Terminal Complement Complex C5b-9 Research Articles

C5b-9 terminal complement complex assembly on apoptotic cells in human arterial wall with atherosclerosis

Apoptosis plays an important role in atherosclerosis. The factors regulating this process are not well defined. We examined the relation of apoptotic cells with the terminal complement complex C5b-9 in human atherosclerotic lesions. The extent of apoptosis was determined using TdT dUTP nick-end labeling (TUNEL) and immunohistochemistry of apoptosis regulators caspase-3, caspase-9, Bax, and Bcl-2. C5b-9 was localized by immunohistochemistry and immunoelectron microscopy. The apoptotic index was higher in fibrous plaques when compared with intimal fatty streaks and intimal thickenings. Bax expression was present in TUNEL+ apoptotic cells, and Bcl-2 was rarely present in the atherosclerotic wall. Active caspase 9 and caspase 3 deposits were present in the same areas, suggesting an involvement of the mitochondrial pathway. C5b-9 deposits colocalized with TUNEL+ cells, and the percent of double-positive cells was 2% in fatty streaks, 12% in intimal thickenings, and 35% in fibrous plaques. Colocalization of apoptotic cells with C5b-9 was also confirmed by immunoelectron microscopy. In conclusion, some apoptotic cells carry C5b-9 deposits, suggesting that complement might be activated by apoptotic cells and involved in the promotion of apoptosis, contributing to the progression of atherosclerotic lesions.

HMG-CoA reductase inhibition reduces the proinflammatory activation of human vascular smooth muscle cells by the terminal complement factor C5b-9

The terminal complement complex C5b-9 is known to participate in inflammatory processes including atherosclerosis. Inflammation appears to be a direct consequence of C5b-9-mediated cell stimulation. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors may exert anti-inflammatory effects on vascular cells independent of lowering plasma cholesterol. Thus, we studied activation of vascular smooth muscle cells (VSMCs) by C5b-9 focusing on whether inhibition of the HMG-CoA reductase can reduce the proinflammatory effects of C5b-9.C5b-9 in sublytic concentrations increased the proliferation of human VSMCs and induced a time-dependent activation of the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK). Proliferation and ERK1/2 activation could be inhibited by the specific ERK inhibitor PD98059. HMG-CoA inhibition with cerivastatin-reduced VSMC proliferation and C5b-9-induced ERK1/2 activation. Cerivastatin also reduced the C5b-9-induced synthesis of the proinflammatory interleukin-6 (IL-6). Furthermore, C5b-9 induced activation of the transcription factors activator protein- 1 (AP-1) and nuclear factor-kappaB (NF-kappaB), which could be inhibited by pretreatment of VSMCs with cerivastatin. L-mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effects of cerivastatin. The present study in VSMCs shows that cerivastatin inhibits IL-6 synthesis and cell proliferation induced by the terminal complement complex C5b-9. This may be an important mechanism contributing to the beneficial effects of HMG-CoA reductase inhibitors beyond lowering of plasma cholesterol.

The terminal complement complex inhibits apoptosis in vascular smooth muscle cells by activating an autocrine IGF-1 loop.

Two counteracting processes determine accumulation of human vascular smooth muscle cells (SMCs) in atherosclerotic lesions: cell proliferation and apoptosis. SMCs synthesize insulin-like growth factor-1 (IGF-1), which potently inhibits apoptosis. The terminal complement complex C5b-9 interacts with SMCs in early human atherogenesis. In this study, we investigated whether C5b-9 may activate the IGF-1 system in SMCs, resulting in the inhibition of SMC apoptosis. C5b-9 generation on SMCs in vitro markedly reduced CD95-mediated apoptosis as assessed by flowcytometric analysis of annexin V binding and in caspase 3 assays. C5b-9 induced both significant IGF-1 release and up-regulation of IGF-1 binding sites in SMCs. Immunoneutralization of IGF-1 with a monoclonal IGF-1 antibody abolished the antiapoptotic effects of C5b-9. We conclude that C5b-9 inhibits apoptosis in SMCs by inducing an autocrine IGF-1 loop. This mechanism may contribute to the accumulation of SMCs in early human atherosclerotic lesions.

Complement activation in early protocol kidney graft biopsies after living-donor transplantation.

