Berit Brusletto

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.

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.

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.

Complement Activation Induced by Purified Neisseria meningitidis Lipopolysaccharide (LPS), Outer Membrane Vesicles, Whole Bacteria, and an LPS-Free Mutant

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker

BackgroundImproved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions.MethodsDifferences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously.ResultsDifferentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC.ConclusionsSeveral miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped.

Plasma interferon-γ and interleukin-10 concentrations in systemic meningococcal disease compared with severe systemic Gram-positive septic shock

ObjectiveTo analyze plasma interferon-&ggr; and interleukin-10 concentrations in patients with systemic meningococcal disease and patients with severe Gram-positive septic shock caused by Streptococcus pneumoniae or Staphylococcus aureus. To study the in vitro cytokine (interferon-&ggr; and interleukin-10) responses in a whole blood model boosted with heat-killed Neisseria meningitidis, S. pneumoniae, and S. aureus before and after treatment with recombinant interleukin-10 or recombinant interferon-&ggr;. DesignExperimental study. SettingLaboratory. SubjectsPlasma samples were collected from patients with systemic meningococcal disease (n = 66) and patients with severe Gram-positive septic shock caused by S. pneumoniae (n = 4) or S. aureus (n = 3). InterventionsWhole blood was boosted with heat-killed N. meningitidis, S. pneumoniae, and S. aureus (1 × 106 colony forming units/mL), and plasmas were analyzed for interleukin-10 or interferon-&ggr; at 0, 5, 12, and 24 hrs. Furthermore, recombinant interleukin-10 or recombinant interferon-&ggr; was added before bacteria, and the effect on the secretion of interferon-&ggr; and interleukin-10, respectively, was analyzed after 24 hrs. Measurements and Main ResultsThe median concentration of interferon-&ggr; was 15 pg/mL and of interleukin-10 was 10,269 pg/mL in patients with meningococcal septic shock (n = 24) compared with median interferon-&ggr; concentration of 3400 pg/mL and interleukin-10 concentration of 465 pg/mL in patients with severe Gram-positive shock (p = .001). Increased interferon-&ggr; concentrations were associated with case fatality (p = .011). In a whole blood model we demonstrated that 1 × 106 colony forming units/mL of N. meningitidis induced more interleukin-10 but less interferon-&ggr; than S. pneumoniae. S. aureus induced minimal secretion of both cytokines. Recombinant interleukin-10 efficiently down-regulated the secretion of interferon-&ggr;, and vice versa, as shown in a whole blood model. ConclusionWe speculate whether high concentrations of interleukin-10 contribute to the low concentrations of interferon-&ggr; in fulminant meningococcal septicemia. In addition, it appears as if interferon-&ggr; plays a minor role in the pathophysiology of meningococcal septic shock.

Identification of Genes Particularly Sensitive to Lipopolysaccharide (LPS) in Human Monocytes Induced by Wild-Type versus LPS-Deficient Neisseria meningitidis Strains

ABSTRACT Lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis plays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-type N. meningitidis (106), LPS-deficient N. meningitidis (106 and 108), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (106) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 106 LPS-deficient N. meningitidis organisms. However, 108 LPS-deficient N. meningitidis organisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 106 wild-type N. meningitidis and 108 LPS-deficient N. meningitidis organisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of “particularly LPS-sensitive” genes (2,288), differentially induced by 106 wild-type N. meningitidis but not by 108 LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-β), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-β may activate the type I IFN signaling pathway, and an unknown number of IFN-β-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 106 wild-type N. meningitidis organisms than when stimulated by 108 LPS-deficient N. meningitidis organisms. Plasma CXCL10, but not CXCL11, was positively correlated (r = 0.67; P < 0.01) to LPS in patients (n = 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock☆ , ☆☆

INTRODUCTION The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis. MATERIALS AND METHODS MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay. RESULTS MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2-2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r(s)=-0.84), time to peak (rs=-0.83), peak (r(s)=0.85) and ETP (r(s)=0.83). CONCLUSIONS MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.

Identification of meningococcal LPS as a major monocyte activator in IL-10 depleted shock plasmas and CSF by blocking the CD14-TLR4 receptor complex

We have examined the in vitro stimulatory effects of lipopolysaccharide (LPS)-containing samples (meningococcal shock plasma, n = 10; non-shock plasma, n = 10; cerebrospinal fluid (CSF), n = 7) before and after immunodepletion of interleukin (IL)-10 in a monocyte target assay. We also studied the stimulatory effects of plasma collected from 3 patients with lethal septicemia caused by Streptococcus pneumoniae without detectable LPS but with 100-fold increased levels of heat-shock protein 70 (HSP70). HSP70 may, like LPS, activate monocytes via the Toll-like receptor 4 (TLR4). The samples were analyzed for LPS, tumor necrosis factor (TNF)-α, IL-10 and HSP70; applied on human monocytes (purity > 95%) before and after IL-10 immunodepletion, in the absence or presence of CD14 blocking mAb (60bca) or the lipid A antagonist, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) which blocks TLR4. Monocyte activation was measured by increased TNF-α secretion and tissue factor (TF) up-regulation by monocyte procoagulant activity (PCA). There was a positive correlation between patientplasma LPS levels (n = 10) and increases in TNF-α secretion by the monocytes after immunodepletion of IL-10 ( r = 0.82). Pretreatment of the monocytes with mAbCD14 or RsDPLA reduced TNF-α secretion to median 5% and 12%, respectively, of the levels before the receptor complex was blocked. The median levels of HSP70 were 543 ng/ml (range, 468—962 ng/ml) in pneumococcal shock plasma, 81.5 ng/ml (range, 41—331 ng/ml) in meningococcal shock plasma and 24 ng/ml (range, < 0.8—41 ng/ml) in meningococcal non-shock plasma. Pneumococcal septic shock plasmas with significantly higher levels of HSP70 (P < 0.05) did not induce TNF-α secretion in the monocytes. The results strongly suggest that LPS in meningococcal shock plasma is the major activator of monocytes whereas HSP70 (in plasma concentrations up to 963 ng/ml) does not activate monocytes in this assay.