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.

Massive Organ Inflammation in Experimental and in Clinical Meningococcal Septic Shock.

Fulminant meningococcal sepsis is characterized by a massive growth of bacteria in the circulation, regarded as the primary inflammatory site, with no specific solid organ focus. Here we aimed to study the local inflammatory response in organs using a porcine model of fulminant meningococcal septic shock challenged with exponentially increasing doses of heat inactivated Neisseria meningitidis. The results were compared with those obtained in organs post mortem from three patients with lethal meningococcal septic shock. Nine patients with lethal pneumococcal disease and 14 patients with sudden infant death syndrome served as controls. Frozen tissue were thawed, homogenized and prepared for quantification of bacterial DNA by real-time polymerase chain reaction, and key inflammatory mediators were measured by ELISA in the pig material and by multiplex in the human material. In addition, gene expression assayed by Affymetrix gene expression profiling was performed in the pig study. The porcine model revealed a major influx of N. meningitidis in lungs, liver, spleen, and kidneys accompanied with major production of cardinal inflammatory mediators including tumor necrosis factor, interleukin (IL)-1ß, IL-6, and IL-8, far exceeding the amount detected in blood. Genes encoding for these mediators revealed a similar profile. By comparing the wild-type with a lipopolysaccharide (LPS) deficient meningococcal strain, we documented that LPS was the dominant group of molecules inducing organ inflammation and was required for IL-8 production. IL-10 production was predominantly stimulated by non-LPS molecules. The massive organ inflammation in the porcine model was present in the three patients dying of meningococcal shock and differed markedly from the patients with lethal pneumococcal infections and sudden infant death syndrome. In conclusion, in meningococcal sepsis, a massive local inflammatory response occurs in specific organs.

miRNA and mRNA expression profiling identifies members of the miR-200 family as potential regulators of epithelial-mesenchymal transition in pterygium.

The current study investigates whether microRNA (miRNA) regulators of epithelial-mesenchymal transition (EMT), tissue fibrosis, and angiogenesis are differentially expressed in human primary pterygium. Genome-wide miRNA and mRNA expression profiling of paired pterygium and normal conjunctiva was performed in the context of conventional excision of pterygium with autotransplantation of conjunctiva (n = 8). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the expression of key molecules previously detected by microarray. In pterygium, 25 miRNAs and 31 mRNAs were significantly differentially expressed by more than two-fold compared to normal conjunctiva. 14 miRNAs were up-regulated (miR-1246, -486, -451, -3172, -3175, -1308, -1972, -143, -211, -665, -1973, -18a, 143, and -663b), whereas 11 were down-regulated (miR-675, -200b-star, -200a-star, -29b, -200b, -210, -141, -31, -200a, -934, and -375). Unsupervised hierarchical cluster analysis demonstrated that members of the miR-200 family were coexpressed and down-regulated in pterygium. The molecular and cellular functions that were most significant to the miRNA data sets were cellular development, cellular growth and proliferation, and cellular movement. qRT-PCR confirmed the expression of 15 of the 16 genes tested and revealed that miR-429 was down-regulated by more than two-fold in pterygium. The concerted down-regulation of four members from both clusters of the miR-200 family (miR-200a/-200b/-429 and miR-200c/-141), which are known to regulate EMT, and up-regulation of the predicted target and mesenchymal marker fibronectin (FN1), suggest that EMT could potentially play a role in the pathogenesis of pterygium and might constitute promising new targets for therapeutic intervention in pterygium.

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 (10-10 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 (10 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.

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.

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.