Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein

Brain Research, 385 (1986) 237-244

237

Elsevier BRE 12076

Molecular Specificity of L2 Monoclonal Antibodies that Bind to Carbohydrate Determinants of Neural Cell Adhesion Molecules and their Resemblance to other Monoclonal Antibodies Recognizing the Myelin-Associated Glycoprotein ANTONIO B. NORONHA l, AMJAD ILYAS 1, HORST ANTONICEK2, MELITI'A SCHACHNER2and RICHARD H. QUARLES 1

1Developmental and Metabolic Neurology Branch, N1NCDS, NIH, Bethesda, MD 20892 (U. S.A.) and 2Department of Neurobiology, University of Heidelberg, D6900 Heidelberg (F. R. G.) (Accepted 18 March 1986)

Key words: Adhesion molecule - - Glycoprotein - - Glycolipid - - L2 monoclonal antibody - Myelin-associated glycoprotein (MAG) - - Neural cell adhesion molecule (N-CAM)

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in bindingto some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipidsof the PNS. INTRODUCTION L2 monoclonal antibodies bind to carbohydrate determinants that are present on a n u m b e r of glycoproteins of the nervous system that have been implicated in cell-cell interactions H. These glycoproteins include neural cell adhesion molecule ( N - C A M ) , the L1 adhesion molecule, and the myelin-associated glycoprotein ( M A G ) . The periaxonal localization of M A G 28 and the correlation of its presence with a normal 12-14 n m periaxonal space in the peripheral nervous system (PNS) 31 strongly suggest that it is involved in the interactions between axons and myelinating cells. Recently, it was d e m o n s t r a t e d that a 160 kDa glycoprotein termed J1 also reacts with the L2 antibodies and that the J1 glycoprotein appears to be involved in n e u r o n - a s t r o c y t e interactions lz. Based

on these findings, it was hypothesized that all molecules expressing the L2 antigen are involved in cell surface interactions and that the carbohydrate moiety identified by the L2 antibodies may be functionally involved in these interactions 11,12. A functional role for this carbohydrate d e t e r m i n a n t was supported by the finding that Fab fragments of L2 antibodies inhibit n e u r o n - a s t r o c y t e and astrocyte-astrocyte adhesion 1°. HNK-1, a mouse monoclonal antibody raised to a lymphoblastoma and used as a marker for a subset of lymphocytes with natural killer function, also reacts with each of the glycoproteins m e n t i o n e d above 1°-12,1s and appears to identify a carbohydrate structure that is closely related to that recognized by L2 antibodies. HNK-1 antibody also reacts with some 19-28 k D a glycoproteins9'19"22 and with some

Correspondence: A. Noronha, Park Bldg., Room 425, NINCDS, NIH, Bethesda, MD 20892, U.S.A. 0006-8993/86/$03.50© 1986 Elsevier Science Publishers B.V. (Biomedical Division)

238 A

B

C ~iiiii!~i~ii! ~iilil

1

2

3

4

1

2

3

4

1

2

3

4

Fig. 1. Western blot analysis of human brain proteins with L2 monoclonal antibodies. The 3 panels show electroblots of 7.5% SDSPAGE gels and the samples loaded in the 4 lanes of each panel are the same. A shows protein staining of the blot with amido black. B and C show immunostaining with L2-336 and L2-334 monoclonal antibodies (diluted l:100), respectively, using the appropriate peroxidase-labeled anti-rat immunoglobulin (diluted 1:100) as second antibody. Lane 1, whole homogenate of white matter from adult human brain, 50 ktg protein; lane 2, human MAG partially purified from brain by the lithium diiodosalicylateprocedure25, 0.8 ktg; lane 3, whole homogenate of gray matter from adult human brain, 50/~g protein; and lane 4, bovine brain N-CAM purified by immunoaffinity chromatography on a monoclonal BSP-2 antibody column 6, 4/~g. The two bands revealed by immunostaining of the isolated MAG in lane 2 are intact MAG and its proteolytic derivative, dMAG, respectively.

