Gut, 1985, 26, 710-717

Rectal mucosal morphologic abnormalities in normalsubjects in southern India: a tropical colonopathy?MINNIE M MATHAN AND V I MATHAN

From the Wellcome Research Unit, Christian Medical College Hospital, Vellore, India

SUMMARY Electron dense bodies and vesicles were increased in undifferentiated crypt cells anddifferentiated colonocytes in the rectal mucosa of healthy volunteers in southern India. Inaddition, in the surface colonocytes lysosomes were increased, the cells were shorter with shortirregularly grouped microvilli, there was evidence of cell immaturity and a high prevalence ofspiral organism infestation. There was also alterations in goblet cell mucus granules, a

reticulohistiocytic response in the subluminal lamina propria and residual evidence of vasculardamage. These alterations indicate a non-specific response to mucosal injury. Such changes havenot been observed in the rectal mucosa of temperate zone controls and could be designatedtropical colonopathy.

Morphologic abnormalities in the small intestinalmucosa in healthy populations of many tropicaldeveloping countries, associated with minor absorp-tive defects, is recognised as tropical enteropathy. -3These widely prevalent structural and functionalalterations are likely to be an adaptation of the smallintestinal epithelium to the contaminated environ-ment of the tropics.4 Although the ultrastructuralmorphology of the rectal mucosa has been studiedextensively in temperate zone controls'11 there areno such reports from tropical regions. This paperreports the ultrastructural morphology of the rectalmucosa in a group of healthy volunteers fromsouthern India. A 'tropical colonopathy', similar tothe small intestinal lesion, was shown by abnormali-ties in the rectal mucosal epithelium and laminapropria in this normal population.

Methods

BIOPSIESRectal mucosal biopsies (two or three pieces) wereobtained, at sigmoidoscopy, 8-10 cm from the analverge, with the Truelove-Salt suction biopsy instru-ment, from 14 adult village volunteers aged 22-50years without gastrointestinal symptoms and normaltests of intestinal absorption. None of them had haddiarrhoea during the two months before biopsy.One or two pieces of the rectal mucosa were

immediately fixed in 2 5% glutaraldehyde with 0 1Address for correspondence: Professor Minnie M Mathan, The WellcomeResearch Unit, CMC Hospital, Vellore 632 004, India.

Received for publication 24 August 1984710

mM CaCl2 in phosphate buffer (pH 7.4), postfixedin 1% phosphate buffered osmium tetroxide andembedded in Araldite. One micron thick sectionswere stained with toluidine blue and examined toassess histological features. Well orientated blocksof tissues where complete longitudinal crypt profilescould be identified were selected for detailedmorphological study. Ultrathin sections were cutwith a diamond knife and stained with saturatedaqueous uranyl acetate and lead citrate for examina-tion with a Philips EM201 electron microscope. Onebiopsy from each patient was processed for paraffinsections stained with haematoxylin and eosin.For comparison with the biopsy material from

southern India, Dr Gregory L Eastwood, Worces-ter, Massachusetts, kindly provided four biopsiesfrom healthy adult volunteers in the United States ofAmerica. Observations on these biopsies as well aspublished reports from temperate climates will bereferred to as temperate zone controls. Measure-ments were done on at least five well orientatedcomplete sections of epithelial cells for each subjectand mean values calculated.

Results

The architecture of the rectal mucosa, with epithe-lial cells organised in tubular glands separated bylamina propria, was similar to that reported fromtemperate regions (Fig. 1). At the base of cryptsundifferentiated and differentiating colonocytes andendocrine cells and higher up differentiated colum-nar colonocytes and mucus producing goblet cells

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Rectal Mucosa in the tropics

Fig. 1 Light micrograph ofa well orientated rectal biopsyfrom a southern Indian control subject. Note increasedcellularity ofthe lamina propria (Paraffin embedded andstained with haematoxylin and eosin, x100 originalmagnification)

characterise the rectal mucosa. Minimal increase inthe cellularity of the lamina propria and shorteningof columnar cells at the luminal area with some lossof nuclear polarity could be appreciated by lightmicroscopy in conventional paraffin sections and inl,u sections. In the 1,u sections, in addition, altera-tions in the staining density of colonocytes andvacuolation of the cytoplasm in the luminal areawere also apparent (Fig. 2). Goblet cells wereapparently normal. Spiral organisms, adherent tothe surface colonocytes, were seen at light micro-scopy in nine of the biopsies.

