The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

OBJECTIVES: Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. METHODS: Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. RESULTS: We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. CONCLUSIONS: Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Fecal calprotectin levels in patients with non-celiac wheat sensitivity: a proof of concept.

Some data suggest the existence of intestinal inflammation in patients with non-celiac wheat sensitivity (NCWS). We aimed to verify whether fecal calprotectin (FCP), a marker of intestinal inflammation, could be used to confirm this inflammatory status and to test its diagnostic performance in differentiating NCWS from irritable bowel syndrome/functional dyspepsia (IBS/FD). We conducted a multicenter study, comparing NCWS patients, diagnosed by a double-blind placebo-controlled wheat challenge, with IBS/FD subjects. In the retrospective phase, FCP values were analyzed to define the prevalence of its positivity and its role as a NCWS diagnostic biomarker. In the prospective phase we tested the effects of a strict 6-month wheat-free diet (WFD) on FCP values. 31.3% (n = 63/201) of NCWS patients had above normal FCP values (NCWS FCP +), whereas all IBS/FD patients proved negative (P = 0.0001). FCP using a cut-off value > 41 µg/g showed a 58.6% sensitivity and a 98.0% specificity (AUC 0.755, 95% C.I. 0.702-0.837) in distinguishing NCWS from IBS/FD patients. Of the 63 NCWS FCP+, 65.1% had negative FCP values after ≥ 6 months of WFD, with a significant reduction in FCP values (P < 0.0001). All NCWS FCP- subjects still preserved negative FCP values after ≥ 6 months of WFD. Our study showed that FCP can be a useful but supplementary diagnostic marker for differentiating between NCWS and IBS/FD. Strict WFD adherence reduced FCP values, normalizing them in 65.1% of NCWS FCP + subjects. These data suggest the existence of two NCWS subgroups: NCWS FCP + characterized by a probable predominantly inflammatory/immunologic pattern and NCWS FCP- featuring non-immuno-mediated etiopathogenetic mechanisms. (Registration number NCT01762579).

Sieving out non-celiac gluten sensitivity amongst patients with irritable bowel syndrome.

OBJECTIVES: It is challenging to make diagnosis of non-celiac gluten sensitivity/non-celiac wheat sensitivity (NCGS/NCWS) in clinical practice, since there is no biomarker and diagnosis is based on response to gluten-free-diet (GFD). We used anti-gliadin antibody (AGA) for screening patients with IBS for gluten-sensitivity. METHODS: 492 Adult-patients with IBS underwent screening for celiac disease and gluten-sensitivity using IgA anti-tissue transglutaminase antibody and IgA-AGA and IgG-AGA, respectively. Patients with positive AGA (IgA and/or IgG) were invited to follow GFD, those willing were put on GFD for 6-weeks. Responsive patients were given gluten re-challenge. Diagnosis of NCGS was confirmed if they had recurrence of symptoms. RESULTS: Of 492 patients with IBS, AGA was positive in 61(12.4 %), hence suspected to have gluten-sensitivity. Of 31 who agreed to participate and followed GFD for 6-weeks, 17 (54.8 %) had complete (>30 % improvement) and 10(32.2 %) had partial (>20 % improvement) response. All 17 complete-responders were given gluten re-challenge for 6-weeks, symptoms recurred in all and hence were confirmed to have NCGS/NCWS. Significant decrease in AGA levels occurred almost in all GFD-responders. CONCLUSIONS: 12.4 % IBS patients have biological evidence of gluten/wheat-sensitivity. Almost 87 % patients with IBS having AGA responded to GFD. The value of AGA may further be explored as a biomarker for screening for the presence of NCGS, before recommending this test for the clinical practice.

No correlation between amylase/trypsin-inhibitor content and amylase inhibitory activity in hexaploid and tetraploid wheat species.

