Complement Regulatory Molecules Research Articles

Evasion of the complement system by Leishmania through the uptake of factor H, a complement regulatory protein

Escaping the complement system is an important step in the establishment of infections. Some pathogens have acquired the ability to inactivate the complement system to ensure successful infection. This has been observed in parasites from the genus Leishmania, which inactivate C3b molecules deposited on their surface through the membrane protease GP63. In the present study, we describe a new mechanism that also acts through C3b inactivation. This mechanism involves the binding of the complement regulatory molecule factor H from serum. Factor H signals a plasma protease (factor I) to inactivate C3b molecules deposited on the surface of the parasites. According to our results, Leishmania infantum, L. amazonensis, and L. braziliensis recruit factor H from human serum. The absorption of factor H by L. infantum was studied in detail to better understand how it works. L. infantum binds factor H from human serum and factor H-like proteins from dog serum. When exposed to purified factor H, promastigotes bind this regulatory molecule and inactivate C3b in the presence of factor I. This indicates the existence of an as yet unidentified factor H-binding outer surface molecule functioning as a receptor. The two mechanisms (GP63 and factor H binding) work independently, as Leishmania promastigotes with inhibited GP63 can easily inactivate C3b molecules on the surface of the parasite. The identification of the factor H receptor could lead to the development of a vaccine target for leishmaniasis control, as blocking antibodies to factor H binding could impair the mechanism of C3b inactivation, making the parasite more susceptible to the complement system.

Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented.Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure.Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, <40% (n=18), 40> - < 90 %(n=18) and >90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of <0.05 were taken to indicate significance.Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot.Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. [Display omitted] DisclosuresArnold:Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Complement initiation and regulation differ by sex in intestinal ischemia reperfusion injury

Abstract Males and females frequently sustain distinct symptoms during ischemic events and subsequent inflammatory events. The inflammatory events include significant complement activation, neutrophil infiltration and significant cytokine secretions. The past literature suggests that complement activation may include a sex-linked protein. Therefore, we hypothesized that complement activation may vary by sex during intestinal ischemia/reperfusion (IR) injury. We subjected male and female mice to 30 min intestinal ischemia followed by 15, 30, 60 or 120 min reperfusion. Additional mice of each sex was subjected to sham surgery. Intestinal sections were stained and complement initiation pathways and time points of deposition determined. Complement regulatory molecules were also examined. Initial results suggest that males expressed more C1q compared to females, while females expressed more MBL than males. In addition, complement regulators varied by sex. Our data demonstrate that complement initiation and regulation differ between sexes during intestinal IR. These response differences in intestinal IR provide insight into the sexual dimorphism of ischemic intestine diseases and may aid in the development of sex-specific therapies that reduce intestinal injury.

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells.

BackgroundTo improve the targeting ability of antitumor drugs, we identified the antigens with high expression on the surface of tumor cells associated with tumor escape, such as the complement regulatory protein CD55 molecule, which is also known as the decay accelerating factor. In this study, phage display technology was used to screen and identify CD55-specific ligand peptide (CD55sp) bound to CD55 molecule on the surface of cervical cancer HeLa cells. We then explored the role of this peptide in inhibiting the growth of cervical cancer cells in vitro. Our characterization of CD55sp will provide implication for tumor target therapy.MethodsThe phage bound to the surface of HeLa cells were isolated by phage display technology. Positive phage clones were identified by ELISA. Phage was then amplified and determined by agarose gel electrophoresis after monoclonal DNA extraction. DNA sequencing and bioinformatical analysis were conducted to obtain specific ligand peptides. Flow cytometry and immunofluorescence were used to measure the expression of CD55 molecule on the surface of tumor and normal cells. Subsequently, the effects of CD55sp on the proliferation and apoptosis of HeLa and SiHa cells were determined by Cell Counting Kit-8 (CCK-8), flow cytometry, and TUNEL assay, respectively. The morphology of apoptotic cells was examined by electron microscope. The distribution of Cleaved caspase-3 was detected by immunofluorescence. The expression of bcl-2 and Cleaved caspase-3 were determined by Western blot.ResultsThe results showed that the peptide (QVNGLGERSQQM) can bind to the CD55 molecule on the surface of cervical cancer HeLa and SiHa cells as a ligand peptide. It can also effectively inhibit the proliferation of cervical cancer cells and induce cell apoptosis.ConclusionThis study demonstrates that CD55sp screened by phage display technology plays a strong antitumor role.

