Gut 1995; 37: 512-518

Attachment of Giardia lamblia trophozoites to acultured human intestinal cell line

P H Katelaris, A Naeem, M J G Farthing

AbstractAttachment of Giardia lamblia tropho-zoites to enterocytes is essential forcolonisation of the small intestine and isconsidered a prerequisite for giardiainduced enterocyte damage. The precisemechanisms involved are still beingdebated and some earlier work has beenperformed in models of uncertain biologi-cal relevance. In this study, co-incubationof giardia with enterocyte-like differenti-ated Caco-2 celis was used as a model tostudy the influence of physical andchemical factors on attachment. Giardiaattachment was maximal between one andeight hours and stable over pH 7.2-8.2 butit was reduced by acidification. Attach-ment was dependent on temperature andwas maximal at 370 and virtually abolishedat 4°C. It was reduced compared withcontrols (p<0.05) by EDTA 2.5 mM (mean(SEM) 32 (4)%/o), colchicine 12.5 ,M (35(5)0/o), mebendazole 10 pg/ml (30 (3)%),and cytochalasin B 1 ,g/ml (34 (3)%/o).Giardia attachment was also diminishedby preincubation with mannose 50 mM ormannose-6-phosphate 35 mM (21 (4); 17(5)0/0) or by preincubating Caco-2 cellswith concanavalin A 100 pug/ml (19 (2)%).Enhanced binding was not evident aftertrypsinisation of trophozoites. Scanningelectron microscopy showed that giardiaseemed to attach to the Caco-2 monolayerpredominantly by its ventral surface butdorsal orientation was also observed. Nodifference in attachment was observedbetween three different giardia isolates or aparent isolate and its clone. Attachment ofgiardia to Caco-2 cells is primarily bycytoskeletal mechanisms, inhibitable byinterference with contractile filamentsand microtubules, while attachment bymannose binding lectin also seems tomediate binding.(Gut 1995; 37: 512-518)

which is considered a specific attachmentorganelle, made up of coiled microtubules con-taining tubulin, cross bridges, and uniquestructures called microribbons that are per-pendicular to the microtubules and crossbridges.3 4 Contractile filaments in the rim ofthe ventral disc may mediate attachment.5Another mechanism of attachment suggestedis a model in which flagellar motion provides ahydrodynamic force sufficient for attachmentvia the disc.6 There is also evidence for lectinmediated receptor-ligand binding.7 8 Previousstudies of attachment have used a variety ofmodel systems including synthetic surfacessuch as plastic and glass, non-human cells suchas isolated rat enterocytes and cultured ratenterocyte cell lines, and human cells.8- 12These models differ in their biological appro-priateness for attachment studies and thediversity of findings from them is probably areflection of this and the different experimentalprotocols and rigor employed. In particular,there is no uniformity of findings regarding theimportance of microtubules, contractile fila-ments, or giardia lectin in the attachmentprocess.The human colonic adenocarcinoma

derived epithelial cell line, Caco-2 undergoesspontaneous differentiation in culture so that itfunctionally and structurally resembles smallbowel enterocytes. 13 Cells develop apical brushborder membranes14 15 which express brushborder enzymes including disaccharidases andalkaline phosphatase.16 17 Disaccharidaseactivity increases with time and is used as amarker of cell differentiation. 17 This cell line isthus useful and appropriate for studies of host-pathogen interactions. In this work dif-ferentiated Caco-2 cells were used as aphysiological model to study the attachment ofhuman isolates ofG lamblia trophozoites undera variety of physicochemical conditions.

Methods

Department ofGastroenterology, StBartholomew'sHospital, LondonEC1A 7BEP H KatelarisA NaeemM J G Farthing

Correspondence to:Dr Peter Katelaris,University of Sydney,Gastroenterology Unit,Concord Hospital, Concord2139, Sydney, Australia.

Accepted for publication24 February 1995

Keywords: Giardia lamblia, attachment, Caco-2 cells,cytoskeleton, lectin.

