Intraepithelial Lymphocyte Counts Research Articles

Histopathological and Immunohistochemical Analysis of Small Intestinal Biopsies in Adults Suspected of Celiac Disease.

To analyse histomorphological and immunohistochemical analysis of small intestinal biopsies in adults suspected of celiac disease. Descriptive study. Department of Histopathology, Army Medical College, Rawalpindi, from November 2014 to December 2015. Fifty cases of small intestinal mucosal biopsies (duodenal and jejunal) were analysed in adult patients aged above 14 years suspected of celiac disease. Their histomorphological data was recorded using Modified Marsh Criteria. Type of intraepithelial lymphocytes was assessed using immunohistochemistry. Intraepithelial lymphocytes were counted both by H&E stain and immunostain CD3 and CD20. Thirty-four percent patients were aged between 21 - 30 years and 22% patients aged between 41 - 50 years. There were 84% (n=42) males. Thirteen (26%) cases showed focal villous atrophy, 32 (64%) cases showed partial villous atrophy and 5 (10%) cases showed complete villous atrophy. Anti-tissue transglutaminase antibody was positive in 21 (42%) cases. CD3 immunomarker was positive for intraepithelial lymphocytes in all 50 cases while CD20 immunomarker showed focal positivity in areas with lymphoid follicle formation. The count of intraepithelial lymphocytes was found to be almost equal (with a difference of 3 - 4 lymphocytes) on both H&E stain and immunostain CD3 and CD20. Males aged 21 - 30 years were the most commonly affected group. The most frequent change in histology was partial villous atrophy along with lymphocytic enteritis. All the intraepithelial lymphocytes were present in crescendo pattern of distribution.

Serum I-FABP Detects Gluten Responsiveness in Adult Celiac Disease Patients on a Short-Term Gluten Challenge.

Response to gluten challenge (GC) is a key feature in diagnostic algorithms and research trials in celiac disease (CD). Currently, autoantibody titers, late responders to GC, and invasive duodenal biopsies are used to evaluate gluten responsiveness. This study investigated the accuracy of serum intestinal-fatty acid binding protein (I-FABP), a marker for intestinal epithelial damage, to predict intestinal damage during GC in patients with CD. Twenty adult CD patients in remission underwent a two-week GC with 3 or 7.5 g of gluten daily. Study visits occurred at day -14, 0, 3, 7, 14, and 28. Serum I-FABP, antibodies to tissue transglutaminase (tTG-IgA), deamidated gliadin peptides (IgA-DGP), and anti-actin (AAA-IgA) were assessed at each visit. Villous-height to crypt-depth ratio (Vh:Cd) and intraepithelial lymphocyte (IEL) count were evaluated at day -14, 3, and 14. Forty-three CD-serology negative individuals were included to compare serum I-FABP levels in CD patients on a gluten-free diet (GFD) with those in healthy subjects. Serum I-FABP levels increased significantly during a two-week GC. In contrast, the most pronounced autoantibody increase was found at day 28, when patients had already returned to a GFD for two weeks. IgA-AAA titers were only significantly elevated at day 28. I-FABP levels and IEL count correlated at baseline (r=0.458, P=0.042) and at day 14 (r=0.654, P=0.002) of GC. Neither gluten dose nor time on a GFD influenced I-FABP change during GC. Serum I-FABP levels increased significantly during a two-week GC in adult CD patients and correlated with IEL count. The data suggest that serum I-FABP is an early marker of gluten-induced enteropathy in celiac patients and may be of use in both clinical and research settings.

