Evaluation of the Complement System and Human Complement 1 Inhibitor (C1-INH) in a Pig Lung Transplant Survival Model.

ABSTRACT Chikungunya virus (CHIKV) is an emerging pathogen capable of causing explosive outbreaks. Prior studies showed that exacerbation in arthritogenic alphavirus-induced pathogenesis is attributed to its interaction with multiple immune components, including the complement system. Viremia concomitant to CHIKV infection makes exposure of the virus to complement unavoidable, yet very little is known about CHIKV-complement interactions. Here, we show that CHIKV activated serum complement to modest levels in a concentration- and time-dependent manner, but the virus effectively resisted complement-mediated neutralization. Heat-inactivated serum from seropositive donors could actively neutralize CHIKV due to the presence of potent anti-CHIKV antibodies. Deposition of key complement components C3 and C4 did not alter the resistance of CHIKV to complement. Further, we identified a factor I-like activity in CHIKV that limited complement by inactivating C3b into inactive C3b (iC3b), the complement component known to significantly contribute to disease severity in vivo, but this activity had no effect on C4b. Inactivation of C3b by CHIKV was largely dependent on the concentration of the soluble host cofactor factor H and the virus concentration. A factor I function-blocking antibody had only a negligible effect on the factor I-like activity associated with CHIKV, suggesting that this activity is independent of host factor I and could be of viral origin. Thus, our findings suggest a complement modulatory action of CHIKV which not only helps the virus to evade human complement but may also have implications in alphavirus-induced arthritogenic symptoms. IMPORTANCE Chikungunya virus is a vector-borne pathogen of global significance. The morbidity associated with chikungunya virus (CHIKV) infection, neurovirulence and adaptability to Aedes albopictus, necessitates a deeper understanding of the interaction of CHIKV with the host immune system. Here, we demonstrate that CHIKV is resistant to neutralization by one of the potent barriers of the innate immune arm, the complement system. Chikungunya virus showed marked resistance to complement despite activation and deposition of complement proteins. Interestingly the C3 component associated with the virion was found to be inactive C3b (iC3b), a key factor implicated in the pathogenesis and disease severity in the mouse model of Ross River virus infection. CHIKV also had an associated unique factor I-like activity that mediated the inactivation of C3b into iC3b. We have unraveled a smart strategy adopted by CHIKV to limit complement which has serious implications in viral dissemination, pathogenesis, and disease.

Download Full-text

Safety and Efficacy of Ex Vivo Donor Lung Adenoviral IL-10 Gene Therapy in a Large Animal Lung Transplant Survival Model

Human Gene Therapy ◽

10.1089/hum.2016.070 ◽

2017 ◽

Vol 28 (9) ◽

pp. 757-765 ◽

Cited By ~ 40

Author(s):

Tiago N. Machuca ◽

Marcelo Cypel ◽

Riccardo Bonato ◽

Jonathan C. Yeung ◽

Yi-Min Chun ◽

...

Keyword(s):

Gene Therapy ◽

Lung Transplant ◽

Ex Vivo ◽

Survival Model ◽

Large Animal ◽

Safety And Efficacy ◽

Transplant Survival ◽

Donor Lung

Download Full-text

Alpha 1 Antitrypsin to Prevent Ischemia Repercusión Injury in a Pig Lung Transplant Survival Model

The Journal of Heart and Lung Transplantation ◽

10.1016/j.healun.2018.01.184 ◽

2018 ◽

Vol 37 (4) ◽

pp. S81

Author(s):

A. Mariscal ◽

L. Caldarone ◽

M. Chen ◽

M. Cypel ◽

J. Tikkanen ◽

...

Keyword(s):

Lung Transplant ◽

Survival Model ◽

Transplant Survival ◽

Alpha 1 Antitrypsin

Download Full-text

A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity

Alternatives to Laboratory Animals ◽

10.1177/0261192920953170 ◽

2020 ◽

Vol 48 (3) ◽

pp. 127-135

Author(s):

Ruby Anne N. King ◽

Fresthel Monica M. Climacosa ◽

Bobbie Marie M. Santos ◽

Salvador Eugenio C. Caoili

Keyword(s):

Guinea Pig ◽

Complement System ◽

Clinical Setting ◽

Human Erythrocyte ◽

Human Erythrocytes ◽

Human Complement ◽

Complement Activity ◽

Functional Assays ◽

The Complement System ◽

Complement Pathways

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

Download Full-text

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646373 ◽

1991 ◽

Vol 66 (01) ◽

pp. 049-061 ◽

Cited By ~ 233

Author(s):

Björn Dahlbäck

Keyword(s):

Vitamin K ◽

Complement System ◽

Protein C ◽

Protein S ◽

High Affinity ◽

Anticoagulant System ◽

Dependent Protein ◽

The Complement System ◽

Β Chain ◽

Dependent Domain

SummaryThe protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF)epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 γ-carboxy glutamic acid residues in the vitamin K-dependent domain, a β-hydroxylated aspartic acid in the first EGF-like domain and a β-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical α-chains and one β-chain. The α-and β-chains are linked by disulphide bridges. The cDNA cloning of the β-chain showed the α- and β-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the β-chain to contain the single protein S binding site on C4BP, whereas each of the α-chains contains a binding site for the complement protein, C4b. As C4BP lacking the β-chain is unable to bind protein S, the β-chain is required for protein S binding, but not for the assembly of the α-chains during biosynthesis. Protein S has a high affinity for negatively charged phospholipid membranes, and is instrumental in binding C4BP to negatively charged phospholipid. This constitutes a novel mechanism for control of the complement system on phospholipid surfaces. Recent findings have shown circulating C4BP to be involved in yet another calcium-dependent protein-protein interaction with a protein known as the serum amyloid P-component (SAP). The binding sites on C4BP for protein S and SAP are independent. SAP, which is a normal constituent in plasma and in tissue, is a so-called pentraxin being composed of 5 non-covalently bound 25 kDa subunits. It is homologous to C reactive protein (CRP) but its function is not yet known. The specific high affinity interactions between protein S, C4BP and SAP suggest the regulation of blood coagulation and that of the complement system to be closely linked.

Download Full-text

Stimulated Glanzmann’s Thrombasthenia Platelets Produce Microvesicles

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649978 ◽

1995 ◽

Vol 74 (06) ◽

pp. 1533-1540 ◽

Cited By ~ 23

Author(s):

Pål André Holme ◽

Nils Olav Solum ◽

Frank Brosstad ◽

Nils Egberg ◽

Tomas L Lindahl

Keyword(s):

Complement System ◽

Shape Change ◽

Annexin V ◽

Platelet Surface ◽

Glanzmann’S Thrombasthenia ◽

Large Numbers ◽

Glanzmann's Thrombasthenia ◽

Series Of Experiments ◽

The Complement System ◽

Number Of Particles

SummaryThe mechanism of formation of platelet-derived microvesicles remains controversial.The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann’s thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 μM), and the thrombin receptor agonist peptide SFLLRN (100 μM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 μM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 ±2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 ± 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 μM SFLLRN gave 16.3 ± 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 ± 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.

Download Full-text