Giardia lamblia colonises the small intestineand localises preferentially to the mid andupper jejunum. Trophozoites attach to thebrush border of enterocytes, a process whichseems to be essential for colonisation and aprerequisite for giardia induced enterocytedysfunction and clinical disease.1 2 Giardiaseem to attach by physical and chemical mech-anisms, although the precise mode of this isstill debated. Trophozoites have a ventral disc,

AXENIC CULTURE OF GIARDIA TROPHOZOITESGiardia trophozoites were routinely cultured in15 ml screw capped glass tubes at 37°C inmodified TYI-S-33 medium18 but without bileor antibiotics. Unattached organisms wereremoved by decanting the culture medium, andonly wall adherent trophozoites were harvestedfor use in co-culture experiments. The humanisolate WB (originally axenised from a travellerto Afghanistan with chronic diarrhoea) wasused in most experiments. Comparative studieswere performed with isolates RW6 and VNB3,both previously axenised from patients withchronic diarrhoea and WB clone 2, obtained

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Attachment ofGiardia

from the parent WB isolate by the technique oflimiting dilution.'9

CACo-2 CELL CULTURECaco-2 cells (passage 88) were kindly providedby Dr I Hassan, Ciba-Geigy Pharmaceuticals,Horsham, UK. Cells were cultured at 37°C in75 cm2 flasks in Dulbecco's modified Eagle'smedium, supplemented by 10% fetal bovineserum, 1% non-essential amino acids, 1%L-glutamine, penicillin 100 IU/ml, andstreptomycin 100 ,ug/ml in an atmosphere of10% CO2 and 90% air.14 15 The medium waschanged every 48 hours and cells werepassaged every seven days in a split ratio of 1:4.For experiments, cells were seeded into 9-6cm2, six-well, tissue culture treated polystyreneplates (Becton Dickinson, UK) in a split ratioof 1:18, and the medium was replaced every 48hours. Cells reached confluency within three tofive days. Cells between passage 92-102 wereused. The number of Caco-2 cells per well wasestimated by counting cells with an invertedmicroscope using a grid lens with a fielddiameter of 0.9 mm and multiplying the cellcount obtained by the number of fields perwell. The median number of cells 16 days afterpassage was 2.0X 106 cells/well (n= 10).

DIFFERENTIATION OF CACo-2 CELLSFunctional differentiation of Caco-2 cells wasdetermined by assay of sucrase and maltaseactivity after a varying duration of culture. Forthis, cell monolayers were washed twice withchilled 0. 15 M phosphate buffered saline(PBS), then harvested in 1 ml ice cold distilledwater. Cells were gently homogenised to a finesuspension by rapid pipetting, and disacchari-dase activity was determined using modifica-tions of the glucose oxidase-peroxidasemethod.20 21 Briefly, 10 RI duplicate aliquots ofappropriately diluted, homogenised Caco-2samples were incubated with 0.056 M disac-charide substrate in 0.1 M phosphate buffersolution in 96 well microtitre plates (BectonDickinson, UK) at 37°C for one hour. TRIS-glucose oxidase reagent (250 1I) was addedand plates were incubated for a further hour. Asubstrate blank was included for each sample.Plates were read on a Titertek Multiscanmicroplate reader (Flow Laboratories, UK) at510 nm and the enzyme activity was expressedas U/jig protein. Protein was estimated withthe bicinchoninic acid protein assay reagentmethod22 (BCA, Pierce, USA).The structural differentiation of Caco-2 cells

was assessed 16 days after passage by scanningelectron microscopy. Cells grown on tissue cul-ture-treated, 10 mm round, glass cover slipswere double fixed in 3% glutaraldehyde in 0 1M phosphate buffer at pH 7-4 followed by 1%aqueous osmium tetroxide. They were dehy-drated, critically point dried using C02, sputtercoated with gold palladium, and examined in aJEOL SEM 5300. Electron micrographs werekindly taken by Dr Alan Phillips, ElectronMicroscopy Department, Queen ElizabethHospital for Sick Children, London.

GIARDIA-CACo-2 CELL CO-INCUBATIONTo determine the optimal medium for co-incubation, preliminary attachment assayswere done (as described below) with four dif-ferent media: supplemented DMEM, modifiedTYI-S-33, modified RPMI-1640, and modi-fied HSP3 (MHSP3). The latter two mediahave been reported to support growth of bothgiardia and mammalian cells.1223 Co-incuba-tion was in a ratio of giardia:Caco-2 cells of1:10, at 37°C in 50/o C02, 95% air for onehour. Giardia motility 24 hours after co-incubation in these media was assessedqualitatively. Giardia attachment was: 24 (5)%in supplemented DMEM, 27 (4)% in modifiedTYI-S-33, 13 (3)% in modified RPMI-1640and 42 (4)% in MHSP3, (n=6 for -eachmedium). On the basis of these results,MHSP3 was selected for use in experiments, asattachment was maximal (p<0.02), giardiamotility was preserved and it provided the bestcompromise between the metabolic needs ofthe micro-organism and the mammalian cell.