Wheat amylase/trypsin-inhibitors (ATI) are known triggers for wheat-related disorders. The aims of our study were to determine (1) the inhibitory activity against different α-amylases, (2) the content of albumins and globulins (ALGL) and total ATI and (3) to correlate these parameters in wholegrain flour of hexaploid, tetraploid and diploid wheat species. The amount of ATI within the ALGL fraction varied from 0.8% in einkorn to 20% in spelt. ATI contents measured with reversed-phase high-performance liquid chromatography (RP-HPLC) revealed similar contents (1.2-4.2 mg/g) compared to the results determined by LC-MS/MS (0.2-5.2 mg/g) for all wheat species except einkorn. No correlation was found between ALGL content and inhibitory activity. In general, hexaploid cultivars of spelt and common wheat had the highest inhibitory activities, showing values between 897 and 3564 AIU/g against human salivary α-amylase. Tetraploid wheat species durum and emmer had lower activities (170-1461 AIU/g), although a few emmer cultivars showed similar activities at one location. In einkorn, no inhibitory activity was found. No correlation was observed between the ATI content and the inhibitory activity against the used α-amylases, highlighting that it is very important to look at the parameters separately.

The Gliadin Hydrolysis Capacity of B. longum, L. acidophilus, and L. plantarum and Their Protective Effects on Caco-2 Cells against Gliadin-Induced Inflammatory Responses.

BACKGROUND: Non-celiac wheat sensitivity (NCWS) is a poorly understood gluten-related disorder (GRD) and its prominent symptoms can be ameliorated by gluten avoidance. This study aimed to determine the effectiveness of a probiotic mixture in hydrolyzing gliadin peptides (toxic components of gluten) and suppressing gliadin-induced inflammatory responses in Caco-2 cells. METHODS: Wheat dough was fermented with a probiotic mix for 0, 2, 4, and 6 h. The effect of the probiotic mix on gliadin degradation was monitored by SDS-PAGE. The expression levels of IL-6, IL-17A, INF-γ, IL-10, and TGF-ß were evaluated using ELISA and qRT-PCR methods. RESULTS: According to our findings, fermenting wheat dough with a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was effective in gliadin degradation. This process also reduced levels of IL-6 (p = 0.004), IL-17A (p = 0.004), and IFN-γ (p = 0.01) mRNA, as well as decreased IL-6 (p = 0.006) and IFN-γ (p = 0.0009) protein secretion. 4 h fermentation led to a significant decrease in IL-17A (p = 0.001) and IFN-γ (p = 0.003) mRNA, as well as reduced levels of IL-6 (p = 0.002) and IFN-γ (p < 0.0001) protein secretion. This process was also observed to increase the expression levels of IL-10 (p < 0.0001) and TGF-ß (p < 0.0001) mRNA. CONCLUSIONS: 4 h fermentation of wheat flour with the proposed probiotic mix might be a good strategy to develop an affordable gluten-free wheat dough for NCWS and probably other GRD patients.

Attenuation of immune activation in patients with multiple sclerosis on a wheat-reduced diet: a pilot crossover trial.

Background: Western lifestyle has been associated with an increase in relapsing-remitting multiple sclerosis (RRMS). In mice, dietary wheat amylase-trypsin inhibitors (ATIs) activate intestinal myeloid cells and augment T cell-mediated systemic inflammation. Objective: The aim of this study was to assess whether a wheat- and thus ATI-reduced diet might exert beneficial effects in RRMS patients with modest disease activity. Methods: In this 6-month, crossover, open-label, bicentric proof-of-concept trial, 16 RRMS patients with stable disease course were randomized to either 3 months of a standard wheat-containing diet with consecutive switch to a > 90% wheat-reduced diet, or vice versa. Results: The primary endpoint was negative, as the frequency of circulating pro-inflammatory T cells did not decrease during the ATI-reduced diet. We did, however, observe decreased frequencies of CD14+ CD16++ monocytes and a concomitant increase in CD14++ CD16- monocytes during the wheat-reduced diet interval. This was accompanied by an improvement in pain-related quality of life in health-related quality of life assessed (SF-36). Conclusion: Our results suggest that the wheat- and thus ATI-reduced diet was associated with changes in monocyte subsets and improved pain-related quality of life in RRMS patients. Thus, a wheat (ATI)-reduced diet might be a complementary approach accompanying immunotherapy for some patients. Registration: German Clinical Trial Register (No. DRKS00027967).

