Increase In Intraepithelial Lymphocytes Research Articles

Diagnosis of celiac disease

Celiac disease is an immune mediated enteropathy initiated by ingestion of gluten, in genetically susceptible individuals. With changing epidemiology, celiac disease initially thought to affect only Europeans, has been increasingly reported from other parts of the world including India. However, its true prevalence in India is still not known, as the diagnosis is being missed. The gold standards for diagnosis have been characteristic small intestinal mucosal changes on gluten and a full clinical remission on its removal from the diet. Presence of serological antibodies, which disappear on gluten free diet further confirms the diagnosis. The understanding of the histopathology of celiac disease has changed over the years. The small bowel mucosal lesion of celiac disease is an evolutionary process with normal mucosal architecture and an increase in intraepithelial lymphocytes at one end of the spectrum and classical flat mucosa at the other. In the Indian subcontinent celiac disease has a heterogeneous histological presentation and the diagnosis may be missed if it is based only on severe mucosal changes or the serology is not considered when moderate or mild mucosal changes are present. The last two decades have shown that antiendomysical (Anti EMA) and anti tissue transglutaminase antibodies (anti-tTGA) have a sensitivity and specificity of more than 95% to diagnose celiac disease. Anti EMA tests being operator dependent are more liable to errors and anti- tTGA may be preferred for large scale screening. However, the different source of tTGA antigen, varied techniques of production and the use of arbitrary units by different commercial kits can influence the diagnostic accuracy of the anti-tTGA assay. There is a strong genetic association of celiac disease with HLA-DQ2 or DQ8. The presence of HLA-DQ2 hetrodimer in more than 97% of a group of North Indian patients with celiac disease indicates that this population has a similar genetic risk for the disease. HLA DQ2 typing can be used for ruling out celiac disease where the diagnosis is equivocal as it has a negative predictive value of greater than 95%. Given the protean clinical manifestation and the heterogeneous histology a standard algorithm for diagnosis of celiac disease is important.

Celiac Disease in India

We have read the article of Bhatnagar et al. (1) with keen interest. The study is relevant in northern India; however, certain points need to be clarified. There is some misinterpretation of Marsh grading (2). In grade I, there is no change in villous architecture; there is only increased intraepithelial lymphocytes (IEL). In grade II, crypts are elongated and in grade III, there is various degree of villous atrophy starting from partial to total (grades IIIa, IIIb and IIIc) (2,3). The authors (1) have separated grade III into mild, moderate and severe mucosal changes. Data should have been interpreted as celiac disease (CD) was diagnosed on the basis of Marsh grade III changes, and depending on the severity of villous atrophy, they were further subclassified as mild, moderate and severe. Accordingly, 86% of their cases had either subtotal (44%) or total (42%) villous atrophy, and the other 14% had partial villous atrophy, and this figure matches perfectly with other studies published from India (subtotal/total villous atrophy in 70%-80% of cases) (4,5). Therefore, it would not be right to state that most Indian children with CD have mild to moderate histological changes (1). The number of CD patients diagnosed is 138 that constitute 53% (138/259) of chronic diarrhea rather than 40% mentioned in the article (1). In this study (1), cases were not from unselected group of children with chronic diarrhea alone as they had also associated abdominal distension and failure to thrive. It has been shown from north India that the prevalence of CD is just 17% in unselected group (5) and 50% in selected group of children with chronic diarrhea (4). The role of antiendomysial antibody (AEA) in the diagnosis of CD has been underplayed in this study (1). According to the authors, approximately 5% of the cases would have been missed if only AEA assay was used for diagnostic purpose. However, they have ignored those 20 of 57 cases with mild mucosal changes where AEA was positive, and 19 of them responded to gluten-free diet (GFD). Had the authors not done AEA, they would have missed the diagnosis in 19 cases. If histology was the sole criteria to decide about GFD trial, then what about the other 37 cases who were not put on GFD? In developing countries, serology plays an important role to distinguish villous atrophy caused by CD from other causes. This fact has been highlighted and emphasized from India (5,6). The above facts raised by us have direct implication on diagnosis of CD in developing countries. Shinjini Bhatnagar, PhD Alan D. Phillips, PhD Maharaj K. Bhan, MD Pediatric Gastroenterologist and Scientist Centre for Diarrhoeal Diseases Research and Nutrition, Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