Validity of Intraepithelial Lymphocyte Count in the Diagnosis of Celiac Disease: A Histopathological Study

The gold standard for diagnosis of Celiac disease (CD) is histological evidence of a small intestinal biopsy together with positive serology. Modified Marsh classification utilizes the histological parameter of intraepithelial lymphocyte (IEL) count in the diagnosis of CD. The reported upper limit of normal IEL in the duodenum varies from 20-40 per 100 epithelial cells (EC). The objectives of the study are to determine the normal upper limit of IEL in the duodenum and assess the diagnostic accuracy of existing criteria of IEL counts to diagnose CD. A retrospective analysis of histopathological records of duodenal biopsies reported as normal (control group, n=38) and consistent with CD (n=37) formed the basis of the study. IEL were counted in an uninterrupted length of surface epithelium and villous tips from formalin fixed, paraffin embedded, Haematoxylin and Eosin stained biopsies under the light microscope (X400 magnification). In the control group the mean IEL/100EC was 3.81(upper limit of normal = 7.78) and the mean IEL/villous tip was 0.96 (upper limit of normal = 3.5). In CD the mean IEL/100EC and mean IEL/villous tip were 20.93 and 6.83 respectively. The upper limit of normal IEL/100EC, mean IEL/100EC in CD and the villous tip IEL count in both the control and CD groups were considerably lower than those reported in other studies. The ethnicity, country of origin and environmental factors may be partly responsible for this observation. If high cut off values of IEL/100EC is taken to diagnose CD many cases may be under diagnosed, particularly when the upper limit of the normal IEL count is lower for that population and region, highlighting the importance of a multidisciplinary approach in the diagnosis of CD.

Intraepithelial Lymphocyte Normal Cut-off Level in Celiac Disease: The Debate Continues

The gold standard for diagnosis of celiac disease is positive serology confirmed by histological evidence of small intestinal biopsy damage as determined by the modified Marsh classification. Intraepithelial lymphocyte count has a pivotal importance in this grading criteria and the normal cut-off density has paramount importance in distinguishing between celiac disease and normal or false+ conditions. However its determination is problematic, complicated and far from being standardized. The localization along the villi or along the small bowel, the section thickness, the mode of detection and analysis, geoepidemiological influences, age dependency, associated infections or diseases and the strictness of the inclusions/exclusion criteria of the normal control group, all affect IEL density and normal cut-off levels. It is hoped that after standardization, the cut-off levels of IELs will better reflect the in-vivo reality to distinguish celiac disease from normal and associated conditions that affect intestinal IEL count.

Pathological and Clinical Correlation between Celiac Disease and Helicobacter Pylori Infection; a Review of Controversial Reports.

There are overwhelming reports and descriptions about celiac associated disorders. Although there is a clear genetic association between celiac disease (CD) and some gastrointestinal disorders, there are controversial reports claiming an association between CD and Helicobacter pylori (H. pylori) infection. Different studies indicated the possible association between lymphocytic gastritis and both CD and H. pylori infection, although this evidence is not consistently accepted. Also it was shown that an increase in intraepithelial lymphocytes count is associated with both H. pylori infection and celiac disease. Therefore the following questions may raise: how far is this infection actually related to CD?, which are the underlying patho-mechanisms for these associations? what are the clinical implications? what is the management? and what would be the role of gluten free diet in treating these conditions? PubMed (PubMed Central), Ovid, ISI of web knowledge, and Google scholar were searched for full text articles published between 1985 and 2015. The associated keywords were used, and papers described particularly the impact of pathological and clinical correlation between CD and H. pylori infection were identified. In this review we tried to answer the above questions and discussed some of the recent developments in the pathological and clinical aspects of CD and H. pylori infection.

May the assessment of baseline mucosal molecular pattern predict the development of gluten related disorders among microscopic enteritis?

