Autoactivation of human complement subcomponent C lr involves ...

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Biochem. J. (1983) 215 369-375 Printed in Great Britain

Autoactivation of human complement subcomponent C lr involves structural changes reflected in modifications of intrinsic fluorescence, circular dichroism and reactivity with monoclonal antibodies Christian L. VILLIERS, Gerard J. ARLAUD and Maurice G. COLOMB* Equipe de Recherche 'Immunochimie-Systeme Complimentaire' du Department de Recherche Fondamentale de Grenoble et de l'Universite Scientifique et Medicale de Grenoble, Associe'e au Centre National de la Recherche Scientifique (Equipe de Recherche Associee n° 695) et a l'INSERM (Unite n° 238), Laboratoire de Biologie Moliculaire et Cellulaire, Centre d'Etudes Nucleaires de Grenoble, B.P. 85 X, 38041 Grenoble Cedex, France

(Received 5 April 1983/Accepted 22 July 1983) Autoactivation of C Ir is closely correlated with an irreversible increase of its intrinsic fluorescence. The activation and the fluorescence increase of C Ir are accelerated on addition of activated Clr. Ca2+, di-isopropyl phosphorofluoridate and Ci inhibitor, which all inhibit, although to different extents, Clr activation, inhibit in parallel the fluorescence increase. C Ir activation is blocked at pH 4.0-5.0, whereas it is accelerated at pH 10.5; under the same conditions the fluorescence increase shows parallel effects. No such fluorescence increase is observed during C ls activation by trace amounts of C ir. Far-u.v. circular-dichroism spectra of C Ir indicate 73 and 78% of unordered form in both the proenzyme and the activated species respectively. The slight changes observed on activation are not restricted to C Ir, as comparable results are obtained for proenzyme and activated C Is. C Ir activation appears thus to involve structural changes leading to an 'activated state' distinct from the 'proenzyme state'. Monoclonal antibody to activated C lr is poorly reactive with proenzyme C Ir, a finding that also supports this hypothesis.

Activation of the classical pathway of complement starts at the level of C 1, a Ca2+-dependent complex of subcomponents C lq, C Ir and C Is (Reid & Porter, 1981). CIr, which exists as a dimer in C1, is the first protein of the complement cascade to undergo a covalent modification upon activation: a single proteolytic cleavage in monochain proenzyme C lr leads to activated subunits, each consisting of the resulting two disulphide-linked polypeptide chains and endowed of a specific proteolytic activity exerted on C Is. Recently it has been shown that Cl reconstructed from its purified subcomponents is able to self-activate and that ClInh controls this activation t

Abbreviations used: The nomenclature of complement components is that recommended by the World Health Organisation (1968); activated components are indicated by an overbar; SDS, sodium dodecyl sulphate; iPr2P-F,

di-isopropyl phosphorofluoridate. * To whom reprint requests should be sent at the following postal address: DRF/BMC, CEN/G, 85 X, 38041 Grenoble Cedex, France.

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(Ziccardi, 1 982a,b). Spontaneous activation of purified Clr in the fluid phase has been described previously (Ziccardi & Cooper, 1976; Takahashi et al., 1976; Assimeh et al., 1978; Arlaud et al., 1980; Villiers et al., 1982), whereas trace contaminant extrinsic proteinases were considered to be responsible for the activation of Clr (Dodds et al., 1978; Bauer & Valet, 1981). Inside CI this autocatalytic potential appears to be modulated by the Ca2+-dependent association of Clr with Cls and Clq, as shown by previous reports; the C1r2-C1S2 subcomplex does not selfactivate, whereas in C 1 the presence of C iq leads to the expression of C Ir autocatalytic potential (Lin & Fletcher, 1980). This modulation is reflected by the difference in the activation energy calculated for C 1 r and Cl autoactivation; a value of 188.26 kJ

(45.0kcal)/mol was found for isolated C lr proteolytic activation (Arlaud et al., 1980), whereas a value of 79.82 kJ (19.1 kcal)/mol was calculated for Cl activation (Ziccardi, 1982a). The high value found for isolated C lr might indicate that its

