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Pathogenic mechanisms in Giardia lamblia infectionB. s. ANAND
In 1681 Anton van Leeuwenhoek, a native of Holland,discovered the motile trophozoite forms of Giardia lambliain his stools, using his own creation the hand lens.' Thisevent was auspicious for two reasons: it led to thedevelopment of the microscope as well as to the birth ofmicrobiology.
Even after 300 years of the discovery of G. lamblia theprecise mechanism by which this parasite causes diarrhoeaand malabsorption is not known. A number of hypotheseshave been suggested, such as altered bowel motility.Icompetition for dietary nutrients and vitamins by a largenumber of trophozoites,? disturbance of exocrine pan-creatic function," and interference with lipolysisvt--butnone of these can satisfactorily explain the pathogenicmechanisms. In this review I will only discuss the moreplausible hypotheses.
THE MECHANICAL BARRIER THEORYIn 1940 Veghelyi suggested that G. lamblia trophozoitesspread out as a layer over the intestinal mucosa, forminga mechanical barrier to the transport of nutrients'? Thiswas supported by Rodrigues et at. on the basis of histo-pathological studies." However, duodenal biopsy speci-mens from patients with symptomatic giardiasis hardlyever show a diffuse layer of G. lamblia trophozoites; theusual picture is of the parasites scattered randomly overthe mucosa." Moreover, a mechanical barrier would beexpected to prevent the transport of all nutrients and notselectively of those that undergo active absorption, as hasbeen observed in animal experiments.
BACTERIAL PROLIFERATIONIt has been proposed that in giardial infection majorbacterial colonization of the small bowel occurs and thisresults in diarrhoea and malabsorption. 10-12The suppres-sion of diarrhoea by tetracycline even though the parasitescontinue to be excreted in the faeces supports thishypothesis. 13
BILE SALT DECONJUGATIONTandon et al. observed high levels of deconjugated bilesalts in the duodenal fluid of patients with giardiasis, someof whom also had bacterial overgrowth. IIThey postulated
G.B. Pant Hospital, New Delhi 110002, IndiaB. S. ANAND Department of Gastroenterology
that the diarrhoea and the malabsorption seen in patientswith giardiasis were related to the bile salt deconjugationinduced by either the parasite or the bacterial over-growth. However, G. lamblia cultured under axenic con-ditions failed to deconjugate glycocholic, taurocholic,deoxycholic or chenodeoxycholic acids.> Recently it hasbeen observed in in vitro studies that G. lambliatrophozoites consume bile salts but do not deconjugatethem, and it was concluded that this may contribute tosteatorrhoea. 14
IMMUNOLOGICAL FACTORSThere is much evidence to suggest that immunologicalmechanisms are deranged in giardial infection.
Humoral ResponseAntibodies have been detected against both the cystic aswell as the trophozoite forms of G. lamblia.15,16The deter-mination of the IgG response, which persists after theprimary response, does not allow past and current infec-tions to be differentiated.Pi'f Hence, detection of IgMantibody is likely to be more useful.'? A specific serumIgA response to G. lamblia has been obtained in aboutone-third of infected individuals.P Moreover, a specificsecretory IgA (SlgA) response can be detected in the milkof infected mice which affords protection to their sucklingoffspring. 21 Similar observations have been made inhumans.P In a recent study, the infants of mothers withhigh SlgA antibody titres in the milk were found to be lessfrequently infected with G. lamblia (16%) than theinfants of mothers with low antibody tit res (63%;p<0.01).23 Using antisera to light and heavy chains ofIgA, it has been detected that a layer of fluorescencesurrounds the dorsal membrane of the trophozoites in thebowel lumen ,24which may contribute to the eradicationof the parasite.
Cell-Mediated ResponseThe cell-mediated immunity in giardiasis appears to bemore pronounced than the humoral response. Increasednumbers of intra-epithelial lymphocytes are seen in theintestinal mucosa ofpatients with G. lamblia infection;25,26similar observations have been made in experimentalanimals.27,28 Intra-epithelial lymphocytes, which aremainly T cells, respond to giardial infection not only by amarked increase in their numbers but also by blast trans-formation.P-" Human peripheral blood mononuclearcells are spontaneously cytotoxic for G. lamblia in
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vitro30,3l and macrophageslocated in the Peyer's patchesof mice have been shown to phagocytose G, muris. 32It ispossible that a similar mechanism operates in humaninfection. Moreover, antibody dependent cytotoxicity forG. Lamblia by resident macrophages has been observed inmice.P Similarly, antibody dependent cytotoxicity for G.lamblia is seen with lamina propria and splenic lympho-cytes, but not with intra-epithelial lymphocytes whichexhibit only direct cytotoxicity." Further evidencesuggesting the role ofT-lymphocytes in the eradication ofgiardial infection is that if T cells are given to nude micethey clear the parasites normally in an infection which isotherwise prolonged. 35
Thus, there is sufficient evidence that immunologicalmechanisms, humoral as well as cell-mediated, have arole both in protection against giardial infection and in theeradication of the parasite. This is further substantiatedby a high incidence of giardial infection in patients withimmunodeficiency disorders such as hypogammaglo-bulinaemia.v Many of these patients suffer from chronicdiarrhoea and malabsorption which improve after eradi-cation of the parasite. Another characteristic feature isthe presence of nodular lymphoid hyperplasia (NLH)seen in about 20 to 60 per cent patients with acquiredhypogammaglobulinaemia.F Histologically, large lym-phoid follicles are seen in the lamina propria of the smallbowel (and less often of the stomach, colon and rectum)and the majority of these patients have giardiasis.Eradication of the parasite reverses the symptoms ofchronic diarrhoea and malabsorption but does not elimi-nate the NLH. Recently, NLH was also observed inpatients with giardiasis with normal serum immunoglobu-lins and it is postulated that the lymphoid hyperplasiarepresented a hypersensitivity response to chronicantigenic stimulation by the parasite. 38
More important from the point of view of aetiopatho-genesis is the possibility that these immunological reactionsmay be involved in the mucosal damage seen in giardia Iinfection. As noted above, the G. lamblia trophozoitesmay elicit a hypersensitivity reaction in which the T cellsreact vigorously to the antigen by releasing enteropatho-genic lymphokines which damage the enterocytes.'? Insupport of this argument is the finding that athymic(nude) mice infected with G. muris do not show anymucosal damage while normal mice do.35Another possi-bility is that G. lamblia induces a Type one hypersensitivityreaction which causes mucosal damage. This hypothesiswas based on the finding of increases in the number ofmast cells in the small bowel mucosa and in intestinal per-meability."
