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PubMed2005

Effects of heparin on bacterial translocation and gut epithelial apoptosis after burn injury in the rat: dose-dependent inhibition of the complement cascade.

This study investigated levels of complement inhibition, apoptosis of gut epithelium, and bacterial translocation (BT) associated with different doses of heparin in rats with severe burns. After burn injury, the animals in Groups 1, 2, 3, and 4 received intravenous tail-vein bolus heparin doses of 150, 300, 600, and 1200 U/kg, respectively. Group 5 received no heparin after burn injury. Group 6 served as control group. According to the results, Group 2 had the highest rate of positive staining for C3, and Group 4 had the lowest rate. There were significant differences between these two groups with respect to distribution of immunoflouresein scores for C3 (p=0.01). Group 5 had the highest mean TUNEL index of all the groups (258/10) (p=0.01). On electron microscopy, the connective tissue cells in the ileal submucosa from Groups 4 and 5 showed more significant apoptotic changes than the corresponding cells in the other groups. The total BT values in Group 4 (129 x 10(4) CFU) and Group 5 (100 x 10(4) CFU) were both significantly higher than those in the other groups (p=0.01). Group 1 had the lowest total BT value (6.1 x 10(2) CFU) (p=0.001). In summary, our results confirm that heparin administration after significant burn injury in rats can reduce BT, and that the effect is related to dose. The findings also indicate that levels of BT after burn injury increase in parallel with the extent of gut epithelial cell apoptosis.

Animals↗
PubMed2005

Complement-inhibiting effect of ovarian cancer antigen CA-125.

Malignant transformation of ovarian cells of surface epithelial origin is associated with expression of a membrane-spanning glycoprotein, cancer antigen (CA)-125. The bulk of the putative CA-125 molecule is comprised a very large, folded, multivalent, mucin-like exodomain. That the extracellular motif of CA-125 exerts immunosuppressive effects which promote tumor progression has been suggested. We report that CA-125 attenuates complement lysis of antibody-sensitized cells. The secreted form of CA-125 derived from culture medium of the human ovarian adenocarcinoma cell line OVCAR-3 caused a dose-response inhibition of sheep erythrocyte hemolysis. Moreover, OVCAR-3 cells became prone to complement attack (trypan blue uptake) mediated by a gonadotropin-releasing hormone receptor antibody when (membrane-bound) CA-125 was excised/removed by trypsin/washing; this effect was counteracted by replacement with (soluble) CA-125. It is conceivable that CA-125 entraps/sheds effectors of the complement cascade.

Animals↗
PubMed2005

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient.

OBJECTIVE: Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. METHODS: We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. RESULTS: DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. CONCLUSIONS: Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.

Animals↗
PubMed2006

Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways.

Studies in rodent models suggested that complement may play a critical role in susceptibility to airway hyperresponsiveness (AHR) and as a mediator of bronchial obstruction and inflammation in asthma. Complement may participate in susceptibility to asthma because of an intrinsic abnormality in complement activation and generation of C3a, C5a, or other products that affect cellular responses, resulting in T(H)2 predominance and asthma susceptibility. Alternatively, an intrinsic abnormality in the cellular response to complement activation products could determine susceptibility to asthma. In this study, the authors investigated whether complement in patients with atopic asthma versus nonatopic controls possesses an increased propensity to become activated. Despite reports that total complement plasma levels in unchallenged asthmatics are normal, an abnormal sensitivity of complement to activation may exist if an isoform or a polymorphic variant of a complement protein was present and resulted in gain or loss of function without associated changes in total complement levels. Therefore, complement activation was induced in vitro in plasma of asthmatics and controls using activators of the classical, alternative, and lectin pathways and measured C3a, other C3 fragments, and C5a. For each pathway, similar amounts of generated fragments, as well as C3a/C3 and C5a/C5 ratios, were found in asthmatics and controls. Also, similar basal plasma levels of C3a and C5a were found in both groups; however, mannan-binding lectin (MBL) levels were moderately elevated in asthmatics. In conclusion, the results suggest that, in asthmatic patients, complement activation does not exhibit an abnormal sensitivity to activation by any of the known activation pathways.

Adult↗
PubMed2004

Serum haemolytic complement activities in 11 different MHC (B) typed chicken lines.

To study the relation between serum complement levels and the chicken MHC (B) complex, complement haemolytic activity was measured in sera from hens from seven pure-bred B-typed White and one Brown Leghorn lines, and three ISA-Warren lines that had been divergently selected for antibody responses to sheep red blood cells (SRBC). Significant differences occurred in the serum haemolytic complement activities, both belonging to the classic (CPW) and the alternative (APW) pathways, among the 11 different haplotyped chicken lines. Hens with high CPW and high APW titres predominantly displayed the B2 or B21 haplotypes. Chickens with low CPW and APW were found in B14 and B15 haplotypes. Haplotype B14 appears to be different in complement levels when present into the pure-bred lines or into the ISA-Warren line selected for low antibody responses to SRBC. Otherwise, the presence of B21 in ISA-Warren line selected for high antibody responses to SRBC does not differ with the B21 in the inbred lines (except in the NL-line for CPW values). In general the haplotypes B2 and B21 are found in chicken lines with enhanced disease resistance, and the B15 haplotype has been connected with enhanced disease susceptibility. Our results suggest that levels of haemolytic complement activity, either from the classical or from the alternative pathways, may underlie part of the immunocompetence ascribed to the MHC (B) complex in chickens.

