6/2/15 What do Clinical Scientists Knead to Know About Gluten? Matthew Schoell, Ph.D., MLS (ASCP)CM Nazareth College A Gluten-Free World Much More Than the Bakery 1
Gluten Itself Major protein component of grains (wheat, barley, rye) GF-grains include: Quinoa, rice, oats Depending on nature of processing Includes two proteins gliadin and glutenin, complexed with starch as well. Provides the elasticity in bread and other doughs. When dough is washed with water, you can shed the starch leaving only the elastic proteins. Gliadin Monomers in the 28-55 kda range Broken into types (α/β, ϒ, ω) by electrophoretic mobility Gln, Pro, Phe rich, often in repeating and overlapping sequence Side By Side Glutenin Protein aggregates in the 0.5-10 mda range Low and High MW varieties, both broken down into domains. Some domains have repetitive sequences, some have homology to gliadin Polymerization occurs through disulfide bonds making cysteine residues key: A substantial amount of the chemistry governing baking revolves around controlling this polymerization two-component glue Herbert Weiser Gluten-Free Market In a state of massive expansion: Mintel (a market research firm) reported the following after their Gluten Free Foods US 2014 study: 68% increase in the gluten free foods market in two years (2012-2014) 8.8 billion estimated sales in 2014 (~5x the size of the market for meat alternatives) On course for 14.2 billion in sales in 2017 41% of those surveyed say gluten free was beneficial for all people, 44% said gluten free was a fad diet 22% of Americans following a gluten free diet in 2014 Let s keep in mind this is one market research study 2
Grain of (Gluten-Free) Salt FDA Labeling Regulations Regulation published August 2013, enforceable August 2014 To be labeled gluten-free or comparable, a food must contain less than 20 ppm gluten Primarily aimed at packaged foods, expectation that restaurants making the claim will meet the standard Doesn t govern other advisory statements: Made in a facility that processes wheat The Health Burden of Gluten Any of the diseases/conditions associated with gluten consumption often present with GI symptoms, especially Irritable Bowel Syndrome While GI symptoms can be very objective, less well defined symptoms are frequently reported, especially fatigue/tiredness Other GI issues (Crohn s, ulcerative colitis) may be relevant based on individual parent s situation. Can present with bowel inflammation, serologic/ autoimmune testing can help differentiate. 3
The Gluten Lineup 1. Celiac Disease, aka Celiac Sprue Well defined autoimmune condition, specific serological and histological testing 2. Wheat Allergy Generally IgE based, classical or exercise induced, traditional allergy symptoms 3. Non-Celiac Gluten Sensitivity Very limited definition, appears derived from phenomenon of mitigation of symptoms in absence of gluten. Celiac Disease Autoimmune Disorder HLA DQ2/DQ8 haplotypes Gliadin peptide Tissue transglutaminase >1 in 200 in most populations, onset at any age Chief risk factors: Primary relation with CD DQ2/DQ8 Haplotype Celiac Symptoms Gastrointestinal/Irritable Bowel Syndrome present a bit more often in children than adults. These can extend into growth/failure to thrive concerns. Adults are more likely to describe malabsorptive symptoms, which link to villous atrophy Iron Deficiency Anemia Osteomalacia Parasthesia Weight Loss Other less nonspecific symptoms include rashes, reproductive complications, psychiatric 4
Villous Atrophy Hematoxilin/ Eosin CD3 Staining Volta and Villanacci. Cellular & Molecular Immunology. 2011 HLA as Risk Factors DQ2/DQ8 haplotypes most responsible linked to increased risk for a variety of autoimmune diseases Molecular Revolution Sequencing will only become more common. Megiorni, F et al. Human Immunolgy. 2009 Coming Back to Gliadin During digestion, the gliadin component of gluten is broken down, yielding a hard to digest peptide. Shan et al., described a 33-mer peptide derived in vitro from exposure of α2-gliadin to digestive enzymes: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF PFPQPQLPY PQPQLPYPQ PYPQPQLPY 3 sequences shown by Shan et al. to stimulate T-cell clones limited to HLA DQ2. This peptide cross the intestinal barrier, where HLA molecules can present it as a possible antigen. 5
