Intraepithelial Lymphocyte Counts Research Articles

A new algorithm for coeliac disease based on the 'long forgotten' TCRγδ+ intra-epithelial lymphocytes detected with an antibody working on FFPE sections.

Diagnosis of coeliac disease (CD) with mild mucosal changes is difficult for all parties involved. We aimed to determine the power of Tcell receptor (TCR)γδ+ intra-epithelial lymphocytes (IELs) in discriminating CD from other causes of intra-epithelial lymphocytosis using a new monoclonal antibody. A total of 167 cases categorised as coeliac (117 untreated CD, classified according to Marsh, updated by Ensari, including 29 type 1, 29 type 2, 39 type 3 and 20 treated CD), and non-coeliac groups (24 controls and 26 non-coeliac IELosis) based on clinical, serological and histological data were studied for IEL counts enumerated per 100 enterocytes using haematoxylin and eosin, CD3, TCR δ-stains. TCRγδ+ IELs were significantly higher in CD (24.83 ± 16.13) compared to non-CD (6.72 ± 6.32) and were correlated with the degree of mucosal damage. Both γδ+ IEL count and ratio showed higher performance in differentiating untreated coeliacs from controls, with a sensitivity of 83.76; 85.57 and specificity of 95.83; 79.17, respectively. TCRγδ+ IEL counts distinguished type 1 CD (20.41 ± 13.57) from non-coeliac IELosis (9.42 ± 7.28) (p = 0.025). Discriminant analysis revealed that villus/crypt ratio, γδ+ and CD3+ IEL counts, γδ+/CD3+IEL ratio, IEL distribution pattern were potent discriminants and correctly classified 82.3% of cases while the algorithm accurately diagnosed 93.4% of cases. The new antibody detecting γδ+ IELs in FFPE sections revealed thresholds of 10.5 for γδ+ IELs and 14% for γδ+/CD3+IEL ratio which distinguished coeliacs from non-coeliacs with high sensitivity and specificity, particularly in cases with normal villus/crypt axis including type 1 CD, non-CD IELosis and controls. A 'coeliac algorithm' based on γδ+ IELs is proposed with the hope that it will be used in the histopathological diagnostic approach by the pathology community.

Growth performance, mucosal immunity and disease resistance in goldfish (Carassius auratus) orally administered with Escherichia coli Strain Nissle 1917.

The current research aimed to shed light on the efficacy of Escherichia coli strain Nissle 1917 (EcN) on goldfish (……) growth, gut immunity, morphology, bacterial nutritional enzyme activity and resistance to Aeromonas hydrophila infection. Fish fed with EcN at 106, 107 and 108CFU/g feed for 80days showed an enhancement in growth better than control fish. The gut innate immunity in terms of lysozyme activity, immunoglobulin and total protein levels was increased in the treatment fish with the best result being observed in fish fed EcN at 108CFU/ g. In addition, an increase was noted in the upregulation of immune-relevant genes, namely lysozyme, interleukin-1β, inducible nitric oxide synthase and tumor necrosis factor α of fish intestine. A marked surge in the number of proteolytic and heterotrophic bacteria was noted in the gut of fish nourished with the probiotic. Histological studies exhibited an improvement in the intestinal absorption surface area, intraepithelial lymphocyte count and goblet cell density. Significantly higher survival rate was obtained in fish fed EcN at 108CFU/g compared with the fish fed with the basal diet. These data exhibited the beneficial effect of EcN on goldfish growth, digestive enzymes, intestine heterotrophic bacteria and resistance against Aeromonas hydrophila challenge. This study confirmed the favorable outcomes resulting from the administration of EcN at108 CFU/g.