Evaluation of gut microbiota of iranian patients with celiac disease, non-celiac wheat sensitivity, and irritable bowel syndrome: are there any similarities?

BACKGROUND AND AIMS: Individuals with celiac disease (CD), non-celiac wheat sensitivity (NCWS), and irritable bowel syndrome (IBS), show overlapping clinical symptoms and experience gut dysbiosis. A limited number of studies so far compared the gut microbiota among these intestinal conditions. This study aimed to investigate the similarities in the gut microbiota among patients with CD, NCWS, and IBS in comparison to healthy controls (HC). MATERIALS AND METHODS: In this prospective study, in total 72 adult subjects, including CD (n = 15), NCWS (n = 12), IBS (n = 30), and HC (n = 15) were recruited. Fecal samples were collected from each individual. A quantitative real-time PCR (qPCR) test using 16S ribosomal RNA was conducted on stool samples to assess the relative abundance of Firmicutes, Bacteroidetes, Bifidobacterium spp., and Lactobacillus spp. RESULTS: In all groups, Firmicutes and Lactobacillus spp. had the highest and lowest relative abundance respectively. The phylum Firmicutes had a higher relative abundance in CD patients than other groups. On the other hand, the phylum Bacteroidetes had the highest relative abundance among healthy subjects but the lowest in patients with NCWS. The relative abundance of Bifidobacterium spp. was lower in subjects with CD (P = 0.035) and IBS (P = 0.001) compared to the HCs. Also, the alteration of Firmicutes to Bacteroidetes ratio (F/B ratio) was statistically significant in NCWS and CD patients compared to the HCs (P = 0.05). CONCLUSION: The principal coordinate analysis (PCoA), as a powerful multivariate analysis, suggested that the investigated gut microbial profile of patients with IBS and NCWS share more similarities to the HCs. In contrast, patients with CD had the most dissimilarity compared to the other groups in the context of the studied gut microbiota.

Anemia in non-celiac wheat sensitivity: Prevalence and associated clinical and laboratory features.

BACKGROUND: Patients suffering from non-celiac wheat sensitivity (NCWS) frequently report extra-intestinal symptoms, such as anemia. AIMS: We investigated the prevalence and associated clinical features of anemia in NCWS patients. METHODS: Data from 244 NCWS patients, diagnosed by double-blind placebo-controlled wheat challenge, were retrospectively reviewed and compared with 2 control groups (celiac disease (CD) and irritable bowel syndrome (IBS)). Furthermore, 31 NCWS anemic patients were prospectively re-evaluated after at least 12 months on the "strict" wheat-free diet (WFD). RESULTS: Anemia prevalence in NCWS patients was 34.8% (mean hemoglobin 10.4 ± 1.4 g/dl), significantly higher than in IBS (17.4%, P = 0.03), but not in CD ones. The NCWS group, on the whole, had sideropenic-like features with low serum iron and altered iron deposits. Both anemia prevalence and sideropenic-like features were more evident in CD than in NCWS patients, whereas only a few IBS subjects showed such features. Significant differences were found in anemic vs non-anemic NCWS patients as regards to female sex, diagnostic delay, poly/hypermenorrhea, iron deficiency, and higher TSH values. A long-term WFD significantly reduced anemia and improved iron metabolism. CONCLUSION: Microcytic/hypochromic anemia and altered iron metabolism occur frequently in NCWS and can be treated with a long-term strict WFD. NCWS should be included in differential diagnosis of anemic patients with "functional gastrointestinal troubles".

Potential tolerability of ancient grains in non-celiac wheat sensitivity patients: A preliminary evaluation.

