Celiac disease (CD) (gluten-sensitive enteropathy) is a chronic gastrointestinal tract disorder in which ingestion of gluten, the protein network present in food products made from wheat, rye, and barley, and their cross-related varieties, leads to damage of the small intestinal mucosa by an autoimmune mechanism in genetically susceptible individuals (1). The gluten network is formed while mixing flour and water by interactions between gliadins and glutenins. Gluten is an unusual protein because it is consumed in relatively large amounts, is partially resistant to luminal digestion by gastric, pancreatic, and brush border enzymes in the human small intestine, and when absorbed, it is susceptible to posttranslational modification (deamidation) by mucosal transglutaminase (1). Proline mainly imparts resistance to digestion because many proteases are unable to cleave peptide bonds located at N- and C-termini of proline (2,3). The variability of the gluten network is extremely large; public databases such as GenBank include more than 200 entries for genes encoding gluten proteins in Triticum aestivum.
Epidemiology of CD is increasing (∼1% of Europeans and North Americans) (1), as well as the prevalence of CD in elderly people (4). During the last decades, cereal food technology changed extensively to modify the dietary habits of entire populations, previously naïve to massive gluten exposure. Cereal baked goods are manufactured by fast processes in which long-time fermentation by sourdough, a cocktail of acidifying and proteolytic lactic acid bacteria, has been almost completely replaced by the use of chemical and/or baker's yeast leavening agents. Under these technological conditions, cereal components (eg, proteins) are not degraded during manufacture (5). The gluten-free diet (GFD) is effective and safe, and at present, it is the only available treatment for CD. Nevertheless, development and strict adherence to GFD are also complicated by formidable economical, cultural, and distribution difficulties in developing countries (6). Despite the proven benefits of the GFD, it may be exceedingly difficult to completely avoid gluten-containing foods, and effective adherence to the GFD is estimated to be only 45% to 80% (7).
Beyond other therapeutic options, oral supplementation with microbial oligopeptidases is proposed as an alternative to the GFD (8–13). In the last decade, several studies (14–19) were carried out in the author's laboratory aimed at showing the capacity of proteolytic enzymes, mainly peptidases, of selected sourdough lactobacilli to degrade gluten during food processing. More recently, it was shown that selected sourdough lactobacilli, in combination with fungal proteases, decreased the residual concentration of gluten (T aestivum and Triticum durum flours) to <10 ppm during sourdough fermentation (13,19).
This study was aimed at showing the safety, for young patients with CD, of sweet baked goods made of wheat flour that were rendered gluten-free during sourdough fermentation. Preliminarily, the hydrolyzed wheat flour was characterized through immunological and ex vivo analyses. Furthermore, sweet baked goods, containing the equivalent of ∼10 g of native gluten, were administered daily to 8 patients with CD for 60 days following a proof-of-concept open study. Hematology, serology, and intestinal permeability analyses were carried out.
MATERIALS AND METHODS
Microorganisms and Enzymes
Lactobacillus sanfranciscensis 7A, LS3, LS10, LS19, LS23, LS38, and LS47, Lactobacillus alimentarius 15 mol/L, Lactobacillus brevis 14G, and Lactobacillus hilgardii 51B were selected based on their peptidase activities, with particular reference to degradation of Pro-rich peptides (14,20). Strains were propagated for 24 hours at 30°C in modified MRS broth (Oxoid, Basingstoke, UK), with the addition of fresh yeast extract (5%, v/v) and 28 mmol/L maltose at a final pH of 5.6 (modified MRS). When used for sourdough fermentation, cells of lactobacilli were cultivated until the late exponential phase of growth was reached (∼12 hours). Fungal proteases from Aspergillus oryzae (500,000 hemoglobin units on the tyrosine basis per gram) and Aspergillus niger (3000 spectrophotometric acid protease units/g), routinely used as improvers in the bakery industry, were purchased from BIO-CAT Inc. (Troy, VA).
