Peter Kierulf

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

The quantitative association of plasma endotoxin, antithrombin, protein C, extrinsic pathway inhibitor and fibrinopeptide a in systemic meningococcal disease

We have evaluated the quantitative relationship between lipopolysaccharide (LPS, endotoxin), fibrinopeptide A (FPA), antithrombin (AT), protein C (PC) and extrinsic pathway inhibitor (EPI) in plasma from 39 consecutively admitted patients with systemic meningococcal disease (SMD). The most severely ill patients with fulminant meningococcal septicemia (n = 13, 6 dead) had significantly (p less than 0.01) higher plasma levels of LPS and FPA and lower levels of PC and AT on admission as compared with the less severe clinical presentations (n = 26, 1 dead). The levels of EPI on admission were significantly (p less than 0.05) higher in nonsurvivors vs survivors with fulminant septicemia. As the disease progressed, the levels of LPS, FPA, AT and PC declined, while the levels of EPI increased. Three of six nonsurviving septicemic patients had levels of EPI greater than 200% within 16 hours of admission vs two of 30 survivors (p = 0.02). The results suggest that increasing levels of LPS in SMD elicit increasing consumption coagulopathy, contributing to the organ pathophysiology. The kinetics of EPI, inhibiting the thromboplastin-FVIIa-FXa complex, differs markedly from the kinetics of AT and PC i.e. increases as opposed to decreases.

Plasminogen activator inhibitor 1 and 2, alpha-2-antiplasmin, plasminogen, and endotoxin levels in systemic meningococcal disease

We have studied the activation state of the fibrinolytic system in 39 patients with systemic meningococcal disease (SMD). Patients defined as having fulminant septicemia (n = 13) with high (greater than 700 ng/L) levels of endotoxin (LPS) in plasma and severe coagulopathy, had significantly lower functional levels of plasminogen (P less than 0.05) and alpha-2-antiplasmin (P less than 0.01) and higher antigen levels of plasminogen activator inhibitor 1 (PAI-1) (P less than 0.01), and fibrin degradation products (FDP) (P less than 0.01), but not of PAI-2 (P greater than 0.1) as compared with less severely ill patients (meningitis and meningococcemia) (n = 25). A positive correlation existed between the admission (maximum) levels of LPS and PAI-1 (r = 0.86, P less than 0.0001). Decreasing admission levels of platelets were associated with increasing levels of PAI-1 (r = -0.55, P less than 0.001). After initiation of treatment with antibiotics and fresh frozen plasma, the PAI-1 levels declined rapidly. PAI-1 levels greater than 360 micrograms/L on admission predicted the development of a severe septic shock combined with renal impairment correctly in 12 of 13 patients (92%). None of 25 patients without multiple organ failure had PAI-1 levels greater than 260 micrograms/L. PAI-1 levels greater than 1850 micrograms/L were associated with 100% fatality. The results suggest that in the early phase of fulminant meningococcal septicemia an extensive plasmin generation occurs. On admission, however, high levels of PAI-1 seem to inhibit the plasmin generation, and thereby promote DIC.

Neisseria meningitidis lipopolysaccharides in human pathology.

Neisseria meningitidis causes meningitis, fulminant septicemia or mild meningococcemia attacking mainly children and young adults. Lipopolysaccharides (LPS) consist of a symmetrical hexa-acyl lipid A and a short oligosaccharide chain and are classified in 11 immunotypes. Lipid A is the primary toxic component of N. meningitidis. LPS levels in plasma and cerebrospinal fluid as determined by Limulus amebocyte lysate (LAL) assay are quantitatively closely associated with inflammatory mediators, clinical symptoms, and outcome. Patients with persistent septic shock, multiple organ failure, and severe coagulopathy reveal extraordinarily high levels of LPS in plasma. The cytokine production is compartmentalized to either the circulation or to the subarachnoid space. Mortality related to shock increases from 0% to > 80% with a 10-fold increase of plasma LPS from 10 to 100 endotoxin units/ml. Hemorrhagic skin lesions and thrombosis are caused by up-regulation of tissue factor which induces coagulation, and by inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1). Effective antibiotic treatment results in a rapid decline of plasma LPS (half-life 1-3 h) and cytokines, and reduced generation of thrombin, and PAI-1. Early antibiotic treatment is mandatory. Three intervention trials to block lipid A have not significantly reduced the mortality of meningococcal septicemia.

Increased activation of coagulation and formation of late deep venous thrombosis following discontinuation of thromboprophylaxis after hip replacement surgery

Hip replacement surgery (HRS) is associated with a high frequency of deep vein thrombosis (DVT). At the same time there is a substantial systemic and local activation of coagulation. This study indicates that discontinuation of thromboprophylaxis one week after surgery may allow a second wave of coagulation and fibrinolysis activation to occur. An almost parallel increase in plasma TAT and D-dimer levels between the 6th and the 35th postoperative day may indicate late DVT formation. Repeated bilateral ascending venography is though to be necessary to evaluate the suitability of using selected activation markers of the coagulation and fibrinolytic systems as indices of DVT formation.