unusual, sulfated, glucuronic acid-containing sphingoglycolipids2"7 which are present in the human peripheral nervous system (PNS). These acidic glycolipids are isolated in the whole 'ganglioside fraction', but are not gangliosides since they do not contain sialic acid. In this report, it is d e m o n s t r a t e d that the L2 monoclonal antibodies used in previous studies 1°-12 as well as subsequently isolated ones also react with these low molecular weight glycoproteins and the glycolipids of the PNS. Conversely, it is r e p o r t e d that some other monoclonal antibodies previously shown to react with M A G and these glycoconjugates of the PNS also react with N - C A M . The results further emphasize the shared antigenicity between M A G , NC A M and other adhesion molecules. Preliminary descriptions of these findings have been r e p o r t e d 2~'24. MATERIALS AND METHODS H u m a n brain tissue was o b t a i n e d at autopsy and

sciatic nerve after limb amputation from persons without evidence of neurological disease. Cat sciatic nerves were obtained from Pelfreeze (Rogers, A R ) . Ten percent h o m o g e n a t e s of human white or gray matter and cat peripheral nerve were p r e p a r e d in deionized water using a Polytron h o m o g e n i z e r (Brinkman Instruments, Ontario, Canada). Partially purified human M A G was p r e p a r e d by the lithium diiodosalicylate (LIS)-phenol m e t h o d as previously described 25. Bovine brain N - C A M , a kind gift from Dr. G. R o u g o n ( N I H ) , was purified by immunoaffinity c h r o m a t o g r a p h y on a monoclonal BSP-2 antibody 6 column. Protein estimations were by the m e t h o d of Lowry 16, using bovine serum albumin ( B S A ) as standard. The L2 monoclonal antibodies were raised against an enriched plasma m e m b r a n e fraction or a glycoprotein fraction from mouse brain and have been described previously 11. The 5 L2 antibodies used in this investigation were: 334 (IgM), 336 ( l g G ) , 344 (IgM),

239 349 (IgM) and 392 (IgM). The stock L2 antibody preparations were ammonium sulfate cuts of serum free hybridoma culture supernatants that were dialyzed exhaustively against phosphate buffered saline and adjusted to a protein concentration of about 5 mg per ml. Mouse monoclonal antibodies produced to MAG that were used in this study have also been described previously4. Rabbit polyclonal antisera to human and rat MAG were prepared as described in Quarles et al. 23. IgG polyclonal and monoclonal antibodies were purified by Protein A (Staphylococcus aureus) sepharose column chromatography (Pharmacia Fine Chemicals, Uppsala, Sweden) by the method of Lindmark et al. 15. IgM monoclonal antibodies were purified by the method of Heide and Schwick5. Affinity purified, peroxidase conjugated anti-rat IgG and anti-rat IgM as well as anti-mouse IgG and anti-mouse IgM were obtained from Cappel Laboratories (Westchester, PA) and Boehringer Mannheim (Indianapolis, IN). Electrophoresis on either 7.5 or 12.5% polyacrylamide slab gels containing sodium dodecyl sulfate (SDS) was according to the method of Laemmli 13. Following SDS-PAGE, proteins were electrophoretically transferred onto nitrocellulose sheets (0.45 #m pore size, Schleicher and Schuell, Keene, N J) essentially as described by Towbin et al. 3°. Electroblots were immunostained using the peroxidase labeled second antibody technique as has been described for MAG 23. An acidic glycolipid fraction was isolated from cat and human sciatic nerve or human brain by DEAESephadex chromatography, followed by base treatment and Unisil (Clarkson Chemical Co., Williamsport, PA) chromatography as described by Ledeen et al. TM. Thin-layer chromatography (TLC) of glycolipids was done on aluminum backed TLC plates (Silica gel 60, E. Merck) obtained from Brinkman Instruments (Westbury, NY). The plates were developed in chloroform:methanol:0.2% KC1, 50:40:10 (v/v/v) and the gangliosides were detected with resorcinol spray 29. L2 antibody binding to glycolipids was demonstrated after overlaying the TLC plates with a 1:200 or 1:600 dilution of the stock solutions. The procedure was the same as that described previously7-8"17 except that a peroxidase conjugated second antibody was used and color development was with o-phenylenediamine (Sigma Chemical Co., St.

