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B P Morgan

Publications and source records attributed to B P Morgan.

At least 37 records · Page 2Linked to original sources
PubMed2000

Role and regulation of pig CD59 and membrane cofactor protein/CD46 expressed on pig aortic endothelial cells.

BACKGROUND: Hyperacute rejection in xenotransplantation is caused by activation of complement (C) on endothelium. We have previously shown that purified C-regulators of the pig (CD59 and membrane cofactor protein [MCP]) are efficient regulators of human C (HuC). The aim of this study was to clarify the role of endogenously expressed C-regulatory molecules on pig endothelium in the protection against hyperacute rejection. METHODS: Porcine aortic endothelial cells (PAEC) were harvested and cultured for various passages. PAEC were examined for the expression of endogenous pig CD59 and MCP by flow cytometry. PAEC were assessed for their susceptibility to lysis by HuC. The effect of phorbol 12-myristate 13-acetate and various cytokines on the expression of MCP and CD59 and C-susceptibility was assessed. RESULTS: Primary PAEC showed an initial high level of expression of pig CD59, however, upon culturing, CD59 levels decreased dramatically to about 20% after five passages. In contrast, levels of MCP doubled upon culturing of PAEC to confluency and remained stable during at least five passages. Primary cells and cells in the early passages were more resistant to HuC than cells that were cultured for longer. Blocking the function of CD59 but not of MCP using monoclonal antibody increased the susceptibility to HuC. Purified human CD59 incorporated to a level of expression similar to that of pig CD59 reversed the increased C-susceptibility, suggesting that pig and human CD59 are similarly protective against HuC. Increase of C-resistance and of expression of pig MCP, but not of CD59, was achieved upon incubation with phorbol 12-myristate 13-acetate. Tumor necrosis factor-alpha, interleukin-1beta, interleukin-4, or interferon-gamma had no effect on C-regulator expression or C-susceptibility. CONCLUSIONS: These data demonstrate the importance of using primary PAEC or cells in the first passages of culturing in in vitro models of xenotransplantation and show that pig MCP and, in particular, pig CD59 play an important role in protection of PAEC from HuC.

Animals↗
PubMed2000

Immunology of reproduction: protecting the placenta.

The propensity of complement to damage self is controlled by expression of regulatory proteins. Recent results demonstrate that deleting just one of these regulators in mice causes complement to attack and destroy the embryo. These findings may have relevance to human pregnancy.

Animals↗
PubMed2000

Identification of mutations in rat CD59 that increase the complement regulatory activity.

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.

Amino Acid Substitution↗
PubMed2000

Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Amino Acid Sequence↗
PubMed2000

Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis.

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)

Animals↗
PubMed2000

Therapeutic efficacy of a novel membrane-targeted complement regulator in antigen-induced arthritis in the rat.

OBJECTIVE: Complement system activation is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis. The objective of this study was to explore the therapeutic potential and local retention of a novel membrane-targeting complement regulatory protein, derived from human complement receptor 1, in the experimental setting of rat antigen-induced arthritis. METHODS: Sensitized animals were treated at the time of arthritis induction with a single intraarticular (IA) dose of the membrane-targeting regulator APT070, a non-membrane-targeting control regulator (APT898), or vehicle control, and disease was assessed clinically and histologically. In addition, immunocytochemical analysis was performed on sections from normal rat knee joints at various time points after IA injection with APT070. RESULTS: Animals treated with APT070 showed a dose-dependent therapeutic effect, with significantly milder clinical and histologic disease compared with both other treatment groups (P < 0.008 at the higher dose) and minimal evidence of erosive disease at study end in the active treatment group. Immunoperoxidase and immunofluorescence studies demonstrated local retention of APT070 on cell surface membranes within the normal joint up to 48 hours after IA injection. CONCLUSION: These results show that IA complement inhibition represents an effective therapeutic strategy in experimental arthritis, by demonstrating that the exogenous delivery of a membrane-targeting complement regulator can result in prolonged synovial cell surface binding and significant clinical benefit in vivo. Complement inhibitory strategies of this type should be considered as novel therapies in human inflammatory arthritis.

Animals↗
PubMed2000

Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis.

This study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 microg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation.

Antibodies, Blocking↗
PubMed2000

Complement components of the innate immune system in health and disease in the CNS.