ULTRASTRUCTURAL MORPHOLOGYUndifferentiated cellsThe undifferentiated cells at the base of crypts hadlarge, indented, basally placed nuclei, poorlyformed microvilli, abundant ribosomes, with manypolyribosomes and a few large mitochondria con-taining small dense bodies. The endoplasmic reticu-lum was scanty and Golgi apparatus was welldeveloped but sparse. Round electron dense bodiesand small vesicles were present in the apicalcytoplasm (Fig. 3). These were usually uniformlyelectron dense, but some showed a clear areasurrounding or to one side, while a few were lessdense and appeared grey. A limiting membrane wasidentifiable on electron dense bodies that were lessdense or had a surrounding clear space. Electrondense bodies were increased in number and moreelectron dense in the southern Indian controlsubjects compared with temperate zone controls. Infour subjects electron dense bodies were markedlyincreased.

Columnar cellsFully differentiated columnar cells on the free

Fig. 2 (a) Rectal biopsy from southern Indian controlsubject. Luminal surface cells showing cytoplasmicvacuolation and variation in staining density. Nucleii areplaced at different levels. Spiral organism are seen attachedto microvillous border. Subepithelial reticular layer is moreprominent. (b) Biopsy from western controlfor comparison(Araldite embedded. I , section stained with toluidene blue,x 930 original magnification).

luminal surface, between the opening of the crypts,develop from the undifferentiated cells in the cryptbase. Cellular.maturation was indicated by morecentrally placed nuclei, with less prominent nuclearindentation, development of microvilli with deepcore rootlets extending into the apical cytoplasm,abundant glycocalyx and microvillar bodies, anincrease in number with decrease in size of supra-and infra-nuclear mitochondria and increasing plica-tions of the lower half of the lateral cell membrane.Electron dense bodies and vesicles increased innumber and size and became less electron densewith larger vesicles containing fine fibrillary oroccasionally crystalloid material (Fig. 4). The finefibrillary material in the vesicles was different fromthe more flocculent contents of the goblet cell mucusgranules (Fig. 5), and resembled the glycocalyx ofthe microvilli and apical border of the columnar cells(Fig. 6). Large lysosomal bodies and R bodies withvesicular structure containing rod-like inclusions,12

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a W b __Fig. 3 (a) Apical cytoplasm of undifferentiated cell in the crypt base ofsouthern Indian control subject with increasedelectron dense bodies and vesicles (x9600 original magnification). (b) Similar cellfrom a western controlfor comparison(x 9600 original magnification).

were seen in the apical cytoplasm of columnar cellsfrom the lower third of the crypt onwards. As thecell matured and progressed towards the upper thirdof the crypt secondary lysosomes and R bodiesincreased in number (Fig. 7). The luminal columnarcells were shorter (35.2±10. 1,) than in the temper-

Fig. 4 Apical border ofcolumnar cellfrom lower third ofcrypt with vesicles containing cystalline material (arrow)(x38 500 original magnification).

ate zone controls (48.3±6.7,u), with irregularlygrouped shorter microvilli (southern India0 83±0*41,u, temperate zone 1.14±0.25,u) andnuclei often arranged at different levels in contrastwith the orderly arrangement near the basal half ofthe cells in the temperate zone controls (Fig. 8). Theelectron density of the columnar cells in the luminalarea varied. Some cells were very electron densewith condensed nuclei and cytoplasm, largemitochondria with a dense matrix and a submicro-villus dense band. Majority of the cells, took a palerstain and were less electron dense with numeroussmall mitochondria. These mitochondria were lessabundant and the interdigitations of lateral cellborders in the basal half less well developed in thesouthern Indian subjects on comparison to temper-ate zone controls.

In nine of the 14 biopsies from southern Indianumerous spiral organisms were seen embedded inthe apical cytoplasm of the luminal cells and cells atcrypt mouths. Microvilli of the cells with spiralorganisms were short and irregularly grouped withincreased spherical microvillus bodies and irregularprotrusions between microvilli (Fig. 9). In fourbiopsies with heavy spiral organism infestation, theproportion of columnar cells compared with gobletcells was increased and they contained more elec-tron dense bodies in the lower third of the crypt andincreased apical vesicles higher up.