Hydrolysis of Gluten During Sourdough Fermentation and Making Sweet Baked Goods
The main characteristics of the wheat (T aestivum cv Appulo) flour used were as follows: moisture, 12.8%; protein, 10.3% of dry matter (d.m.); fat, 1.8% of d.m.; ash, 0.6% of d.m.; and total carbohydrates, 74.5% of d.m. Wheat flour and tap water (ratio 1:4), containing ca 1 × 109 CFU/g of lactobacilli cells and 200 ppm of both fungal proteases, were used for sourdough fermentation at 37°C for 48 hours under stirring conditions (∼200 rpm). After fermentation, wheat sourdough was subjected to spray drying to remove water, and the resulting hydrolyzed flour was further milled and used. The spray drying method allows to obtain dry powder from liquid or semiliquid matrices by rapidly drying with a hot gas, and it is commonly applied to thermally sensitive materials (eg, foods, pharmaceuticals).
The following formula was used for making sweet baked goods at the pilot plant of the Department of Plant Protection and Applied Microbiology: fermented wheat flour (50%, w/w), sucrose (11.3%, w/w), eggs (8.8%, w/w), pasteurized yolk (1.2%, w/w), egg whites (13%, w/w), butter (7.3%, w/w), milk cream (7.3%, w/w), sodium bicarbonate (1%, w/w), vanilla bean, and lemon peel as flavoring agents. The dough was whipped with a continuous high-speed bakery mixer (dough mixing time, 5 minutes) (Chopin and Co, Boulogne, Sur-Seine, France), and sweet baked goods (weight of ∼100 g) were baked at 160°C for 40 minutes (Combo 3, Zucchelli, Verona, Italy).
Immunological Analyses and Characterization of Wheat Flour
Immunological analyses were carried out by using R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assays (ELISAs) and R5 antibody-based Western blotting. The R5 monoclonal antibody and the horseradish peroxidase-conjugated R5 antibody were used for gluten analysis. The R5-based sandwich ELISA was carried out with the Transia plate detection kit following the instructions of the manufacturer (Diffchamb, Västra, Frölunda, Sweden). The R5-based competitive ELISA was carried out at the gluten unit of the Centro National de Biotecnologia (Madrid, Spain). For R5-based Western blot analysis, after 1-dimensional SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated directly with R5-horseradish peroxidase antibody, and the blots were developed by immunodetection with ECL Western blotting analysis system (Amersham Pharmacia, Uppsala, Sweden).
Proteins were selectively extracted from flour according to the method of Weiss et al (21). Two-dimensional electrophoresis of proteins extracted from various fractions was carried out with the immobiline-polyacrylamide system (14). Gels were silver stained, and spot intensities were normalized as reported by Bini et al (22). Multidimensional HPLC (MDLC) coupled with electrospray ionization (ESI)-ion trap mass spectrometry (MS) was used to detect epitopes (13). The protein concentration of the flours used for making baked goods was determined by the Bradford method (23). The organic nitrogen concentration was determined using the Kjeldahl method. Free amino acids were analyzed by a series 30 Amino Acid Analyzer (Biochrom Ltd, Cambridge Science Park, UK).
Ex Vivo Assays
Gliadins and glutenins, extracted from fermented and nonfermented wheat flour, were subjected to sequential pepsin-trypsin (PT) hydrolysis to simulate the in vivo digestion (18). After digestion, PT digests were heated at 100°C for 30 minutes to inactivate enzymes and freeze-dried for further analysis.
Duodenal biopsies were obtained from 10 CD patients on a GFD (age range 8 to 17 years). All of the patients with CD expressed the HLA-DQ2 phenotype. CD was diagnosed according to European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) criteria (24). Immediately after excision, all of the biopsies were placed into RPMI-1640 (Gibco-Invitrogen, Paisley, UK) ice-chilled culture medium, which is used largely for cultivating mammalian cells and/or for handling and transport of biopsies (30–60 minutes) (13,25). Within 30 minutes, duodenal biopsies were cultured for 4 hours using the organ culture method originally described by Browing and Trier (26). Biopsies from each patient with CD were cultured with PT digest (5 mg/mL) of gliadin/glutenins from fermented or nonfermented wheat flour or with medium alone (negative control).
