Screening and monitoring coeliac disease: multicentre trial of a new serum antibody test kit.

A multicentre trial was conducted to evaluate a new test for anti-gliadin antibodies (AGA) in serum (Coeliac Screening Kit, CSK, Medical Innovations Limited, Artarmon, NSW, Australia). The test showed excellent reproducibility for both anti-gliadin IgA and IgG detection. The average intraassay coefficient of variation (CV) was 3.0% for IgA and 2.4% for IgG (n = 6), while the average interassay CV was 6.4% for IgA and 4.3% for IgG (n = 3). By defining a positive test as both IgA and IgG elevated, a sensitivity of 93% in untreated coeliacs (n = 75) was observed. The corresponding specificities in healthy adults (n = 130) and healthy children (n = 77) were > 99% and 100% respectively, while in patients with other gastrointestinal disorders (disease controls) the specificity was 94% (n = 129). The test was also useful in monitoring patients, with anti-gliadin IgA and IgG falling for up to a year after commencing a gluten-free diet (GFD) (12 adults). In some patients however, antibody levels did not reach the normal cutpoint after many months on a GFD, which may reflect the patients' poor adherence to their gluten free diet. The test was superior to the Pharmacia anti-gliadin ELISA, and should be useful as an aid to the diagnosis of coeliac disease, as well as in the follow-up of treated patients.

Studies of intestinal lymphoid tissue. IV--The predictive value of raised mitotic indices among jejunal epithelial lymphocytes in the diagnosis of gluten-sensitive enteropathy.

It has been established that considerable blast-transformation and mitotic activity occurs among epithelial lymphocytes of untreated coeliac mucosa. This paper is concerned solely with the proliferative activity of epithelial lymphocytes (expressed as percentage "mitotic index") in the prospective diagnosis of coeliac disease, in comparison with other conditions such as lymphoma. Crohn's disease and immunodeficiency which are often associated with malabsorption and flattening of jejunal mucosa. The results demonstrate that a high mitotic index (greater than 0.2%) clearly distinguishes, and hence predicts, gluten-associated enteropathies (including dermatitis herpetiformis and malignant histiocytosis) from others in which gluten plays no aetiological role and where the mitotic index differs insignificantly from normal control mucosae (much less than 0.2%). Furthermore, it has been demonstrated that the mitotic index is raised in so-called "non-responsive coeliacs," thus suggesting that such patients may also be gluten-sensitive despite their subsequent failure to respond morphologically to dietary gluten restriction.

Morphometric analysis of intestinal mucosa. VI--Principles in enumerating intra-epithelial lymphocytes.

The mucosal response by intra-epithelial lymphocytes (IEL) to antigenic challenge is a useful monitor of local immune activity. Conventional counts of IEL (determined as profile ratios of IEL to enterocyte nuclei) are inaccurate, and over-estimate values by a factor of two, both for disease-control mucosae and untreated 'flat' gluten-sensitized mucosae. Two further proofs are advanced in this paper which expose the inaccuracy of conventional profile-density IEL counts. New ranges (log-transformed data) indicate a disease-control mean of 11 IEL per 100 enterocytes (95% confidence limits 5-27) and 29 IEL per 100 enterocytes (95% confidence limits 14-61) for untreated flat gluten-sensitive mucosae. For simplicity, if conventional IEL "counts" are halved, correct values (based on precise morphometric analyses) are easily obtained for comparative and other purposes.

Studies of intestinal lymphoid tissue. VI--Proliferative response of small intestinal epithelial lymphocytes distinguishes gluten- from non-gluten-induced enteropathy.

Several diseases of the small intestine, including gluten-sensitivity, present with malabsorption and a "flat" mucosa. Determination of the mitotic index of epithelial lymphocytes provides a simple, objective method of assessing, and thus of predicting, whether a flat mucosa is due to gluten-sensitivity (index greater than 0.2%), or not (index less than 0.2%). The use of this index in circumstances especially likely to cause diagnostic confusion--for example, intestinal lymphoma; Crohn's jejunitis of immunodeficiency--is illustrated in this paper. Of seven cases, five (two primary lymphoma, three immunodeficiency) had been treated with a gluten-free diet without benefit; a mitotic index performed on the initial biopsy in each of these patients could have predicted from the outset that none was gluten-sensitive. Of the remaining two cases, determination of the mitotic index on the biopsy initially obtained from a man with severe hypogammaglobulinaemia would have indicated that he was also gluten-sensitive. Empirical use of a gluten-free diet was avoided in the other patient (with flat small intestinal mucosa and low mitotic index) in whom the diagnosis was ultimately shown to be due to Crohn's disease of jejunum.