Additive Expression of hTBM on hCD46 Stable Cell Line Has Synergistic Effect on Complement Regulation

Background Coagulation dysregulation and intravascular thrombosis during acute humoral xenograft rejection (AHXR) still remains the major obstacle in solid organ xenotransplantation. TBM or CD46 have been reported as an anti-coagulant or complement regulatory molecules and also potent anti-inflammatory molecules. In this study, we showed whether additional TBM expression on CD46 stable cell has synergistic effects on anti-coagulant or complement regulation. Methods & Results Fetal pig fibroblasts were transfected with hTBM-hCD46 vector and colonies expressing high amounts of the fusion protein were selected and used as nucleus donor for somatic cell nuclear transfer (SCNT) and three gilts became pregnant and one farrowed one live male piglet, which was no healthy and moved to the SPF facility but died one day after and two of the pregnant gilts, however, aborted during gestation. We isolated fibroblast from the ear of TG pig expressing hTBM-hCD46 and check the expression levels of each genes and the regulatory functions on complement and coagulation. Serum toxicity was significantly reduced in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast at 10% or 20% serum treatment. Thrombin generation was dramatically reduced and coagulation time was prolonged in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast. Conclusion The fibroblast from hTBM-hCD46 TG pig has synergistic effects on anti-coagulant or complement regulation therefore, genetically modifying pigs expressing TBM and CD46 would be a promising candidate for xenotransplantation to prolong the graft survival.

Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

ABSTRACTA subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis.

Cancer cells release anaphylatoxin C5a from C5 by serine protease to enhance invasiveness.

Anaphylatoxin C5a indirectly fosters cancer cells through recruitment of myeloid-derived suppressor cells (MDS) for inhibiting antitumor CD8+ T cells and induction of neovascularization. We recently found activation of cancer cells by C5a directly via the C5a-receptor (C5aR; CD88) to enhance invasiveness. Thus, C5a possibly contributes to cancer progression rather than elimination. C5a generation in cancer tissues has been reported; however, the mechanism is not fully elucidated. Cancer cell expression of complement regulatory molecules suggests inefficient C5a generation through activation of the complement system in response to cancer cells. To explore another C5a generation mechanism in cancer tissues, we examined cancer cells for C5a-releasing activity from C5. C5a was present in C5-supplemented culture media of cancer cells including C5aR-expressing cells, and the media enhanced C5aR-expressing cancer cell invasion, which was abolished by anti-C5a antibody. The C5a-releasing activity was absent in the supernatants of the media and was inhibited by aprotinin, a serine protease inhibitor, and decanoyl-Arg-Val-Lys-Arg-chloromethylketone but not by inhibitors specific for cysteine, acid, or metal proteases. These results indicated C5a release from C5 by a cancer cell membrane-bound serine protease that can cleave peptide bonds at the carboxy-terminal site of paired basic amino acid residues. Cancer cell C5a release from the complement-immobilized plasma supported feasibility of this cancer cell protease-dependent C5a generation in cancer tissues. The new mechanism of C5a generation suggests self-activation of C5aR-expressing cancer cells to enhance invasiveness and induction of MDS recruitment and neovascularization to create a microenvironment favorable for cancer progression.

Modulation of immune responses to porcine xenoantigen by targeting stimulatory and inhibitory signalling pathways