CO-INCUBATION AND ATTACHMENT ASSAYGiardia were decanted and the remainingattached trophozoites were re-fed with modi-fied MHSP3 media and chilled on ice untildetached. Trophozoites were then centrifugedat 1000Xg for 10 minutes, the supernatantwas decanted, and the pellet resuspended inMHSP3 medium warmed to 37°C. An aliquotwas counted using a haemocytometer and thevolume was adjusted to give the desiredconcentration of trophozoites per ml. Giardiawere then co-incubated with Caco-2 cellsusing modifications of a method previouslydescribed.12 Medium was aspirated fromCaco-2 cells and the monolayers gently washedwith warmed MHSP3 medium to remove anycells that had not adhered or debris. Giardiawere then added and the volume adjusted to 3ml per well. Plates were incubated at 37°C in5% CO2 and 95% air. The percentage attach-ment of giardia to Caco-2 cell monolayers wasestimated by determining the ratio of attached:total giardia seeded. At the end of the incuba-tion period unattached trophozoites wererecovered by gently rinsing culture plates threetimes with MHSP3 medium warmed to 37°C.Adherent trophozoites were then recovered byrepeated washes with ice cold Ca2+ and Mg2+free 0.15 M PBS (Dulbecco's formula, FlowLaboratories, UK) over 15-20 minutes untilno trophozoites were visible when checked bylight microscopy. An aliquot from each ofthese samples was counted in a haemocyto-meter and attachment was expressed as thepercentage attached of the total numberrecovered.The time course of attachment was deter-

mined over 24 hours and the effect of varyingthe number of giardia was studied over a rangeof giardia:Caco-2 cell ratios from 1:100 to 1:2.The impact of physicochemical factors onattachment was studied by varying the incuba-tion temperature (40, 210, 37°C) and byadjusting the pH of the medium with HClor NaOH over a pH range of 5.5-9-2. The

513

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120-

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80 _

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. 0

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Figure 1: The time course for the expression ofdisaccharidase activity in Caco-2 cells (n= 6for eachpoint).

divalent cation dependency of attachment wasdetermined by co-incubating giardia in thepresence of EDTA 2.5 mM. These experi-ments and those following, unless otherwisestated, were done with isolate WB, on Caco-2cells 15-17 days after passage in 5% CO2 and95% air. The percentage attachment wascompared between three different giardia iso-lates and a clone. As bile promotes growthof trophozoites in vitro,'8 24 an effect onattachment was sought by comparing attach-ment of trophozoites passaged in the presenceand absence of bovine bile in the culturemedium.To determine the role of components of the

giardia cytoskeleton in attachment, assayswere done in the presence of microtubuleinhibitors colchicine (dissolved in PBS)and mebendazole (1.0-100 ,ug/ml, SigmaChemical Co, St Louis, USA) and after 15minutes' preincubation of trophozoites withthe microfilament inhibitor, cytochalasin B(dissolved in DMSO 1%). The role of giardialectin in attachment was studied by preincu-bating giardia for 15 minutes with D-mannose(5-100 mM), mannose-6-phosphate (3.5-70mM), and D-glucose (5-300 mM). Glucosestudies were done using PBS for co-incuba-tion. Attachment was also determined aftergiardia had been pre-incubated in trypsin(0.01-10-0 mg/ml) for 20 minutes (Sigma typeXIII) to determine whether previouslyreported lectin activation by trypsin in giardiasonicate is also evident in whole tropho-zoites.25 Further studies were done pre-incu-bating Caco-2 cells for 15 minutes withconcanavalin A (10-100 ,ug/ml), which bindsmannosyl residues.

CONTROLSFor all experiments, the results were comparedwith an equal number of control attachmentassays. These assays were done at the sametime as test wells, under identical conditionsbut without the alteration of co-culture para-meter on the compound being tested. Wherepossible controls were included in the sameculture plates as test wells. Further controlexperiments were done in medium containingDMSO 1%.

SCANNING ELECTRON MICROSCOPYScanning electron micrographs of giardia afterco-incubation with Caco-2 cells for one hourand 16 hours were obtained, using themethods described above.

STATISTICAL ANALYSISResults are expressed as mean (SEM).Differences between means were comparedusing two-tailed Student's t tests or analysis ofvariance where appropriate. All data representa minimum of n=6 for each data point andexperiments were carried out on at least threeseparate occasions.

Results

CACO-2 CELLS MODELThe time course for the expression of disac-charidase activity is shown in Figure 1.Enzyme activity rose sharply from day 9 afterpassage and reached a plateau after day 14,indicating functional differentiation.'7 Thepresence of confluent monolayers with micro-villi on the apical surface of cells providedevidence of structural differentiation. As hasbeen previously observed,'5 17 the density ofthe microvilli varied between cells indicatingthe heterogeneity of the cell population. Somecells showed dense microvilli, others hadclusters, while a few cells had sparsely presentmicrovilli (Fig 2). On the basis of thesefindings, cells 15-17 days after passage wereused for attachment experiments as functionaland structural differentiation was evident.At the end of the co-incubation period,

Caco-2 cell monolayers remained confluentand appeared intact with normal morphologywhen examined by light microscopy and scan-ning electron microscopy. Giardia remainedviable after co-incubation as trophozoites weremotile and could be successfully subculturedafter recovery from culture wells.

PHYSICOCHEMICAL FACTORS IN ATTACHMENTUsing an inoculum of 2.OX 105 trophozoites at37°C and pH 7.2, giardia attachment to Caco-2 cells increased with time up to 60 minutes,then reached a plateau. The range of attach-ment over one to eight hours was 40-46 (6)%of the total number added. Attachment wasstill evident by 24 hours. The time course ofattachment is shown in Figure 3. The totalnumber of organisms recovered was between75-1 15% of the estimated inoculum added fortime periods up to eight hours. After 24 hoursco-incubation, giardia numbers had increasedto 5.1 x 105 indicating multiplication oftrophozoites (generation time= 17 hours).Multiplication was not evident after shorterperiods of co-incubation. Attachment wastemperature dependent, being maximal at37°C (43.5 (4)%, reduced by 59% at 21°C(17.8 (3)%) and virtually abolished at 40C (0 3(0.3)%). Attachment occurred over a range ofpH but was maximal at pH 7.2-8.2 (Fig 4).The total number of trophozoites attached

514

Attachment of Giardia

N _U y .. ...

Figure 2: Scanning electron micrographs of Caco-2 cell monolayers 16 days after passage. (A) Structural differentiation isevidenced by the presence of microvilli on the apical surface of the cells. These cells have a dense array of microvilli.(Magnification X 7500; bar= I ,m.) (B) These cells display clusters of microvilli on the apical surface. (MagnificationX 7500; bar= 1 ,m.)

after 60 minutes at pH 7.2 and 37°C increasedwith increasing numbers of giardia seeded,whereas the percentage attached was similar atall giardia:Caco-2 cell ratios (Fig 5). The fore-going experiments established the optimal con-ditions for the attachment assay. Unlessotherwise stated, the results following werederived using a parasite:Caco-2 cell ratio of1:10, over 60 minutes at 37°C, pH 7-2, in 5%CO2 and 95% air.

CYTOSKELETAL INHIBITION AND CHELATION OFDIVALENT CATIONSChelation of divalent cations with EDTA 2.5mM reduced attachment by 32 (4)% com-pared with controls (p<0 04). Higher concen-trations of EDTA (-5 mM) resulted invacuolation and disruption of the Caco-2 cellmonolayer. Co-incubation of giardia in thepresence of colchicine or mebendazole resultedin a concentration dependent reduction inattachment compared with controls (Fig 6).Preincubation of giardia with cytochalasinB also significantly inhibited attachmentcompared to controls. This did not seem tobe concentration dependent as the degree ofinhibition of attachment was similar with1 ,ug/ml and 20 [Lg/ml of cytochalasin B. Asecond control, using medium containing 1%DMSO (the solvent for the cytochalasin B)

had no detrimental effect on attachment(Fig 6).

