In this trial, a protracted intake of gluten from wheat flour treated with mTG and K-CH3 was associated to a reduced number of relapses in challenged GFD CD patients.
The possibility of preventing immune activity against gluten has been underscored by the finding that the digestive resistance of gliadin may play a role in the pathogenesis of CD [26
]. Gliadin can be cleaved by bacterial PEPs into short peptides that lose their immune activity [26
]. Accordingly, oral PEP therapy has been proposed as a possible treatment [10
]. Recently, protein engineering has been exploited to improve PEP activity [28
]. However, a very long fermentation is required to reduce the intolerance; therefore, it is still a challenge to prepare bread for CD patients [29
We studied the transamidation activity of food-grade mTG, an alternative enzymatic approach to detoxify gluten [30
]. Our previous experiments indicated that consequent to the transamidation with K-CH3
, but not with lysine, the immune reactivity of gliadin was completely suppressed in intestinal T-cell lines isolated from CD patients [15
It is widely accepted that both gliadins and glutenins are responsible for the toxicity in CD [31
]. Therefore, it was an important finding that both protein types are substrates for mTG. Moreover, the dough from transamidated flour rose similarly to the control dough, which gave us the opportunity to produce normal slices of the double-baked bread used in the trial. Patients received 50
g/day of bread, corresponding to a 3.7
g daily gluten intake. This quantity is considered sufficient to reinduce the disease; in previous studies, 1–5
g gluten/day caused CD relapse on a clinical, laboratory and histological level both in children and in adults [32
]. In a subsequent study, 50
mg gluten/day was considered the minimum dose required to produce measurable damage to the mucosa in CD patients [35
]. Notably, the gluten content, detectable by R5-ELISA, drastically dropped following transamidation. We interpreted this data to mean that the enzymatic reaction masked the epitopes of the gliadins. In agreement with these observations, we detected increased levels of lysine moieties bound to transamidated gliadins and glutenins. Given the considerations described, a single-blinded, randomized, controlled trial was performed to verify the safety of the enzyme treatment in treated CD patients. We applied the GSRS, a disease-specific instrument, developed to evaluate common symptoms of gastrointestinal disorders [16
]. We found that the dose was sufficient for producing a significant difference between the experimental and control groups in terms of clinical relapse after only 15 days. In fact, 37% and 75% of the experimental and control groups, respectively, exhibited clinical symptoms.
The intestinal permeability was investigated by the noninvasive C/M test [17
] because, in CD, there is a decreased absorption of small molecules (mannitol) and a paradoxically increased absorption of large molecules (cellobiose). The test was valuable for monitoring dietary lapses in patients on a GFD [36
]. Interestingly, although the C/M ratio was not significantly different between the experimental group and the control group after 15 days of treatment, the 90-day challenge showed that abnormal values essentially developed early, within 15 days, concomitantly with the timing of clinical relapse. In particular, only 2 patients in the experimental group dropped out later (), suggesting the existence of two distinct subsets of CD patients with different sensitivity to transamidated gluten. In line with these findings, we also registered unchanged haematological values after the 90-day challenge. On the other hand, these results indicated that our primary endpoint was only partially fulfilled.
The Q-K isopeptide, the final product of transamidation, is largely metabolized in the kidney, where ε
-glutamyl)-lysine represents a substrate for γ
-glutamylamine cyclotransferase (EC 126.96.36.199) [21
]. The cleavage of the isopeptide bond by this enzyme results in the formation of free L-lysine and 5-oxoproline, which is metabolized to glutamic acid by 5-oxoprolinase. However, 5-oxoprolinase is an ATP-dependent enzyme, and ingestion of transamidated proteins would increase the consumption of ATP in the kidney, thus, potentially influencing renal function. Accordingly, we examined creatinine clearance in the experimental group and found that the end values were not different from the baseline values. Also, no differences were observed in the normal subjects undergoing the same challenge.
No variation was reported for the anti-tTG titres after 90 days, which are a good noninvasive indicator to assess the compliance in CD patients [18
]. The morphometry and IEL counts of duodenal biopsies are considered hallmarks for a quantitative assessment of gluten-induced damage in CD [19
]. Therefore, we compared the baseline and end values in 10 consenting patients who completed the 90-day challenge. The statistical evaluation of the morphological data highlighted a difference only in the crypt depth. Notably, the overall number of IELs, a very sensitive signal of mucosal damage, was unchanged. Accordingly, no significant changes in the Marsh-Oberhuber grade occurred in the experimental group. Taken together, the serological and morphological results confirmed that transamidated gluten was tolerated in this subset of CD patients. Finally, the IFN-γ
mRNA levels in the intestinal biopsies at baseline and after the 90-day challenge did not show a significant difference, thus confirming our previous in vitro