Biomedical subjects

Claire L Harris

Publications and source records attributed to Claire L Harris.

At least 19 recordsLinked to original sources

PubMed2007

In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick Ornithodoros moubata.

The involvement of complement (C) in inflammatory diseases has driven the search for agents capable of inhibiting dysregulated complement activation. Many of these reagents inhibit the C3 convertases during the early stages of the cascade. However, a drawback of total systemic C inhibition, particularly in longterm treatment of chronic disease, is potentiation of infection and immune complex disease due to an inability to opsonize complexes and foreign cells and to lyse pathogens. Recent identification of a C5-binding protein in the salivary gland of the soft tick Ornithodoros moubata has enabled development of a terminal pathway-specific reagent, OmCI, with potential to ameliorate disease while leaving key physiological processes unaffected. Here we demonstrated that OmCI has broad cross-species activity. When given intravenously to rodents, OmCI totally ablated complement hemolytic activity, which gradually restored as C5 was resynthesized. The circulating half-life of OmCI was 30 h, demonstrating a much slower clearance than other small, biological agents. Using C5-sufficient and C5-deficient mice we showed that prolonged half-life was due to binding to plasma C5. Surface plasmon resonance analysis of C5 binding to OmCI confirmed a high binding affinity with a slow dissociation rate. OmCI was effective in preventing experimental autoimmune myasthenia gravis induced by passive transfer in normal Lewis rats. OmCI ablated clinical disease, reduced C3 and C9 deposition at the neuro-muscular junction, and effected a marked reduction in cellular infiltration at this site. These data offer exciting prospects for targeted treatment of complement-mediated diseases without the detrimental inhibition of the opsonic roles of complement.

Animals↗

PubMed2007

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay.

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.

Biological Assay↗

PubMed2006

Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation.

CD55 Antigens↗

PubMed2006

CD55 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.

CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.

Acrosome↗

PubMed2006

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa.

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

Acrosome↗

PubMed2006

Holding T cells in check--a new role for complement regulators?

Complement is not only part of the innate immune system, but has also been implicated in adaptive immunity. The role of complement and its regulatory proteins in modulating T cell activity has been the focus of several recent studies. These, which have included work on the membrane co-factor protein (MCP or CD46), decay accelerating factor (DAF or CD55) and CD59, indicate that complement regulators can influence the proliferative capacity of T cells and their ability to produce cytokines, influencing the outcome of a T cell response to a given antigen. Here we review these studies, which reveal another important link between the innate and the adaptive immune system.

Animals↗

PubMed2006

Complement and complement regulators in the male reproductive system.

Spermatozoa are almost unique among cells in that they must survive transplantation into a foreign host in order to perform their physiological role. The biggest hurdle to overcome is innate immune defence that will target the invaders in the female genital tract. Complement is a major player in innate immunity and is present in the female genital tract. Spermatozoa must therefore evade complement attack if they are to reach their goal. Complement evasion is achieved by the presence of complement regulators both in seminal plasma and on the spermatozoa. Here we discuss the parts played by complement and complement regulators in permitting spermatozoa to survive long enough to reach the oocyte, in clearance of the excess spermatozoa that have outlived their usefulness and in aiding activation of spermatozoa to engage the oocyte. In particular, we focus on the unique distribution patterns of complement regulators on spermatozoa, patterns that strongly suggest roles in spermatozoal development and oocyte binding. An understanding of these roles will inform studies of their contribution to fertility and infertility in man.

Animals↗

PubMed2005

Spermatogenic cells distal to the blood-testis barrier in rats lack C3 convertase regulators and may be at risk of complement-mediated injury.

On most tissues, multiple membrane complement regulators (CReg) protect self-cells from damage by complement. An exception is the brain, where the blood-brain barrier provides a protected environment within which cells survive with little or no protection from complement. The testis has a functionally similar structure, the blood-testis barrier (BTB). Here, we have investigated the expression of C3/C5 convertase CReg and C3 in the normal rat testis at different ages and different spermatogenetic stages, as well as in rats in which spermatogenesis and the BTB were impaired due to a developmental deficit. Immature testis, prior to BTB formation at puberty, displayed broad expression of the ubiquitous rodent CReg Crry on all elements and no expression of CD46 or CD55. Within days of BTB formation, CReg expression was dramatically altered; Crry was expressed only in the spermatogenetic cells external to the BTB in basal layers of adult seminal epithelium. Spermatogenic cells immediately distal to the BTB at first expressed no C3/C5 convertase regulators but later acquired expression of CD46 and CD55. Staining for C3 was widespread pre-puberty, but absent distal to the BTB in mature rats. In rats with defects in spermatogenesis and BTB integrity, expression patterns of CReg and C3 resembled those in pre-pubertal normals. The relative paucity of CReg and absence of C3 synthesis distal to the BTB suggest the presence of a complement-protected environment analogous to that described in the brain, and suggest also that cells enclosed by the BTB may be susceptible to complement damage when the barrier is breached.

Animals↗

PubMed2005

Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf-/- mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.

Adult↗

PubMed2005

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells.