Louis, MO) as substrate. Binding of glycolipids was also demonstrated with an ELISA procedure described previouslys. RESULTS

Reactivity of L2 antibodies with proteins of human CNS Immunoblots of human CNS stained with L2 monoclonal antibodies are shown in Fig. 1. The figure compares the staining in white matter and gray matter and includes MAG and N-CAM standards. The amounts of the purified standards were too small to be seen clearly on the protein stained blot (Fig. 1A). The antibodies bound very strongly to the 100 kDa MAG in human white matter (Fig. 1, lane 1) and also with its proteolytic derivative, dMAG. In gray matter (Fig. 1, lane 3), the principal proteins binding the L2 antibodies were of higher M r and presumably included N-CAM, L1 and J1 adhesion molecules. The L2 antibodies differed somewhat in the staining patterns produced. L2-336 shown in Fig. 1B stained very little besides MAG in white matter and only two bands were visible in gray matter, whereas L2-334 shown in Fig. 1C stained a large number of minor components as well. The other L2 antibodies (not shown) gave staining patterns intermediate between the two that are shown. In general, there was a positive correlation between the intensity of N-CAM staining in Fig. 1, lane 4 and the capacity to detect minor components in the whole tissue homogenates. However, all of the antibodies stained MAG very intensely. These variations in staining patterns probably reflect differences in affinity between the antibodies and/or slight qualitative differences in the carbohydrate epitopes recognized. The staining pattern with HNK-1 (not shown) was similar to that in Fig. 1C.

Reactivity of anti-MA G antibodies with N-CAM F7F7 is one of a group of mouse monoclonal antibodies raised in response to human MAG that have been shown to recognize carbohydrate determinants 4. Fig. 2 shows that F7F7 also resembles HNK-1 and the L2 antibodies by binding to purified bovine N-CAM on immunoblots. A rabbit polyclonal antiserum raised to human MAG and which is known to have substantial activity against carbohydrate 22 also

240

Reactivity of L2 antibodies with glycoconjugates of the PNS Since HNK-1 and the anti-MAG antibodies such as F7F7 are known to react strongly with some 19 to 28 kDa glycoproteins in human and cat peripheral nerve 9'19'22, experiments were done to see if the L2 antibodies also reacted with these glycoproteins. Fig. 3 demonstrates that the L2 antibodies do react with these components of the PNS, but, as was the case in the CNS, the specificity of the staining varied between antibodies. L2-336 was relatively specific for M A G and the 19-28 kDa glycoproteins (Fig. 3, lane 1), while L2-392 stained numerous other components in addition to these (Fig. 3, lane 2). L2-334 which was

9.4 K 68K

43 K

30K

21 K

:: i¸ '14K

1

2

3

!!

Fig. 2. Western blot analysis of purified N - C A M with L2, HNK-1 and a m o u s e a n t i - h u m a n M A G monoclonal antibody, F7F7. Each lane contains 4/~g of N - C A M . Immunostaining was done with 1:100 dilutions of L2-344 in lane 1, HNK-1 in lane 2, and F7F7 in lane 3. 1

immunostained the N-CAM (not shown). However, monoclonal and polyclonal antibodies recognizing polypeptide epitopes in M A G did not react with the purified N-CAM suggesting that the immunological similarity between M A G and N-CAM is confined to the carbohydrate domains of the molecules.

2

3

4

Fig. 3. Western blot analysis of glycoproteins of peripheral nerve with L2 monoclonal antibodies. The first 3 lanes are immunoblots obtained from 50t~g protein of a cat sciatic nerve homogenate. Immunostaining was done with L2-336 in lane 1, L2-392 in lane 2, and a m o u s e anti-human M A G monoclonal antibody (F7F7) in lane 3. The thin arrow at the left of the figure points to M A G , and the broad arrow points to the 19-28 k D a glycoproteins that react with these antibodies. The major PO myelin glycoprotein does not react and appears as a negative image at about 30 k D a in lanes 2 and 3. Protein stained M, standards are shown in lane 4 for reference.

