Gluten Induces Subtle Histological Changes in Duodenal Mucosa of Patients with Non-Coeliac Gluten Sensitivity: A Multicentre Study.

Background: Histological changes induced by gluten in the duodenal mucosa of patients with non-coeliac gluten sensitivity (NCGS) are poorly defined. Objectives: To evaluate the structural and inflammatory features of NCGS compared to controls and coeliac disease (CeD) with milder enteropathy (Marsh I-II). Methods: Well-oriented biopsies of 262 control cases with normal gastroscopy and histologic findings, 261 CeD, and 175 NCGS biopsies from 9 contributing countries were examined. Villus height (VH, in µm), crypt depth (CrD, in µm), villus-to-crypt ratios (VCR), IELs (intraepithelial lymphocytes/100 enterocytes), and other relevant histological, serologic, and demographic parameters were quantified. Results: The median VH in NCGS was significantly shorter (600, IQR: 400−705) than controls (900, IQR: 667−1112) (p < 0.001). NCGS patients with Marsh I-II had similar VH and VCR to CeD [465 µm (IQR: 390−620) vs. 427 µm (IQR: 348−569, p = 0·176)]. The VCR in NCGS with Marsh 0 was lower than controls (p < 0.001). The median IEL in NCGS with Marsh 0 was higher than controls (23.0 vs. 13.7, p < 0.001). To distinguish Marsh 0 NCGS from controls, an IEL cut-off of 14 showed 79% sensitivity and 55% specificity. IEL densities in Marsh I-II NCGS and CeD groups were similar. Conclusion: NCGS duodenal mucosa exhibits distinctive changes consistent with an intestinal response to luminal antigens, even at the Marsh 0 stage of villus architecture.

From 2-dimensional to 3-dimensional: Overcoming dilemmas in intestinal mucosal interpretation.

The purpose of this review is to provide a definitive account of small intestinal mucosal structure and interpretation. The coeliac lesion has been well known, but not well described to date and this review aims to identify the interpretative difficulties which have arisen over time with the histological assessment of coeliac disease. In early coeliac interpretation, there were significant inaccuracies, particularly surrounding intraepithelial lymphocyte counts and the degree of villous flattening which occurred in the tissue. Many of these interpretive pitfalls are still encountered today, increasing the potential for diagnostic errors. These difficulties are mostly due to the fact that stained 2-dimensional sections can never truly represent the 3-dimensional framework of the intestinal tissue under investigation. Therefore, this review offers a critical account occasioned by these 2-dimensional interpretative errors and which, in our opinion, should in general be jettisoned. As a result, we leave a framework regarding the true 3-dimensional knowledge of mucosal structure accrued over the 70-year period of study, and one which is available for future reference.

Diagnosing celiac disease: A critical overview.

The diagnosis of celiac disease (CD) no longer rests on a malabsorptive state or severe mucosal lesions. For the present, diagnosis will always require the gold-standard of a biopsy, interpreted through its progressive phases (Marsh classification). Marsh classification articulated the immunopathological spectrum of gluten-induced mucosal changes in association with the recognition of innate (Marsh I infiltration) and T cell-based adaptive (Marsh II, and the surface re-organisation typifying Marsh III lesions) responses. Through the Marsh classification the diagnostic goalposts were considerably widened thus, over its time-course, permitting countless patients to begin a gluten-free diet but who, on previous criteria, would have been denied such vital treatment. The revisions of this classification failed to provide additional insight in the interpretation of mucosal pathology. Morever, the subclassification of Marsh 3 imposed an enormous amount of extra work on pathologists with no aid in diagnosis, treatment, or prognosis. Therefore, it should now be apparent that if gastroenterologists ignore these sub-classifications in clinical decision-making, then on that basis alone, there is no need whatsoever for pathologists to persist in reporting them. Since new treatments are under critical assessment, we might have to consider use of some other higher level histological techniques sensitive enough to detect the changes sought. A promising alternative would be to hear more voices from imaginative histopathologists or morphologists together with some more insightful approaches, involving molecular-based techniques and stem cell research may be to evaluate mucosal pathology in CD.

Exploring the villus.

