©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chimeras of Human Complement C9 Reveal the Site Recognized by Complement Regulatory Protein CD59 (*)

(Received for publication, November 8, 1994; and in revised form, December 12, 1994)

Thomas Hüsler (1) Dara H. Lockert (1) Kenneth M. Kaufman (2) James M. Sodetz (2) Peter J. Sims (1)(§)

From the  (1)Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201-2178 and (2)Department of Chemistry and Biochemistry and the School of Medicine, University of South Carolina, Columbia, South Carolina 29208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex, thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective and is most effective toward C9 derived from human or other primate plasma. By contrast, rabbit C9, which can substitute for human C9 in the membrane attack complex, mediates unrestricted lysis of human cells. To identify the peptide segment of human C9 that is recognized by CD59, rabbit C9 cDNA clones were isolated, characterized, and used to construct hybrid cDNAs for expression of full-length human/rabbit C9 chimeras in COS-7 cells. All resulting chimeras were hemolytically active, when tested against chicken erythrocytes bearing C5b-8 complexes. Assays performed in the presence or absence of CD59 revealed that this inhibitor reduced the hemolytic activity of those chimeras containing human C9 sequence between residues 334-415, irrespective of whether the remainder of the protein contained human or rabbit sequence. By contrast, when this segment of C9 contained rabbit sequence, lytic activity was unaffected by CD59. These data establish that human C9 residues 334-415 contain the site recognized by CD59, and they suggest that sequence variability within this segment of C9 is responsible for the observed species-selective inhibitory activity of CD59.


INTRODUCTION

Human CD59 antigen is a 18-21-kDa plasma membrane protein that functions as an inhibitor of the C5b-9 membrane attack complex (MAC) (^1)of human complement(1) . CD59 interacts with both the C8 and C9 components of MAC during its assembly at the cell surface, thereby inhibiting formation of the membrane-inserted C9 homopolymer responsible for MAC cytolytic activity (2, 3) . This serves to protect human blood and vascular cells from injury arising through activation of complement in plasma. CD59's inhibitory activity is dependent upon the species of origin of C8 and C9, with greatest inhibitory activity observed when C9 is from human or other primates. By contrast, CD59 exerts little or no inhibitory activity toward C8 or C9 of most other species, including rabbit (4, 5, 6) . Because the activity of CD59 is largely restricted to regulating human C9, and the activity of analogous complement inhibitors expressed by cells of other species is likewise generally selective for homologous C9, xenotypic cells and tissue are particularly susceptible to complement-mediated destruction due to unregulated activity of MAC. This phenomenon underlies hyperacute immune rejection after xenotransplantation(7) .

Analysis of the physical association of CD59 with components of MAC suggested that separate binding sites for CD59 are contained within the alpha-chain of human C8 and within human C9(8) . Within C9, this site(s) appears to reside in the C-terminal ``C9b'' fragment obtained by cleavage with alpha-thrombin (residues 245-538)(8, 9) . Whereas CD59 binds to human C9 and to peptides derived from human C9, such binding is not detected with rabbit C9, consistent with the apparently unrestricted lytic activity of rabbit complement toward human cells containing CD59(4, 5, 6, 8) . Unresolved is whether the human C9-derived peptide fragments that bind CD59 actually correspond to the site in the intact protein through which CD59's inhibitory function is exerted during MAC assembly at the cell surface. The capacity of rabbit C9 to functionally substitute for human C9 within MAC, but not be recognized by the inhibitor CD59, suggested that hemolytically active C9 chimeras containing human and rabbit sequence would permit experimental mapping of the residues within human C9 that confer its particular sensitivity to inhibition by membrane CD59.


EXPERIMENTAL PROCEDURES

Materials

Human complement proteins C5b6, C7, C8, and C9 and human erythrocyte membrane glycoprotein CD59 were purified and assayed as described previously(3, 10, 11) . Full-length cDNA for human C9 in the vector pSVL was a generous gift from Dr. J. Tschopp (University of Lausanne, Epalinges, Switzerland)(12) . Chicken erythrocytes (chE) were from Cocalico Biologics, Inc. (Reamstown, PA), COS-7 cells were from American Tissue Culture Collection (Rockville, MD), and Escherichia coli strain JS5 was from Bio-Rad. Dulbecco's modified Eagle's medium was from Mediatech Inc. (Herndon, VA), Opti-MEM I was from Life Technologies, Inc., and heat-inactivated fetal calf serum was from Biocell (Rancho Dominguez, CA). Oligonucleotides were synthesized by the Molecular Biology Core Laboratories, Blood Research Institute.