370

activation involves large structural changes. Surface iodination has been used by different laboratories as a tool for investigating these changes (Arlaud et al., 1980; Bauer & Valet, 1981; Villiers et al., 1982); the results do not seem to be in good agreement, but this could be related to the quality of the C Ir used. The present paper reports that autoactivation of isolated C Ir is associated with intrinsic-fluorescence changes, modifications of c.d. spectrum and of antigenic reactivity of the protein. The other serine proteinase of C1, C is, which is devoid of any autocatalytic activity, was used as a reference. Experimental Materials Activated C is and C lr were prepared from human serum as described by Arlaud et al. (1979); proenzyme C Is was purified as described by Arlaud et al. (1980); proenzyme C Ir was prepared either as described by Arlaud et al. (1980) or by Villiers et al. (1982). Purified C ir (C lr) and C is (C is) were estimated from their A280 by using respectively A 1Im = 11.5 and 9.5 (Sim et al., 1977). Molecular weights were taken as 85 000 for monomeric C Ir (C ir) and C ls (C is). Monoclonal antibodies against C ir, prepared from mouse hybridomas, were kindly provided by Dr. Jane Skok (Imperial Cancer Research Fund Laboratory, Lincoln's Inn Fields, London W.C.2,

U.K.). Yeast alcohol dehydrogenase, horse spleen apoferritin and ox liver catalase were obtained from Calbiochem.

Methods Fluorescence studies. Kinetic measurements and fluorescence-emission spectra were obtained at 370C in an Aminco SPF 500 spectrofluorimeter, in the quantum-corrected mode. C.d. measurements. Spectra were recorded in a Jobin-Yvon Dichrographe III. Samples were dialysed against 5mM-Mops (4-morpholinepropanesulphonic acid)/145 mM-NaCl, pH 7.4, and the dialysis fluid was used as a solvent blank. All measurements were performed at 200C under a constant N2 purge. Results were expressed in terms of mean residue ellipticity [OIm.r.w. in units of degrees cm2 dmol-h. From the amino acid composition of C Ir (Arlaud et al., 1982), a mean residue weight of 114 was calculated for Clr and Cir. The percentages of a-helix and «sheet were calculated as described by Chen et al. (1974), using [01m.r.w. values of -9210 degrees . cm2 . dmol for 100% fl-sheet at 216nm and -30000 degrees cm2* dmol for 100% a-helix at 222 nm. Iodination. Lactoperoxidase-catalysed iodination was used to label proteins with 1251 as described by

C. L. Villiers, G. J. Arlaud and M. G. Colomb

Heusser et al. (1973); labelled proteins were kept at 4oC. SDS/polyacrylamide-gel electrophoresis. Samples were reduced and treated as described by Villiers et al. (1982). Gels containing 6% (w/v) acrylamide were prepared by the method of Fairbanks et al. (1971); protein staining with Coomassie Blue and destaining was as described by Weber & Osborn (1969); gels were scanned at 550nm. Sucrose - density -gradient ultracentrifugation. Samples were sedimented at 40C for 15 h (1 f0000g-, rav 9cm), as described by Martin & Ames (1961). After centrifugation, fractions (120,1) were collected from the top of the tubes and their radioactivity was determined in a gamma counter. s2O,w calculation was based on three standards, namely yeast alcohol dehydrogenase (7.6 S), ox liver catalase (11.4S) and horse spleen apoferritin (17.6 S). Results Intrinsic-fluorescence changes upon Cir activation The emission fluorescence spectrum of proenzyme CIr showed a maximum at 333nm (Fig. la); after activation of the same sample for 40min at 370C, the observed maximum was 332nm, with a net increase of fluorescence intensity (Fig. la). Thus activation of Clr was clearly associated with an increase of the intrinsic fluorescence emitted by the protein. The evolution of Clr intrinsic fluorescence upon incubation at 370C is given in Fig. l(b); starting from a minimal value corresponding to C ir entirely in its proenzyme form, the fluorescence increased for a period of 30-45 min and reached a plateau corresponding to a fluorescence increase of 13% over the starting value; at this stage C lr was fully activated, as shown by analysis of the reduced protein on SDS/polyacrylamide-gel electrophoresis. Between the two above limits, the increase in fluorescence was closely correlated with the activation of C Ir, as shown by the superposition of the two curves of evolution (Fig. lb) and a shift of the fluorescence maximum within the 1 nm range reported above (Fig. 1 a). This fluorescence increase was not ,reversible and appeared as a reliable probe of C Ir activation. Factors affecting Cir activation also affect the fluorescence increase Previous observations (Villiers et al., 1982) have shown that activated C ir is able to stimulate the activation of proenzyme C Ir. As shown in Fig. 2, the fluorescence increase observed upon incubation of proenzyme C Ir at 370C was also accelerated by the addition of activated C ir, and the effect varied as a function of the amount of activated C lr added.

