l’lacuntrc (1992), 13, 475-487

Galactose Alpha 1-3 Galactose and Antialpha Galactke Antibody in Normal and Pathological Pregnancies Y. CHRISTIANE”,“, M. AGHAYAN”,“, H. EMONARD”?“, A. LALLEMANDd, PH. MAHIEU’ & J. M. FOIDART”L~ nLaboratory of Biology (ProfessorJ. M. FOIDART), Universit~~I!/ Liege, Tour de Pathologie (B 2.?), Sart T&an, B 4000 Liqe. Belgium b Department of Obstetrics and Gynecology (Professor R. LAXBOTTE), University ofliege, HOpita de la Citadelle, B 4000 Liege, Belgium ’ CNRS URA 1459, Institut Pasteur, .Ivenue F-69007 LJJO~,France

Toq~ Gamier,

’ Laboratoire Pol Bouin, H&pitalMaison Blanche, rue CognaqJq 45, F-51 100 Reims, France ”LaboratoQl of Experimental Nephrology, Universi[y of Liege CHL; (B35), Sart Tilman, B 4000 Liege, Belgium .f To whom correspondence should be addressed Paper accepted 28.2.1992

SUMMARY The galactose alpha l-3 galactose (Gal a I-3 Gal) residue is a carbohydrate widely distributed in many non-human mammals. Since Gal a 1-3 Gal residues are described on the cell surface of tumor cells, we have examined the possibility of their expression on human trophoblastic cells at drrerent stages ofplacental implantation and in various pregnancy-associated conditions. Using immunohistochemical methods, Gal a l-3 Gal was demonstrated on interstitial and vascular trophoblast during pregnancy. For cillous trophoblast, the staining disappeared in second trimester pregnancies. The density of staining for Gal a I-3 Gal was increased in highly invasive trophoblast (mole and choriocarcinoma) and decreased in poorly invasivespecimens (spontaneous abortion, X0 monosomia). No cells displaying Gal a I-3 Gal at their surface were identified in some segments of spiral arteries from preeclamptic women. The anti-Gal antibody titer increased in the first trimester of pregnancy and in the sera of pre-eclamptic and eclamptic patients. These findings suggest that Gal a l-3 Gal residues could be considered as markers& trophoblast invasive capacity and that the binding of maternal anti-Gal antibodies to the trophoblast could contribute to limit trophoblastic invasion and thus participate to the immunological control of implantation. 0143-4004/92/050475

+ 13 $08.00/O

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Placenta(1992), FOl.13

INTRODUCTION Glycoconjugates are ubiquitous components found at the surface of eucaryotic cells. They play a major role in cellular recognition, differentiation and invasion (Gabius, 1987). Malignant transformation induces drastic qualitative and quantitative changes in cell surface glycoconjugates (Hakomori, 1985; Feizi, 1985). Recently, galactose (r l-3 galactose (Gal cx l-3 Gal) residues were identified in glycoconjugates of mammals except humans (Peters and Goldstein, 1979). In man, these residues are expressed at the surface of group B erythrocytes and senescent or pathological (sickle cells anemia, thalassemia) red blood cells (RBC) (Galili et al, 1983, 1986a, 1986b). This carbohydrate is normally absent from other adult human tissues or cells tested. We have described the presence of Gal u l-3 Gal at the surface of human malignant tumour cells with metastasizing capacity (Castronovo et al, 1989). The invasiv-e and metastatic potential of several murine tumour cell lines is closely correlated with the density of Gal (x l-3 Gal residues at the cell surface (McCoy, Goldstein and Varani, 1985). &‘e have also shown that anti-Gal Q 1-3 Gal antibody (anti-Gal) drastically reduces the local invasive potential and the metastasizing capacity of human and murine malignant cells (Castronovo et al, 1987, 1989). These results indicate that Gal u l-3 Gal and anti-Gal antibody may be important for invasion by tumour cells. Galili et al (1984) have identified a new natural IgG antibody, present in the serum of all humans, that specifically recognizes Gal a. l-3 Gal residues. It probably contributes to the prevention of the penetration of those intestinal microorganisms which bear accessible Gal u l-3 Gal residues. This human anti-Gal antibody could also participate in the clearance, by the macrophage system of pathological or senescent erythrocytes (Galili et al, 1986b). During embryo implantation, the human trophoblastic cells invade the maternal endometrium and infiltrate the arterial walls of the spiral arteries in a process that somewhat resembles local tumour invasion (Robertson, Brosens and Dixon, 1975). However, in contrast to malignant invasion, the trophoblast invasion is restricted to the decidua and inner myometrium. Considering a possible analogy between tumour and trophoblast invasion, we have novv studied the distribution of Gal GLl-3 Gal residues at the surface of trophoblast cells during early and third trimester pregnancy and in various pregnancy-associated conditions. Robertson, Brosens and Dixon (1975) described a decreased invasion or a lack of invasion of the uterine spiral arteries by the vascular and interstitial trophoblast in placental bed biopsies from pre-eclamptic or eclamptic patients. \Ve have therefore attempted to correlate this pattern of decreased trophoblast invasion with possible changes in the distribution of Gal (;Ll-3 Gal residues in this disease.