AIMTo evaluate mucosal baseline mRNA expression of tissue transglutaminase 2 (tTG2), interferon gamma (IFNγ), toll-like receptor 2 (TLR2) and Myeloid Differentiation factor 88 (MyD88) in patients with microscopic enteritis (ME).METHODSWe retrospectively enrolled 89 patients with ME of different etiology, which was defined within a 2-year mean period of follow-up. Baseline histological examination was performed on Hematoxylin-Eosin stained sections and CD3 lymphocyte immunohistochemistry was used for intraepithelial lymphocyte count (IELs). ME was defined according to the criteria of Bucharest Consensus Conference. For each patient, formalin embedded biopsy samples of the duodenum referred to the period of ME diagnosis were retrieved. Real-time polymerase chain reaction (RT-PCR) was used to detect the amount of mRNA coding for tTG2, IFNγ, TLR2 and MyD88, and the quantity was expressed as fold change compared to controls. Control group was represented by duodenal normal specimens from 15 healthy subjects undergoing endoscopy for functional symptoms. Comparisons among continuous variables were performed by One way analysis of variance (ANOVA) and Bonferroni’s test. The χ2 test was used for categorical variables. Pearson’s test was used to evaluate correlations. Receiver operating curves were drawn for all four markers to estimate sensitivity and specificity in discriminating the development of CD and GS.RESULTSAfter a period of follow up of 21.7 ± 11.7 mo, the following diagnoses were achieved: gluten related disorders in 48 subjects (31 CD; 17 GS) and non-gluten related ones in 41 (29 Irritable Bowel Syndrome - IBS; 12 Others). CD patients had the highest tTG2 levels (8.3 ± 4.5). The ANOVA plus Bonferroni analysis showed that CD > Other ME > GS = IBS > negative controls. A cut off value of 2.258 was able to discriminate between CD and GS with a sensitivity of 52.94% and a specificity of 87.1%. Additionally, CD patients had the highest IFNγ levels (8.5 ± 4.1). ANOVA plus Bonferroni demonstrated CD > Other ME > GS = IBS > negative controls. A cut off of 1.853 was able to differentiate CD and GS with a sensitivity of 47.06% and a specificity of 96.77%. Patients with non gluten-related causes of ME exhibited the highest TLR2 levels (6.1 ± 1.9) as follows: Other ME > CD = GS = IBS > negative controls. TLR2 was unable to discriminate CD from GS. Patients with CD overexpressed MyD88 levels similarly to non gluten-related causes of DL (7.8 ± 4.9 and 6.7 ± 2.9), thus CD = Other ME > GS = IBS > negative controls. A cut off of 3.722 was able to differentiate CD from GS with a sensitivity of 52.94% and a specificity of 74.19%. IELs count (15-25 and more than 25/100 enterocytes) strongly correlated with mRNA levels of all tested molecules (P < 0.0001).CONCLUSIONOur results confirm that a single marker is unable to predict a discrimination among ME underlying conditions as well as between CD and GS. Mucosal high levels of tTG and IFNγ mRNA may predict the development of CD more than GS with high specificity, despite an expected low sensitivity. TLR2 does not discriminate the development of CD from GS. MyD88 levels indicate that intestinal permeability is more increased when a severe intestinal damage underlies ME in both gluten related and unrelated conditions. Therefore, the results of the present paper do not seem to show a clear translational value.

INTRAEPITHELIAL LYMPHOCYTES IN THE DUODENAL MUCOSA OF MEAT QUAILS RECEIVING DIETS CONTAINING OR NOT CONTAINING PROBIOTIC AND DIFFERENT LEVELS OF CRUDE PROTEIN

The aim of this study is to evaluate the effect of probiotic supplementation associated to different levels of crude protein (CP) on the count of intraepithelial lymphocytes (IELs) in the duodenum of meat quails. A total of 2304 quails were distributed in a completely randomized design in a 2 x 4 factorial scheme (with and without probiotic and four levels of CP - 15, 20, 25 and 30%), with two replicates per treatment in two experimental periods, in a total of 32 experimental units. At seven days old, two quails from each experimental unit were euthanized to harvest the duodenum segment. Semi-serial 7-μm histological sections were obtained, which were subsequently stained with hematoxylin-eosin to perform the IEL count. For the calculation, first 2500 epithelial cells were counted from the mucosa of each animal, and then the IELs present between these cells were counted, with the results expressed in amounts of IELs/100 epithelial cells. No differences were found in the IEL count among the treatments. Under the experimental conditions, it can be concluded that the use of probiotic associated to different levels of CP supplementation does not alter the IEL count in the duodenum of meat quails.

Seronegative Celiac Disease and Immunoglobulin Deficiency: Where to Look in the Submerged Iceberg?