Animals↗
PubMed2002

Associations between Fc gamma receptor IIA polymorphisms and the risk and prognosis of meningococcal disease.

BACKGROUND: In vitro studies have shown that the neutrophil Fc gamma receptor IIA (FcgammaRIIA) polymorphism influences the phagocytic capacity of neutrophils and the removal of encapsulated bacteria from the bloodstream. In particular, the R/R131 allotype is associated with less phagocytic activity. SUBJECTS AND METHODS: We performed a case-control study to determine the influence of the FcgammaRIIA polymorphism (R/R131, R/H131, H/H131) on the risk and outcome of meningococcal disease. The polymorphisms were measured in 130 patients with microbiologically proven meningococcal disease diagnosed from 1987 to 1998 (cases) and 260 asymptomatic sex-matched blood donors (controls). Clinical manifestations and complications of meningococcal disease were recorded, and a prognostic score (based on age, hemorrhagic diathesis, neurologic signs, and the absence of preadmission antibiotic) therapy was calculated. RESULTS: The distributions of FcgammaRIIA allotypes were similar in cases and controls. However, among patients with meningococcal infection, fulminant meningococcal disease (odds ratio [OR] = 3.9; 95% confidence interval [CI]: 1.0 to 16; P = 0.04) and meningococcemia without meningitis (OR = 3.0; 95% CI: 1.4 to 7.8; P = 0.004) were more common in those with the FcgammaRIIA-R/R131 allotype. Complications were also significantly more frequent in these patients. Of the 42 patients with the R/R131 allotype, 31 (74%) had an adverse prognostic score, compared with 7% (4 of 59) of those with the R/H131 allotype and 3% (1 of 29) of those with the H/H131 allotype (P <0.0001). CONCLUSION: The FcgammaRIIA-R/R131 allotype is associated with more severe forms of meningococcal disease.

Adolescent↗
PubMed1997

Effect of whole and fractionated intravenous immunoglobulin on complement in vitro.

Intravenous immunoglobulins (IVIG) are increasingly used for treatment of inflammatory diseases, and the modulation of complement may contribute to some of its beneficial effects. IVIG may bind C1q and activated C3 and C4, and enhance inactivation of C3b. We have previously shown that IVIG inhibited complement-mediated lysis solely via its Fc part through interaction with the classical pathway. In the present study we have investigated whole IVIG (Octagam, and Sandoglobulin) and the monomer, dimer and multimer fractions of Octagam with respect to complement activation in serum and inhibition of complement lysis of red cells. The isolated fractions were found to be stable, homogeneous (monomer, dimer or multimer) and pure (virtually only IgG). Both whole IVIG and its fractions significantly activated complement in serum and inhibited hemolysis compared with human albumin. These effects were most pronounced in the monomer, less in the multimer and least in the dimer fraction. The complement activation was shown to be mediated through the classical pathway since formation of C1rs-C1inh complexes and C4bc were increased, in contrast to Bb. Surprisingly, heat aggregation of Octagam was not followed by a corresponding increase in complement activation, as would be expected, unless it was dialysed before heating, suggesting that it is stabilized to avoid excess activation. In conclusion, the results support the hypothesis that IVIG causes a mild activation of complement in vitro. We suggest that this effect may contribute to the complement inhibitory properties of IVIG by diverting complement deposition from the target to the fluid phase.

Animals↗
PubMed1999

Inhibition of human and mouse complement-dependent hemolytic activity by mouse fibronectin.

Not only does mouse complement (C) have low hemolytic activity, but mouse serum has an inhibiting effect on hemolysis by human C. To purify and identify the putative mouse serum factor inhibiting human C activity, a sequential procedure of fractionated precipitation by PEG, followed by chromatographies with a heparin-Sepharose column, a phenyl-Sepharose column, a Protein G column, and a gel-filtration column was performed. The amino acid sequence analyses of two polypeptides obtained by digestion of the purified serum factor with TPCK-trypsin revealed that it was mouse fibronectin (FN). Highly purified mouse FN, but not human FN, has an inhibiting effect on human C-dependent hemolysis. Moreover, the hemolysis of sensitized rabbit erythrocytes by mouse C was also inhibited by the addition of mouse FN in a dose-dependent fashion, but not by the addition of human FN. These results suggest that FN is the putative internal C inhibitor in the mouse system.

Animals↗
PubMed2001

Effect of oral administration of high vitamin C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system.

