In vitro activation of complement and contact system by lactic acidosis.

The activation of complement and contact systems occurs in reperfusion injuries with initial tissue hypoxia, and lactic acidosis such as mycardial infarction and birth asphyxia. The aim of our experiment was the formal proof of activation by sole lactic acidosis. Lactic acid was added to blood and plasma samples from 10 healthy volunteers. C5a and factor XIIa were measured by EIA after incubation at 37 degrees C for 1 h. Both concentrations increased (P < 0.0001 by Friedman analysis) in blood and plasma samples with increasing amount of added lactic acid. Lactic acidosis can activate C5 from the complement system and factor XII from the contact system directly, even in the absence of cellular components.     

Acidosis activates complement system in vitro.

We investigated the in vitro effect of different forms of acidosis (pH 7.0) on the formation of anaphylatoxins C3a and C5a. Metabolic acidosis due to addition of hydrochloric acid (10 micromol/ml blood) or lactic acid (5.5 micromol/ml) to heparin blood (N=12) caused significant activation of C3a and C5a compared to control (both p=0.002). Respiratory acidosis activated C3a (p=0.007) and C5a (p=0.003) compared to normocapnic controls. Making blood samples with lactic acidosis hypocapnic resulted in a median pH of 7.37. In this respiratory compensated metabolic acidosis, C3a and C5a were not increased. These experiments show that acidosis itself and not lactate trigger for activation of complement components C3 and C5.     

Inhibition by epsilon-aminocaproic acid of the activation of the first component of the complement system.

The influence of EACA on C1 in whole human serum and on C1 and C (see article) as isolated molecules was assessed hemolytically. There was selective inhibition of C1 without effect on the levels of C4, C2, C3, and C9 in whole human serum that was reversed by dialysis. EACA was found to inhibit the intrinsic activation of C1 without inhibiting the already active molecule. This was confirmed by the capacity of trypsin to uncover C1 activity in cellular intermediates formed by C1 treated with EACA that did not evolve in the absence of this extrinsic activating mechanism. Inasmuch as the trypsin-dependent recovery of C1 was incomplete, an effect on binding cannot be excluded.     

Non-linear dynamics of the complement system activation

The complement system (CS) plays a prominent role in the immune defense. The goal of this work is to study the dynamics of activation of the classic and alternative CS pathways based on the method of mathematical modeling. The principal difficulty that hinders modeling effort is the absence of the measured values of kinetic constants of many biochemical reactions forming the CS. To surmount this difficulty, an optimization procedure consisting of constrained minimization of the total protein consumption by the CS was designed. The constraints made use of published data on the in vitro kinetics of elimination of the Borrelia burgdorferi bacteria by the CS. Special features of the problem at hand called for a significant modification of the general constrained optimization procedure to include a mathematical model of the bactericidal effect of the CS in the iterative setting. Determination of the unknown kinetic constants of biochemical reactions forming the CS led to a fully specified mathematical model of the dynamics of cell killing induced by the CS. On the basis of the model, effects of the initial concentrations of complements and their inhibitors on the bactericidal action of the CS were studied. Proteins playing a critical role in the regulation of the bactericidal action of the CS were identified. Results obtained in this work serve as an important stepping stone for the study of functioning of the CS as a whole as well as for developing methods for control of pathogenic processes.     

Anti-inflammatory drugs, eicosanoids and the annexin A1/FPR2 anti-inflammatory system

The action of anti-inflammatory and anti-allergic drugs on the eicosanoid system is briefly reviewed. In addition to the aspirin-like drugs, which directly inhibit the cyclo-oxygenase enzymes, other drugs such as the glucocorticoids and the cromones also inhibit the formation of eicosanoids. In the latter cases this is bought about through the release of a protein factor that acts through formyl peptide receptors on the target cell surface. Of growing interest, is the observation that this receptor is also a target for other eicosanoids, such as lipoxins and resolvins that modulate host defence systems.     

Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo.

Complement plays a role in activating the inflammatory response and has been implicated in the pathogenesis of some inflammatory diseases. With a view toward controlling unwanted C activation, we evaluated the C regulator, human decay accelerating factor (DAF). Three forms of recombinant DAF were purified from transfected Chinese hamster ovary cells: glycophosphatidylinositol (GPI)-linked membrane DAF (mDAF) extracted from cell membranes; spontaneously shed soluble DAF (sDAF) derived from mDAF; and a novel secreted protein (seDAF), generated by deletion of the signal for GPI attachment. We show that all three molecules inhibit both the classical and alternative pathways of C activation. The following observations indicate that mDAF extracted from Chinese hamster ovary cells reincorporates into RBC membranes via its GPI anchor: 1) cells that are preincubated with mDAF and then washed remain fully protected from C-mediated hemolysis; 2) incubation with phosphatidylinositol-specific phospholipase C abolishes this protection; and 3) sDAF and seDAF, which lack a GPI anchor, do not associate with cell membranes. mDAF is a more potent inhibitor of C-mediated hemolysis than either sDAF or seDAF, suggesting that incorporation into cell membranes greatly enhances the efficiency with which DAF inhibits C activation on the cell surface. In contrast, C activation in the fluid phase is inhibited by sDAF and seDAF, but not by mDAF, possibly due to interference by serum lipoproteins. A reversed passive Arthus reaction in guinea pigs was used to evaluate the ability of recombinant seDAF to inhibit C activation in vivo. When administered at dermal sites, seDAF reduced the severity of immune complex-mediated inflammatory reactions induced by a reversed passive Arthus reaction, as judged by both gross and histologic examination. These data indicate that seDAF may be useful as an anti-inflammatory therapeutic.     