A36 EFFECT OF FIBRE SUPPLEMENTATION IN CELIAC DISEASE: IMPLICATIONS FOR MUCOSAL HEALING

Abstract Background Celiac disease (CeD) is a T-cell mediated enteropathy driven by gluten in genetically susceptible individuals. Mucosal inflammation persists for years even when treated with a gluten-free diet (GFD). Although patients may be advised to increase dietary fibre, some fibres are poorly tolerated leading, potentially, to suboptimal fibre intake and hampering dietary recommendations. Aims Our project aims to investigate whether and how the supplementation of two fibre types with distinct functional profiles affects the recovery of gluten-immunopathology in a mouse model of gluten-sensitivity. We hypothesize that: (1) HylonVII, but not inulin, accelerates mucosal recovery by increasing microbial carbolytic activity and production of short-chain fatty acids (SCFA); (2) fibre intake is decreased in CeD patients, leading to decreased SCFA production. Methods NOD-DQ8 mice were immunized to gliadin and then placed on a gluten-containing diet. Three weeks later, mice were returned to GFD supplemented with or without HylonVII or inulin to 15% total fibre, for 6, 10, or 12 weeks. Recovery from gluten-induced pathology was evaluated by counting CD3+ intraepithelial lymphocytes (IELs) in villi tips and measuring villus-to-crypt (V/C) ratios in the small intestine. Adult CeD patients (newly diagnosed n=6 and GFD-treated n=7) and healthy controls (n=5) completed a food frequency questionnaire (Victoria DQES v2) and provided fecal samples for evaluation of SCFA production. Fecal SCFA were measured with targeted single ion monitoring gas-chromatography/mass-spectrometry (Agilent) as markers of fibre metabolism. Results In gluten immunized mice, 3 weeks of wheat diet led to increased CD3+ IEL counts and decreased V/C ratios (pampersand:003C0.01), which remained abnormal after 6 weeks of GFD. Mice consuming GFD+inulin, but not HylonVII, normalized CD3+ IELs and V/C ratios, had higher fecal SCFAs (pampersand:003C0.05) (primarily acetate), and a higher carbolytic-proteolytic metabolite ratio (pampersand:003C0.05) compared with mice consuming a GFD alone for six weeks. Only 1/7 patients with CeD on GFD met the minimum recommendation for fibre intake (25 g/day, Health Canada), compared with 5/11 participants on gluten-containing diets. SCFA production was impaired in active but not in treated CeD patients (2563 vs 3744 μg/g, pampersand:003C0.01). Conclusions In gluten-immunized NOD-DQ8 mice, GFD+inulin accelerates mucosal recovery, which is associated with increased fecal SCFA. In our pilot clinical data, patients on a GFD did not meet the recommendation for fibre intake although they were capable of metabolizing fibre to produce SCFA that could facilitate mucosal recovery. Together these results suggest that CeD on GFD may benefit from increasing fibre, such as inulin, during the GFD. Funding Agencies CIHRCeliac Canada

Evaluating the Effects of Chronic Oral Exposure to the Food Additive Silicon Dioxide on Oral Tolerance Induction and Food Sensitivities in Mice.

The increasing prevalence of food sensitivities has been attributed to changes in gut microenvironment; however, ubiquitous environmental triggers such as inorganic nanoparticles (NPs) used as food additives have not been thoroughly investigated. We explored the impact of the NP-structured food-grade silicon dioxide () on intestinal immune response involved in oral tolerance (OT) induction and evaluated the consequences of oral chronic exposure to this food-additive using a mouse model of OT to ovalbumin (OVA) and on gluten immunopathology in mice expressing the celiac disease risk gene, HLA-DQ8. Viability, proliferation, and cytokine production of mesenteric lymph node (MLN) cells were evaluated after exposure to . C57BL/6J mice and a mouse model of OT to OVA were orally exposed to or vehicle for 60 d. Fecal lipocalin-2 (Lcn-2), anti-OVA IgG, cytokine production, and immune cell populations were analyzed. Nonobese diabetic (NOD) mice expressing HLA-DQ8 (NOD/DQ8), exposed to or vehicle, were immunized with gluten and immunopathology was investigated. MLN cells exposed to presented less proliferative T cells and lower secretion of interleukin 10 (IL-10) and transforming growth factor beta () by T regulatory and cells compared to control, two factors mediating OT. Mice given exhibited intestinal Lcn-2 level and interferon gamma () secretion, showing inflammation and less production of IL-10 and . These effects were also observed in OVA-tolerized mice exposed to , in addition to a breakdown of OT and a lower intestinal frequency of T cells. In NOD/DQ8 mice immunized with gluten, the villus-to-crypt ratio was decreased while the intraepithelial lymphocyte counts and the Th1 inflammatory response were aggravated after treatment. Our results suggest that chronic oral exposure to blocked oral tolerance induction to OVA, and worsened gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between exposure and food sensitivities in humans. https://doi.org/10.1289/EHP12758.

Supplementing infant milk formula with a multi-strain synbiotic and osteopontin enhances colonic microbial colonization and modifies jejunal gene expression in lactating piglets.

