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A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease.
Hardy, MY, Goel, G, Russell, AK, Chen Yi Mei, SLG, Brown, GJE, Wang, S, Szymczak, E, Zhang, R, Goldstein, KE, Neff, KM, et al
Frontiers in immunology. 2021;:661622
Abstract
Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial ("RESET CeD") of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 μg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.
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2.
Poor Sensitivity of Fecal Gluten Immunogenic Peptides and Serum Antibodies to Detect Duodenal Mucosal Damage in Celiac Disease Monitoring.
Laserna-Mendieta, EJ, Casanova, MJ, Arias, Á, Arias-González, L, Majano, P, Mate, LA, Gordillo-Vélez, CH, Jiménez, M, Angueira, T, Tébar-Romero, E, et al
Nutrients. 2020;(1)
Abstract
A lifelong gluten-free diet (GFD) is the only current treatment for celiac disease (CD), but strict compliance is complicated. Duodenal biopsies are the "gold standard" method for diagnosing CD, but they are not generally recommended for disease monitoring. We evaluated the sensitivity and specificity of fecal gluten immunogenic peptides (GIPs) to detect duodenal lesions in CD patients on a GFD and compared them with serum anti-tissue transglutaminase (tTG) IgA antibodies. A prospective study was conducted at two tertiary centers in Spain on a consecutive series of adolescents and adults with CD who maintained a long-lasting GFD. Adherence to a GFD and health-related quality of life were scored with validated questionnaires. Mucosal damage graded according to the Marsh-Oberhüber classification (Marsh 1/2/3) was used as the reference standard. Of the 97 patients included, 27 presented duodenal mucosal damage and 70 had normal biopsies (Marsh 0). The sensitivity (33%) and specificity (81%) of GIPs were similar to those provided by the two assays used to measure anti-tTG antibodies. Scores in questionnaires showed no association with GIP, but an association between GIPs and patients' self-reported gluten consumption was found (p = 0.003). GIP displayed low sensitivity but acceptable specificity for the detection of mucosal damage in CD.
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Serum cytokines elevated during gluten-mediated cytokine release in coeliac disease.
Goel, G, Daveson, AJM, Hooi, CE, Tye-Din, JA, Wang, S, Szymczak, E, Williams, LJ, Dzuris, JL, Neff, KM, Truitt, KE, et al
Clinical and experimental immunology. 2020;(1):68-78
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Abstract
Cytokines have been extensively studied in coeliac disease, but cytokine release related to exposure to gluten and associated symptoms has only recently been described. Prominent, early elevations in serum interleukin (IL)-2 after gluten support a central role for T cell activation in the clinical reactions to gluten in coeliac disease. The aim of this study was to establish a quantitative hierarchy of serum cytokines and their relation to symptoms in patients with coeliac disease during gluten-mediated cytokine release reactions. Sera were analyzed from coeliac disease patients on a gluten free-diet (n = 25) and from a parallel cohort of healthy volunteers (n = 25) who underwent an unmasked gluten challenge. Sera were collected at baseline and 2, 4 and 6 h after consuming 10 g vital wheat gluten flour; 187 cytokines were assessed. Confirmatory analyses were performed by high-sensitivity electrochemiluminescence immunoassay. Cytokine elevations were correlated with symptoms. Cytokine release following gluten challenge in coeliac disease patients included significant elevations of IL-2, chemokine (C-C motif) ligand 20 (CCL20), IL-6, chemokine (C-X-C motif) ligand (CXCL)9, CXCL8, interferon (IFN)-γ, IL-10, IL-22, IL-17A, tumour necrosis factor (TNF)-α, CCL2 and amphiregulin. IL-2 and IL-17A were earliest to rise. Peak levels of cytokines were generally at 4 h. IL-2 increased most (median 57-fold), then CCL20 (median 10-fold). Cytokine changes were strongly correlated with one another, and the most severely symptomatic patients had the highest elevations. Early elevations of IL-2, IL-17A, IL-22 and IFN-γ after gluten in patients with coeliac disease implicates rapidly activated T cells as their probable source. Cytokine release after gluten could aid in monitoring experimental treatments and support diagnosis.