Solutions

MBS consisted of 150 mM NaCl, 10 mM MOPS, pH 7.4. GVBS consisted of 150 mM NaCl, 3.3 mM sodium barbital, 0.1% (w/v) gelatin, pH 7.4. GVBE consisted of 150 mM NaCl, 3.3 mM sodium barbital, 0.1% (w/v) gelatin, pH 7.4.

Isolation of Rabbit C9 cDNA Clones

A 1.8-kilobase pair XbaI-SacI fragment from human C9 cDNA was used to screen a rabbit liver cDNA library(13) . Nucleotide sequence (Sequenase kit, U. S. Biochemical Corp.) was obtained from both strands of a single full-length 2034-base pair clone. N-terminal amino sequencing of the mature protein purified from rabbit plasma was performed by the Protein Microanalysis Core Facility of the University of South Carolina.

Construction of Chimeric C9 cDNAs

Chimeric constructs were designed by aligning the amino acid sequences of rabbit and human C9 using the PALIGN program (PC Gene). Based on this alignment, polymerase chain reaction was performed to generate defined segments of rabbit and human C9 cDNAs. Primers corresponding to 5` or 3` untranslated sequence with XbaI (5` end) or SacI (3` end) recognition sites were paired with chimeric primers (29-39 base pairs) and used to generate cDNA fragments that contained the desired overlapping sequence at either 5` or 3` ends. These fragments were purified, mixed, and used with primers located in the 5` or 3` untranslated region to produce full-length chimeric C9 cDNAs. Fragments were cloned into the XbaI/SacI sites of pSVL for sequencing and expression. Polymerase chain reaction fidelity was confirmed by sequencing 3` coding sequence in each construct starting from the stop codon and continuing through all junctions of rabbit and human sequence.

Transfection of COS-7 Cells

Plasmids were propagated in E. coli JS5. Plasmid DNA was isolated using Qiagen tips (Qiagen Inc., Chatsworth, CA), and used with or without further purification over a CsCl gradient. COS-7 cells were transfected using DEAE-dextran. After transfection, cells were grown for 24 h in Dulbecco's modified Eagle's medium (Mediatech Inc., Herndon, VA) supplemented with 10% fetal bovine calf serum, after which this medium was replaced by Opti-MEM I (Life Technologies, Inc.). Cell supernatants were harvested after 48-65 h, phenylmethylsulfonyl fluoride (1 mM), benzamidine (1 mM) and EDTA (10 mM) were added, and the supernatants concentrated at 4 °C (Centricon 30, Amicon). Aggregated protein in the concentrated supernatants was removed by centrifugation (100,000 times g, 1 h), and the supernatants were used for assay within 48 h.

Immunoblotting

C9 in the COS-7 supernatants was analyzed by Western blotting and quantitative dot blotting using murine monoclonal antibody P9-2T recognizing an epitope common to the C9a domains of human and rabbit C9. (^2)To quantitate C9, dilutions of each supernatant were adsorbed onto nitrocellulose (50 µl/well), the fatty acid- and globulin-free bovine serum albumin-blocked membranes incubated with 5 µg/ml I-labeled P9-2T (1,000 cpm/ng), and specifically bound radioactivity quantitated by photon counting (AMBIS 4000). C9 concentration in each transfected COS-7 supernatant was determined by reference to standard curves obtained with known quantities of plasma-derived human or rabbit C9, added to COS-7-conditioned medium. These experiments confirmed that antibody P9-2T identically quantitated rabbit and human C9 antigen present in the COS-7 supernatants (not shown). For Western blotting, each COS-7 supernatant was denatured by heating (100 °C, 1 min) in 2% (w/v) SDS under non-reducing conditions, proteins separated by 8% polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the fatty acid- and globulin-free bovine serum albumin-blocked membranes probed with either 5 µg/ml P9-2T, or 10 µg/ml of goat IgG against human C9. Human and rabbit C9 from plasma served as standards and the supernatants of COS-7 transfectants containing circular pSVL (without insert) served as a nonspecific control. After washing, membranes were developed by incubation with alkaline phosphatase conjugates of the appropriate secondary antibody (against mouse or goat IgG).