Complement is the canonical innate immune system involved in host defense and tissue repair with the clearance of cell debris. In contrast to the robust armory mounted against microbial nonself-pathogens, complement is selectively activated on altered self (i.e. apoptotic and necrotic cells) to instruct the safe demise by poorly characterized mechanisms. Our data shed new light on the role of complement C1q in sensing nucleic acids (NA) rapidly exposed on apoptotic Jurkat T cell membranes and in driving C3 opsonization but without the lytic membrane attack complex. DNA/RNase-treated apoptotic cells failed to activate complement. We found that several other apoptotic cell models, including senescent keratinocytes, ionophore-treated sperm cells, and CMK-derived platelets, stained for cleaved caspase 3 were rapidly losing the key complement regulator CD46. CD46 from nuclear and membrane stores was found to cluster into blebs and shed into microparticles together with NA, phosphatidylserine, C1q, and factor H. Classical and alternative pathways of complement were involved in the recognition of H2O2-treated necrotic cells. Membrane attack complex was detected on necrotic cells possibly as a result of CD46 and CD59 shedding into soluble forms. Our data highlight a novel and universal paradigm whereby the complement innate immune system is using two synergistic strategies with the recognition of altered self-NA and missing self-CD46 signals to instruct and tailor the efficient removal of apoptotic and necrotic cells in immunoprivileged sites.

Apoptosis↗

PubMed2005

Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation.

There is compelling evidence that a unique innate immune response in the CNS plays a critical role in host defense and clearance of toxic cell debris. Although complement has been implicated in neuronal impairment, axonal loss, and demyelination, some preliminary evidence suggests that the initial insult consequently activates surrounding cells to signal neuroprotective activities. Using two different models of experimental autoimmune encephalomyelitis, we herein demonstrate selective C1q complement activation on neuron cell bodies and axons. Interestingly, in brains with chronic but not acute experimental autoimmune encephalomyelitis, C3b opsonization of neuronal cell bodies and axons was consistently associated with robust neuronal expression of one of the most effective complement regulators, decay-accelerating factor (CD55). In contrast, levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46), and CD59 were largely unaffected on neurons and reactive glial cells in both conditions. In vitro, we found that proinflammatory stimuli (cytokines and sublytic doses of complement) failed to up-regulate CD55 expression on cultured IMR32 neuronal cells. Interestingly, overexpression of GPI-anchored CD55 on IMR32 was capable of modulating raft-associated protein kinase activities without affecting MAPK activities and neuronal apoptosis. Critically, ectopic expression of decay-accelerating factor conferred strong protection of neurons against complement attack (opsonization and lysis). We conclude that increased CD55 expression by neurons may represent a key protective signaling mechanism mobilized by brain cells to withstand complement activation and to survive within an inflammatory site.

Acute Disease↗

PubMed2005

Glycation of CD59 impairs complement regulation on erythrocytes from diabetic subjects.

Type 1 diabetes is associated with anaemia. Although the underlying mechanisms remain unclear, the accompanying reticulocytosis implies that erythrocyte lifespan in the circulation is shortened. Among the factors that permit prolonged survival of erythrocytes are the membrane complement regulators. In conditions such as paroxysmal nocturnal haemoglobinuria, where erythrocyte expression of these regulators is reduced, erythrocyte survival is compromised and anaemia follows. Recent in vitro evidence indicates that one of the key membrane complement regulators, CD59, is inactivated by glycation in the presence of high concentrations of glucose or other glycating sugars. To ascertain whether glycation-induced inactivation of CD59 occurrs in vivo we examined CD59 surface expression and function on erythrocytes from a cohort with poorly controlled type 1 diabetes (hyperglycaemic) and from matched normoglycaemic controls. Although expression of CD59, assessed using polyclonal anti-CD59 antiserum, was similar in the two groups, erythrocytes from hyperglycaemic individuals were more susceptible to lysis by complement, entirely as a result of the loss of functional CD59. These data implicate glycation-induced inactivation of CD59 as a factor contributing to anaemia in type 1 diabetes.

Aged↗

PubMed2005

''Homologous restriction'' in complement lysis: roles of membrane complement regulators.

The complement system is a powerful bactericidal immune defence with the potential to damage self cells. Protection of self is provided by expression on cells of a battery of membrane regulators that inhibit activation of complement. Roles of complement in the rejection of transplanted organs have long been recognized, and are particularly relevant in xenotransplantation, where hyperacute rejection is complement-driven. Inhibiting complement was therefore considered early in the history of xenografting, and the use of membrane complement regulators to this end was proposed more than two decades ago. For each of the membrane regulators in humans, early studies implied a species-specificity of action, inhibiting human complement but not that from other species. The dogma of species-specificity dictated strategies for inhibiting complement in xenografts and drove the creation of donor transgenic pigs expressing human regulators. Here we critically evaluate the evidence for species-specificity in membrane complement regulators from humans and other animals. We challenge the dogma and show that there is considerable cross-species activity for each of the membrane regulators of complement. Acceptance of the fact that species selectivity is not a limitation will open new avenues for protection of the xenograft from complement damage.