The innate immune system and notably the complement (C) system play important roles in host defense to recognise and kill deleterious invaders or toxic entities, but activation at inappropriate sites or to an excessive degree can cause severe tissue damage. C has been implicated as a factor in the exacerbation and propagation of tissue injury in numerous diseases including neurodegenerative disorders. In this article, we review the evidence indicating that brain cells can synthesise a full lytic C system and also express specific C inhibitors (to protect from C activation and C lysis) and C receptors (involved in cell activation, chemotaxis and phagocytosis). We also summarise the mechanisms involved in the antibody-independent activation of the classical pathway of C in Alzheimer's disease, Huntington's disease and Pick's disease. Although the primary role of C activation on a target cell is to induce cell lysis (particularly of neurons), we present evidence indicating that C (C3a, C5a, sublytic level of C5b-9) may also be involved in pro- as well as anti-inflammatory activities. Moreover, we discuss evidence suggesting that local C activation may contribute to tissue remodelling activities during repair in the CNS.

Central Nervous System↗
PubMed2000

Complement mediated cell death is associated with DNA fragmentation.

In this study, we demonstrate for the first time that complement attack of target cells, in the presence of suitably high levels of serum, can induce the oligonucleosomal DNA fragmentation characteristic of apoptosis. This phenomenon requires membrane permeabilisation induced by formation of the complete membrane attack complex and relies on physiologically relevant levels of serum. TUNEL analysis detected complement mediated DNA fragmentation as early as 30 min after the addition of serum and electron microscopy confirmed that chromatin became condensed after complement attack. Various experiments implicate serum DNase I as the mediator of this DNA fragmentation. Intriguingly, membrane permeability induced by melittin gave rise to similar serum dependent DNA fragmentation. The implications of these results for the study of apoptosis in vitro and in vivo are discussed.

Animals↗
PubMed2000

Comparison of the suppressive effects of soluble CR1 and C5a receptor antagonist in acute arthritis induced in rats by blocking of CD59.

We investigated the effects of suppression of complement activation at C3 level and inhibition of C5a on acute synovitis in rats. Acute synovitis was induced in Wistar rats by intra-articular (i.a.) injection into one knee of 0.3 mg of MoAb 6D1 (anti-rat CD59 antibody). In the treatment groups, soluble CR1 (sCR1) or C5a receptor (C5aR) antagonist was administered intra-articularly or intravenously and effects on the course of the acute synovitis were monitored. Synovitis induced by 6D1 was characterized by joint swelling, thickening of synovial tissue, cellular infiltration and deposition of membrane attack complex (MAC) on the synovial surface. Neither inflammatory change nor MAC deposition was found in rats which received an i.a. injection of sCR1 to suppress complement activity in the joint. Intra-articular injection of sCR1 did not reduce plasma complement activity. Intravenous administration of sCR1 suppressed plasma complement activity but had no effect on the course of the arthritis and synovitis with MAC deposition was observed. Neither i.a. nor i.v. injection of C5aR antagonist had any suppressive effects on inflammatory change or MAC deposition in synovium. The data show that inflammatory change induced by 6D1 was mediated by local complement activation and was not accompanied by systemic complement activation. C5a generation was not responsible for the observed inflammation, suggesting that other complement activation products, possibly MAC, mediate the inflammatory change observed in this model of acute synovitis in rats.

Acute Disease↗
PubMed2000

Soluble complement receptor one (sCR1) inhibits the development and progression of rat collagen-induced arthritis.

We set out to determine whether inhibition of complement using sCR1 could influence the development and progression of collagen arthritis in the Lewis rat. Collagen arthritis was successfully established in the Lewis rat, using a novel immunization schedule. In separate experiments, cobra venom factor (CVF) and sCR1 were used to achieve systemic complement inhibition. Their respective effects on disease onset and on the progression of established disease compared with saline-treated control animals was explored. Arthritis was assessed by measurement of clinical score, paw diameter and paw volume. Complement inhibition using either CVF or sCR1, prior to the onset of clinical signs of inflammation, delayed the development of disease. CVF was ineffective in the treatment of established disease, whereas sCR1 delayed the progression of disease in affected joints and prevented the recruitment of further joints while the animals were complement-depleted. In the control saline-treated groups the disease continued to progress relentlessly. We conclude that complement activation is important in the initiation and maintenance of inflammation in collagen arthritis. The potent disease-modulating effect of sCR1 provides persuasive evidence that specific complement inhibiting agents may be an effective approach to the treatment of inflammatory joint diseases

Animals↗
PubMed2000

Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack.