Tissue samples were taken and kept in RNAlater (Qiagen GmbH, Hilden, Germany) to preserve RNA. Total RNA was extracted from tissue using the Rneasy Mini Kit (Qiagen GmbH) according to the manufacturer's instructions. Concentration of mRNA was estimated by ultraviolet absorbance at 260 nm. Aliquots of total RNA (500 ng) were reverse transcribed using random hexamers and the TaqMan Reverse Transcription Reagents (Applied Biosystems, Monza, Italy) with 3.125 U/μL of MultiScribe Reverse Transcriptase in a final volume of 50 μL. cDNA samples were stored at −20°C.
Quantitative real-time polymerase chain reaction (RT-PCR) for interferon (IFN)-γ gene was carried out in 96-well plates on the ABI Prism 7500HT Fast Sequence Detection System (Applied Biosystems). TaqMan Gene Expression Assay for IFN-γ gene and Pre-Developed TaqMan Assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, endogenous control) gene (Applied Biosystems) with the provided primers (Appled Biosystems) were used. The assays were carried out using 20× mix of PCR primers and TaqMan Minor Groove Binder 6-FAM dye labeled probes with a nonfluorescent quencher at the 3′ end of the probe as described by De Angelis et al (13). Six serial dilutions (20–0.1 ng/μL) of IFN-γ cDNA were used as a template for each primer/probe set. The standard curve was generated by plotting the threshold cycle (CT) values against the log of the amount of cDNA. The average value of the target gene was normalized using the GAPDH gene. A normal duodenal biopsy was used as calibrator in all of the experiments. IFN-γ proteins secreted in the supernatant were quantified by ELISA in 96-well, round-bottomed plates (Tema Ricerca, Milan, Italy), according to the manufacturers' recommendations (13).
Protocol of the Clinical Challenge
Eight patients with CD (age median 13, range 8–17 years) were admitted to the study. Because 2 patients interrupted the clinical challenge (see below), the age median of individuals who followed the trial was 11 years. CD was diagnosed according to ESPGHAN (24). At recruitment all of the patients with CD showed normal values for total serum IgA (0.34–3.48 g/L) (Fig. 1A). All of the patients were on a GFD for at least 3 years and did not report any other associated diseases. The protocol for recruitment of patients with CD and the clinical challenge were approved by the University Hospital Umberto I, Sapienza University of Rome, Italy. The parents of patients qualified for the study gave their informed consent.
All of the enrolled patients with CD were asked to maintain a strict GFD during challenge; the only permitted gluten-containing dietary products were those provided in the study. During the first assessment, volunteers underwent clinical and nutritional evaluation. All patients with CD consumed daily ∼200 g of sweet baked goods, corresponding to 100 g of processed wheat flour, which contained ∼10 g of hydrolyzed gluten. The challenge lasted 60 days.
Blood samples were collected as venous blood in evacuated tubes containing K3EDTA (Becton-Dickinson, Rutherford, NJ) and were analyzed within 4 hours. The following parameters were considered: WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW-SD, RBC distribution width standard deviation; PLT, platelet count; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit; NEUT, neutrophils; LYMPH, lymphocytes; MONO, monocytes; EO, eosinophils; BASO, basophils; LUC, large unstained cells; VES, red blood cell sedimentation rate; PCR, C-reactive protein; glucose, creatinine; total proteins; albumins; α-amylase; lipases; AST/GOT, aspartate aminotransferase/glutamic-oxaloacetic transaminase; ALT/GPT; alanine aminotransferase/glutamic-pyruvic transaminase; γ-GT, γ-glutamyl-transferase; total bilirubin; direct bilirubin; ALP, alkaline phosphatase; total cholesterol; HDL cholesterol, cholesterol high-density lipoprotein; LDL cholesterol, cholesterol low-density lipoprotein; triglycerides; ferritin; TSH, thyroid-stimulating hormone; FT4, free thyroxine; FT3, free triiodothyronine; anti-TPO, anti-thyroid peroxidase antibodies; and anti-TG, anti-thyroglobulin antibodies.
