Molecular and Biochemical Parasttology, 23 (1987) 47-53 47 Elsevier

MBP 00771

Amino-sugars inhibit the in vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells

B a l b i r S i n g h 1, M i c h e l M o n s i g n y 2 a n d M a r c e l H o m m e l 1

1Wolfson Tropical Immunology Umt, Department of Parasitology, Ltverpool School of Troptcal Me&cine, Liverpool, U.K. and 2Centre de Biophystque Moleculmre, Orleans, France

(Received 20 July 1986, accepted 24 October 1986)

Twenty-two sugars and related compounds, nine neoglycoproteins, dopamine, four polyamines and oligomers of glucosamine were examined for their effect on the cytoadherence of Plasmodium falc~parum-infected erythrocytes to melanoma cells. Inhibi- tion of cytoadherence was high in the presence of the amino-sugars, glucosamine, galactosamme and mannosamme, and dopa- mine, and significant, although lower, in the presence of the polyamines, spermine, spermidine and putrescine N-acetylated amino- sugars and the other compounds were not sigmficant inhibitors of cytoadherence.

Key words: Plasmodium falciparum; Cerebral malaria; Melanoma cell; Amino-sugars

Introduction

Cerebral malaria is the most important,~severe manifestation of Plasmodium falciparum infec- tions and despite the advances in drug therapy it still carries a mortality of between 20 and 50% [1,2]. One of the mechanisms which has been proposed for the pathogenesis of cerebral ma- laria is the mechanical obstruction of cerebral blood vessels resulting from the sequestration of infected erythrocytes containing mature tropho- zoites and schizonts in these vessels. Evidence for this has recently been provided by Macpherson and co-workers [3] who examined post mortem samples of brains from patients dying with P. fal- ciparum malaria, with or without cerebral ma- laria. They found that the proportion of infected erythrocyte in cerebral vessels and the erythro-

Correspondence address: B. Singh, Wolfson Tropical Immu- nology Unit, Liverpool School of Tropical Medicine, Pem- broke Place, Liverpool L3 5QA, U.K.

Abbrev,ations: PBS, phosphate buffered saline; TC, tissue culture; MCBA, melanoma cell binding assay; BSA, bovine serum albumin.

cyte packing of these vessels was significantly higher in patients with cerebral malaria.

Sequestration of infected erythrocytes occurs not only in cerebral vessels, but also in venules of other organs in the body during falciparum infec- tions [4]. In these vessels erythrocytes infected with mature trophzoites and schizonts bind to en- dothelial cells [5] and it has been suggested that by doing so the parasites are protected from the filtering action of the spleen [6]. The parasites may also develop better in the hypoxic venular envi- ronment and indeed it has been reported that a hypoxic environment favours the in vitro growth of P. falciparum [7]. Means of inhibiting or re- versing sequestration may therefore hinder par- asite development and alleviate the clinical se- verity of cerebral malaria.

The findings of David et al. [8] that P. falci- parum-infected Saimiri erythrocytes have in- creased reactivity for certain lectins suggested that the interaction between infected erythrocytes and endothelial cells may be lectin-ligand in nature. Therefore the effect of sugars and related com- pounds on cytoadherence was studied using an in vitro correlate of sequestration, the melanoma cell binding assay [9].

0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

48

Materials and Methods

Animals. Female Bolivian squirrel monkeys (Sai- miri sciureus) were used throughout the study.

The parasite. The Ugandan Palo Alto 1/PLF-3 strain of P. falciparum adapted to the squirrel monkey [10] was used. Animals were infected by intravenous injection of infected blood. Infected blood was obtained by venipuncture of anaesth- etised infected monkeys and was cryopreserved in liquid nitrogen and thawed when required by the method of Diggs et al. [11]. Short term cul- tures of infected erythrocytes were performed as described by Trager and Jensen [12], except that foetal bovine serum was used instead of human serum.