Reassessment of faecal alpha-1-antitrypsin excretion for use as screening test for intestinal protein loss.

Faecal alpha-1-antitrypsin and 51Cr-albumin losses in 42 patients with either gastrointestinal or hepatic disease were compared. The reference range was derived from measurements in 20 controls without gastrointestinal disease. Alpha-1-antitrypsin excretion was increased in patients with excessive 51Cr-albumin loss, and correlations were found between alpha-1-antitrypsin clearance and 51Cr-albumin excretion. Because of the considerable overlap of faecal alpha-1-antitrypsin excretion between controls and patients, sensitivity and specificity of the test were only 58% and 80%, respectively. This poor reliability could not be explained by sampling error or temporal variations in alpha-1-antitrypsin excretion. These results show that although faecal alpha-1-antitrypsin excretion correlates with 51Cr-albumin excretion when whole groups of patients are studied, its poor sensitivity makes it an unreliable measure of enteric protein loss.

Celiac disease : a brief overview.

Historically, the term celiac disease evolved within pediatric practice during the nineteenth century, defining children with severe wasting and putrid stools (1). In the earlier twentieth century, similar complaints in adults were categorized as "intestinal insufficiency" or "idiopathic steatorrhea." It was also realized at that time that, for many of these adult patients, celiac-like features had been present since early childhood.

Morphometric analysis of intestinal mucosa : the measurement of volume compartments and cell volumes in human intestinal mucosa.

The widespread use of peroral (capsule) and, more recently, endoscopically obtained mucosal biopsies from jejunum and duodenum provides an easy source of material for diagnostic (clinical) and investigative scientific study. The basis of our understanding of small intestinal diseases has stemmed directly from these sources since, in addition to the purely morphological (and pathological/immunopathological) domain, these biopsies have helped to elucidate other types of mucosal disease (e.g., the "disaccharidase" deficiencies, defects in water and electrolyte transfer, and amino acid transport and lipid metabolism).

Morphometry of rectal mucosa.

The diagnosis of many gastrointestinal disorders is made by assessing lesions in an endoscopic mucosal biopsy subjectively, or by objective parameters and morphometric techniques (1-4). Morphometry was first introduced into pathology 80 yr ago. It arose from doubts about qualitative observations and the need to correlate changes in morphology with function (5,6). The quality of the results of morphometry is mainly affected by the preparation of the specimens. Since biological structures such as mucosae are highly organized structurally and, in mathematical terms, are extremely anisotropic, a proper allowance for the orientation of the measured structures must be made. Thus, in comparative morphometric studies, it is essential to ensure that only sections obtained in proper and consistent orientation are used (2,7,8).

Studies of intestinal lymphoid tissue. XIII. Immunopathology of the evolving celiac sprue lesion.

Observations, by computerised image-analysis on the evolution of flat mucosae in two celiac sprue patients, are described. Initial immunopathologic features comprised infiltration of epithelium of normal villi by small, non-mitotic lymphocytes, accompanied by crypt hypertrophy and increased crypt cell mitotic activity. The subsequent development, several years later, of flat mucosae was accompanied by mitotic, "immunoblastoid" EL thus fulfilling the diagnostic criteria for celiac sprue. These sequential phases in the evolving flat celiac mucosa parallel experimental graft-versus-host reactions, suggesting that they are fundamentally cell-mediated in type. In becoming flat, it appears obligatory for the mucosa to pass through the early "proliferative-infiltrative" stage in which crypt hypertrophy is a prominent feature.

Small intestinal mucosal histology in the syndrome of persistent diarrhoea and malnutrition: a review.

