Biomedical subjects

B P Morgan

Publications and source records attributed to B P Morgan.

At least 19 recordsLinked to original sources

PubMed2006

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats.

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.

Animals↗

PubMed2006

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Animals↗

PubMed2004

Complement regulation at the molecular level: the structure of decay-accelerating factor.

The human complement regulator CD55 is a key molecule protecting self-cells from complement-mediated lysis. X-ray diffraction and analytical ultracentrifugation data reveal a rod-like arrangement of four short consensus repeat (SCR) domains in both the crystal and solution. The stalk linking the four SCR domains to the glycosylphosphatidylinositol anchor is extended by the addition of 11 highly charged O-glycans and positions the domains an estimated 177 A above the membrane. Mutation mapping and hydrophobic potential analysis suggest that the interaction with the convertase, and thus complement regulation, depends on the burial of a hydrophobic patch centered on the linker between SCR domains 2 and 3.

CD55 Antigens↗

PubMed2004

Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis.

OBJECTIVE: To investigate the roles of CD59a in the protection of joint tissue in the context of murine antigen-induced arthritis (AIA). METHODS: AIA was triggered in CD59a-deficient (CD59a(-/-)) mice and in CD59a-sufficient (CD59a(+/+)) controls; the course and severity of disease were compared between groups. The effects on arthritis of restoring CD59 to the joint in CD59a(-/-) mice by use of a membrane-targeted recombinant CD59 were also explored. RESULTS: Disease, as assessed clinically by measurement of joint swelling on day 1 (P < 0.0001), day 2 (P < 0.01), and day 7 (P < 0.02) and histologically from indicators of joint damage on day 21 (P < 0.02), was significantly enhanced in CD59a(-/-) mice compared with CD59a(+/+) wild-type controls. Membrane attack complex (MAC) deposition in the arthritic joints of CD59a(-/-) mice was also increased compared with that in the joints of CD59a(+/+) controls. Restitution of CD59 activity in joints of CD59a(-/-) mice was attempted with soluble recombinant rat CD59 (sCD59) or with a novel membrane-targeted rat CD59 derivative (sCD59-APT542). Strong immunohistochemical staining of the synovial membrane and subsynovial tissue was apparent in sCD59-APT542-injected joints, but not in joints injected with untargeted sCD59. Intraarticular administration of sCD59-APT542 markedly ameliorated disease severity in CD59a(-/-) mice, knee swelling was significantly reduced over the time course of the disease, and joint damage, assessed histologically, was significantly milder on day 21 (P < 0.05). CONCLUSION: These data firmly implicate the MAC of complement as a major effector of joint damage in the murine AIA model of rheumatoid arthritis (RA), and they provide a rationale for the inhibition of MAC assembly as a therapeutic strategy for RA.

Adjuvants, Immunologic↗

PubMed2004

Blockade of the C5a receptor fails to protect against experimental autoimmune encephalomyelitis in rats.

Complement activation contributes to inflammation and tissue damage in human demyelinating diseases and in rodent models of demyelination. Inhibitors of complement activation ameliorate disease in the rat model antibody-dependent experimental autoimmune encephalomyelitis and rats unable to generate the membrane attack complex of complement develop inflammation without demyelination. The role of the highly active chemotactic and anaphylactic complement-derived peptide C5a in driving inflammation and pathology in rodent models of demyelination has been little explored. Here we have used a small molecule C5a receptor antagonist, AcF-[OPdChaWR], to examine the effects of C5a receptor blockade in rat models of brain inflammation and demyelination. C5a receptor antagonist therapy completely blocked neutrophil response to C5a in vivo but had no effect on clinical disease or resultant pathology in either inflammatory or demyelinating rat models. We conclude that C5a is not required for disease induction or perpetuation in these strongly complement-dependent disease models.

Animals↗

PubMed2004

The possibilities and pitfalls for anti-complement therapies in inflammatory diseases.