DIRECT MUCOSAL DAMAGEG. Lamblia trophozoites are capable of directly damagingthe intestinal epithelium. Invasion of the small bowelmucosa by the parasite, though uncommon, is welldocumented and G. lamblia trophozoites have beenobserved at all levels; within epithelial cells, in the laminapropria and even down to the muscle layer. 41
In vitro G. Lamblia trophozoites have been shown tocause damage to a variety of mammalian cells .. In one
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report human fibroblasts cultured in the presence of G.lamblia trophozoites showed degenerative changes after24 hours and were almost completely destroyed by 96hours.P Cytopathogenic abnormalities have also beenobserved in cultured epithelial cell mono layers by twodifferent axenic strains of G. lamblia.v Intestinal mucosalscrapings of mice incubated with human G. Lambliatrophozoites show cell damage as judged by trypan bluestaining and fall in the level of brush border disaccharidaseswithin 4 hours of incubation.f Also G. Lamblia has beenshown by light microscopy and scanning electron micro-scopy to cause damage to HeLa and Vero cell lines."
G. lamblia trophozoites can attach themselves to thesurface of the small intestinal mucosa by the sucker disclocated on their ventral surface. It is believed that theattachment is effected by an applied suction force producedby the movement of the ventro-Iateral flagellae whichresults in the expulsion of fluid from under the suctiondisc." Recently, it has been suggested that the attachmentis receptor mediated. The site of attachment on the para-site is believed to be a trypsin-sensitive lectin like surfaceligand which comes into contact with intestinal receptorswhich are rich in D-mannosyl residues." However, thesuction force generated by G. Lamblia is so strong that it iscapable of attaching itself to any surface, even glass andplastic,48,49and an additional mechanism of attachmentmay not be essential.
The final step resulting in cell damage is not clear andvarious hypotheses have been forwarded. One possibilityis that the close physical contact of Giardia damages theepithelial cell layer by a purely mechanical process.Indeed electron microscopic studies show circular impres-sions and damaged microvilli at the site of attachment ofthe ventral disc;50,51such an abnormality can explain thefall in brush border enzymes and the derangement ofactive transport mechanisms in the presence of normalcellular enzymes.52,53A further possiblity is that thedamage is chemically mediated. The role of enterotoxinshas been suggested since some patients present with sec-retory diarrhoea. 54But Smith et al. failed to identify anyenterotoxin using the rabbit ileal loop, infant mouse andrabbit skin capillary factor assay techniques. 55Further-more in vitro studies have shown that a cell-free extract ofG. Lamblia or the supernatant of the giardial culturemedium does not produce any deleterious effect on themammalian cell lines, indicating the lack of anycytopathogenic toxin liberation."
Ganguly et al. obtained high levels of prostaglandins E(PGE) and F(PGF) in the small intestinal mucosa of Swissalbino mice infected with G. lamblia= These workerspostulated the stimulation of the adenylate cyclase-cAMPsystem by PGE and PGF in the pathogenesis of diarrhoeasince the intestinal cAMP levels were found to beincreased in giardiasis. 57 Recently, the same workersfound increased levels of calmodulin, a calcium bindingregulatory protein, in the brush border membrane ofanimals infected with G. lamblia= This abnormality wasaccompanied by an increase in the uptake of Ca+ anddecreased uptake of Na" and Cl=. These workers con-cluded that in giardial infection there was stimulation of
ANAND: PATHOGENIC MECHANISMS IN G. LAMBLIA INFECTION
calmodulin by cAMP which caused a decrease in thetransport of Na" and Cl: from the bowel lumen into theenterocyte resulting in diarrhoea. However, the problemwith the chemically mediated mechanisms is that althoughthey can explain the acute diarrhoea, the pathogenesis ofmucosal damage remains unclear.
CONCLUSIONSThere is sufficient evidence that G. lamblia trophozoitescan damage the intestinal epithelial cells. However, theprecise mechanism by which the damage occurs is still notclear. Most studies indicate that the parasite does notrelease any cytopathogenic enterotoxins. Moreover,stimulation of chemical agents such as cyclic AMP, pros-taglandins E and F, and Calmodulin cannot explain themalabsorption and the histological abnormalties. Thus,the most likely explanation for the mucosal damageappears to be a direct mechanical effect of the activelymotile trophozoites. Certainly, in vitro studies favoursuch a possibility. However, in vivo factors such as over-growth of bacteria, presence of deconjugated bile saltsand stimulation of cytopathogenic immunologicalresponses may also contribute to the mucosal damage.
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