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3 galactosyltransferase) [1] enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by monocytes/macrophages, natural killer (NK) cells, and T cells.Research in our laboratory focuses on receptor‐ligand interactions regulating the responses of human cells to porcine tissues and thus could be targets for immunomodulation. NK cell activation is tightly regulated by different inhibitory and activating receptors and their ligands. Transgenic pigs overexpressing HLA‐E have been generated [2]. In vitro experiments revealed that cells from these pigs are only partially protected from NK cell‐mediated lysis. This might be due to the fact that the inhibitory receptor for HLA–E (CD94/NKG2A) is not expressed on all human NK cells. We are currently exploring the concept of downregulating NK cell activity by enhancing CD161/KLRB1‐mediated signalling. CD161 is a C‐type lectin‐like receptor which is expressed on the great majority of human NK cells and can transmit inhibitory signals after binding of its ligand LLT1. The data obtained so far suggest, that porcine cells genetically engineered to overexpress human LLT1 posses reduced potential to activate human NK cells.Activation of T cells by antigen presenting cells (APC) requires interactions between T cell receptor (TCR) and MHC/peptide (“signal one” of T cell activation) as well as interactions between costimulatory receptors and their ligands (“signal two” of T cell activation). T cells receiving “signal one” alone without “signal two” are not activated but achieve a state of unresponsiveness (anergy). In vitro and in vivo data suggest that effective inhibition of human T cell activation can be obtained by blocking the costimulatory CD28‐CD80/CD86 interaction using monoclonal antibodies (mAb) or an antagonistic CTLA‐4.Ig fusion protein [3]. Furthermore, blocking of TCR‐MHC and CD154(CD40L)‐CD40 interactions by mAb to porcine MHC class‐II or CD40 significantly reduced human T cell activation. New approaches for the modulation of anti‐pig immune responses may result from the finding that enhancement of inhibitory signals diminishes T cell activation. We have recently shown that overexpression of the human inhibitory ligand PD‐L1 on porcine APC markedly decreased their capacity to activate human T cells and promotes regulatory T cells [4, 5]. Current studies suggest that enhanced inhibitory signalling does not only downregulate T cell reactivity but also dampens induced antibody responses to porcine xenoantigen. Thus, disruption of stimulatory receptor‐ligand interactions (e.g. by blocking antibodies or “knock‐out/down” technologies) combined with transgenic overexpression of inhibitory ligands in porcine cells and tissues could be an effective approach to downregulate human anti‐pig immunity.

Inhibition of complement component C5 protects porcine chondrocytes from xenogeneic rejection

Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Microvesicles and pre-eclampsia

The maternal syndrome of pre-eclampsia is characterised by an excessive inflammatory response associated with endothelial dysfunction, brought about by the release of multiple factors from the placenta into the maternal circulation. While some of these factors are released as soluble molecules it is now apparent that many of them are associated with syncytiotrophoblast microvesicles and exosomes (collectively termed STBM), which are present in increased amounts in the circulation of women with pre-eclampsia. We have shown that STBM have proinflammatory, anti-endothelial and procoagulant activities in vitro, all of which are features of the maternal syndrome. We propose that the different effects of STBM result from different types of vesicles within the circulation the smaller exosomes being immunoregulatory and the larger microvesicles being proinflammatory, with a shift to the latter in pre-eclampsia. In support of this, we have demonstrated using a novel technique, Nanoparticle Tracking Analysis, that pre-eclampsia STBM are indeed larger than those from normal placentas. Furthermore, the range and types of factors they carry (and hence their functions) may differ in pre-eclampsia, where the syncytiotrophoblast is subjected to oxidative and inflammatory stress. We have carried out proteomic analysis on vesicles prepared from normal and pre-eclampsia placentas by perfusion and have identified differences in the repertoire of molecules they carry. Candidates include immunoregulatory molecules (B7-H1, CD200 and Galectin 1), complement and complement regulatory molecules (C1q, C3, CD55, CD59 and vitronectin), proinflammatory molecules (HSP70, HMGB1, Galectin 3 and Synctin 1), anti-angiogenic molecules (CD49e, CD51, CD26, Flt-1 and endoglin) and procoagulant molecules (tissue factor and phosphatidylserine). Characterising the molecular cargos of the STBM may lead to the discovery of new biomarkers for pre-eclampsia and inform future treatments.

SY12-4 - Molecular Mechanisms of Rituximab Resistance and Strategies to Overcome Them

ABSTRACT Rituximab is the first anti-CD20 chimeric-monoclonal antibody utilized for patients, and is now a key molecular targeting drug for CD20-positive B-cell lymphomas (BCL). Although combination chemotherapies with rituximab have surely improved the clinical outcomes of BCL patients, many clinicians recognize that relapse/refractory disease, which occurs in almost 50% of BCL patients, is still a major problem. Confirming the molecular mechanisms of rituximab resistance is crucial for further improvement of overall survival and progression-free survival in BCL patients. Several mechanisms of rituximab resistance have recently been reported as follows: (i) downregulation of CD20 expression after using rituximab, (ii) low expression level of CD20 at diagnosis, (iii) CD20 genetic mutations, (iv) aberrant expression of complement regulatory molecules (e.g. CD55, CD59) on tumor cells), (v) disruption of lipid raft formation, (vi) abnormality of signal transduction pathway, and (vii) Fc-gamma receptor polymorphisms of effector cells. To overcome these phenomena, several strategies are considered: (i) utilization of newer generation anti-CD20 antibodies (e.g. type I and II antibodies), (ii) new monoclonal antibodies for other targets (e.g. anti-CD22, -CD23, -CD79), and (iii) combination therapy with other molecular targeting drugs (e.g. demethylating and deacetylating agents, BH3 mimetics). In this session, recent progress in molecular analyses of resistant mechanisms and the strategies to overcome them will be summarized. Understanding these mechanisms may provide quite important information for not only improving clinical outcomes of BCL patients but also confirming the resistance mechanisms against newer monoclonal antibodies for BCL and solid tumors.