SEVERITY OF HISTOLOGICAL CHANGES IN CELIAC DISEASE CORRELATES WITH IGA ENDOMYSIAL AND TISSUE TRANSGLUTAMINASE ANTIBODY LEVELS

Background: Although IgA endomysial antibodies (EMA) and tissue transglutaminase (TG) are sensitive and specific serologic tests for the diagnosis of celiac disease, there is limited information on the association of the magnitude of antibody level with the severity of the histological abnormalities of the intestine. Purpose: To determine if EMA and TG titers correlate with the severity of histological changes in patients with celiac disease. Methods: We identified 148 children from our laboratory database that had EMA, TG and intestinal biopsies performed. IgA EMA was determined by indirect immunofluorescence with results expressed as a dilutional titer with positivity determined at 1:5. IgA TG was determined by an enzyme linked human immunosorbent ELISA assay with results expressed in standardized units. A modified Marsh histological grading system was used to describe the duodenal biopsies: Type 0 normal, I increased intraepithelial lymphocytes (IEL), II hyperplastic crypts, IIIa partial villus atrophy, IIIb subtotal villus atrophy, IIIc total villous atrophy. Results: Mean values for EMA (Table 1) and TG (Table 2) progressively increased with increasing Marsh score.TABLE 1: Endomysial antibody titer and Marsh GradeTABLE 2: Tissue transglutaminase level and Marsh GradeConclusion: There was considerable variability in EMA and TG levels for each Marsh grade, so that an individual level could not be utilized to predict histological severity. The data show that as a group, increasing severity of the histological lesion in celiac disease was associated with increased levels of both IgA EMA and TG antibodies.

Pathological and clinical significance of increased intraepithelial lymphocytes (IELs) in small bowel mucosa

Intestinal intraepithelial lymphocytes (IELs) belong to a unique T-cell population interspersed between epithelial cells of both the small and large intestine. It is becoming increasingly recognised that an increased number of IELs with a normal villous architecture is within the wide spectrum of histological abnormalities observed in coeliac disease. An increased number of IELs is the earliest pathological change following gluten challenge and a high IEL count may be the only sign of gluten sensitivity. Therefore, the finding of a raised IEL count with normal villous architecture is of sufficient clinical importance to be reported in routine small bowel biopsies. However, it is evident that not all small intestinal biopsy specimens showing increased IELs are explained by gluten sensitivity. Increased IELs in small bowel mucosa have also been associated with autoimmune disorders, tropical sprue, food protein intolerance, Helicobacter pylori-associated gastritis, peptic duodenitis, parasitic and viral infections, as well as the development of intestinal lymphoma. Histological examination of a biopsy specimen of the small bowel remains the diagnostic gold standard for coeliac disease. There will be an ever increasing demand for histological confirmation of gluten sensitivity in patients in whom the classic microscopic appearance of flattened villi may not have fully developed. The more widespread recognition by histopathologists of the pattern of injury manifested by increased numbers of IELs in intestinal biopsy specimens will certainly help in early diagnosis of coeliac disease, lessen diagnostic confusion and influence the modern practice of gastrointestinal tract medicine. This review discusses some of the recent developments in clinical pathology pertaining to increased IELs in small bowel mucosal biopsies.