In the present narrative review, we analyzed the relationship between seronegative celiac disease (SNCD) and immunoglobulin deficiencies. For this purpose, we conducted a literature search on the main medical databases. SNCD poses a diagnostic dilemma. Villous blunting, intraepithelial lymphocytes (IELs) count and gluten “challenge” are the most reliable markers. Immunohistochemistry/immunofluorescence tissue transglutaminase (tTG)-targeted mucosal immunoglobulin A (IgA) immune complexes in the intestinal mucosa of SNCD patients may be useful. In our experience, tTG-mRNA was similarly increased in seropositive celiac disease (CD) and suspected SNCD, and strongly correlated with the IELs count. This increase is found even in the IELs’ range of 15–25/100 enterocytes, suggesting that there may be a “grey zone” of gluten-related disorders. An immune deregulation (severely lacking B-cell differentiation) underlies the association of SNCD with immunoglobulin deficiencies. Therefore, CD may be linked to autoimmune disorders and immune deficits (common variable immunodeficiency (CVID)/IgA selective deficiency). CVID is a heterogeneous group of antibodies dysfunction, whose association with CD is demonstrated only by the response to a gluten-free diet (GFD). We hypothesized a familial inheritance between CD and CVID. Selective IgA deficiency, commonly associated with CD, accounts for IgA-tTG seronegativity. Selective IgM deficiency (sIgMD) is rare (<300 cases) and associated to CD in 5% of cases. We diagnosed SNCD in a patient affected by sIgMD using the tTG-mRNA assay. One-year GFD induced IgM restoration. This evidence, supporting a link between SNCD and immunoglobulin deficiencies, suggests that we should take a closer look at this association.

Helicobacter pylori in celiac disease and in duodenal intraepithelial lymphocytosis: Active protagonist or innocent bystander?

Helicobacter pylori (H. pylori) infection influences duodenal inflammation. Consequently, in celiac disease and in duodenal intraepithelial lymphocytosis, the bacterium could affect the clinical-histological manifestations. The aim of this work was to evaluate the prevalence and the potential role of H. pylori infection in celiac disease and duodenal intraepithelial lymphocytosis. H. pylori status was reviewed in 154 patients with celiac disease or duodenal intraepithelial lymphocytosis and in a control population. This retrospective study was performed at Molinette hospital, university of Torino, Italy. H. pylori prevalence was 36% in celiac disease patients, 19% in case of duodenal intraepithelial lymphocytosis and 41% in controls (P<0.05 vs. duodenal intraepithelial lymphocytosis). H. pylori prevalence was not significantly different between celiac disease patients with or without iron deficiency anemia (22% vs. 39%) and it was higher in patients with milder duodenal lesions: 50% in Marsh-Oberhuber classification type 1-2 vs. 33% in type 3. Celiac disease patients had a mean intraepithelial lymphocytes count greater than that of duodenal intraepithelial lymphocytosis patients (52 vs. 44 intraepithelial lymphocytes per 100 epithelial cells). Both in celiac disease and in duodenal intraepithelial lymphocytosis patients, H. pylori infection was associated with an increase in intraepithelial lymphocytes count, but this difference was not significant. H. pylori prevalence was similar in celiac disease patients and in controls and higher in patients with milder duodenal lesions. There was no association between H. pylori infection and duodenal intraepithelial lymphocytosis.

Coeliac-like duodenal pathology in orthotopic liver transplant patients on mycophenolic acid therapy.