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Fig. 5 Cells from upper third ofcrypt with goblet cellmucus granules (G) showingflocculent material andcolumnar cell vesicles (C) containingfine fibrillary material(x38 500 original magnification).

Goblet cellsIn four subjects there was marked variation in thedensity of the mucus granules of goblet cells withsome of the granules being extremely electron dense(Fig. 10). In these four patients there was also a

marked increase in electron dense bodies andvesicles in the columnar epithelial cells along the

Fig. 7 Apical cytoplasm ofcolumnar cells from upperthird ofcrypt with increase in electron dense bodies. Alsoseen are R bodies (R), secondary lysosomes (L), and manymicrovillous bodies (x27 200 original magnification).

Fig. 6 Luminal border ofsame columnar cell as in Fig. 5,shows the vesicles emptying onto the surface with contentssimilar to that ofthe glycocalyx ofthe microvilli (x38 500original magnification).

length of the crypt and at the free luminal border.Spiral organisms were found in only two of thesebiopsies.

Lamina propriaThe cellularity of the lamina propria was increasedcompared with temperate zone controls, with in-crease in plasma cells, large subluminal macro-phages with heterogenous lysosomal bodies, eosino-phils and mucosal mast cells. In three of thevolunteers occasional neutrophil polymorphs werealso present. The plasma cells showed increasedactivity and plasmacytolysis in the deeper layers ofthe lamina propria. There was also degranulation ofscattered eosinophils and mucosal mast cells. Sub-luminal reticulin was increased compared withtemperate zone controls.

Striking changes were seen in the blood vessels ofthe lamina propria with reduplication of the basallamellae in a varying number of venules andcapillaries (Fig. 11). While these changes were seenin a small number of vessels in all the biopsies, inthree biopsies a few blood vessels near the base ofthe crypts also showed organised thrombi occludingthe lumen (Fig. 12). In one subject there was focalrupture of venules with platelet adhesion andeffusion of red blood cells into the lamina propriawith dehaemoglobinisation. In addition, in thisbiopsy, in occasional capillaries damaged endothe-lial cells were present.

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Fig. 8 (a) Luminal border away from site of extrusion.The cells show variation in electron density. Nuclei are

placed in different levels. Microvilli are short and irregular.Lateral membrane shows few plications (x2900 originalmagnification). (b) Similar area from a western control.Note the abundant mitochondria, supra and infra nuclear,and well developed microvilli and lateral membraneplications (x2900 original magnification).

Discussion

Increased electron dense bodies and vesicles in theapical cytoplasm of undifferentiated crypt cells anddifferentiated columnar cells, with increase in lyso-somes and R bodies in the columnar epithelial cells

even in the lower third of the crypts and heteroge-nous electron density of the mucus granules ofgoblet cells found in rectal mucosal epithelial cells inbiopsies obtained from volunteer control subjects insouthern India were not seen in temperate zonecontrols. In addition, the luminal epithelial cellswere shorter with shorter microvilli and less maturewith decrease in the plication of the lower part of thelateral cell membrane and fewer mitochondria.There was also a high prevalence of spiral organismsattached to the surface of the luminal epithelial cellsand alterations in the lamina propria vasculature,cellularity and subluminal reticulin content,

It has been suggested that there is only oneepithelial cell type in the colon and that the darkstaining epithelial cells are goblet cells that havedischarged their mucus granules." The lighterstained epithelial cells are presumed to be derivedfrom these cells when they absorb water from thelumen. Dark staining epithelial cells are foundscattered among lighter staining cells in the hencoprodeum. Freeze fracture studies showed rodshaped organelles in the cell membranes of darkstaining cells in the coprodeum and it has beensuggested that these cells are specialised for sodiumabsorption. 13 Similar intramembrane organelleshave been demonstrated in about a fifth of colonicepithelial cells in primates'4 although no functionalcorrelates were made. Further work is necessary onthe morphogenesis of these dark staining epithelialcells and to understand their significance andfunction.

