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J D Lambris

Publications and source records attributed to J D Lambris.

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

Complement inhibitors: a resurgent concept in anti-inflammatory therapeutics.

In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is a pressing need to develop therapeutically effective complement inhibitors to prevent these adverse effects. This concept, though old, received little scientific attention until recently. Data from animal models of diseases that have been produced using complement-deficient, knockout, and transgenic animals, as well as data demonstrating that complement proteins are produced in many important tissue sites (including the brain) have attracted the interest of many basic research scientists and applied scientists from the biotechnology field and larger pharmaceutical firms. This resurgence of interest has generated a wealth of new information in the field of complement inhibition. In this article, we comprehensively review up-to-date information in the field of complement inhibitors.

Animals↗
PubMed1999

In vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gC.

Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack.

Animals↗
PubMed1999

Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activation in vivo on the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activation in vivo but exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activation in vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.

Animals↗
PubMed1999

Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts.

Compstatin, a newly described C3-binding peptide, inhibits complement activation by blocking C3 convertase-mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Pig kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid-phase and in the tissue were examined. The survival of the Compstatin-perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs-C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immune electron microscopy showed less terminal SC5b-9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62L (L-selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P-selectin or beta-thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.

Animals↗
PubMed1999

Inhibition of herpes simplex virus gD and lymphotoxin-alpha binding to HveA by peptide antagonists.

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-alpha (LT-alpha), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-alpha, we found that BP-1 inhibited the interaction of cellular LT-alpha with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-alpha and gD, the virus-specific protein involved in HSV entry into cells.

Amino Acid Sequence↗
PubMed1998

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles.

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.

Ambystoma↗
PubMed1998

Cloning, structure, and function of two rainbow trout Bf molecules.

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+ EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.

Amino Acid Sequence↗
PubMed1998

Compstatin inhibits complement and cellular activation in whole blood in two models of extracorporeal circulation.

Recently, a C3-binding cyclic synthetic peptide (Compstatin) has been identified that binds to complement component C3 and inhibits complement activation. Here we have examined the influence of Compstatin on complement activation and its indirect effects on cellular responses in whole blood in two models for extracorporeal circulation. Compstatin effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/ C3 fragments to the polymer surface. As a result of the inhibition of complement activation, the activation of polymorphonuclear leukocytes (PMNs; as assessed by the expression of CD11b) and the binding of these cells (CD16(+)) to the polymer surface were almost completely lost. In contrast, blood cell counts were not affected. Using surface plasmon resonance technology, we have confirmed that Compstatin exerts its inhibitory effect on complement activation by binding to native C3. These data show that complement activation, leading to activation and binding of PMNs to the biomaterial surface, can be abolished by the addition of Compstatin. The properties of Compstatin make Compstatin a promising drug for use in extracorporeal circuits to avoid bioincompatibility reactions, eg, during cardiopulmonary bypass, but also a favorable precursor peptide for the development of an anticomplement drug for oral use.

Amino Acid Sequence↗
PubMed1998

Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Arginine↗
PubMed1998

Sea urchin coelomocytes specifically express a homologue of the complement component C3.

A homologue of complement component C3 (SpC3) has been cloned and sequenced from the purple sea urchin, Strongylocentrotus purpuratus. The preprocessed, deduced protein size is estimated to be 186 kDa with a short leader and two chains, alpha and beta. There are cysteines in conserved positions for interchain disulfide bonding, and there is a conserved thioester site in the alpha-chain with an associated histidine. There are five consensus N-linked glycosylation sites, and putative cleavage sites for factor I and C3 convertase. Partially purified SpC3 on protein gels shows a nonreduced size of 210 kDa and, under reducing conditions, reveals an alpha-chain of 130 kDa and a beta-chain of 80 kDa. These sizes are larger than the deduced sizes, suggesting that the protein has carbohydrates added to most of the consensus N-linked glycosylation sites. Phylogenetic analysis of SpC3 compared with other members of the thioester protein family, which includes C3, C4, C5, and alpha2-macroglobulin, shows that SpC3 is the first divergent complement protein, falling at the base of the complement protein clade. Transcripts from the SpC3 gene (Sp064) are 9 kb, and the gene is expressed specifically in coelomocytes, which are the immunocytes in the sea urchin. Genome blots suggest that SpC3 is encoded by a single copy gene per haploid genome. This is the first identification of a complement component in an invertebrate, and suggests homology of the innate immune system within the deuterostome lineage of animals.