LECTIN STUDIESPreincubation of trophozoites with D-mannose(5-100 mM) reduced attachment by between28 (4) and 35 (5)% (p<003). Preincubationwith mannose-6-phosphate (35-70 mM)reduced attachment by between 17 (5) and 24(5)%/o (p<0.04). A lower concentration ofman-nose-6-phosphate (3.5 mM) did not reduceattachment. With D-glucose, attachment wassignificantly reduced only at high concentra-tion (300 mM, p<0.02), where diminishedattachment is likely to be a result of raisedosmolarity. Preincubation of Caco-2 cellmonolayers with concanavalin A, 10 and 100,ug/ml reduced giardia attachment by 11 (3)O/o(p=NS) and 19 (2)% (p<002), respectively.

INFLUENCE OF HOST FACTORSPreincubating trophozoites for 20 minutes inmedium containing trypsin (0-0 1-10.0 mg/ml)did not enhance subsequent attachment. Onthe contrary, attachment was reduced by 9-13(5)% compared with controls. Attachment of

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05.0 6.0 7.0 8.0 9.0 10.0

pHFigure 4: The pH dependency of attachment of Giardialamblia under standard assay conditions, (*p<o002compared with pH 5.5 andpH 9-2).

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giardia was not differenthad been grown in the piabsence of bile (45 (3)%,

ISOLATE VARIATIONThe proportion of trophnot vary significantly betisolates. Using the standsattachment was: WB 46 (and VNB3 47 (5)%. Theattached to the same disolate (44 (5)%).

SCANNING ELECTRON MIC.Giardia were seen in c

apparently attached tomost trophozoites were

25;: 5c* 1251225 125- 10X 00

Figure 6: The effect of EDTA, colchicine, mebendazole, cytochal.

dimercaptosuccinic acid (DMSO) 1 on attachment of Giardia

point, *p<0 04 **p<0 02, tp<0001 compared with controls).

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ventral surfaces applied to the monolayer,trophozoites were also seen with their dorsalsurfaces opposed to Caco-2 cells, apparentlybound by means other than the ventral suckerdisc (Fig 7). Trophozoites did not seem toshow a predilection for Caco-2 cells with moredense microvilli.

, O Discussion_15 These experiments confirm giardia trophozoite1.0 attachment to differentiated Caco-2 cells in

< vitro and support a role for both cytoskeletal05 and lectin mediated mechanisms. This Caco-2

i ' 0 cell model is an appropriate model of attach-1:5 1:2

0ment as it involves human isolates of giardia ona human enterocyte-like cell line. The assay is

ratio and the percentage simple and reproducible. This is a morepoint). physiological model of attachment than those

used in previous studies, where syntheticbetween isolates that surfaces such as plastic or glass have beenresence (43 (3)%/o) or used.9 10 These models are useful but have thep=NS). obvious limitation that they are not biological.

In other studies, animal cells were used.Human giardia isolates co-incubated with iso-lated rat enterocytes have been used to study

Lozoites attached did attachment, particularly the role of giardiaween three different surface membrane lectin.8 This system has theard assay conditions, disadvantage that adherence of trophozoites(4)%, RW6 42 (3)%, occurs to both the apical and basolateralclone of isolate WB surfaces of enterocytes, a situation that does

Legree as the parent not occur in vivo. Another model of attach-ment using radiolabelled human giardia andIEC-6 cells, a cultured rat enterocyte line, hasalso been described."l

ROSCOPY This study supports a primary role forlose apposition and mechanical attachment via the ventral discCaco-2 cells. While with a prominent role for microtubules andobserved with their contractile microfilaments. Attachment was

inhibited by divalent cation depletion and*5 ^- iit cytochalasin B, which suggests interference

with the actin-myosin system. Attachment wasalso reduced with colchicine and mebendazole,which affect microtubular function.26 Someprevious reports have differed from these

. <i- -** ** g findings. In view of the different models andexperimental procedures used to study attach-

; 4>S ment it is not surprising that experimentalresults have not always been concordant. Both

i Inge et al8 and Magne et al 27 describeddecreased attachment of giardia to cells after

,,_:microtubular inhibition, and of a similar mag-nitude to that found in the current study.