The complement system is a key component of innate immunity, acting to protect the host from micro-organisms such as bacteria and other "foreign" threats, including tumor cells. However, excessive activation of complement can injure the host and can even be life threatening. These toxic effects are caused primarily by the excessive production of the anaphylatoxins C3a and C5a during complement activation and excessive formation of membrane attack complex on the host cell membrane. Many inflammatory diseases, including rheumatoid arthritis and glomerulonephritis, are thought to involve excessive activation of complement, both for their development and perpetuation. Uncontrolled complement activation is also implicated in post-ischemic inflammation and tissue damage and in sepsis. Therefore, it is important to regulate the complement system to treat disease. There are still no broadly applicable agents for the therapeutic regulation of excessive complement activation. However, there are now some agents in the development that might provide useful anti-complement therapies in the near future. Current strategies include the use of neutralizing antibodies, small synthetic antagonists, soluble recombinant forms of the natural complement regulators, and gene therapies to control excessive complement activation. Here we describe these new agents, their strengths and weaknesses and progress in testing the agents in relevant animal models.

Animals↗

PubMed2002

Tailoring anti-complement therapeutics.

Complement is a core component of the immune system, which performs vital roles in immune surveillance. However, the active products that enable complement to perform its physiological roles can inappropriately target self tissues and cause pathology. Complement-mediated inflammation and tissue destruction is an important drive to pathology in diseases as diverse as rheumatoid arthritis and dementia. Two decades ago there were no agents that could be used therapeutically to inhibit the activation of complement, but increased understanding of the natural control of complement in vivo has markedly changed this situation. The realization that drugs mimicking the action of the complement regulatory proteins present on self cells, and in plasma, could effectively control pathological activation of complement has opened the door to the use of anticomplement therapy in disease. Here we will review the development of anticomplement therapeutics from the first generation agents, which are unmodified recombinant forms of natural regulators, to recent strategies for making better drugs. We will describe strategies for targeting the anticomplement activity to the site of disease, and for extending the plasma half-life of the agent. Finally, we will illustrate a novel approach to the delivery of anticomplement agents, making prodrugs that are activated only at disease sites thus minimizing the deleterious effects of systemic complement inhibition.

Animals↗

PubMed2002

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo.

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.

Animals↗

PubMed2002

Complement activation in postpartum thyroiditis.

BACKGROUND: Postpartum thyroid dysfunction (PPTD) develops in 50% of pregnant women who have raised levels of circulating thyroid peroxidase autoantibodies (TPOAb) at booking. Although these antibodies are able to activate the complement cascade in vitro, it is not known whether complement activation plays any role in the pathogenesis of this disease. AIM: To investigate potential and actual activation of the complement system in women with postpartum thyroiditis. DESIGN: Complement activation was monitored on a weekly basis in 24 postpartum women who had raised TPOAb at 16 weeks gestation, attending an antenatal clinic in Mid-Glamorgan, Wales. METHODS: ELISA procedures were used to measure both in-vitro complement C3 activation by TPOAb and circulating terminal complement complexes (TCC) in serum. RESULTS: Higher levels of bioactive TPOAb activity were seen in women who developed PPTD when compared to those who did not. However, TCC remained undetectable in serum throughout the period of study. CONCLUSIONS: In PPTD, despite the presence of circulating bioactive TPOAbs, the extent of complement activation is inadequate to cause detectable increases in peripheral blood TCC, suggesting that the complement system may not play a major role in PPTD pathogenesis.

Autoantibodies↗

PubMed2001

Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria.

The glycolipid-anchored glycoprotein CD59 inhibits assembly of the lytic membrane attack complex of complement by incorporation into the forming complex. Absence of CD59 and other glycolipid-anchored molecules on circulating cells in the human hemolytic disorder paroxysmal nocturnal hemoglobinuria is associated with intravascular hemolysis and thrombosis. To examine the role of CD59 in protecting host tissues in health and disease, CD59-deficient (CD59(-/-)) mice were produced by gene targeting in embryonic stem cells. Absence of CD59 was confirmed by staining cells and tissues with specific antibody. Despite the complete absence of CD59, mice were healthy and fertile. Erythrocytes in vitro displayed increased susceptibility to complement and were positive in an acidified serum lysis test. Despite this, CD59(-/-) mice were not anemic but had elevated reticulocyte counts, indicating accelerated erythrocyte turnover. Fresh plasma and urine from CD59(-/-) mice contained increased amounts of hemoglobin when compared with littermate controls, providing further evidence for spontaneous intravascular hemolysis. Intravascular hemolysis was increased following administration of cobra venom factor to trigger complement activation. CD59(-/-) mice will provide a tool for characterizing the importance of CD59 in protection of self tissues from membrane attack complex damage in health and during diseases in which complement is activated.