To gain insight into complement activation in kidney grafts, we studied the deposition of components from all complement pathways in protocol biopsies from living-donor recipients that were taken 1 week (median 7 days) after transplantation. Graft protocol biopsies (n=37) were taken consecutively and stained for two-color immunofluorescence, with antibodies to C4d, C3, C1q, factor B, C6, terminal C5b-9 complement complex, mannose-binding lectin (MBL), and MBL-associated serine protease-1, combined with an endothelial marker. Light and electron microscopy were performed in all cases. Clinical acute rejection (AR), graft loss, and long-term kidney function were recorded. Baseline biopsies from 15 of the patients served as controls. Endothelial C4d deposition was demonstrated in peritubular capillaries in 11 of 37 cases (30%), of which 9 of 11 (82%) experienced clinical AR but only 6 of 11 (55%) experienced AR as defined by histopathologic criteria. Biopsies from three patients, two with early graft loss, showed diffuse global C4d in the glomerular endothelium with codeposition of C3 in all patients and MBL-associated serine protease-1 in one patient. Focal peritubular capillary C3 deposition was found in two additional C4d-positive cases with AR. No posttransplant deposition was demonstrated for the other components. Early diffuse C4d deposition in the kidney graft capillaries is closely related to acute humoral rejection, whereas focal staining may occur with mild AR or, rarely, without rejection. Codeposition of C3 indicates early AR with a higher risk of graft loss. In most cases, activation was limited to C4d, indicating efficient in situ regulation of complement activation.

How safe is the port access technique in minimally invasive coronary artery bypass grafting?

Background This study compares conventional coronary artery bypass grafting (CABG) with port access CABG via a left anterior small thoracotomy in patients requiring surgical multivessel revascularization. Clinical, neuropsychological, and angiographic outcomes were studied, as well as parameters of myocardial and cerebral protection. Pathogenicity of cardiopulmonary bypass (CPB) was further evaluated by measuring parameters of peripheral limb ischemia and inflammatory whole-body response. Methods In a prospective randomized study, 40 patients who required multivessel CABG were assigned to either conventional CABG via complete median sternotomy (group A) or port access CABG via minithoracotomy (group B). Control angiograms were performed in group B only. In addition, patients underwent neuropsychological testing after the operation. CK, CK-MB, and Troponin T levels were documented. S-100B protein and neuron-specific enolase (NSE) served to quantify cerebral injury. The terminal complement complex (C5b-9) and myeloperoxidase concentrations were determined to analyze inflammatory whole-body response after CPB. Results There was no mortality. One patient suffered a retrograde aortic dissection immediately after onset of CPB, but had an uneventful postoperative course after surgical repair. Troponin T and CK-MB showed no difference between groups. CK and myoglobin were significantly higher in the minimally invasive cohort. Changes in complement activation (C5b-9) and myeloperoxidase during CPB markers of the whole-body inflammatory response were similar in both groups. S-100B concentrations in the port access group were significantly higher, whereas NSE levels were similar in both groups. Both groups did not display any significant difference in neuropsychological testing. Conclusions Minimally invasive multivessel CABG via minithoracotomy using port access technology is feasible and safe. Though prolonged operating and CPB times with significantly higher S-100B concentrations were observed in group B, equivalent myocardial and cerebral protection and similar whole-body inflammatory response were documented.

Preeclampsia Associated With Antiphospholipid and Anti-β2-Glycoprotein-I Antibodies and Postpartum Thrombocytopenia, Cardiac Dysfunction, and Blindness: A Case Report

We describe a patient with severe preeclampsia and postpartum complications in whom we investigated a panel of autoimmune reactions. Case Report: A 27-year-old primigravida was referred at 37 weeks' gestation because of severe preeclampsia. A cesarean section was performed. Postpartum the patient developed severe cardiac dysfunction, thrombocytopenia, eclamptic seizures, and blindness. Immunohistochemical studies of the placenta showed marked deposition of terminal C5b-9 complement complex in intervillous fibrin and in intravillous stroma. Conclusion: The generation of C5b-9 complement complex may be involved in the pathogenesis ofpreeclampsia-eclampsia.

Capillary leak syndrome after cardiopulmonary bypass in elective, uncomplicated coronary artery bypass grafting operations: Does it exist?

