Hans Christian Dalsbotten Aass

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.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

ABSTRACT Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Recovery from major depressive disorder episode after non‐pharmacological treatment is associated with normalized cytokine levels

Several lines of evidence show that the immune system is implicated in the pathophysiology of major depressive disorder (MDD) and that treatment with antidepressants affects cytokine and C‐reactive protein (CRP) levels. Few studies have investigated immune markers during non‐pharmacological treatment. In this follow‐up study, we investigated whether CRP and elevated plasma cytokine levels observed before treatment of an acute episode of MDD are normalized during non‐pharmacological treatment.

Serum Levels of Cytokines, Glucose, and Hemoglobin as Possible Predictors of Poststroke Depression, and Association With Poststroke Fatigue

ABSTRACT Depression is a frequent and potentially disabling sequela of stroke. In the present study, we investigated the ability of stroke type, infarct volume, and laterality, and the levels of various cytokines and other blood components in the acute phase of acute ischemic stroke (AIS) in 45 patients, to predict the level of depression (Beck Depression Inventory [BDI] score) at 6, 12, and 18 months after its onset. The BDI score at 12 months poststroke was positively correlated with the acute serum level of glucose (r = 0.32, p = .038). When excluding the patients using antidepressants, the correlation between glucose level and later depression became significant at all three time points. A general association was found between depression and fatigue. Novel findings are that high acute serum levels of glucose may predict depression after AIS, a glucose level of approximately 126 mg/dL at admission might be a critical limit. Furthermore, depression and fatigue are two generally related—although independent—sequelae of stroke. Our findings did not support a causal immunological etiology for poststroke depression (PSD), as has been suggested previously for poststroke fatigue (PSF) in the same study sample.

Inflammation-Induced Catabolism of Tryptophan and Tyrosine in Acute Ischemic Stroke

Whether the inflammatory response that accompanies acute ischemic stroke induces the kynurenine pathway is currently a matter of conjecture. Activation of this pathway may disturb active metabolites. The aim of this study was thus to characterize the catabolism of tryptophan and tyrosine in acute ischemic stroke (AIS) patients, and its association with cytokines, C-reactive protein, and glucose. Serum levels of 5-hydroxytryptamine, tryptophan catabolites, and competing amino acids and significant ratios of these were measured in 45 AIS patients and compared to those of 40 control subjects. Furthermore, associations between the serum levels of these biomarkers and serum levels of cytokines, C-reactive protein, and glucose were determined. Significantly lower levels of tryptophan and tyrosine in the stroke group indicate increased tryptophan and tyrosine oxidation in acute ischemic stroke, while significantly lowered tryptophan index and tyrosine index indicate a reduced capacity for the synthesis of 5-hydroxytryptamine and catecholamines in the brain, respectively. Furthermore, our findings indicate that the proinflammatory response in acute ischemic stroke may be responsible for a reduced capacity for the biosynthesis of brain catecholamines and mediate neurotoxic effects. Meanwhile, the anti-inflammatory IL-10 may exert a neuroprotective effect and prevent the putative reduced capacity for 5-hydroxytryptamine synthesis in the brain. These mechanisms may be involved in several sequelae following stroke, such as cognitive impairment, depression, and fatigue.

Chromosomal 20q gain in the DNA diploid component of aneuploid colorectal carcinomas

The order of appearance of different genetic aberrations during the shift from diploidy/near‐diploidy to aneuploidy in colorectal cancers is not yet clear. We studied genetic alterations in flow cytometrically‐sorted DNA diploid and corresponding aneuploid epithelial cell populations from each of 20 colorectal tumors using comparative genomic hybridization, FISH, and PCR. Analysis of the 19 cases in which aberrations were found in the flow‐sorted diploid population indicated that large‐scale aneuploidization in colorectal cancer was preceded by amplification of oncogene(s) localized to chromosome 20q13.2 and by KRAS mutations, but not by TP53 deletions or losses of large chromosomal regions such as 4q, 8p and 18q.

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.

