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B Paul Morgan

Publications and source records attributed to B Paul Morgan.

At least 19 recordsLinked to original sources
PubMed2003

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites.

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

ADAM Proteins↗
PubMed2003

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59.

Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59, but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.

Antibodies, Monoclonal↗
PubMed2003

Complement therapeutics; history and current progress.

Complement (C) performs vital roles in immune surveillance, from killing of bacteria to generation of an optimal antibody response. However, the mediators responsible for this protective role can inappropriately target self tissues and cause pathology in many inflammatory diseases, in ischaemia-reperfusion injuries and also as a result of therapeutic intervention, such as in cardiopulmonary bypass. Here we review the history of anti-complement therapeutics and describe the plethora of reagents that have evolved to treat complement-mediated pathologies. These agents range from small compounds, including natural products isolated from plants and synthetic peptides designed to target and inhibit the complement cascade, to large, intricately engineered biological reagents. Recombinant, humanised antibody fragments which inhibit at specific points in the complement cascade have been generated and used successfully in man. Other reagents, mimicking the action of the natural complement regulatory proteins present on the surface of self cells, have also been developed and extensively tested. We discuss the pros and cons of these different reagents and describe recent advances in the field, such as specific targeting of drugs to sites of inflammation, which have opened the door to the use of anti-complement therapy in both acute and chronic inflammatory conditions.

Animals↗
PubMed2003

Beyond lysis: how complement influences cell fate.

Complement is a central component of the innate immune system involved in protection against pathogens. For many years, complement has been known to cause death of targets, either indirectly by attracting and activating phagocytes or directly by formation of a membrane pore, the membrane attack complex. More recently, it has been recognized that complement may cause other 'non-classical' effects that may not directly be aimed at killing of pathogens. Products of complement activation collaborate with the adaptive immune system to enhance responses to antigens. The membrane attack complex of complement, apart from lysing cells, can also trigger diverse events in target cells that include cell activation, proliferation, resistance to subsequent complement attack and either resistance to, or induction of, apoptosis. Various complement products play important roles in signalling for clearance by phagocytes of apoptotic self cells. Here we review some of these non-classical activities of complement and stress the roles that they may play in maintaining the integrity of the organism.

Apoptosis↗
PubMed2003

An abnormal but functionally active complement component C9 protein found in an Irish family with subtotal C9 deficiency.

Two independently segregating C9 genetic defects have previously been reported in two siblings in an Irish family with subtotal C9 deficiency. One defect would lead to an abnormal C9 protein, with replacement of a cysteine by a glycine (C98G). The second defect is a premature stop codon at amino acid 406 which would lead to a truncated C9. However, at least one of two abnormal proteins was present in the circulation of the proband at 0.2% of normal C9 concentration. In this study, the abnormal protein was shown to have a molecular weight approximately equal to that of normal C9, and to carry the binding site for monoclonal antibody (mAb) Mc42 which is known to react with an epitope at amino acid positions 412-426, distal to 406. Therefore, the subtotal C9 protein carries the C98G defect. The protein was incorporated into the terminal complement complex, and was active in haemolytic, bactericidal and lipopolysaccharide release assays. A quantitative haemolytic assay indicated even slightly greater haemolytic efficiency than normal C9. Epitope mapping with six antihuman C9 mAbs showed the abnormal protein to react to these antibodies in the same way as normal C9. However, none of these mAbs have epitopes within the lipoprotein receptor A module, where the C98G defect is located. The role of this region in C9 functionality is still unclear. In conclusion, we have shown that the lack of a cysteine led to the production of a protein present in the circulation at very much reduced levels, but which was fully functionally active.

Aged↗
PubMed2003

Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison.

CD59, the sole membrane regulator of the membrane attack complex of complement, is broadly and abundantly expressed in man and other mammals. In mouse, CD59 is encoded by two homologous genes. The expression patterns and functional roles of the proteins encoded by these genes, mCD59a and mCD59b, have not been well characterized. Here we describe the generation of monoclonal and polyclonal antibodies detecting specifically mCD59a and mCD59b. These reagents have been used to study function and to ascertain the cell and tissue distributions of mCD59a and mCD59b. mCD59a was broadly distributed on endothelia, erythrocytes, platelets, and on numerous other cell types in organs, a distribution pattern resembling that of CD59 in other species. In marked contrast, expression of mCD59b was restricted to germ cell elements in the testis and mature spermatozoa. Both mCD59a and CD59b inhibited human and rodent complement with similar efficiency. These findings demonstrate that the broadly distributed mCD59a is the key regulator of the terminal complement pathway in mice whereas CD59b, expressed only in testis and on sperm, probably plays other roles in vivo.

Animals↗
PubMed2003

CD59a deficiency exacerbates accelerated nephrotoxic nephritis in mice.