A total of ninety-six weaned piglets were assigned to four dietary treatments in a 2 × 2 design. The treatments included: a standard milk formula (CTR); CTR + probiotics (6.4 × 108 cfu L-1Bifidobacterium longum subsp. infantis CECT 7210 and 1.1 × 108 cfu L-1Lactobacillus rhamnosus NH001) + prebiotics (galacto-oligosaccharides 4.36 g L-1 and human-milk-oligosaccharide 0.54 g L-1) (SYN); CTR + osteopontin (0.43 g L-1) (OPN); and CTR + SYN + OPN (CON). Daily records including feed intake, body weight, and clinical signs, were maintained throughout the 15-day trial. At the end of the study samples from blood, digestive content, and gut tissues were collected to determine serum TNF-α, intestinal fermentative activity (SCFA and ammonia), colonic microbiota (16S rRNA Illumina-MiSeq), histomorphology, and jejunal gene expression (Open-Array). No statistical differences were found in weight gain; however, the animals supplemented with osteopontin exhibited higher feed intake. In terms of clinical signs, synbiotic supplementation led to a shorter duration of diarrhoea episodes. Regarding gut health, the sequenced faecal microbiota revealed better control of potentially dysbiotic bacteria with the CON diet at day 15. In the colon compartment, a significant increase in SCFA concentration, a decrease in ammonia concentration, and a significant decrease in intraepithelial lymphocyte counts were particularly observed in CON animals. The supplemented diets were also associated with modified jejunal gene expression. The synbiotic combination was characterized by the upregulation of genes related to intestinal maturation (ALPI, SI) and nutrient transport (SLC13A1, SLC15A1, SLC5A1, SLC7A8), and the downregulation of genes related to the response to pathogens (GBP1, IDO, TLR4) or the inflammatory response (IDO, IL-1β, TGF-β1). Osteopontin promoted the upregulation of a digestive function gene (GCG). Correlational analysis between the microbiota population and various intestinal environmental factors (SCFA concentration, histology, and gene expression) proposes mechanisms of communication between the gut microbiota and the host. In summary, these results suggest an improvement in the colonic colonization process and a better modulation of the immune response when milk formula is supplemented with the tested synbiotic combined with osteopontin, benefiting from a synergistic effect.

Intestinal Barrier and Gut Microbiota in Patients with Overlapping Irritable Bowel Syndrome and Functional Dyspepsia.

Disturbances in the intestinal barrier and gut dysbiosis have been observed in patients with functional bowel diseases. To investigate the correlation between biomarkers of intestinal barrier disorders at different layers and the severity of symptoms in patients with overlapping diarrhea-predominant irritable bowel syndrome and functional dyspepsia (IDFO), as well as with gut microbiota taxa. This study included 45 patients with IDFO and 16 healthy controls. Endoscopy with biopsy of the duodenum and sigmoid colon (SC) was performed to count intraepithelial lymphocytes (IELs) and mucosal eosinophils (subepithelial layer), assess fatty acid binding protein (FABP; epithelial layer) level, and stain for mucin-2 (MUC-2; pre-epithelial layer). Composition of the gut microbiota was evaluated using 16S rRNA gene sequencing. Patients with IDFO exhibited an increase in biomarkers of intestinal barrier disorders at all layers studied. IEL count in the duodenum was correlated with the severity of bloating (r = 0.336; p = 0.024) and, in the SC, was correlated with tenesmus severity (r = 0.303; p = 0.042). FABP-1 level in the SC was correlated with the severity of diarrhea (r = 0.577; p = 0.001), and FABP-5 concentration in the SC was correlated with abdominal distension (r = 0.477; p = 0.010). MUC-2 concentration in the duodenum was correlated with the severity of heartburn (r = 0.572; p = 0.025) and burning sensation in the epigastrium (r = 0.518; p = 0.048). All biomarkers of intestinal barrier permeability were correlated with the abundance of some gut microbiota taxa. Patients with IDFO exhibited disrupted intestinal barrier function in all layers, which was associated with clinical symptom severity and changes in the gut microbiota.

Effects of dietary supplementation with purple garlic powder and oregano essential oil on intestinal health in post-weaning piglets from commercial farms

This work studied the effects of the inclusion of Purple Garlic Powder (PGP) and Oregano Essential Oil (OEO) in the feed, at different doses and combinations, on intestinal health and the growth performance of 140 and 3000 piglets, respectively, weaned at 21 days of age. Seven dietary treatments were used: a negative control group (basal diet), a positive control group with ZnO (3000 mg/Kg of feed), two groups with OEO at 0.4% and 1.2% respectively, two groups with PGP at 0.4% and 2% respectively and one group with OEO at 1.2% combined with PGP at 2%. Only the positive control group received ZnO in the diet. Each group of piglets received the treatment for seven weeks, from weaning, and were later sacrificed to obtain jejunum and ileum samples for counting of goblet cells, intraepithelial lymphocytes, and IgA-producing cells. The growth performance were measured at the beginning and at the end of the seven weeks. In jejunum and ileum, the number of goblet cells increased in the groups with ZnO, PGP 2%, OEO 1.2% and PGP 2% + OEO 1.2%, presenting significant differences with the rest of the groups. The results obtained for the intraepithelial lymphocyte count were in line with those obtained for the count of goblet cells. Regarding IgA-producing cells, the groups that showed significantly favourable results in the jejunum and ileum were OEO 1.2%, PGP 2% and their combination, but the groups that showed the most similar means to ZnO were the OEO 0.4% and the PGP 0.4%. Regarding the growth performance, PGP 2%, OEO 1.2% and their combination had similar results to ZnO. The intestinal health of piglets could be improved, without harming the growth performance, by means of the supplementation of PGP 2%, OEO 1.2% and PGP 2% + OEO 1.2% offering a natural alternative to the use of ZnO.