Amino Acid Sequence↗
PubMed1998

Solution structure of Compstatin, a potent complement inhibitor.

The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13-residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure-function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry-restrained simulated-annealing methodology, using distance, dihedral angle, and 3J(NH-Halpha)-coupling constant restraints. The Compstatin structure contained a type I beta-turn comprising the segment Gln5-Asp6-Trp7-Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3J(NH-Halpha)-coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100-fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side-chain interactions and the beta-turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.

Binding, Competitive↗
PubMed1998

Structure, functions, and evolution of the third complement component and viral molecular mimicry.

The third component of the complement system, C3, is a common denominator in the activation of the classical, alternative, and lectin pathways. The ability of C3 molecule to interact with at least 20 different proteins makes it the most versatile component of this system. Since these interactions are important for phagocytic, immunoregulatory, and immune evasion mechanisms, the analysis of its structure and functions has been a subject of intense research. Here we review our current work on the C3-ligand interactions, C3-related viral molecular mimicry, evolution of the complement system, and identification of C3-based complement inhibitors.

Animals↗
PubMed1998

Complement diversity: a mechanism for generating immune diversity?

Unlike mammalian species, several cold-blooded species have been shown to possess multiple forms of complement components. The multiple forms of C3 characterized in several fish species can bind with different specificities to various complement-activating surfaces. Here, Oriol Sunyer, Ioannis Zarkadis and John Lambris explore the possible advantages conferred by having multiple forms of individual complement proteins in a single organism.

Animals↗
PubMed1998

Evolution and diversity of the complement system of poikilothermic vertebrates.

In mammals the complement system plays an important role in innate and acquired host defense mechanisms against infection and in various immunoregulatory processes. The complement system is an ancient defense mechanism that is already present in the invertebrate deuterostomes. In these species as well as in agnathans (the most primitive vertebrate species), both the alternative and lectin pathway of complement activation are already present, and the complement system appears to be involved mainly in opsonization of foreign material. With the emergence of immunoglobulins in cartilaginous fish, the classical and lytic pathways first appear. The rest of the poikilothermic species, from teleosts to reptilians, appear to contain a well-developed complement system resembling that of homeothermic vertebrates. However, important differences remain. Unlike homeotherms, several species of poikilotherms have recently been shown to possess multiple forms of complement components (C3 and factor B) that are structurally and functionally more diverse than those of higher vertebrates. It is noteworthy that the multiple forms of C3 that have been characterized in several teleost fish are able to bind with varying efficiencies to various complement-activating surfaces. We hypothesize that this diversity has allowed these animals to expand their innate capacity for immune recognition.

Animals↗
PubMed1998

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Animals↗
PubMed1998

Structural and antigenic analysis of a truncated form of the herpes simplex virus glycoprotein gH-gL complex.

The herpes simplex virus (HSV) gH-gL complex is essential for virus infectivity and is a major antigen for the host immune system. The association of gH with gL is required for correct folding, cell surface trafficking, and membrane presentation of the complex. Previously, a mammalian cell line was constructed which produces a secreted form of gHt-gL complex lacking the transmembrane and cytoplasmic tail regions of gH. gHt-gL retains a conformation similar to that of its full-length counterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL. We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of one molecule each of gH and gL. The N-linked carbohydrate (N-CHO) site on gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results suggest that the complex is processed to the mature form via the Golgi network prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclonal antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted with the N-terminal portion of gH between amino acids 19 and 276. One of the gH MAbs, H12, reacted with the middle portion of gH (residues 476 to 678). Nine gL MAbs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped within amino acids 168 to 178 with overlapping synthetic peptides. Finally, plasmids expressing the gH and gL truncations were employed in cotransfection assays to define the minimal regions of both gH and gL required for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hetero-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a complex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.

Amino Acid Sequence↗