Gliadin in HLA Blue/Purple Cartoon Form α/β chains of DQ2 Spacefill LQPFPQPELPY Proline in Yellow, Glutamine in Green Kim et al. PNAS. Mar 23, 2004. The image was generated with Jmol: an opensource Java viewer for chemical structures in 3D. http://www.jmol.org/ Tissue Tranglutaminase (TTG) AKA Tranglutaminase 2 78 kda, Ca dependent Wide expression in a variety of tissues, including the small intesting Can regulate intracellular matrix, regulates programmed cell death and differentiation Tissue Transglutaminase Blue N-terminal domain Green Catalytic domain Red/Yellow C-terminal domains Liu et al. PNAS. 2002 The image was generated with Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/ Activities of ttg Cross-Linking Transamidation Deamidation Di Sabatino et al. Autoimmunity Reviews. 2012 6
Two Interactions of ttg and Gliadin Cross-Linking Gliadin peptides get crosslinked directly to tissue transglutaminase. This results in production of anti-ttg antibodies These antibodies suppress the physiologic functions of ttg Deamidation Deamidation of gliadin peptides (recall these are Gln rich) produces highly potent antigens T cell activation results in cytokine signaling, and immune/inflammatory response that drives villous atrophy Clinical Lab Testing for CD HLA Sequencing Symptoms or Family Serologies Histology/ Biopsy Serologies Past antibody tests include: Anti-Gliadin antibodies A series of autoimmune markers including antireticulin and antiendomysial antibodies Current antibody tests: Anti-TTG (sensitivity/ specificity > 95%) Anti-DGP (deamidated gliadin peptide) as reflext with neg anti-ttg + low IgA Antibody testing is IgA based IgG testing is called for when IgA is deficient (global or selective) or very young age (<2). IgA deficiency more prevalent in CD than in general population Most of this testing is ELISA based, Anti-EMA is an exception as an immunofluorescent test. Testing accuracy does depend on diet containing gluten. 7
Histology (Gold Standard) Ludvigsson et al. United European Gastroenterol J. 2015 Non-Celiac Gluten Sensitivity (NCGS) Recognized within the last decade, despite an initial report in 1980. Symptoms with gluten, symptoms abate on GFD, CD and allergies ruled out. Hypotheses regarding development of wheat strains, influence of genetic modification Symptoms generally limited to gastrointestinal, psychological (co-morbidities?) Non-IgE, limited IgA component, no villous atrophy, not autoimmune No biomarkers analysis limited to subjective criteria. Immunologic Basis Pair of articles from Sapone et al. Differential Mucosal IL-17 Expression Int Arch Allergy Immunol. 2010 7 dyspeptic controls, 13 CD, 11 GS patients EMA-IgA/tTG-IgA only present in CD, 92 vs 36% DQ2/ DQ8 phenotype Divergence of gut permeability and mucosal immune gene expression BMC Medicine. 2011 39 DC, 42 CD, 26 GS 8
Intestinal Lymphocytes CD3 Staining Small intestine biopsy Real Time PCR of intestinal biopsy More comparisons Intestinal Permeability Markers Cytokine Expression (PCR) More comparisons Toll-Like Receptors (PCR) Immune Regulatory Genes (PCR) 9
Nature of Immune Function in NCGS? Sapone et al. offer a scenario where NCGS is mediated by an inflammatory mechanism driven by innate, rather than adaptive immunity. Some reports with in vitro evidence of wheat proteins stimulating immune response Control (n =?) NCGS (n = 48) Valerii MC, et al. Food Chemistry. 2015 Biesiekierski vs Biesiekierski Two publications (2011, 2013) with intent on using a placebo controlled gluten challenge to demonstrate NCGS. 2011: Double-blinded, 34 patients (IBS pos, CD neg) Start on GFD, switch to 6 weeks rechallenge 19 on GFD plus 2 slices bread, 1 muffin/day 15 in placebo group 2013: Self-reported NCGS, double-blind, 37 patients 2-weeks reduced FODMAPs 1 week challenge (high gluten, low gluten, placebo) 3 day rechallenge (gluten, whey, placebo) 2011 Results Biesiekierski JR, et al. Gastroenterology. 2011 10
