Duodenal Intraepithelial Lymphocytes Research Articles

Intraepithelial Lymphogram in the Diagnosis of Celiac Disease in Adult Patients: A Validation Cohort.

Celiac disease is a gluten-related pathology, highly prevalent and heterogeneous in its clinical presentation, which leads to delays in diagnosis and misdiagnosis. The analysis of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (lymphogram) is emerging as a discriminative tool in the diagnosis of various forms of celiac disease (CD). The aim of this study was to validate IEL lymphogram performance in the largest adult series to our knowledge, in support of its use as a diagnostic tool and as a biomarker of the dynamic celiac process. This was a retrospective study including 768 adult patients (217 with active CD, 195 on a gluten-free diet, 15 potential CD patients, and 411 non-celiac controls). The IEL subset cut-off values were established to calculate the diagnostic accuracy of the lymphogram. A complete celiac lymphogram profile (≥14% increase in T cell receptor [TCR]γδ IELs and simultaneous ≤4% decrease in surface-negative CD3 [sCD3-] IELs) was strongly associated with active and potential forms in over 80% of the confirmed patients with CD, whereas the remaining patients with CD had partial lymphogram profiles (≥14% increase in TCRγδ or ≤4% decrease in sCD3- IELs), with lower diagnostic certainty. None of these patients had a non-celiac lymphogram. Quantifying the TCRγδ versus sCD3- imbalance as a ratio (≥5) is a discriminative index to discard or suspect CD at diagnosis. We have validated the IEL lymphogram's diagnostic efficiency (79% sensitivity, 98% specificity), with an LR+ accuracy of 36.2. As expected, the increase in TCRγδ IELs is a reliable marker for celiac enteropathy, while changes in sCD3- IEL levels throughout the dynamic CD process are useful biomarkers of mucosal lesions.

Clinicopathologic Characterization of Lymphocytic Colitis in the Pediatric Population.

Lymphocytic colitis (LC) in the pediatric population has been associated with immune dysregulation. Single-center retrospective study of pediatric LC. 50 patients (35 female, 70%) with a median age of 12 years at diagnosis (interquartile range: 5.7-15.8) of LC were identified. At presentation, 11 patients (22%) had malnutrition, 16 (32%) had a known underlying immune dysregulation, 4 (8%) had celiac disease (CD), and none had a diagnosis of inflammatory bowel disease. The most common medications prior to diagnosis were non-steroidal anti-inflammatory drugs, proton pump inhibitor, and selective serotonin reuptake inhibitors (10% each). Colonic biopsies showed a median number of intraepithelial lymphocytes (IELs)/100 epithelial cells of 48 (range: 25-85), and only 10% of cases had neutrophilic cryptitis. Upper gastrointestinal tract findings included lymphocytic esophagitis (4%), and duodenal IELs without and with villous blunting (9% each) (n: 47). Ten patients (23%) had increased IELs in the terminal ileum (n: 43). Treatments including 5-ASA, budesonide, prednisone, and gluten-free diet improved symptoms in <50% of patients (n: 42), and all follow-up colonoscopies showed persistent LC (n: 13). Our study supports the association of LC with immune-mediated conditions, most commonly celiac disease. Symptomatic improvement was seen in <50% of patients with none of the patients with repeat colonoscopy showing histologic improvement.

Flow cytometric analysis of duodenal intraepithelial lymphocytes (celiac lymphogram): A diagnostic test for celiac disease.

Celiac disease (CD) diagnosis in adults and certain cases of children mainly relies on the assessment of histopathological features in duodenal biopsies. However, none of the histological findings that characterize CD are pathognomonic. This, in addition to the clinical heterogeneity of the disease and the presence of seronegative forms, makes the diagnosis of CD still a challenge. A hallmark of the celiac mucosa is the elevated number of TCRγδ intraepithelial lymphocytes (IEL) in the epithelium, which may remain increased even long after gluten withdrawal. Active disease is also characterized by the decreased CD3- IEL subset. The use of flow cytometry enables a precise cell counting and phenotyping, allowing the ascertainment of both TCRγδ+ and CD3- IEL subsets, what is known as the "IEL lymphogram." Although determination of this lymphogram has become a routine evaluation tool in numerous hospitals, standardization of the technical method will guarantee an accurate performance in order to become a pivotal technique for CD diagnosis. Here we describe the protocol to process duodenal biopsies in order to obtain the IELs from the mucosa and to characterize lymphocyte populations by flow cytometry to obtain the IEL lymphogram.