Serum samples were kept frozen at 30°C until the assays were carried out. Quantitative determination of total serum IgA was carried out using the UniCAP System (Pharmacia & Upjohn Diagnostics, Uppsala, Sweden). IgG and IgA AGA (anti-gluten antibodies) were determined by commercially available ELISA kits (Eurospital, Trieste, Italy). The cutoff values provided by the manufacturer were 15.0 and 50.0 UA/mL for IgA and IgG AGA, respectively (Fig. 1B and C). Endomysial antibodies (EMA) were determined by an indirect immunofluorescence assay on sections of monkey esophagus (Eurospital). Tissue transglutaminase (tTG) IgA antibodies were measured by commercially available ELISA kits (Eurospital). The cutoff value provided by the manufacturer was 16.0 UA/mL (Fig. 1D).
Enrolled patients with CD were hospitalized at the University Hospital Umberto I, La Sapienza University of Rome, Italy, and followed a lactulose- and mannitol-free diet 24 hours before intestinal permeability assay. After an overnight fast, patients with CD voided a urine sample and 1 mL of chlorhexidine 20% (wt/vol) was added to inhibit bacterial growth. Further more, they drank 120 mL of deionized water containing 5.0 g of lactulose and 1.0 g of mannitol. One hour after the test was started, patients were encouraged to drink up to 150 mL of tap water to enhance diuresis. Urine was collected during the next 6 hours following the solution administration. Also in this case, 1 mL of chlorhexidine 20% (wt/vol) was added to samples. The total volume of collected urine was measured, and a 10-mL aliquot was stored at −20°C until analysis. Carbohydrates were determined by HPLC according to the method previously described by Lostia et al (27,28). The fractional excretion of lactulose was calculated from the ratio milligrams of lactulose excreted/milligrams of lactulose assumed. The milligrams of lactulose excreted were obtained from milligrams per liter lactulose per liters of urine. The same occurred with mannitol. The values of lactulose and mannitol from samples before administration were subtracted from those of samples collected for 6 hours. Results were expressed as the ratio of the fractional excretion of lactulose to the fractional excretion of mannitol (L/M ratio) (27,28). As reported for healthy children or patients with CD under strict GFD (pediatric age) (27–29), values of L/M ratio in the range 0.00 to 0.03 were considered to be normal.
Hydrolysis of Gluten During Sourdough Fermentation
The native concentration of gluten of the wheat (T aestivum cv Appulo) flour was ∼103,127 ± 123 ppm. After sourdough fermentation by selected sourdough lactobacilli and fungal proteases, the concentration of residual gluten was <10 ppm. This was shown by sandwich and competitive ELISA analyses. The absence of gluten was confirmed by R5 antibody-based Western blotting (data not shown). Two-dimensional electrophoresis of the gliadin fraction did not reveal the presence of protein spots in the fermented wheat flour (data not shown). Only a few spots were detectable for the glutenin fraction (data not shown). No gliadin/glutenin epitopes were found at the lowest detectable concentration (20 ppm) by nano-ESI-MS/MS analysis (data not shown). The lowest detectable concentration was estimated by injecting different concentrations (1–100 ppm) of the synthetic epitope QLQPFPQPQLPY together with the fermented wheat flour (19).
After fermentation, wheat flour was subjected to spray drying. Compared to nonfermented wheat flour (Table 1), the water-/salt-soluble fraction increased from 16.1% to 87.5% of the total protein fractions (total organic nitrogen content of 1.80% that approximately corresponded [N × 5.70] to the protein concentration of 10.26%). This fraction consisted of low-molecular-mass peptides and free amino acids mainly derived from the degradation of all wheat proteins. Indeed, no organic nitrogen was detectable in the gliadin fraction of fermented wheat flour, and the level of glutenin fraction decreased from 47% to 12.5%. The concentration of total free amino acids confirmed these differences (∼1032 ± 32 versus 15,320 ± 102 mg/kg for nonfermented and fermented wheat flour, respectively) (P < 0.01).