The effect of the oral administration of high dosages of vitamins C and E on the innate immune system of the seabream was investigated. Gilthead seabream (Sparus aurata L.) were fed four different diets for 45 days: a commercial diet as control, a 3g/kg vitamin C-supplemented diet, a 1.2g/kg vitamin E-supplemented diet or a diet containing both vitamin supplements. After 15, 30 and 45 days, serum ascorbic acid and alpha-tocopherol levels, growth, complement titers and head-kidney leucocyte phagocytic and respiratory burst activities were evaluated. The results showed that serum vitamin levels reflected dietary input. Fish fed the vitamin C-supplemented diet showed an enhanced respiratory burst activity, while fish fed the vitamin E-supplemented diet exhibited increased complement and phagocytic activities. All of these positive effects were found in fish fed the joint diet, as well as a synergistically enhanced respiratory burst activity at day 30. The results demonstrate that in vivo vitamins C and E exert a synergistic effect enhancing the respiratory burst activity of seabream phagocytes.

Administration, Oral↗
PubMed1997

Complement activates neutrophils during PTCA procedure in patients with unstable angina pectoris.

We estimated adherence, aggregation and chemiluminescence of neutrophils as well as concentrations of C3c, C4 and C5 complement components and complement haemolytic activity (CH50) in 27 patients with unstable angina pectoris subjected to percutaneous transluminal coronary angioplasty (PTCA). The control group consisted of 12 patients with unstable angina pectoris, in whom coronary angiography was performed but PTCA was decided against for various reasons. Blood samples for examination were taken from coronary sinus and peripheral vein just before, 1 min and 20 min after PTCA or coronary angiography. We observed enhancement of neutrophil adherence, aggregation and chemiluminescence, and decrease in concentrations of C3c, C5 and complement haemolytic activity (CH50) after PTCA procedure. In conclusion we think that ischemia resulting from PTCA causes complement activation in an alternative pathway which seems to be connected with neutrophil activation.

Acute-Phase Reaction↗
PubMed1998

Effect of soluble complement receptor type 1 on porphyrin-induced phototoxicity in guinea pigs.

The effect of recombinant human soluble complement receptor type 1 (sCR1) on the porphyrin-mediated phototoxic reaction was evaluated in guinea pigs. Phototoxicity was induced in the animals by intraperitoneal injection of hematoporphyrin derivative (HpD), followed by irradiation at a wavelength range of 320-450 nm. sCR1 administration decreased CH50 titers in a dose-dependent fashion, while it only moderately suppressed HpD/radiation-induced ear swelling at a high dose. These findings suggest that phototoxic dermatological changes in cutaneous porphyrias are not solely due to complement activation.

Animals↗
PubMed1997

Leishmania amazonensis promastigotes evade complement killing by interfering with the late steps of the cascade.

During their growth in vitro, promastigotes of Leishmania amazonensis undergo differentiation from complement-susceptible to complement-resistant forms. Here, we demonstrate that both forms bind comparable amounts of C3 on their surfaces, with the predominant molecule species being the haemolytically active C3b. Likewise, equivalent amounts of C9 are deposited on both forms of promastigotes. However, while C9-bearing complexes are exposed on the cell surface of resistant promastigotes, they are cryptic in the susceptible stage of the parasites. The membrane fraction of complement-resistant promastigote lysates has the ability to inhibit complement-mediated haemolysis, blocking C9, but not C3 deposition to complement-activating complexes. Moreover, the membrane fraction of complement-resistant promastigote lysates can inhibit the late steps of guinea-pig erythrocyte lysis much more efficiently than complement-susceptible ones. Our results indicate that L. amazonensis promastigotes evade complement killing by inhibiting the cytolytic pathway of the complement cascade.

Animals↗
PubMed2000

An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma.

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.

Amino Acid Sequence↗
PubMed1998

Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation.

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.

Amino Acid Sequence↗
PubMed1999

Chimeric and truncated forms of human complement protein C8 alpha reveal binding sites for C8 beta and C8 gamma within the membrane attack complex/perforin region.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.

Animals↗
PubMed2003

Demonstration of alternative and classical complement pathway activity in colostrum from buffalo (Bubalus bubalis).

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50 degrees C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg(2+) in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81-6.77 CH50/ml.

Animals↗
PubMed2004

Different serum haemolytic complement levels in indigenous chickens from Benin, Bolivia, Cameroon, India and Tanzania.

Titres of classical (CPW) and alternative (APW) complement pathways were measured in clinically healthy local chicken populations (ecotypes) from Africa (Benin, n = 78; Cameroon, n = 299; Tanzania, n = 101), Asia (India, n = 96) and South America (Bolivia, n = 64). A wide variation was found in haemolytic complement levels between the various ecotypes. Distributions of the classical and alternative complement titres were not normal but were skewed to the right. Differences in complement were found both within and between ecotypes. Furthermore, CPW titres of the indigenous chickens were lower than those determined in commercial layer chickens. This suggests that complement levels background and husbandry. The relationships between complement levels, the chicken MHC(B) complex, environmental antigenic pressure, and survival of the scavenging local chickens are discussed.

Africa↗