Diarrhoea following orthotopic liver transplantation (OLT) is a significant clinical problem associated with mycophenolic acid (MPA). The histological injury pattern associated with MPA in the large bowel is well documented in the literature; however, that in the duodenum is less extensively documented. The aim of this study was to investigate the histological spectrum of duodenal injury specifically in symptomatic OLT patients on MPA, and to compare this with the spectrum in patients with coeliac disease and in normal controls. We reviewed our pathology database for all duodenal biopsies from patients on the OLT list over a period of 19 years. Medical records, anti-tissue transglutaminase IgA serology and histology were reviewed. Of the 667 patients who underwent endoscopy, 127 had duodenal biopsies (152 biopsies). Of these, 87.5% were normal. Sixteen showed abnormal histology, and seven (43.8%) of these were on MPA at the time of biopsy. Significant features included coeliac-like changes (shortened villi and increased intraepithelial lymphocyte counts), and novel findings included increased endocrine cell counts, apoptotic counts and lamina propria eosinophil counts in comparison with normal duodenal biopsies. Pathologists should be aware of the features of MPA-associated duodenal injury, including coeliac-like changes and increased apoptotic counts. In those with abnormal histology, discontinuation or a reduction in the dose of MPA should be discussed.

Evolution of ideas on microscopic colitis

The literature review gives the present-day views of the definition, etiology, pathogenesis, diagnosis, and treatment of microscopic colitis (MC). In the present view, MC is an inflammatory bowel disease of unknown etiology, which is characterized by chronic watery diarrhea, no macroscopic signs of large bowel involvement in the presence of specific pathomorphological changes. There are two major forms of MC, which are similar in its clinical picture, yet, heterogeneous in histological criteria: collagenous colitis (CC) and lymphocytic colitis (LC). As of now, the prevalence of MC is about 100 cases per 100,000 population, which is similar with that in other inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. MC generally prevails in women aged over 50 years. The etiology and pathogenesis of MC have not fully investigated. Watery diarrhea is as a predominant pathognomonic symptom in all the patients with MC. The major histological criterion for the diagnosis of CC is subepithelial collagen lining thickening (more than 10 pm) and that for LC is higher intraepithelial lymphocyte counts (more than 20 intraepithelial lymphocytes/100 epitheliocytes). The topical glucocorticosteroid budesonide is currently the only agent, the efficacy of which has been proven in both inducing and maintaining remission in patients with MC in many clinical trials.

Histological Correlation Between Intra-Epithelial Lymphocyte Counts Using Hematoxylin and Eosin and CD3

Histological Correlation Between Intra-Epithelial Lymphocyte Counts Using Hematoxylin and Eosin and CD3

Mutatis mutandis: Are we diagnosing too many people with non-celiac gluten sensitivity? Multiple case report

We report three patients presenting with gluten-related signs and symptoms. Since villous height/crypt depth ratio, intraepithelial lymphocyte count, and serum antibody tests were not diagnostic for celiac disease (CD), a diagnosis of non-celiac gluten sensitivity (NCGS) was suggested. On the other hand, antibodies suggestive for CD surprisingly showed positive results in the duodenal biopsy organ culture of all three cases. The reported cases suggest the precious potential role that organ culture systems may play in differentiating CD from NCGS. This method should be recommended when gluten-related disorders are suspected in order to reduce the inappropriate diagnosis of NCGS.

Tu1750 Microbial, Metabolomic, and Immunologic Dynamics During Disease Onset, Remission, and Relapse in a Mouse Model of Colitis Involving a Genetically Impaired Mucus Barrier