CD59 is a complement regulatory protein that inhibits the terminal part of the complement system, the membrane attack complex (MAC), a mediator of renal injury. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in experimentally induced accelerated nephrotoxic nephritis, a model of immune complex-mediated glomerulonephritis. After accelerated nephrotoxic nephritis was induced by administration of sheep nephrotoxic globulin, mCd59a-/- mice and strain-matched controls on two genetic backgrounds, 129/Sv x C57BL/6 and 129/Sv, were examined. For both, mCd59a-/- mice developed significantly greater glomerular cellularity than wild-type (WT) mice at day 5 after administration. At day 10 post-administration, mCd59a-/- mice exhibited more glomerular thrombosis than WT mice (thrombosis score, 1.8 [range, 1.4 to 4.0] versus 0.8 [range, 0.2 to 1.5] quadrants thrombosed per glomerulus, respectively; P = 0.0006). In the majority of experiments, mCd59a-/- mice also had significantly more proteinuria than controls; however, there was no difference in serum creatinine or albumin. Quantitative immunofluorescence of kidney sections revealed significantly more C9 (as a marker of MAC) deposition within glomeruli of mCd59a-/- mice than WT controls (P < 0.001). There was no difference in deposition of C3 and sheep IgG between the two experimental groups. The lack of CD59a, by allowing unregulated MAC deposition, exacerbates the renal injury in this model of immune complex-mediated glomerulonephritis.

Animals↗
PubMed2003

Complement regulation by Kaposi's sarcoma-associated herpesvirus ORF4 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three types of human tumor: Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. The virus encodes a number of proteins that participate in disrupting the immune response, one of which was predicted by sequence analysis to be encoded by open reading frame 4 (ORF4). The predicted ORF4 protein shares homology with cellular proteins referred to as regulators of complement activation. In the present study, the transcription profile of the ORF4 gene was characterized, revealing that it encodes at least three transcripts, by alternative splicing mechanisms, and three protein isoforms. Functional studies revealed that each ORF4 protein isoform inhibits complement and retains a C-terminal transmembrane domain. Consistent with the complement-regulating activity, we propose to name the proteins encoded by the ORF4 gene collectively as KSHV complement control protein (KCP). KSHV ORF4 is the most complex alternatively spliced gene encoding a viral complement regulator described to date. KCP inhibits the complement component of the innate immune response, thereby possibly contributing to the in vivo persistence and pathogenesis of this virus.

Alternative Splicing↗
PubMed2002

Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions.

Monoclonal antibody production (mAb) first requires the availability of large amounts of pure immunogen for animal immunisation and fusion screening procedures. To overcome this obstacle, we have developed a simple method for rapid generation of pure antigen by generation of recombinant protein containing the antigen of interest fused to the hinge and Fc domains of human immunoglobulin (Ig). The Fc domain forms a convenient 'tag' to enable detection of the protein in supernatant of transfected cells and for purification of immunogen by protein A affinity chromatography. The only requirement for immunogen preparation using this methodology is that a DNA sequence encoding a portion of the molecule of interest is known and that a suitable PCR template is available. Antibody production can be tailored to specific protein domains, for example functional domains, by expressing solely those domains in the fusion protein. We illustrate the technique with two different fusions used to raise antibodies against the porcine and human analogues of a complement (C) regulatory protein, decay accelerating factor (DAF) (CD55). Use of the specific Ig-fusion protein and a control protein facilitated screening of fusions by ELISA. We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains. Low levels of expression required subcloning into a high expression vector and resulted in yields of fusion protein at between 2 and 10 mg per litre of supernatant. The second expression system utilised the high expression vector directly, Ig domains of the chosen immunoglobulin isotype were amplified from peripheral blood mononuclear cell (PBMC) RNA and ligated into the vector in frame with DNA encoding the antigen. We describe potential pitfalls that may be encountered while using Ig fusion proteins as immunogen and demonstrate ways in which to tailor their design for optimal mAb production.

Animals↗
PubMed2002

Expression of human complement receptor type 2 (CD21) in mice during early B cell development results in a reduction in mature B cells and hypogammaglobulinemia.

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43(+)CD25(-) late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220(int)CD43(+)CD25(-) B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.

Agammaglobulinemia↗
PubMed2002

Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts.

Despite the clinical success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about its mechanism of action. Here we show that the ability of mAbs to translocate CD20 into low-density, detergent-insoluble membrane rafts appears to control how effectively they mediate complement lysis of lymphoma cells. In vitro studies using a panel of anti-B-cell mAbs revealed that the anti-CD20 mAbs, with one exception (B1), are unusually effective at recruiting human complement. Differences in complement recruitment could not be explained by the level of mAb binding or isotype but did correlate with the redistribution of CD20 in the cell membrane following mAb ligation. Membrane fractionation confirmed that B1, unlike 1F5 and rituximab, was unable to translocate CD20 into lipid rafts. In addition, we were able to drive B1 and a range of other anti-B-cell mAbs into a detergent-insoluble fraction of the cell by hyper-cross-linking with an F(ab')(2) anti-Ig Ab, a treatment that also conferred the ability to activate lytic complement. Thus, we have shown that an important mAb effector function appears to be controlled by movement of the target molecule into membrane rafts, either because a raft location favors complement activation by mAbs or because rafts are more sensitive to complement penetration.

Antibodies, Monoclonal↗
PubMed2002

Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind C1q.

