Turning Swords Into Plowshares: Transglutaminase to Detoxify Gluten

Abstract

See “Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease” by Gianfrani C, Siciliano RA, Facchiano AM, et al, on page 780. Celiac disease (CD) is a small intestinal enteropathy caused by an inflammatory response to wheat gluten and similar proteins of barley and rye. Expression of HLA-DQ2 or HLA-DQ8 is the most important predisposing genetic factor because certain gluten peptides are preferentially bound to HLA-DQ2 or -DQ8 on antigen-presenting cells, such as macrophages, dendritic cells, or B cells. Thereby, proliferation of T cells in the lamina propria and production of proinflammatory cytokines, such as interferon-γ, is induced. The subsequent inflammation can lead to complete destruction of the microscopic resorptive villi, with consequent malabsorption and diarrhea. This classical malabsorptive presentation of CD has become relatively rare, with prevalences ranging from 1:2,000 to 1:10,000 in the West. However, the use of highly sensitive and specific serologic screening tests revealed that atypical (eg, affecting other organ systems) and oligosymptomatic (eg, showing iron deficiency) manifestations of CD or CD that lacks any symptoms are far more frequent (with prevalences between 1:75 and 1:200 in the United States, Europe, North Africa, and the Near and Middle East). These high prevalences make CD one of the most common inherited disorders.1Schuppan D. Current concepts of celiac disease pathogenesis.Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 2Ciclitira P.J. King A.L. Fraser J.S. AGA technical review on celiac sprue American Gastroenterological Association.Gastroenterology. 2001; 120: 1526-1540Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3Green P.H. Jabri B. Coeliac disease.Lancet. 2003; 362: 383-391Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 4Fasano A. Catassi C. Current approaches to diagnosis and treatment of celiac disease: an evolving spectrum.Gastroenterology. 2001; 120: 636-651Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 5Maki M. Mustalahti K. Kokkonen J. et al.Prevalence of celiac disease among children in Finland.N Engl J Med. 2003; 348: 2517-2524Crossref PubMed Scopus (834) Google Scholar Almost all patients with untreated CD develop autoantibodies to tissue transglutaminase (tTG).6Dieterich W. Ehnis T. Bauer M. et al.Identification of tissue transglutaminase as the autoantigen of celiac disease.Nat Med. 1997; 3: 797-801Crossref PubMed Scopus (1774) Google Scholar tTG is a ubiquitous enzyme stored intracellularly but released from cells, mainly fibroblasts and endothelial cells but also immune and epithelial cells, upon activation, such as during inflammation and wound healing.7Lorand L. Graham R.M. Transglutaminases: crosslinking enzymes with pleiotropic functions.Nat Rev Mol Cell Biol. 2003; 4: 140-156Crossref PubMed Scopus (1198) Google Scholar Being dependent on calcium, tTG can cross-link certain glutamine residues in proteins with primary amines such as the amino group of lysine.8Dieterich W. Esslinger B. Trapp D. et al.Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease.Gut. 2006; 55: 478-484Crossref PubMed Scopus (68) Google Scholar In the absence of such amines, the enzyme causes deamidation of the target glutamine residues to glutamic acid, which introduces a negative charge. Gluten, which is the storage protein of wheat, is a preferential target for tTG because it is extremely rich in glutamine residues. Gluten can be separated into the ethanol-insoluble glutenins, which are responsible for the desired baking properties of this cereal, and the alcohol-soluble gliadins. Each wheat variant produces an estimated 40–50 gliadins, structurally related proteins that contain approximately 250–500 amino acids, and a lower number of high- and low-molecular-weight glutenins, with approximately 650–800 and approximately 270–320 amino acids, respectively. Structurally related molecules are found in barley and rye, which have to be strictly avoided by patients with CD as well; storage proteins of corn and oats are more distantly related.9Wieser H. Chemistry of gluten proteins.Food Microbiol. 2007; 24: 115-119Crossref PubMed Scopus (1118) Google Scholar Only a subset of certain glutamines in the glutamine-rich gliadins (and to a lesser degree in the glutenins), preferentially near proline residues, are substrates for the highly selective tTG.6Dieterich W. Ehnis T. Bauer M. et al.Identification of tissue transglutaminase as the autoantigen of celiac disease.Nat Med. 1997; 3: 797-801Crossref PubMed Scopus (1774) Google Scholar, 10Schuppan D. Dieterich W. Ehnis T. et al.Identification of the autoantigen of celiac disease.Ann N Y Acad Sci. 1998; 859: 121-126Crossref PubMed Scopus (33) Google Scholar, 11Arentz-Hansen H. Korner R. Molberg O. et al.The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.J Exp Med. 2000; 191: 603-612Crossref PubMed Scopus (555) Google Scholar, 12Molberg O. McAdam S.N. Korner R. et al.Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.Nat Med. 1998; 4: 713-717Crossref PubMed Scopus (974) Google Scholar The negative charge that is introduced into the gluten peptides by tTG-mediated deamidation usually increases the binding affinity of the gluten peptides to HLA-DQ2 or HLA-DQ8 on antigen-presenting cells, creating strong T-cell stimulatory (immunodominant) gluten epitopes.12Molberg O. McAdam S.N. Korner R. et al.Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.Nat Med. 1998; 4: 713-717Crossref PubMed Scopus (974) Google Scholar, 13van de Wal Y. Kooy Y. van Veelen P. et al.Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity.J Immunol. 1998; 161: 1585-1588PubMed Google Scholar The current treatment of CD is a life-long adherence to a strict gluten-free diet. Because gluten is a common ingredient in the human diet and trace amounts of gluten can contaminate foods that are considered or labeled “gluten free,” strict adherence poses a big challenge for patients with CD, especially in young adults and those with only few symptoms.14Sollid L.M. Khosla C. Future therapeutic options for celiac disease.Nat Clin Pract Gastroenterol Hepatol. 2005; 2: 140-147Crossref PubMed Scopus (146) Google Scholar On the other hand, untreated or insufficiently treated CD poses a hazard because patients may have an increased risk of secondary autoimmune disorders and gastrointestinal or hematologic malignancies.15Logan R.F. Rifkind E.A. Turner I.D. et al.Mortality in celiac disease.Gastroenterology. 1989; 97: 265-271Abstract PubMed Google Scholar, 16Howdle P.D. Jalal P.K. Holmes G.K. et al.Primary small-bowel malignancy in the UK and its association with coeliac disease.QJM. 2003; 96: 345-353Crossref PubMed Scopus (162) Google Scholar, 17Ventura A. Magazzu G. Greco L. Duration of exposure to gluten and risk for autoimmune disorders in patients with celiac disease SIGEP Study Group for Autoimmune Disorders in Celiac Disease.Gastroenterology. 1999; 117: 297-303Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar Thus, there is an obvious need to eliminate detrimental gluten peptides from the celiac diet or to blunt their immune stimulatory effects. To be acceptable for treatment of a mainly not life-threatening disease for which a dietary alternative is available, these therapies have to be safe, effective, and affordable. The most advanced strategy is oral enzyme therapy exploiting the properties of oral prolyl endopeptidases (PEPs). PEPs are proteolytic enzymes that, in contrast to the human gastrointestinal proteases, are capable of cleaving the proline- and glutamine-rich immunostimulatory gluten peptides.18Hausch F. Shan L. Santiago N.A. et al.Intestinal digestive resistance of immunodominant gliadin peptides.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G996-G1003Crossref PubMed Scopus (297) Google Scholar A pilot clinical study using a PEP from Flavobacterium meningosepticum has demonstrated some clinical efficacy in ameliorating the development of steatorrhea of celiacs in remission who were challenged with 5–10 g of oral gluten (which equals one third to two thirds of a normal daily gluten load).19Pyle G.G. Paaso B. Anderson B.E. et al.Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue.Clin Gastroenterol Hepatol. 2005; 3: 687-694Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Questions remain regarding the efficacy of PEP therapy to degrade the immunodominant gliadin peptides. These include the limited PEP stability at low gastric pH and incomplete mixing with gastric contents, as well as the high enzyme concentrations necessary to achieve complete gluten peptide degradation before gluten reaches the duodenum.20Koning F. Vader W. Gluten peptides and celiac disease.Science. 2003; 299 (author reply 513–515): 513-515Crossref PubMed Scopus (10) Google Scholar, 21Matysiak-Budnik T. Candalh C. Cellier C. et al.Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease.Gastroenterology. 2005; 129: 786-796Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Cerf-Bensussan N. Matysiak-Budnik T. Cellier C. et al.Oral proteases: a new approach to managing coeliac disease.Gut. 2007; 56: 157-160Crossref PubMed Scopus (37) Google Scholar In addition, the intestinal epithelium of patients with CD facilitates a more efficient transepithelial transport of gluten peptides into the lamina propria (where T-cell activation mainly occurs) than that of normal controls, as determined in Ussing chamber studies,21Matysiak-Budnik T. Candalh C. Cellier C. et al.Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease.Gastroenterology. 2005; 129: 786-796Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar stressing the need for a highly efficient enzyme therapy that eliminates essentially every trace of toxic gluten. PEP from Aspergillus niger, which is stable at low pH, has a 60-fold higher enzymatic rate, and which is less costly to produce, is a promising alternative.23Stepniak D. Spaenij-Dekking L. Mitea C. et al.Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G621-G629Crossref PubMed Scopus (233) Google Scholar Another strategy exploits certain lactobacilli that can be added to sourdough for fermentation and are able to proteolyze the proline/glutamine-rich gluten peptides.24Di Cagno R. De Angelis M. Lavermicocca P. et al.Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance.Appl Environ Microbiol. 2002; 68: 623-633Crossref PubMed Scopus (237) Google Scholar A pilot challenge suggested that CD patients can tolerate a carefully prepared sourdough bread containing 30% wheat flour, but the 2 days of challenge in this study are far too short to draw conclusions.25Di Cagno R. De Angelis M. Auricchio S. et al.Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients.Appl Environ Microbiol. 2004; 70: 1088-1096Crossref PubMed Scopus (229) Google Scholar Similarly, proteases in germinating wheat can degrade toxic gluten, opening the possibility that flour based on germinating wheat, barley, or rye may be used to create cereal products for patients with CD.26Kiyosaki T. Matsumoto I. Asakura T. et al.Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins.FEBS J. 2007; 274: 1908-1917Crossref PubMed Scopus (44) Google Scholar Endoprotease (EP)-B2 has been identified as the responsible protease from germinating barley, and digestion of gluten in a mixture that contained EP-B2:PEP:gluten in a ratio of 3:1:75 for 1 hour at acidic pH completely abrogated its T-cell stimulatory capacity.27Siegel M. Bethune M.T. Gass J. et al.Rational design of combination enzyme therapy for celiac sprue.Chem Biol. 2006; 13: 649-658Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Despite their drawbacks, these enzymatic strategies appear more feasible than projects aimed at genetic modification of wheat via mutation of DNA that encodes toxic gluten sequences. Apart from the anticipated reluctance of the public to ingest genetically modified foods, it has been estimated that there are ≥50 immunodominant gluten epitopes, both in gliadins and glutenins, that would have to be eliminated.28Vader L.W. Stepniak D.T. Bunnik E.M. et al.Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.Gastroenterology. 2003; 125: 1105-1113Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar In this context, the elimination of certain glutenin epitopes could negatively impact on the desired baking properties. On the other hand, it is possible to select wheat variants that contain fewer of the immune-dominant T-cell epitopes and that could be used as a better starting material for detoxification.29Spaenij-Dekking L. Kooy-Winkelaar Y. van Veelen P. et al.Natural variation in toxicity of wheat: potential for selection of nontoxic varieties for celiac disease patients.Gastroenterology. 2005; 129: 797-806Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar How far an inhibitor of zonulin, which is implicated in the opening of intestinal epithelial tight junctions thus facilitating gluten influx into the lamina propria,30Watts T. Berti I. Sapone A. et al.Role of the intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-prone rats.Proc Natl Acad Sci U S A. 2005; 102: 2916-2921Crossref PubMed Scopus (241) Google Scholar can prevent gluten-mediated T-cell stimulation in CD is the subject of an ongoing multicenter clinical trial (see www.albatherapeutics.com). Other approaches are more remote from potential clinical application; despite a sound theoretical basis, proof of principle is missing, and the hurdles for even pilot clinical studies and approval by the Food and Drug Administration are higher than for enzyme therapy.31Schuppan D. Hahn E.G. Biomedicine Gluten and the gut-lessons for immune regulation.Science. 2002; 297: 2218-2220Crossref PubMed Scopus (62) Google Scholar Thus inhibitors of tTG could block deamidation and subsequent immunologic potentiation of gluten peptides.32Choi K. Siegel M. Piper J.L. et al.Chemistry and biology of dihydroisoxazole derivatives: selective inhibitors of human transglutaminase 2.Chem Biol. 2005; 12: 469-475Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Alternatively, inhibitors of HLA-DQ2 (DQ8) could prevent antigenic presentation of gluten peptides and lead to silencing of gluten-reactive T cells.33Kim C.Y. Quarsten H. Bergseng E. et al.Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease.Proc Natl Acad Sci U S A. 2004; 101: 4175-4179Crossref PubMed Scopus (360) Google Scholar Cytokine or anti-cytokine therapy, for example, using interleukin (IL)-10 or a blocking antibody to IL-15 could counteract the T helper type 1 (TH1)–dominated immune response in CD.34Salvati V.M. Mazzarella G. Gianfrani C. et al.Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa.Gut. 2005; 54: 46-53Crossref PubMed Scopus (116) Google Scholar, 35Villadsen L.S. Schuurman J. Beurskens F. et al.Resolution of psoriasis upon blockade of IL-15 biological activity in a xenograft mouse model.J Clin Invest. 2003; 112: 1571-1580Crossref PubMed Scopus (167) Google Scholar All these strategies are based on agents that would likely have to be given for long periods and may have side effects, which would be unacceptable for a disease that can usually be treated by diet and that is not life threatening. In this issue of Gastroenterology, Gianfrani et al36Gianfrani C. Siciliano R.A. Facchiano A.M. et al.Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease.Gastroenterology. 2007; 133: 780-789Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar demonstrate an interesting and novel approach to detoxifying gluten by exploiting the same substrate specificity of transglutaminase that leads to more potent immunostimulatory gluten peptides via deamidation. They incubated the potent immunostimulatory peptide α-gliadin p56-6837Anderson R.P. Degano P. Godkin A.J. et al.In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope.Nat Med. 2000; 6: 337-342Crossref PubMed Scopus (481) Google Scholar, 38Shan L. Molberg O. Parrot I. et al.Structural basis for gluten intolerance in celiac sprue.Science. 2002; 297: 2275-2279Crossref PubMed Scopus (1245) Google Scholar and a peptic–tryptic digest of gliadin that contains several immune-stimulatory peptides (PT-gliadin) with tTG and lysine or lysine methyl ester, which resulted in quantitative cross-link formation between these gliadin peptides and the terminal amino group of the lysines. The lysine-modified gliadin peptides, especially those modified with lysine methyl ester, inhibited IFN-γ production (a hallmark of a TH1-weighted immune response as observed in CD) by intestinal T-cell lines derived from HLA-DQ2 positive CD patients. The T-cell lines were otherwise strongly stimulated by tTG-deamidated p56-68 and PT-gliadin, and even by nondeamidated PT-gliadin. The modified peptides had lost their affinity to bind to HLA-DQ2 and computer simulation confirmed steric hindering of the binding of lysine-modified peptide p56-68 to HLA-DQ2, being in full agreement with the experimental data. Furthermore, the authors demonstrated that treatment of whole wheat flour with a low-molecular-weight (Mr 38,000) microbial transglutaminase (mTG) derived from Streptomyces moboraensis abolished the flour’s stimulatory effect on gluten-reactive T-cell lines derived from 12 patients with CD. mTG has a broader range of substrate specificity, a higher reaction rate, and does not depend on calcium.39Pasternack R. Dorsch S. Otterbach J.T. et al.Bacterial pro-transglutaminase from Streptoverticillium mobaraense—purification, characterisation and sequence of the zymogen.Eur J Biochem. 1998; 257: 570-576Crossref PubMed Scopus (126) Google Scholar, 40Yokoyama K. Nio N. Kikuchi Y. Properties and applications of microbial transglutaminase.Appl Microbiol Biotechnol. 2004; 64: 447-454Crossref PubMed Scopus (471) Google Scholar It is used by the food industry all over the world, as a dough improver, for restructuring meat, or for improving the texture of foods,40Yokoyama K. Nio N. Kikuchi Y. Properties and applications of microbial transglutaminase.Appl Microbiol Biotechnol. 2004; 64: 447-454Crossref PubMed Scopus (471) Google Scholar owing to its ability to gel concentrated solutions with proteins such as soybean proteins, milk proteins, beef, pork, chicken, and fish gelatin, and myosins.40Yokoyama K. Nio N. Kikuchi Y. Properties and applications of microbial transglutaminase.Appl Microbiol Biotechnol. 