Animals↗

PubMed2004

Expression of CD46 in developing rat spermatozoa: ultrastructural localization and utility as a marker of the various stages of the seminiferous tubuli.

Identification of the various stages of the seminal tubule epithelium that are important in spermatogenesis in humans and rodents requires considerable expertise for analysis of ultrastructural appearance under light microscopy. Few good stage-specific markers have been reported to facilitate the process. We recently described characterization of the expression of CD46 (membrane cofactor protein) in the rat using a novel monoclonal antibody. Expression of CD46 was restricted to spermatozoa and their immediate precursors in the testis. In the present study, we used a combination of morphological analyses, known acrosome markers, actin staining, direct nuclear staining, and staining for CD46 to delineate precisely the subcellular location of CD46. Staining of CD46 colocalized with known acrosome markers in late spermatids and mature spermatozoa and was confirmed by electron microscopy to be acrosome-restricted. Expression was first detected in step 7 spermatids, whereas known markers were not expressed until step 9. The CD46 staining pattern differed through spermatid development, and distinct patterns of staining could be identified that, when combined with 4'-6-diamino-2-phenylindole-2HCl nuclear staining, enabled the accurate staging of the seminiferous tubule epithelium in different profiles. This detailed description of the spatiotemporal expression patterns of CD46 provides a valuable tool for analysis of spermatogenesis in the rat. Furthermore, this information will aid ongoing studies regarding the roles of CD46 in acrosome-related spermatozoal functions.

Acrosome↗

PubMed2004

Complement: central to innate immunity and bridging to adaptive responses.

The complement system, a pillar of innate immunity, has belatedly become recognised as a key modulator of adaptive immunity, acting to direct, modulate and modify the responses of lymphocytes to stimuli. These effects are mediated by interactions between complement components or activation-derived fragments and specific binding proteins--complement receptors and regulators--on the target cells. This review will describe the current state of knowledge in this swiftly moving field. It is hoped that the recognition of these properties will help to establish complement in the role it richly deserves as the lynchpin of immunity.

Animals↗

PubMed2004

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55).

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.

Biosensing Techniques↗

PubMed2004

CD59a is the primary regulator of membrane attack complex assembly in the mouse.

Gene-deleted mice have provided a potent tool in efforts to understand the roles of complement and complement-regulating proteins in vivo. In particular, mice deficient in the membrane regulators complement receptor 1-related gene/protein y, decay-accelerating factor, or CD59 have demonstrated homeostatic relevance and backcrossing between the strains has revealed cooperativity in regulation. In mouse, genes encoding decay-accelerating factor and CD59 have been duplicated and show differential expression in tissues, complicating interpretation and extrapolation of findings to man. The first described form of CD59, CD59a, is broadly distributed and deletion of the cd59a gene causes a mild hemolytic phenotype with increased susceptibility in complement-mediated disease models. The distribution of the second form, CD59b, was originally described as testis specific, but later by some as widespread. Deletion of the cd59b gene caused a severe hemolytic and thrombotic phenotype. To apply data from these mouse models to man it is essential to know the relative distribution and functional roles of these two forms of CD59. We have generated new specific reagents and used them in sensitive quantitative analyses to comprehensively characterize expression of mRNA and protein and functional roles of CD59a and CD59b in wild-type (wt) and CD59a-negative mice. cd59b mRNA was detected only in testis and, at very low levels, in bone marrow. CD59b protein was present on mature spermatozoa and precursors and, in trace amounts, erythrocytes. Erythrocyte CD59b did not inhibit complement lysis except when CD59a was absent or blocked. These data confirm that CD59a is the primary regulator of complement membrane attack in mouse.

Animals↗

PubMed2004

Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3.

The rat analogue of the complement regulator membrane cofactor protein (MCP; CD46) was recently cloned and analysis at the mRNA level suggested that expression was restricted to testis. In light of the proposed roles of human MCP in sperm-egg interaction, we undertook to analyze rat MCP expression at the protein level in order better to address its putative role in fertilization. Recombinant fusion proteins comprising antibody Fc and specific domains of rat MCP were generated and used to develop a monoclonal antibody, MM.1, specific for rat MCP. Immunohistochemistry using these reagents confirmed the reported testis-specific expression of MCP in sexually mature rats and demonstrated that MCP was expressed only by spermatozoa and their immediate precursors in spermiogenesis, spermatids. Prepubertal male rats did not express MCP, and there was no evidence of MCP expression at any site in the embryo. Spermatozoal MCP expression was restricted to the inner acrosomal membrane, exposed only after fixation or induction of the acrosome reaction. Acrosome-reacted but not unreacted spermatozoa bound methylamine-activated C3 immobilized on plastic. The retention of MCP at this subcellular site, which is probably crucial to sperm-egg interaction, and the functional demonstration of binding to activated C3 strengthen suggestions from human studies that MCP may play an important role in fertilization. The reagents and results described here will enable studies of the role of spermatozoal MCP in sperm-egg interaction using a relevant animal model system.

Acrosome↗