Role of C5 in the development of airway inflammation, airway hyperresponsiveness, and ongoing airway response.

The role of complement component C5 in asthma remains controversial. Here we examined the contribution of C5 at 3 critical checkpoints during the course of disease. Using an mAb specific for C5, we were able to evaluate the contribution of C5 during (a) the initiation of airway inflammation, (b) the maintenance of airway hyperresponsiveness (AHR), and (c) sustainment of an ongoing airway response to allergen provocation.

Laboratory studies in cross-species lung transplantation.

The lack of sufficient suitable human donor lungs for the many patients requiring pulmonary transplantation as life-saving therapy for end-stage lung diseases has generated extensive interest in cross-species lung transplantation. Ethical concerns and those of animal rights advocates have prompted studies of nonprimate species as potential solid organ donors for humans. This paper provides an overview of some of the laboratory studies of cross-species pulmonary transplantation performed over the past 20 years and focuses, in particular, on more recent work (from our laboratory and others) in the area of porcine-to-primate pulmonary xenotransplantation.

Genetically modified animal organs for human transplantation.

The major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection (HAR). Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Here we describe our strategy to overcome HAR in the pig-to-primate transplant setting, which includes the genetic incorporation into transgenic organs and high level expression of both a novel human bifunctional complement inhibitor and a human blood group enzyme.

Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed.

Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9.

Xenogeneic organ transplantation.

Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplanted cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP).

Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model.

The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours.

Retroviral vector producer cell killing in human serum is mediated by natural antibody and complement: strategies for evading the humoral immune response.

The introduction of retroviral vector producer cells (VPC) into tumors as a means of increasing transduction efficiency has recently been employed in human gene therapy trials. However, the fate of these xenogeneic cells in humans is not well understood. In the present study, we used an in vitro model to examine the survival of commonly used VPC lines in serum from humans and various other species.

Complement inhibition with an anti-C5 monoclonal antibody prevents acute cardiac tissue injury in an ex vivo model of pig-to-human xenotransplantation.

Prevention of hyperacute xenograft rejection in the pig-to-primate combination has been accomplished by removal of natural antibodies, complement depletion with cobra venom factor, or prevention of C3 activation with the soluble complement inhibitor sCR1. Although these strategies effectively prevent hyperacute rejection, they do not address the relative contribution of early (C3a, C3b) versus late (C5a, C5b-9) activated complement components to xenogeneic organ damage. To better understand the role of the terminal complement components (C5a, C5b-9) in hyperacute rejection, an anti-human C5 mAb was developed and tested in an ex vivo model of cardiac xenograft rejection.

Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis.

The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope).

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells.

A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody.

Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope.

A novel bifunctional chimeric complement inhibitor that regulates C3 convertase and formation of the membrane attack complex.

Human cells express cell surface complement regulatory molecules that inhibit the activity of the C3/C5 convertases (DAF, MCP, CR1) or inhibit the membrane attack complex (CD59). A single molecule that inhibits both the convertase activity and formation of the membrane attack complex has never been characterized. To this end, we have developed two reciprocal chimeric complement inhibitors (CD, NH2-CD59-DAF-GPI; and DC, NH2-DAF-CD59-GPI) that contain the functional domains of decay accelerating factor (DAF; CD55) and CD59.

Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression).

Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.

Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed.

Human capillary endothelial cells from abdominal wall adipose tissue: isolation using an anti-pecam antibody.

We have developed a novel isolation technique for harvesting human capillary endothelial cells. We compared the use of either Ulex Europaeus Agglutinin (UEA) lectin or anti-platelet endothelial cell adhesion molecule (PECAM) antibody conjugated to magnetic beads for the ability to isolate and maintain pure cultures of human capillary endothelial cells. Cells isolated using either method actively scavenged DiI-acetylated-low density lipoprotein and expressed von Willebrand factor (vWf) up to four passages as assessed by immunofluorescent labeling.

The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity.

The herpesvirus saimiri genome encodes a complement control protein homolog (CCPH). Stable mammalian cell transfectants expressing a recombinant transmembrane form of CCPH (mCCPH) or a 5'FLAG epitope-tagged mCCPH (5'FLAGmCCPH) conferred resistance to complement-mediated cell damage by inhibiting the lytic activity of human serum complement. The function of CCPH was further defined by showing that the mCCPH and the 5'FLAGmCCPH transfectants inhibited C3 convertase activity and effectively reduced cell surface deposition of the activated complement component, C3d.

Protection of retroviral vector particles in human blood through complement inhibition.

The rapid inactivation of murine-derived retroviral vectors in human or nonhuman primate sera is largely attributed to the activity of complement mediated through the classical pathway. In this study, we have further investigated the relationship between the human complement cascade and retrovirus inactivation. Preincubation in normal human serum effectively inactivated LXSN retroviral vector particles, whereas the vector maintained the ability to transduce cells following incubation in sera deficient in either the C1, C2, C3, C5, C6, C8, or C9 human complement proteins.

A BDNF autocrine loop in adult sensory neurons prevents cell death.

During the initial phase of their development, sensory neurons of the dorsal root ganglion (DRG) require target-derived trophic support for their survival, but as they mature they lose this requirement. Because many of these neurons express BDNF (brain-derived neurotrophic factor) messenger RNA, we hypothesized that BDNF might act as an autocrine survival factor in adult DRG neurons, thus explaining their lack of dependence on exogenous growth factors. When cultured adult DRG cells were treated with antisense oligonucleotides to BDNF, expression of BDNF protein was reduced by 80%, and neuronal survival was reduced by 35%.

Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection.

The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation. However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours.