The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

A monoclonal antibody which blocks the function of factor D of human complement.

Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Role of complement and perspectives for intervention in ischemia-reperfusion damage

Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

The role of the complement system in ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a common clinical event with the potential to seriously affect, and sometimes kill, the patient. Interruption of blood supply causes ischemia, which rapidly damages metabolically active tissues. Paradoxically, restoration of blood flow to the ischemic tissues initiates a cascade of pathology that leads to additional cell or tissue injury. I/R is a potent inducer of complement activation that results in the production of a number of inflammatory mediators. The use of specific inhibitors to block complement activation has been shown to prevent local tissue injury after I/R. Clinical and experimental studies in gut, kidney, limb, and liver have shown that I/R results in local activation of the complement system and leads to the production of the complement factors C3a, C5a, and the membrane attack complex. The novel inhibitors of complement products may find wide clinical application because there are no effective drug therapies currently available to treat I/R injuries.

Inflammation, complement activation and endothelial function in stable and unstable coronary artery disease

Background: Endothelial dysfunction plays an important role in the pathogenesis of coronary artery disease (CAD). Apart from traditional risk factors complement activation and inflammation may trigger and sustain endothelial dysfunction. We sought to assess the association between endothelial function, high sensitivity C-reactive protein (hs-CRP) and markers of complement activation in patients with either stable or unstable coronary artery disease. Methods: We prospectively recruited 78 patients, 35 patients with stable angina pectoris (SAP) and 43 patients with unstable angina pectoris (UAP). Endothelial function was assessed as brachial artery reactivity (BAR). Hs-CRP, C3a, C5a, and C1-Inhibitor (C1 inh.) were measured enzymatically. Results: Patients with IJAP showed higher median levels of hs-CRP and C3a compared to patients with SAP, while BAR was not significantly different between patient groups. In UAP patients, hs-CRP was significantly correlated with cholesterol (r = 0.27, p < 0.02), C3a (r = 0.32, p < 0.001) and C1 INH.(r = 0.41, p < 0.003), but not with flow mediated dilatation (r = 0.09, P = 0.41). Hs-CRP and C1 INH.were found to be independant predictors of IJAP in a backward stepwise logistic regression model. Conclusions: We conclude that both hs-CRP, a marker of inflammation and C3a, a marker of complement activation are elevated in patients with UAP, but not in patients with SAP. (c) 2005 Elsevier B.V. All rights reserved.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation

Purpose: To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Design: Multicenter, cross-sectional study. Participants: We recruited 223 Western European patients ≥55 years old who had undergone LT ≥5 years previously. Methods: We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). Main Outcome Measures: We evaluated AMD status and recipient and donor CFH Y402H genotype. Results: In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Conclusions: Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. © 2013 by the American Academy of Ophthalmology Published by Elsevier Inc.

Protein-energy malnutrition alters the C3 complement factor in response to lipopolysaccharide in a murine model

A desnutrição proteico-energética modifica a resistência à infecção, modificando diversos processos fisiológicos, incluindo a hematopoiese e as funções imunológicas. Neste estudo, avaliamos as concentrações séricas do fator C3 e do Sistema Complemento total (CH50) em um modelo no qual camundongos submetidos à desnutrição proteico-energética são estimulados com lipopolissacarídeo (LPS), e avaliamos a celularidade periférica, medular e esplênica. Camundongos Swiss, machos, adultos, com dois meses de idade foram submetidos ao processo de desnutrição proteica com uma dieta contendo 4% de proteína em comparação aos animais controles com uma dieta contendo 20% de proteína. Quando os animais do grupo desnutrido alcançaram aproximadamente 20% de perda de peso, em relação ao inicial, foram inoculados endovenosamente com LPS. As células do sangue, da medula óssea e do baço foram quantificadas, bem como as concentrações circulantes de C3 e CH50 em animais estimulados com LPS. Os animais desnutridos apresentaram anemia e leucopenia, além de redução significativa da celularidade da medula óssea e do baço. Os animais desnutridos apresentaram menor taxa de sobrevivência, bem como das concentrações do fator C3 do complemento e do complemento total em relação aos animais controles. Estes resultados sugerem que animais desnutridos apresentam uma resposta deficiente aos LPS. A síntese menor do complemento pode ser em parte responsável pela imunodeficiência observada. Estes resultados conduzem-nos a inferir que a desnutrição proteico-energética interfere na ativação da resposta imune

Biomass Refinery as a Renewable Complement to the Petroleum Refinery

Biomass Refinery is a sequential of eleven thermochemical processes and one biological process with two initial basic treatments: prehydrolysis for lignocellulosics and low temperature conversion for biomass with medium-to-high content of lipids and proteins. The other ten processes are: effluent treatment plant, furfural plant, biodiesel plant, cellulignin dryer, calcination, fluidized bed boiler, authotermal reforming of cellulignin for syngas production, combined cycle of two-stroke low-speed engine or syngas turbine with fluidized bed boiler heat recovery, GTL technologies and ethanol from cellulose, prehydrolysate and syngas. Any kind of biomass such as wood, agricultural residues, municipal solid waste, seeds, cakes, sludges, excrements and used tires can be processed at the Biomass Refinery. Twelve basic products are generated such as cellulignin, animal feed, electric energy, fuels (ethanol, crude oil, biodiesel, char), petrochemical substitutes, some materials (ash, gypsum, fertilizers, silica, carbon black) and hydrogen. The technology is clean with recovery of energy and reuse of water, acid and effluents. Based on a holistic integration of various disciplines Biomass Refinery maximizes the simultaneous production of food, electric energy, liquid fuels and chemical products and some materials, achieving a competitive position with conventional and fossil fuel technologies, as well as payment capacity for biomass production. Biomass Refinery has a technical economical capability to complement the depletion of the conventional petroleum sources and to capture its GHGs resulting a biomass + petroleum ""green"" combination.

Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children`s sera. Cls was undetectable, while in the parents` sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients` fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3` splice site within intron I which increases the size of exon 2 by 87 nucleotides. (c) 2007 Elsevier Ltd. All rights reserved.

Complement-mediated regulation of Trichomonas vaginalis infection in mice

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract, The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T, vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.