Diagnosing coeliac disease by rectal gluten challenge: a prospective study based on immunopathology, computerized image analysis and logistic regression analysis.

The purpose of this study was to evaluate the use of rectal gluten challenge in the diagnosis of coeliac disease. A total of 103 patients with features suggestive of this diagnosis were prospectively enrolled into the study; a diagnosis of coeliac disease was based on strictly defined criteria used in judging the proximal jejunal biopsy. On that basis, 45 out of the 103 patients were deemed to have coeliac disease. A slurry of gluten powder in physiological saline was introduced into the rectum, and biopsies taken before and at 2 h or 4 h after the challenge were examined immunohistochemically by computerized image analysis. Cell counts were analysed by logistic regression, and the best equations were obtained for each challenge group. The 2 h challenge yielded diagnostic sensitivity and specificity of 69.6% and 78.6% respectively. The 4 h challenge provided sensitivity and specificity of 100% and 100% respectively. These results were compared with other clinical diagnostic predictors,including anti-endomysial antibodies, which yielded diagnostic sensitivity and specificity of 70% and 98% respectively. It is concluded that a 4 h rectal challenge is a highly sensitive means of identifying gluten-sensitized individuals, and would be of particular value in cases showing negative antibody screening or equivocal biopsy appearances.

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).

Morphology of the mucosal lesion in gluten sensitivity.

Gluten sensitivity is associated with a spectrum of mucosal lesions, arbitrarily termed pre-infiltrative, infiltrative-hyperplastic, flat-destructive and atrophic-hypoplastic. Histologically and immunohistologically these lesions are all compatible with T-cell-driven events operative at a local mucosal level. They are classifiable either in terms of antibody titres (pre-infiltrative) (see Chapter 10) or by the characteristic disposition of IELs throughout the surface and crypt epithelium. From in-vivo challenges, it has been demonstrated: (i) that all these lesions comprise a dynamically interrelated series of events, culminating in the severe flat-destructive lesion; and (ii) that gluten evokes a dose-responsive infiltration of IELs (CD3+ CD8+ and TCR alpha beta + or gamma delta +) into the epithelium. Apart from that, little is known of the functions of IELs; it is possible they may have little to do with the evolving mucosal pathology of gluten sensitivity. Increasing work seems to support a view, proposed from this laboratory over 10 years ago, that the immune-mediated responses in jejunal tissue in gluten sensitivity arise in the lamina propria, in association with DR+ macrophages and an abundance of CD4(+)-activated lymphocytes. Many other inflammatory consequences flow from these interactions, involving activation of mast cells, eosinophils and neutrophils, elaboration of cytokines and other products of inflammation, and increased hyperpermeability of the microvasculature with upregulation of adhesion molecules. The result is a doubling of lamina propria volumes in the severe flat lesion. Evidence is also given to show that measurable changes in lamina propria inflammation occur with the infiltrative-hyperplastic lesion. Symptomatology is not related to the degree of proximal mucosal pathology, but to the extent of the mucosal lesion. Data, although scanty, suggests that lesional pathology involves only 30-50% of the entire small bowel mucosa. Thus, most patients, irrespective of proximal mucosal damage, have latent (or asymptomatic) gluten sensitivity. Symptom development requires additional environmental triggers, of which infection is a major contributor. It should also be noted that, while these various environmental triggers may precipitate symptomatology, they do not advance the severity of the mucosal lesion.

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.

Morphometric analysis of small intestinal mucosa. IV. Determining cell volumes.

This study was concerned with the measurement of volumes of surface epithelial cells in human small intestine, both in disease-control subjects and, for comparison, in patients with gluten sensitivity. Four procedures were employed, of which two were geometrical, based on cylindrical or truncated conoid models. The third method evolved from the proportionality of area to volume, and required determination of cellular and nuclear profile areas, and an estimation of nuclear volume based on models conforming to (1) a prolate spheroid, or (2) a cylinder with hemispherical caps. This procedure appeared to underestimate enterocyte volumes and failed to reveal volume differences between controls and gluten-sensitive individuals. Finally, a fourth method was devised, based on traditional intraepithelial profile counts per hundred enterocyte nuclei, calculation of surface epithelial volume and of the absolute number of lymphocytes contained therein. Enterocyte volumes appeared to be overestimated twofold by this procedure compared with the first two geometric methods. The results of this study indicate that the cuboidal-type enterocyte profiles typical of the untreated mucosa in gluten sensitivity are a reflection of cells with a reduced volume. From the number of enterocytes and the absolute lymphocyte population present within a morphometrically defined volume of surface epithelium, the ratios of intraepithelial lymphocytes to enterocytes were found to be 50% less than conventional density-profile counts.

Persistent diarrhea and malnutrition--the impact of treatment on small bowel structure and permeability.

Previous studies using dual sugar permeability tests suggested that damage to the small intestinal mucosa plays an important part in the development of persistent diarrhea in The Gambia. The present study has extended these findings by examining the effect of nutritional rehabilitation on intestinal permeability and mucosal morphology. Intestinal permeability, measured by lactulose:mannitol (L:M) absorption, and mucosal structure, measured by a quantitative, computerised morphologic technique, were evaluated in 20 children before and after such treatment. L:M ratios were high on admission, (0.66 +/- 0.36) and, despite some temporary improvement, did not significantly improve (0.49 +/- 0.30) following rehabilitation for one month. The changes in L:M ratio were largely due to an increase in lactulose absorption, showing that the small intestinal mucosa becomes more "leaky" as a result of nutritional rehabilitation. Although no correlation was found between measures of intestinal permeability and mucosal morphology, nutritional restitution was associated with a significant increase in size of the mucosal crypt cell compartment, but not in villous epithelial volumes during the same period. It is necessary to establish, by further prospective studies, the interval required for full restitution of small intestinal structure and function during treatment for persistent diarrhea.