Operations coupled with cardiopulmonary bypass may provoke a systemic inflammatory response, and it has been suggested that this responses causes capillary leakage of proteins, edema formation, and even organ failure. However, capillary leak syndrome is mainly a clinical diagnosis and has not been verified as yet by actual demonstration of protein leakage from the circulation. We have therefore measured the disappearance of labeled plasma protein before and after cardiopulmonary bypass. Sixteen patients scheduled for elective coronary artery bypass grafting were enrolled in a prospective controlled study. The cardiopulmonary bypass circuit was primed with crystalloids only. Tumor necrosis factor alpha, interleukin 6, interleukin 8, anaphylatoxin C3a, and terminal complement complex C5b9 levels were determined before, during, and 3 hours after cardiopulmonary bypass. The transvascular escape rate of plasma protein from the intravascular compartment was assessed by measuring the disappearance of intravenously injected Evans blue dye before and during the third hour after cardiopulmonary bypass. A significant inflammatory response could be demonstrated by means of the 5 measured mediators after bypass. The maximal increase, as compared with the baseline value, was found for interleukin 6 (36-fold). The transvascular escape rate of Evans blue dye was similar before and after bypass (7.6 +/- 0.6%/h vs 7.3 +/- 0.6%/h). The above data confirm the systemic inflammatory response induced by cardiopulmonary bypass. Contrary to expectations, the transvascular escape rate of Evans blue dye did not change when comparing values before and after bypass. The data do not support the concept of increased protein leakage in the exchange vessels after bypass. We were unable to demonstrate a capillary leak syndrome.

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Serum complement activation in congestive heart failure

Background Although activation of the complement system in myocardial infarction and cardiopulmonary bypass has been shown to contribute to myocardial injury, its role in congestive heart failure (CHF) is unknown. The purpose of this study was to determine the presence of terminal complement activation and its relation to clinical outcomes in patients with CHF. Methods We measured serum levels of the terminal complement complex C5b-9 in 36 patients with symptomatic heart failure and left ventricular ejection fraction <40%. We compared the serum C5b-9 levels of these patients with CHF with a group of 12 age-matched control patients. Combined clinical outcomes (death, urgent heart transplantation, or hospitalization with worsening heart failure) at 6 months were determined. Results The serum C5b-9 [median (25th to 75th percentiles)] levels in 36 patients with CHF [101.5 ng/mL (40 to 164)] were significantly (P =.003) higher than in the 12 control patients [36.5 ng/mL (22 to 50)]. Significantly more of the patients with CHF with the highest levels of C5b-9 (highest 50th percentile) had New York Heart Association class IV symptoms (67% vs 33%; P =.04) and adverse clinical outcomes by 6 months (56% vs 17%; P =.02) compared with the patients with CHF with lower levels (lowest 50th percentile). Conclusions We have described a significant elevation in circulating C5b-9, the terminal complement complex, in patients with symptomatic heart failure and have observed an association between high levels of C5b-9 and near-term adverse events. (Am Heart J 2001;141:684-90.)

The terminal complement complex C5b-9 stimulates interleukin-6 production in human smooth muscle cells through activation of transcription factors NF-kappa B and AP-1.

Activation of the complement system plays an important role in the pathogenesis of atherosclerosis. The proinflammatory cytokine interleukin (IL)-6 is potentially involved in the progression of the disease. We therefore investigated whether the terminal complement complex C5b-9 affects IL-6 production from vascular smooth-muscle cells (VSMC) and set out to determine the underlying signal transduction pathway. Stimulation of human VSMC with C5b-9 resulted in an increase of IL-6 transcript and production of IL-6 protein. Pretreatment with pertussis toxin or pyrrolidine dithiocarbamate inhibited complement-dependent IL-6 mRNA expression and IL-6 release, suggesting the involvement of Gi-proteins and nuclear factor-kB (NF-kB). C5b-9 also induced formation of reactive oxygen species, which, along with IL-6 release, was inhibited by the antioxidant N-acetylcysteine. C5b-9 activated the redox-sensitive transcription factors NF-kB and activator protein-1 (AP-1), which were both involved in the induction of IL-6 by C5b-9, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). The results demonstrate that activation of the complement system induces IL-6 release from human VSMC by a Gi-dependent pathway involving the generation of oxidative stressand the activation of the redox sensitive transcription factors NF-kB and AP-1. Our data support a new mechanism for the proatherogenic effect of the terminal complement complex.