STEC-HUS, atypical HUS and TTP are all diseases of complement activation

Haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP) are diseases characterized by microvascular thrombosis, with consequent thrombocytopaenia, haemolytic anaemia and dysfunction of affected organs. Advances in our understanding of the molecular pathology led to the recognition of three different diseases: typical HUS caused by Shiga toxin-producing Escherichia coli (STEC-HUS); atypical HUS (aHUS), associated with genetic or acquired disorders of regulatory components of the complement system; and TTP that results from a deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. In this Review, we discuss data indicating that complement hyperactivation is a common pathogenetic effector that leads to endothelial damage and microvascular thrombosis in all three diseases. In STEC-HUS, the toxin triggers endothelial complement deposition through the upregulation of P-selectin and possibly interferes with the activity of complement regulatory molecules. In aHUS, mutations in the genes coding for complement components predispose to hyperactivation of the alternative pathway of complement. In TTP, severe ADAMTS13 deficiency leads to generation of massive platelet thrombi, which might contribute to complement activation. More importantly, evidence is emerging that pharmacological targeting of complement with the anti-C5 monoclonal antibody eculizumab can effectively treat not only aHUS for which it is indicated, but also STEC-HUS and TTP in some circumstances.

OS045. Multi-dimensional protein identification technology analysis of syncytiotrophoblast vesicles released from perfused preeclampsia placentas

In pre-eclampsia, the consequences of poor placentation lead to the second stage of pre-eclampsia, which involves activation of a maternal systemic inflammatory response (MSIR). Endothelial and other inflammatory cellular dysfunction cause the diverse features which characterise the disorder. We have previously shown that syncytiotrophoblast microvesicles (STBM) are pro-inflammatory and circulate in increased amounts in pre-eclamptic women. We hypothesise that multiple placental "danger signals" are carried by STBM into the maternal circulation in increased amounts in PE with pro-inflammatory, anti-angiogenic and pro-coagulant activity, implicating STBM in the pathophysiology of PE. To characterise the proteins carried by STBM from normal and PE placentas. For the first time multi-dimensional protein identification technology (MudPIT) was used to derive the proteome profiles of normal and PE placenta STBM. STBM were prepared from placentas (normal term: n=9 and PE: n=5) by dual lobe perfusion, isolated by ultracentrifugation and stored at -80°C. Normal and PE derived placenta STBM pools were then subjected to MudPIT analysis. 538 proteins unique to PE STBM, 604 proteins unique to normal STBM and 1421 proteins common to both preparations were found. Preliminary analysis indicates the presence of alarmins (HSP70, and galectin 3), exosomal proteins (CD63,CD9,CD81), immunoregulatory molecules (CD26,CD200,CD47,Galectin 1), complement and complement regulatory molecules (C1q,C3,CD55,CD59 and vitronectin), amino acid transporters (CD98) and anti-angiogenic molecules (endoglin). Our analysis also reveals that proteins known to be elevated in blood before, or at, the time of pre-eclampsia are elevated or unique in STBM from PE placentas, including Fetuin A, Inter-alpha (globulin) inhibitor H4, Serum amyloid P component, Apolipoprotein H (or B2GP1) and Apolipoprotein AII. Thus, as predicted, a large number of circulating molecules are associated with STBM. The inter-relationships between proteins that are unique to either PE or normal pregnancy and the processes in which they are involved are being determined by Ingenuity Pathways Analysis software. In terms of biofunctions, preliminary analysis shows that proteins unique to PE STBM have a highly significant association (p<10(-11)) with 6 disease pathways including inflammatory, immunological, cardiovascular and reproductive system diseases and organ injury, whereas for proteins unique to normal STBM only protein synthesis was significant at the same level. STBM contain a heterogeneous population of vesicles that convey a large repertoire of placental proteins into the maternal circulation. The profound differences between PE and normal STBM indicate their pro-inflammatory potential.