It has been suggested that the human C1qRp is a receptor for the complement component C1q; however, there is no direct evidence for an interaction between C1q and C1qRp. In this study, we demonstrate that C1q does not show enhanced binding to C1qRp-transfected cells compared with control cells. Furthermore, a soluble recombinant C1qRp-Fc chimera failed to interact with immobilized C1q. The proposed role of C1qRp in the phagocytic response in vivo is also unsupported in that we demonstrate that this molecule is not expressed by macrophages in a variety of human tissues and the predominant site of expression is on endothelial cells. Studies on the rodent homolog of C1qRp, known as AA4, have suggested that this molecule may function as an intercellular adhesion molecule. Here we show that C1qRp is the Ag recognized by several previously described mAbs, mNI-11 and two anti-CD93 Abs (clones X2 and VIMD2b). Interestingly, mNI-11 (Fab') has been shown to promote monocyte-monocyte and monocyte-endothelial cell adhesive interactions. We produced a recombinant C1qRp-Fc chimera containing the C-type lectin-like domain of C1qRp and found specific binding to vascular endothelial cells in sections of inflamed human tonsil, indicating the presence of a C1qRp ligand at this site. This interaction was Ca(2+) independent and was not blocked by our anti-C1qRp mAb BIIG-4, but was blocked by the proadhesive mAb mNI-11. Collectively, these data indicate that C1qRp is not a receptor for C1q, and they support the emerging role of C1qRp (here renamed CD93) in functions relevant to intercellular adhesion.

Animals↗
PubMed2002

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.

Animals↗
PubMed2002

The membrane attack complex of complement causes severe demyelination associated with acute axonal injury.

Complement is implicated in pathology in the human demyelinating disease multiple sclerosis and in animal models that mimic the demyelination seen in multiple sclerosis. However, the components of the complement system responsible for demyelination in vivo remain unidentified. In this study, we show that C6-deficient (C6-) PVG/c rats, unable to form the membrane attack complex (MAC), exhibit no demyelination and significantly reduced clinical score in the Ab-mediated experimental autoimmune encephalomyelitis model when compared with matched C6-sufficient (C6+) rats. In C6+ rats, perivenous demyelination appeared, accompanied by abundant mononuclear cell infiltration and axonal injury. Neither demyelination nor axonal damage was seen in C6- rats, whereas levels of mononuclear cell infiltration were equivalent to those seen in C6+ rats. Reconstitution of C6 to C6- rats yielded pathology and clinical disease indistinguishable from that in C6+ rats. We conclude that demyelination and axonal damage occur in the presence of Ab and require activation of the entire complement cascade, including MAC deposition. In the absence of MAC deposition, complement activation leading to opsonization and generation of the anaphylatoxins C5a and C3a is insufficient to initiate demyelination.

Animals↗
PubMed2002

Rat T cells express neither CD55 nor CD59 and are dependent on Crry for protection from homologous complement.

All human blood cells express decay-accelerating factor (DAF, CD55), CD59, and, with the exception of erythrocytes, membrane cofactor protein (MCP, CD46) to protect themselves from damage by the constant low-level activation of complement in serum. In rats and mice MCP is expressed only in testis, whereas DAF and CD59 are broadly distributed. Rats and mice also express a unique complement regulator, Crry. Previously we have shown that DAF was absent from at least 75% of rat T cells. To further investigate this surprising finding, we assessed the expression levels of DAF, CD59 and Crry on all blood cell types in the rat. We found that Crry was abundantly expressed on all blood cells. CD59 was expressed abundantly on erythrocytes and granulocytes but was absent from all T cellsand platelets and a minority of B cells and NK cells. Double staining and depletion studies showed that T cells in all rat strains tested were DAF-CD59-. Neutralization of Crry using a blocking monoclonal antibody rendered T cells susceptible to lysis by homologous complement, indicating that Crry was solely responsible for protecting DAF-CD59- T cells from complement damage in the rat.

Animals↗
PubMed2002

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.

BACKGROUND: Decay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF. METHODS: Function-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats. RESULTS: DAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab')2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were "planted" on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model. CONCLUSION: In cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC.

Animals↗
PubMed2002

Coxsackie B viruses that use human DAF as a receptor infect pig cells via pig CAR and do not use pig DAF.

Coxsackie B viruses (CVB) are enteroviruses belonging to the family Picornaviridae. Serotypes 1, 3 and 5 of CVB bind to the human membrane complement regulator decay-accelerating factor (DAF) and the coxsackievirus/adenovirus receptor (CAR), using either or both as receptors. These viruses are known to infect pig cell lines, but the receptor(s) involved has not been identified. We have recently characterized the pig homologue of DAF and here explore the interactions of human DAF-binding CVB with pig homologues of DAF and CAR. CVB infection of three pig cell lines resulted in cytolysis, which could be not be blocked by anti-pig DAF antibodies. CVB bound to CHO cells transfected with human DAF, but not pig DAF. Modification of pig DAF by incorporation of the fourth short consensus repeat of human DAF did not confer CVB-binding capacity. CVB did bind CHO cells expressing pig or human CAR, and pre-incubation of pig cells with anti-CAR antibody blocked CVB infection.

Animals↗