Reintroduction of Gluten Following Flour Transamidation in Adult Celiac Patients: A Randomized, Controlled Clinical Study

Reintroduction of Gluten Following Flour Transamidation in Adult Celiac Patients: A Randomized, Controlled Clinical Study Giuseppe Mazzarella,1 Virginia M. Salvati,2 Gaetano Iaquinto,3 Rosita Stefanile,1 Federica Capobianco,4 Diomira Luongo,1 Paolo Bergamo,1 Francesco Maurano,1 Nicola Giardullo,3 Basilio Malamisura,2 and Mauro Rossi1 1Institute of Food Sciences, CNR, 83100 Avellino, Italy 2Center for Coeliac Disease S. Maria dell'Olmo Hospital, 84013 Cava de' Tirreni Salerno, Italy 3Gastroenterology Department, San G. Moscati Hospital, 83100 Avellino, Italy 4IPALC Research & Development, 83040 Frigento, AV, Italy Received 10 April 2012; Accepted 28 May 2012 Academic Editor: Francisco J. Pérez-Cano Copyright © 2012 Giuseppe Mazzarella et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract A lifelong gluten-free diet (GFD) is mandatory for celiac disease (CD) but has poor compliance, justifying novel strategies. We found that wheat flour transamidation inhibited IFN-γ secretion by intestinal T cells from CD patients. Herein, the primary endpoint was to evaluate the ability of transamidated gluten to maintain GFD CD patients in clinical remission. Secondary endpoints were efficacy in prevention of the inflammatory response and safety at the kidney level, where reaction products are metabolized. In a randomized single blinded, controlled 90-day trial, 47 GFD CD patients received 3.7 g/day of gluten from nontransamidated (12) or transamidated (35) flour. On day 15, 75% and 37% of patients in the control and experimental groups, respectively, showed clinical relapse ( ) whereas intestinal permeability was mainly altered in the control group (50% versus 20%, ). On day 90, 0 controls and 14 patients in the experimental group completed the challenge with no variation of antitransglutaminase IgA ( ), Marsh-Oberhuber grading ( ), or intestinal IFN-γ mRNA ( ). Creatinine clearance did not vary after 90 days of treatment ( ). In conclusion, transamidated gluten reduced the number of clinical relapses in challenged patients with no changes of baseline values for serological/mucosal CD markers and an unaltered kidney function. 1. Introduction Celiac disease (CD) is caused by the ingestion of wheat gluten and related prolamins in genetically predisposed subjects [1], influencing 1% of population in developed countries [2, 3]. Currently, a lifelong gluten-free diet (GFD) is mandatory to alleviate the symptoms of CD and to normalize the antibodies in the intestinal mucosa [4]. Recovery is often observed after only a few weeks on a GFD [5]. CD is mainly characterized by the activation of intestinal gluten-specific CD4+ T cells [6]. In particular, gluten becomes a better antigen following its deamidation, which is catalyzed by tissue transglutaminase (tTG) [7]. Furthermore, proline residues protect against digestive proteolysis and direct tTG-mediated deamidation of glutamines [8]. A noteworthy finding was that gliadin can be cleaved by bacterial prolyl endopeptidases (PEPs) into short peptides that lose their activity [9]. Accordingly, oral PEP therapy has been proposed as a possible treatment [10, 11]. PEPs have also been evaluated as a technological tool for the preparation of detoxified gluten. Selected sourdough lactobacilli have specialized PEPs, and baked products from sourdough following 24 h of fermentation did not produce any alteration in intestinal permeability in 13 out of 17 patients [12]. A 60-day diet of baked goods made from hydrolyzed wheat flour, which was manufactured with sourdough lactobacilli and fungal proteases, was not toxic to patients with CD [13]. We tested a different enzymatic approach: the transamidation activity of food-grade microbial transglutaminase (mTG), a transamidase of the endo- -glutamine: -lysine transferase type [14]. We recently found that mTG exhibited the same site specificity as tTG but lacked the deamidase activity [15]. Most importantly, the transamidation of gliadin by treatment of wheat flour with mTG and lysine methyl ester (K-CH3) caused a dramatic downregulation of IFN- production in vitro in the intestinal T cells of CD patients [15]. In this first clinical study, we examined the safety of (K-CH3)-transamidated wheat flour in CD patients. The primary outcome measures included the appearance of clinical symptoms, by applying the gastrointestinal symptoms rating scale (GSRS) [16], and an altered intestinal permeability [17]. The second outcome was to evaluate the tolerance of treated flour by analyzing serum IgA antitissue transglutaminase (tTG) antibodies [18], changes in the Marsh degree of intestinal biopsies [19], and intestinal IFN- mRNA after 90 days [20]. In addition, by considering that the Q-K isopeptide, the final product of transamidation is largely metabolized in the kidney [21], we also determined creatinine clearance to monitor the integrity of renal function. 2. Materials and Methods2.1. Patients and Study Design The study was a randomized, controlled clinical trial. 65 asymptomatic celiac patients on a GFD for almost two years were enrolled at two investigator sites: S. Maria Incoronata dell’Olmo Hospital, Cava de’ Tirreni-SA, and the Gastroenterology Department of S. G. Moscati Hospital, Avellino. Nine of them declined participation after elucidation of the protocol study; another nine patients were excluded because of an antiendomysial antibodies (EMA)-positive test. Finally, 47 CD patients were enrolled for a 90-day trial. Their demographic characteristics, symptoms at diagnosis, and baseline laboratory data are reported in Table 1. Patients were randomized to receive 50 g/day of twice-baked bread slices produced from either nontransamidated wheat flour containing K-CH3 (control group, n.12) or (K-CH3)-transamidated wheat flour (experimental group, n.35). A simple randomization scheme was generated using a web site resource (http://www.randomization.com/). Patients were monitored for the appearance of clinical symptoms by using the gastrointestinal symptoms rating scale (GSRS) [16] and intestinal permeability [17] every 15 days throughout the study. On day 90, upper-gastrointestinal endoscopy and duodenal biopsies were performed to evaluate alterations. Figure 1 is a flow chart of the study. Table 1: Demographic data, clinical symptoms (GSRS) at diagnosis, and baseline laboratory investigations in 47 GFD CD patients. Figure 1: Flow chart of the study. Patients with appearance of clinical symptoms related to gluten exposure or altered intestinal permeability were considered for withdrawn at any time. The following outcome measures were also considered for relapsed CD: changes in serum anti-tTG IgA antibodies [18], intestinal IFN- mRNA [20], and changes in the Marsh degree of examined intestinal biopsies [19]. (...truncated)