MATERIALS

AND

METHODS

Purification of circulating anti-galactosyl antibodies Anti-Gal antibody was purified as described (Galili et al, 1984). Briefly, anti-Gal antibodies from heat-inactivated sera (100 ml) of normal individuals of ,4B, Rh+ blood group were affinity-purified on a melibiose-agarose column (16 x 0.9 cm) of 10 ml (Sigma Chemical Co., St Louis, MO, USA). Bound antibodies were eluted from the affinity column with 20 ml of a 0.5 M D-galactose solution. The IgG was then purified from the eluate by passage through a 5 ml column (8 x 0.9 cm) of protein A-sepharose (Sigma). The IgG eluted in 10 ml of 100 rnhl glycine-HCl buffer, pH 2.6 was immediately neutralized with 1 LI Tris-

F@K~ I. Immunoelectrophoresis of anti-Gal antibodies purified from human serum in presence of (A) anti-human serum proteins or (B) anti-human IgG serum. One single precipitation line is obsewed with material bound to and subsequently eked from the protein .I column.

I-ICI buffer, pH 8.4. After extensive dialysis against PBS, purified IgG was absorbed with Ci’brio clzolerae neuraminidase (Sigma)-treated human RBC, to remove any eventual antiThomsen Fridenreich antibody activity (Galili et al, 1984). After centrifugation at 2,000 rpm for 5 min, the IgG content of supernatants was assessed by quantitative radial immunodiffusion (Alancini, Carbonara and Heremans, 1965). About 3 mg of IgG were recovered from 100 ml of serum. Testing of antibodies The class of antibodies was determined either by immunoelectrophoresis (Figure 1) performed using monospecific goat antisera directed against human IgG, IgA and Ig1l (Nordic Laboratories, Tilburg, The Netherlands), by indirect immunofluorescence or by slab gel electrophoresis (Laemmli, 1970) (Figure 2). Immunoelectrophoresis in 1.5 per cent (a-/v) agarose was performed in electrophoresis base and agar gel plates (Hyland Laboratories, Costa Mesa, CA, LSA). Anti-Gal antibody was applied in the well and electrophoresed for 50 min at 7 V/cm in a 75 m.M sodium barbital buffer system pH 8.2. Antisera to human IgG or to human serum proteins were then applied in the trough. The proteins were allowed to diffuse overnight at room temperature in a moist chamber. For immunofluorescence microscopy, a solution of 10,ug of bound or unbound antibodies in 100~1 of PBS was mixed for lh at 37°C with 100 ~1 of a 1 per cent (v/v) rabbit RBC suspension in PBS. .After three u-ashings with PBS, the last pellet resuspended in 100~1 ofPBS was incubated for lh at 37°C vIith 100 ,ul of fluoresceinated goat anti-human IgG, IgA or IgM antibodies (Nordic Laboratories) diluted 10 times in PBS. After three further washings with PBS, the red cells N-ere examined under an UV microscope (Leitz Ortholux epi-illuminated fluorescent microscope equipped with a Ploemopak barrier filter system). Specificity of antibodies was determined by immunofluorescence labelling of various tissues. Indirect immunofluorescence studies performed on fresh frozen sections of murine tissues (skin, li\-er, kidney, spleen) established that the purified antibodies reacted only with