Overview and pathogenesis of celiac disease

Celiac disease is characterized by small intestinal mucosal injury and nutrient malabsorption in genetically susceptible individuals following the dietary ingestion of “gluten.” The pathogenesis of disease involves interactions between environmental, genetic, and immunologic factors. 1 This brief overview of celiac disease and its pathogenesis is designed to place this disease, and current views of its pathogenesis, in a context appropriate for further consideration within the framework of questions being posed by the NIH Consensus Conference on Celiac Disease. Pathology and Clinical Symptoms First, a brief overview of disease pathology and clinical symptoms. The luminal surface of a small intestinal mucosal biopsy specimen from a healthy subject and an individual with celiac disease with total villous atrophy, as viewed through a dissecting microscope, are shown in Figure 1A and 1B, respectively. The normal biopsy specimen shows abundant villi (looking much like the shag household carpets of a prior era), whereas the biopsy specimen from the patient with celiac disease shows a complete loss of villi, with a flat mucosal surface accentuated by ridges and numerous openings of the crypts onto the luminal surface. Microscopic analysis of a normal H&E-stained, small intestinal mucosal biopsy specimen is shown in Figure 1C. Characteristic features include tall villi lined by a single layer of columnar epithelial cells with basally oriented nuclei, a “smattering” of intraepithelial lymphocytes (IEL) occurring at a ratio of approximately 1 per 6 –10 epithelial cells, a normal complement of lymphocytes and plasma cells in the lamina propria consistent with the normal “physiologic inflammation” of the small intestine, and a villous to crypt ratio of 4 –5:1. Contrast that biopsy specimen with the biopsy specimen from a celiac disease patient with total villous atrophy as shown in Figure 1D. The latter reveals a complete loss of villi, markedly abnormal squamoid surface epithelial cells, an increase in intraepithelial lymphocytes, a chronic lymphocyte and plasma cell infiltrate in the lamina propria, and marked crypt hyperplasia with increased crypt mitoses. The pathology shown in this celiac biopsy specimen reflects the more severe end of the pathologic spectrum of disease, which, as indicated in Figure 2, can vary markedly among affected individuals. Just as the pathology can vary markedly among patients, so can the spectrum of clinical symptoms, which can range from severe to subtle (Figure 3). The proportion of individuals presenting with more dramatic symptoms of diarrhea, severe weight loss, and malnutrition is decreasing when compared with those presenting with mild or minimal symptoms or asymptomatic individuals in which disease is discovered only when investigating nongastrointestinal symptoms, for example, iron deficiency anemia or osteopenia. 2 A more detailed explanation of the clinical presentation of celiac disease in children and adults is presented in later articles in this series. Pathogenesis of Celiac Disease

Lymphomatous infiltration of the peripheral nervous system in enteropathy‐associated T‐cell lymphoma

Clinical record A 53-year-old woman with a 10-year history of coeliac disease controlled by a gluten-free diet presented with a 6-month history of dramatic weight loss and pyrexia. Physical examination showed wasting and peripheral oedema. Plasma albumin level was 23g/L (normal, 34-48 g/L). Computed tomography of head, neck, chest, abdomen and pelvis, gallium and positron emission tomography scans, liver biopsy and bone marrow biopsy were normal. The serum lactate dehydrogenase (LDH) level was 466 U/L (normal, 110-230 U/L), raising the possibility of occult malignancy, especially enteropathy-associated T-cell lymphoma (EATL). Endoscopic duodenal biopsy revealed mild increase in intraepithelial lymphocytes, crypt hyperplasia and villous abnormality in keeping with coeliac disease. Molecular clonality studies of the lymphoid cells in the duodenal biopsy yielded a monoclonal band of T-cell receptor (TCR) γ gene rearrangement in two separate series of biopsies taken 6 weeks apart. Refractory sprue was diagnosed, and the patient received sequential trials of prednisolone, cyclosporin A and infliximab without benefit. High dose cyclophosphamide (2 g/m 2 ) was administered 3 months later for presumed EATL. This resulted in immediate resolution of the fever and improved weight gain. The patient subsequently complained of paraesthesiae in her feet. Neurological examination was normal. Nerve conduction studies suggested an early axonal sensory peripheral neuropathy. Vitamin B 12 , folate, vitamin A-tocopherol, red blood cell transaminase and transketolase studies, vasculitic screen, glycated haemoglobin, syphilis serology and thyroid function tests were normal. Paraprotein was not detected in serum electrophoresis. Magnetic resonance imaging (MRI) of the brain showed non-specific patchy periventricular areas of high signal not suggestive of the suspected lymphoma. Her systemic symptoms of fever, sweats and weight loss responded to two further doses of cyclophosphamide (1 g/m 2 ), despite worsening neurological symptoms. After 3 months, the patient relapsed with recurrence of pyrexia, and the serum LDH level progressively rose to 838 U/L. Neuropathy progressed to the point where she could not walk. Reflexes were lost, with a glove-and-stocking pattern of sensorimotor disturbance. A repeated nerve conduction study confirmed severe axonal sensorimotor peripheral neuropathy affecting both upper and lower limbs. Cerebrospinal fluid was normal, and there were no malignant cells. Repeated investigations, including brain MRI and upper gastrointestinal endoscopy, did not reveal any further evidence of malignancy. A right sural nerve biopsy showed severe loss of myelinated nerve fibres, and many of the surviving fibres showed varying stages of axonal degeneration (Box 1) associated with a sparse, patchy endoneurial mononuclear cell infiltrate in some fascicles and prominent endoneurial fibrosis on trichrome stain. The CD45RB immunostain showed increased numbers of immunopositive endoneurial lymphoid cells that showed immunopositivity with the T-cell markers CD3 (Box 2) and CD5. The CD68 immunostain showed increased endoneurial macrophages, and ultrastructural assessment confirmed macrophage phagocytosis of degenerate myelinated fibres. CD20 immunostain was negative. The Congo red stain for amyloid was negative.