A Potential Role for Immune Complex Pathogenesis in Drusen Formation

Drusen are abnormal extracellular deposits that accumulate between the retinal pigmented epithelium and Bruch's membrane and are commonly associated with age-related macular degeneration. Our recent work has identified a number of plasma proteins as molecular components of drusen. Of interest is the fact that many of these drusen-associated molecules are acute phase reactant proteins and some have established roles in mediating immune responsiveness. As immune and inflammatory responses appear to play a role in the formation of other pathologic age-related deposits, we examined the distribution of immunoglobulin molecules and terminal complement complexes at sites of drusen deposition. Here, we report that concentrations of immunoglobulin G and terminal C5b-9 complement complexes are present in drusen. In addition, we observe that retinal pigmented epithelial cells overlying or directly adjacent to drusen, as well as some within apparently normal epithelia, exhibit cytoplasmic immunoreactivity for immunoglobulin and the C5 component of complement. Taken together, these results suggest that drusen biogenesis may be a byproduct of immune responsiveness, and they implicate immune complex-mediated pathogenesis involving retinal pigmented epithelial cells as an initiating event in drusen formation.

Biocompatibility of silicone-coated oxygenator in cardiopulmonary bypass

Background. This study was designed to analyze the biocompatibility of silicone-coated oxygenators using inflammatory response as the outcome measure, and to investigate whether the silicone-coated oxygenators perform better in terms of postoperative organ dysfunction.Methods. The 32 patients who underwent cardiopulmonary bypass (CPB) were divided into 3 groups: group A (n = 10), heparin-coated circuit with silicone-coated oxygenator; group B (n = 11), whole heparin-coated circuit; and group C (n = 11), whole untreated circuit. The plasma concentrations of the proinflammatory markers, made of inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, interleukin-8), terminal complement complex (C5b-9), and polymorphonuclear elastase (PMN-E), were measured by enzyme-linked immunosorbant assay.Results. All proinflammatory markers were significantly lower in groups A and B than in group C, especially C5b-9 and PMN-E concentrations, which were significantly lower in group A than in group B. The alveolar-arterial oxygen gradients (A-aDO2) and the respiratory index were significantly better in group A than in group C. In group B, however, only the A-aDO2 was significantly better than in group C. The duration of intubation and the length of stay in the intensive care unit stay were significantly shorter in groups A and B than in group C.Conclusions. Silicone-coated oxygenators are biocompatible and prevent postoperative organ dysfunction.

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues.

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Up-regulation of intracellular calcium, cyclic adenosine monophosphate and fibronectin synthesis in tubuar epithelial cells by complement.

The terminal complement complex C5b-9 is known to participate in inflammatory processes including glomerular or tubulointerstitial injury. Injury appears to be a direct consequence of C5b-9-mediated cell stimulation. In that context we studied activation of tubular epithelial cells by C5b-9 particularly with regard to fibronectin synthesis and the transmembrane signals involved. C5b-9 in sublytic concentrations caused a rise of intracellular calcium and of cAMP, followed by an increase in abundance of fibronectin-specific mRNA and accumulation of protein. Stabilized cAMP or increasing the cAMP level by forskolin enhanced fibronectin synthesis with similar kinetics. The effect of cAMP could be enhanced by adding a calcium ionophore. Since the fibronectin gene is known to have a cAMP-responsive element, the data suggest that C5b-9 increases fibronectin synthesis via generation of cAMP.

Effects of cardiopulmonary bypass on neonatal and paediatric inflammatory profiles

Cardiopulmonary bypass (CPB) causes significant morbidity in paediatric patients, yet the mechanisms involved in the related inflammatory processes (resulting in capillary leak and edema) are poorly understood. Moreover, earlier palliative and corrective intervention in neonates and infants has provided the cohorts of patients about whom little is known of their proinflammatory response. In the present two group study, 14 neonates (age 1-28 days, 2.5-4.5 kg) and 13 infants (2-12 months, 3-7 kg), undergoing CPB for congenital heart disease were consecutively recruited. The two cohorts were well matched in terms of CPB and aortic cross-clamp times (P > 0.1). Blood samples were collected on induction of anaesthesia, 5 min following onset of CPB, at the end of CPB, and 30 min, 2 and 24 h post-protamine (PP) administration. Plasma concentration of cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), terminal complement complex (C5b-9) neutrophil counts and leucocyte elastase were measured. Plasma levels of all inflammatory markers significantly increased in both groups during and following CPB as compared to baseline. During and following CPB the change in IL-8 level was more pronounced in neonates (peak 30 min PP, median(range): 1062 (182-3872) pg/ml) than in infants 568 (172-1368) pg/ml), P = 0.01. Changes in IL-6 level were indistinguishable between groups intraoperatively, but remained significantly higher at 24 h in neonates (P = 0.02). Peri and postoperative levels of C5b-9 were significantly higher in infants than in neonates (peak 30 min PP, median (range): 984 (118-1142) ng/ml vs 458 (22 1340) ng/ml in neonates respectively, P = 0.01) but were similar at 24 h. Despite this, leucocyte elastase profiles did not differ significantly between the respective cohorts. These results indicate that there may be differences between neonates and infants with regard to the inflammatory response to CPB and neonatal patients merit further investigation in order to elucidate whether the pathophysiology of their CPB related inflammatory response and its clinical sequelae differs from their older counterparts.