The small intestinal villus and its associated epithelium includes enterocytes as the main cell type and differentiated goblet and argentaffin cells, while the invaginated crypt epithelium is the site of cell division and hence the origin of all epithelial components. Enterocytes form a cohesive monolayer which acts both as a permeability barrier between lumen and the interior, and an important gateway for nutrient digestion, absorption and transport. Differentiation and polarisation of enterocytes depends on cytoskeletal proteins that control cell shape and maintain functionally specialised membrane domains; extracellular matrix (ECM) receptors; channels and transporters regulating ion/solute transfer across the cell. The mesenchymally-derived basement membrane dynamically controls morphogenesis, cell differentiation and polarity, while also providing the structural basis for villi, crypts and the microvasculature of the lamina propria so that tissue morphology, crucially, is preserved in the absence of epithelium. Mucosal re-organisation requires immense cooperation between all elements within the lamina, including marked revisions of the microvasculature and extensive alterations to all basement membranes providing support for endodermal and mesenchymal components. In this context, subepithelial myofibroblasts fulfil important regulatory activities in terms of tissue morphogenesis; remodelling; control of epithelial cell development, polarity and functional attributes; and an intimate involvement in repair, inflammation and fibrosis. This paper reviews the main structural and functional aspects of the villus, including the epithelium and its outer glycocalyx and microvillous border; and subjacent to the epithelium, the basement membrane with its attached web of myo-fibroblasts together with the lamina propria core of the villi, and its microvasculature and lacteals. Finally, some comments on the rapidity with which the overall structure of the villi changes in their response to both external, and internal, influences.

ROC-king onwards: intraepithelial lymphocyte counts, distribution & role in coeliac disease mucosal interpretation.

OBJECTIVES: Counting intraepithelial lymphocytes (IEL) is central to the histological diagnosis of coeliac disease (CD), but no definitive 'normal' IEL range has ever been published. In this multicentre study, receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off between normal and CD (Marsh III lesion) duodenal mucosa, based on IEL counts on >400 mucosal biopsy specimens. DESIGN: The study was designed at the International Meeting on Digestive Pathology, Bucharest 2015. Investigators from 19 centres, eight countries of three continents, recruited 198 patients with Marsh III histology and 203 controls and used one agreed protocol to count IEL/100 enterocytes in well-oriented duodenal biopsies. Demographic and serological data were also collected. RESULTS: The mean ages of CD and control groups were 45.5 (neonate to 82) and 38.3 (2-88) years. Mean IEL count was 54±18/100 enterocytes in CD and 13±8 in normal controls (p=0.0001). ROC analysis indicated an optimal cut-off point of 25 IEL/100 enterocytes, with 99% sensitivity, 92% specificity and 99.5% area under the curve. Other cut-offs between 20 and 40 IEL were less discriminatory. Additionally, there was a sufficiently high number of biopsies to explore IEL counts across the subclassification of the Marsh III lesion. CONCLUSION: Our ROC curve analyses demonstrate that for Marsh III lesions, a cut-off of 25 IEL/100 enterocytes optimises discrimination between normal control and CD biopsies. No differences in IEL counts were found between Marsh III a, b and c lesions. There was an indication of a continuously graded dose-response by IEL to environmental (gluten) antigenic influence.

Evolutionary Developments in Interpreting the Gluten-Induced Mucosal Celiac Lesion: An Archimedian Heuristic.

The evolving history of the small intestinal biopsy and its interpretation-and misinterpretations-are described in this paper. Certain interpretative errors in the technical approaches to histological assessment are highlighted-even though we may never be rid of them. For example, mucosal "flattening" does not reduce individual villi to their cores, as still seems to be widely believed. Neither is the mucosa undergoing an atrophic process-since it can recover structurally. Rather, the intestinal mucosa manifests a vast hypertrophic response resulting in the formation of large plateaus formed from partially reduced villi and their amalgamation with the now increased height and width of the inter-villous ridges: this is associated with considerable increases in crypt volumes. Sections through mosaic plateaus gives an erroneous impression of the presence of stunted, flat-topped villi which continues to encourage both the continued use of irrelevant "atrophy" terminologies and a marked failure to perceive what random sections through mosaic plateaus actually look like. While reviewing the extensive 40+ year literature on mucosal analysis, we extracted data on intraepithelial lymphocytes (IEL) counts from 607 biopsies, and applied receiver-operating characteristic (ROC)-curve analysis. From that perspective, it appears that counting IEL/100 enterocyte nuclei in routine haematoxylin and eosin (H&E) sections provides the most useful discriminator of celiac mucosae at histological level, with an effective cut-off of 27 IEL, and offering a very high sensitivity with few false negatives. ROC-curve analysis also revealed the somewhat lesser accuracies of either CD3+ or γδ+ IEL counts. Current official guidelines seem to be somewhat inadequate in clearly defining the spectrum of gluten-induced mucosal pathologies and how they could be optimally interpreted, as well as in promoting the ideal manner for physicians and pathologists to interact in interpreting intestinal mucosae submitted for analysis. Future trends should incorporate 3-D printing and computerised modelling in order to exemplify the subtle micro-anatomical features associated with the crypt-villus interzone. The latter needs precise delineation with use of mRNA in-section assays for brush border enzymes such as alkaline phosphate and esterase. Other additional approaches are needed to facilitate recognition and interpretation of the features of this important inter-zone, such as wells, basins and hypertrophic alterations in the size of inter-villous ridges. The 3-D computerised models could considerably expand our understandings of the microvasculature and its changes-in relation both to crypt hypertrophy, in addition to the partial attrition and subsequent regrowth of villi from the inter-villous ridges during the flattening and recovery processes, respectively.