Antibodies Directed Against AB Blood Antigens or Human Leukocyte Antigen Molecules Activate Different Intracellular Pathways Explaining Their Differential Effects Toward Accommodation

The limited availability of organs for transplantation has driven the search for new strategies, such as the use of ABO and human leukocyte antigen (HLA) incompatible donor grafts for which antidonor antibodies (Abs) preexist in the recipient. The control of T-cell alloresponses has considerably improved over the last two decades thanks to the discovery of novel anti-T-cell molecules, dramatically reducing the incidence of acute cellular rejection. On the other hand, control of the humoral response remains a considerable challenge. Nevertheless, recent strategies, such as the use of plasma exchange, intravenous immune globulin, rituximab, bortezomib, or anti-C5 mAb (eculizumab), have helped to improve the success rate for so-called incompatible transplants. However, ABO and HLA incompatibility seems to be different. In fact, ABO-incompatible transplantation has improved considerably, with results now comparable with ABO-compatible transplants. On the contrary, HLA-incompatible transplantation with donor-specific Abs poses a higher risk of Ab-mediated rejection (ABMR), with a significant negative impact on graft survival. Graft survival despite the presence of antigraft Abs is known as “accommodation,” a phenomenon first described in the context of successful ABO-incompatible liver transplantation performed for acute liver failure (1). The first graft tissue with which antidonor Abs come into contact is the endothelium, whose response seems to be Ab-dependent. For example, endothelial cells (EC) seem to be particularly resistant to anti-A/B Abs in the context of accommodation, which is not the case for anti-HLA Abs, suggesting that differential EC pathways are involved in defining response to antidonor Abs. The study by Iwasaki et al. (2) aimed to compare the in vitro signal transduction pathways triggered in EC upon binding of anti-A/B and anti-HLA Abs, particularly in the context of accommodation. They showed that in resistance to complement-dependant cytotoxicity (CDC), the effect of EC preincubation with high-titer anti-A/B Abs was comparable with that of low-titer anti-HLA class I Abs. They additionally showed that a 1-hr incubation of EC with high-titer anti-A/B Abs up-regulated CD55 and CD59 by means of ERK1/2 pathway inactivation. However, a 1-hr incubation of EC with low-titer anti-HLA Abs resulted in reduced CDC, associated with an AKT pathway-dependent increase in mRNA expression of the cytoprotective proteins HO-1 and ferritin H. Given that CDC is the major (but not unique) mechanism by which Abs acutely aggress graft tissue, complement staining in graft biopsies, particularly C4d (or C3c/C3d), is considered as a good indicator of ABMR, especially in the context of donor-specific Abs. On the contrary, in ABO-incompatible transplantation, C4d or C3d staining is found in graft biopsies of stable nonrejected grafts without any correlation with poor outcome (3). The up-regulation of CD55 and CD59 shown by Iwasaki et al. (2) could explain the endothelial resistance to A/B Abs despite deposition of C4d and C3d, because the complement activation pathway is blocked at this step and cannot progress to full activation. In fact, the complement regulatory molecules CD55 and CD59 exert their inhibitory roles at multiple levels of the complement cascade: dissociation of C3 convertases by CD55 and prevention of membrane attack complex assembly by CD59. These roles are acted out locally at the site of Ab binding to EC, and a correlation exists between their up-regulated expression and the efficacy of CDC blockade. Their action is so efficient locally that in the field of xenotransplantation, this has spurred the generation of human CD55 or CD59 transgenic pigs expressing high levels of the human molecules on their EC, thereby preventing hyperacute ABMR in primate models. On another hand, the finding of reduced CDC through activation of the PI3K/AKT pathway and expression of cytoprotective genes upon EC preincubation with low-titer anti-HLA Abs well confirms the work of Reed et al. (4). The latter authors also reported on an induction of the antiapoptotic genes A20, Bcl-2, and Bcl-xL, driving EC to an accommodated profile. The identification of specific signaling pathways that could render EC resistant to Ab-mediated cytotoxicity would be of great value in transplantation, because it would help to understand how the status of accommodation may be acquired. Two parameters are obviously critical in this phenomenon referred here: Ab preincubation and Ab titer. So far, encouraging data such as these have only been observed in vitro and their confirmation in whole organs could justify strategies such as ex vivo preconditioning of donor organs with high-titer A/B Abs before A/B-incompatible transplantation or low-titer recipient anti-HLA Abs. Such strategies would aim to induce these particular protective pathways before transplantation in the context of prepared recipients (plasma exchange and immunosuppression) in an attempt to obtain accommodation in the context of HLA incompatibility.