Materials. Neoglycoproteins were prepared by the reaction of glycosidophenyl isothiocynate onto bovine serum albumin as previously described [13]. The oligomers of glucosamine were pre- pared by acetolysis of chitin as previously de- scribed [14], fractionation on silica gel column [15], deacetylation by hydrazinolysis of the oli- gomers comprising pentamers and higher oligo- mers, and finally purified by gel filtration on a column of Ultrogel GF05 (IBF-Reactif, Ville- neuve-La-Garenne, France). All the other com- pounds were purchased from Sigma Chemical Company. These compounds were made up to ten times the final test concentration in phosphate- buffered saline (PBS) immediately before use.

Tissue culture medium. The tissue culture (TC) medium used for both the culture of melanoma cells and malaria parasites was RPMI 1640 me- dium, 25 mM HEPES buffer, gentamycin at 0.1 mg m1-1 and 10% heat-inactivated foetal bovine serum.

Melanoma cells. Amelanotic human melanoma cells (American Type Culture Collection No. CRL 1685, designation C32r) were cuRured and plated onto 22 mm square glass cover slips as described by David et al. [9].

Melanoma cell binding assay (MCBA). The assay used was as described previously [9] with the fol-

lowing modifications: 0.5 mCi of sodium chro- mate (New England Nuclear; 1 mCi m1-1 sterile saline) was added to 4 ml of a 5% (v/v) erythro- cyte suspension in TC medium and incubated for 1.5 h at 37°C in humidified gas mixture of 6% CO2, 1% 0 2 and 93% N 2. The chromium-la- bel led erythrocytes were washed three times with TC medium and a 15% (v/v) suspension of cells was prepared of the pellet with TC medium. Me- dium was aspirated from the petri dishes contain- ing melanoma cell-coated cover slips and re- placed with 0.8 ml of TC medium. 100 p.1 of either PBS, 5 mg m1-1 bovine serum albumin (BSA) in PBS or a solution of ten times the final concen- tration of the test compound in PBS was added, followed immediately by the addition of 100 ~1 of the 51Cr- labelled erythrocyte suspension. Dupli- cate petri dishes were prepared for each test sam- ple, incubation was carried out at 370C in a hum- idified gas mixture of 6% CO2, 1% O2 and 93% N 2 for 1.5 h on a rocking platform with gentle swirling by hand every 15 min. The cover slips were then washed in RPMI-bicarbonate medium, treated with detergent, and the amount of 5~Cr released by the bound erythrocytes was meas- ured with a gamma counter (Packard Multi-Prias Auto Gamma, Packard Instrument Interna- tional). The radioactivity counts were corrected by subtracting the background count and % bind- ing was calculated according to the following for- mula: % binding = 100 x a/b where a = cor- rected counts for test compounds and b = corrected counts for 0.5 mg m1-1 BSA control (for the neoglycoproteins) or for 100 lzl PBS and TC medium control (for the other compounds).

Effect of glucosamine on the detachment of mel- anoma cells from cover slips. Melanoma cells were labelled with 51Cr by incubating 4 x 106 cells in 2 ml TC medium with 0.1 mCi sodium chromate for 1.5 h at 37°C. The cells were washed three times with TC medium and resuspended in medium to a concentration of 2 x 10 s m1-1. 0.5 ml of this suspension was pipetted onto a 22 mm square glass cover slip in a 35 mm diameter petri dish. Cells were allowed to settle and attach to the cover slip overnight at 37°C in a humidified gas mixture of 6% CO 2, 1% 02 and 93% N 2. 1.5 ml of TC medium was then added to each petri dish.

24 h later each cover slip was washed 4 times by the addition and subsequent removal of 2 ml PBS to remove any unbound melanoma cells. Each petri dish in a group of eight then received 1 ml of a particular concentration of glucosamine in TC medium. Dishes were incubated for 1.5 h and cover slips removed for washing as described for the MCBA. The remaining contents of each petri dish were transferred into counting vials and ra- dioactivity was determined with a gamma counter to ascertain whether any melanoma cells had been detached from the cover slips during incubation with glucosamine. The melanoma cells on the cover slips were lysed with detergent and the ra- dioactivity determined as described for the MCBA. The radioactivity counts indicated whether any melanoma cells had been washed off the cover slips during the washing procedure.