Background and aims: A wheat-free diet (WFD) represents the elective treatment for Non-celiac Wheat Sensitivity (NCWS) patients. Preliminary reports have shown a possible better tolerability of ancient grains in these subjects. The aim of this observational study was to evaluate the frequency of consumption of ancient grains and its correlation with clinical manifestations in NCWS patients. Methods: 223 NCWS patients were recruited, and their consumption of ancient grains was monitored. Participants were first administered a modified version of the Pavia/Biagi questionnaire to investigate their adherence to "modern WFD." The appearance/exacerbation of symptoms after ingestion of ancient grains was then assessed with WHO toxicity grading scale. Results: 50.2% of the recruited patients reported consuming ancient grains before NCWS diagnosis; the diagnostic delay in this group was significantly higher than in non-consumers [median (range) 72 (6-612) vs. 60 months (3-684), P = 0.03] and these patients reported lower frequency of constipation (P = 0.04). Of the 107 patients with optimal adherence to modern WFD, 14 reported eating ancient wheat after NCWS diagnosis. Among them, 5 reported milder symptoms than those caused by modern wheat intake and 3 had an excellent tolerability without symptoms. Timilia/Tumminia variety was the most frequently used ancient grain. Conclusions: NCWS patients who consume ancient grains may receive a late diagnosis due to the possible clinical benefit (tolerability) obtained with these grains. Even after diagnosis, 10% of the patients still consumed ancient grains and had mild or no symptoms. Further studies are required to define the pathophysiological mechanism behind their putative greater tolerability.

The duodenal mucosa associated microbiome, visceral sensory function, immune activation and psychological comorbidities in functional gastrointestinal disorders with and without self-reported non-celiac wheat sensitivity.

Frequently, patients with functional gastrointestinal disorders (FGIDs) report intolerance of wheat products. We compared gastrointestinal symptoms, sensory function, psychiatric comorbidities, gut-homing immune cells, and duodenal mucosa-associated microbiome (d-MAM) in FGID patients and controls with and without self-reported wheat sensitivity (SR-NCWS). We recruited 40 FGID patients and 20 controls referred by GPs for treatment. Gastrointestinal/extraintestinal symptoms, visceral sensory function, psychological comorbidities, and SR-NCWS were assessed in a standardized approach. Peripheral gut homing T-cells (CD4+α4+ß7+CCR9+/CD8+α4+ß7+CCR9+) were quantified, and the d-MAM was assessed by DNA sequencing for 46 subjects. Factors of bacterial genera were extracted utilizing factor analysis with varimax rotation and factors univariately associated with FGID or SR-NCWS included in a subsequent multivariate analysis of variance to identify statistically independent discriminators. Anxiety scores (p < .05) and increased symptom responses to a nutrient challenge (p < .05) were univariately associated with FGID. Gut homing T-cells were increased in FGID patients with SR-NCWS compared to other groups (p all <0.05). MANOVA revealed that anxiety (p = .03), visceral sensory function (p = 0.007), and a d-MAM factor comprise members of the Alloprevotella, Prevotella, Peptostreptococcus, Leptotrichia, and Veillonella lineages were significantly (p = .001) associated with FGID, while gut homing CD4+α4+ ß7+CCR9+ T-cells were associated (p = .002) with SR-NCWS. Compared to controls, patients with and without SR-NCWS show that there are shifts in the amplicon sequence variants within specific bacterial genera between the FGID subgroups (particularly Prevotella and Streptococcus) as well as distinct bacterial taxa discriminatory for the two different FGID subtypes. Compared to controls, both FGID patients with and without SR-NCWS have an increased symptom response to a standardized nutrient challenge and increased anxiety scores. The FGID patients with SR-NCWS - as compared to FGID without SR-NCWS (and controls without SR-NCWS) - have increased gut homing T-cells. The d-MAM profiles suggest species and strain-based variations between the two FGID subtypes and in comparison to controls.

Digestion of protein and toxic gluten peptides in wheat bread, pasta and cereal and the effect of a supplemental enzyme mix.