Prolonged injury to the small intestinal mucosa is probably the final common pathway by which a variety of noxious influences--nutritional, infective and possibly allergic--perpetuate the syndrome of persistent diarrhoea in children in developing countries. Animal studies have helped to separate the individual effects of malnutrition and diarrhoea on the gut in a way that is not possible in the clinical situation. Early studies in children provided somewhat subjective or semi-quantitative data on intestinal morphology. More recently, the application of computer-assisted quantitative morphological techniques to intestinal mucosae from children with persistent diarrhoea have revealed a spectrum of changes consistent with a cell-mediated immune form of damage. The nature of the antigens that provoke these responses remains to be elucidated. Several reports indicate that in children with persistent diarrhoea clinical severity and prognosis do not necessarily correlate with the degree of small intestinal mucosal damage. Nutritional rehabilitation can be shown to produce a demonstrable improvement in small intestinal crypt cell proliferative activity in children with persistent diarrhoea. It is not yet known for how long nutritional rehabilitation should be continued to ensure complete recovery of the intestinal damage following persistent diarrhoea.

Studies of intestinal lymphoid tissue: the cytology and electron microscopy of gluten-sensitive enteropathy, with particular reference to its immunopathology.

Knowledge of the cytology, ultrastructure and histochemistry of the small intestinal mucosa has advanced considerably over the last 20 years, particularly in regard to the changes associated with coeliac disease. Many of the known structural and cytological changes in coeliac mucosa are probably non-specific, and not directly related to its pathogenesis. It seems important to move away from purely descriptive images of mucosal abnormality such as "villous atrophy', and static measurements, such as crypt-villous ratios. Rather, the mucosa should be viewed as a dynamic, three-dimensional structure and evaluated in terms of total villous cell counts, crypt cell production rates, and so on. The organisation of the lamina propria is still poorly documented, and requires further exploration at the ultrastructural level. More thought should be given to the meaning of mucosal permeability and to its structural counterparts. The immunocytopathology of the coeliac lesion is far from understood; it is questionable whether local humoral activity is central to the pathogenesis of the condition. More needs to be learned of the role of T cells, not only in local mucosal reactions, but also in terms of possible regulatory effects on crypt cell kinetics, villous shape and hence mucosal structure. Increased mitotic activity of epithelial lymphocytes in coeliac disease appears to correlate exclusively with gluten-sensitivity and the use of this presumed immunological marker in the histological diagnosis, and thus prediction, of gluten-sensitised individuals is proposed in this paper.

Functional and structural aspects of the epithelial lymphocyte, with implications for coeliac disease and tropical sprue.

Epithelial lymphocytes comprise a compartmentalised and specialised population of presumed effector cells which, in general, express the surface phenotypes (Lyt-2+; OX2+; OKT8+) of suppressor/cytolytic (Ts/c) cells. Granular cells within this population (gEL) morphologically resemble the circulating large granular lymphocytes (LGL) which subserve spontaneous (NK) cytolytic activity. Recent in vitro results indicate that gEL can develop this function after prolonged in vitro culture; the relevance of this, in vivo, remains to be decided. EL also appear to be able to mediate ADCC with sIgA against enteric micro-organisms. This is the kind of integrated activity that might be anticipated from local immunocytes within the intestinal mucosa. Other recent work suggests that gEL are not precursors of mucosal mast cells. EL also appear to be capable of inducing Ia-like expression in surface and crypt enterocytes, a property enjoyed both by highly purified Th, but also Ts/c, cells as well. This raises the interesting prospect that enterocytes may display antigen in macrophage-like fashion to other adjacent cells within the inter-epithelial cell spaces. These latter observations might be more consistent with the presence of 'activated' and 'blast-transformed' lymphocytes in such conditions as coeliac disease and tropical sprue. Another emergent view that demands appropriate attention is that the infiltrate of Ts/c cells into surface, and crypt, epithelium of coeliac mucosa does not necessarily cause injury or damage to the jejunal tissues. Nevertheless the role, either primary or secondary, that EL play either in coeliac disease or tropical sprue still remains obscure.

Studies of intestinal lymphoid tissue. III. Quantitative analyses of epithelial lymphocytes in the small intestine of human control subjects and of patients with celiac sprue.