CD4+CD8+ human small intestinal T cells are decreased in coeliac patients, with CD8 expression downregulated on intra-epithelial T cells in the active disease.

The intestinal lesion of coeliac disease is thought to be initiated and exacerbated by dysregulation of local T-lymphocyte sub-populations. This study examines changes in intestinal T cells from coeliac patients, with a particular focus on CD4CD8 T cells, immunoregulatory cells normally found in relatively high proportions in the small intestine. Cells were obtained from duodenal biopsies from active and treated coeliac patients using chelating and reducing agents (epithelial layer) followed by collagenase treatment (lamina propria). Cell yield and viability were assessed and flow cytometric analysis was used to examine CD4CD8 T cells and to quantify CD8 expression. Surprisingly, total T-cell yields in the epithelial layer did not increase in active coeliac disease although enterocyte counts decreased significantly, giving an appearance of infiltration. In active coeliac patients, CD4CD8 T cell percentages were significantly decreased in both the epithelial layer and lamina propria. Levels of CD8 expression by CD4CD8 T cells in the epithelial layer were decreased significantly in patients with active coeliac disease. CD4CD8 T cell proportions did not return to normal in treated coeliac patients whose villous architecture had responded to gluten withdrawal. No increase of intra-epithelial lymphocytes in the coeliac lesion may require us to reconsider the definition of coeliac disease as an inflammatory condition. Low CD4CD8 populations in treated as well as untreated coeliac patients indicate that these T cells are inherently absent in individuals genetically predisposed to coeliac disease.

BCG vaccine modulates intestinal and systemic response to beta-lactoglobulin.

Beta-Lactoglobulin (BLG) is a clinically important antigen in cow's milk and one of the major allergens causing cow's milk allergy. Bacillus Calmette-Guerin (BCG) vaccination has been suggested to modify immune response possibly decreasing the risk of allergy to some antigens in both human and experimental animals. In the present study, we have analyzed whether the early BCG vaccination has any effect on the markers of systemic and gastrointestinal (GI) sensitization to BLG. We immunized two groups of Hooded-Lister rat puppets with intraperitoneal injections of native BLG at 43 and 62 days with pertussis vaccine as adjuvant, one group receiving additionally BCG. The animals were then fed native and denatured milk products twice weekly from 73 to 131 days of age, when they were killed. Control group was not vaccinated and received normal rat forage. Total immunoglobulin E (IgE) levels and BLG-specific IgG(1) and IgG(2a) concentrations were determined in serum samples. Spontaneous interleukin (IL)-4 and interferon (IFN)-gamma production from duodenal specimens were measured, and the inflammatory cells were quantitated in specimens from different sections of the GI tract. Administration of BCG simultaneously with BLG resulted in reduced IgE concentration in serum, while the specific IgG(1) and IgG(2a) antibody responses and the spontaneous secretion of IL-4 and IFN-gamma were not affected. Furthermore, BCG-induced eosinophilic infiltration and increase of intraepithelial lymphocytes (IEL) in the GI mucosa, and a trend toward increased number of lamina propria mononuclear inflammatory cells in the colon (BCG compared with BLG, p = 0.09; BCG compared with controls, p = 0.02). Controls showed increment of IgG(1) response in comparison with the BLG group (p = 0.04) and increase of mucosal eosinophilic infiltration. The BCG modified the response to BLG both at the systemic level as shown by decrease of total IgE and at GI mucosa where increase of eosinophilic infiltration and increased number of IEL were seen. Increment of IgG(1) level and eosinophils in the controls might be related with the lack of modulatory effect of pertussis vaccination. A shift of response toward the lower GI tract after BCG immunization as shown by a trend for increase of mononuclear inflammatory cells in colon lamina propria mimics disease development in some cases of clinical food allergy, and emphasizes the need for evaluation of the changes in the whole GI tract in food allergy models.