Active hexose transport across cultured human Caco-2 cells: characterisation and influence of culture conditions.

Human Caco-2 cells (passage 80 to 100) were seeded onto collagen-coated Millipore filter assemblies and these were maintained in culture either (a) floated on the surface of the medium or (b) submerged within the body of the medium. Structural and functional assessments were made over a 30-day period. After seeding, all cells assumed a flattened, squamous configuration and rapidly became confluent. Cells submerged within the medium formed polarised monolayers with well developed junctional complexes, abundant apical microvilli and increasing levels of alkaline phosphatase activity. Cells grown floated on the surface of the medium formed complex multilayers in which polarisation was confined to the surface layer. Junctional complexes and apical microvilli were similar to those seen in submerged monolayers but alkaline phosphatase activities were higher. Transepithelial electrical resistance increased rapidly from day 1, as the layers became confluent. Electrical resistance was higher and short-circuit current and potential differences were lower across monolayers than across multilayers. After 10 days in culture, the addition of D-glucose to the apical bathing solution, of all cell layers, caused a rapid rise in short-circuit current and potential difference. These changes were sodium-dependent and phlorizin-sensitive. Galactose and 3-O-methylglucose induced similar changes and the affinity constants for these hexoses ranked in the order reported for rat jejunum (Km glucose 2.44 +/- 0.52 mM; Km galactose 8.05 +/- 1.33 mM; Km 3-O-methylglucose 22.0 +/- 5.2 mM). Culture conditions had a marked effect on hexose maximum transport rates (glucose Vmax: submerged 2.94 +/- 0.20 microA/cm2; floated 9.94 +/- 0.82 microA/cm2, P less than 0.05) but affinity constants were unchanged. Apical to basolateral mannitol fluxes, used as an index of paracellular permeability, decreased from day 1 to day 5 and then remained steady. Fluxes across monolayers and multilayers were not significantly different. We conclude that sodium-dependent hexose transport occurs in cultured Caco-2 cell layers grown on permeable supports. Culture conditions, however, have a marked effect on both cell layer structure and function, and should be an important factor when considering Caco-2 cells as an in vitro model of enterocyte function.

Rectal gluten challenge and diagnosis of coeliac disease.

44 patients referred consecutively for jejunal biopsy underwent rectal gluten challenge with 2 g peptic-tryptic digest (Frazer's fraction III; FF3). Rectal biopsy was done before the challenge and 6 h afterwards. Total intraepithelial lymphocytes (IEL) overlying a 10(4) micron 2 test area of muscularis mucosae were quantified by computerised image analysis. The subjects comprised 21 controls with disorders other than coeliac disease and 23 patients (14 treated, 9 untreated) with coeliac disease diagnosed by strict jejunal biopsy gold standard criteria. There was no difference between the groups in IEL numbers before challenge. Coeliac disease patients but not controls responded to FF3 with a rise in mucosal IEL (median 60.5% rise for treated, 63.0% for untreated). There was no response to challenge with beta-lactoglobulin in coeliac disease or control subjects. When a predefined, post-challenge IEL "predictive index" of more than 10% above baseline was used to indicate a diagnosis of coeliac disease, it gave a sensitivity of 90% and specificity of 91% (95% confidence intervals 78-93%). Rectal gluten challenge is a simple, safe, and reliable test of gluten sensitivity, both as a screening test for untreated coeliac disease and as a confirmatory test in patients with treated coeliac disease.

Studies of intestinal lymphoid tissue. XII. Epithelial lymphocyte and mucosal responses to rectal gluten challenge in celiac sprue.

The immunopathologic, structural, and functional changes within rectal mucosa of known celiac sprue subjects were quantitated during local challenge with a peptic-tryptic digest of gluten. In the celiac sprue patients challenged with 2 g of digest, major effects occurred in lamina propria, submucosa, and local microvasculature. The lamina propria swelling was biphasic, starting 1-2 h after challenge with widespread extravascular deposition of fibrinogen, indicative of increased microvascular permeability, receding by 24 h postchallenge. A rapid fall in mast cells together with granule discharge suggested their involvement in this response. The late-phase swelling (48-72 h) was preceded by a rapid influx of neutrophils and basophils, the latter showing evidence of degranulation beyond 72 h. Reestablishment of vessel lumina, a rise in mast cells, and loss of neutrophils indicated tapering of the inflammatory cellular cascade by 96 h. Lymphocytes, first seen to enter the lamina by 2 h postchallenge, increased progressively, thereby resulting in substantial infiltration between 36 and 96 h. A marked rise in epithelial lymphocytes, maximal at 6-8 h, waned by 24 h. Volumes of surface and crypt epithelium remained constant throughout. In another challenge series with 4 g of gluten digest, electrical potential difference across rectal mucosa decreased significantly 12 h postchallenge, but the associated decreases in net sodium and chloride absorptive fluxes were insignificant. It is concluded that rectal mucosa is sensitized to gluten in celiac sprue disease and thus offers a promising and convenient in vivo substrate for investigative and diagnostic purposes.