Cytokine Expression in Duodenal Biopsies of Patients With CD
Gliadins and glutenins were extracted from fermented and nonfermented wheat flour, subjected to PT hydrolysis, pooled, and used to treat duodenal biopsies from patients with CD. Compared to the negative control, the exposure of biopsies to PT digest from nonfermented wheat flour resulted in a significant (P < 0.05) increase of the production of IFN-γ mRNA (Fig. 2A and B). The release of IFN-γ mRNA by biopsies treated with the PT digest of fermented wheat flour did not significantly (P > 0.05) differ from that of the negative control (medium alone) (Fig. 2A and C). Increased levels of IFN-γ protein were found in the culture supernatant of all biopsies. A good agreement was found between proteins and level of mRNA. The highest synthesis of IFN-γ proteins was found in biopsies treated with PT digest from nonfermented wheat flour (data not shown).
Eight patients with CD underwent a clinical challenge daily consuming 200 g of sweet baked goods, which contained the equivalent of ∼10 g of native gluten. At recruitment all of the patients had normal values of hematology and serology parameters. Due to difficulties in the compliance of the daily consumption, 1 patient interrupted the trial after 15 days and another after 30 days of challenge. The other 6 patients reached the endpoint of 60 days. None of the 8 patients with CD had clinical complaints during the challenge. All hematology parameters were within the normal values at recruitment. For 60 days, none of the 42 hematology parameters (see Materials and Methods) varied with respect to the initial values.
At recruitment all of the patients with CD showed normal values for total serum IgA (0.34–3.48 g/L) (Fig. 1A). IgG- and IgA-AGA and IgA-tTG antibodies of all of the patients with CD were within normal values at all times (Fig. 1B–D). The data distribution was highly homogeneous; no outliers were found for any serology parameters over time. In particular, the values for IgA-AGA and IgA-tTG antibodies were <6.0 and 8.0 UA/mL, respectively. Response for EMA antibodies was always negative for all of the patients at all times. When levels of gluten (0.2–4.3 g), markedly lower than those of this study, were administered daily to numerous children with CD for 60 days, abnormal values of IgG- and IgA-AGA, IgA-tTG, and EMA antibodies were found in 90% of individuals (10,30–32).
The intestinal permeability was determined at recruitment and during clinical challenge up to 60 days. Daily consumption of 200 g of sweet baked goods made with fermented wheat flour (corresponding to ∼10 g of native gluten) did not cause variation of the intestinal permeability of all of the patients with CD for all times (Fig. 3). All L/M ratios were <0.03. The median value decreased over time. Several reports (33,34) showed abnormal values of intestinal permeability in patients with CD already after a single intake of gluten (∼30–50 g).
Bacterial-derived endopeptidases are recommended as a supplement in the diet to abolish the antigenicity of the 33-mer and other Pro-rich putative immunotoxic epitopes (11,19,35). Problems with the stability of endopeptidases in an acidic gastric environment and efficient mixing with gluten are frequently associated with the oral supplementation (36). Besides, administration of peptidases with the meal did actually enhance the immunoresponse, by exposing more rapidly the small intestinal mucosa to immunopeptides from fragmented gluten proteins (36). The use of microbial peptidases during food processing is not associated with these problems and guarantees the easiest and noninvasive tool to eliminate, in a dose-dependent manner, the toxicity of gliadins and glutenins from wheat (37). This study showed that the combination of fungal proteases, routinely used as bakery improvers, and lactobacilli peptidases completely degrades gluten (<10 ppm) during sourdough fermentation of wheat flour. Fungal proteases are indispensable to generate polypeptides of intermediate dimensions (eg, 4–40 amino acids) from native proteins that are suddenly transported inside the lactobacilli cells to be hydrolyzed through the complex peptidase system of lactobacilli (19). A large number of intracellular peptidases (eg, PepN, PepO, PEP, PepX, PepT, PepV, PepQ, PepR) are needed to hydrolyze the 33-mer or other synthetic immunogenic polypeptides to free amino acids (13). After hydrolysis, the wheat flour was spray dried and used for making sweet baked goods. Bread, pasta, or sweet baked goods may be manufactured with the wheat flour rendered gluten-free. From a technological point of view, the use of this spray-dried flour has the same technical problems as other naturally gluten-free raw materials (13,16,19). On the contrary, the use of the wheat flour rendered gluten-free has indubitably economical, nutritional, social, and sensory advantages compared with the naturally gluten-free counterpart used in the GFD. As shown by sensory analysis, the properties of sourdough baked goods manufactured with wheat flour rendered gluten-free were almost comparable to those of the full-gluten counterpart; these goods were highly superior to GF baked goods (19).