Impact of Dietary Supplementation with Sodium Butyrate Protected by Medium-Chain Fatty Acid Salts on Gut Health of Broiler Chickens.

Simple SummaryNutritional strategies to improve gut health are under research to reduce antibiotic use in poultry production. This study investigated the effect of dietary supplementation with sodium butyrate protected by sodium salts of medium-chain fatty acids as a feed additive on broiler gut health. A first trial was conducted to assess the effectiveness of this feed additive supplemented at a dose range of 0.5, 1, and 2 kg/t to promote a good health status on broilers raised under optimal conditions. Supplementation at 0.5 and 1 kg/t maintained the number of mucin-secretory cells contained in the gut barrier of young chickens, and the use of 1 kg/t improved the intestinal immune system of aged broilers. However, the beneficial effects of some feed additives are not detected under non-challenged conditions. Therefore, the second experiment was performed to evaluate the effect of the feed additive at 1 kg/t in coccidiosis-challenged broilers. In this context, sodium butyrate protected by sodium salts of medium-chain fatty acids restored the number of mucin-secretory cells as well as impacted on the intestinal morphometry and microbiota. The results of the present study suggest that this feed additive could be a useful strategy to reinforce the gut barrier, especially for birds with coccidiosis.Nutritional strategies to improve gut health of broilers are under research. This study investigated the effect of dietary supplementation with sodium butyrate protected by sodium salts of medium-chain fatty acids as a feed additive on broiler gut health. The first experiment was conducted to evaluate the effect of supplementing at 0.5, 1, and 2 kg/t in broilers housed under optimal conditions. Supplementation at 0.5 and 1 kg/t maintained goblet cell counts at 10 days of age (p ≤ 0.05), and supplementation at 1 kg/t decreased intraepithelial lymphocyte counts compared to 2 kg/t at 39 days (p ≤ 0.10). Abdominal fat pad levels of lauric and myristic acids were gradually increased by supplement dose (p ≤ 0.05). In the second experiment, the feed additive at 1 kg/t was evaluated in coccidiosis-challenged broilers. Experimental treatments were as follows: non-challenged, control-challenged, and supplemented-challenged treatments. Coccidiosis negatively impact performance and modify histomorphometry and microbiota (p ≤ 0.05). The feed additive increased crypt depth at 7 days post-inoculation and goblet cell count at 14 days post-inoculation (p ≤ 0.05). Further, supplementation interacted with the microbiota modification led by the coccidiosis (p ≤ 0.05). These results suggest that this feed additive could be a useful strategy to reinforce the gut barrier, especially for birds under coccidiosis-challenge treatments.

Immunohistochemical analysis of intestinal biopsies in individuals with celiac disease.

Background and AimsThe immunohistochemical application of CD3 (T lymphocytes) and CD20 (B lymphocytes) markers in duodenal biopsy can facilitate the detection of the number and distribution of intraepithelial lymphocytes along the villi, which is regarded as a key factor for accurate diagnosis of celiac disease. This study aims at finding a relationship between CD3 and CD20 immunohistochemical and histopathological alterations of celiac disease, and at investigating whether the application of those immunohistochemical stainings would improve the detection of lymphocytosis within the epithelium and add advantages to celiac disease diagnosis.MethodsBiopsies were obtained from 100 individuals and stained with hematoxylin and eosin (H&E). They were then evaluated according to the Marsh classification. After that, staining for CD3 and CD20 was individually done and assessed.ResultsThe overall mean intraepithelial lymphocyte count per 100 enterocytes for H&E was 23.1 (95% confidence interval [CI] = 19.52–26.68), and for immunohistochemistry by CD3 and CD20 was 27.84 (95% CI = 24.31–31.38). The difference was highly significant, P = 0.001. The expression of CD3 immunohistochemically was as follows: Less‐than‐half staining pattern was reported in 16% cases, and half staining pattern was seen in 26%, while most cases 58% had more than half staining pattern. This discovery was consistent with the histological classification of March III among most cases. The expression of CD20 immunohistochemically was as follows: mild crypt involvement was observed in 16% of cases, while moderate crypt involvement and intense crypt involvement were seen in 43% and 41% of cases, respectively.