Effect of glucosamine and N-acetyl glucosamine on the retention of 51Cr by 51Cr-labelled erythro- cytes. Infected erythrocytes (2% parasitaemia) were labelled with 51Cr and added to petri dishes as described for the MCBA except that there were no melanoma cells on the coverslips. Each petri dish in a group of eight received 100 Vd of eryth- rocyte suspension, 800 v~l of TC medium and 100 ixl of a particular concentration of glucosamine or N-acetyl glucosamine in PBS. After incubating for 1.5 h as described for the MCBA, the cell sus- pension from each petri dish was transferred into a plastic tube and centrifuged at 800 × g for 10 min. 0.5 ml of the supernatant was transferred into counting vials and the radioactivity deter- mined using a gamma counter.

Glutaraldehyde fixation of melanoma cells. Mel- anoma cells were plated onto glass coverslips in petri dishes as above. 48 h after plating out the cells, 2 ml of 0.5% glutaraldehyde in 0.1 M so- dium cacodylate (pH 7.4) was added to each petri dish. After incubating at room temperature for 15 min the fixative was aspirated and 2 ml of PBS was added. The PBS was replaced with fresh PBS after a minute and this washing procedure was repeated twice. 2 ml of 5% BSA with 0.02 M gly- cine in 0.1 M sodium cacodylate was then added to each petri dish. Following a 45 min incubation at room temperature, each cover slip was washed

49

lt.lt,tt i 'ol | V,?%. ,

,,, \ ",,] Ftg. 1. Effect of sugars, amino-sugars and N-acetylated amino- sugars on the binding of P. falclparum-mfected erythrocytes to melanoma cells. These compounds were tested at a final concentration of 100 mM and % binding was calculated as de- scribed in Materials and Methods. The heights of the columns represent the means and the bars represent 1 SD of values obtmned from at least two binding assays with duplicate val- ues for each assay.

,20 °°;'LLT: :

30-

0 - I o 2'5 ~o 1~o SUGAR [ZONCENTRATION (raM)

Fig. 2. Effect of varying concentrations of amino-sugars and N-acetyl glucosamine on the bmchng of P. falczparum-in- fected erythrocytes to melanoma cells. % binding was calcu- lated as described in Materials and Methods. The points rep- resent the means and the bars represent +- SD of values obtained from at least two binding assays with duplicate val- ues for each assay.

three times with PBS as described previously. These glutaraldehyde-flxed melanoma cells were used immediately in the MCBA.

Results

Effect of neoglycoproteins, hexoses and related compounds on binding. Of the 22 compounds and

50

9 glycoproteins tested, only the amino sugars, glucosamine, galactosamine and mannosamine inhibited the binding of P. falciparum-infected erythrocytes to melanoma cells by 70% or more at a final concentration of 100 mM (Fig. 1 and Table I). When tested at lower concentrations there was still a significant difference between binding in the presence of these amino sugars compared to binding in the presence of N-acetyl glucosamine (Fig. 2).

In order to examine the possibility that the amino sugars may have caused the detachment or lysis of melanoma cells from the cover slips dur- ing the MCBA, the effect of glucosamine on the retention of 51Cr by SlCr-labelled melanoma cells grown on cover slips was studied. There was no significant increase in the amount of 51Cr re- leased by these cells when exposed to increasing concentrations of glucosamine (Table II). When the cover slips were then washed in medium, as

TABLE I

Effect of neoglycoproteins, hexoses and related compounds on binding

Compound % Binding

a-L-Fucose 89 (± 11) a-D-Fucose 81 (± 17) 2-Deoxy-D-galactose 87 (± 15) a-Methyl-D-galactose 87 (± 8) 13-Methyl-D-galactose 87 (± 17) 6-Deoxy-o-glucose 60 (± 7) 2-Deoxy-D-glucose 65 (± 11) ct-MethyI-D-glucoside 87 (± 5) [3-Methyl-o-glucoside 84 (± 11) 3-o-Methyl-D-glucopyranose 95 (± 16) a-Methyl-mannoside 76 (± 13) a-L-Rhamnose 95 (± 3) D-Sorbitol 90 (± 17) ct-Glucose-BSA 102 (± 10) a-L-Rhamnose-BSA 72 (± 5) Ct-L-Fucose-BSA 103 (± 8) a-D-Galactose-BSA 97 (± 5) ct-Mannose-BSA 97 (-+ 8) Mannose-6-phosphate-BSA 86 (± 11) Lactose-BSA 78 (± 4) N-Acetyl-glucosamine-BSA 94 (± 5) Chitobiose-BSA 93 (± 1)