1983

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Activation of proenzyme C Ir

Ca2+ has been shown previously to inhibit C Ir autoactivation very efficiently (Ziccardi & Cooper, 1976; Arlaud et al., 1980; Villiers et al., 1982). In the presence of 2.5 mM-Ca2+ the fluorescence of proenzyme C Ir remained stable upon incubation for 15 min at 370C (Fig. 3a), but subsequent addition of 20mM-EGTA led to a fluorescence increase. The fluorescence of activated C 1r was not affected either by Ca2+ or EGTA (results not shown). CiInh has been previously shown to partially inhibit proenzyme Clr activation (Villiers et al., 1982); in the experiments reported in Fig. 3(b), ClInh slowed down the fluorescence increase of Clr incubated at 370C. This increase reached a plateau at 38% of the maximum value reached in the absence of C lInh; in correlation with these findings, only 50% of C Ir was found activated after 2h at 370C as judged from SDS/polyacrylamide-gel electrophoresis of the reduced protein. The slight decrease of fluorescence observed when C lInh reacts with activated C ir (Fig. 3c) cannot account for the above observed effect. iPr2P-F has also been shown to inhibit partially C Ir autoactivation (Villiers et al., 1982); it was also able to inhibit the fluorescence increase on incubation of C lr at 370C.

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Fig. 1. Intrinsic-fluorescence increase upon activation of proenzyme Cl r (a) The emission fluorescence spectra of proenzyme Clr (0.15mg/ml) was recorded in 145mM-NaCl/ 5mM-triethanolamine, pH7.4; Clr was then activated for 40min at 370C, and the fluorescence emission spectra of activated C ir was recorded after adjustment of the temperature of the sample to 200 C. Excitation was at 289 nm, with excitation and emission slits of 2 nm; spectra were recorded at lOnm/min. (b) Clr (0.14mg/ml) was incubated at 370C in 145 mM-NaCI/5 mM-triethanolamine, pH7.4, and the intrinsic-fluorescence emission was measured (excitation at 289 nm, emission at 333 nm with 2nm excitation and emission slits); portions Vol. 215

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Fig. 2. Increase of Clr intrinsic fluorescence upon activation: effect of activated Clr Proenzyme C lr (0.14mg/ml) was incubated at 370C under conditions described in Fig. l(b), in the absence (a) or in the presence of 8% (b) and 26% (c) of activated C lr. Activated C ir alone (0.14mg/ml) was incubated under the same conditions (d). Intrinsic-fluorescence emission was recorded as a function of time as described in Fig. 1 (b).

(50,u1) were withdrawn at various times, then reduced and submitted to SDS/polyacrylamide-gel electrophoresis, in order to evaluate the state of activation of C ir.

372

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Fig. 3. Increase of Clr intrinsic-fluorescence emission upon activation: effect of Ca2+ and ClInh (a) Clr (0.14mg/ml) was incubated at 37°C in 145 mM-NaCI/5 mM-triethanolamine, pH 7.4, containing 2.5 mM-CaCi2. EGTA was added to 20mM as indicated by the arrow. (b) A mixture of C lr (0.14mg/ml) and ClInh (0.14mg/ml) was incubated at 37°C in 145 mM-NaCI/5 mM-triethanolamine, pH 7.4. (c) C Ir (0.14mg/ml) was incubated at 370C in the same medium as in (b). ClInh (0.14mg/ml) was added as indicated by the arrow. Intrinsic-fluorescence emission was recorded as a function of time as described in Fig. 1 (b).