Evaluation of High Doses of Phytase in a Low-Phosphorus Diet in Comparison to a Phytate-Free Diet on Performance, Apparent Ileal Digestibility of Nutrients, Bone Mineralization, Intestinal Morphology, and Immune Traits in 21-Day-Old Broiler Chickens.

Simple SummaryPhytic acid is the main storage form for phosphorus in plants. Although it is the main source of organic phosphorus in broiler diets, its utilization by monogastric animals is limited. In addition, phytic acid displays antinutritional effects, impacting optimal nutrient and energy digestibility. The supplementation of feed with phytases enables broilers to use phytate phosphorus more efficiently. Superdosing of phytase has been reported to have additional beneficial effects on the animal beyond the ones derived from the improved phosphorus utilization. This study tried to elucidate the mechanisms related to the immune response and mucosal morphology contributing to those overall beneficial effects. Although addressing performance was not the primary target of the study, performance improved linearly with increasing levels of phytase. The use of increasing doses of phytase showed linear increases in apparent ileal digestibility of crude protein, crude ash, and phosphorus. Jejunal crypt depth decreased with the addition of phytase to a low-protein diet, as well as the content of CD3-positive intraepithelial lymphocytes, but the results could not demonstrate clear differences between phytase doses in these specific parameters.The supplementation of feed with phytases enables broilers to utilize more efficiently phosphorus (P) from phytic acid (IP6), the main storage form of P in plants. The current study evaluated the addition of 500, 1000, and 3000 FTU of phytase per kg to a phytate-containing diet with low P level (LP) fed to broilers from 1 to 21 days of age and compared it to a hypoallergenic phytate-free diet (HPF). There was a linear improvement in performance parameters with increasing levels of phytase in the LP diet (p < 0.001). Apparent ileal digestibility of crude protein, P, and some amino acids, increased with phytase. Crude ash, P, and the calcium content of tibia bones linearly increased with increasing levels of phytase (p < 0.001). Crypt depth (related to body weight) in the jejunum epithelium linearly decreased with phytase addition (p < 0.001). Cecal crypt depth decreased with phytase supplementation (p = 0.002). Cecum tissue showed lower counts of CD3-positive intraepithelial lymphocytes in broilers receiving the phytase in comparison to LP (p < 0.001), achieving similar counts to HPF-fed broilers. Although results from the current study seem to point out some mechanisms related to the immune response and mucosal morphology contributing to those overall beneficial effects, no clear differences between different phytase doses could be demonstrated in these specific parameters.

The combination of clinical parameters and immunophenotyping of intraepithelial lymphocytes allows to assess disease severity in refractory celiac disease

BackgroundFlow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis. AimsTo investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease. MethodsFlow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data. ResultsFlow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients’ allocation (sensitivity 100%, specificity 96%). ConclusionImmunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

Clinicopathological and Microbiological Profile of Patients with Chronic Diarrhoea and Malabsorption with Special Emphasis on Duodenal Biopsies: A Study from Tertiarycare Hospital in South India

Background: Chronic diarrhoea and malabsorption continue to be a major health problem worldwide. Due to conflicting reports regarding the clinicohistological spectrum of malabsorption, this study was conducted to study the spectrum of malabsorption.&#x0D; Objective: To study the morphologic spectrum of duodenal biopsies and stool microbiological profile in patients with chronic diarrhoea and malabsorption.&#x0D; Materials and Methods: The study included 106 patients aged above 18 years, presenting with chronic diarrhoea or malabsorption. Duodenal mucosal biopsy obtained was evaluated for villous atrophy, crypt architecture and Intraepithelial lymphocyte count (IEL) by H&amp;E and immunohistochemistry for CD3. Stool samples collected were subjected to wet mount and culture examination. Serological testing for antibodies to coeliac sprue were also performed.&#x0D; Results: This descriptive study included 57 males and 49 females. Duodenal biopsy spectrum consisted of chronic duodenitis (48), duodenitis associated with autoimmune conditions (20), histomorphology of coeliac sprue (5), duodenitis with villous atrophy (14), only increased IEL (11) and miscellaneous (8). CD3 stained sections (n=90) offer a slightly better advantage over H &amp; E-stained sections in detecting IEL. Stool microscopy and culture were positive in 6 cases. Coeliac sprue antibodies were positive in 1 case with Marsh type 1 lesion.&#x0D; Conclusion: Duodenal biopsy is a useful parameter in the study of malabsorption which must be adjuncted by microbiological and serological tests. Coeliac sprue is a rare entity in our population as per this study.&#x0D; Key words: Chronic diarrhoea, Malabsorption, Duodenal biopsy, Serological testing

Increase of Deep Intraepithelial Lymphocytes in the Oxyntic Mucosa of Patients With Potential and Overt Autoimmune Gastritis.