Global Effect of Interleukin-10 on the Transcriptional Profile Induced by Neisseria meningitidis in Human Monocytes

ABSTRACT In meningococcal septic shock, the dominant inducer of inflammation is lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis, while interleukin-10 (IL-10) is the principal anti-inflammatory cytokine. We have used microarrays and Ingenuity Pathway Analysis to study the global effects of IL-10 on gene expression induced by N. meningitidis, after exposure of human monocytes (n = 5) for 3 h to N. meningitidis (106 cells/ml), recombinant human IL-10 (rhIL-10) (25 ng/ml), and N. meningitidis combined with rhIL-10. N. meningitidis and IL-10 differentially expressed 3,579 and 648 genes, respectively. IL-10 downregulated 125 genes which were upregulated by N. meningitidis, including NLRP3, the key molecule of the NLRP3 inflammasome. IL-10 also upregulated 270 genes which were downregulated by N. meningitidis, including members of the leukocyte immunuglobulin-like receptor (LIR) family. Fifty-three genes revealed a synergistically increased expression when N. meningitidis and IL-10 were combined. AIM2 (the principal molecule of the AIM2 inflammasome) was among these genes (fold change [FC], 18.3 versus 7.4 and 9.4 after stimulation by N. meningitidis and IL-10, respectively). We detected reduced concentrations (92% to 40%) of six cytokines (IL-1b, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], macrophage inflammatory protein alpha [MIP-α], MIP-β) in the presence of IL-10, compared with concentrations with stimulation by N. meningitidis alone. Our data analysis of the effects of IL-10 on gene expression induced by N. meningitidis suggests that high plasma levels of IL-10 in meningococcal septic shock plasma may have a profound effect on a variety of functions and cellular processes in human monocytes, including cell-to-cell signaling, cellular movement, cellular development, antigen presentation, and cell death.

CD59 Efficiently Protects Human NT2-N Neurons Against Complement-mediated Damage

The complement regulatory protein CD59 controls cell survival by the inhibition of C5b–9 formation on the cell membrane. Loss of CD59 increases the susceptibility of cells to complement‐mediated damage and lysis. Deposition of IgM can induce complement activation with subsequent cell death. We have previously demonstrated the presence of CD59 on human NT2‐N neurons. In this study, we investigated the functional role of CD59 for NT2‐N cell survival after IgM‐mediated complement activation. Complement activation was induced on NT2‐N neurons with human serum following incubation with the IgM monoclonal antibody A2B5 reacting with a neuronal cell membrane epitope. Deposition of C1q and C5b–9 was detected on the cell membrane and sC5b–9 in the culture supernatant. Specific inhibition of complement was obtained by the C3 inhibitor compstatin, and by anti‐C5/C5a MoAb. CD59 was blocked by the MoAb BRIC 229. Membrane damage of propidium iodide‐stained NT2‐N cells was confirmed by immunofluorescence microscopy and degeneration of neuronal processes was shown with crystal violet staining. A2B5, but not the irrelevant control IgM antibody, induced complement activation on NT2‐N neurons after incubation with a human serum, as detected by the deposition of C1q. A marked membrane deposition of C5b–9 on NT2‐N neurons with accompanying cell death and axonal degeneration was found after the blocking of CD59 with MoAb BRIC 229 but not with an isotype‐matched control antibody. Compstatin and anti‐C5 monoclonal antibodies which blocked C5 activation efficiently inhibited complement activation. In conclusion, CD59 is essential for protecting human NT2‐N neurons against complement‐mediated damage, which is known to occur in a number of clinical conditions including stroke.

Microparticle-associated tissue factor activity is reduced by inhibition of the complement protein 5 in Neisseria meningitidis-exposed whole blood.

Neisseria meningitidis causes fulminant meningococcal sepsis with a massive activation of the coagulation and complement cascades. Bacterial cell envelope molecules from N. meningitidis, particularly lipopolysaccharide (LPS), induce tissue factor (TF) expression. In meningococcal sepsis, TF can be detected on circulating monocytes and microparticles (MPs) within the bloodstream. During infection, Nm activates C5 and C5a, which also is able to induce TF. We evaluated the effect of eculizumab, a C5-blocking monoclonal antibodies (mAb), on cell- and MP-associated TF. Using a lepirudin-anticoagulated whole blood model, we activated the coagulation and complement cascades by N. meningitidis, and investigated the interaction between the cascade systems with special focus on cell-associated TF-expression (mRNA and protein) and MP-associated TF-dependent thrombin and fibrin generation in platelet-free plasma. We also examined the ability of TF-positive MPs to support clot formation in whole blood. In addition, the effect of corn trypsin inhibitor and time-dependent changes on MP-associated functional TF activity was examined. Inhibition of C5 reduced cell-associated TF expression at both gene and protein level, and reduced MP-associated TF-dependent thrombin and fibrin generation in platelet-poor plasma, MP-induced TF-dependent clot formation in whole blood, implying that the complement and coagulation cascades are interplayers in N. meningitidis-mediated activation of these cascades.