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those structures previously shown to contain these molecules (Peters and Goldstein, 1979). The exact specificity of anti-Gal antibodies has been determined by the immunostaining of rabbit red cell neutral glycolipids with known carbohydrate structures separated by thin layer chromatography as described by Galili (1988). We also verified that the purified anti-Gal did not bind to the Br antigen purified from B-type red cells. This antigen also contains the Gal a l-3 Gal epitope, but with an additional fucosyl branch, joined in an a l-2 linkage to the penultimate P-galactose (Galili, 1988). This lack of reactivity was not surprising, because anti-Gal was isolated from AB-type individuals, who lack antibodies to blood group B antigen. Prior incubation of the purified anti-a-Gal antibody with rabbit erythrocytes or with a ceramide pentahexoside (Gal a l-3 Gal p l-4 Glc NAc /I l-3 Gal p, l-4 Glc l-l Cer) abolished its subsequent binding to murine tissues or to a melibiose (a Gal glucoside) column or to Gal a l-3 Gal /3 l-4 Glc immunoadsorbent (Synsorb 90, Chem. Biomed., Edmonton, Canada). Prior incubation of rabbit erythrocytes with 100 yg/ml of purified anti-a-Gal antibodies in PBS prevented the subsequent binding of F.I.T.C.-Gri&ziu simplicifolia isolectin B4 (GS IB4). Histological techniques Samples. Human placental villi and basal plate biopsies were obtained from ten uncomplicated term normotensive pregnancies immediately after delivery and from ten therapeutic

I

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Figure

(.%ristitrw tt 01: Gal CLI -.? Gal: R .Warker of Trophohlast Imasion

479

abortions for psychological reasons (weeks 8-13) or for maternal persistent heart disease (two cases, weeks 12, 14) (blood group: 11 0, 7 A, 2 AB, 2 B). Placental bed biopsies were obtained from ten normotensive term pregnant women during caesarean sections performed for obstetrical reasons (six breech deliveries, four elective repeat sections). Placental bed biopsies were also performed during caesarean section of five se\-ere pre-eclamptic and three eclamptic women (gestational age 35-38 weeks). Placental bed biopsies of first trimester pregnancies (weeks 8-10) were obtained byechoguided chorionic villi sampling forceps. Pathologic specimens (placental villi and basal plate biopsies) were obtained from six spontaneous or eight therapeutic abortions for a well-known pathology (3 X0 monosomias and five molar placentae). In addition, biopsies of four tubal pregnancies and one choriocarcinoma were also analysed. Ir~lnlll~~2nhist~che~?zist~~, The anti-o-Gal

antibody was coupled aith peroxidase (Tijssen and Kurstak, 1984) or conjugated with fluorescein isothiocyanate as described previously (Unanue and Dixon, 1967). GS IB4 is a lectin commonly used to demonstrate Gal u l-3 Gal residues (Hayes and Goldstein, 1974). The specificity of the natural anti-Gal IgG seems to partially parallel that of isolectin B+ However, unlike anti-Gal antibody. isolectin B4 binds to the B blood group antigens (Galili et al, 1985). Peroxidase-labelled or fluorescein isothioc!.anatc conjugated GS IB_+was purchased from Sigma (Saint-Louis, 510, USA). Histolo@zl

trchnipes. Fresh frozen tissue sections (10 pm thick) were cut in an international cryostat and reacted with fluorescein-isothiocyanate conjugated human anti-Gal IgG antibody (25 pg/ml in PBS) or fluorescein-isothiocyanate conjugated GS IB4 for 30 min at room temperature in a moist chamber. After extensive washing, sections were mounted and examined under an epiilluminated Leitz Ortholux UT7 microscope. In some studies, peroxidase-labelled isolectin B4 (25 pug/ml in PBS) and peroxidase-conjugated Gal u l-3 Gal IgG (10 pug/ml in PBS) were used to localize this carbohydrate on paraformaldehyde fixed tissues according to previously described techniques (Sternberger et al, 1970).

Double

labelling studies. Peroxidase-conjugated anti-human placental lactogen p subunit antibody (50 pg/ml in PBS) was applied for 1 h at room temperature in a moist chamber on paraformaldehyde fixed sections. After extensive washing, the sections were incubated in the presence of diaminobenzidine with 0.1 per cent (v/v) Hz02 for 5 min. They were then incubated for 1 h at room temperature in methanol (80 per cent) in order to block the peroxidase activity. _lfter washing in PBS, tissues were incubated for 30 min at room temperature in a moist chamber with either peroxidase-conjugated anti-Gal antibody or peroxidase-conjugated GS IB+ After washing, the sections were incubated for 10 min in the dark in the presence of amino-ethyl-carbazole (0.01 per cent, w/v-).