Animals↗

PubMed2001

Membrane complement regulators protect against the development of type II collagen-induced arthritis in rats.

OBJECTIVE: To investigate changes in the distribution patterns of membrane complement regulators (MCRs) during the development of type II collagen-induced arthritis (CIA) and to examine the protective effects of these molecules against the augmentation of CIA in the knee joint. METHODS: Immunohistochemistry was used to examine the distribution of the MCRs Crry, DAF, and CD59 in the synovium of knee joints before and 2, 4, and 10 weeks after induction of CIA by immunization with type II collagen. In addition, at 2 or 10 weeks after induction of CIA, rats were injected intraarticularly with anti-Crry and/or anti-CD59 as the F(ab')2 fraction of monoclonal antibodies (mAb). Knee joint swelling and histologic changes in the synovium were examined 2 weeks after mAb injection. RESULTS: Synovial expression of Crry, DAF, and CD59 decreased in parallel with increased inflammation. When Crry and CD59 were functionally blocked at 2 weeks after the induction of CIA, swelling of the knee joints was markedly increased. Blocking of either regulator alone had no effect on swelling. Thickening of the synovial surface and proliferation of subsynovial tissue were all increased after blocking Crry and CD59, whereas blocking of either MCR alone had no effect. When both Crry and CD59 were blocked, deposits of membrane attack complex were found in the synovium. CONCLUSION: Our findings indicate that in rats with CIA and severely inflamed synovium, local expression of MCR is reduced. The MCRs Crry and CD59 appear to suppress the development of CIA.

Animals↗

PubMed2001

Immunopathology of secondary-progressive multiple sclerosis.

Twenty-three plaques obtained at early autopsy from 2 patients with secondary-progressive multiple sclerosis were examined immunohistochemically for microglia/macrophages, and for immunoglobulins and components of activated complement. Most of the lesions examined in both cases exhibited evidence of low-grade active demyelination of an unusual type (frustrated phagocytosis) in periplaque white matter. This included linear groups of microglia engaging short segments of disrupted myelin that were associated with deposits of C3d, an opsonin formed during complement activation. Similar microglia/C3d/myelin profiles were not observed in newly forming lesions in cases of acute multiple sclerosis or other central white matter diseases. As C3d coupling is known to increase the immunogenicity of potential antigens enormously, present findings point to disrupted myelin close to plaques as a possible source of the putative multiple sclerosis antigen. Ongoing myelin destruction found in a high proportion of old, established plaques was surprising. It suggests that slowly expanding lesions (progressive plaques), in which ongoing myelin breakdown occurs in the absence of florid perivascular cell cuffing or other histological signs of acute inflammation, contribute to disease progression in cases of secondary-progressive multiple sclerosis.

Adult↗

PubMed2001

Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity.

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol (GPI)-linked membrane inhibitor of complement activation. While human and other mammalian species contain only one DAF gene, two distinct DAF genes, referred to as GPI-DAF and transmembrane (TM)-DAF, respectively, have been identified in the mouse. Using several independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TM-DAF gene products, we have examined the expression of the two DAF genes in tissues of the wild-type and a strain of knockout mouse whose GPI-DAF gene has been inactivated. By fluorescence-activated cell sorting (FACS) analysis, we found that DAF protein is present on the wild-type mouse erythrocytes and lymphocytes but no signal was detectable on the same cells of GPI-DAF gene knockout mice. Both T and B lymphocytes and splenic macrophages express the GPI-DAF gene but the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4+ and CD8+ T cells are positive. DAF protein was detected by immunohistochemistry at high levels on wild-type mouse spermatids and mature sperm. In contrast, only mature sperm stained positive in the GPI-DAF gene knockout mouse testis, suggesting that GPI-DAF but not the TM-DAF gene is expressed on spermatids. Examination of the fetoplacental unit at the day 7.5 stage revealed that GPI-DAF but not the TM-DAF gene is expressed in the maternal decidua cells surrounding the trophoectoderm of the embryo. No DAF expression was detected on trophoblast or the embryo proper. These findings suggest that although the TM-DAF gene is irrelevant on mouse blood cells, the two DAF genes may have different roles in germ cell development and/or mature sperm function. Because complement receptor 1-related gene/protein y (Crry) has been shown to be expressed on early mouse embryos, the complete lack of GPI-DAF and TM-DAF gene expression in early mouse development may explain the observed sensitivity of Crry-deficient embryos to maternal complement attack.