The dependence of the fluorescence variation on pH was investigated, as previous experiments had shown an unusual effect of pH on Clr activation: the activation rate decreases progressively in the acid range (pH4.5-6.5), which coincides with the dissociation of the C Ir dimer, whereas above pH 6.5 the rate increases slightly, without showing any clear maximum (Arlaud et al., 1980). The fluorescence of Clr was found to be stable at pH4.0, whereas at pH 10.5 it increased and reached a plateau after only 15 min, in complete agreement with the activation results.

Specificity of thefluorescence variations observed When incubated at 370C, activated Clr, proenzyme Cls or activated Cis did not show any

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Fig. 4. Variation of intrinsic fluorescence upon incubation of activated Cis and Cir, or activation of proenzyme Cls and Clr All proteins were at a concentration of 0.14mg/ml and incubated at 370C in 145 mM-NaCI/5 mmtriethanolamine, pH 7.4. (a) Activated C is; (b) proenzyme Cls incubated alone, then mixed with activated C ir (7.7,g/ml) as indicated by the arrow; (c) activated C ir; (d) proenzyme C lr. Kinetic variations of intrinsic fluorescence were recorded as described in Fig. l(b). The extent of activation of C Is (b) and Clr (d), at the end of the incubation, was measured by SDS/polyacrylamide-gel electrophoresis as in Fig. 1 (b).

fluorescence variation. Also, incubation of proenzyme C is with trace amounts of activated C 1r at 370C, under conditions which lead progressively to total activation of Cls, did not lead to fluorescence changes (Fig. 4).

C.d. changes upon Cl r activation Far-u.v. c.d. spectra of proenzyme and activated C Ir are shown in Fig. 5. The most significant differences were found in the 212-230nm region, 1983

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Fig. 6. Binding of monoclonal anti-Cir antibody to proenzyme and activated Cl r 1251-labelled proenzyme or activated C Ir (40jug) was incubated with monoclonal anti-C ir antibody (40,ug) for 3h at 0°C in ImM-EDTA/l0OmMNaCl/l0mM-Tris/HCI, pH 8.3. Samples (150,ul) were layered on top of a 5-20% sucrose gradient prepared in the same buffer. Apoferritin (40,g) was added to each sample as an internal standard.

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Fig. 5. Far-u.v. c.d. spectra of proenzyme and activated .Cir The c.d. spectra of proenzyme and activated C Ir were recorded at 200C on the same sample before or after activation for 40min at 370C. 0.1- and 0.5 cmpathlength cuvettes were used between 200 and 230nm and between 220 and 250nm respectively; all other conditions were as described under 'Methods'.

where activation of C Ir resulted in less negative values of ellipticity, and also below 212 nm, where this activation resulted in more negative values. In terms of secondary structure, calculations by the method of Chen et al. (1974) with values of [1m of -3350 and -2875 degrees.cm2-dmol-h at 216 and 222 nm respectively gave the following estimate: pure a-helix, 10%; fl-form, 17%; unordered form, 73%. The same calculation for activated C ir with values of [Ol..,rW. of -3075 and -2562 degrees * cm2* dmol-h at the same wavelengths gave: pure a-helix, 1 1%; fl-form, 1 1%; unordered form, 78%. Activation thus apparently resulted in a net disappearance of fl-form structure without appreciable variation in a-helix content. C.d. spectra of proenzyme and activated C Is also showed differences; calculations indicated amounts of 8% pure a-helix,' 20% f-form and 72% unordered form in .r.w.

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,) 1125i-Cir.

C Is, with corresponding values in activated C is of 6, 12 and 82% respectively.

proenzyme

Interaction between anti-Cir monoclonal antibodies and proenzyme or activated Clr Surface modification of C Ir upon activation was not detected by polyclonal antibodies to activated C ir, which react with both forms of C ir, but could be revealed by a monoclonal antibody purified from mouse ascites fluid. The antibody-antigen interaction was studied by sucrose-gradient ultracentrifugation (Fig. 6), which showed that, when activated Clr (7.1S) was mixed with an equal amount of antibody (7.0 S), as much as 40% of the total activated C ir was bound to the antibody, appearing as an 11.2 S peak, whereas 60% was found as a 7.0-7.1S peak corresponding to proteins that had not reacted. In contrast, proenzyme C Ir (7.1 S) did not react with the antibody under the same conditions. In agreement with these results, separate experiments showed that polyclonal antibodies to Clr were able to block Clr activation, whereas monoclonal anti-C ir antibodies were without effect.