Measurement

of circulating

Prctients. Sera from 27 eclamptic,

anti-a-Gal

antibody

titers

59 pre-eclamptic patients, 92 normal pregnant women with the same gestational age (30 to 38 weeks) and 75 normotensive women (8 to 18 weeks of pregnancy) were used. The sera of 20 eclamptic patients and 40 normotensive pregnant women with the same gestational age were obtained from Mamayemo Maternity Hospital in Kinshasa (Zaire) after approval of a University Ethics Committee.

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Figure 3. Immunoperoxidase staining with GS IB.+ on a placental basal plate from a 8 weeks gestation specimen. Gal u l-3 Gal is clearly demonstrated on the villous (arrow head) and interstitial (arrow) trophobiast (120x).

Pre-eclampsia was defined according to the criteria of the American cians and Gynecologists as described previously (Foidart et al, 1986).

College of Obstetri-

Determination ofthe anti-a-Gal antibody titers. The a-Gal antibody titers were measured by passive haemagglutination using rabbit RBC as previously described (Malaise et al, 1986). The titers of anti-Gal antibodies were expressed as the greatest dilution of serum that induced complete macroscopic agglutination. Each serum was tested at least in triplicate.

RESULTS In first trimester pregnancies (< 11 weeks) we localized the Gal a l-3 Gal residues at the surface ofvillous, interstitial and vascular trophoblastic cells (Figure 3). Identical results were using affinityobtained when the Gal a l-3 Gal was localized by immunohistochemistry purified peroxidase conjugated anti-Gal antibody or by lectin histochemistry using fluorescein-isothiocyanate conjugated GS IB+ Using first trimester placental villi as substrate, immunofluorescence blocking and inhibition studies confirmed the specificity of the staining reaction. No binding of the FITC-GS IB4 was observed on sections pretreated with purified anti-Gal antibody. Conversely prior incubation of the tissue section with GS IB4 prevented the subsequent binding of the peroxidase-conjugated anti-Gal antibody. No reaction occurred when FITC-GS IB4 or peroxidase-conjugated anti-Gal antibody incubated with Gal a l-3 Gal at the surface of rabbit erythrocytes or linked to SynsorbR (CHEM. BIOMED.) were used (not shown). From the 11th week of pregnancy, the staining intensity of villous trophoblast became progressivelyweaker and disappeared almost completely in 13-14 week aged specimens. Gal a l-3 Gal residues were absent from term placental villi (Figure 4). The presence of Gal CL l-3 Gal residues in interstitial and endovascular trophoblastic cells was then tested in placental bed biopsies of normal first trimester or term pregnancies. Some cells present in the interstitium and in the walls of utero-placental arteries simultaneously displayed an immuno-

(~‘bC&tw ct al: Gal CLI-.? Gal: a .2larkrr

ofTrophoblast Itxasion

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reactivity with anti-HPL and anti-Gal antibodies in double labelling studies (not shon-n). These results indicate that the normal interstitial and endovascular trophoblast retains the capacity of exhibiting the Gal u l-3 Gal epitope throughout pregnancy. The trophoblastic shell also reacted strongly up to the term ofpregnancy (Figure 4). In ectopic pregnancies, the trophoblast which is similar to that of intra-uterine pregnancies also displayed these residues (not shown). The poorly developed villous trophoblast from specimens of spontaneous abortion and of X0 monosomia (Figure 5) did not express Gal (x l-3 Gal residues. In molar placenta (two cases) (Figure 6) and choriocarcinoma (one case) villous trophoblastic cells highl! expressed Gal a l-3 Gal residues. In placental bed biopsies from term normotensive women, the decidual and inner myometrial segments of the utero-placental arteries were infiltrated by a high density of cells exhibiting Gal (1 l-3 Gal residues (Figure 7). These cells also exhibited HPL reactivi% as demonstrated by double labelling studies using purified human anti-Gal IgG and rabbit antiI IPL IgG. They acre thus identified respectively as endovascular and interstitial trophoblast cells. In all sections from pre-eclamptic women, the decidual segments of the arterial walls w-ere infiltrated by some trophoblast cells also displaying Gal u l-3 Gal residues. However, the myometrical segments and some segments of the inner decidua were surprisingly characterized by an absence of endovascular trophoblast with a persistent invasion of the interstitial trophoblast surrounding the adventitial layer of the spiral arteries. No cells displaying Gal (1 l-3 Gal at their surface were identified in the internal or medial layers of these arteries (Figure 8). It-e next compared the anti-Gal antibody titers in normal pregnancies and in patients with pre-eclampsia or eclampsia. In the normotensive non-pregnant control group (40 women) [Figure 9(a)] the mean antibody titer was 40. In early pregnancy (75 normotensive women; 8-18 weeks of pregnancy) [Figure 9(b)] a significant rise in the antibody titer was observed (mean titer, SO). At the end of pregnancy (36-40 weeks-92 women) [Figure 9(c)] the antiGal antibody titer had returned to values observed in non-pregnant women (titer, 40). In