Neoglycoproteins were tested at a final concentration of 0.5 mg ml -~ and the other compounds at 100 mM. % binding was calculated as described in Materials and Methods. The figures are the means ± SD from at least two binding assays with du- phcate determinations in each assay.

TABLE II

Effect of glucosamme on the detachment of melanoma cells from cover slips

Glucosamme Radioactivity Radioactivity concentration associated with associated with (raM) detached cells undetached cells

(clam) (cpm)

0 2223 ± 247 21330 ± 291 25 2462 ± 170 20527 ± 316 50 2885 ± 291 20335 ± 549

100 2431 ± 451 20589 ± 551 200 2443 ± 394 20807± 511

51Cr-labelled melanoma cells grown on cover slips were in- cubated with various concentrations of glucosamine. 51Cr re- leased by these cells and detached cells was measured as was the 51Cr content of cells remaining on the cover slips Figures represent means of 8 values ± SEM from a single experi- ment

for the MCBA, and the cells lysed with deter- gent, there was no difference between the result- ant radioactive counts (Table II). Taken together these results strongly suggest that glucosamine did not cause the lysis or detachment of melanoma cells from the cover slips during incubation in the presence of glucosamine or during washing at the end of the MCBA. The inhibition of binding by glucosamine was also observed when glutaralde- hyde-fixed melanoma cells were used (Tabel III), providing further evidence that the effect of glu- cosamine on binding was not due to the detach- ment of melanoma cells from the cover slips dur- ing the MCBA.

TABLE III

Effect of glucosamine and N-acetyl glucosamine on the bind- mg of P falctparum-infected erythrocyte to glutaraldehyde- fixed melanoma cells

Compound Concentration % Binding (mM)

Glucosamme 12.5 70 ± 9 N-Acetyl glucosamine 12.5 95 ± 13 Glucosamine 25 27 ± 2 N-Acetyl glucosamme 25 95 ± 19 Glucosarmne 50 19 ± 5 N-Acetyl glucosamine 50 83 ± 18 Glucosamine 100 11 ± 4 N-Acetyl glucosamme i00 65 ± 12

% Binding was calculated as described in Materials and Methods. The figures represent mean ± SD from two binding assays wlth duplicate determinations m each assay

TABLE IV

Effect of glucosamine and N-acetyl glucosamine on the release of 5~Cr by 5~Cr-labelled erythrocytes

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Compound Concentration (mM)

Radioactivity released by cells (clam)

Experiment 1 Experiment 2

- - - - 1515 ± 123 1485 --- 220 Glucosamine 25 1310 ± 140 1502 --- 116 Glucosamine 50 1089 ... 33 1657 ± 189 Glucosamine 100 1149 --+ 54 1758 ± 64 Glucosamine 200 1217 --- 47 1816 ± 119 N-Acetyl glucosamine 100 1525 --- 51 1434 --- 105

51Cr-labelled infected erythrocytes (2% parasitaemia) were subjected to various concentrations of the compounds. The amount of radioactivity released by the cells is given as the means of 8 values (Exp. 1) and 6 values (Exp. 2) ± SD. The maximum amount of 51Cr released by the cells was 0.23% (Exp. 1) and 0.19% (Exp. 2) of the total 51Cr content of the cells.

Increasing the concentration of glucosamine had no significant effect on the release of 51Cr by 51Cr- labelled e ry throcytes (Table IV) , thereby sug- gesting that the observed inhibit ion of binding in the presence o f g lucosamine was no t due to the release of 51Cr f rom ery throcytes by glucosamine dur ing the M C B A .