Protein Radiolabeling

Monoclonal anti-C9 antibody P9-2T was radioiodinated to a specific radioactivity of 1,000 cpm/ng with IODO-GEN (Pierce).

Analysis of the Inhibitory Function of CD59 toward C9 Chimeras

Hemolytic activity of each chimera was assayed using as target cells chE that were reconstituted with purified human CD59. chE were washed extensively and suspended in GVBS, and the membrane C5b67 complex assembled by mixing cells (1.4 times 10^9/ml final concentration) with C5b6 (13 µg/ml final concentration) followed by addition of C7 (1 µg/ml). After 10 min, the C5b67 chE were diluted to 1.4 times 10^8/ml in GVBE and incubated (10 min, 37 °C) with 0 or 400 ng/ml CD59. In each case, the final concentration of Nonidet P40 was 0.0005% (v/v). After washing in ice-cold GVBE, 2.8 times 10^6 of these cells were incubated (37 °C) in a total volume of 100 µl with 1 ng of rabbit C8 plus 0-50 ng of recombinant C9 (human, rabbit, or chimeric), serially diluted in Opti-MEM I. Hemolysis was determined after 30 min at 37 °C, with correction for nonspecific lysis in the absence of C9.


RESULTS AND DISCUSSION

To undertake construction of human/rabbit C9 chimeras, it was necessary to first isolate and sequence a cDNA encoding rabbit C9. A full-length clone was characterized and found to contain a putative leader sequence of 21 amino acids, a deduced N-terminal sequence (GPTPSYVHEPIQR) that was identical to that of C9 purified from rabbit plasma, a translation termination codon, and a 3` poly(A) tail. The cDNA sequence predicts that mature rabbit C9 contains 536 residues and exhibits 72% identity (78% similarity) to human C9, including conservation of all cysteines.

Human C9, rabbit C9, and various chimeras containing human and rabbit C9 sequence were produced by cDNA expression in COS-7 cells. Western blotting (not shown) indicated that each recombinant protein secreted from the C9-transfected COS-7 cells conformed to the expected molecular weight of C9 isolated from human or rabbit plasma. As illustrated in Fig. 1, the lytic activity of full-length recombinant human C9 (panel C) was markedly inhibited by CD59, whereas that of full-length recombinant rabbit C9 (panelD) was virtually unaffected, consistent with the species-selective regulation by CD59 observed for the corresponding plasma-derived proteins (panels A and B). We next analyzed human/rabbit C9 chimeras in which segments that approximate the alpha-thrombin-derived C9b domain (245-538 in human C9) were interchanged (panels E and F). Both chimeras expressed similar hemolytic activity to that of human and rabbit C9, measured in the absence of CD59. In the presence of CD59, the chimera containing rabbit N-terminal and human C-terminal sequence (panel E) was inhibited to the same extent as recombinant or plasma-derived human C9. By contrast, a C9 chimera containing human N-terminal sequence and rabbit C-terminal sequence (panel F) shared the property of recombinant or plasma-derived rabbit C9, exhibiting hemolytic activity that was virtually unaffected by CD59. These data imply that CD59 inhibits MAC lytic function by recognizing a site between residues 268-538 in human C9, consistent with observed binding of CD59 to the alpha-thrombin-derived C9b fragment (residues 245-538), but not the C9a fragment (residues 1-244) of human C9(8, 9) .


Figure 1: Effect of CD59 on hemolytic activity of recombinant C9. Hemolytic activity of human, rabbit, and human/rabbit chimeras of C9 measured in the absence (down triangle) or presence () of cell-surface CD59. The concentration of each recombinant C9 (abscissa) was determined by quantitative dot blotting using a monoclonal antibody against an epitope common to human and rabbit C9, and the hemolytic activity of each (ordinate) was determined using C5b-8 chE target cells reconstituted with human CD59 (see ``Experimental Procedures``). Results shown are for human plasma-derived C9 (panel A), rabbit plasma-derived C9 (panel B), full-length recombinant human C9 (H1-538; panel C), full-length recombinant rabbit C9 (R1-536; panel D), or chimeric C9s containing rabbit residues R1-272 joined to human residues H268-538 (panel E) or human residues H1-267 joined to rabbit residues R273-536 (panel F). Results shown are from a single experiment and are representative of results obtained in at least six separate experiments.