Spermine prevents endonuclease activation and apoptosis in thymocytes.

Glucocorticoid hormones, Ca2+ ionophores, and some toxic chemicals activate a suicide process in thymocytes, known as apoptosis or programmed cell death. A crucial event in apoptosis is the activation of a Ca(2+)- and Mg(2+)-dependent endonuclease that promotes extensive DNA fragmentation. In this study, we investigated the effect of various polyamines on endonuclease activation leading to thymocyte apoptosis. We found that both glucocorticoid- and Ca2+ ionophore-induced DNA fragmentation and apoptosis were prevented by spermine. Other polyamines such as putrescine or spermidine had moderate or no effect. Moreover, spermine, and to a lesser extent spermidine, but not putrescine, prevented endonuclease activation in permeabilized liver nuclei incubated in the presence of Ca2+ and Mg2+, indicating that spermine efficiency in blocking DNA fragmentation was related to the interaction of this polyamine with the endonuclease or its substrate, DNA. Experiments with the fluorescent dye, ethidium bromide, and a purified preparation of liver endonuclease revealed that the protective effect of spermine on DNA fragmentation was related to its ability to modify the chromatin arrangement. Thymocytes incubated with methyl glyoxal bis(guanylhydrazone) to deplete intracellular spermine exhibited spontaneous DNA fragmentation, which suggests that modulation of the intracellular polyamine content and regulation of chromatin structure may play a critical role in the early phases of apoptosis. Finally, these results demonstrate that inhibition of DNA fragmentation also prevents the onset of apoptosis, directly linking endonuclease activation and cell death.     

In-vitro activation of complement system by lactic acidosis in newborn and adults

INTRODUCTION: Complement activation occurs secondary to a variety of external stimuli. Lactic acidosis has been previously shown to activate the complement factors C3a and C5a. In the present investigation we examined the differential effect of lactic acidosis on anaphylatoxin levels in cord and adult blood. Furthermore we aimed to determine if the entire complement cascade could be activated by lactic acidosis. METHODS: Cord and adult blood samples (n = 20 each) were collected and incubated for one hour in either untreated condition or with the addition of lactate in two concentrations (5.5 mmol/l vs. 22 mmol/l). Following incubation, levels of C3a, C5a and sC5b-9, and blood gas parameters were determined. RESULTS: Anaphylatoxin (C3a and C5a) and sC5b-9 levels increased with the addition of lactate in a dose-dependent manner in cord and adult blood (C3a: 1 h, 5.5 mmo/l, 22 mmol/l: 418/498/622 microg/l in cord blood; 1010/1056/1381 microg/l in adult blood, p<0,05; similar results were found for C5a and sC5b-9). CONCLUSION: Lactic acidosis leads to an activation of the entire complement system in neonates and in adults. This activation is dose-dependent and more pronounced in adults as compared to neonates.     

Activation of the alternative complement pathway by Newcastle disease virus. I) Effect of the virus on the complement system in vivo and in vitro

The authors have studied the effect of the paramyxovirus of Newcastle disease on the complement system in the mouse. The results obtained show that NDV activate both in vivo and in vitro the alternative complement pathway, suggesting that the intervention of complement system during viral infections represents a general biological phenomenon.     

Reactivity of Candida albicans in the system of the alternative way of complement activation

The neutrophil-stimulating activity of C. albicans before and after opsonization in the system of the alternative way of complement activation (AWCA) was studied. The study revealed that, in comparison with zymosan, C. albicans exhibited a considerably lower index of AWCA-dependent opsonic effect in reaction with neutrophils. This did not correlate with the capacity of substrates to activate the alternative cascade and with the intensity of phagocytosis. The suggestion on the involvement of C. albicans thermolabile cell-wall components into the negative regulation AWCA-dependent opsonic effect was substantiated.     

A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.     

Detrimental effects of complement activation in hemorrhagic shock.

The complement system has been implicated in early inflammatory events and a variety of shock states. In rats, we measured complement activation after hemorrhage and examined the hemodynamic and metabolic effects of complement depletion before injury and worsening of complement activation after hemorrhage and resuscitation [with a carboxypeptidase N inhibitor (CPNI), which blocks the clearance of C5a]. Rats were bled to a mean arterial pressure of 30 mmHg for 50 min and were then resuscitated for 2 h. Shock resulted in significant evidence of complement consumption, with serum hemolytic activity being reduced by 33% (P < 0.05). Complement depletion before injury did not affect hemorrhage volume (complement depleted = 28 +/- 1 ml/kg, complement intact = 29 +/- 1 ml/kg, P = 0.74) but improved postresuscitation mean arterial pressure by 37 mmHg (P < 0.05) and serum bicarbonate levels (complement depleted = 22 +/- 3 meq/ml, complement intact = 13 +/- 8 meq/ml, P < 0.05). Pretreatment with CPNI was lethal in 80% of treated animals vs. the untreated hemorrhaged group in which no deaths occurred (P < 0.05). In this model of hemorrhagic shock, complement activation appeared to contribute to progressive hypotension and metabolic acidosis seen after resuscitation. The lethality of CPNI during acute blood loss suggests that the anaphylatoxins are important in the pathophysiological events involved in hemorrhagic shock.