Pathological correlates of potential autoimmune gastritis (AIG), defined by anti-parietal cell antibody (PCA) positivity in the absence of gastric atrophy, have never been described. We herein aimed to assess intraepithelial lymphocyte (IEL) infiltration in gastric corpus of AIG patients. From 2000 to 2021, among 53 potential AIG patients, we focused on nine (median age 61 years, IQR 53-82; four females) who subsequently developed overt AIG. IEL infiltration of the oxyntic mucosa was assessed before and after developing overt AIG by measuring deep and superficial CD3+ IEL. AIG patients with different degrees of corpus atrophy, healthy controls (HC), active H. pylori gastritis, celiac disease (CD), and Hashimoto’s thyroiditis patients were included as controls. Of note, deep, but not superficial, CD3+ IEL count was higher (p<0.001) in potential AIG compared to HC and H. pylori gastritis. Deep CD3+ IEL infiltration did not change before or after the evolution into atrophy (median 9.6, IQR 8.8-12.4, vs 11.3, IQR 9.4-12.9). No difference was found in deep CD3+ IEL infiltration among potential, mild, and severe AIG, and compared to Hashimoto’s thyroiditis or CD. A deep CD3+ IEL cut-off of >7/100 epithelial cells allowed discrimination of any AIG stage and severity (AUC=0.842). We conclude that an increased deep CD3+ IEL infiltration of the oxyntic mucosa could represent a marker of potential AIG. Prospective studies including a larger number of potential AIG patients are needed.

A10 EARLY LIFE SENSITIZATION TO GLUTEN INDUCES SUSTAINED IMMUNOPATHOLOGY IN DR3-DQ2 MICE

Abstract Background Celiac disease (CeD) is an autoimmune T-cell mediated enteropathy, triggered by gluten, a group of proteins found in wheat, barley, and rye. The defined role of gluten as a dietary trigger, necessary genes (HLA-DQ2 and/or DQ8), and tissue transglutaminase (TG2) as the autoantigen together, are unique features of CeD. Although CeD onset can occur at any age, first dietary introduction of gluten during infancy is a critical window of exposure, especially in infants homozygous for the HLA DQ2.5 allele. While adult sensitization studies have been recently performed in DR3-DQ2 mice, the consequences of early life gluten sensitization timing remain unexplored. Aims Our aim was to characterize gluten-immunopathology and CeD-specific serology in specific pathogen free (SPF) DR3-DQ2 transgenic mice sensitized to gluten, 1 week before weaning. Methods Seven-week-old SPF DR3-DQ2 transgenic mice, kept on a gluten-free diet (GFD), were paired for breeding. At post-natal day 3, pups were standardized to 4 per litter (n=2 male, n=2 female) to ensure equal nutrition across litters. At 14 days of age, pups were sensitized with pepsin-trypsin digested gliadin and cholera toxin (CT) three times in one week (n=15). At 21 days of age, pups were weaned and placed either on a gluten-containing diet (n=7), (equivalent of 20g/d of gluten in a human diet -high dose-) or an isocaloric GFD (n=8) until 10 weeks of age. Non-sensitized controls (n=7) received only CT and were kept on the GFD. At sacrifice, serum was collected for anti-TG2 and anti-gliadin antibodies (AGA). Jejunal tissue was collected for histological analysis using villus-to-crypt (V/C) ratios and CD3+ intraepithelial lymphocytes (IEL) counts. Results Gluten-sensitized mice placed on a gluten-containing diet post-weaning had lower V/C ratios and higher CD3+ IEL counts compared with controls (p&amp;lt;0.01). Pre-weaning sensitized mice that were kept on a GFD post-weaning had sustained decreases in V/C ratios and higher CD3+ IEL counts (p&amp;lt;0.01). Out of 15 sensitized mice, 7 developed positive anti-gliadin IgA (p=0.02) and 4 had positive anti-TG2 IgA antibodies (p=0.01) in intestinal contents, irrespective of gluten in the diet. None of the controls had detectable AGA or anti-TG2 antibodies. Conclusions Pre-weaning gluten sensitization of DR3-DQ2 mice induced prolonged gluten immunopathology that did not reverse after 5 weeks on a GFD. Our results indicate that young DR3-DQ2 mice are susceptible to gluten sensitization, with sustained immunopathology, suggesting a critical window of vulnerability in familial carriers of DQ2.5. This novel model will be useful to investigate environmental cofactors at the first time of gluten introduction to the diet. Funding Agencies CIHR