Discussion The intrinsic fluorescence of human C lr increases as a function of its activation and is clearly

374

influenced by factors affecting this activation; activated C lr, which was previously shown to accelerate proenzyme Clr activation (Villiers et al., 1982), also accelerates the fluorescence increase; Ca2+ and C lInh are respectively able to block totally and partially both activation and fluorescence increase; iPr2P-F slows down the activation and fluorescence variations, which are also sensitive to pH; Clr intrinsic fluorescence is stable at pH4.0, most likely due to monomerization of the protein and its subsequent inability to autoactivate (Arlaud et al., 1980), whereas at pH10.5 activation and fluorescence increases proceed faster than at pH 7.4. The increase of C lr intrinsic fluorescence is irreversible; it can be considered as specific for proenzyme C lr, as it is not observed with proenzyme C Is. However, the interesting question is: does the fluorescence increase relate to a precise phase of the activation process?, as this change may precede or follow the proteolytic cleavage of the proenzyme. On the basis of the amino acid sequence of the C ir b-chain (Arlau,d & Gagnon, 1983) and of the activation mechanism of other serine proteinases, it is likely that the formation of the C Ir active site involves an ionic pair between the N-terminal isoleucine residue of the C lr b-chain and the aspartic acid residue at position 190 of this chain (Arlaud & Gagnon, 1983). Such an interaction could bring closer the tryptophan residues located at positions 14 and 205 in the C ir b-chain (Arlaud & Gagnon, 1983), and the coupling of these residues could account for the fluorescence increase observed during C Ir activation. This hypothesis is consistent with the observation that C Is intrinsic fluorescence is stable on activation and that tryptophan-205 is not found at an homologous position in the C is b-chain (Carter et al., 1982). In this view, the increase of intrinsic fluorescence observed during C Ir activation would take place after the proteolytic cleavage and coincide with the formation of the active site. This change would therefore be different from early alteration(s) preceding the proteolytic cleavage, reported by Kasahara et al. (1982), on the basis of extrinsic probe fluorescence. C.d. spectra of proenzyme and activated C ir and C Is indicate low percentages of a-helix and ,florms in these proteins, comparable with other serine proteinases such as chymotrypsin (Chen et al., 1972) or trypsin (Jirgensons, 1970). For C ir, the estimate of a-helix and fl-forms calculated as described by Chou & Fasman (1974) from the complete sequence of the b-chain (Arlaud & Gagnon, 1983) is compatible with the evaluation based on c.d. spectra of the whole molecule. The c.d. variations observed on activation of both C Ir and C Is could be interpreted in terms of secondary-structure changes, but as tryptophan residues are likely to be involved in structural rearrange-