MAKING A STAT DIAGNOSIS IN A PATIENT PRESENTING WITH VILLOUS ATROPHY

<h3>Introduction</h3> Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) gene mutations are highly penetrant, resulting in enhanced STAT1-dependent responses to interferon-alpha/beta and interferon-gamma signaling, and impaired Th17 development. This manifests as recurrent infections, cytopenias, and autoimmune disease. Herein, we present a case of a child diagnosed with a STAT1 mutation. <h3>Case Description</h3> A 3-year-old female presented for secondary evaluation of persistent gastrointestinal and respiratory symptoms, recurrent sinopulmonary and skin infections, failure to thrive, and developmental delay. An esophagogastroduodenoscopy performed elsewhere showed increased duodenal intraepithelial lymphocytes with crypt hyperplasia and villous atrophy. Testing for Celiac disease and autoimmune enteritis were negative. Chest computed tomography showed bilateral bronchiectasis with mucous plugging. Bronchoalveolar lavage culture was positive for Streptococcus pneumoniae. Laboratory workup was notable for anemia and T-cell lymphopenia. Quantitative lymphocytes showed a low CD3+ count of 446 cells/mcL, low CD4+ count of 185 cells/mcL, and low CD8+ count of 194 cells/mcL. Lymphocyte proliferation to antigens showed essentially absent total CD45+ lymphocytes and CD3+ T cell responses to Candida and Tetanus toxoid antigens. Genetic testing later revealed a heterozygous, pathogenic c.1154C>T (p.Thr385Met) variant in the STAT1 gene. Additional testing for Th17 enumeration showed a decreased IL17 and Th17 cell percentage. She was treated with supportive care, antibiotics, and referred to Medical Genetics for additional education and counseling. <h3>Discussion</h3> STAT1-GOF gene mutations are rare and associated with variable phenotypes and disease severity. Genetic testing can help determine the cause of complex, multisystem diseases. Stem cell transplant, immunoglobulin replacement therapy, and JAK inhibitors are potential treatment considerations.

Assessment of Duodenal Intraepithelial Lymphocyte Composition (Lymphogram) for Accurate and Prompt Diagnosis of Celiac Disease in Pediatric Patients.

INTRODUCTION:Quantitative and phenotypic analyses of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (IEL lymphogram) confer specificity and enable the diagnosis even in unconventional presentations of celiac disease (CD). To evaluate the validity of the IEL lymphograms in the pediatric population for new insights into their use as biomarkers in the natural history of CD.METHODS:We retrospectively included 1,211 children (602 with active CD, 92 on a gluten-free diet, 47 with potential CD, and 470 nonceliac controls) who required duodenal biopsies in this study. The cutoff values for IEL subsets were established to calculate the probability of disease according to the lymphogram.RESULTS:A celiac lymphogram (a ≥15% increase in gamma-delta T-cell receptor IELs and a simultaneous ≤6% decrease in CD3 surface-negative [sCD3−]) IELs was strongly associated with the diagnosis of active CD, which was present in 89.7% of the confirmed patients. The remaining 10% of the celiac patients had a partial celiac lymphogram (≥15% increase gamma-delta T-cell receptor IELs or ≤6% decrease in sCD3− IELs), with lower diagnostic certainty. On a gluten-free diet, nearly 20% of the patients were indistinguishable from nonceliac subjects based on the lymphogram. In potential CD, a decrease in sCD3− IELs was a risk marker of progression to villous atrophy and a diagnosis of active CD.DISCUSSION:If a biopsy is clinically indicated, the IEL lymphogram adds specificity to the histological findings, reducing diagnostic delays and misdiagnoses. The lymphogram is useful for monitoring the natural progression of the disease and predicting the transition from potential celiac to overt CD.

Mucosal eosinophils, mast cells, and intraepithelial lymphocytes in youth with rumination syndrome.