241 TABLE I Binding of L2 antibodies to the acidic glycolipid fractions from human and cat nervous tissue

Binding was measured by ELISA as previously describeds using peroxidase labeled anti-rat IgM (~-chain specific) as second antibody, except in the case of L2-336 for which the second antibody was peroxidase labeled anti-rat IgG (y-chain specific). Each well contained 10 ng sialic acid of the whole ganglioside fraction from the tissue indicated. The CNS ganglioside fraction was from brain and the PNS fractions were from sciatic nerve. Each value is the mean optical density at 492/~m obtained for 3 separate wells in the ELISA. L2 antibody

334 336 344 349 392

Dilution

(1:600) (1:200) (1:600) (1:600) (1:600)

GM

1

GD la

Optical density Human CNS

Human PNS

Cat PNS

0.02 0.01 0.01 0.00 0.01

0.72 0.18 1.0 1.2 1.0

0.84 0.37 1.1 1.2 0.89

shown to stain n u m e r o u s c o m p o n e n t s in the brain homogenates (Fig. 1C) also gave a h e t e r o g e n e o u s staining pattern of p e r i p h e r a l nerve which was similar to that of L2-392. The latter staining p a t t e r n is similar to that p r o d u c e d by F7F7 (Fig. 3, lane 3) and to that previously r e p o r t e d for HNK-1 (ref. 9). If the amount of protein on the blot was greatly reduced, these antibodies gave a p a t t e r n similar to that in Fig. 3, lane 1 in which M A G and the 19-28 k D a glycoproteins were the principal stained c o m p o n e n t s (see ref. 9). O t h e r a n t i - M A G c a r b o h y d r a t e monoclonal antibodies such as G7C8 p r o d u c e a m o r e specific staining pattern, similar to that in Fig. 3, lane 1, even on heavily l o a d e d blots 22. The L2 antibodies were also tested for reactivity with the acidic sphingoglycolipids of the h u m a n PNS that have been shown to react with HNK-1 and the a n t i - M A G monoclonal antibodiesT-q The E L I S A data in Table I shows that each of the L2 antibodies did react with the acidic glycolipid fraction from human peripheral nerve but not with that from h u m a n brain. Cat nerve also contains these glycolipids that react with the antibodies as shown by the E L I S A data. This is similar to results o b t a i n e d with HNK-1 and other a n t i - M A G c a r b o h y d r a t e monoclonal antibodies 7-s. T L C overlay e x p e r i m e n t s (Fig. 4) d e m o n strated that the L2 antibodies were binding to two principal glycolipids of the PNS, one migrating be-

GD l b GTlb 1

2

3

4

Fig. 4. Immunostaining of acidic glycolipids of peripheral nerve on thin layer chromatograms. The acidic glycolipid fractions from human brain (lane 1) and cat sciatic nerve (lanes 2-4) were chromatographed on aluminum backed-silica gel TLC plates as previously describeds. Each lane contains 10/~g of sialic acid. Lanes 1 and 2 were stained for sialic acid with resorcinol reagent. Lanes 3 and 4 were immunostained with L2334 (1:600) and L2-336 (1:200), respectively, using peroxidase labeled second antibodies. The major CNS gangliosides are labeled to the left of the figure for reference.

tween GM1 and G D l a and a second migrating slightly ahead of G D l b . These are the same glucuronic acid-containing sphingoglycolipids that have previously been shown to react with H N K - 1 and o t h e r a n t i - M A G monoclonal antibodies 2'7's. The minor, m o r e rapidly migrating c o m p o n e n t seen in Fig. 4, lane 3 is an unidentified lipid that also reacts with some of human IgM paraproteins. DISCUSSION The results described here show that the L2 antibodies, like H N K - 1 , belong to a family of monoclonal antibodies that bind to c a r b o h y d r a t e epitopes that are shared b e t w e e n M A G , some 19-28 k D a glycoproteins of the PNS, and some sulfated, glucuronic acid containing sphingoglycolipids of the PNS. A substantial n u m b e r of mouse monoclonal antibodies raised in response to human M A G also react with these same glycoconjugates 4'22'24. The similarity be-