Microscopic enteritis: Bucharest consensus.

Microscopic enteritis (ME) is an inflammatory condition of the small bowel that leads to gastrointestinal symptoms, nutrient and micronutrient deficiency. It is characterised by microscopic or sub-microscopic abnormalities such as microvillus changes and enterocytic alterations in the absence of definite macroscopic changes using standard modern endoscopy. This work recognises a need to characterize disorders with microscopic and submicroscopic features, currently regarded as functional or non-specific entities, to obtain further understanding of their clinical relevance. The consensus working party reviewed statements about the aetiology, diagnosis and symptoms associated with ME and proposes an algorithm for its investigation and treatment. Following the 5(th) International Course in Digestive Pathology in Bucharest in November 2012, an international group of 21 interested pathologists and gastroenterologists formed a working party with a view to formulating a consensus statement on ME. A five-step agreement scale (from strong agreement to strong disagreement) was used to score 21 statements, independently. There was strong agreement on all statements about ME histology (95%-100%). Statements concerning diagnosis achieved 85% to 100% agreement. A statement on the management of ME elicited agreement from the lowest rate (60%) up to 100%. The remaining two categories showed general agreement between experts on clinical presentation (75%-95%) and pathogenesis (80%-90%) of ME. There was strong agreement on the histological definition of ME. Weaker agreement on management indicates a need for further investigations, better definitions and clinical trials to produce quality guidelines for management. This ME consensus is a step toward greater recognition of a significant entity affecting symptomatic patients previously labelled as non-specific or functional enteropathy.

Diagnosis of coeliac disease.

The diagnosis of coeliac disease depends on the finding of characteristic, though not specific changes, of intraepithelial lymphocytosis, crypt hyperplasia and various degrees of villous height reduction identified in duodenal biopsies of individuals ingesting a gluten containing diet, together with symptomatic and histologic improvement on gluten withdrawal. Serologic testing has become the main mode of determining who will undergo biopsy. The IgA endomysial antibody and IgA tissue transglutaminase antibody have approximately 90% sensitivity and specificity, though there are reports of lower sensitivity and specificity in the clinical practice setting. This is due to lower titers of these antibodies in the presence of lesser degrees of mucosal damage. The widespread availability of serologic tests for coeliac disease allows the diagnosis to be considered by any physician. Gastroenterologists will be required to interpret the results of serologic tests and perform duodenal biopsies when indicated. Pathologists likewise need to be better acquainted with the more subtle changes of cell mediated immunity within the mucosa that are suggestive of underlying gluten sensitivity.

Celiac disease.

Clinically, celiac disease has always been regarded as a wasting, malabsorptive disorder due to disease of the small intestinal mucosa. It has been difficult for clinicians to recognize that this condition is primarily due to sensitization of mesenteric T lymphocytes to wheat protein (gluten) in genetically predisposed (DQ2+) individuals. On contact with dietary-derived gluten in the upper intestine, these sensitized T lymphocytes are activated leading to inflammation of and morphologically altered mucosal architecture: the latter reverts to normal with a gluten-free diet. The circulation of sensitized T lymphocytes to other parts of the intestinal mucosa explains why identical immunopathological inflammation can be induced in ileal and rectal mucosa. It appears, then, that in predisposed DQ2+ subjects, mesenteric T lymphocytes recognize gluten as foreign (non-self) antigen, thereby inducing mucosal pathology secondary to the initiating lymphocyte-protein interaction, analogously to the mucosal lesions that typify graft-vs-host reactions, or nematode or Giaraia infestations. Today, as this article describes, we recognize that celiac disease often exists in a subclinical, or "compensated-latent," form, or with symptoms that do not immediately suggest an origin in the gastrointestinal tract.