Noé Ontiveros

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell‐mediated disease diagnosed by the presence of gluten‐dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten‐free diet (GFD) mobilizes gluten‐reactive T cells measurable by interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA‐DQ2·5. We aimed to develop whole blood assays to detect gluten‐specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA‐DQ2·5+), GFD donors confirmed not to have CD (n = 6 HLA‐DQ2·5+, 11 HLA‐DQ2·5−) and donors with CD not following GFD (n = 4, all HLA‐DQ2·5+). Plasma IFN‐γ and IFN‐γ inducible protein‐10 (IP‐10) were measured by enzyme‐linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN‐γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN‐γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA‐DQ2·5+ CD patients; the whole blood IP‐10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten‐reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.

Self-Reported Prevalence of Symptomatic Adverse Reactions to Gluten and Adherence to Gluten-Free Diet in an Adult Mexican Population

The prevalence of symptomatic adverse reactions to gluten and adherence to gluten-free diet in Latin American countries is unknown. These measurements are strongly linked to gluten-related disorders. This work aimed to estimate the prevalence of adverse reactions to oral gluten and the adherence to gluten-free diet in the adult Mexican population. To reach this aim, a self-administered questionnaire was designed and tested for clarity/comprehension and reproducibility. Then, a self-administered questionnaire-based cross-sectional study was conducted in the Mexican population. The estimated prevalence rates were (95% CI): 11.9% (9.9–13.5) and 7.8 (6.4–9.4) for adverse and recurrent adverse reactions to gluten respectively; adherence to gluten-free diet 3.7% (2.7–4.8), wheat allergy 0.72% (0.38–1.37); celiac disease 0.08% (0.01–0.45), and NCGS 0.97% (0.55–1.68). Estimated pooled prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.88% (0.49–1.5), and 93.3% respondents reported adherence to gluten-free diet without a physician-diagnosis of gluten-related disorders. Symptom comparisons between those who reported recurrent adverse reactions to gluten and other foods showed statistically significant differences for bloating, constipation, and tiredness (p < 0.05). Gluten-related disorders may be underdiagnosed in the Mexican population and most people adhering to a gluten-free diet are doing it without proper diagnostic work-up of these disorders, and probably without medical/dietician advice.

Prevalence of Self-Reported Gluten Sensitivity and Adherence to a Gluten-Free Diet in Argentinian Adult Population

Background: Previous studies suggest that the prevalence of wheat/gluten sensitivity and adherence to a gluten-free diet (GFD) are high in Latin population despite a poor diagnosis of celiac disease. However, these prevalence rates still remain unknown in most Latin American countries. Methods: A cross-sectional survey study was conducted in Santa Fe, Argentina. Results: The estimated self-reported prevalence rates were (95% Confidence Interval [CI]): self-reported gluten sensitivity (SR-GS) 7.61% (6.2–9.2), SR-GS currently following a GFD 1.82% (1.2–2.7), celiac disease 0.58% (0.3–1.2), wheat allergy 0.33% (0.12–0.84), self-reported non-celiac gluten sensitivity (SR-NCGS) 6.28% (5.1–7.8), SR-NCGS currently following a GFD 0.91% (0.5–1.6), and adherence to a GFD 6.37% (5.1–7.9). SR-GS was more common in women (6.0%; p < 0.001) and associated with irritable bowel syndrome (p < 0.001). Among the GFD followers, 71.4% were doing it for reasons other than health-related benefits and 50.6% without medical/dietitian advice. In the non-SR-GS group, the main motivations for following a GFD were weight control and the perception that a GFD is healthier. Conclusion: In Argentina, gluten sensitivity is commonly reported and it seems that physicians/gastroenterologists are aware of celiac disease diagnosis. Trustable information about the benefits and potential consequences of following a GFD should be given to the general population.

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background: Celiac disease is a chronic immune‐mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T‐cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten‐specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten‐specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten‐specific CD4+ T‐cell response, we paired traditional IFN‐&ggr; ELISpot with an assay to detect antigen‐specific CD4+ T cells that does not rely on tetramers, antigen‐stimulated cytokine production, or proliferation but rather on antigen‐induced coexpression of CD25 and OX40 (CD134). Results: Numbers of circulating gluten‐specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten‐specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten‐specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion: This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten‐specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten‐specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.

Self-Reported Prevalence of Gluten-Related Disorders and Adherence to Gluten-Free Diet in Colombian Adult Population

Background. Celiac disease seems to be rare in Colombians, but there are currently no data about the prevalence rates of symptomatic adverse reactions to gluten or adherence to gluten-free diet (GFD) in this population. Aim. to evaluate the self-reported prevalence rates of adverse reactions to gluten, adherence to GFD, and gluten-related disorders at population level in Colombia. Methods. A self-administered questionnaire-based cross-sectional study was conducted in a population from Northwest Colombia. Results. The estimated prevalence rates were (95% CI) 7.9% (6.5–9.6) and 5.3% (4.1–6.7) for adverse and recurrent adverse reactions to wheat/gluten, respectively, adherence to GFD 5.9% (4.7–7.4), wheat allergy 0.74% (0.3–1.4), and nonceliac gluten sensitivity 4.5% (3.5–5.8). There were no self-reported cases of celiac disease. Prevalence of self-reported physician-diagnosis of gluten-related disorders was 0.41% (0.17–0.96). Most respondents reported adherence to GFD without a physician-diagnosis of gluten-related disorders (97.2%). The proportion of gluten avoiders was 17.2% (15.2–19.5). Most of them did not report recurrent adverse reactions to wheat/gluten (87.0%). Conclusions. Nonceliac gluten sensitivity is rarely formally diagnosed in Colombia, but this population has the highest prevalence rate of adherence to GFD reported to date. Consequently, most respondents were avoiding wheat- and/or gluten-based products for reasons other than health-related symptoms.