Centrifugal pump and heparin coating improves cardiopulmonary bypass biocompatibility

BACKGROUND Centrifugal pumps are being used increasingly for short-term extracorporeal circulation purposes such as during heart operations. Whether the centrifugal pump improves the cardiopulmonary bypass biocompatibility has not been fully documented. METHODS A roller pump (n = 20) was compared in vivo with a centrifugal pump (n = 20) in groups of patients in which cardiopulmonary bypass circuits that were either totally heparin coated (Carmeda BioActive Surface; n = 20) or uncoated (n = 20) were used. We expected the heparin coating to attenuate blood activation, thus possibly making the comparison of the two pumps easier with respect to their different blood activation potentials. Samples of blood plasma, obtained during cardiopulmonary bypass from low-risk coronary artery bypass grafting patients, were analyzed for hemolysis (plasma haemoglobin), complement activation (C3bc and the terminal complement complex), a complement lytic inhibitor (vitronectin), coagulation activation (fibrinopeptide A), granulocyte activation (lactoferrin), and platelet activation (beta-thromboglobulin). RESULTS The concentrations of terminal complement complex, lactoferrin, and beta-thromboglobulin were significantly lower in association with heparin-coated surfaces. The concentration of plasma hemoglobin was significantly lower in association with the centrifugal pump. In uncoated circuits, the beta-thromboglobulin level was significantly higher in association with the roller pump than with the centrifugal pump, but this significant reduction in the beta-thromboglobulin level did not hold true for the heparin-coated circuit group. CONCLUSIONS A heparin-coated cardiopulmonary bypass surface reduces the blood activation potential during cardiopulmonary bypass, and the centrifugal pump causes less hemolysis than the roller pump.

Sequential intrapulmonary and systemic activation of coagulation and fibrinolysis during and after total hip replacement surgery

Hip joint replacement surgery, using acrylic cement for prosthesis fixation, is associated with intraoperative cardiorespiratory dysfunction, and a high frequency of postoperative proximal deep vein thrombosis (DVT). Levels of prothrombin fragments 1+2 (F1+2), tissue plasminogen activator antigen (t-PA), plasminogen activator inhibitor 1 activity (PAI-1), D-dimer and interleukin 6 (IL-6) were measured in arterial (AB) and mixed venous blood (MVB) in five patients during and after total hip replacement operation with acrylic cement prosthesis fixation. Sequential peaks of F1+2, t-PA, PAI-1 and IL-6 appeared, starting with activation of coagulation during preparation of bone, closely followed by activation of fibrinolysis. Later, this was counteracted by an antifibrinolytic response and increase of IL-6. After a fibrinolytic shutdown on the third postoperative day as evidenced by a drop in t-PA and D-dimer concentrations, a second wave of coagulation was seen at the end of the first week. The present model, with frequent sampling of blood entering and leaving the lungs, confirms our earlier findings of the lung as a key organ in promoting coagulation following traumatic activation.

Chemokine patterns in meningococcal disease

Chemokines are important in regulating leukocyte traffic during infection. We analyzed plasma chemokine levels of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha , interleukin (IL)-8, and RANTES in patients with meningococcal infection and correlated these to plasma lipopolysaccharide (LPS) levels, which are closely associated with clinical presentation. In patients with fulminant meningococcal septicemia, versus distinct meningitis or mild systemic meningococcal disease, MCP-1 (both P<.0001), MIP-1 alpha (both P<.0001), and IL-8 (P<.0001 and P=.011) were significantly higher and RANTES significantly lower (P=.007 and P=.021). MCP-1 (r=.88), MIP-1 alpha (r=.82), and IL-8 (r=.89) were positively correlated to plasma LPS levels, whereas RANTES was negatively correlated (r=-.49). In an ex vivo whole-blood model, heat-inactivated wild-type Neisseria meningitidis, purified meningococcal LPS, and (to a negligible extent) heat-inactivated LPS-deficient mutant N. meningitidis induced these chemokines. N. meningitidis LPS is the major cause of chemokine release in meningococcal disease.

Elevated VIP and endotoxin plasma levels in human gram-negative septic shock

Vasoactive intestinal polypeptide (VIP) and endotoxin (lipopolysaccharides, LPS) were measured in plasma samples from 11 patients with bacteriologically verified meningococcal disease. Five patients suffered fulminant septicaemia, developed severe septic shock, and 2 died due to circulatory collapse. Initially, all 5 had levels of VIP above 4 pM and plasma endotoxin above 200 ng/liter. Five patients were diagnosed as meningitis and 1 as having meningococcaemia, all with a normal circulatory state. None of these 6 patients had initially levels of VIP above 2.5 pM or endotoxin levels above 25 ng/liter (P less than 0.001). A correlation existed between plasma endotoxin and VIP levels (r = 0.735, P = 0.01). Sequentially collected samples from 3 patients showed rapidly declining VIP levels after initiation of antibiotic and fluid treatment. These results are in agreement with previous animal experiments, suggesting that endotoxin directly or indirectly stimulates the VIP-ergic nervous system in the initial phase of gram-negative septic shock in man.

Invited review: Neisseria meningitidis lipopolysaccharides in human pathology:

Neisseria meningitidis causes meningitis, fulminant septicemia or mild meningococcemia attacking mainly children and young adults. Lipopolysaccharides (LPS) consist of a symmetrical hexa-acyl lipid A and a short oligosaccharide chain and are classified in 11 immunotypes. Lipid A is the primary toxic component of N. meningitidis . LPS levels in plasma and cerebrospinal fluid as determined by Limulus amebocyte lysate (LAL) assay are quantitatively closely associated with inflammatory mediators, clinical symptoms, and outcome. Patients with persistent septic shock, multiple organ failure, and severe coagulopathy reveal extraordinarily high levels of LPS in plasma. The cytokine production is compartmentalized to either the circulation or to the subarachnoid space. Mortality related to shock increases from 0% to > 80% with a 10-fold increase of plasma LPS from 10 to 100 endotoxin units/ml. Hemorrhagic skin lesions and thrombosis are caused by up-regulation of tissue factor which induces coagulation, and by inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1). Effective antibiotic treatment results in a rapid decline of plasma LPS (half-life 1—3 h) and cytokines, and reduced generation of thrombin, and PAI-1. Early antibiotic treatment is mandatory. Three intervention trials to block lipid A have not significantly reduced the mortality of meningococcal septicemia.