Diagnostic evaluation of microscopic enteritis in duodenal biopsies of suspected malabsorption cases with clinico-immunohistological correlation

Microscopic Enteritis (ME) is characterized by increase in intraepithelial lymphocytes (IEL) in intestinal mucosa. It represents a common feature of broad group of diseases including gluten mediated and non gluten related diseases. Duodenal biopsies play an important role in diagnosing these group of disorders. To compare IEL counts in Hematoxylin and eosin (H &amp; E) stain and CD3 immunohistochemical (IHC) stain in duodenal biopsies of suspected malabsorption cases and compare them with clinical, immunological and biochemical parameters. This was a prospective study of two years. 164 patients were studied. IEL counted at villous tip and base in H&amp;E sections and IHC were compared in duodenal biopsy. Data of clinical history, other parameters were collected and correlated whenever available. To compare any two variables, Chi - square test and independent T test was used. Statistical significance was defined as p &amp;#60; 0.05. Out of 164 cases,105 cases had increased IEL. The age range was 4 to 94 years with mean age of 43.29 ± 17.96 years. Males (56, 53.3%) were affected more than females (49, 46.6%). The clinical and histological parameters showing statistical significance with raised IEL were pallor, dyspepsia, loss of appetite, crypt architecture, blunting, ulcer, villous crypt ratio, exudates, reactive atypia and edema with P&amp;#60; 0.05. IEL at villous tip in CD3 had highest sensitivity (100%) and specificity (92.20%) in our study.ME should be investigated and diagnosed in correlation with a detail clinical history, complete haematological, biochemical and serological findings.

The Place of Histochemical Stains for Differential Diagnosis in Duodenal Biopsies

The biopsy is the primary examination method in diagnosing duodenal pathologies and Gluten enteropathy that can cause symptoms such as gas, diarrhea, weight loss, anemia, osteoporosis. Allows monitoring of response to therapy. Histochemical studies are inevitable in specific findings such as reactive changes, giardia, subepithelial collagen thickening, fibrosis, and gastric metaplasia. We aimed to understand the contribution of histochemical applications to differential diagnosis and to show their superiority compared to hematoxylin-eosin sections in routine use by simultaneously making histomorphological evaluations on hematoxylin and eosin sections. This research is a retrospective study conducted in 2011, using the archives of Yüzüncü Yıl University, Faculty of Medicine, Department of Pathology, between 2001-2010. Ten normal, 50 Gluten enteropathy and 50 duodenitis samples; Along with histopathological examinations, we applied Masson Trichrome, Periodic acid-Schiff, Periodic acid-Schiff with diastasis, Periodic acid Schiff-Alcian Blue, High Iron Diamine-Alcian Blue. Chi-square, likelihood ratio tests, and SPSS (ver: 13) statistical package program used. We detected microscopic findings parallel to diagnostic criteria. While the number of goblet cells and intraepithelial neutrophils did not differ in the groups (P =0.176 and P=0.096), there was a significant variation in the intraepithelial lymphocyte count (P=0.010). The frequency of flattening and blunting of the villi, crypt hyperplasia, and Brunner gland hyperplasia were significant in gluten enteropathies (P=0.000). Conclusion We could not find any difference in specific pathologies associated with histochemical studies in our group, so hematoxylin-eosin sections are more valuable in the differential diagnosis.

Neutrophils, eosinophils, and intraepithelial lymphocytes in the squamous esophagus in subjects with and without gastroesophageal reflux symptoms

Whilst intraepithelial lymphocytes (IELs) are considered normal within the distal esophageal mucosa, they have an increasingly recognised role in the pathogenesis of reflux esophagitis, and IEL quantification establishes the diagnosis of lymphocytic esophagitis. Knowledge regarding the upper limit of a normal IEL count in health is lacking. We studied 117 non-healthcare seeking adult volunteers from a random community sample (the Kalixanda study) with esophageal biopsies 2 cm above the gastroesophageal junction. Subjects were divided into four groups based on the presence or absence of gastro-esophageal reflux symptoms and/or esophagitis on endoscopy. Asymptomatic subjects with no endoscopic esophagitis were selected as controls, and the cell counts in this group were used to define the upper limit of normal of IELs, eosinophils and neutrophils. The entire sample was used to identify independent predictors of increased cellular counts by logistic regression analysis. None of the healthy controls had an IEL count of more than three per five high power fields (HPF), and therefore this was considered as the upper limit of normal; no controls had eosinophils or neutrophils in esophageal biopsies. Independent predictors of an elevated IEL count were spongiosis on histology (OR 11.17, 95% CI 3.32-37.58, P < 0.01) and current smoking (OR 4.84, 95% CI 1.13-2.71, P = 0.03). A receiver operating characteristics analysis concluded that a threshold of 3 IELs/5HPFs performs best in predicting reflux symptoms when a normal esophageal mucosa is visualized on endoscopy (sensitivity = 100.0%, specificity = 35.2%). The healthy esophageal mucosa does not contain more than three IELs per five HPF in the distal esophagus.