Histopathology of Pediatric Celiac Disease (Response)

We appreciate the interest in our recent paper on celiac disease (CD) [1], and Dr. Drut’s sharing his perspective on tissue handling and the details of his grading system for villous atrophy [2]. As our paper indicated, and a recent flurry of activity on the Pediatric Pathology listserve has reiterated, there is currently no consensus for optimal tissue handling of small intestinal biopsies. While in our hands, orientation of tissue at the time of embedding by an experienced histotechnologist and examination of multiple levels for selected cases works best, in Dr. Drut’s institution, orientation of tissue on a substrate is preferred. We discuss the advantages and disadvantages of both methods in our paper [1]. We agree with Dr. Drut that a standardized grading scheme for small intestinal biopsies for CD is helpful to the clinician. The Marsh classification is widely used and the modified Marsh classification includes an infiltrative lesion lacking architectural abnormalities and subdivision of villous atrophy into three grades of severity [3]. As CD is currently considered to be predominantly an immune-mediated process, a grading scheme that includes lymphocytic infiltration seems appropriate. As Oberhuber et al. state, “An increase in the number of IEL is the first and most sensitive index of the effects of gluten on the mucosa [their ref. 20], and is therefore the single most important histological feature in CD” [3]. In contrast to Dr. Drut’s experience, our group and others have seen small intestinal biopsies from patients with CD show increased intraepithelial lymphocytes (IEL) and no architectural abnormalities (modified Marsh type I) [3,4], as well as cases of CD with patchy involvement of only some of the biopsy fragments [5]. Thus, although a wider net is cast, we favor a grading system which will call to attention possible cases of CD that would be missed if architectural abnormalities alone were required to suggest diagnosis. Whatever grading scheme is employed, biopsy findings alone are not specific for CD and should be interpreted within the clinical context of each individual case.

Chronic diarrhea

Chronic diarrhea

P0390 VARIABILITY OF HISTOLOGIC LESIONS IN RELATION TO BIOPSY SITE IN CELIAC DISEASE

Introduction: Histologic lesions of celiac disease (CD) range from increased intraepithelial lymphocytes (IEL) to crypt hyperplasia (CH) to villous atrophy (VA). It is believed that the optimal biopsy site for detection of VA is the distal duodenum or proximal jejunum and lesions may have a patchy distribution which could lead to misdiagnosis. We wanted to verify these concepts and establish whether biopsies should always be taken from these areas in order to make a diagnosis of CD. Methods: One-hundred and 12 consecutive children with positive anti-endomysium (EMA) or anti-tissue transglutaminase (tTGA) antibodies who had been referred for suspected CD were enrolled in a prospective fashion. All patients underwent upper GI endoscopy where 4–5 biopsies were taken sequentially with standard biopsy forceps from: Treitz and/or distal duodenum (T-D3), intermediate duodenum (D2), proximal duodenum (D1) and duodenal bulb (B). Biopsies were oriented, fixed in formalin, stained with H&E and subjected to immunohistochemistry with anti-CD3 antibodies for IEL count. Histologic lesions were classified according to Marsh criteria modified by Oberhuber. Results: One-hundred and 10 of 112 patients, all HLA DQ2 or DQ8 positive, had a final diagnosis of CD (59 classic, 28 atypical, 23 silent). Of these 110, 102 (92.7%) had VA with lesion type 3: a) mild, b) moderate or c) severe in at least one site and 94/110 (85.4%) had VA in all sites though with variable degree. VA of identical degree was present in all biopsy sites in 55/110 (50%) patients. Severe VA (type 3c) was present in at least one site in 85/110 (75%), in all sites in 50/110 (45.4%) and was distributed as follows: B 50/110 (45.4%), D1 48/110 (43.6%), D2 70/110 (63.6%), D3/T 83/110 (75.4%), with a significant increase in aborad direction (chi2>26.22 with a=0.01 and g.l.=12). Eight/110 (7.2%) CD patients had exclusively type 1 or 2 lesions (increased IEL ± CH), 9/110 (8.1%) had lesion variability = 1 degree (e.g. type 1 and 2, type 2 and 3a) but no patient had lesion variability >1 degree and none had normal biopsies. There was no statistically significant correlation between type or distribution of histologic lesions and clinical presentation of CD, though interestingly all patients with silent CD had type 3c lesions throughout the duodenum. Conclusion: Mucosal atrophy is present in 85% of CD patients and TVA is significantly more frequent in more distal areas (D3/T). However, in half of the patients VA has the same degree throughout the duodenum including B, and no duodenal areas are histologically normal. Therefore, in genetically susceptible children with positive serology, a diagnosis of CD can reliably be made aven if biopsies are not taken from the distal duodenum or jejunum.