242 TABLE II Reactivity of anti-carbohydrate monoclonal antibodies with glycoconjugates of the nervous system

Numbers in parentheses are references where reactivity is demonstrated. CS, current study; +, some, but not all, of these antibodies show reactivity; ?, not tested; + ?, reference suggests reactivity. Monoclonal antibodies

MA G (100 kDa)

Low M r PNS glycoproteins (19-28 kDa)

Sulfated glucuronyl glycolipids

N-CAM (180, 140, 120 kDa)

L1 (200, 140 kDa)

Jl (160 kDa)

L2

+ (11, CS) + (18,19) + (4, 22, 24) + (1, 8, 19, 22, 26, 27) + (20) +? (32)

+ (CS) + (9,19,22) + (22) + (19, 22) + (20) +? (32)

+ (CS) + (2,7) + (24) + (2, 8) + (20) ?

+ (11, CS) + (11, CS) + (CS) _+ (CS) + (20) +? (32)

+ (11) + (11) ?

+ (11, 12) + (11) 9

?

?

'~

?

'~

?

HNK-1 Anti-MAG Human IgM paraprotein Anti-melanoma (10C5, 11B5) NC-1

tween some of these a n t i - M A G monoclonal antibodies and HNK-1 was also indicated by their ability to immunostain the same subset of human lymphocytes with natural killer function as HNK-1 (ref. 3). A n o t h e r group of monoclonal antibodies falling into this family that has attracted substantial interest in recent years are the human IgM p a r a p r o t e i n s associated with n e u r o p a t h y 1'2'8'19'22'26'27. In addition, it has been shown that rabbit polyclonal antisera raised in response to M A G from species that express a high level of this c a r b o h y d r a t e structure in M A G (e.g. humans) contain a high titer of antibodies reacting with the same glycoconjugates 22. Since all of these antibodies react with each of the protein and lipid antigens mentioned above it is likely they are recognizing the same or closely related c a r b o h y d r a t e structures. The frequency with which antibodies of this type occur suggests that the c a r b o h y d r a t e structure or structures that they recognize are highly immunogenic. Table II summarizes the reactivity of these and o t h e r anti-carbohydrate monoclonal antibodies with glycoconjugates of nervous tissue. Two other mouse monoclonal antibodies that fall into this class are 10C5 and l l B 5 which were raised to a human m e l a n o m a and react with a 100 k D a glycoprotein released into the culture m e d i u m by this cell line as well as by other tumors of n e u r o e c t o d e r mal origin 2°. Experiments similar to the ones described here showed that these antibodies also react with a c a r b o h y d r a t e d e t e r m i n a n t in M A G and the

protein and lipid glycoconjugates of the PNS 2°. Small cell lung carcinoma is another example of a t u m o r of n e u r o e c t o d e r m a l origin which expresses some surface glycoproteins that react with HNK-1 and some of the other antibodies described in this p a p e r 33. Still another monoclonal antibody that p r o b a b l y falls into this family of antibodies is NC-1 which was raised to quail ciliary ganglion and resembles HNK-1 in a n u m b e r of respects 32. W e s t e r n blots of chick dorsal root ganglion with NC-1 showed p r o m i n e n t 20, 100 and 200 k D a bands 32, which is similar to the pattern obtained when chicken sciatic nerve h o m o g e n a t e s were immunostained with HNK-1 and other antibodies in this family 22. Kruse et al. 11 were the first to describe an antigenic similarity between M A G and several cell adhesion molecules including N - C A M revealed by the L2 antibodies and HNK-1. The results described here further document this antigenic similarity by showing that a monoclonal antibody (F7F7) and polyclonal antisera raised to human M A G bind to purified NC A M . A n o t h e r antibody that we have found to react well with both M A G and N - C A M is the anti-melanoma antibody (10C5) described in the previous paragraph (unpublished result). H o w e v e r , it appears that not all the antibodies reacting with c a r b o h y d r a t e in M A G and the PNS glycoconjugates described in this paper also bind to N - C A M . F o r example, G7C8 is one of the mouse lgM antibodies raised to human M A G 4 which also stains the PNS glycoconjugates 2z