Rumination syndrome has been associated with increased duodenal eosinophils and intraepithelial lymphocytes in adults. The aims of the current study were to assess densities of antroduodenal eosinophils and mast cells and duodenal intraepithelial lymphocytes in youth with rumination syndrome and to compare cell densities in those with and without abdominal pain or early satiety. Twenty-eight youth fulfilling Rome IV criteria for rumination syndrome who had undergone endoscopy were identified and compared to 10 controls. Antral and duodenal biopsies were assessed to determine densities of eosinophils, mast cells, and intraepithelial lymphocytes. Cell densities were also compared between rumination patients with and without abdominal pain and those with and without early satiety. Antral mast cell (peak 18.5±6.5 vs. 12.5±2.7) and eosinophil (peak 9.6±5.2 vs. 4.9±2.1) densities were significantly greater in patients with rumination syndrome as compared to controls. Duodenal intraepithelial lymphocyte densities were also increased in rumination syndrome (18.9 ± 5.1 vs. 11.7 ± 1.5; p<.001). Associations were independent of the presence of abdominal pain or early satiety. In conclusion, we found an increase in eosinophil and mast cell densities in the gastric antrum and an increase in intraepithelial lymphocytes in the duodenum in youth with rumination syndrome which was independent of the presence of abdominal pain or early satiety. These findings suggest a potential role for inflammation in the pathophysiology of rumination syndrome. Future studies should address whether treatment directed at these cells are beneficial in treating rumination syndrome.

Duodenal intraepithelial lymphocytosis in Helicobacter pylori gastritis: comparison before and after treatment

Our aims were to assess performance of duodenal intraepithelial lymphocyte counting for diagnosis of Helicobacter pylori (H. pylori) gastritis, and effects of eradication therapy on intraepithelial lymphocytosis. Paired duodenal and gastric biopsies from subjects with a pathologic diagnosis of H. pylori gastritis were reviewed. Higher duodenal intraepithelial lymphocyte counts were observed in 40 subjects with H. pylori gastritis (26 ± 5 per villus) than 52 subjects negative for H. pylori (12 ± 2 per villus). After successful eradication therapy, duodenal lymphocytes were indistinguishable from H. pylori-negative subjects, whereas they remained elevated after failed eradication therapy. This study confirms previous reports of increased duodenal intraepithelial lymphocytes in patients with concurrent Helicobacter pylori gastritis. Intraepithelial lymphocyte counts of > 15 per villus or > 10 per 100 enterocytes were predictive of infection. Duodenal lymphocytosis decreases significantly after successful eradication therapy but remains elevated when treatment fails.

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in pediatric population

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in pediatric population

Influence of low FODMAP and gluten-free diets on disease activity and intestinal microbiota in patients with non-celiac gluten sensitivity

Non-celiac gluten sensitivity (NCGS) is characterized by intestinal and extra-intestinal symptoms triggered by ingestion of gluten. However, non-gluten triggers have recently been implicated, and a FODMAP (fermentable oligo-, di-, monosaccharides and polyols)-reduced diet can partially improve symptoms in NCGS. Our aim was to analyze the effect of a low FODMAP versus a gluten-free diet (GFD) on clinical symptoms, psychological well-being, intestinal inflammation and integrity, and stool microbiota. Nineteen patients with NCGS and ten healthy controls consumed a gluten-containing standard diet before starting a two-week low FODMAP diet; after a five day transition period, participants ingested a GFD for another two weeks. The primary outcome measure was the improvement of clinical symptoms in NCGS patients under the different diets. Secondary outcomes were the determination of dietary effects on intestinal inflammation, psychological well-being, and differences in stool microbiota between NCGS patients and controls. The low FODMAP diet and especially the GFD led to a significant improvement of clinical and psychological symptoms in NCGS. A clear reduction in duodenal intraepithelial lymphocytes and mucin-producing Goblet cells was found after the GFD in these patients. Significant microbial differences between NCGS patients and controls were noticed in stool samples at every time point. Both diets caused microbial shifts in all participants, with a greater variability on genus level and metabolisms groups in NCGS patients. Our findings suggest a multifactorial etiology of NCGS, due to a functional effect caused by FODMAPs, combined with a mild gluten-triggered immune reaction, and a microbiota dysbalance. CLINICALTRIAL. NCT03268720.

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases

Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases.