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[Frequency of determining markers of casein's inhability and gluten in children with disorders of autistic spectrum].
Bavykina, IA, Popov, VI, Zvyagin, AA, Bavykin, DV
Voprosy pitaniia. 2019;(4):41-47
Abstract
The most optimal approach to the problem of managing children with autism spectrum disorders (ASD) is a complex one that involves a pediatric gastroenterologist, a nutritionist, a neurologist, a psychiatrist. Currently, there are studies that confirm the effectiveness of diet in the correction of neuropsychiatric status and gastroenterological disorders in ASD. Evidence supporting the therapeutic value of diets is limited and inconclusive. Diet therapy should be used only if food allergy or gluten or casein intolerance is diagnosed. Aim. To study the frequency of detection of markers of gluten and casein intolerance in children with ASD. Material and methods. The study involved 51 children (39 boys and 12 girls) aged 3 to 15 years with a diagnosis of ASD. Among the study participants, 20 children used gluten-free diet and casein-free diet for more than 6 months. The material for the study was venous blood taken from the elbow vein in the morning on an empty stomach. Determination of specific IgG-antibodies to casein and gliadin, IgA-antibodies to deamidized gliadin peptides was carried out by enzyme immunoassay. The level of total IgA to exclude selective deficiency was also determined. Results and discussion. Most children with ASD (79.5%) had increased levels of specific IgG antibodies to casein. The increase in IgG antigliadin antibodies was determined in 19.3% of children who do not follow a gluten-free diet, and antibodies to deamidized gliadin Ig peptides were not detected in any patient. Gluten intolerance in children with ASD is characterized by sensitivity to it and occurs in 40-50%. Conclusion. According to the literature and the results of own studies, some children with ASD have gluten and casein intolerance. Before the appointment of diet therapy for children with ASD, it is necessary to conduct a survey to clarify the nature of intolerance and the choice of optimal tactics of diet therapy.
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Non-Celiac Gluten Sensitivity among Patients Perceiving Gluten-Related Symptoms.
Capannolo, A, Viscido, A, Barkad, MA, Valerii, G, Ciccone, F, Melideo, D, Frieri, G, Latella, G
Digestion. 2015;(1):8-13
Abstract
BACKGROUND Non-celiac gluten sensitivity (NCGS) is a recently recognized disorder, characterized by the occurrence of symptoms following gluten ingestion. It is often self-diagnosed by the patient, but should be confirmed by the response to a gluten-free diet, followed by a gluten challenge. Celiac disease (CD) and wheat allergy (WA) must first be ruled out. AIMS (1) to determine the frequency of visits performed for symptoms self-perceived as gluten-related; (2) to assess in this cohort, the proportion of patients satisfying the diagnostic criteria for NCGS. METHODS A two-year prospective study including all consecutive patients complaining of gluten-related symptoms. NCGS was diagnosed on the basis of the disappearance of the symptoms within 6 months of a gluten-free diet, followed by their reappearance with the reintroduction of gluten in the diet for 1 month. RESULTS Three hundred and ninety two patients complaining of gluten-related symptoms were enrolled; 26 of these (6.63%) were affected by CD, 2 (0.51%) by WA and 27 were diagnosed with NCGS (6.88%). The remaining 337 patients (85.96%) did not experience any change of symptoms with a gluten-free diet. The PPV of the gluten-related symptom was found to be 7%. CONCLUSION Eighty six percent of patients reporting gluten-related symptoms have neither NCGS, nor CD, nor WA. Self-perceived gluten-related symptoms are rarely indicative of the presence of NCGS.
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[Treatment and prevention of gluten-sensitive celiac disease].
