Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4.

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Effect of Cardiac Resynchronization Therapy on Inflammation in Congestive Heart Failure: A Review.

Congestive heart failure is associated with increased levels of several inflammatory mediators, and animal studies have shown that infusion of a number of cytokines can induce heart failure. However, several drugs with proven efficacy in heart failure have failed to affect inflammatory mediators, and anti-inflammatory therapy in heart failure patients has thus far been disappointing. Hence, to what extent heart failure is caused by or responsible for the increased inflammatory burden in the patient is still unclear. Over the past couple of decades, resynchronization therapy with a biventricular pacemaker has emerged as an effective treatment in a subset of heart failure patients, reducing both morbidity and mortality. Such treatment has also been shown to affect the inflammation associated with heart failure. In this study, we review recent data on the association between heart failure and inflammation, and in particular how resynchronization therapy can affect the inflammatory process.

Atrial fibrillation: inflammation in disguise?

Atrial fibrillation is highly prevalent, and affected patients are at an increased risk of a number of complications, including heart failure and thrombo-embolism. Over the past years, there has been increasing interest in the role of inflammatory processes in atrial fibrillation, from the first occurrence of the arrhythmia to dreaded complications such as strokes or peripheral emboli. As the standard drug combination which aims at rate control and anticoagulation only offers partial protection against complications, newer agents are needed to optimize treatment. In this paper, we review recent knowledge regarding the impact of inflammation on the occurrence, recurrence, perpetuation and complications of the arrhythmia, as well as the role of anti-inflammatory therapies in the treatment for the disease.

LDL apheresis and inflammation--implications for atherosclerosis.

Low-density lipoprotein (LDL) apheresis is an extracorporeal treatment modality used in high-risk patients when LDL cholesterol levels cannot be reduced adequately with medication. The treatment is highly effective, but could be affected by potential unwanted effects on pro- and anti-inflammatory biomarkers. In this paper, we review the literature regarding the effect of LDL apheresis on pro- and anti-inflammatory biomarkers important in atherosclerosis, also as patients in LDL apheresis have high risk for atherosclerotic complications. We discuss the effect of LDL apheresis on complement, cytokines and finally a group of other selected pro- and anti-inflammatory biomarkers. The complement system is affected by LDL apheresis, and there are differences between different LDL apheresis systems. The plasma separation columns seem to trigger the formation of proinflammatory complement factors including C3a and C5a, while the same anaphylatoxins are adsorbed by the LDL apheresis columns, however, to varying degree. Proinflammatory cytokines are to some extent adsorbed by the LDL apheresis columns, while some of the anti-inflammatory cytokines increase during treatment. Finally, we discuss the effect of apheresis on different biomarkers including C-reactive protein, fibrinogen, adhesion molecules, myeloperoxidase and HDL cholesterol. In conclusion, even if there are differences between pro- and anti-inflammatory biomarkers during LDL apheresis, the consequences for the patients are largely unknown and larger studies need to be performed. Preferably, they should be comparing the effect of different LDL apheresis columns on the total inflammatory profile, and this should be related to clinical endpoints.

The artificial surface-induced whole blood inflammatory reaction revealed by increases in a series of chemokines and growth factors is largely complement dependent.

Exposing blood to an artificial surface results in a systemic inflammatory response, including cytokine release and complement activation. We studied the artificial surface-induced inflammation in human whole blood using an extensive panel of inflammatory mediators including proinflammatory cytokines, chemokines and growth-factors and investigated the role of the complement system in the induction of this response. Using multiplex technology, 27 different inflammatory mediators were measured after circulating blood for 4 hours in polyvinyl chloride tubing. The C3 inhibitor compstatin was used to block complement activation. A significant (p < 0.05) increase in 14 of the 27 mediators was induced by the surface, of which 7 were chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, RANTES, eotaxin and IP-10) and 5 were growth-factors (G-CSF, GM-CSF, VEGF, PDGF and FGF). The traditional proinflammatory cytokines like IL-1beta, TNFalpha and IL-6 were not induced, although IL-6, as well as IL-15 and IL-17 increased if the surface was coated with highly bioincompatible laminaran. Inhibition of complement activation with compstatin significantly (p < 0.05) reduced the formation of 12 of the 14 mediators. For 10 of the 12 mediators, the inhibition was by 2/3 or more, for the remaining two the inhibition was more moderate. A highly biocompatible heparin-coated PVC surface was used as negative control and completely abolished the whole inflammatory response. The artificial surface PVC markedly induced a broad spectrum of chemokines and growth-factors, which was largely dependent on activation of complement.

Changes in serum levels of E-selectin correlate to improved glycaemic control and reduced obesity in subjects with the metabolic syndrome.