C. L. Villiers, G. J. Arlaud and M. G. Colomb ments, as discussed above for fluorescence, it could not be excluded that tryptophan re-alignments are responsible for the change in c.d. spectra (Adler et al., 1973). However, on the basis of the absence of tryptophan-205 in Cls b-chain, it should be envisaged that tryptophan residues other than those thought to be responsible for the fluorescence variations are involved in the c.d. changes. Our results show that modifications of c.d. previously observed on activation of whole C 1 bound to aggregated immunoglobulin (Tschopp, 1982) are unlikely to be restricted to Clr itself, but probably represent an average variation including at least C Is. Our findings point to two different conformational states for proenzyme and activated Clr, and this is supported by the use of monoclonal antibody, which binds preferentially to activated C Ir and does not recognize the proenzyme form. From the reactivity of polyclonal antibodies with activated Cir, the major antigenic sites have been previously indirectly assigned to the b-chain of this protein (Ziccardi & Cooper, 1978; Arlaud et al., 1980). In contrast, the monoclonal antibody used in the present study appears to react with an antigenic site located in the a-chain of activated C lr, as it interferes with the Ca2+-dependent interaction between Clr and Cis, which is known to involve the a-chain moiety of C lr (Arlaud et al., 1980). Moreover, the monoclonal antibody does not inhibit activation of proenzyme C Ir by activated C Ir. Three different criteria (intrinsic fluorescence, c.d. and antigenic reactivity) point to structural modifications of C lr between its proenzyme and activated states, one of these criteria (intrinsic fluorescence) being likely to be associated with the formation- of the active site. Further studies on sequence analysis of C I r, and crystallization of this protein (Haupt & Baudner, 1981), will help in locating the detailed structural changes associated with its activation. We are indebted to Ms. Jane Skok for generously providing the monoclonal anti-C ir antibodies. The excellent technical assistance of Monique Lacroix is gratefully acknowledged. This work was partly supported by the Fondation pour la Recherche Medicale.

References Adler, A. J., Greenfield, N. J. & Fasman, G. D. (1973) Methods Enzymol. 27, 675-735 Arlaud, G. J. & Gagnon, J. (1983) Biochemistry 22, 1758-1764 Arlaud, G. J., Sim, R. B., Duplaa, A. M. & Colomb, M. G. (1979) Mol. Immunol. 16,445-450 Arlaud, G. J., Villiers, C. L., Chesne, S. & Colomb, M. G. (1980) Biochim. Biophys. Acta 616, 116-129 Arlaud, G. J., Gagnon, J. & Porter, R. R. (1982) Biochem. J. 201, 49-59

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Activation of proenzyme C Ir Assimeh, S. N., Chapuis, R. M. & Isliker, H. (1978) Immunochemistry 15, 13-17 Bauer, J. & Valet, G. (1981) Biochim. Biophys. Acta 670, 129-133 Carter, P. E., Dunbar, B. & Fothergill, J. E. (1982) Biochem. Soc. Trans. 10, 441-442 Chen, Y. H., Yang, J. T. & Martinez, U. M. (1972) Biochemistry 11, 4120-4131 Chen, Y. H., Yang, J. T. & Chau, K. H. (1974) Biochemistry 13, 3350-3359 Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 222-245 Dodds, A. W., Sim, R. B., Porter, R. R. & Kerr, M. A. (1978) Biochem. J. 175, 383-390 Fairbanks, G., Stech, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617 Haupt, H. & Baudner, S. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 1147-1150 Heusser, C., Boesman, M., Nordin, J. H. & Isliker, H. (1973) J. Immunol. 110, 820-828 Jirgensons, B. (1970) Biochim. Biophys. Acta 200, 9-17 Kasahara, Y., Takahashi, K., Nagasawa, S. & Koyama, J. (1982) FEBS Lett. 141, 128-131

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375 Lin, T. Y. & Fletcher, D. S. (1980) J. Biol. Chem. 255, 7756-7762 Martin, R. G. & Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379 Reid, K. B. M. & Porter, R. R. (1981) Annu. Rev. Biochem. 50,433-464 Sim, R. B., Porter, R. R., Reid, K. B. M. & Gigli, I. (1977) Biochem. J. 163, 219-227 Takahashi, K., Nagasawa, S. & Koyama, J. (1976) FEBS Lett. 65, 20-23 Tschopp, J. (1982) Mol. Immunol. 19, 651-657 Villiers, C. L., Duplaa, A. M., Arlaud, G. J. & Colomb, M. G. (1982) Biochim. Biophys. Acta 700, 118-126 Weber, K. & Osborn, M. (1969) J. Bio. Chem. 244, 4406-4412 World Health Organisation (1968) Bull. W.H.O. 39, 935-936 Ziccardi, R. J. (1982a) J. Immunol. 128, 2500-2504 Ziccardi, R. J. (1982b) J. Immunol. 128, 2505-2508 Ziccardi, R. J. & Cooper, N. R. (1976) J. Immunol. 116, 504-509 Ziccardi, R. J. & Cooper, N. R. (1978) J. Immunol. 121, 2148-2152