Introduction: Disturbances of the intestinal microbiota due to enhanced hygienic conditions or antibiotic use have been linked to the increasing prevalence of obesity, allergy, autoimmunity, and functional and inflammatory disorders of the gut, including celiac disease (CD). It is unknown whether perturbation of normal gut colonization plays a causal role in subsequent development of immune responses to gluten. Thus, our aim was to determine and characterize whether absence of gut commensals modifies the response to gluten using germ-free (GF), specific pathogen free (SPF) and altered Schaedler flora (ASF) colonized NOD/DQ8 mice. Methods: GF, SPF, and ASF 8-10 week old male and female NOD/DQ8 mice were mucosally sensitized with peptic tryptic (PT) gliadin (500μg) and cholera toxin (CT; 25μg) by oral gavage once a week for three weeks. Control mice were sensitized with CT alone. Following sensitization, mice were challenged three times a week for two weeks with 2mg of gluten by oral gavage. All materials for GF experiments were aseptically prepared and administered to mice housed in gnotobiotic isolators. Twenty-four hours following the final gluten challenge enteropathy was evaluated by intraepithelial lymphocyte (IEL) counts and villus/crypt (V/C) ratios. Anti-gliadin IgA antibodies (AGA) in intestinal washes were determined as markers of sensitization and specificity towards gliadin was tested by Western blot. Gliadin-specific T cell responses were tested using in vitro proliferation assays. Results: V/C ratios in GF NOD/DQ8 mice decreased by 52% following sensitization, which was significantly greater than SPF (40%) andASF (38%) gliadin-sensitizedmice (p<0.01). Gliadinsensitized GF mice also had the greatest increase in IELs compared to gliadin-sensitized SPF and ASF mice (p<0.01). No control GF, SPF or ASF mouse had a positive AGA result. In gliadin-sensitized mice, positive AGA was detected in 75% of GF mice, 33% of SPF mice and no ASF mice. The specificity of AGA towards gliadin was confirmed by Western blot. T cells isolated from mesenteric lymph nodes of GF gliadin-sensitized mice responded to PT-gliadin stimulation in vitro, but not to PT-zein stimulation. T cells from non-sensitized mice did not respond to PT-gliadin stimulation. Conclusions: Gluten-induced enteropathy and development of gliadin-specific AGA was enhanced under germ-free conditions in NOD/ DQ8 mice and T cell responses are gliadin-specific. These results suggest that the commensal intestinal microbiota may have a protective effect during gliadin-sensitization and challenge in NOD/DQ8mice. Themechanisms leading to this protective effect are under current investigation.

Tu1751 The Convergence of Stasis-Induced Dysbiosis, Colonic Metaplasia, and Genetic Susceptibility in Increasing Risk of Pouchitis and Ulcerative Colitis

Introduction: Disturbances of the intestinal microbiota due to enhanced hygienic conditions or antibiotic use have been linked to the increasing prevalence of obesity, allergy, autoimmunity, and functional and inflammatory disorders of the gut, including celiac disease (CD). It is unknown whether perturbation of normal gut colonization plays a causal role in subsequent development of immune responses to gluten. Thus, our aim was to determine and characterize whether absence of gut commensals modifies the response to gluten using germ-free (GF), specific pathogen free (SPF) and altered Schaedler flora (ASF) colonized NOD/DQ8 mice. Methods: GF, SPF, and ASF 8-10 week old male and female NOD/DQ8 mice were mucosally sensitized with peptic tryptic (PT) gliadin (500μg) and cholera toxin (CT; 25μg) by oral gavage once a week for three weeks. Control mice were sensitized with CT alone. Following sensitization, mice were challenged three times a week for two weeks with 2mg of gluten by oral gavage. All materials for GF experiments were aseptically prepared and administered to mice housed in gnotobiotic isolators. Twenty-four hours following the final gluten challenge enteropathy was evaluated by intraepithelial lymphocyte (IEL) counts and villus/crypt (V/C) ratios. Anti-gliadin IgA antibodies (AGA) in intestinal washes were determined as markers of sensitization and specificity towards gliadin was tested by Western blot. Gliadin-specific T cell responses were tested using in vitro proliferation assays. Results: V/C ratios in GF NOD/DQ8 mice decreased by 52% following sensitization, which was significantly greater than SPF (40%) andASF (38%) gliadin-sensitizedmice (p<0.01). Gliadinsensitized GF mice also had the greatest increase in IELs compared to gliadin-sensitized SPF and ASF mice (p<0.01). No control GF, SPF or ASF mouse had a positive AGA result. In gliadin-sensitized mice, positive AGA was detected in 75% of GF mice, 33% of SPF mice and no ASF mice. The specificity of AGA towards gliadin was confirmed by Western blot. T cells isolated from mesenteric lymph nodes of GF gliadin-sensitized mice responded to PT-gliadin stimulation in vitro, but not to PT-zein stimulation. T cells from non-sensitized mice did not respond to PT-gliadin stimulation. Conclusions: Gluten-induced enteropathy and development of gliadin-specific AGA was enhanced under germ-free conditions in NOD/ DQ8 mice and T cell responses are gliadin-specific. These results suggest that the commensal intestinal microbiota may have a protective effect during gliadin-sensitization and challenge in NOD/DQ8mice. Themechanisms leading to this protective effect are under current investigation.