F/~urr 4. Immunoperoxidase staining with GS IB4 on a placental bed biopsy from a term pregnancy. Gal u 1-3 Gal is completely absent from villous trophoblast (arrow head) while the trophoblastic strongly (arrow) (60X).

At this time, shell reacted

482

Figure 5. Immunoperoxidase i-3 Gal is not demonstrated

staining with GS IB.+ on chorionic rilli from X0 monosomia. on the trophoblastic layer (120x).

In this pathology,

Gal u

contrast, the anti-Gal antibody titer remained considerably elevated in pre-eclamptic (mean titer, 160) [Figure 9(d)] or eclamptic patients (mean titer, 320) [Figure 9(e)]. Comparison between the different groups of pregnant women by using non-parametric statistical methods (Mann-Whitney test) concluded that they were statistically different (Z’< 0.005 in each case).

DISCUSSION This study demonstrates that the human trophoblast cells express Gal CLl-3 Gal glycosidic structures. The Gal a 1-3 Gal epitopes are prevalent and easily detected as terminal

Figure 6. Immunofluorescence Gal residues (300x).

stainingwith anti-Gal IgG on molar placenta. Trophoblastic

cells display Gal a l-3

Christrune et al: Gal a I-.i’ Gal: a .Marker

ofTrophoblast Inrasion

483

Fi&rru i. Immunoperoxidase staining with G.S IB4 of a maternal uteroplacental artery. The vascular trophoblast infiltrating the arterial wall (%\? exhibits Gal a l-3 Gal residues (arrow) (L: arterial lumen) (300x).

carbohydrate structures on glycoproteins and glycolipids of many mammalian species and all New World monkeys. They have not been detected in Old World monkeys, apes or man. This structure resembles but is distinct from the blood group B antigen (Galili, 1988). In the villous trophoblast, the reactivity disappeared after 10 weeks gestation. In the extravillous trophoblast, a positive reaction persisted throughout pregnancy. In first trimester of term placental bed biopsies, cells exhibiting Gal c( 1-3 Gal residues also displayed HPL reactivity indicating that they were indeed trophoblast cells. They were localized in the interstitium and in the utero-placental arterial walls. The expression of Gal c1 l-3 Gal residues was strongly decreased or absent in poorly developed trophoblast (X0 monosomia)

Figure 8. Immunoperoxidase staining with GS IB4 of an uteroplacental No cells displaying Gal c( 1-3 Gal residues were identified (240x).

artery from a patient with pre-eclampsia.

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Figure 9. Anti-Gal antibody titers in the sera of: (a) 40 normotensive non-pregnant women (control group); (b) 75 normotensive pregnant women (8-18 weeks gestation); (c) 92 normotensive pregnant women (30-38 weeks gestation); (d) 58 pregnant women with pre-eclampsia (30-38 weeks gestation); (e) 27 pregnant women with eclampsia (30-38 weeks gestation). Statistically significant differences are observed between groups b, c, d and e (Mann-Whitney test) (p < 0.005).