P0581 CASE REPORT: ONE YEAR OLD CHILD WITH ARTHROGRYPOSIS AND COLLAGENOUS ENTEROPATHY

Introduction: The diffuse deposition of collagen in the intestine without the presence of an inflammatory infiltrate (collagenous enteropathy) is rarely encountered, especially in children, and its occurrence with arthrogryposis has not been previously reported. Methods: HD was hospitalized at 12 months of age for diarrhea requiring persistent IV rehydration and nutritional support. Because of the inability to tolerate oral feeds, she had become TPN dependent. Results: On admission, her weight was 6.71 kg (<5%ile). Her physical exam was most striking for a severely limited range of motion with severe joint contractures. While hospitalized, she had large volume secretory diarrhea of 115 cc/kg/day. Small bowel histology showed mild villous blunting with intact enterocytes and goblet cells without increased intraepithelial lymphocytes. Trichrome staining showed a patchy deposition of a thick band of collagen. Histology of the colon showed intact architecture with full crypts. The lamina propria showed a massive increase in collagen, which was also present as a large thickened table sub-epithelially and extended to involve deeper portions of the lamina propria. There was no significant increase in intraepithelial lymphocytes, nor significant active inflammation. She was maintained solely on TPN and had a good weight gain. Octreotide was started with improvement of her secretory diarrhea and with the addition of loperamide, her stool output fell to 30 cc/kg/day. Her TPN was adjusted to account for stool replacement, and she was discharged home. Conclusion: Arthrogryposis is a phenotype marked by multiple joint contractures, secondary to many unrelated disorders. It has been associated in the past with intestinal atresias, but never with collagenous enteropathy. It is our hypothesis that this collagen deposition may be similar to that seen in the joints of patients with arthrogryposis. The fact that the patient tolerated enteral feeds early in infancy, and then had worsening intolerance over the first year of life indicates that this was a progressive deposition of collagen. It appears that this patient may have a defect in the regulation of her connective tissue matrix leading to diffuse collagen deposition.

Significance of intraepithelial lymphocytosis in small bowel biopsy samples with normal mucosal architecture

Objectives The aim of this study was to determine the specificity of increase in intraepithelial lymphocytes (IELs) with normal villous architecture in small bowel biopsy samples for diagnosis of gluten sensitivity (GS) and its significance in the absence of GS. Methods Small bowel biopsy samples from 43 patients with increased IELs and no other pathology were reviewed. Patients with prior diagnosis of GS were excluded. A group of 46 patients with normal duodenal biopsy during the same period served as controls. The clinical records of patients and controls were examined for presenting symptoms, laboratory tests, and final clinicopathological diagnosis. Immunohistochemical characterization of IELs was performed in 13 cases. Results Four (9.3%) patients had GS based on positive IgA antiendomysial antibodies (n = 3) and favorable response to gluten-free diet (n = 4). One patient (2.2%) had partially treated tropical sprue; six patients (14%) had disorders of immune regulation including Hashimoto's thyroiditis (n = 2) and one case each of Graves' disease, rheumatoid arthritis, psoriasis, and multiple sclerosis; and six patients (14%) were on nonsteroidal anti-inflammatory drugs (NSAIDs). In contrast, none of the control subjects had GS ( p = 0.05), tropical sprue, or immunoregulatory disorders ( p = 0.011), and one (2.2%) was on NSAIDs ( p = 0.04). Increased IELs were also observed in Crohn's disease, lymphocytic/collagenous colitis, and bacterial overgrowth, but the association did not reach statistical significance. Histological features (number and distribution of IELs, crypt mitoses) and immunophenotypic analysis of IELs did not reliably distinguish GS-related from non–GS-related causes of increased IELs. Conclusions Intraepithelial lymphocytosis in an otherwise normal small bowel biopsy is somewhat nonspecific, but in nearly 10% of cases can be the initial presentation of GS. Therefore all patients with this finding should be investigated for GS. Increased IELs may also be associated with autoimmune disorders and NSAIDs.