Earlier analysis of the binding of CD59 to synthetic peptides suggested two possible binding sites for CD59 in human C9: residues 247-261 within the proposed hinge domain (14) and a site between residues 359-411(9) . Accordingly, we next analyzed the capacity of CD59 to inhibit the lytic function of C9 chimeras containing either rabbit or human sequence in these regions. These chimeras were analyzed in multiple hemolytic titrations performed under the conditions of Fig. 1, and the cumulative data for each are summarized in Fig. 2. Inspection of these data reveals that the inhibitory activity of CD59 was unrelated to the presence of either human or rabbit sequence in the hinge domain of C9 (cf. constructs 1 and 5 and constructs 2 and 6), suggesting that this segment of C9 does not contribute to the inhibitory interaction of CD59 with human MAC (discussed below). These data also reveal that inhibition of lytic activity by CD59 was observed only for those C9 chimeras retaining human residues 334-415, irrespective of whether the remainder of the protein contained rabbit or human sequence (cf. constructs 7-11). Furthermore, since the inhibitory activity of CD59 toward a C9 chimera containing human residues 334-415 flanked by only rabbit sequence (construct 11; Fig. 2) was indistinguishable from that observed for human C9, we suggest that this segment is the only segment that is recognized by CD59. Consistent with this conclusion, exchanging rabbit sequence for residues 334-415 of human C9 (construct 10; Fig. 2), yielded a chimera that like rabbit C9, was unaffected by CD59, even though it retained 85% of human C9 sequence.


Figure 2: Inhibitory function of CD59 requires human C9 residues 334-415. Bar graph (right panel) summarizes combined results of all experiments performed under conditions of Fig. 1. From each C9 titration, the inhibitory activity of CD59 (expressed as the percent inhibition of hemolysis due to CD59, ordinate) was calculated at the concentration of C9 resulting in 50% hemolysis in the absence of CD59. Error bars denote mean + S.D.; parentheses indicate number of independent experiments; asterisks indicate significance (p < 0.02) compared to rabbit C9 from plasma. To the left of each data bar, the protein tested is depicted so as to designate those portions containing human (open) or rabbit (shaded) C9 sequence. Human C9 and rabbit C9 denote C9 purified from human and rabbit plasma, respectively. Recombinant C9 proteins (designated 1-11) contain human (H) or rabbit (R) sequence numbered according to the deduced mature primary structure of human and rabbit C9. In some chimeras, numbering appears discontinuous because of gaps in the alignment of the rabbit and human C9 sequences: 1, H1-538; 2, R1-536; 3, H1-267R273-536; 4, R1-272H268-538; 5, H1-226R228-272H268-538; 6, R1-227H227-267R273-536; 7, H1-333R339-536; 8, R1-338H334-538; 9, H1-415R425-536; 10, H1-333R339-424H416-538; 11, R1-338H334-415R425-536. Domains depicted in the proposed structure of C9 include thrombospondin type 1 (TS), LDL-receptor (LDLR), hinge (Hinge), membrane binding (MB), and epidermal growth factor precursor (EGFP). Domain structure of C9 was adapted from (23) . , due to low recovery, results for this construct were determined at 10% hemolysis.



Comparison of the amino acid sequence of human C9 to the two other mammalian C9s for which sequence is now available (Fig. 3) reveals two prominent regions of sequence divergence: one between human C9 residues 230 and 270, which contains the putative hinge domain (residues 228-271) of the protein (14, 15) ; and one between residues 360 and 390, a segment of unknown function. The portion of human C9 that we now identify as the CD59 recognition domain encompasses this second segment of highly divergent sequence. Although our preliminary analysis suggests that most of the interaction of CD59 with human C9 is mediated through recognition of sequence contained between residues 360-390, our data also suggest a contribution of more highly conserved flanking residues to full expression of the CD59 binding site in human C9(9) .^2 Sequence variability in this segment of C9 comprising the CD59 recognition site provides an explanation for the species-selectivity that is characteristic of CD59's MAC inhibitory function, and for why the lytic activity of human, but not rabbit C9, is restricted toward cells that express human CD59.(5)


Figure 3: Sequence comparison of mammalian C9s. Human C9 amino acid sequence was aligned with rabbit or mouse C9 using the Gap program of the University of Wisconsin Genetics Computer Group. At each position, a similarity score was generated using the Plotsimilarity program with a window = 20. The average similarity across the alignment is indicated by a dashed line. Brackets define residues 334-415 in human C9, which correspond to 339-424 in the aligned sequence of rabbit C9 (upper panel) or 333-418 in mouse C9 (lower panel).