There has been an increasing interest in the relationship between wheat digestibility and potential toxicity to the host. However, there is a lack of understanding about temporal profile of digestion of wheat proteins from different food matrices under physiologically relevant conditions. In this study, digestion of three wheat-based foods (bread, pasta and cereal) was conducted based on the INFOGEST semi-dynamic protocol in the absence and presence of a commercial supplemental enzyme preparation (a Glutalytic® based supplement, which will be marketed as Elevase®). Protein hydrolysis (OPA- ortho-phthalaldehyde - assay), molecular weight distribution (SEC-HPLC) and potential toxicity (R5 antibody-based competitive ELISA), were assessed. Our results demonstrated that under normal conditions, the complexity of the food influenced the temporal profile of protein hydrolysis and gluten breakdown throughout simulated gastric and intestinal digestion. However, treatment with the enzyme supplement significantly and acutely increased protein hydrolysis and gluten degradation in the gastric stage, and this enhanced digestion was maintained into the intestinal environment. These findings highlight the limitations of temporal gastric proteolysis and gluten degradation under normal conditions to different food types. They also show that supplemental enzyme mixes can effectively accelerate the breakdown of protein and hydrolysis of toxic gliadin fractions from the early stages of gastric digestion, thereby reducing intestinal exposure and potentially limiting the sensitization of the host.

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

Diploid Wheats: Are They Less Immunogenic for Non-Celiac Wheat Sensitive Consumers?

Non-celiac wheat sensitivity (NCWS) is a clinical entity induced by the ingestion of gluten that leads to intestinal and/or extraintestinal symptoms, and is diagnosed when celiac disease and wheat allergy have been ruled out. In addition to gluten, other grains' components, including amylase trypsin inhibitors (ATIs) and fermentable short-chain carbohydrates (FODMAPs), may trigger symptoms in NCWS subjects. Several studies suggest that, compared with tetraploid and hexaploid modern wheats, ancient diploid wheats species could possess a lower immunogenicity for subjects suffering from NCWS. This review aims to discuss available evidence related to the immunological features of diploid wheats compared to common wheats, and at outlining new dietary opportunities for NCWS subjects.

No Difference in Tolerance between Wheat and Spelt Bread in Patients with Suspected Non-Celiac Wheat Sensitivity.

Individuals with suspected non-celiac wheat sensitivity (NCWS) often report better tolerance of spelt (Triticum aestivum ssp. spelta) compared to wheat (Triticum aestivum ssp. aestivum) bakery products. This experience has neither been validated nor explained on a molecular level. Therefore, we performed blinded wheat and spelt bread challenge in this patient group. Twenty-four adults with a history of NCWS but suspected spelt tolerance were challenged in a single-blinded crossover design over six weeks with six different study breads each at 300 g per day for 4 days followed by a washout phase of 3 days. Study breads comprised spelt and wheat breads made either after a traditional (T) or a current (C) recipe, resulting in four bread types plus a gluten-free bread with 1.5% added oligosaccharides (+FODMAP) and a gluten-free bread with 5% added wheat gluten (+Gluten). The main outcome parameter was the Irritable Bowel Syndrome-Severity Scoring System, which was higher than self-estimated by the participants after spelt bread consumption (p = 0.002 for T; p = 0.028 for C) and lower for wheat bread (p = 0.052 for T; p = 0.007 for C), resulting in no difference between wheat and spelt bread tolerance. The +FODMAP bread was better tolerated than both T breads (p = 0.003 for spelt; p = 0.068 for wheat) and equally well tolerated as both C breads and +Gluten breads after normalization to the washout scores. Neither signs of inflammation nor markers for intestinal barrier integrity were influenced. Our data do not confirm, on an objective basis, the differences in expected symptoms resulting from wheat and spelt products, suggesting a strong nocebo effect for wheat and a placebo effect for spelt.

[Food sensitivities of the digestive tract-Part 2: Food intolerances]. / Nahrungsmittelunverträglichkeiten des Verdauungstraktes ­ Teil 2: Nahrungsmittelintoleranzen.