2013 Results Biesiekierski JR, et al. Gastroenterology. 2013 Summary Celiac Disease Well defined auto-immune condition driven by interaction of HLA, gliadin peptide, ttg Specific biomarkers and diagnostic criteria Villous atrophy, GI symptoms, nutrient malabsorption Gluten Sensitivity Defined chiefly by alleviation of symptoms after gluten free diet No biomarkers Inconsistent results comparing NCGS to placebo Adaptive immune response excluded, possible innate immune response with inflammation. A Final Thought Priven M, et al. J Acad Nutri Diet. 2015 11
References Biesiekierski JR, Newnham ED, Irving PM, Barrett JS, Haines M, Doecke JD, Shepherd SJ, Muir JG, Gibson PR. Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trial. American Journal of Gastroenterology. Mar 2011. 106(3): 508-514 Biesiekierski JR, Peters SL, Newnham ED, Rosella O, Muir JG, Gibson PR. No Effects of Gluten in Patients with Self-Reported Non-Celiac Gluten Sensitivity After Dietary Reduction of Fermentable, Poorly Absorbed, Short-Chain Carbohydrates. Gastroenterology. 2013. 145:320-328 Di Sabatino A, Vanoli A, Giuffrida P, Luinetti O, Solcia E, Corazza GR. The function of tissue transglutaminase in celiac disease. Autoimmunity Reviews. 2012. 11: 746-753 Kim CY, Quarsten H, Bergseng E, Kholsa C, Sollid L. Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease. PNAS. Mar 23, 2004. 101(12):4175-4179 Liu S, Cerione RA, Clardy J. Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity. PNAS. Mar 5, 2002. 99(5): 2743-2747 Ludvigsson JF, Card T, Ciclitira PJ, Swift GL, Nasr I, Sanders DS, Ciacci C. Support for patients with celiac disease: A literature review. United European Gastroenterology Journal. 2015. 3(2):146-159 Marsh MN. Gluten, Major Histocompatibility Complex, and the Small Intestine. Gastroenterology. 1992. 102:330-354 Megiorni F, Mora B, Bonamico M, Barbato M, Nenna R, Maiella G, Lulli P, Mazzilli MC. HLA-DQ and risk gradient for celiac disease. Human Immunology. 2009. 70:55-59 Gluten Free Foods US 2014. Mintel Market Research References Priven M, Baum J, Vieria E, Fung T, Herbold N. The Influence of a Factitious Free-Form Food Product Label on Consumer Perceptions of Healthfulness. Journal of the Academy of Nutrition and Dietetics. In Press 2015. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. ACG Clinical Guidelines: Diagnosis and Management of Celiac Disease. The American Journal of Gastroenterology. May 2013. 108:656-676 Sapone A, Lammers KM, Mazzarella G, Mikhailenko I, Carteni M, Casolaro V, Fasano A. Differential Mucosal IL-17 Expression in Two Gliadin-Induced Disorders: Gluten Senstivity and the Autoimmune Enteropathy Celiac Disease. International Archives of Allergy and Immunology. 2010. 152:75-80 Sapone A, Lammers KM, Casolaro V, Cammarota M, Giuliano MT, De Rosa M, Stefanile R, Mazzarella G, Tolone C, Russo MI, Esposito P, Ferraraccio F, Carteni M, Riegler G, de Magistris L, Fasano A. Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity. BMC Medicine. 2011. 9(23): 1-11 Shan L, Molberg O, Parrot I, Huasch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural Basis for Gluten Intolerance in Celiac Sprue. Science. Sep 27, 2002. 297:2275-2279 Valerii MC, Ricci C, Spisni E, Di Silvestro R, De Fazio L, Cavazza E, Lanzini A, Campieri M, Dalpiaz A, Pavan B, Volta U, Dinelli G. Reponses of peripheral blood mononucleated cells from non-celiac gluten sensitive patients to various cereal sources. Food Chemistry. 2015. 176:167-174 Volta U, Villanacci V. Celiac disease: Diagnostic criteria in progress. Cellular & Molecular Immunology. 2011 8:96-102 References Volta U, Caio G, Tovoli F, De Giorgio R. Non-celiac gluten sensitivity: questions still to be answered despite increasing awareness. Cellular & Molecular Immunology. 2013. 10:383-392 Weiser H. Chemistry of Gluten Proteins. Food Microbiology. 2007. 24:115-119 12