Assessing of Celiac Disease and Nonceliac Gluten Sensitivity

The publication of papers on the topic of gluten related disorders has substantially increased over the last few years. This has motivated healthcare professionals to pay attention not only to celiac disease and wheat allergy but also to a condition termed nonceliac gluten sensitivity (NCGS). Until now this condition has been diagnosed clinically on the basis of exclusion criteria and clinical response to gluten withdrawal. In addition, recent research in this field has shown that other food components distinct from gluten are implicated in NCGS cases, thereby changing our general understanding of NCGS diagnosis in either individuals on gluten containing diets or those already following a gluten-free diet with no proper diagnostic work-up of celiac disease. With this in mind, the assessment of NCGS will require extensive knowledge of celiac disease manifestations and the laboratory tests commonly performed during diagnosis of celiac disease.

Prevalence of Self-Reported Gluten-Related Disorders and Adherence to a Gluten-Free Diet in Salvadoran Adult Population

Gluten-related disorders are not considered of relevance at public health level in Central America. The prevalence of gluten-related disorders, and adherence to a gluten-free diet, remain unknown in the Central American region. We conducted a cross-sectional survey of the Central American population from San Salvador, El Salvador, to estimate the prevalence rates of self-reported gluten-related disorders and adherence to a gluten-free diet. 1326 individuals were surveyed. Self-reported prevalence rates were (95% Confidence Interval): gluten sensitivity 3.1% (2.3–4.2); physician-diagnosed celiac disease 0.15% (0.04–0.5); wheat allergy 0.75% (0.4–1.3); non-celiac gluten sensitivity 0.98% (0.5–1.6). The prevalence rate of adherence to a gluten-free diet was 7.0% (5.7–8.5). Seven self-reported physician diagnosed gluten-sensitive cases informed the co-existence of non-celiac gluten sensitivity with celiac disease and/or wheat allergy. Among the non-self-reported gluten sensitivity individuals following a gluten-free diet, 50% reported that they were seeing a health professional for gluten-free dietary advice. Gluten sensitivity is commonly reported in Salvadoran population, but some health professionals acknowledge the coexistence of wheat allergy, celiac disease, and non-celiac gluten sensitivity. Among studies at population level, the prevalence of adherence to a gluten-free diet in Salvadoran population is the highest reported until now. However, just a few of the gluten-free diet followers were doing it for health-related benefits; the others reported weight control and the perception that the diet is healthier as the main motivation for adopting such a diet.

The gluten-free diet: access and economic aspects and impact on lifestyle

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Assessment of the Sensitizing Potential of Proteins in BALB/c Mice: Comparison of Three Protocols of Intraperitoneal Sensitization

Most food allergy cases are associated with a limited group of allergens. This could be attributed to an increased ability of some foods to sensitize and trigger allergic reactions. However, there are no validated animal models to evaluate the sensitizing or allergenic potentials of proteins. Our aim was to evaluate three protocols of adjuvant-free intraperitoneal sensitization that differ in the time points for sample collection (days 14, 28 and 35 from beginning of the sensitization) and also in the number of immunizations (2, 5 and 3, respectively). Ovalbumin (OVA; 0.05 mg), cow milk proteins (CMP; 0.025, 0.05 and 0.25 mg), and potato acid phosphatase (PAP; low allergenic protein; 250.0 mg) were administered intraperitoneally (ip) to BALB/c mice (n = 4–6) and the protein-specific IgE and IgG antibody responses were evaluated using ELISA. Additional serum protein-specific IgE antibodies evaluations were carried out after IgG depletion. Anti-OVA IgE antibodies were detected in mice from all three protocols. The responses were higher in the group of mice that underwent the 28-day protocol than in those that underwent the 14- or 35-day protocols (p < 0.01 and p < 0.05, respectively). Anti-CMP IgE antibodies were detected in both the 14- and 28-day protocols, but the response was higher in the group that underwent the 28-day protocol (p < 0.001). The anti-CMP IgE antibody response detection was improved after serum IgG depletion (p < 0.001). Anti-PAP IgE antibodies were not detected. Mice with undetectable serum levels of protein-specific IgE triggered anti-OVA, -CMP, and -PAP IgG responses. An adjuvant-free 28-day protocol with five ip immunizations seems appropriate for evaluation of the inherent sensitizing or allergenic capacity of the studied proteins. Reproducible results were obtained utilizing the BALB/c mouse strain. Inter-laboratory studies including a larger number of proteins should be carried out to validate this model.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA‐DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)‐based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA‐DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP‐based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA‐DQ2.2. Here we test this two‐step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA‐DQ2.2 enabled HLA‐DQ2.2 to be accurately typed. This technique is a simple addition to a SNP‐based typing strategy and enables comprehensive definition of all at‐risk HLA genotypes in CD in a timely and cost‐effective manner.