Effect of glucosamine oligomers and other amines on binding. Since the c o m p o u n d s which showed the greatest inhibi tory activity were the amino-

sugars glucosamine, galactosamine and manno- samine, other amines were also examined for their effect on the binding of P. falciparum-infected erythrocytes to m e l a n o m a cells. Glucosamine ol- igomers were found to be no more effective than m o n o m e r s in inhibiting binding (Table V). Fur- the rmore , inhibition o f binding similar to that ob- served with amino sugars was observed in the presence o f dopamine and to a lesser extent with the polyamines spermine, spermidine and putres- cine.

TABLE V

Effect of glucosamine ohgomers and other amines on the binding of P. falciparum-infected erythrocytes to melanoma cells

Compound Concentration % Binding

Glucosamine oligomers 0.1 mg m1-1 98 ... 1 1.0 mg m1-1 95 - 4

10.0mgml -la 78± 8

Dopamine 100 mM 15 ± 1 50 mM 43 ± 2 25 mM 63 --- 7 12.5 mM 90 --- 10

Spermine 100 mM 47 - 10

Spermidine 100 mM 52 ± 18

Putrescine 100 mM 46 ± 8

Cadaverine 100 mM 64 ± 8

% Binding was calculated as described in Materials and Methods. The figures are means --- SD of not more than two binding assays with duplicate determinations in each assay. a 35 raM, expressed on the basis of glucosamine content.

D i s c u s s i o n

There is considerable evidence which suggests that cell-cell interactions are media ted by the ca rbohydra te moities of cell surface glycoproteins and glycolipids, and that the specificity of these carbohydra tes largely determines the specificity o f the cellular interactions [16]. Studies with P. falciparum, for example, have indicated that gly- cophor ins and their associated sugars may be of considerable impor tance in the interact ion of merozoi tes with the surface of the h u m a n eryth- rocyte m e m b r a n e [17,18]. It was demons t r a t ed that N-acetyl glucosamine inhibited the penet ra- t ion of P. falciparum merozoi tes into erythro- cytes in vitro and that this acetylated amino-sugar coupled to B S A was more effective in blocking invasion of merozoites than its unconjugated form [19,20]. We have examined various sugars, neo- glycoproteins and o ther c o m p o u n d s for their ability to inhibit the interact ion be tween P. fal- ciparum-infected erythrocytes and melanoma cells and have found that, in contrast to the mero-

52

zoite-erythrocyte interaction, only the sugars containing a free amino group and certain other amines were effective inhibitors. To circumvent the low inhibitory effect of free monosaccharides related to a low affinity to their receptors, neo- glycoproteins have been developed [13] because they compensate for the low affinity of simple sugars by an avidity effect [21,22]. But none of the neoglycoproteins tested were found to be effec- tive inhibitors of binding, indicating that the in- volvement of neutral sugars or of N-acetylated hexosamines is quite unlikely. Amino-sugars linked to BSA are currently being prepared and they will be examined to determine whether they are more effective than uncoupled amino-sugars at inhibiting binding.

The results of the control experiments strongly suggest that the observed inhibitory effect of glu- cosamine was not due to a generalised cytotoxic effect of this compound on either the melanoma cells or the infected erythrocytes. Although glu- cosamine has been reported to be toxic to the in- tracellular growth and development of P. falci- parum [23], this effect was observed by subjecting ring forms of the parasite to glucosamine for 24 h, whereas in our study trophozoite and schizont- infected erythrocytes were exposed to glucosa- mine for only 90 min. Furthermore, fucose was also found to have a similar toxic effect as glu- cosamine on the intracellular growth and devel- opment of the parasite in the 24 h incubation as- say [23], yet it did not inhibit cytoadherence in our relatively short incubation assay. Taken together these observations strongly suggest that the ob- served inhibition of cytoadherence by the amino- sugars is not due to their toxic effect on either the infected erythrocytes or melanoma cells. Finally, while infected erythrocytes are selectively perme- able to D-sorbitol [24], this compound did not in- hibit binding, thereby excluding selective perme- ability as a possible explanation for the observed inhibitory effect of the amino-sugars on cytoad- herence.