Intraepithelial lymphocyte-derived interferon-γ evokes enterocyte apoptosis with parenteral nutrition in mice

Total parenteral nutrition (TPN) results in an increase in intraepithelial lymphocyte (IEL)-derived interferon-gamma (IFN-gamma) expression as well as an increase in epithelial cell (EC) apoptosis. This study examined the role that IEL-derived IFN-gamma has in the increase in EC apoptosis. Mice received either TPN or oral feedings for 7 days. Small bowel EC apoptosis significantly rose in mice receiving TPN. The administration of TPN also significantly increased IEL-derived IFN-gamma and Fas ligand (FasL) expression. EC apoptosis in IFN-gamma knockout (IFNKO) mice that received TPN was significantly lower than in wild-type TPN mice. Sensitivity of EC to Fas-mediated apoptosis in IFNKO mice was significantly lower than in wild-type TPN mice. Apoptosis in Fas-deficient and FasL-deficient mice that received TPN was significantly lower than in wild-type mice that received TPN. The TPN-induced increase in IFN-gamma expression appears to result in an increase in Fas-L expression and EC sensitivity to Fas, with a resultant increase in EC apoptosis. This may well be one of the mediators of increased EC apoptosis observed with TPN administration.

Is a raised intraepithelial lymphocyte count with normal duodenal villous architecture clinically relevant?

A raised intraepithelial lymphocyte (IEL) count with normal villous architecture is a recognised finding in latent coeliac disease. Little information is available in cases without gluten sensitive enteropathy in adults. To assess the frequency of such a finding in routine practice and to determine whether it is clinically relevant. Patients with subjectively increased IELs as the only abnormality were identified prospectively from a routine duodenal biopsy series over a 12 month period. The biopsy specimens in these index cases were re-examined together with two controls with normal histology for each case, and three counts of IEL/100 epithelial cells were made in all samples. The index cases were then contacted and interviewed to obtain clinical information, approximately 12 months from the initial biopsy. Further data were obtained from their clinical records. Fourteen of 626 (2.2%) patients who had duodenal biopsies over the 12 month period had a subjective increase in IELs with normal villous architecture. Fifteen patients with newly diagnosed gluten sensitive enteropathy were also identified during the study period. Formal counting of the index cases and controls revealed a significant difference in IELs/100 epithelial cell counts between the two (mean, 38 (SD, 6.2) v 12.4 (4.6); p < 0.0001). Three of the 14 index cases tested had a positive coeliac antibody test compared with 12 of 15 newly diagnosed patients with coeliac disease and 10 of 93 patients with normal histology. The major clinical diagnostic categories in raised IEL cases were those with positive coeliac serology (n = 3), unexplained anaemia (n = 3), and chronic liver disease (n = 3). Six of 10 patients who were interviewed had ongoing gastrointestinal symptoms one year later. Three patients had had follow up duodenal biopsies, at the discretion of their responsible clinicians, with no change in IEL counts despite the commencement of a gluten free diet in two patients. A raised IEL count with normal villous architecture is not uncommon. Six of the 14 patients may have had latent coeliac disease. The cause in at least half of cases is not obvious at present. The finding of a raised IEL count with normal villous architecture is of sufficient clinical importance to be highlighted in routine duodenal biopsy reports.

Quantitation of intraepithelial lymphocytes in human duodenum: what is normal?

An increase in intraepithelial lymphocytes (IELs) is mandatory for the histological diagnosis of coeliac disease (CD). Currently, duodenal biopsies are used almost exclusively to establish the diagnosis, yet published work continues to cite an upper limit of 40 lymphocytes/100 epithelial cells, a figure derived from jejunal biopsies over 30 years ago. To establish the normal range for IEL counts in distal duodenal biopsies. Twenty subjects (seven men, 13 women; median age, 34 years; range, 20-65) with a normal sugar permeability test and concurrent distal duodenal biopsies were identified. The number of IELs and epithelial cell nuclei in an uninterrupted length of surface (villous) epithelium (> 500 cells) was counted. An image analysis system was used to assess villous architecture by calculating the villous height to crypt depth ratio. The range of IEL counts in 20 subjects was 1.8-26/100 villous epithelial cells, with a mean value of 11 and SD of 6.8. The mean villous to crypt ratio was 1.82 (SD, 0.38; range, 1.22-2.46). There was no correlation between IEL counts and villous to crypt ratio (Spearman rank correlation, -0.066; p = 0.80). These results suggest that 25 IELs/100 epithelial cells (mean +2 SD) should be taken as the upper limit of the normal range for duodenal mucosa.