OBJECTIVE: Inflammation plays an essential role in the atherosclerotic process. Cellular adhesion molecules (CAMs) such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are involved in the rolling, adhesion and extravasation of monocytes and T-lymphocytes into the atherosclerotic plaque. In the present study the effect of physical exercise and pravastatin (40 mg daily) on serum levels of CAMs and a possible role of adipose tissue in regulating serum levels of CAMs were investigated. MATERIAL AND METHODS: The study was designed as an unmasked randomized 2x2 factorial trial of 12 weeks duration in 32 subjects with the metabolic syndrome. Changes from baseline were studied, and correlations between changes in CAMs, anthropometric measures, regional fat distribution, glycaemic control and the adipocytokine tumour necrosis factor-a (TNF-a) and adiponectin were investigated. RESULTS: No significant changes in CAMs were observed in any of the intervention groups. However, when examining the whole study population regardless of intervention, changes in serum E-selectin were significantly correlated to changes in body mass index (r=0.48, p=0.006), waist circumference (r=0.48, p=0.006), fasting glucose (r=0.43, p=0.02) and HbA1c (r=0.45, p=0.01), but not to changes in visceral fat, subcutaneous fat, TNF-a or adiponectin. CONCLUSION: Changes in glycaemic control and obesity, rather than regional fat distribution, seem to influence the levels of E-selectin in subjects with the metabolic syndrome.

Thoracic myelitis as a possible cause of myocardial infarction.

During the course of an inflammatory process in the thoracic part of the spinal cord, a previously healthy male suffered two myocardial infarctions in separate coronary territories. A coronary angiogram revealed only minor wall changes in one coronary artery. We hypothesize that the myocardial infarctions may have been caused by vasospastic reactions secondary to his spinal cord pathology, and present the case report and a review of the literature.

Statin drugs do not affect serum complement activation in vitro.

Statin drugs prevent coronary heart disease through anti-inflammatory mechanisms in addition to the well-known reduction of low-density lipoproteins. The complement system plays an essential role in the inflammatory response and has been postulated to be modified by statins. A direct role for statins in complement activation, however, has not been previously investigated. We therefore studied the effect of statins on in vitro complement activation. Pravastatin, atorvastatin and the active metabolite of the latter, ortho-hydroxy atorvastatin, were added to normal human serum and incubated for 1 h in the absence or presence of aggregated immunoglobulin (classical pathway activation) or cobra venom factor (alternative pathway activation). The degree of complement activation, as detected by specific complement-activation products for the classical pathway (C1rs-C1-inhibitor complexes), the combined classical and lectin pathway (C4bc), the alternative pathway (C3bBbP) and the final common pathway (C3bc and TCC), was not affected by pre-incubation of the serum with any of the statins. Statins do not affect complement activation directly, but indirect effects in vivo may well be operative.

Complement activation in patients with congestive heart failure: effect of high-dose intravenous immunoglobulin treatment.

BACKGROUND: Increasing evidence implicates innate immunity in the pathogenesis of congestive heart failure (CHF). In the present study, we examined the possible role of complement, an important part of innate immunity, in CHF. METHODS AND RESULTS: Complement activation was analyzed in systemic and coronary circulation in 39 patients with CHF and 20 healthy control subjects. In a double-blind, placebo-controlled study, we have recently reported that high-dose intravenous immunoglobulin (IVIG) improves left ventricular ejection fraction (LVEF) in these patients. To examine if this improvement was related to IVIG-induced effects on complement, we also examined complement activation during induction (first week) and maintenance therapy (6 months) with IVIG or placebo. Our main findings were: (1) We found enhanced systemic complement activation involving classic, alternative, as well as terminal pathway in patients with CHF compared with healthy control subjects. (2) Particularly enhanced complement activation was found in coronary sinus, representing venous drainage from the heart. (3) The systemic complement activation was further enhanced during IVIG but not during placebo therapy, particularly during induction therapy. (4) Although IVIG improved LVEF in patients with CHF, the degree of IVIG-mediated complement activation was negatively correlated with this improvement of LVEF. CONCLUSIONS: This study further supports the involvement of innate immunity in the pathogenesis of CHF. Our findings suggest that complement may be added to the list of possible therapeutic targets in CHF and that future studies with specific complement inhibitors may be of interest in this disorder.

A novel enzyme immunoassay for plasma thrombospondin. Comparison with beta-thromboglobulin as platelet activation marker in vitro and in vivo.

A novel enzyme immunoassay for plasma thrombospondin (TSP) based on commercially available monoclonal antibodies was established. The following conditions for correct collection and preservation of blood samples were required: venipuncture directly into a vacutainer containing citrate, theophylline, adenosine and dipyridamole, storage on ice, and separation of plasma within 30 minutes. Thereafter, the plasma TSP concentration remained constant at room temperature and after five times of freezing and thawing. Both inter- and intraassay variation coefficients were 5%. The lower detection limit was 20 microg/L. Median TSP concentration among 40 healthy blood donors was 43 microg/L, slightly lower than previously published. The assay is valid, reliable, and has certain advantages compared with previously published methods. TSP and beta-thromboglobulin (BTG) were then compared as platelet activation and biocompatibility markers in vivo: 23 patients undergoing cardiopulmonary bypass (CPB); and in vitro: effect of coating polyvinyl chloride with heparin. The kinetic patterns of TSP and BTG were markedly different in vivo but virtually identical in vitro, explained by different in vivo clearance mechanisms during CPB. We conclude that BTG is superior to TSP for evaluation of platelet activation during in vivo CPB, whereas TSP and BTG are virtually identical as markers in vitro.