Christime et al: Gal u l-3 Gal: a Marker of Trophoblast Invasion

485

and in spontaneous abortion specimens. On the contrary, a prominent staining was demonstrated at the surface of the trophoblast cells (molar placenta, choriocarcinoma). The decrease or the lack of trophoblast invasion in the spiral arteries in pre-eclampsia or eclampsia was also correlated wiaa decreased arterial colonization by cells exhibiting Gal a l-3 Gal residues. Since the metastatic and invasive potential of malignant cells is strongly correlated with the density of Gal a l-3 Gal at their surface (McCoy, Goldstein and Varani, 1985; Castronovo et al, 1989; Petryniak et al, 1991), it is tempting to speculate that these glycosidic structures also represent a marker for the trophoblast invasive capacity. The anti-Gal IgG molecule is a natural antibody that represents 1 per cent of total circulating IgG. The ubiquitous presence of anti-Gal in human sera was also demonstrated by other investigators who purified this antibody from normal sera using a-Gal immunoadsorbents (Malaise et al, 1986; Etienne-Decerf et al, 1987; Kay and Bosman, 1985). The ubiquitous presence of anti-Gal in man in high titers suggests that, analogous with the synthesis of anti-blood groups antibodies against bacterial antigens (Springer and Horton, 1969) Gal a l-3 Gal epitopes on bacteria of the intestinal flora provide constant antigenic stimulation for the synthesis of the anti-Gal antibody. The anti-Gal antibody titers increased in first trimester pregnancies when the density of Gal a l-3 Gal residues was maximal. Binding of maternal antibody to the trophoblast could contribute to limit trophoblast invasion and to the immunological control of implantation. The significant rise in the anti-Gal antibody titer in pre-eclampsia and eclampsia could also contribute to the decreased infiltration of the uteroplacental arteries by the trophoblast cells in this condition. Our study suggests that in humans, a carbohydrate epitope on trophoblast cells is recognizable by natural antibodies that are probably present as a protective measure against intestinal flora. Sharing of antigenic determinants between bacterial antigens and selfantigens has been previously reported. This was demonstrated in acetylcholine receptors and bacterial components (Stefansson, Dieperink and Richman, 1985) or between group A streptococcus and myocardial membrane components (Van de Rijn, Zabriskie and McCarthy, 1977). It has been proposed as an etiologic background for autoimmune phenomena concerning neural or cardiac tissues. Thus it is possible that in the absence of anti-Gal, human trophoblast cells might infiltrate deeper into the myometrium and that placental implantation might later be limited by other mechanisms. Hence it is evolutionary possible that the human and Old World monkey trophoblast may have been challenged by factors not directly related to the process of implantation which are, nevertheless, responsible for the limitation of placental anchorage. The defective development of the physiological changes in the spiral arteries during preeclampsia is probably related to their decreased invasion by endovascular trophoblast cells (Brosens, 1988). The placental bed invasion by interstitial trophoblast remains, however, both in normal (Brosens, 1988). Since the Gal a l-3 Gal residues are demonstrated interstitial and vascular trophoblast, it might appear improbable that an anti-Gal antibody could contribute to decrease solely the colonization of the spiral arteries and not of the interstitium. However, cell-antibody mediated immune reactions are complex and require a sufficient density of cell surface antigens. Below a critical level, complement-mediated cytoxicity cannot be elicited despite antibody binding to the antigen. It is thus possible that differences in density of Gal a l-3 Gal residues in vascular versus interstitial trophoblast may explain the apparent discrepancy between their distinct invasive capacities.

486

Placenta (1992), Vol. 13 ACKNOWLEDGEMENTS

This work was supported by grants of the CGER in Belgium, of the FRSM in Belgium (no 3.4514.88), of the ‘Association contre le Cancer’ (Belgium), of the ‘Fonds de la Recherche Facultaire’ (a grant of the Faculty of Medicine), of the ‘Oeuvre Belge du Cancer’ (Belgium) and of the ‘Communaute Francaise de Belgique’ (‘Action de Recherche Concertee’ no 90/94-139).