Krums, LM, Parfenov, AI, Sabel'nikova, EA, Gudkova, RB, Vorob'eva, NN
Eksperimental'naia i klinicheskaia gastroenterologiia = Experimental & clinical gastroenterology. 2011;(2):86-92
Abstract
In this article presented results of examination and treatment of 207 patients with glutensensitive celiac disease. And also you can find comprehensive method for the treatment of patients with varying severity of disease. The fundamental method of therapy glutensensitive celiac disease is strict lifelong adherence to a gluten-free diet (GFD), which eliminated protein cereal--gluten contained in wheat, rye and barley. Recently, there is large number of products which include the "hidden gluten". Its elimination from the diet of GFD is mandatory for patients with celiac disease. In the presence of diarrhea and malabsorption syndrome are used adsorbents, astringents, enzymes, intestinal antiseptic, probiotics. With the purpose of correction of metabolic disorders intravenous electrolyte mixture containing potassium, calcium and magnesium. To eliminate protein deficiency drugs used solid protein mixture of pure amino acids, gluten-free mixes for enteral feeding. Clinical examination of patients with celiac disease is aimed at monitoring compliance with GFD, early detection of cancer, autoimmune and other related diseases.
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Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients.
Ráki, M, Fallang, LE, Brottveit, M, Bergseng, E, Quarsten, H, Lundin, KE, Sollid, LM
Proceedings of the National Academy of Sciences of the United States of America. 2007;(8):2831-6
Abstract
Tetramers of MHC-peptide complexes are used for detection and characterization of antigen-specific T cell responses, but they require knowledge about both antigenic peptide and the MHC restriction element. The successful application of these reagents in human diseases involving CD4+ T cells is limited. Celiac disease, an intestinal inflammation driven by mucosal CD4+ T cells recognizing wheat gluten peptides in the context of disease-associated HLA-DQ molecules, is an ideal model to test the potential clinical use of these reagents. We investigated whether gluten-specific T cells can be detected in the peripheral blood of celiac disease patients using DQ2 tetramers. Nine DQ2+ patients and six control individuals on a gluten-free diet were recruited to the study. Participants consumed 160 g of gluten-containing bread daily for 3 days. After bread-challenge, gluten-specific T cells were detectable in the peripheral blood of celiac patients but not controls both directly by tetramer staining and indirectly by enzyme-linked immunospot. These T cells expressed the beta(7) integrin indicative of gut-homing properties. Most of the cells had a memory phenotype, but many other phenotypic markers showed a heterogeneous pattern. Tetramer staining of gluten-specific T cells has the potential to be used for diagnosis of celiac disease.
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Dietary treatment of gluten neuropathy.
Hadjivassiliou, M, Kandler, RH, Chattopadhyay, AK, Davies-Jones, AG, Jarratt, JA, Sanders, DS, Sharrack, B, Grünewald, RA
Muscle & nerve. 2006;(6):762-6
Abstract
We studied the effect of a gluten-free diet in patients with idiopathic sensorimotor axonal neuropathy and circulating antigliadin antibodies. Consecutive patients underwent baseline neurophysiological assessment and were offered a gluten-free diet. Those who went on the diet formed the intention-to-treat group and those who did not were the control group. Repeat neurophysiological assessment and subjective evaluation of neuropathy symptoms were performed at 1 year. A total of 35 patients participated in the study, with 25 patients going on the diet and 10 not doing so. There was a significant difference in the change of sural sensory action potentials (pre-defined primary endpoint), with evidence of improvement in the intention-to-treat group and deterioration in the control group. Subjective change in neuropathy symptoms also showed significant differences, with patients in the intention-to-treat group reporting improvement and those in the control group reporting deterioration. Gluten-free diet may thus be a useful therapeutic intervention for patients with gluten neuropathy.
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Endoscopic and histological findings in the duodenum of adults with celiac disease before and after changing to a gluten-free diet: a 2-year prospective study.