,;-b8X 1 X However, Feely and Erlandsen9 did not findthat colchicine inhibited attachment of giardiato plastic; Similarly, McCabe et al," using aphysiological surface, also failed to find an

effect, probably because the concentration ofcolchicine used (1 pug/ml) was too low.Inge et al8 did not show that divalent cationdepletion or cytochalasin B affected attach-

i 1 20 1 ment, perhaps because in that model, in whiche; isolated rat enterocytes were used, lectin

mediated attachment to basolateral as well as*'.'',',T :,,.:''', apical surfaces of enterocytes was marked.

8.-. Gillin and Reiner'0 found little effect ofg; lk cytochalasins on adherence of trophozoites to

asin B and glass but it is unclear whether other mechani-lamblia (n28for each cal forces are involved in attachment to this

surface. Curiously, Magne et al,27 who used

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Attachment of Giardia 517

Figure 7: Scanning electron micrographs ofGiardia lamblia coincubated with Caco-2 cells. (A) G lamblia trophozoite inclose apposition and apparently attached to a Caco-2 cell. The ventral surface of the trophozoite is applied to themonolayer, which displays sparse microvilli (1 h co-incubation). (Magnification X 5000; bar= 1 ,gm.) (B) G lambliatrophozoites in dorsal and ventral orientation to Caco-2 cells (16 h co-incubation). (Magnification X 5000; bar= 1 ,gm.)

Caco-2 cells as a model for attachment,reported a biphasic increase of attachment toCaco-2 cells after inhibition of contractileproteins with cytochalasin B, a finding which isat odds with other previous studies and thecurrent work and may be the result of a lownumber of repeat experiments. This alsoconflicts with a previous report from the samegroup using this model, which describedreduced attachment to Caco-2 cells afterinhibition of contractile proteins by chelationof divalent cations.12 The current study showsthe importance of both divalent cation deple-tion with EDTA and microfilament inhibitionwith cytochalasin B in reducing attachment ofgiardia to a human cell line. This is consistentwith data from models using plastic surfacesand cell lines that are not human9 11 andsupports the role of contractile filaments inattachment.

Lectin mediated attachment was evident inthis system, inhibitable by D-mannose, man-nose-6-phosphate, and concanavalin A, consis-tent with a mannosyl target for binding. Noenhancement of attachment was seen afterexposure to trypsin which suggests that thetrypsin activation of giardia lectin described ingiardia sonicate25 28 does not have biologicalimportance for attachment. Evidence suggeststhat lectin mediated binding is not the primarymode of attachment of giardia. Firstly, attach-ment to synthetic surfaces is avid and is notdependent on receptor-ligand mediated bind-ing. Secondly, the magnitude of the reductionin attachment is greater after inhibition ofcytoskeletal function than with competitiveinhibitin of lectin mediated binding. Lastly,although giardia are found in various orienta-tions to epithelial cells, most trophozoites areobserved ventral surface down. Avidity forbinding of giardia to rat intestinal cells hasbeen shown to be greater with more proximalintestinal cells.8 It is possible that lectinmediated attachment serves to localise giardiato its preferred intestinal site and that tropho-zoites subsequently reorientate and attach viathe ventral disc.

This model allows physiological manipula-tion of the co-culture medium to mimic someconditions in the small intestine. For example,

pancreatic bicarbonate secretion into theduodenum results in periodically elevatedluminal pH and giardia survive in thisalkalinised milieu. It is not known whether thisis because giardia can resist a raised pH orbecause trophozoites may avoid exposure toluminal contents by burrowing under themucus layer. With this model, attachment hasbeen demonstrated to be maintained despitedirect exposure to alkaline conditions up to apH of 8.2.

In summary, the giardia-Caco-2 cell co-incubation model seems to be a relativelysimple and useful vehicle for the study of theattachment of giardia in vitro. A predominantrole for mechanical attachment via cytoskeletalmechanisms is suggested by these studies,although lectin associated binding is presentand may also have a role in vivo.This study was supported by the Wellcome Trust, London. Partof this work was presented at the American GastroenterologicalAssociation meeting in Boston 1993 (Gastroenterology 1993;104: A721).

1 Erlandsen SL. Scanning electron microscopy of intestinalgiardiasis: Lesions of the microvillous border of the villusepithelial cells produced by trophozoites of Giardia,pp. 775-82. In: Johari 0, ed. Scanning electron microscopy.Chicago: IIT Research Institute, 1974.