As noted above, there is also marked sequence variability in the segment of C9 corresponding to the hinge domain. It has been proposed that CD59 interrupts MAC assembly and inhibits complement lysis by binding to this segment of C9, based on evidence that a small synthetic peptide (residues 247-261) derived from the hinge domain exhibits binding affinity for CD59 and increases MAC-induced hemolysis of human erythrocytes(14) . As previously reported, we were unable to detect specific binding of CD59 to recombinant fusion proteins or to peptides containing sequence derived from the hinge domain of human C9, whereas such binding was observed to fusion proteins containing human C9 sequence spanning residues 320-415(9) . Importantly, this latter study also found that the enhanced lytic activity of MAC observed in the presence of hinge domain peptide 247-261 was unrelated to interaction with CD59, but instead reflected a direct membrane-perturbing effect of the peptide itself(9) . Combined with the current data (Fig. 2; constructs 5 and 6), we conclude that the species-selective MAC inhibitory function of CD59 is not related to sequence unique to the hinge domain of human C9.

CD59 is also known to interact with human C8alpha, a polypeptide that exhibits sequence similarity to human C9 and to several other components of MAC(8, 13) . Although the segment of human C8alpha recognized by CD59 has not been experimentally determined, it has been proposed that residues 349-385 might contain this site, based on the different susceptibility of human and rabbit C8 to inhibition by CD59 and the particularly low conservation of sequence in this region when the two species are compared(13) . It is of interest to note that from the aligned sequences of human C8alpha and human C9, the proposed site in C8alpha corresponds to C9 residues 359-398, which are contained in the segment of C9 that has now experimentally been shown to encompass the CD59 recognition domain.

CD59 plays a key role in protecting blood cells from complement in plasma. Its deletion from blood cells in paroxysmal nocturnal hemoglobinuria results in episodic intravascular hemolysis due to unregulated incorporation of C9 into MAC(16) . Recent identification of analogous proteins with homologous C9 regulatory function on erythrocytes of other species(17, 18) , and of MAC-inhibitors expressed by pathogenic microorganisms that share structural and functional features with CD59(19, 20, 21) , underscores the selective advantage conferred by inhibiting MAC directly at the cell surface. Insight into how CD59's interaction with that portion of C9 contained between residues 334-415 confers its MAC inhibitory function awaits further information as to how this region participates in the conformational transitions required to convert plasma C9 into a membrane-inserted protein. Interestingly, the segment recognized by CD59 is adjacent within the primary structure to the proposed membrane-interacting domain in C9 (residues 292-333; see Fig. 2)(22) . Whereas this domain is presumed buried in the conformer of C9 found in plasma, binding to membrane C5b-8 is thought to initiate unfolding so as to expose this domain for direct interaction with membrane lipid (15, 22) . By binding next to this domain, CD59 might prevent its exposure and thereby interrupt C9 insertion into the membrane.


FOOTNOTES

*
This work was supported by Grants HL36061 (to P. J. S.) and GM42898 (to J. M. S.) from the National Institutes of Health. A preliminary report of these data was presented to the 36th Annual Meeting of the American Society of Hematology, Dec. 2-6, 1994, Nashville, TN, and in abstract form (Hüsler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., and Sims, P. J.(1994) Blood84 (Suppl. 1), 235a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U2005.

§
To whom correspondence should be addressed: Blood Center of Southeastern Wisconsin, P. O. Box 2178, Milwaukee, WI 53201-2178. Tel.: 414-937-3850; Fax: 414-937-6284.