Adverse reactions to food affect about one third of the population. They are based on very different mechanisms and are triggered by specific foods. They are divided into food intolerances, which manifest mainly in the gastrointestinal tract and food allergies, which can also cause extraintestinal symptoms and have an immunological genesis. In adults, food intolerances are significantly more common than food allergies with a prevalence of approximately 10-20%. The most important food intolerances are sugar intolerances, such as lactose and fructose intolerance but intolerances to wheat also play an increasing role. The diagnostics of food intolerances require extensive exclusion diagnostics, whereby in particular irritable bowel syndrome and intestinal dysbiosis must be differentiated. The therapy of food intolerance is primarily based on a targeted elimination diet. In this advanced education article the most important food intolerances are discussed.

LC-MS/MS quantitation of α-amylase/trypsin inhibitor CM3 and glutathione during wheat sourdough breadmaking.

AIMS: This study aimed to quantify α-amylase/trypsin inhibitor (ATI) CM3 and glutathione (GSH) during wheat sourdough breadmaking. METHODS AND RESULTS: Breads were made with two wheat cultivars and fermented with Fructilactobacillus sanfranciscensis, F. sanfranciscensis ΔgshR or Latilactobacillus sakei; chemically acidified and straight doughs served as controls. Samples were analysed after mixing, after proofing and after baking. GSH and CM3 were quantified by multi-reaction-monitoring-based methods on an LC-QTRAP mass spectrometer. Undigested ATI extracts were further examined by SDS-PAGE. CONCLUSIONS: GSH abundance was similar after mixing and after proofing but increased after baking (p < 0.001), regardless of fermentation. In breads baked with cv. Brennan, the samples fermented with lactobacilli had higher GSH abundance (p < 0.001) than in the controls. CM3 relative abundance remained similar after mixing and after proofing but decreased after baking (p < 0.001) across all treatments. This trend was supported by the SDS-PAGE analysis in which ATI band intensities decreased after baking (p < 0.001) in all experimental conditions. The overall effect of baking exerted a greater effect on the abundances of GSH and CM3 than fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to quantify ATI over the course of breadmaking by LC-MS/MS in sourdough and straight dough processes.

Frequency and Clinical Aspects of Neurological and Psychiatric Symptoms in Patients with Non-Celiac Wheat Sensitivity.

BACKGROUND: Non-Celiac Wheat Sensitivity (NCWS) is characterized by both intestinal and extra-intestinal symptoms. The study aims to investigate the frequency of neuropsychiatric manifestations in NCWS patients and identify their clinical and demographic characteristics. METHODS: 278 clinical records of NCWS patients, diagnosed by a double-blind placebo-controlled wheat challenge between 2006 and 2020, were retrospectively revised. Fifty-two patients with Celiac Disease (CD) and 54 patients with Irritable Bowel Syndrome (IBS) served as controls. RESULTS: 87% of the NCWS patients had an IBS-like clinical presentation. The NCWS group showed a longer duration of symptoms, a higher frequency of positive serum anti-nuclear antibodies than CD and IBS patients, and a higher frequency of DQ2/DQ8 haplotypes and duodenal mucosa lymphocytosis than IBS controls. In addition, 50% of NCWS patients showed neuropsychiatric manifestations, while lower percentages were observed in CD (25%) and IBS (28%) controls. Neuropsychiatric symptoms in NCWS were more frequently associated with the male sex, longer duration of symptoms, and IBS-diarrhea-like clinical presentation. CONCLUSIONS: Our data suggest that in patients with IBS-like symptoms and neuropsychiatric manifestations of unknown cause, it could be useful to investigate a correlation of these symptoms with wheat ingestion to identify NCWS patients with this 'atypical' manifestation.

Triticum monococcum amylase trypsin inhibitors possess a reduced potential to elicit innate immune response in celiac patients compared to Triticum aestivum.

SCOPE: Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. METHODS AND RESULTS: ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. CONCLUSION: TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.