2 Smith PD. Pathophysiology and immunology of giardiasis.Ann Rev Med 1985; 36: 295-307.

3 Holberton DV. Arrangement of subunits in microribbonsfrom Giardia. J Cell Sci 1981; 47: 167-85.

4 Peattie DA, Alonso RA, Hein A, Caulfield JP.Ultrastructural localisation of giardins to the edges of diskmicroribbons of Giardia lamblia and the nucleotide anddeduced protein sequence of alpha giardin. J Cell Biol1989; 109: 2323-35.

5 Feely DE, Schollmeyer JV, Erlandsen SL. Giardia: distri-bution of contractile proteins in the attachment organelle.Exp Parasitol 1982; 53: 145-54.

6 Holberton DV. Attachment of Giardia - a hydrodynamicmodel based on flagellar activity. J Exp Biol 1974; 60:207-21.

7 Farthing MJG, Periera MEA, Keusch GT. Description andcharacterization of a surface lectin from Giardia lamblia.Infect Immun 1986; 51: 661-7.

8 Inge PMG, Edson CM, Farthing MJG. Attachment ofGiardia lamblia to rat intestinal epithelial cells. Gut 1988;29: 795-801.

9 Feely DE, Erlandsen SL. Effect of cytochalasin-B, lowCa' concentration, iodoacetic acid and quinacrine-HClon the attachment of Giardia trophozoites in vitro.J Parasitol 1982; 68: 869-73.

10 Gillin FD, Reiner DS. Attachment of the flagellate Giardialamblia: role of reducing agents, serum, temperature, andionic composition. Mol Cell Biol 1982; 2: 369-77.

11 McCabe RE, Yu GSM, Conteas C, Morrill PR, McMorrowB. In vitro model of attachment of Giardia intestinalistrophozoites to IEC-6 cells, an intestinal cell line.Antimicrob Agents Chemother 199 1; 35: 29-35.

12 Favennec L, Chochillon C, Meillet D, Magne D, Savel J,Raichvarg D, et al. Adherence and multiplication of

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Giardia intestinalis on human enterocyte-like differentiatedcells in vitro. Parasitol Res 1990; 76: 581-4.

13 Pinto M, Robine-Leon S, Appay MD, Kerdinger M,Triadou N, Dussaulx E, et al. Enterocyte-like differentia-tion and polarisation of the human colon carcinoma cellline Caco-2 culture. Biol Cell 1983; 47: 323-30.

14 Hidalgo IJ, Raub TJ, Borchardt RT. Characterisation of thehuman colon carcinoma cell line (Caco-2) as a modelsystem for intestinal epithelial permeability.Gastroenterology 1989; 96: 736-49.

15 Wilson G, Hassan IF, Dix CJ, Williamson I, Shah R, MackayM, Transport and permeability properties of humanCaco-2 cells: an in vitro model of the intestinal epithelialcell barrier. Journal of Controlled Release 1990; 11: 25-40.

16 Matsumoto H, Erickson RH, Gum JR, Yoshioka M, GumE, Kim YS. Biosynthesis of alkaline phosphatase duringdifferentiation of the human colon cancer cell line Caco-2.Gastroenterology 1990; 98: 1199-207.

17 Vachon PH, Beaulieu J-F. Transient mosaic patterns ofmorphological and functional differentiation in the Caco-2 cell line. Gastroenterology 1992; 103: 414-23.

18 Keister DB. Axenic culture of Giardia lamblia in TYI-S-33media supplemented with bile. Trans Roy Soc Trop MedHvg 1983; 77: 487-8.

19 Baum KF, Berens RL, Jones RH, Marr JJ. A new methodfor cloning Giardia la?nblia, with a discussion of thestatistical considerations of limiting dilution. _7 Parasitol1988; 74: 267-9.

20 Belosevic M, Faubert GM, MacLean JD. Disaccharidase

activity in the small intestine of gerbils (Merionesunguiculatus) during primary and challenge infectionswith Giardia lamblia. Gut 1989; 30: 1213-9.

21 Dahlqvist A. Assay of intestinal disaccharidases. AnalBiochem 1968; 22: 99-107.

22 Smith PK, Krohn RI, Hermanson GT, Mallai AK, GartnerFH, Provenzano MD, et al. Measurement of protein usingbicinchoninic acid. Anal Biochemn 1985; 150: 76-85.

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