Tu1749 Gluten-Induced Responses in NOD/DQ8 Mice Are Influenced by Bacterial Colonization

Introduction: Disturbances of the intestinal microbiota due to enhanced hygienic conditions or antibiotic use have been linked to the increasing prevalence of obesity, allergy, autoimmunity, and functional and inflammatory disorders of the gut, including celiac disease (CD). It is unknown whether perturbation of normal gut colonization plays a causal role in subsequent development of immune responses to gluten. Thus, our aim was to determine and characterize whether absence of gut commensals modifies the response to gluten using germ-free (GF), specific pathogen free (SPF) and altered Schaedler flora (ASF) colonized NOD/DQ8 mice. Methods: GF, SPF, and ASF 8-10 week old male and female NOD/DQ8 mice were mucosally sensitized with peptic tryptic (PT) gliadin (500μg) and cholera toxin (CT; 25μg) by oral gavage once a week for three weeks. Control mice were sensitized with CT alone. Following sensitization, mice were challenged three times a week for two weeks with 2mg of gluten by oral gavage. All materials for GF experiments were aseptically prepared and administered to mice housed in gnotobiotic isolators. Twenty-four hours following the final gluten challenge enteropathy was evaluated by intraepithelial lymphocyte (IEL) counts and villus/crypt (V/C) ratios. Anti-gliadin IgA antibodies (AGA) in intestinal washes were determined as markers of sensitization and specificity towards gliadin was tested by Western blot. Gliadin-specific T cell responses were tested using in vitro proliferation assays. Results: V/C ratios in GF NOD/DQ8 mice decreased by 52% following sensitization, which was significantly greater than SPF (40%) andASF (38%) gliadin-sensitizedmice (p<0.01). Gliadinsensitized GF mice also had the greatest increase in IELs compared to gliadin-sensitized SPF and ASF mice (p<0.01). No control GF, SPF or ASF mouse had a positive AGA result. In gliadin-sensitized mice, positive AGA was detected in 75% of GF mice, 33% of SPF mice and no ASF mice. The specificity of AGA towards gliadin was confirmed by Western blot. T cells isolated from mesenteric lymph nodes of GF gliadin-sensitized mice responded to PT-gliadin stimulation in vitro, but not to PT-zein stimulation. T cells from non-sensitized mice did not respond to PT-gliadin stimulation. Conclusions: Gluten-induced enteropathy and development of gliadin-specific AGA was enhanced under germ-free conditions in NOD/ DQ8 mice and T cell responses are gliadin-specific. These results suggest that the commensal intestinal microbiota may have a protective effect during gliadin-sensitization and challenge in NOD/DQ8mice. Themechanisms leading to this protective effect are under current investigation.

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.

Glutenase ALV003 Attenuates Gluten-Induced Mucosal Injury in Patients With Celiac Disease

Gluten ingestion leads to small intestinal mucosal injury in patients with celiac disease, necessitating strict life-long exclusion of dietary gluten. Despite adherence to a gluten-free diet, many patients remain symptomatic and still have small intestinal inflammation. In this case, nondietary therapies are needed. We investigated the ability of ALV003, a mixture of 2 recombinant gluten-specific proteases given orally, to protect patients with celiac disease from gluten-induced mucosal injury in a phase 2 trial. We established the optimal daily dose of gluten to be used in a 6-week challenge study. Then, in the intervention study, adults with biopsy-proven celiac disease were randomly assigned to groups given ALV003 (n= 20) or placebo (n= 21) together with the daily gluten challenge. Duodenal biopsies were collected at baseline and after gluten challenge. The ratio of villus height to crypt depth and densities of intraepithelial lymphocytes were the primary end points. A daily dose of 2 g gluten was selected for the intervention study. Sixteen patients given ALV003 and 18 given placebo were eligible for efficacy evaluation. Biopsies from subjects in the placebo group showed evidence of mucosal injury after gluten challenge (mean villus height to crypt depth ratio changed from 2.8 before challenge to 2.0 afterward; P= .0007; density of CD3(+) intraepithelial lymphocytes changed from 61 to 91 cells/mm after challenge; P= .0003). However, no significant mucosal deterioration was observed in biopsies from the ALV003 group. Between groups, morphologic changes and CD3(+) intraepithelial lymphocyte counts differed significantly from baseline to week 6 (P= .0133 and P= .0123, respectively). There were no statistically significant differences in symptoms between groups. Based on a phase 2 trial, the glutenase ALV003 appears to attenuate gluten-induced small intestinal mucosal injury in patients with celiac disease in the context of an everyday gluten-free diet containing daily up to 2 g gluten. Clinicaltrial.gov, NCT00959114 and NCT01255696.