Several investigators have reported that aggre- gation or agglutination of different cells is blocked by compounds which we have found to inhibit the binding of P. falciparum-infected erythrocytes to melanoma cells. Garber [25] for instance showed that glucosamine inhibited the aggregation of

neural-retina cells and Glaeser et al. [26] re- ported that the aggregation of dissociated retina and liver cells was blocked by glucosamine, gal- actosamine, mannosamine, dopamine and some other amines. The haemagglutinating activity of thrombin-activated platelets has also been shown to be inhibited by glucosamine, galactosamine and mannosamine, and these compounds were also found to inhibit thrombin-induced platelet aggre- gation [27]. Thrombospondin has been recently identified as the endogenous platelet agglutinin that is responsible for the haemagglutinating ac- tivity of activated platelets, and the amino-sugars glucosamine, galactosamine and mannosamine were found to inhibit the haemagglutinating ac- tivity of purified thrombospondin [28]. Throm- bospondin has also recently been implicated as the glycoprotein receptor on melanoma and endoth- elial cells for binding of P. falciparum-infected erythrocytes containing mature throphozoites and schizonts [29]. While the exact mechanism by which the amino-sugars inhibit the haemagglutin- ating activity of thrombospondin remains un- known, it is possible that the inhibition of bind- ing between P. falciparum-infected erythrocytes and melanoma cells that we have observed with the amino-sugars and other amines may be due to these compounds interfering with the action of thrombospondin.

Acknowledgements

This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, by the European Economic Community 'Medicine in the Tropics' Programme, and by the Wolfson Foundation. We wish to thank Professor J. Schr6vel for helpful discussions.

References

1 Daroff, R.B., Deller, J.J., Kastl, A.J. and Blocker, W (1976) Cerebral malaria. J. Am. IVied. Assoc. 202, 679--682.

2 Harinasuta, T., Dixon, K.E., Warrell, D.A. and Dober- styn, E.B. (1982) Recent advances in malaria with special reference to Southeast Asia. Southeast Asian J. Trop. Med. Public Health 13, 1-34.

3 Maepherson, G.G., WarreU, M.J., White, N.J., Looar- eesuwan, S. and Warrell, D.A. (1985) Human cerebral

malaria. A quantitative ultrastructural analys~s of parasi- tised erythrocyte sequestration. Am. J. Pathol. 119, 385--401.

4 Clark, H.C. and Tomlinson, W.J. (1949) The pathologic anatomy of malaria. In: Malariology (Boyd, M.F., ed.), pp. 874-903, W.B. Saunders, London

5 Miller, L.H. (1969) Distributaon of mature trophozoites and schizonts of Plasmodium falaparum in the organs of Aotus tnwrgams, the mght monkey. Am. J. Trop. Med. Hyg. 18, 860-865.

6 Kreler, J.P. and Green, T.J. (1980) The vertebrate host's immune response to Plasmodia. In: Malaria, Vol. 3 (Kreier, J.P. ed.), pp. 111-162, Academic Press, New York.

7 Scheibel, L.W., Ashton, S.H. and Trager, W. (1979) Plasmodium falc~parum: microaerophilic reqmrements in human red blood cells. Exp Parasitol. 47, 410-418.

8 David, P.H., Hommel, M. and Ohgmo, L.D. (1981) In- teractions of Plasmodium falc~parum-infected erythro- cytes with ligand-coated agarose beads. Mol. Bmchem Parasitol. 4, 195-204.

9 David, P.H., Hommel, M., Miller, L.H., Udeinya, I.J. and Oligmo, L.D. (1983) Parasite sequestration in Plasmo- drum falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl. Acad. Sci. USA 80, 5075-5079.

10 Gysin, J., Hommel, M. and Pereira da Sdva, L.H. (1980) Experimental infection of the squirrel monkey, Saimiri sciureus with Plasmodtum falciparum. J. Parasitol. 66, 1003-1009.