After hydrolysis, some glutenin polypeptides persisted in the wheat-fermented flour. Glutenins may contain sequences that activate T cells from the small intestine and result in the secretion of large amounts of IFN-γ (12). Based on these findings, the diagnostic value of R5 antibody to detect toxic epitopes may be in part limited. Ex vivo assays based on organ culture of treated intestinal biopsies from 10 patients with CD were used to confirm the absence of toxicity (38). As expected, the PT digest from nonfermented wheat flour caused a marked increase in IFN-γ. IFN-γ coordinates many aspects of the innate and adaptive immune responses, and it is the principal cytokine produced by α-β CD4+ reactive T cells upon gluten activation (12,39). The PT digests of fermented wheat flour induced IFN-γ as the negative control.
Based on the above results, a clinical challenge was started daily administering 200 g of sweet baked goods made with wheat flour rendered gluten-free (corresponding to ∼10 g of native gluten). Standardized gluten challenges based on the daily intake of 10 to 20 g were frequently considered, independently of the body weight of enrolled patients with CD (32). The daily intake of 10 to 20 g of gluten was shown to shorten the time of challenge and minimize discomfort (32). Eight patients with CD in remission were enrolled, and hematology, serology, and intestinal permeability analyses were carried out. Even though intestinal biopsy showing the typical picture of flat mucosa is still considered the criterion standard for CD diagnosis, the importance of serology is growing, thanks to reliable tests that were shown to be highly predictive for this condition (37). All of the most complementary serology analyses were carried out in this study. For ethical reasons, no positive controls (patients with CD fed with native gluten) were included in this proof-of-concept open study. Nevertheless, several reports showed that pathological titers of celiac antibodies were detectable within 2 to 7 weeks after reintroduction of gluten in patients with CD (10,30,31,40). Laurin et al (32) demonstrated that levels of gluten (1.7 g/day) caused the deterioration of the intestinal mucosa, and especially the appearance of abnormal values of celiac antibodies. All of the patients with CD enrolled in this study showed normal values of hematology and serology during 60 days of clinical challenge.
The measurement of the intestinal permeability following an oral dose administration of usually nonabsorbable versus absorbable sugars is the most widely accepted method for monitoring the small intestinal mucosal (tight junctions) leakiness, as in Crohn disease and CD (10,28,41). Even after a single-dose gluten challenge, remarkable abnormal values of the L/M ratio were found in patients with CD under GFD (33,34). All of the patients with CD who daily consumed 200 g of sweet baked goods made with fermented wheat flour showed values of L/M ratio that were ∼0.03. Overall, this value is considered to be the threshold for normal intestinal permeability in clinical practice (28,29,33).
The findings of this study provide the rationale for exploring tools that could reduce the toxicity of gluten for patients with CD beyond the standard GFD. This is the first time that a wheat flour–derived product has been shown to be nontoxic after administration for 60 days to patients with CD; nevertheless, these foods should not be recommended for patients with CD until a formal trial has been done. There is no doubt that a period of 60 days, although repeatedly shown to be sufficient to evaluate gluten toxicity in the majority of patients, may not be long enough to evaluate toxicity in all patients with CD who occasionally may show different sensitivity to gluten. Prolonged trials must be planned to state the safety of the baked goods manufactured by applying this promising and rediscovered biotechnology.
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