Diarrhoeal diseases are major causes of morbidity, with attack rates ranging from 2 to 12 or more illnesses per person per year in developed and developing countries. With improved management of acute episodes of infectious diarrhoea, increased attention is now being given to persistent diarrhoea and its nutritional consequences and associated mortality. Descriptive epidemiology indicates that 3%-20% of episodes of acute diarrhoea in children in developing countries become persistent and cause about one-third to one-half of all deaths from diarrhoea.
PATHOGENESIS OF PERSISTENT DIARRHOEA
AND MALNUTRITION
The pathogenesis of persistent diarrhoea and malnutrition (PDM) remains uncertain but whatever the relative importance of nutritional, infective or allergic factors, it is likely that the underlying basis of this syndrome is prolonged injury to the small intestinal mucosa. An understanding of this process and of the complex interactions that take place at the small intestinal luminal-mucosal interface is central to an understanding of the nature of persistent diarrhoea. Protein-energy malnutrition (PEM) has a severe impact on small intestinal growth, development, structure and function. PEM impairs the normal proliferative response of the small intestinal crypt epithelium to villous damage : this ineffective villous repair process perpetuates the syndrome. Animal work has demonstrated the effect of starving and refeeding on intestinal cell kinetics. Luminal factors are now known to be essential for the maintenance of normal intestinal structure and function and the promotion of mucosal repair following nutritional rehabilitation in malnourished subjects. Such luminal factors combine both exogenous (eg nucleotides, zinc, vitamin A etc) and endogenous substances (eg epidermal growth factor, enteroglucagon) with a defined physiological role in the maintenance of intestinal structure and function.
CLASSICAL TECHNIQUES FOR STUDYING THE INTESTINAL MUCOSA
The majority of morphological studies of the intestinal mucosa have been based on linear measurements of mucosal thickness and on simple visual assessment of the histological appearances of the villous pattern. However, Da Costa et al (1987) used quantitative stereological techniques, and demonstrated in malnourished rats a 60% reduction of total jejunal absorptive surface which occurred despite any obvious change in villous height. The implication of this is that biopsy specimens may be interpreted to be normal when assessed by villous height alone, although there may be very significant decreases in absorptive surface.
COMPUTERISED IMAGE-ANALYSIS TECHNIQUE FOR STUDYING THE INTESTINAL MUCOSA
More recent studies in The Gambia have used a computerised image-analysis technique
to evaluate jejunal mucosae in 40 children with PDM3. In this study, glutaraldehyde-fixed,
Epon-processed small bowel biopsies were sectioned with glass knives using a
Reichert OMU-3 ultramicrotome and stained with toluidine blue. Five or six
one micron sections were mounted per slide and 10 micrometer (µm) steps of tissue
were discarded between successive sections. Selected sections (one per slide)
that were perfectly oriented perpendicular to the mucosal surface were observed
through a X 100 oil-immersion objective with an Olympus BHS-2 research microscope.
For quantitative microscopy, sections were projected through a high-resolution
colour television camera to a MOP-Videoplan (Reicher-Kontron) image-analysis
system. All measurements were carried out relative to a standard test area of
100 µm2 of muscular mucosae. The volumes of both the surface and
crypt epithelial compartments were determined, together with the number and
size of inter-epithelial lymphocytes in both areas.
Using this computerised image analysis technique, measurements of surface epithelial
volume (villous compartment), crypt epithelial volume (crypt compartment) and
the absolute numbers of intra-epithelial lymphocytes (IEL) within these compartments
were obtained. In the majority (29/40) of specimens there was marked
hypertrophy of the crypts which invariably contained an elevated
population of IEL (NV, CR). In 13 of these specimens
surface epithelial volumes (VSE)
were normal, although villous epithelium contained an increased number of IEL.
The absolute number of IEL in the villous epithelium was proportional to the
surface epithelial volume, such that flat mucosae contained fewer lymphocytes
than more normally-structured mucosae. Six specimens at the extreme right hand
of the display were "flat" and lay within the range of VSE
for untreated coeliac sprue mucosae : all demonstrated crypt hypertrophy except
the last biopsy from the sole patient with kwashiorkor. Note that
although all remaining crypts carried high IEL populations, the absolute numbers
of surface IEL fell to within the control range as values for VSE
entered the flat coeliac-sprue range.