Hepatic parenchymal cells respond in many different ways to acute-phase cytokines. Some responses may protect against damage by liver-derived inflammatory mediators. Previous investigations have shown that cytokines cause increased secretion by hepatoma cells of soluble complement regulatory proteins, perhaps providing protection from complement attack. More important to cell protection are the membrane complement regulators. Here we examine, using flow cytometry and Northern blotting, the effects of different cytokines, singly or in combination, on expression of membrane-bound complement regulators by a hepatoma cell line. The combination of tumour necrosis factor-alpha, IL-1beta, and IL-6 caused increased expression of CD55 (three-fold) and CD59 (two-fold) and decreased expression of CD46 at day 3 post-exposure. Interferon-gamma reduced expression of CD59 and strongly antagonized the up-regulatory effects on CD59 mediated by the other cytokines. Complement attack on antibody-sensitized hepatoma cells following a 3-day incubation with the optimum combination of acute-phase cytokines revealed increased resistance to complement-mediated lysis and decreased C3b deposition. During the acute-phase response there is an increased hepatic synthesis of the majority of complement effector proteins. Simultaneous up-regulation of expression of CD55 and CD59 may serve to protect hepatocytes from high local concentrations of complement generated during the acute-phase response.

Acute-Phase Reaction↗
PubMed2000

Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities.

Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

Animals↗
PubMed2000

Production and functional characterization of a soluble recombinant form of mouse CD59.

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.

Animals↗
PubMed2000

Production and functional analysis of rat CD59 and chimeric CD59-Crry as active soluble proteins in Pichia pastoris.

Crry (CR1-related gene/protein) is a rodent complement regulator that inhibits C3 convertases. CD59 is a conserved protein inhibitor active towards C8 and C9. We have previously produced rat Crry as a recombinant soluble (rs) protein in Pichia pastoris. In this study we produced functionally active rat rsCD59 and a chimeric rsCD59-Crry protein in P. pastoris. The GPI anchor addition site of rat CD59 (Asn-79) was replaced either by a stop codon to produce rsCD59, or with the sequence of the first five short consensus repeats of Crry to produce rsCD59-Crry. Proteins were generated by fermentation and purified by affinity chromatography on an anti-CD59 column. In a standard classical pathway haemolysis assay, all three rs proteins had inhibitory activity, with 50% inhibition at 0.5 microM (rsCrry and rsCD59-Crry) and 4.4 microM (rsCD59). In an assay examining inhibition of C5b-9, in which C5b-7 was first formed, followed by purified C8 and C9, rsCD59 and rsCD59-Crry were active with 50% inhibition at 0.8 microM (rsCD59-Crry) and 1.3 microM (rsCD59). The degree of inhibition was independent of whether the C8 and C9 were of rat or human origin. Therefore, we have produced rsCD59 and rsCD59-Crry in P. pastoris. The rsCD59 retains its inhibitory activity towards C5b-9, while rsCD59-Crry appears to have the combined activities of Crry and CD59. In a haemolytic assay, the inclusion of CD59 to Crry is of no additional benefit to Crry, which may illustrate the overall importance of the C3 convertase step. Yet, inclusion of Crry to CD59 increases the potency of CD59 towards C5b-9.

Animals↗
PubMed2000

CD59 protects rat kidney from complement mediated injury in collaboration with crry.

BACKGROUND: As previously reported, the membrane-bound complement regulator at the C3 level (Crry/p65) is important in maintaining normal integrity of the kidney in rats. However, the role of a complement regulator at the C8/9 level (CD59) is not clear, especially when activation of complement occurs at the C3 level. The aim of this work was to elucidate the in vivo role of CD59 under C3 activating conditions. METHODS: Two monoclonal antibodies, 5I2 and 6D1, were used to suppress the function of Crry and CD59, respectively. In order to activate alternative the pathway of complement, the left kidney was perfused with 5I2 and/or 6D1 and was recirculated. RESULTS: In the kidneys perfused with 5I2 alone, deposition of C3 and membrane attack complex (MAC) was observed in the peritubular capillaries, vasa recta, and tubular basement membranes. Cast formation, tubular dilation and degeneration, and cellular infiltration were observed at days 1 and 4, and they recovered by day 7. Further suppression of CD59 by 6D1 significantly enhanced the deposition of MAC and worsened the already exacerbated tubulointerstitial injury. These effects of 6D1 were dose dependent. Perfusion with 6D1 alone did not induce histologic damage or MAC deposition in the tubulointerstitium. CONCLUSIONS: In rats, CD59 maintains normal integrity of the kidney in collaboration with Crry in rats against complement-mediated damage in vivo.

Animals↗