REFERENCES Brosens, I. A. (1988) The utero-placental vessels at term. The distribution and extent of physiological changes. In TrophoblustResearch (Ed.) Miller. R. K. & Thiede. H. A.. vol. 3 (Ed.) Kaufmann. P.. Miller. R. K. IDD. L 61-67. New York: London: Plenum Medical Book Co. Castronovo, V., Colin, C., Parent, B., Foidart, J. M., Lambotte, R. & Mahieu, P. (1989) Possible role of human natural anti-Gal antibodies in the natural antitumor defense system. Jotdrnal of the National Cancer Institute, 81, 212-215. Castronovo, V., Foidart, J. M., Li Vecchi, M., Foidart, J. B., Bracke, M., Mareel, M. & Mahieu P. (1987) Human anti-a-galactosyl IgG reduces the lung colonization by murine MO4 cells. Invasion andMetastasis, 7,325345. Etienne-Decerf, J., Malaise, M., Mahieu, P. & Winand, R. (1987) Elevated anti-a-galactosyl antibody titers. A marker of progression in autoimmune thyroid disorders and in endocrine ophthalmopathy. Acta Endxxrinologica, 115,67-74. Feizi, T. (1985) Demonstration by monoclonal antibodies that carbohydrates structures of glycoproteins and glycolipids are onto-developmental antigens. Nature, 314, 53-57. Foidart, J. M., Hunt, J., Lap&e, Ch. M., Nusgens, B., De Rycker, C., Bruwier, M., Lambotte, R., Bernard, A. & Mahieu, Ph. (1986) Antibodies to laminin in preeclampsia. KidnqZnternational, 29,10X-1057. Gabius, H. J. (1987) Vertebrate lectins and their possible role in fertilization development and tumor biology (review). In Vivo, 1, 75-84. Galili, U. (1988) The natural anti-Gal antibody, the B-like antigens, and human red cell aging. Blood Cells, 14,205220. GaIili, U., Clark, M. & Shohet, S. B. (1986b) Excessive binding of natural anti-a-galactosyl immunoglobulin G to sickle erythrocytes may contribute to extravascular cell destruction.Journal of Clinical Investigation,77, 27-33. Galili, U., Flechner, I., Knyszynski, A., Danon, D. & Rachmilewitz, E. (1986a) The natural anti-a-galactosyl IgG on human normal senescent red blood cells. BritishJournal ofHaematology, 62, 317-324. Galili, U., Korkesh, A., Kahane, I. & Rachmilewitz, E. A. (1983) Demonstration of a natural antigalactosyl 1gG antibody on thalassemic red blood cells. Blood, 61, 1258-1264. GaIili, U.. Macher, B., Buehler, J. & Shohet. S. (1985) Human natural anti-a-galactosyl 1gG: the specific recognition of a (1-3)-linked galactose residues.Jomnal ofExperimental.tiedicine, 162, 573-582: Galili. U.. Raclunilewitz. E. A.. Pelen. A. & Flechner. I. (1984) A uniaue natural human IeG L, antibodv with antia-galactosyl specificity.~ofourt& ofE~~erimentalMedi&e, i60, i5 19-15’3 1. Hakomori, S. I. (1985) Aberrant glycosylation in cancer cell membranes as focused on glycolipids. Overview and perspectives. Cancer Research, 45,2405-2414. Hayes, C. E. & Goldstein, I. J. (1974) An a-D-galactosyl binding lectin from Bundeiraea simplicijbliaseeds. Isolation by affinity chromatography and characterization. journal ofBiological Chemistry, 249, 1904-1914. Kay, M. M. B. & Bosman, G. J. C. G. M. (1985) Naturally occurring human “antigalactosyl” IgG antibodies are heterophile antibodies recognizing blood group-related substances. Experimental Hematology, 13, 1103-I 112. Laemmli. U. K. (1970) Cleavage of structural proteins during the assemblv of the head of bacteriophage . I T4. Nature, !227, 68Ok85: Malaise, M. G., Davin, J. C., Mahieu, P. R. & Franchimont, P. (1986) Elevated anti-galactosyl antibody titers reflect a renal iniurv after gold or d-uenicillamine in rheumatoid arthritis. ClinicalImmunology .I and Immuno~atho. ” _ logy, 40,356-364. Mancini, G., Carbonara, A. D. & Heremans, J. F. (1965) Immuno-chemical quantitation of antigens by single Immunochemistry, 2, 235-254. radial immunodiffusion. McCoy, J., Goldstein, I. J. & Varani, J. (1985) A review of studies in our laboratory regarding Ella methodology for the study of cell surfaces carbohydrates from tumors ofvarying metastatic potential. TumourBiology, 6,99-l 14. Peters, B. & Goldstein, I. (1979) The use of fluorescein-conjugated Bandeiraea simplictjidiaBd-isolectin, as a histochemical reagent for the detection of a-D-galactopyrannosyl group. Experimental Cell Research, 120, 321339. Petryniak, J., Varani, J., Ervin, P. R. & Goldstein, I. J. (1991) Differential expression of glycoproteins containing alpha-D-galactosyl groups on normal human breast epithelial cells and MCF-7 human breast carcinoma cells. Cancer Letters, 60, 59-65. Robertson, W. B., Brosens, I. & Dixon, G. (1975) Uteroplacental vascular pathology, European Journal of Obstetrics,Gynecologyand ReproductiveBiology, 5, 47-65.

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