Celiac disease in duodenal biopsies; 2 year experience at a tertiary care hospital

Background: Duodenal endoscopic biopsy is a common useful test advised for patients presenting with various malabsorption syndromes. Aim: To study the histopathological changes observed in suspected malabsoption cases including Celiac disease (CD) with emphasis upon the Intraepithelial Lymphocytes (IELs) and to have a clinico-pathologic correlation. Settings & Design: This was a 2 year prospective study of cases presenting to gastrology OPD with suspected malabsorption selected for duodenal biopsy. Materials and Methods : Haematoxylin and Eosin (H&E) stain was used for study of histological parameters. IEL counts were correlated in CD3 immunohistochemical (IHC) stain. Clinical and laboratory parameters were correlated wherever available. Statistical Analysis: The variables were expressed as mean SD. Chi - square test or independent T test was used for comparison of variables. p value was < 0> Results: Out of 164 duodenal biopsies,105 cases had raised IEL.11 patients had CD (10.47%), rest were non celiac disease (NCD) of differen t aetiologies.Liver function tests, Endoscopy, Architecture of mucosa, crypt architecture, blunting, villous crypt ratio showed statistically significant correlation between CD and nonceliac disease groups with p values (P~0.034), (P~0.000),P~0.002),(P~0.000), (P~0,000) and (P~0.000) respectively. The specificity and positive predictive value (PPV) of villous tip IEL in both H&E & CD3 IHC were 100% in CD cases in our study. Conclusion: A complete clinical, endoscopic, serological and histopathological evaluation is essential for diagnosis of CD cases. Raised IEL in duodenal biopsies can be a very useful initial indicator for detecting many latent or subclinical CD cases. Keywords: Biopsy, Celiac Disease, Intraepithelial lymphocytes, Malabsorption.

Normal intraepithelial lymphocyte counts in duodenal biopsies and histological features of celiac disease among Jordanian adults

Normal intraepithelial lymphocyte counts in duodenal biopsies and histological features of celiac disease among Jordanian adults

Ileocolonic Histopathological and Microbial Alterations in the Irritable Bowel Syndrome: A Nested Community Case-Control Study.

INTRODUCTION:Histopathological alterations in the ileum and colon in irritable bowel syndrome (IBS) are controversial, and normal values are poorly established. We hypothesized that changes in mucosal immune cells characterize IBS and key changes in immune composition are associated with the mucosa-associated microbiota (MaM).METHODS:A nested case-control study (48 IBS and 106 controls included) from 745 colonoscopy participants in a random population sample. Intraepithelial lymphocytes (IELs)/100 enterocytes and eosinophils/5 nonoverlapping high-power fields counted; mast cells identified by immunocytochemistry (CD117)/5 high-power fields. Paneth cells quantified per 5 crypts. 16S rRNA gene amplicon sequencing performed on available sigmoid MaM, n = 55 and fecal microbiota, n = 20. Microbiota profiles compared between samples with high and low IEL counts.RESULTS:IBS had increased IELs in the terminal ileum (relative risk ratio = 1.70, 95% confidence interval 1.08–2.76, P = 0.022 adjusted for age, sex, and smoking). Cecal IELs were increased in IBS—diarrhea (relative risk ratio = 2.03, 95% confidence interval 1.13–3.63, P = 0.017). No difference was observed in alpha diversity of MaM or fecal microbiota based on IEL count. There was no difference in beta diversity of the MaM according to IEL count in the terminal ileal (TI) (P = 0.079). High TI IEL counts associated with a significant expansion of the genus Blautia (P = 0.024) and unclassified Clostridiales (P = 0.036) in colon MaM.DISCUSSION:A modest but significant increase in IELs was observed in IBS vs. controls in a population-based setting. Subtle TI and cecal inflammation may play a pathogenic role in IBS but needs confirmation. Modest but discernible differences in the colonic MaM were seen according to TI IEL count but not IBS status.