Increased electron dense bodies and vesicles anddiffering electron density of goblet cell granulesreported here are similar to changes reported intransitional mucosa adjacent to colonic carcinomaand polyps in temperate zone subjects.15 Similarchanges were found in the normal epithelium of theascending and proximal transverse colon but wereseldom seen in the rectal mucosa of temperate zonecontrols. 1" In the monkey colon electron densebodies in the crypt basal cells gradually matures toless dense vesicles and finally discharges materialwith a striking resemblance to glycocalyx.'6 Silvermethanamine and immunohistochemical stainssuggest that electron dense bodies may containglycoproteins and immunoglobulins and may alsocontribute to the surface coat material.8 1649 Theincreased electron dense bodies and vesicles in thesouthern Indian rectal mucosa would thereforeappear to be a response of the epithelial cells todamage, by increasing secretions and production ofglycocalyx.The prevalence of spiral organisms in rectal

biopsies is extremely low in the temperate zonepopulations, although it was high in a West African

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Rectal mucosa in the tropics

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Fig. 9 Apical border ofluminal cells heavily infested with spiral organisms. Microvilli are short and sparse. Cross sectionof bacteria seen in the surface mucus. Apical cytoplasm shows many vesicles (x9500 original magnification).Fig. 10 Columnar cells from upper third showing increase in electron dense bodies. Goblet cells (G) show variation inelectron density ofmucus granules (x4300 original magnification).

Fig. 11 Longitudinal section ofa blood vessel showingprominent reduplication of basal lamella (arrow) (x 7100original magnification).

study.2'22 The clinical significance of these organ-isms is not known although there are suggestionsthat they may give rise to rectal symptoms andincreased vacuolation of rectal surface epithelialcells.23 24 The spiral organisms present in southernIndian biopsies are likely to be a part of the alteredflora of the colon reflected by the wide prevalence ofpathogens in otherwise asymptomatic subjects.25 Inthe present series of subjects the presence of spiralorganisms were not associated with any symptoms.Striking morphological alterations in colonocyteswere found in subjects with and without spiralorganisms. The nature of the injury responsible forthese reactive changes is not clear but is likely to bebecause of a variety of intraluminal factors of whichthe microbial flora is only one.

Increased reticulin and activation of histiocytesand plasma cells in the lamina propria was a strikingfeature of these biopsies. In southern India, inaddition to episodes of acute infectious diarrhoeaasymptomatic infection by presumed pathogens iscommon25 and there is also a high prevalence ofenteroviruses (Patel and Mathan, unpublished). The'reticulohistiocytic complex' of the submucosa of thecolon is a primary defence against the microbial

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Fig. 12 Cross section ofa blood vessel occluded byorganised thrombus (x 4300 original magnification).

flora of the lumen5 and the observed changes are aresponse to the luminal environment in tropicalcountries.The changes in the microvasculature with redupli-

cation of the basal lamellae in scattered bloodvessels in all the biopsies, organised thrombus inisolated vessels in three biopsies and evidence ofendothelial damage and haemorrhage in one biopsyare similar to the vascular damage described in thejejunal mucosa in Crohn's disease.26 Similar vascu-lar alterations were not found in at least three othercareful studies of the rectal mucosal lamina propriain temperate zone controls.5 11 27 We have foundendothelial and platelet mediated vascular damagewith intravascular thrombosis and basement mem-brane reduplication as a significant feature of severeepisodes of acute diarrhoea in adults in this popula-tion (unpublished observations). As the averagesouth Indian adult has at least one episode of acutediarrhoea every three years, it is reasonable topostulate that the vascular alterations found in allthe biopsies may be the fingerprints of earlierdiarrhoeal episodes.The morphological alterations in the rectal mu-

cosa of healthy volunteers in southern India de-scribed here suggest a response to non-specificinjury similar to that in the small intestine in thetropics. They should be kept in mind in interpretingultrastructural pathology in rectal mucosal biopsies.Further studies are necessary to delineate theirgeographic distribution, extent of colonic involve-

ment and to understand the mechanism ofpathogenesis in which the microbial flora, dietaryconstituents and factors as yet not understood maybe important.

The Wellcome Research Unit is supported by TheWellcome Trust, London.

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