2004; 64: 447-454Crossref PubMed Scopus (471) Google Scholar Importantly, loaf volume and crumb texture of breads are positively influenced, especially for flours with weak gluten and poor baking properties, making pretreatment of flour with mTG attractive for patients with CD (Figure 1). The presented strategy to exploit the substrate specificity of tTG or mTG to inactivate the immunodominant antigenic epitopes in gluten proteins is a logical and highly targeted approach of cereal modification that could benefit the large population of patients with CD. It would only be effective in controlled settings, such as in places where these modified cereals and flours are used. In addition, it needs to be shown that these modifications retain the desired consistency and baking properties that patients with CD miss in gluten-free products. Moreover, several other questions have to be resolved, such as how reliable complete transamidation of gluten peptides can be achieved in flours by treatment with mTG and lysine methyl ester, because some patients react to trace amounts of gluten. Also, the practicality of large-scale industrial production and costs of these flours compared with a gluten-free diet are unclear. Furthermore, safety issues regarding the novel products, for example, the possible generation of novel antigenic epitopes and the nutritional value of the cross-linked peptides, need to be addressed, although as mentioned mTG has been used for many years to improve food quality.40Yokoyama K. Nio N. Kikuchi Y. Properties and applications of microbial transglutaminase.Appl Microbiol Biotechnol. 2004; 64: 447-454Crossref PubMed Scopus (471) Google Scholar Another possible downside is that the transamidated peptides did not compete with the native, nondeamidated gluten peptides for binding to HLA-DQ2. Although intestinal T-cell–mediated inflammation in adults appears to be mainly driven by deamidated peptides,41Vader W. Kooy Y. Van Veelen P. et al.The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides.Gastroenterology. 2002; 122: 1729-1737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar this is not the case in infants and young children whose T-cell repertoire is prominently directed against nondeamidated gluten and sequences that are not substrates for tTG.12Molberg O. McAdam S.N. Korner R. et al.Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.Nat Med. 1998; 4: 713-717Crossref PubMed Scopus (974) Google Scholar, 13van de Wal Y. Kooy Y. van Veelen P. et al.Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity.J Immunol. 1998; 161: 1585-1588PubMed Google Scholar, 28Vader L.W. Stepniak D.T. Bunnik E.M. et al.Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.Gastroenterology. 2003; 125: 1105-1113Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 38Shan L. Molberg O. Parrot I. et al.Structural basis for gluten intolerance in celiac sprue.Science. 2002; 297: 2275-2279Crossref PubMed Scopus (1245) Google Scholar Another uncertainty regards the yet ill-defined involvement of innate immunity in the pathogenesis of CD. The innate response is mainly triggered in macrophages, monocytes, natural killer cells, dendritic cells, and intestinal epithelial cells in response to gluten peptides that are different from those peptides that drive adaptive immunity (which involves HLA-DQ2/DQ8 and antigen-specific T cells).42Maiuri L. Ciacci C. Ricciardelli I. et al.Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar Innate immunity appears to be independent of gluten deamidation, and even a near-complete abrogation of adaptive immunity by gluten transamidation may not be sufficient to control CD. Final answers to these questions can only be obtained from long-term clinical studies with sizable numbers of patients and sensitive readouts, such as cytokine expression measured in intestinal biopsies before and after challenge. Development of alternative diets and treatments for CD would be speeded up if a rodent animal model of CD was at hand. However, such a model is still not available. Considering the problems associated with a life-long gluten-free diet, the availability of “nontoxic” bread and other cereal products would already mean an enormous improvement of life quality for CD patients. Nonetheless the social problems at public events (dinner parties, receptions, or invitations) would remain. Therefore, apart from the availability of cereal products that have been modified preingestion, a drug that can neutralize unaltered gluten upon ingestion remains an important goal of future translational research in CD. Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac DiseaseGastroenterologyVol. 133Issue 3PreviewBackground & Aims: Celiac disease is characterized by activation of HLA-DQ2/DQ8–restricted intestinal gluten-specific CD4+ T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Methods: Enzyme modifications of the immunodominant α-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Full-Text PDF