A wide spectrum of pathologic changes was observed in the small intestinal mucosa of 40 PDM subjects who underwent jejunal biopsy. These changes ranged from near "normal" appearances to a "flat", coeliac sprue-like lesion. Jejunal biopsies from each of 40 Gambian children studied were arranged in descending order of surface epithelial volumes (VSE x 106 mm3) (left hand axis), with their corresponding crypt epithelial volumes (VCR) displayed below the horizontal zero reference. (Reproduced with permission from the Journal of Pediatric Gastroenterology and Nutrition 3)
RESULTS OBTAINED WITH THE COMPUTERISED IMAGE-ANALYSIS TECHNIQUE
The spectrum of changes observed using the computerised image-analysis of jejunal
mucosae from Gambian children with persistent diarrhoea was reminiscent of those
seen in other human enteropathies associated with gluten sensitivity, giardiasis
and tropical sprue. The mucosae from some of these children revealed features
of what has been termed a "hyperplastic" lesion. This comprises crypt hyperplasia
and hypertrophy with infiltration of CD8+ IEL into crypt and villous epithelium,
the villi in these specimens being of reasonable size. These appearances are
similar to those seen in explants of fetal small intestine following T cell
stimulation in which crypt cell hyperplasia can occur in the absence of damage
to surface enterocytes. The "hyperplastic" lesion contrasts with the milder
"infiltrative" lesion, in which small lymphocytes occupy villous epithelium
only. IEL represent one of the first components of the immune system to encounter
intestinal pathogens or dietary antigens. Some of the remaining children demonstrated
a flat, or "destructive" lesion similar to that seen in coeliac sprue and dermatitis
herpetiformis characterised by complete lack of villi and infiltration
of crypt epithelium. In all these various conditions a stereotyped progression
of mucosal change and injury is seen but in which there is a striking parallelism
to the intestinal lesions evoked by various models of allograft and graft-versus
host reactions in experimental animals: these are T lymphocyte-dependent phenomena
brought about by lymphokine-activated cells and restricted by MHC Class 2 alloantigen
expression.
In summary, this quantitative mucosal morphometric study has shown that Gambian
children with PDM have an enteropathy characterised by a spectrum of immunopathologic
changes suggestive of T cell-mediated mucosal injury. It is not possible to
state whether or not these responses are directed against dietary, protozoal
or bacterial antigens: further work is necessary to elucidate the precise nature
of the luminal antigens that provoke these responses. It has been suggested
that intractable diarrhoea in infancy may be caused by autoreactive T cells
(probably TCR alpha-ß+) directed to a self antigen, and that a study of
the local T cell repertoire might furnish additional proof of this. Furthermore,
demonstration of lymphokine production (eg Interleukin 6, gamma-interferon,
tumour necrosis factor) by small intestinal mucosa would support the contention
that T cells do play a role in the mucosal changes described.
CLINICAL RELEVANCE OF THE STUDY OF SECRETION OF INTESTINAL MUCUS
To date all studies of the intestinal mucosa in persistent diarrhoea have focused on either the morphology or the permeability of the epithelium, overlooking the important mucus-gel layer. A major function of the gastrointestinal adherent mucus gel layer is to act as a stable unstirred layer creating a microenvironment at the mucosal surface which does not mix with the bulk luminal contents. It is noteworthy that decreased mucus secretion has been demonstrated in malnourished rats. The combination of depressed mucus production and consumption of feeds contaminated with faecal micro-organisms may account for the development of upper intestinal bacterial overgrowth in infants with PDM. A deficit in this defence system may not only facilitate bacterial contamination of the small intestine but also lead to a breakdown in "immune exclusion". This may allow microbial pathogens and dietary antigens increased access to the intestinal mucosa with adverse effects on intestinal function. Recent methodological advances in the study of mucus glycoproteins have led to a greater understanding of their importance and role in mucosal defence. Application of these new techniques in individuals with malnutrition and diarrhoeal disease may prove to be a fruitful area of research in the future.