Complement C3 is a novel plasma clot component with anti-fibrinolytic properties

Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes.

Levels of complement C3 and C4 components in Amerindians living in an area with high prevalence of tuberculosis

The levels of complement C3 and C4 components were determined in non-indigenous (creoles) and indigenous (Warao) populations, the latter with an extremely high tuberculosis (TB) rate. Serum samples from 209 adults were studied and classified in 4 groups taking into account tuberculin skin tests (TST): (1) the group of Warao patients (58 positive for the TST, WP TST+ and 9 negative for the TST, WP TST-), (2) the group of creole patients (34 positive for the TST, CP TST+ and 9 negative for the TST, CP TST-), (3) the group of healthy Warao controls (38 positive and 14 negative for TST, WC TST+ and WC TST-, respectively), (4) the creole controls (26 positive and 21 negative for the TST, CC TST+ and CC TST-, respectively). With respect to the results concerning the measurement of both complement C3 and C4 components with the exception of the WC TST and the CC groups, the WP TST+ and WP TST- as well as WC TST+ groups showed a significant frequency of individuals with decreased levels of complement C3 component (20.6, 33.3, and 26.3%, respectively) and also C4 component (12.0, 11.1, and 13.3%, respectively) in comparison to both creole patients (CP TST+, 8.82% and CP TST-, 0% and CP TST+, 5.88% and CP TST-, 0%) for C3 and C4, respectively. The study of these parameters carried out in 15 Warao subjects with active infection, before and after anti-TB chemotherapy,statisticallyconfirmedthat the effective chemotherapy did not restore normal levels of the complement C3 and C4 components among Warao patients. Aditional tests for hepatitis B or hepatitis C infection, and the profile of the hepatic proteins were not associated to the deficiency in production of the complement components.In conclusion, the results show that within the Warao population, a high percentage of subjects exhibit decreased levels of both complement C3 and C4 components independent of latent or active infection and the status of TST.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband`s FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient`s fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient`s C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

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

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.

A novel mechanism for hypofibrinolysis in diabetes: the role of complement C3

Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies.

Gender-specific modulation of immune system complement gene expression in marine medaka Oryzias melastigma following dietary exposure of BDE-47

BDE-47 is one of the most widely found congeners of PBDEs in marine environments. The potential immunomodulatory effects of BDE-47 on fish complement system were studied using the marine medaka Oryzias melastigma as a model fish. Three-month-old O. melastigma were subjected to short-term (5 days) and long-term (21 days) exposure to two concentrations of BDE-47 (low dose at 290 +/- 172 ng/day; high dose at 580 +/- 344 ng/day) via dietary uptake of BDE-47 encapsulated in Artemia nauplii. Body burdens of BDE-47 and other metabolic products were analyzed in the exposed and control fish. Only a small amount of debrominated product, BDE-28, was detected, while other metabolic products were all under detection limit. Transcriptional expression of six major complement system genes involved in complement activation: C1r/s (classical pathway), MBL-2 (lectin pathway), CFP (alternative pathway), F2 (coagulation pathway), C3 (the central component of complement system), and C9 (cell lysis) were quantified in the liver of marine medaka. Endogenous expression of all six complement system genes was found to be higher in males than in females (p < 0.05). Upon dietary exposure of marine medaka to BDE-47, expression of all six complement genes were downregulated in males at day 5 (or longer), whereas in females, MBl-2, CFP, and F2 mRNAs expression were upregulated, but C3 and C9 remained stable with exposure time and dose. A significant negative relationship was found between BDE-47 body burden and mRNA expression of C1r/s, CFP, and C3 in male fish (r = -0.8576 to -0.9447). The above findings on changes in complement gene expression patterns indicate the complement system may be compromised in male O. melastigma upon dietary exposure to BDE-47. Distinct gender difference in expression of six major complement system genes was evident in marine medaka under resting condition and dietary BDE-47 challenge. The immunomodulatory effects of BDE-47 on transcriptional expression of these complement components in marine medaka were likely induced by the parent compound instead of biotransformed products. Our results clearly demonstrate that future direction for fish immunotoxicology and risk assessment of immunosuppressive chemicals must include parallel evaluation for both genders.