Animals↗

PubMed2001

The lipopolysaccharide co-receptor CD14 is present and functional in seminal plasma and expressed on spermatozoa.

CD14 is a 54 000-molecular weight (MW) glycolipid-anchored membrane glycoprotein, expressed on myeloid cells, which functions as a member of the lipopolysaccharide (LPS) receptor complex. Soluble forms of CD14 have been reported in plasma, cerebrospinal fluid, amniotic fluid and breast milk. In plasma and breast milk, soluble CD14 has been implicated as a regulator of T- and B-cell activation and function. Expression of CD14 in the male reproductive system has not previously been investigated. We here show that soluble CD14 is present in seminal plasma at levels comparable to those in serum. Spermatozoa expressed CD14 on their membranes, as demonstrated by fluorescence microscopy and flow cytometry. Post-vasectomy, the levels of seminal plasma CD14 (spCD14) were much reduced, implying an origin distal to the point of transection of the vas deferens. Ultracentrifugation analyses demonstrated that spCD14 was not associated with lipid complexes, indicating that it lacks the glycolipid anchor. Purified spCD14 mediated activation by LPS of CD14-negative cells. These findings suggest that CD14 may play a hitherto unexplored role in immune defence and cell activation in the male reproductive tract.

Blotting, Western↗

PubMed2001

Glomerular complement regulation is overwhelmed in passive Heymann nephritis.

BACKGROUND: An injection of anti-Fx1A antibodies in rats leads to passive Heymann nephritis (PHN), a model of membranous nephropathy. Fx1A is a crude extract of renal cortex that contains megalin as a principal component. However, when rats are given anti-megalin antibodies, abnormal proteinuria does not occur. Because of the established complement dependence of PHN, we hypothesized that antibodies neutralizing complement regulatory proteins in the rat glomerulus also were required to induce PHN. Two likely targets are Crry and CD59, proteins abundant on the rat podocyte and contained within Fx1A that inhibit the C3 convertase and C5b-9 assembly, respectively. METHODS: Rats were injected with anti-megalin monoclonal antibodies, followed by anti-Crry and/or anti-CD59 F(ab')(2) antibodies five days later. In a second group of experiments, rats were injected with anti-Fx1A or anti-Fx1A immunodepleted of reactivity against Crry and/or CD59. RESULTS: In the setting of podocyte-associated anti-megalin monoclonal antibodies, simultaneous neutralization of Crry and CD59 function led to the development of significant proteinuria (11.0 +/- 2.1 mg/day, P < 0.001 vs. all other groups). In contrast, animals that had neither or only one of these complement regulators inhibited had normal urinary protein excretion (< or =6 mg/day). In animals given anti-Fx1A depleted of anti-Crry and/or anti-CD59, all groups developed typical PHN, characterized by heavy proteinuria and extensive glomerular deposition of C3 and C5b-9. CONCLUSION: Crry and CD59 play an important role in restraining complement-mediated injury following subepithelial immune complex deposition; however, in PHN, their regulatory capacity is overwhelmed.

Animals↗

PubMed2000

Human factor H-related protein 5 (FHR-5). A new complement-associated protein.

A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.

Amino Acid Sequence↗

PubMed2000

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement.

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

Animals↗

PubMed2000

Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats.

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.

Amino Acid Sequence↗