Intraepithelial lymphocytes in category IV prostatitis

Category IV chronic prostatitis (CP) is an asymptomatic inflammatory prostatitis. Different injury agents can caused damage of the prostate epithelial cells and initiate an immune response. The main type of the cells involved in the immunity of the prostate is lymphocytes, particulary intraepithelial lymphocytes (IEL). <h3>Aim</h3> A quantitative analysis of the IEL population in prostate regarding a presence of CP. <h3>Methods</h3> Fifty-nine non-consecutive autopsy prostate glands were retrospectively evaluated for presence of CP. The density of IELs was scored using CD3 immunohistochemistry. Cell count was performed in two steps: within inflammatory zones and in areas around inflammation. <h3>Results</h3> Patients' ages ranged from 19 to 87 years (mean 50.9). The vast majority of the IEL were T-cells. There was significant difference (<i>p</i><0,001) in the mean IELs number between non-CP group (0.61±0.34; n=19) and group with CP (3.43±2.38) around; 8.53±3.25 with inflammation (n=40). In patients with mild (n=13) and moderate (n=16) grade of CP the mean IELs count was significantly higher around inflammatory areas (9.51±2.72 and 13.67±3.72) than within them (2.15±1.02 and 2.89±1.89); <i>p</i>=0.0009 and <i>p</i>=0.0064, respectively. <h3>Conclusion</h3> IELs have a fundamental role in the development of CP, suggesting an autoimmune character of the process.

Colonic Mucosal Immune Activity in Irritable Bowel Syndrome: Comparison with Healthy Controls and Patients with Ulcerative Colitis

Mucosal immune activity may participate in irritable bowel syndrome (IBS) pathogenesis. Mast- and T cell numbers from patients with IBS or ulcerative colitis (UC) and healthy controls were determined. Between November 2007 and May 2012, patients with diarrhea-predominant IBS (D-IBS, n = 83), 49 patients with UC, and 25 healthy controls were recruited. Of the UC group, 28 were in remission and 21 had mildly active UC. Biopsies from each colon segment were subjected to immunohistochemical analysis. The mast cells, intraepithelial lymphocytes (IELs), and lamina proprial lymphocytes (LPLs) were counted. Compared to the healthy controls, the patients with D-IBS, UC in remission, and mildly active UC had significantly higher mean colorectal mucosal mast-cell, IEL, and LPL counts. Comparison with the colon segments (ascending, transverse, descending, and sigmoid segments) that had once been involved in UC (in the patients with remission) revealed that the D-IBS colons had similar immune-cell counts. However, they had significantly fewer immune cells than the colon segments that presently showed involvement in the patients with mildly-activated UC. The mast-cell and IEL counts were similar in the D-IBS rectums and once-involved UC rectums but significantly higher in the presently-involved UC rectums. However, both the once-involved and presently-involved UC rectums had significantly higher LPL counts than the D-IBS rectums. Patients with D-IBS had significantly higher colonic mucosal immune-cell counts than healthy controls but had similar counts to patients with UC in remission. The symptoms in both conditions may originate from low-grade inflammation in the colonic mucosa.