Cellular immunological barriers in xenotransplantation: current status and prospects for avoidance of graft destruction

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig-to-primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by macrophages, T cells and NK cells. Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind the inhibitory receptor SIRPα (signal regulatory protein α). Thus, one approach to impair the ability of human macrophages to phagocyte porcine cells is the overexpression of the human ligand for SIRPα in porcine cells. To inhibit human NK cell reactivity after xenotranslantation transgenic expression of HLA-E in pigs has been shown to be a promising concept. Cells from these pigs were partially protected from lysis by human NK cells. Our group focuses on manipulation of human anti-pig T cell responses by negative costimulatory signals. Thus, we asked whether overexpression of PD-L1 on porcine cells can (i) downregulate human anti-pig cellular responses in vitro, and (ii) inhibit rat anti-pig cellular immune responses in vivo. Pig cells overexpressing PD-L1 triggered reduced proliferation and low amounts of IL-2, IFNγ, TNF-alpha, IL-4, and IL-5 in human CD4+ T cells compared to control pig cells. The concentration of IL-10, however, was increased. In long-term cultures of human CD4+ T cells and PD-L1 transfectants a high frequency of CD4+ CD25high FoxP3+ cells showed up which had the capacity to suppress the activation of conventional CD4+ T cells. Cytotoxic CD8+ T cells and NK cells lysed pig control cells very efficiently. In contrast, PD-L1 transfected pig cells were partially protected from lysis by human effector cells. Overexpression of PD-L1 on porcine cells was not sufficient to prevent rejection after transplantation under the rat kidney capsule. However, in rats that had been grafted with PD-L1 expressing cells we observed reduced cellular infiltrates in the kidneys and lower antibody responses compared to rats grafted with control cells. Together these observations support the assumption that PD-1/PD-Ligand pathways are interesting targets to prevent cellular immune responses after xenotransplantation. PD-L overexpression might not only impede the initiation of an anti-pig T cell response by suppressing CD4+ T cells but may also protect pig cells from destruction by cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).

Adenovirus 11p downregulates CD46 early in infection

Adenovirus 11 prototype (Ad11p), belonging to species B, uses CD46 as an attachment receptor. CD46, a complement regulatory molecule, is expressed on all human nucleated cells. We show here that Ad11p virions downregulate CD46 on the surface of K562 cells as early as 5min p.i. Specific binding to CD46 by the Ad11p fiber knob was required to mediate downregulation. The complement regulatory factors CD55 and CD59 were also reduced to a significant extent as a consequence of Ad11p binding to K562 cells. In contrast, binding of Ad7p did not result in downregulation of CD46 early in infection. Thus, the presumed interaction between Ad7p and CD46 did not have the same consequences as the Ad11p–CD46 interaction, the latter virus (Ad11p) being a promising gene therapy vector candidate. These findings may lead to a better understanding of the pathogenesis of species B adenovirus infections.