Intraepithelial lymphocytes, scores, mimickers and challenges in diagnosing gluten-sensitive enteropathy (celiac disease).

The upper digestive tract is routinely scoped for several causes of malabsorption, and the number of duodenal biopsy specimens has increased notably in the last 10 years. Gluten-sensitive enteropathy (GSE) is an autoimmune disease, which shows an increasing prevalence worldwide and requires a joint clinico-pathological approach. The classical histopathology of GSE with partial or total villous blunting is well recognized, but the classification of GSE is not straightforward. Moreover, several mimickers of GSE with intraepithelial lymphocytosis have been identified in the last 20 years, with drug interactions and medical comorbidities adding to the conundrum. In this review, we report on the normal duodenal mucosa, the clinical presentation and laboratory diagnosis of GSE, the duodenal intraepithelial lymphocytes and immunophenotype of GSE-associated lymphocytes, the GSE mimickers, the differences “across oceans” among guidelines in diagnosing GSE, and the use of a synoptic report for reporting duodenal biopsies in both children and adults in the 21st century.

Novel variant of EATL evolving from mucosal γδ-T-cells in a patient with type I RCD

ObjectivesEnteropathy associated T-cell lymphoma (EATL) is a rare non-Hodgkin lymphoma that may complicate coeliac disease and typically occurs in patients with refractoriness to the gluten-free diet. The majority of these...

OP‐23 FUNCTIONAL DYSPEPSIA IS ASSOCIATED WITH DUODENAL EOSINOPHILIA IN A PEDIATRIC COHORT

Objectives:Functional dyspepsia (FD) is a highly prevalent gastrointestinal disorder with unknown etiology. We aimed to assess the association between dyspeptic symptoms and duodenal eosinophilia in children referred for upper endoscopy.Methods:In this retrospective cohort study, all histopathology reports of normal upper endoscopies performed at a single tertiary center between 2010 and 2014 were reviewed. FD was defined as epigastric pain or discomfort for more than 2 months with no response to acid suppressants. Controls were those with non‐erosive reflux disease, dysphagia or rumination syndrome. Duodenal biopsies were analyzed by pathologists blinded to indication. Intramucosal eosinophils were counted per mm2 on haematoxylin and eosin stained digital imaged sections using Aperio eSlide Manager. Demographics, clinical variables, family history and histologic findings were compared between cases and controls.Results:In total, 36 cases and 36 non‐matched controls were identified of which 56% and 53% were female (p = 0.81). Mean (± SD) age was higher in cases compared to controls (13.6 (± 3.1) vs. 10.5 (± 4.0) years; p = .001). Dyspeptic symptoms (epigastric pain 81% and/or upper abdominal discomfort 33%) were food‐related in 69% and nocturnal in 31% of cases. Self‐reported nausea (64% vs. 17%; p &lt; .0001), lethargy (19% vs. 0%; p = .005) and a family history of functional gastrointestinal disorders (28% vs. 3%; p = .003) were more common in cases than controls. There was a higher but not significantly different rate of atopic history (39% vs. 25%; p = .21) and psychological co‐morbidity (53% vs. 39%; p = .24) in FD cases versus controls. Duodenal intraepithelial lymphocytes per 100 enterocytes were similar in cases and controls (median (IQR) 10 (8–13) vs. 12 (8–18); p = 0.12). Duodenal eosinophil counts per mm2 were significantly increased in cases compared to controls (151 (118–207) vs. 76 (60–106); p &lt; 0.001).Conclusions:This study confirms recent reports of duodenal eosinophilia in FD. The high rate of atopic and psychological co‐morbidity in FD suggests multi‐factorial mechanisms and may explain why current therapeutic options aimed at symptom control are largely unsatisfactory. Duodenal eosinophilia in FD should be considered a therapeutic target.