Proinflammatory cytokine release during pediatric cardiopulmonary bypass: Influence of centrifugal and roller pumps

Objective: It has been proposed that nonocclusive centrifugal pumps may elicit less blood cell trauma and hence a reduced inflammatory response than standard roller pumps. However, there have been no reports describing the impact of such pumps on proinflammatory cytokine release in pediatric cohorts. Design: A prospective randomized study was undertaken. Setting: A regional cardiothoracic center of a university hospital. Participants: Thirty-four pediatric patients undergoing cardiopulmonary bypass (CPB) for the correction of complex congenital heart defects were recruited. Interventions: Either standard twin roller (n = 17), or centrifugal vortex (Biopump, Medtronic Biomedicus Inc, MN) (n = 17) blood pumping. Measurements and Main Results: Venous blood was drawn (1) on induction of anesthesia, (2) 5 minutes on bypass, (3) end of CPB, (4) 30 minutes post-protamine, (5) 2 hours and (6) 24 hours postoperation. Neutrophil count, level of plasma leukocyte elastase, terminal complement complex (C5b-9), interleukin-6 (IL-6) and interleukin-8 (IL-8) were increased during and after CPB compared with the postinduction baseline. C5b-9 levels in both groups peaked at the end of CPB before returning to baseline at 24 hours: (median [range]), 564 (16 to 1,136) ng/mL in centrifugal group versus 508 (0 to 1,128) ng/mL in the roller group. IL-6 in both groups reached its peak level at 2 hours postprotamine (208 [98 to 411] pg/mL in centrifugal versus 205 [60–327] pg/mL in the roller group), before coming back to baseline at 24 hours. Plasma leukocyte elastase and IL-8 reached their maximum level 15 minutes after protamine administration: 215 (64 to 375) pg/mL in centrifugal versus 235 (87 to 410) pg/mL in roller group; and 700 (90 to 5,925) ng/mL versus 362 (120 to 3,400) ng/mL, respectively. Conclusions: The current study confirms the proinflammatory nature of pediatric CPB surgery, but failed to show a significant advantage of centrifugal pumping over roller perfusion in terms of the inflammatory response.

Acquired C3 deficiency in patients with alcoholic cirrhosis predisposes to infection and increased mortality.

Acquired deficiencies of certain complement proteins and impaired opsonisation activity have been implicated in the pathogenesis of the increased susceptibility to infections of patients with alcoholic cirrhosis. Serum concentrations of C3 and C4, plasma concentrations of C3bc, C9, and the terminal C5b-9 complement complex (TCC), and haemolytic complement activity (classic and alternative pathway) of serum, and serum opsonic activity were determined in 46 patients with compensated alcoholic cirrhosis, 31 who were decompensated, and in 15 healthy subjects. After 19 months (median) the investigated variables were analysed for their use in prognosis of recurrent infections and survival. C3 and C4 concentrations and the haemolytic complement activity of the alternative pathway were decreased in decompensated cirrhotic patients compared with controls (p < 0.01). Univariate analysis (log rank test) showed that low concentrations (< or = lower quartile) of C3 (p < 0.001) and C3bc (p < 0.05), haemolytic complement activity of the alternative pathway (p < 0.01) and classic pathway (p < 0.05), and decompensated cirrhosis (p < 0.001) were associated with an increased risk of infection and increased mortality. Multivariate (Cox) analysis showed that low C3 concentrations and decompensation of cirrhosis were significant predictors of infections and mortality (p < 0.02). Low serum C3 concentrations and decreased haemolytic complement function predisposes to infection and increased mortality in patients with alcoholic cirrhosis.