11 Diggs, C.L., Joseph, K., Hemmins, B., Snodgrass, R. and Hines, F. (1975) Protein synthesis in vitro by cryopres- erred Plasmodmm falciparum. Am. J. Trop. Med. Hyg. 24, 760-763.

12 Trager, W. and Jensen, J.B (1976) Human malaria par- asite m continuous culture. Science NY 193,673--675.

13 Monslgny, M., Roche, A.C. and Mldoux, P. (1984) Up- take of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytofluorometry and drug targetting. Biol. Cell 51,187-196.

14 Barker, S.A., Foster, A.B., Stacey, M. and Webber, J.M. (1958) Amino-sugars and related compounds. Part IV. Isolation and properties of oligosacchandes obtained by controlled fragmentation of chitin. J. Chem. Soc. 2218,2227.

15 Delmotte, F. and Monsigny, M. (1974) Synthese des p-m- trobenzyl 1-thlo-~-chitobioside et 1-thio-13-clutotnoside et du p-nitrophenyl-I~-chltotrioside. Carbohydr Res. 36, 219-226

16 Winzler, R.J. (1970) Carbohydrates in cell surfaces. Int. Rev Cytol. 29, 77-125

53

17 Perkins, M.J. (1981) Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malarial parasite (Plasmodium falciparum) into its host cell J. Cell. Biol. 90, 563--567

18 Pasvol, G., Jungery, M., Weatherall, D.J., Parsons, S.F., Anstee, D.J. and Tanner, M J A. (1982) Glycophorin as a possible receptor for Plasmodium falciparum. Lancet n, 947-950.

19 Jungery, M., Pasvol, G., Newbold, C.I. and Weatherall, D.J. (1983) A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum Proc. Natl. Acad. Sci. USA 80, 1018--1022.

20 Schulman, S., Lee, Y.C. and Vanderburg, J.P. (1984) Ef- fects of neoglycoproteins on penetration of Plasmodium falciparum merozoites mto erythrocytes in vitro. J. Par- asitol. 70, 213-216.

21 Monsigny, M., Kieda, C. and Roche, A.C. (1983) Mem- brane glycoproteins/glycolipids and membrane lectins as recognition signals in normal and mahgnant cells. Biol. Cell 47, 95-110.

22 Townsend, R. and Stab.l, P.D. (1981) Isolation and char- acterisation of a mannose/N-acetylghicosamine/fucose- binding protein from rat hver. Blochem. J. 194, 209-214.

23 Weiss, M.M., Oppenheim, J.D. and Vandenberg, J.P. (1981) Plasmodium falciparum: assay in vitro for inhibl- tors of merozoite penetration of erythrocytes. Exp. Par- asitol. 51,400--407.

24 Ginsburg, H., Kutner, S., Krugliak, M. and Cabantchik, Z I. (1985) Characterisation of permeation pathways ap- pearing in the host membrane of Plasmodium falciparum infected red blood cells. Mol. Biochem. Parasitol. 14, 313-322.

25 Garber, B. (1963) Inhibition by glucosamme of aggrega- uon of dissociated embryonic cells. Dev. Biol. 7, 630-641.

26 Glaeser, R.M., Richmond, J.E. and Todd, P.W. (1968) Histotypic self-organisation by trypsin-dissociated and EDTA-dissociated chick embryo cells Exp Cell Res. 52, 71-85.

27 Gartner, T.K., Williams, D C., Minion, F.C. and Philips, D.R (1978) Thrombin-induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin Science 200, 1281-1283.

28 Jaffe, E.A., Leung, L.L.K., Nachman, R.L., Levm, R.I. and Mosher, D.F. (1982) Thrombospondin is the endog- enous lectm of human platelets. Nature 295,246-248.

29 Roberts, D.D., Sherwood, J.A., Spitalnik, S.L., Panton, L.J , Howard, R.J., Dixit, V.M., Frazier, W.A., Miller, L.H. and Ginsburg, V. (1985) Thrombospondm brads fal- ciparum malaria parasitlsed erythrocytes and may mediate cytoadherence. Nature 318, 64--66.