Clinical expression of lymphocytic duodenosis in “mild enteropathy” celiac disease and in functional gastrointestinal syndromes

Objective. Abnormally high number of duodenal intraepithelial lymphocytes is frequently found in many conditions including mild enteropathy celiac disease (CD) and functional gastrointestinal syndromes, but is unclear whether lymphocytosis affects the clinical phenotype particularly in functional syndromes. Materials and methods. We compared clinical characteristics of celiac patients with lymphocytic duodenosis and normal villous structure with those of patients with functional gastrointestinal syndromes with and without lymphocytic duodenosis. We retrospectively identified 3 cohorts among patients referred for suspected CD: (1) “CoelD”, 135 patients (age 36 ± 14 years) with mild enteropathy CD; (2) “LymD”, 245 patients (38 ± 12 years) with functional gastrointestinal syndromes and lymphocytic duodenosis; and (3) “NorD”, 147 patients (37 ± 15 years) with functional syndromes and normal duodenal histology. Results. Prevalence of gastrointestinal symptoms was similar in the three cohorts, but prevalence of extra-intestinal manifestations (42% vs. 27% vs. 18%, p < 0.003) and of associated diseases (35% vs. 15% vs. 14%, p < 0.0001) was higher in “CoelD” than in “LymD” and “NorD”, respectively. Prevalence of Helicobacter pylori infection was similar in the three cohorts. The proportion of patients with final diagnosis of irritable bowel syndrome-diarrhea (38% vs. 37%), dyspepsia (31% vs. 27%), functional pain (14% vs. 19%), and functional diarrhoea (14% vs. 11%) was virtually the same in the cohorts with (LymD) and without (NorD) lymphocytic duodenosis. Conclusions. Lymphocytic duodenosis has different clinical presentation in patients with mild enteropathy CD than those with functional gastrointestinal syndromes, and is not specific for any particular functional syndrome.

Duodenal intraepithelial lymphocytosis is common in children without coeliac disease, and is not meaningfully influenced by Helicobacter pylori infection

Increased numbers of duodenal intraepithelial lymphocytes (IELs) characterise coeliac disease (CD) but have also been described in noncoeliacs. Controversy exists regarding an association between increased IELs and infection with Helicobacter pylori, which is commonly found in children. To assess the relationship between H. pylori infection and duodenal IELs in a large cohort of children, with and without CD. We reviewed gastric and duodenal biopsies of children who underwent esophagogastroduodenoscopy between January 2006 and February 2013 because of either recurrent abdominal pain (RAP) or suspected CD at Schneider Children's Medical Center of Israel, a referral centre for Israel's largest Health Maintenance Organization. The duodenal IEL count and H. pylori presence in antral biopsies were determined for each specimen. Children with RAP (n=693) or CD (n=306) were included. Among children with RAP, H. pylori was present in 33.8%. The mean IEL count in the H. pylori positive RAP group was 17.8(±8.8)/100 enterocytes, vs. 15.8(±8.3) in the H. pylori negative patients (P=0.004). Increased IEL counts (≥25 IELs/100 enterocytes) were found in 15.7% of H. pylori negative, noncoeliac children. Among children with CD, there was no significant difference in IEL counts according to H. pylori status: 73.1(±26.1) vs. 72.6 (±26.5) in H. pylori positive and negative patients respectively. Our study suggests that slightly elevated duodenal intraepithelial lymphocyte counts are common in the paediatric population. Helicobacter pylori infection has no major influence on the intraepithelial lymphocyte counts in children with recurrent abdominal pain or children with coeliac disease.

Immunohistochemical stains for CD3 and CD8 do not improve detection of gluten-sensitive enteropathy in duodenal biopsies

Patients with gluten-sensitive enteropathy usually have increased numbers of duodenal intraepithelial lymphocytes even if the villous architecture is normal. Some authors advocate the use of CD8 and CD3 immunohistochemical stains to improve detection of intraepithelial lymphocytosis, yet the added value of immunohistochemistry when biopsies appear normal remains unproven. The purpose of this study was to evaluate the utility of CD3 and CD8 immunostains in detecting intraepithelial lymphocytosis among duodenal biopsies originally interpreted to be normal based on routine evaluation. We identified 200 duodenal biopsies from 172 patients, all of which were accompanied by a clinical question of gluten-sensitive enteropathy. Five well-oriented villi from each biopsy were assessed. Intraepithelial lymphocytes present in hematoxylin and eosin (H&E)-stained slides were counted and compared with the number of CD3 and CD8 immunopositive cells present in the villous epithelium. Results were expressed as the mean number of intraepithelial lymphocytes or immunopositive cells present per 20 villous tip enterocytes. Review of H&E-stained slides revealed a mean of 2.1±0.1 intraepithelial lymphocytes, compared with 3.2±0.1 CD3-positive and 2.1±0.1 CD8-positive intraepithelial cells (P=<0.001 and 1, respectively), although none of the cases displayed sufficient numbers of intraepithelial lymphocytes to be considered abnormal (ie, ≥12/20 enterocytes) by any method. The number of intraepithelial lymphocytes detected by H&E evaluation or immunohistochemistry did not correlate with results of serologic studies for markers of gluten sensitivity. We conclude that immunostains for T cell markers do not improve detection of gluten-sensitive enteropathy when H&E-stained sections are normal.