Negative costimulation to prevent human anti‐pig T cell responses

Genetic modification of pigs [e.g. transgenic expression of human complement regulatory molecules or inactivation of α(1,3)‐galactosyltransferase] enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Our group focuses on the inhibition of human anti‐pig T cell responses by targeting ‘‘negative’’ costimulatory pathways. We tested the hypothesis that over‐expression of the human negative costimulatory ligands PD‐L1 and PD‐L2 on pig cells will result in reduced human anti‐pig T cell responses. To this end, pig cell lines stably over‐expressing human PD‐Ligands (L23‐PD‐L1/L2 cells) and appropriate controls (L23‐GFP cells) were generated and used for in vitro stimulation of purified human CD4+ T cells. Furthermore, the effect of PD‐L expression on cellular cytotoxicity was tested by 51Cr‐release assays using L23‐PD‐L1/L2 and L23‐GFP cells as targets and IL‐2‐activated human CD8+ T cells and NK cells as effector. CD4+ T cells responded with significantly reduced proliferation to L23‐PD‐L1 or L23‐PD‐L2 cells and produced less IL‐2, IFNγ, TNFα, IL‐4, and IL‐5 than cells stimulated with mock‐transfected B cells. The concentration of IL‐10, however, was increased in CD4+ T cells responding to stimulation with PD‐L1 or PD‐L2 transfectants. Furthermore, in cultures of CD4+ T cells stimulated for 3 weeks with PD‐L1 or PD‐L2 transfectants a CD4+CD25highFoxp3+ subset showed up that effectively suppressed the activation of conventional CD4+ T cells. CD8+ T cells and NK cells acquired cytolytic potential and up‐regulated the PD‐1 receptor during cultivation with IL‐2. Both cell types lysed L23‐GFP cells very effectively but were less potent on L23‐PD‐L1 targets (reduced cytotoxicity of about 50%). When cytotoxic effector cells were used that do not express PD‐1 (293‐95L cells), L23‐GFP and L23‐PD‐L1 cells were lysed with similar intensity suggesting that PD‐1/PD‐L1 interactions are required for PD‐L1 mediated protection. Flow cytometric studies revealed that a proportion of activated CD8+PD‐1+ T cells underwent apoptosis when exposed to PD‐L1 expressing L23 cells. Together these observations support the assumption that PD‐1/PD‐Ligand pathways are interesting targets to prevent human anti‐pig cellular immune responses after xenotransplantation [1]. PD‐L over‐expression might not only empede the initiation of a human anti‐pig T cell response by suppressing CD4+ T cells but may also protect pig cells from destruction by human cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forscher‐gruppe ‘‘Xenotransplantation’’, FOR 535).Reference1. Plege A, Borns K, Baars W, Schwinzer R. Suppression of human T‐cell activation and expansion of regulatory T cells by pig cells overexpressing PD‐ligands. Transplantation 2009; 87: 975–982.

Cloning of the sixth complement component and, spatial and temporal expression profile of MAC structural and regulatory genes in chicken

Humoral cytotoxicity results from the assembly of terminal components of complement, called membrane attack complex (MAC), which lead to the formation of pores on pathogen membranes. The complement components involved in MAC formation are C5b, C6, C7, C8alpha, C8beta, C8gamma and C9. Among them, C6 protein interacts with C5b through a metastable binding site to form a soluble C5b-6 dimer in the vicinity of the activating cell. Formation of the MAC is controlled by complement regulatory molecules, such as CD59, vitronectin and clusterin. Here, we report the molecular characterization of the C6 complement component, as well as the spatial and temporal expression profile of MAC structural (C6, C7, C8alpha, C8beta, C8gamma) and regulatory (CD59, vitronectin and clusterin) genes in chicken (Gallus gallus). The deduced polypeptide sequence of chicken C6 consists of 935 amino acid residues and exhibits 81%, 58%, 56% and 44% identity with zebra finch, human, frog and trout orthologs, respectively. The 'domain' architecture of chicken C6 resembles that of mammalian counterparts and the cysteine backbone is also conserved. MAC structural and regulatory genes are expressed in a wide range of adult chicken tissues, with the liver being the major source of their produced transcripts. The developmental expression profile of chicken MAC structural genes shows that their transcripts initially appear in the 12th embryonic day in the liver, exhibiting a pick in the 17th, while no expression was detected in the early whole embryo (day 4 and 6), as well as in the 2-day old neonate chicken liver. On the other hand, MAC regulatory genes are expressed in all the developmental stages investigated.

Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues

Gangliosides are considered to be essential in the maintenance and repair of nervous tissues; however, the mechanisms for neurodegeneration caused by ganglioside defects are unknown. We examined gene expression profiles in double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes and showed that the majority of complement genes and their receptors were up-regulated in cerebellum in DKO mice. Inflammatory reactions were demonstrated in those tissues by measuring up-regulated inflammatory cytokines, indicating the presence of complement activation and inflammation as reported in Alzheimer's disease. Immunoblotting of fractionated membrane extracts by sucrose density gradient revealed that complement-regulatory molecules such as decay-accelerating factor and CD59 were dispersed from glycolipid-enriched microdomain/rafts in DKO cerebellum. Immunohistostaining of these molecules showed disordered membrane localization. These results suggested that dysfunction of complement-regulatory molecules may be due to abnormal glycolipid-enriched microdomain/rafts that triggered complement activation, subsequent inflammation, and neurodegeneration in DKO mice. Generation of the triple KO mice lacking complement activity in addition to the two glycosyltransferases suggested that complement activation is involved in the inflammatory reactions and neurodegeneration caused by the ganglioside deficiency.