Anne-Marie Siebke Trøseid

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis.

Tissue factor (TF)‐positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP‐associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF‐protein and TF‐activity, which have been explained by antibody binding to “encrypted” or degraded forms of inactive TF‐protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte‐derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP‐associated TF‐protein (flow cytometry) and TF‐activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL−1) had ∼14 times higher TF‐activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti‐TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF‐positive events as MPs secernated from LPS‐stimulated monocytes [∼45,000 events mL−1 (American Diagnostica); ∼15,000 events mL−1 (BD)]. These TF‐positive events did not exert any TF‐activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP‐associated TF‐protein, and interpreted as a discrepancy between TF‐protein and TF‐activity.

Critical roles of complement and antibodies in host defense mechanisms against Neisseria meningitidis as revealed by human complement genetic deficiencies.

ABSTRACT Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log10 increase of CFU and 4- to 5-log10 increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed.

Reduced Platelet Function and Role of Drugs in Acute Gastrointestinal Bleeding

Gastrointestinal (GI) bleeding may be caused by a constitutive bleeding disposition or drug-induced inhibition of hemostasis. Platelet function in patients with ongoing GI bleeding is unknown. The aim of this study was to investigate platelet function in patients with acute GI bleeding. Patients (n=35) presenting with acute GI bleeding (hematemesis or melena) were recruited. For comparison, 13 patients treated with aspirin and 11 patients treated with clopidogrel without GI bleeding and 27 healthy controls were studied. Platelet function was measured by whole-blood aggregation and flow cytometry. Coagulation function was measured with calibrated automated thrombography. Platelet aggregation and P-selectin expression were significantly lower after arachidonic acid stimulation in GI bleeding patients than in healthy subjects (p≤0.05). Collagen-induced P-selectin expression was significantly reduced in patients using anti-platelet drugs (p=0.02) and in many patients not using anti-platelet drugs. Thrombin generation, measured by calibrated automated thrombography, was only reduced in patients on warfarin treatment. In conclusion, platelet function is reduced in acute GI bleeding patients and a considerable proportion appears to be related to drug use.

Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay.

There is increasing clinical interest for measuring microparticle (MP)-associated tissue factor (TF) activity owing to its possible role as a prothrombotic biomarker in a variety of diseases. However, the methods used are to various extents hampered by lack of (pre)analytical standardization as well as limited published documentation. The objective of this study was to evaluate the performance of the Zymuphen MP-TF kit and the calibrated automated thrombogram (CAT) assay in measuring MP-associated TF activity in plasma using a Neisseria meningitidis (Nm)-stimulated whole blood model. In addition, (pre)analytical variables like centrifugation procedures, freezing/thawing and the effect of addition of exogenous phosphatidylserine in plasma were evaluated in the CAT assay. Citrate-anticoagulated blood was stimulated with Nm bacteria for 4 h before platelet-poor plasma (PPP) or platelet-free plasma (PFP) were prepared and assayed with either of the two methods. Nm dose-dependently (104–108 bacteria/ml) induced TF-specific activity, measured as decreased lagtimes, in the CAT assay. The Zymuphen MP-TF kit also detected TF activity, although much higher Nm doses (108 bacteria/ml) were required to achieve measurable levels. Neither freezing/thawing nor the use of PPP vs. PFP influenced the TF activity, measured over a broad range of lagtimes, in the CAT assay. In conclusion, changes in lagtime in the CAT assay reflected levels of MP-associated TF activity in a more sensitive manner than the Zymuphen MP-TF kit did, in our Nm-stimulated whole blood system.

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression:

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.

Immunomodulatory effects of the Agaricus blazei Murrill-based mushroom extract AndoSan in patients with multiple myeloma undergoing high dose chemotherapy and autologous stem cell transplantation: a randomized, double blinded clinical study.

Forty patients with multiple myeloma scheduled to undergo high dose chemotherapy with autologous stem cell support were randomized in a double blinded fashion to receive adjuvant treatment with the mushroom extract AndoSan, containing 82% of Agaricus blazei Murrill (19 patients) or placebo (21 patients). Intake of the study product started on the day of stem cell mobilizing chemotherapy and continued until the end of aplasia after high dose chemotherapy, a period of about seven weeks. Thirty-three patients were evaluable for all study endpoints, while all 40 included patients were evaluable for survival endpoints. In the leukapheresis product harvested after stem cell mobilisation, increased percentages of Treg cells and plasmacytoid dendritic cells were found in patients receiving AndoSan. Also, in this group, a significant increase of serum levels of IL-1ra, IL-5, and IL-7 at the end of treatment was found. Whole genome microarray showed increased expression of immunoglobulin genes, Killer Immunoglobulin Receptor (KIR) genes, and HLA genes in the Agaricus group. Furthermore, AndoSan displayed a concentration dependent antiproliferative effect on mouse myeloma cells in vitro. There were no statistically significant differences in treatment response, overall survival, and time to new treatment. The study was registered with Clinicaltrials.gov NCT00970021.