Sa2003 Increased Duodenal Intra Epithelial Lymphocytes (IELs) are Associated With Recurrent Abdominal Pain and Parasite Infection but Not Helicobacter pylori in a Paediatric Chilean Cohort

Introduction Functional Recurrent Abdominal Pain (RAP) is a paediatric functional gastrointestinal disorder with poorly investigated pathophysiology. Proposed aetiology varies and the diagnosis is characterised by Rome III criteria. Some studies consider Helicobacter pylori to be a cause of RAP, while others disagree. The aim of this study was to investigate upper gastrointestinal pathology in a cohort of 123 children in Chile with respect to RAP, H pylori infection and other concurrent infection. Methods This blinded retrospective and IRB-approved study analysed biopsies taken from the gastric antrum and body and the duodenum in 123 Chilean children referred to endoscopy (with informed parental consent). Histopathology was evaluated against a clinical database which included symptoms, symptom duration and endoscopy findings. Rome III criteria were used to assign RAP to the relevant cases. All patients had stool microbiology and parasitology. H pylori infection was assessed by serology and histology. In the duodenum, routine histopathology and also eosinophil counts (in 5HPF ×40 magnification), were performed by microscopy. IELs/100 enterocytes were counted. Independently those patients with IELs >25 had serology performed for coeliac disease. Results Overall 105 patients were diagnosed with RAP and 12 patients were able to act as controls, having no symptoms of RAP or concurrent infection. The Rome III diagnosis of RAP was significantly associated with higher IEL counts (>20 in 74 patients) compared to controls (p=0.04). Furthermore, a higher IEL count was also positively associated with parasitic infection (nine with parasites) (p=0.02). Of 16 patients with lymphocytic duodenosis, (>25 IELs per 100 enterocytes) only three were infected by H pylori . One had coeliac disease with positive serology. Antral nodularity was observed in association with lymphoid follicles (p≤0.01) and H pylori infection (p H pylori but infection was not significantly associated with RAP (p=0.55) or parasite infection, as concurrent infection was present in only 2 patients (p=0.24). Conclusion From this study, low grade inflammation, manifest by increased IELs, may be associated with RAP and also parasitic infection. H pylori is not associated with parasite infection. However, as eosinophilia was not significantly associated with the condition further investigation is required to elucidate the potential involvement of innate immunity, including mast cells. Furthermore, there is no association between H pylori infection and RAP. Funded by EU CONTENT Project (INCO-CT-2006-032136), CONICYT/BM (RUE #29) and Fondecyt #1100654 (Chile). Competing interests None declared.

Lymphocyte subpopulations in blood and duodenal epithelium of broilers fed diets contaminated with deoxynivalenol and zearalenone

The effects of two dietary levels of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) on subpopulations of peripheral blood and duodenal intraepithelial lymphocytes in Ross 308 hybrid broiler chickens of both sexes were examined. From the day of hatching to the age of two weeks three groups of chickens were fed the same uncontaminated control diet. After 14 days, broilers in two of the groups were switched to otherwise identical experimental diets containing DON and ZEA. The diet of Group 2 contained DON and ZEA, each at a level of 3.4 mg/kg and the Group 3 diet contained DON and ZEA at 8.2 and 8.3 mg/kg, respectively. Contaminated diets were prepared in the laboratory using maize cultivated with Fusarium graminearum. Group 1 (control) continued with intake of uncontaminated diet for entire experiment. At the age of four weeks samples of blood and duodenal tissue were collected. Intake of both contaminated diets reduced blood phagocytic activity (P<0.001) whereas numbers of various types of peripheral white blood cells were not affected. Among different blood lymphocyte populations investigated, only the counts of CD3 + cells were lowered by diet with higher mycotoxin levels (P=0.021). In duodenal epithelium, the large increase in cells expressing MHC II was observed due to intake of both diets contaminated with lower and higher mycotoxins contents (P<0.001) while the IgA + cell subpopulation was decreased (P=0.018). The other intraepithelial lymphocyte subpopulations in duodenum examined (CD3 +, CD4 +, CD8 + and CD44 +) were not influenced by intake of contaminated diets (P>0.05). Results suggest that intake of diets contaminated with DON and ZEA at medium levels may affect some blood and intraepithelial duodenal lymphocyte subpopulations and compromise blood phagocytic activity in broilers.

