considered histologically stable on gluten with AN-PEP. Also, after the efficacy phase, the team saw no significant deterioration in immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, in four out of seven patients on placebo, IgA-tTG deposit staining increased after two weeks of gluten intake compared to baseline. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. AN–PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN–PEP. The research team included Greetje J Tack, Jolanda MW van de Water, Chris J Mulder of the Department of Gastroenterology and Hepatology, VU University Medical Centre in Amsterdam, The Netherlands; Engelina MC Kooy-Winkelaar, Jeroen van Bergen, and Frits Koning of the Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre in Leiden, The Netherlands; Petra Bonnet, B Mary E von Blomberg, and Marco WJ Schreurs from the Department of Pathology, VU University Medical Centre, in Amsterdam, The Netherlands; Anita CE Vreugdenhil, with Department of Paediatrics, University Hospital Maastricht in Maastricht, The Netherlands; and Ilma Korponay-Szabo, with the Department of Paediatrics, University of Debrecen in Hungary, and the Paediatric Research Centre, University of Tampere, in Tampere, Finland. Source: World Journal of Gastroenterology, 10/03/2013