243 and binds to the same subset of h u m a n lymphocytes 3. H o w e v e r , this a n t i b o d y does not bind to bovine NC A M (unpublished results). In our experiments, some of the h u m a n I g M p a r a p r o t e i n s associated with n e u r o p a t h y stained purified bovine N - C A M faintly on i m m u n o b l o t s (not shown), but the intensity of staining was much less than that o b t a i n e d with the mouse m o n o c l o n a l antibodies described in this paper. The differences in staining intensity p r o d u c e d by the various m o n o c l o n a l antibodies and even among the several L2 antibodies (see Results) p r o b a b l y reflect differences in affinities as well as small qualitative differences a m o n g the epitopes recognized by the antibodies. The exact c a r b o h y d r a t e structures on the different glycoconjugates that bind the various antibodies need not be chemically identical in o r d e r to cross react immunologically. Much additional work is r e q u i r e d to precisely define the c a r b o h y d r a t e structures present on the different glycoconjugates and the exact r e q u i r e m e n t s for binding the various monoclonal antibodies. Nevertheless, it is likely that

REFERENCES 1 Braun, P.E., Frail, D.E. and Latov, N., Myelin-associated glycoprotein is the antigen for a monoclonal IgM in polyneuropathy, J. Neurochem., 39 (1982) 1261-1265. 2 Chou, K.H., Ilyas, A.A., Evans, J.E., Quarles, R.H. and Jungalwala, F.B., Structure of a glycolipid reacting with monoclonal IgM in neuropathy and with HNK-1, Biochim. Biophys. Res. Commun., 128 (1985) 383-388. 3 Dobersen, M.J., Gascon, P., Trost, S., Hammer, J.A., Goodman, S., Noronha, A.B., O'Shannessy, D.J., Brady, R.O. and Quarles, R.H., Murine monoclonal antibodies to the myelin-associated glycoprotein react with large granular lymphocytes of human blood, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 552-555. 4 Dobersen, M.J., Hammer, J.A., Noronha, A.B., Macintosh, T.D., Trapp, B.D., Brady, R.O. and Quarles, R.H., Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG), Neurochem. Res., 10 (1985) 423-437. 5 Heide, K. and Schwick, H.G., Salt fractionation of immunoglobulins. In D.M. Weir (Ed.), Handbook of Experimental Immunology, Blackwell Scientific Publications, Oxford, 1973, pp. 6.1-6.11. 6 Hirn, M., Ghandour, M.S., Deagostini-Bazin, H. and Goridis, C., Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2, Brain Research, 265 (1983) 87-100. 7 Ilyas, A.A., Quarles, R.H. and Brady, R.O., The monoclonal antibody HNK-1 reacts with a peripheral nerve ganglioside, Biochim. Biophys. Res. Commun., 122 (1984) 1206-1211.

the c a r b o h y d r a t e structures involved are similar, and it may be significant that several of the molecules in which they are present ( N - C A M , L1, J1 and M A G ) have b e e n implicated in c e l l - c e l l interactions in the nervous system 1°-12,28,31. Since it has been hypothesized that all molecules expressing the L2 epitope could be involved in adhesion 1°-12, experiments to elucidate possible functions in c e l l - c e l l interactions for the 19-28 k D a glycoproteins and the glucuronic acid-containing sphingoglycolipids of the PNS will be of interest.

ACKNOWLED GEMENTS This w o r k was s u p p o r t e d in part by a postdoctoral fellowship from the National Multiple Sclerosis Society ( U . S . A . ) to A . B . N . and by G r a n t (SFB 317) from the Deutsche Forschungsgemeinschaft. T h e authors thank Lottie Garfinkle for assistance in p r e p a ration of the manuscript.

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