Redefining the intraepithelial lymphocytes threshold to diagnose gluten sensitivity in patients with architecturally normal duodenal histology

Accuracy of intraepithelial lymphocytes counts for diagnosing mild enteropathy coeliac disease in absence of villous atrophy can be limited by inappropriate controls included in the studies. To determine the diagnostic accuracy of intraepithelial lymphocytes counts utilising controls lacking HLA coeliac disease-associated alleles. Intraepithelial lymphocytes counting at villus tip and per 100 enterocytes was performed at haematoxylin and eosin (H&E) and CD3-stainings in: 29 cases (21 with potential coeliac disease and 8 affected by latent coeliac disease) representing the patient population and 14 noncoeliac controls lacking HLA-DQ2/DQ8 alleles. Threshold (mean+2 s.d.) of duodenal intraepithelial lymphocytes at villus tip and per 100 enterocytes in noncoeliac controls was respectively: 3.5 and 18 at H&E, 3.2 and 17 following CD3-staining. Considering the whole patient population, the sensitivity of tip intraepithelial lymphocytes in detecting mild enteropathy coeliac disease was 90% (95% CI=72.6-97.8) both at H&E and CD3-stainings. The sensitivity of intraepithelial lymphocytes per 100 enterocytes was 93% (95% CI=77.2-99.2) both at H&E and CD3-staining. Specificity of both intraepithelial lymphocytes counts was 100% (95% CI=76.8-100). Using a threshold of 25 intraepithelial lymphocytes per 100 enterocytes could miss 59% of cases at H&E and 48% following CD3-staining. Intraepithelial lymphocytes counts are diagnostic feasible tools to detect mild enteropathy coeliac disease. Threshold of duodenal intraepithelial lymphocytes may be lower than currently accepted.

Duodenal Intraepithelial Lymphocytes of Children with Cow Milk Allergy Preferentially Bind the Glycan-Binding Protein Galectin-3

A breakdown in intestinal homeostasis results in inflammatory bowel diseases including coeliac disease and allergy. Galectins, evolutionarily conserved beta-galactoside-binding proteins, can modulate immune-epithelial cell interactions by influencing immune cell fate and cytokine secretion. In this study we investigated the glycosylation signature, as well as the regulated expression of galectin-1 and -3 in human duodenal samples of allergic and non-allergic children. Whereas galectin-1 was predominantly localized in the epithelial compartment (epithelial cells and intraepithelial lymphocytes) and the underlying lamina propria (T cells, macrophages and plasma cells), galectin-3 was mainly expressed by crypt epithelial cells and macrophages in the lamina propria. Remarkably, expression of these galectins was not significantly altered in allergic versus non-allergic patients. Investigation of the glycophenotype of the duodenal inflammatory microenvironment revealed substantial alpha2-6-linked sialic acid bound to galactose in lamina propria plasma cells, macrophages and intraepithelial lymphocytes and significant levels of asialo core 1 O-glycans in CD68+ macrophages and enterocytes. Galectin-1 preferentially bound to neutrophils, plasma cells and enterocytes, while galectin-3 binding sites were mainly distributed on macrophages and intraepithelial lymphocytes. Notably, galectin-3, but not galectin-1 binding, was substantially increased in intraepithelial gut lymphocytes of allergic patients compared to non-allergic subjects, suggesting a potential role of galectin-3-glycan interactions in shaping epithelial-immune cell connections during allergic inflammatory processes.