To investigate the morphology and proliferation of small intestinal lymphocytes, 1-micrometer sections of Epon-embedded jejunum from 14 control subjects and 5 celiac sprue patients were studied by light microscopy. Results showed that: (a) Epithelial lymphocytes were of significantly greater diameter in untreated, compared with treated, celiacs (P < 0.001), and comprised 5% more cells > 9 micrometer diameter compared with control mucosa. (b) Mitotic indices of epithelial lymphocytes were considerably raised in untreated sprue, and a brisk rate in accumulation of metaphase-arrests was observed during a 4-hr period of colchicine administration; after treatment, proliferative activity fell steeply, but not to the zero levels found in controls. (c) "Areal densities" (number of lymphocytes subtended by unit area of muscularis mucosae) were reduced in untreated patients (P < 0.02) and increased to control levels after treatment. "Flux ratios" (number of epithelial lymphocytes penetrating basal lamina/unit area muscularis divided by "areal density") were greater in untreated, than treated, specimens (P < 0.005) suggesting that in untreated sprue, there is increased traffic of lymphocytes across basal lamina. Thus celiac epithelium contains fewer, but significantly larger lymphocytes that exhibit heightened mitotic activity; greater numbers of lymphocytes also appear to be in transit across the basal lamina. With the exception of mitotic activity, all other observed changes were completely reversed by gluten withdrawal.

Studies of intestinal lymphoid tissue. XV. Histopathologic features suggestive of cell-mediated reactivity in jejunal mucosae of patients with dermatitis herpetiformis.

Peroral jejunal mucosae from 32 patients with untreated DH were quantitated by computerized image-analysis in terms of surface (villous) and crypt epithelial volumes and their corresponding lymphoid infiltrates, together with lamina propria volumes, neutrophils, mast cells and basophils. Three distinctive patterns of mucosal abnormality were identified: (a) the "infiltrative" lesion in which normal villus epithelium was infiltrated by small, non-mitotic lymphocytes: (b) the "hyperplastic" type, in which crypt hyperplasia and hypertrophy together with lymphoid infiltration of crypt epithelium was additional to the "infiltrative" lesion, and in which lamina propria was swollen and contained modest neutrophilic and basophilic infiltration: and (c) the "destructive" lesion, identical to the classic celiac sprue appearances with effacement of villi, crypt hypertrophy and more intensive polymorph infiltration of lamina propria. These progressive lesions parallel those seen in experimental graft-versus-host reactions, so that the entire spectrum of changes described here in DH appear consistent with a cell-mediated mucosal response to gluten. The extent of mucosal abnormality was unrelated to individual HLA status.

Studies of intestinal lymphoid tissue. I. Electron microscopic evidence of 'blast transformation' in epithelial lymphocytes of mouse small intestinal mucosa.

The morphology of epithelial lymphocytes in osmium-fixed, Epon-embedded jejunum of adult mice was studied by light and electron microscopy. Toluidine blue-stained 1 mum 'thick' plastic sections were compared with adjacent thin sections, thereby permitting precise ultrastructural identification and description of selected epithelial lymphocytes. Their size and appearances varied considerably, ranging from typical small lymphocytes through medium-sized lymphocytes to large immunoblasts. A high proportion of medium-sized epithelial lymphocytes (mean diameter 6-9 +/- 1-1 mum) contained several lysosomes, extensive Golgi complexes, prominent centrioles and abundant ribosomes. Their appearances, therefore, corresponded directly to mitogen-stimulated lymphocytes. In contrast, immunoblasts were big cells (mean diameter 11-0 +/- 0-8 mum) with large, euchromatic nuclei and prominent nucleoli. The majority had pale-staining, ribosome-studded cytoplasm and thus resembled type I, or T blasts. Very rarely, densely staining blasts containing ribosomes and well developed rough endoplasmic reticulum were observed; these corresponded to type II or B blasts. These observations indicate that transformation of lymphocytes occurs within the interepithelial cell spaces of the small intestinal mucosa, suggesting that epithelial lymphocytes are immunocompetent cells which may be responsive to local antigenic stimulation.