288. IGA ANTIBODY TO TISSUE TRANSGLUTAMINASE IS A HIGHLY SENSITIVE PREDICTOR OF ADULT ONSET COELIAC DISEASE IN CLINICAL PRACTICE

Introduction: Anti-endomysial antibodies have high specificity (90%) for the presence of coeliac disease, but there are ethical and cost issues associated with using monkey oesophagus for clinical tests. Tissue transglutaminase...

Histopathology of celiac disease

The diagnosis of celiac disease (CD) is based upon histologic findings in duodenal or jejunal biopsies. In the past few years it has turned out that the development of CD lesion in the small bowel is a dynamic process which may present in various histologic forms. At one end of the spectrum is a mucosa with normal architecture and an increase in intraepithelial lymphocytes (IEL), at the other end is the classical flat mucosa. Histologic features supporting the diagnosis of CD are architectural changes of the villi and/or crypts, an increase in lamina propria cell density and an increase in IEL counts. For diagnostic purposes and for monitoring CD patients an exact histologic classification of the histologic findings has to be given. This has become possible by using a modified Marsh classification. In the present paper the histologic presentation of CD is presented as well as the modified Marsh classification.

Differential intra-epithelial lymphocyte phenotypes following Cryptosporidium parvum challenge in susceptible and resistant athymic strains of mice

The lack of immunocompetent laboratory animal models has limited our understanding of functional immune responses to Cryptosporidium parvum infection, but such responses have been studied in susceptible laboratory rodents with genetic, acquired, or induced immunodeficiencies. We previously observed that athymic C57BL/6J nude mice inoculated with C. parvum oocysts had lower or absent fecal oocyst excretion when compared to inoculated athymic BALB/cJ nude mice. This discrepancy prompted us to explore potential differences in intestinal immune responses in both strains. Prior to and after C. parvum challenge, BALB/cJ nude and C57BL/6J nude mice did not differ in either spleen cell numbers or in parasite-specific proliferation. However, both strains of mice exhibited a significant increase in intra-epithelial lymphocyte (IEL) numbers prior to and following C. parvum inoculation when compared to uninoculated controls ( P<0.05). Prior to challenge, C57BL/6J nude mice had a higher percentage of both CD8 + and CD8 +γδ + IEL than BALB/cJ nude mice. Following challenge, resistant C57BL/6J nude mice had a higher percentage of γδ +, CD4 +, and CD8 +γδ + IEL than uninoculated C57BL/6J nude mice and than susceptible BALB/cJ nude mice ( P<0.05). Conversely, inoculated C57BL/6J nude mice had a significantly lower percentage of αβ + IEL than inoculated BALB/cJ nude mice ( P<0.05). We conclude that γδ +, CD4 +, and/or CD8 +γδ + IEL may influence responses to cryptosporidiosis in athymic murine models, and that the increased percentage of αβ + IEL in susceptible BALB/cJ nude mice could reflect a preferential expression during chronic C. parvum infection and/or might downregulate local protective responses.

Screening by anti-endomysium antibody for celiac disease in diabetic children and adolescents in Austria.

Unrecognized celiac disease (CD) may be found in a substantial proportion of patients with type I diabetes mellitus. A cohort of 403 Austrian children and adolescents with type I diabetes mellitus (210 males and 193 females; age range, 1-22 years) was screened for celiac disease using the IgA anti-endomysium antibody test (EMA) and the immunoglobulin (Ig)G anti-gliadin (AGA-IgG) and IgA anti-gliadin (AGA-IgA) antibody test. Twelve patients' sera (2.98%) yielded positive EMA results and two patients' sera (0.49%) with IgA deficiency had high AGA-IgG values. All but one of these patients underwent intestinal biopsy. Six (1.49%) had clear histologic evidence of CD (flat mucosa), whereas three (0.74%) showed minor histologic changes (increase in intraepithelial lymphocytes) and four (0.99%), including the EMA-negative patients with IgA deficiency, had a normal mucosa. When the cases with silent and potential CD were combined, the overall prevalence in the current cohort was 2.98%. There was no difference in the hemoglobin (Hb)A1c level between antibody-positive and -negative patients, and subsequent gluten-free diet did not change this metabolic parameter. The prevalence of clinically unrecognized CD, found by EMA screening, is much higher in Austrian children with diabetes than in a comparable population without diabetes. The prevalence of CD in diabetic children in Austria is distinctly lower, however, than in several other countries.