Tissue trauma after laparoscopic and abdominal hysterectomy measuring inflammatory response

It is suggested that laparoscopic surgery reduces postoperative pain and shortens hospital stay and convalescence because of the small amount of tissue trauma. We evaluated the inflammatory response during abdominal hysterectomy (AH, 12 women) and laparoscopic hysterectomy (LH, 12 women) by measuring interleukin (IL)-6, neopterin and terminal C5b9 complement complex (TCC). Blood samples were drawn preoperatively, perioperatively, 1 minute, 24 hours, and 7 days postoperatively. Levels of IL-6 were determined to evaluate cytokine release, neopterin was determined as a marker for macrophage-monocyte activation, and TCC was determined to assess complement activation. The IL-6 concentrations, as a percentage of preoperative level, were significantly elevated postoperatively in both groups, and also perioperatively in the LH group. Neopterin concentrations, as a percentage of perioperative level, were significantly increased in the LH group preoperatively and postoperatively. No elevation was seen in the AH group. There was no sign of complement activation in either group. Our results indicate significant tissue trauma during both LH and AH. The extent of trauma might be greater in laparoscopic surgery. Despite this, the LH group had a shorter hospital stay and convalescence than the AH group. The proposed advantages to the patient of laparsocopic surgery thus seem to be attributable to other factors than the amount of tissue trauma.

Increased release of tumor necrosis factor-alpha and interleukin-6 in women with the syndrome of hemolysis, elevated liver enzymes, and low platelet count.

Complement is activated in preeclampsia and complement products are known to activate macrophages. The aim of this study was to determine whether the macrophage derived cytokines, interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha, are released in patients with a form of severe preeclampsia characterized by the syndrome of hemolysis, elevated liver enzymes, and low platelet count (HELLP syndrome). Complement activation and plasma levels of cytokines were studied in 11 women with HELLP syndrome and in 11 controls with uncomplicated pregnancies. To further evaluate the connection between complement activation and cytokine release an in vitro study on heparinized whole blood incubated with recombinant C5a was performed. In the HELLP group, complement anaphylatoxin C5a was increased in plasma at delivery (p < 0.01) and one day after delivery (p < 0.05), terminal C5b-9 complement complex was elevated in plasma at delivery (p < 0.001) and one day after (p < 0.01), plasma levels of interleukin-6 were increased one day after delivery (p < 0.01), and plasma concentrations of tumor necrosis factor-alpha were elevated at delivery (p < 0.01), compared with corresponding levels in controls. All parameters normalized within one week. Interleukin-I beta did not differ between the groups. In vitro, recombinant C5a incubated in whole blood gave a dose-dependent release of interleukin-6. No increased release of interleukin-1 or tumor necrosis factor-alpha was seen after incubation. Since cytokine release occurs in severe preeclampsia, inflammatory mechanisms may participate in the pathophysiology of severe preeclampsia.

Glycoprotein IIb-IIIa on platelet-derived microparticles, and microparticle structures studied by electron microscopy, confocal laser microscopy and crossed radio-immunoelectrophoresis

Shedding of microparticles from the platelet surface is usually associated with exposure of platelet procoagulant activity. Platelet-derived microparticles have been detected in blood in various disease states. In vitro, platelet stimulation with a number of different agonists results in formation of microparticles. In the present study, microparticles induced by platelet stimulation by calcium ionophore or by membrane incorporation of the terminal complement complex C5b-9 were studied using electron microscopy, confocal laser microscopy, flow cytometry and radio-immunoelectrophoresis. When studied by electron microscopy, microparticle morphology was found to be dependent upon the induction method. Platelet stimulation with the calcium ionophore resulted in smaller, more homogeneous and electron dense microparticles than those induced by insertion of the terminal complement complex. With flow cytometry and confocal laser immunofluorescence microscopy, microparticle GPIIb-IIIa was demonstrated using a FITC-conjugated antibody to GPIIIa. Surface-bound GPIIb-IIIa was demonstrated on the microparticles by immunoelectron microscopy. Crossed immunoelectrophoresis of detergent-solubilized microparticles visualized a very prominent GPIIb-IIIa immunoprecipitate arc, and binding of [(125)1]fibrinogen to microparticle GPIIb-IIIa was demonstrated by radio-immunoelectrophoresis. This suggests that the activated GPIIb-IIIa complex is preserved intact during the shedding of microparticles from the platelet surface.