Tursi, A, Brandimarte, G, Giorgetti, GM, Elisei, W, Inchingolo, CD, Monardo, E, Aiello, F
Endoscopy. 2006;(7):702-7
Abstract
BACKGROUND AND STUDY AIMS Published follow-up data on small-intestinal recovery in patients with celiac disease are scarce and contradictory. This is especially the case for adult patients, who often show incomplete histological recovery after starting a gluten-free diet (GFD). We conducted a 2-year prospective study to evaluate the effectiveness of a GFD in improving the endoscopic and histological duodenal findings in adults with celiac disease. PATIENTS AND METHODS We studied 42 consecutive adults with newly diagnosed celiac disease (13 men, 29 women; mean age 32.7 years, range 15 - 72 years). All the patients underwent esophagogastroduodenoscopy and small-bowel biopsy. We devised our own grading system for the endoscopic appearance of the duodenum, which ranged from "normal" appearance to "mild", "moderate", or "severe" alterations. Small-bowel biopsies were obtained from the second part of the duodenum (and from the duodenal bulb when it had a micronodular appearance). The histopathological appearances were described according to modified Marsh criteria. RESULTS A normal endoscopic appearance in the duodenum was found in 5/42 patients (11.9 %) at entry and in 32/42 patients (76.2 %) after 2 years on a GFD. Subdividing the patients according to age, patients aged from 15 years to 60 years showed significant improvement within 12 months ( P < 0.0001 for patients aged from 15 years to 45 years; P < 0.003 for patients in the 46 years to 60 years group), whereas the improvement in endoscopic findings in patients older than 60 years was not statistically significant, even 24 months after starting the GFD. "Normal" histology was reported in none of the patients at entry, but in 25 patients (59.5 %) after 24 months on a GFD, but this parameter did not show a significant improvement until the patients had been on the GFD for 12 months ( P < 0.0001). Only the younger patients (5 - 30 years) showed significant improvement of histology within 12 months ( P < 0.034); older patients (>30 years) showed histological improvement but this was not statistically significant, even after 24 months on a GFD. CONCLUSIONS This study shows for the first time that endoscopic recovery is faster than histological recovery in adults with celiac disease who go on a GFD. Moreover, older patients showed incomplete endoscopic and histological recovery even 24 months after starting a GFD. We therefore advise, as a minimum recommendation, that follow-up biopsies should be taken 1 - 2 years after starting a GFD in adults with celiac disease.
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Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study.
Kaukinen, K, Peräaho, M, Collin, P, Partanen, J, Woolley, N, Kaartinen, T, Nuutinen, T, Halttunen, T, Mäki, M, Korponay-Szabo, I
Scandinavian journal of gastroenterology. 2005;(5):564-72
Abstract
OBJECTIVE In coeliac disease, autoantibodies directed against transglutaminase 2 are produced in small-bowel mucosa, and they have been found to be deposited extracellularly. The aim of this study was to investigate whether such mucosal IgA deposits are important in the diagnostic work-up of early-stage coeliac disease without small-bowel mucosal villous atrophy. MATERIAL AND METHODS Forty-one adults suspected of coeliac disease owing to increased density of mucosal gamma(delta)+ intraepithelial lymphocytes but normal villous morphology were randomized to gluten challenge or a gluten-free diet for 6 months. Clinically and histologically verified gluten dependency was compared with existence of small-bowel mucosal transglutaminase 2-specific extracellular IgA deposits and (coeliac disease-type) HLA DQ2 and DQ8; 34 non-coeliac subjects and 18 patients with classical coeliac disease served as controls. RESULTS Of the 41 patients, 5 in the challenge group and 6 in the gluten-free diet group were clinically gluten sensitive; all 11 had HLA DQ2 or DQ8. Ten of these 11 patients showed transglutaminase 2-targeted mucosal IgA deposits, which were dependent on gluten consumption. Minimal IgA deposits were seen in only 3 out of 30 patients with suspected coeliac disease without any clinically detected gluten dependency. The deposits were found in all classical coeliac patients and in none of the non-coeliac control subjects. CONCLUSIONS Clinically pertinent coeliac disease exists despite normal small-bowel mucosal villous architecture. Mucosal transglutaminase 2-specific IgA deposits can be utilized in detecting such patients with genetic gluten intolerance.