Hypofibrinolysis in type 2 diabetes: the role of the inflammatory pathway and complement C3

AIMS/HYPOTHESIS Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes. METHODS Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay. RESULTS Clot lysis time correlated with C3 and PAI-1 plasma levels (r = 0.24, p < 0.001 and r = 0.22, p < 0.001, respectively). In a multivariable regression model involving age, sex, BMI, C3, PAI-1, CRP and fibrinogen, and using log-transformed data as appropriate, C3 was associated with clot lysis time (regression coefficient 0.227 [95% CI 0.161, 0.292], p < 0.001), as was PAI-1 (regression coefficient 0.033 [95% CI 0.020, 0.064], p < 0.05) but not fibrinogen (regression coefficient 0.003 [95% CI -0.046, 0.051], p = 0.92) or CRP (regression coefficient 0.024 [95% CI -0.008, 0.056], p = 0.14). No correlation was demonstrated between plasma levels of C3 and PAI-1 (r = -0.03, p = 0.44), consistent with previous observations that the two proteins affect different pathways in the fibrinolytic system. CONCLUSIONS/INTERPRETATION Similarly to PAI-1, C3 plasma levels are independently associated with fibrin clot lysis in individuals with type 2 diabetes. Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fibrinolysis potential in this population.

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. ^

Leptospirosis pulmonary haemorrhage syndrome is associated with linear deposition of immunoglobulin and complement on the alveolar surface

Leptospirosis is a zoonotic infection associated with severe diseases such as leptospirosis pulmonary haemorrhage syndrome (LPHS). The cause of pulmonary haemorrhage is unclear. Understanding which mechanisms and processes are involved in LPHS will be important in treatment regimens under development for this life-threatening syndrome. In the present study, we evaluated 30 lung specimens from LPHS patients and seven controls using histology and immunohistochemistry (detection of IgM, IgG, IgA and C3) in order to describe the pathological features associated with this syndrome. Immunoglobulin deposits were detected on the alveolar surface in 18/30 LPHS patients. Three staining patterns were observed for the immunoglobulins and C3 in the lung tissues of LPHS patients: AS, delicate linear staining adjacent to the alveolar surface, which was indicative of a membrane covering the luminal surface of type I and II pneumocyte cells; S, heterogeneous staining which was sporadically distributed along the alveolar septum; and IA, weak, focal intra-alveolar granular staining. Human LPHS is associated with individual and unique histological patterns that differ from those of other causes of pulmonary haemorrhage. In the present study, it was found that the linear deposition of immunoglobulins (IgA, IgG and IgM) and complement on the alveolar surface may play a role in the pathogenesis of pulmonary haemorrhage in human leptospirosis.

In vitro and in situ activation of the complement system by the fungus Lacazia loboi

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 ºC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.

Immunologic evaluation in infective endocarditis

OBJECTIVE: To analyze the immune response in peripheral blood of patients with infective endocarditis. METHODS: We studied 10 patients with infective endocarditis, age range from 20 to 50 years-old, males and females, and 20 healthy subjects in the same age range. The diagnosis of the disease was based on the clinical picture, echocardiogram, and hemoculture based upon samples drawn and tested before the treatment started. The were no history of atopy or malnutrition, no autoimmune disease, and they were not using any immunosuppressant or antibiotic medication. RESULTS: The patients with endocarditis had significantly higher T and B lymphocyte, CD4+ and CD8+ cell counts, IgM and IgG serum levels, and C4 component of the complement than the control group; no significant difference concerning serum IgA and neutrophil oxidative metabolism; a significant decrease in C3, chemotaxis, and monocyte phagocytosis;cryoglobulins were detected in 66.6% of patients and they were formed by IgG, IgM, IgA, C3, and C4. CONCLUSION: The patients with infective endocarditis were immunocompetent in most sectors of immune response and, at a certain moment, an autoimmune component may be present.

Complement facilitates early prion pathogenesis.

New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

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.