The Neisseria meningitidis lpxL1 mutant induces less tissue factor expression and activity in primary human monocytes and monocyte-derived microvesicles than the wild type meningococcus:

Neisseria meningitidis (N. meningitidis) may cause sepsis and meningitis. N. meningitidis with a mutated lpxL1 gene has five, instead of six, acyl chains in the lipid A moiety. Compared with patients infected with the wild type (wt) meningococcus, patients infected with the lpxL1 mutant have a mild meningococcal disease with less systemic inflammation and less coagulopathy. Circulating tissue factor (TF), the main initiator of coagulation, has a central role in the development of coagulation disturbances during sepsis. To study how TF was influenced by the lpxL1 mutant, human primary monocytes and whole blood were incubated with the lpxL1 mutant or the wt meningococcus (H44/76). Monocyte and microvesicle (MV)-associated TF expression and TF-dependent thrombin generation were measured. In both purified monocytes and whole blood, our data show that the lpxL1 mutant is a weaker inducer of monocyte and MV-associated TF compared with the wt. Our data indicate that low levels of circulating TF may contribute to the reduced coagulopathy reported in patients infected with lpxL1 mutants.

Whole-blood incubation with the Neisseria meningitidis lpxL1 mutant induces less pro-inflammatory cytokines than the wild type, and IL-10 reduces the MyD88-dependent cytokines

Levels of bacterial LPS, pro-inflammatory cytokines and IL-10 are related to the severity of meningococcal septicaemia. Patients infected with a Neisseria meninigitidis lpxL1 mutant (Nm-mutant) with penta-acylated lipid A present with a milder meningococcal disease than those infected with hexa-acylated Nm wild type (Nm-wt). The aim was to compare the pro-inflammatory responses after ex vivo incubation with the heat-inactivated Nm-wt or the Nm-mutant in citrated whole blood, and the modulating effects of IL-10. Concomitantly, we measured intracellular IL-6, IL-8 and TNF-α to elucidate which cell types were responsible for the pro-inflammatory responses. Incubation with Nm-wt (106/ml;107/ml;108/ml) resulted in a dose-dependent increase of the MyD88-dependent pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), which were mainly derived from monocytes. In comparison, only 108/ml of the Nm-mutant significantly increased the concentration of these cytokines. The MyD88-independent cytokines (IP-10, RANTES) were evidently increased after incubation with the Nm-wt but were unaffected by the Nm-mutant. Co-incubation with IL-10 significantly reduced the concentrations of the MyD88-dependent cytokines induced by both the Nm-wt and the Nm-mutant, whereas the MyD88-independent cytokines were almost unaffected. In summary, the Nm-mutant is a weaker inducer of the MyD88-dependent/independent cytokines than the Nm-wt in whole blood, and IL-10 attenuates the Nm-stimulated increase in MyD88-dependent pro-inflammatory cytokines.

Large-scale reduction of tyrosine kinase activities in human monocytes stimulated in vitro with N. meningitidis

N. meningitidis induces extensive gene expression changes in human monocytes, suggesting that complex networks of signaling pathways are activated during meningococcal sepsis. These effects are modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). To further study changes in signal transduction suggested by mRNA data, we used kinase substrate arrays to identify composite kinase activities induced by lysates from a primary human monocyte model system. Cell lysates were prepared from monocytes treated with the following experimental conditions: 106 N. meningitidis/mL, 25 ng/mL IL-10, 106 N. meningitidis/mL in combination with 25 ng/mL IL-10, and vehicle. Lysates were subjected to kinase activity profiling with Tyrosine Kinase PamChip® arrays containing 144 kinase peptide substrates. In our experimental model, we were not able to detect a statistically significant large-scale change in ex vivo array peptide phosphorylation by lysates from monocytes treated for 15 minutes. Targets of the IL-10 anti-inflammatory response were not identified. A profound inhibition of array peptide phosphorylation by monocytes treated for 60 minutes was identified, suggesting low activity of a large number of kinases associated with different signaling pathways and immune cell functions, including STAT3 activity, Nf-κB and VEGF signaling, and PTEN signaling activity. The peptide representing ZBTB16, which was reduced in phosphorylation by lysates from all three experimental conditions, was in Ingenuity Pathway Analysis identified to be linked to reduced cytokine release and mRNA levels of tumor necrosis factor (TNF), IL-6, and CXCL10. Further studies should investigate changes in tyrosine kinase-mediated signal transduction in human immune cells, in order to evaluate the potential clinical application of kinome profiling in the study of systemic inflammatory responses to pathogens.