[Hereditary warfarin resistance]. / Arvelig warfarinresistens.

BACKGROUND: Warfarin is the major oral anticoagulant drug used in Norway. The effect is monitored by measuring the patient's prothrombin time expressed as international normalized ratio (INR). The dose necessary to achieve a therapeutic INR varies. A few patients require extremely high doses, and hereditary warfarin resistance has been reported. MATERIAL AND METHODS: The present paper describes two brothers with reduced sensitivity to warfarin. A review of the literature is given. RESULTS: Both patients achieved therapeutic levels of INR, but required daily doses of 25-35 mg warfarin. INTERPRETATION: Hereditary warfarin resistance is a rare cause of reduced sensitivity to warfarin. It is not associated with increased thromboembolic risk.

[Cardiac manifestations in Fabry disease]. / Kardiale manifestasjoner ved Fabrys sykdom.

BACKGROUND: Fabry's disease is an X-linked inborn error of metabolism. The patients lack or have very low activity of the enzyme alpha-galactosidase A. This results in deposition of sphingolipids in endothelial cells and vascular smooth muscle cells; thus the disease can affect nearly every organ in the body. Renal failure is the most common cause of death, but cardiac involvement is frequent. MATERIAL AND METHODS: We describe two brothers with Fabry's disease and provide a review of the literature in the field. RESULTS: Both patients had extensive electro- and echocardiographic findings. INTERPRETATION: Fabry's disease should be suspected in men with unexplained electro- and/or echocardiographic signs of left ventricular hypertrophy and a short PQ interval in the ECG.

Modulation by interferons of human neutrophilic granulocyte migration.

The effect of various interferons (IFN) on neutrophilic granulocyte (PMN) random and directed migration is incompletely understood. We, therefore, investigated PMN migration with a novel micropore membrane technique. No chemotactic effect of either 10-10000 U/ml IFN-alpha or IFN-beta, or 1-1000 U/ml IFN-gamma was observed on PMN isolated from normal human venous blood. However, when present on both sides of the micropore membrane, all the IFN (1000 U/ml IFN-alpha and IFN-beta, 100 U/ml IFN-gamma) inhibited both random and directed migration toward zymosan-activated serum (ZAS). IFN-gamma was the most potent inhibitory agent and produced an inhibition of about 30%. When the bacterial peptide fMLP was used as a chemoattractant, IFN-gamma also depressed chemotaxis. Taking the reduced random migration of IFN-gamma treated cells into account, however, chemotaxis per se-toward both ZAS and fMLP-was not significantly affected. Random migration and directed migration assessed simultaneously with PMN from the same donor were clearly correlated for both control and IFN-gamma treated cells, suggesting that a general antimotility effect of IFN-gamma might explain both reduced random migration and chemotaxis. The antimotility effect of IFN-gamma was not dependent on protein synthesis or on tyrosine kinase activity. In fact, inhibition of tyrosine kinase with herbimycin A increased the ZAS-stimulated motility of both control and IFN-gamma-inhibited PMN. In conclusion, our data indicate that IFN depress both random and directed PMN migration by mechanisms that do not involve protein synthesis or protein tyrosine kinase activity.

[Chronic granulomatous disease. A disease model with new therapeutic possibilities]. / Kronisk granulomatøs sykdom. Modellsykdom med nye behandlingsmuligheter.

Chronic granulomatous disease is an inherited disorder characterized by recurrent infections from early childhood. The disease is due to reduced production of reactive oxygen intermediates in host phagocytes, causing inefficient killing of bacteria. Recently it has been shown that the immuno-modulating compound interferon-gamma can stimulate oxygen metabolism in the phagocytes of these patients. Immuno-modulating treatment with recombinant proteins may prove to be important in the treatment of this and other related disorders.

Interferons affect oxygen metabolism in human neutrophil granulocytes.

Human polymorphonuclear neutrophil granulocytes (PMN) were incubated with recombinant interferons (IFNs) and tested for O2 consumption, hydrogen peroxide formation, and chemiluminescence. N-formyl-methionyl-leucyl-phenylalanine (f-MLP, a bacterial peptide analogue) and phorbol myristate acetate (PMA, a protein kinase C activator) were used as PMN stimuli. An increase in O2 consumption after f-MLP-stimulation was seen when PMN had been incubated 2-4 h with either 1000 IU/ml IFN-alpha or 100 IU/ml IFN-gamma, but this increase in O2 consumption was not observed with 1000 IU/ml IFN-beta. Likewise, 100 U/ml IFN-gamma enhanced f-MLP stimulated chemiluminescence, whereas IFN-alpha or IFN-beta (1000 U/ml) had no detectable effects. None of the interferons affected baseline or PMA-stimulated O2 consumption and chemiluminescence, nor did they influence the H2O2-dependent oxidation of intracellular dichlorofluorescein (DCFH) (baseline, f-MLP-stimulated or PMA-stimulated). Our data indicate that some--but not all--aspects of oxygen metabolism in PMN can be affected by IFN, and that there are differences between various subtypes of IFNs regarding their neutrophil priming potential.