(^1)
The abbreviations used are: MAC, membrane attack complex; chE, chicken erythrocytes; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
T. Hüsler, D. H. Lockert, and P. J. Sims, unpublished data.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Diana Schick, Lilin Li, and Randal Orchekowski.


REFERENCES

  1. Davies, A., and Lachmann, P. J. (1993) Immunol. Res. 12, 258-275 [Medline] [Order article via Infotrieve]
  2. Meri, S., Morgan, B. P., Davies, A., Daniels, R. H., Olavesen, M. G., Waldmann, H., and Lachmann, P. J. (1990) Immunology 71, 1-9 [Medline] [Order article via Infotrieve]
  3. Rollins, S. A., and Sims, P. J. (1990) J. Immunol. 144, 3478-3483 [Abstract/Free Full Text]
  4. Okada, N., Harada, R., Fujita, T., and Okada, H. (1989) Int. Immunol. 1, 205-208 [Medline] [Order article via Infotrieve]
  5. Rollins, S. A., Zhao, J., Ninomiya, H., and Sims, P. J. (1991) J. Immunol. 146, 2345-2351 [Abstract/Free Full Text]
  6. Zhao, J., Rollins, S. A., Maher, S. E., Bothwell, A. L., and Sims, P. J. (1991) J. Biol. Chem. 266, 13418-13422 [Abstract/Free Full Text]
  7. Dalmasso, A. P. (1992) Immunopharmacology 24, 149-160 [CrossRef][Medline] [Order article via Infotrieve]
  8. Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680 [Abstract/Free Full Text]
  9. Chang, C.-P., Hüsler, T., Zhao, J., Wiedmer, T., and Sims, P. J. (1994) J. Biol. Chem. 269, 26424-26430 [Abstract/Free Full Text]
  10. Wiedmer, T., and Sims, P. J. (1985) J. Membr. Biol. 84, 249-258 [Medline] [Order article via Infotrieve]
  11. Wiedmer, T., and Sims, P. J. (1985) J. Biol. Chem. 260, 8014-8019 [Abstract/Free Full Text]
  12. Dupuis, M., Peitsch, M. C., Hamann, U., Stanley, K. K., and Tschopp, J. (1993) Mol. Immunol. 30, 95-100 [Medline] [Order article via Infotrieve]
  13. White, R. V., Kaufman, K. M., Letson, C. S., Platteborze, P. L., and Sodetz, J. M. (1994) J. Immunol. 152, 2501-2509 [Abstract/Free Full Text]
  14. Tomlinson, S., Whitlow, M. B., and Nussenzweig, V. (1994) J. Immunol. 152, 1927-1934 [Abstract/Free Full Text]
  15. Stanley, K. K. (1989) Curr. Top. Microbiol. Immunol. 140, 49-65 [Medline] [Order article via Infotrieve]
  16. Rosse, W. F. (1992) Curr. Top. Microbiol. Immunol. 178, 163-173 [Medline] [Order article via Infotrieve]
  17. Hughes, T. R., Piddlesden, S. J., Williams, J. D., Harrison, R. A., and Morgan, B. P. (1992) Biochem. J. 284, 169-176 [Medline] [Order article via Infotrieve]
  18. van den Berg, C. W., Harrison, R. A., and Morgan, B. P. (1993) Immunology 78, 349-357 [Medline] [Order article via Infotrieve]
  19. Braga, L. L., Ninomiya, H., McCoy, J. J., Eacker, S., Wiedmer, T., Pham, C., Wood, S., Sims, P. J., and Petri, W. A., Jr. (1992) J. Clin. Invest. 90, 1131-1137 [Medline] [Order article via Infotrieve]
  20. Rother, R. P., Rollins, S. A., Fodor, W. L., Albrecht, J.-C., Setter, E., Fleckenstein, B., and Squinto, S. P. (1994) J. Virol. 68, 730-737 [Abstract]
  21. Parizade, M., Arnon, R., Lachmann, P. J., and Fishelson, Z. (1994) J. Exp. Med. 179, 1625 [Abstract]
  22. Peitsch, M. C., Amiguet, P., Guy, R., Brunner, J., Maizel, J. V., Jr., and Tschopp, J. (1990) Mol. Immunol. 27, 589-602 [CrossRef][Medline] [Order article via Infotrieve]
  23. Esser, A. F. (1994) Toxicology 87, 229-247 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.