Transcriptomic data from two primary cell models stimulating human monocytes suggest inhibition of oxidative phosphorylation and mitochondrial function by N. meningitidis which is partially up-regulated by IL-10.

BACKGROUND: Biological interpretation of DNA microarray data may differ depending on underlying assumptions and statistical tests of bioinformatics tools used. We used Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to analyze previously generated DNA microarray data from human monocytes stimulated with N. meningitidis and IL-10 ("the model system"), and with meningococcal sepsis plasma before and after immunodepletion of IL-10 ("the patient plasma system"). The objectives were to compare if the two bioinformatics methods resulted in similar biological interpretation of the datasets, and to identify whether GSEA provided additional insight compared with IPA about the monocyte host response to meningococcal activation. RESULTS: In both experimental models, GSEA and IPA identified genes associated with pro-inflammatory innate immune activation, including TNF-signaling, Toll-like receptor signaling, JAK-STAT-signaling, and type I and type II interferon signaling. GSEA identified genes regulated by the presence of IL-10 with similar gene sets in both the model system and the patient plasma system. In the model system, GSEA and IPA in sum identified 170 genes associated with oxidative phosphorylation/mitochondrial function to be down-regulated in monocytes stimulated with meningococci. In the patient plasma system, GSEA and IPA in sum identified 122 genes associated with oxidative phosphorylation/mitochondrial dysfunction to be down-regulated by meningococcal sepsis plasma depleted for IL-10. Using IPA, we identified IL-10 to up-regulate 18 genes associated with oxidative phosphorylation/mitochondrial function that were down-regulated by N. meningitidis. CONCLUSIONS: Biological processes associated with the gene expression changes in the model system of meningococcal sepsis were comparable with the results found in the patient plasma system. By combining GSEA with IPA, we discovered an inhibitory effect of N. meningitidis on genes associated with mitochondrial function and oxidative phosphorylation, and that IL-10 partially reverses this strong inhibitory effect, thereby identifying, to our knowledge, yet another group of genes where IL-10 regulates the effect of LPS. We suggest that relying on a single bioinformatics tool together with an arbitrarily chosen filtering criteria for data analysis may result in overlooking relevant biological processes and signaling pathways associated with genes differentially expressed between compared experimental conditions.

Increased ID2 levels in adult precursor B cells as compared with children is associated with impaired Ig locus contraction and decreased bone marrow output.

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2.

Authorship: attitudes and practice among Norwegian researchers.

BACKGROUND: Attitudes to, and practices of, scientific authorship vary. We have studied this variation among researchers in a university hospital and medical school in Norway. METHODS: We invited all faculty, researchers and PhD students at Oslo University Hospital and the Faculty of Medicine, University of Oslo (approximately 2700) by e-mail to answer a web-based questionnaire in January 2013. We asked the researchers to report their authorship experiences and to score their agreement with, and ability to practice according to, 13 statements on authorship qualifications and criteria on a five-point Likert scale (1 = completely agree, 5 = completely disagree). The statements were taken from the International Committee of Medical Journal Editors (ICMJE) and other recommendations on authorship. RESULTS: 654 questionnaires were returned (response rate 24%); 25% of the respondents had published less than five scientific articles, 43% five to 49, and 32% more than 50 articles. 97% reported knowledge of defined authorship criteria, and 68% regarded breaches of these as scientific misconduct. 36% had experienced pressure to include undeserved authors in their papers, more in basic science (46%) than in community medicine (25%). 29% reported that they had been denied authorship they believed they deserved. Researchers with less than six years of research experience found authorship decisions more difficult than more experienced researchers (48% vs 30%).The respondents' agreement with the statements on authorship was higher than their self-reported ability to follow them for all statements. Average scores for agreement and practice for all statements combined were 1.4 vs 2.3. The discrepancy between attitude and practice declined with publishing experience. For the core ICMJE authorship requirements the average difference between attitude and practice was 1.2 among those who had published less than 5 articles and 0.7 among those who had published 50 articles or more (p < 0.05). CONCLUSIONS: Almost all the responding researchers had knowledge of formal authorship requirements. Most of them agreed with the criteria, but found it harder to put them into practice. More experienced researchers found decisions on authorship and about the order of authors easier than less experienced researchers.

Global effect of interleukin-10 on the transcriptional profile induced by Neisseria meningitidis in human monocytes.

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 (10(6) 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-ß) 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.

Long-term occupational outcomes of endotoxin exposure and the effect of exposure cessation.

OBJECTIVE: To study the possible respiratory and haematological effects of endotoxin exposure to bacterial single-cell protein (BSCP) in workers during a follow-up period of 5 years including 4 years of exposure and 1 year without exposure. METHODS: The study included 28 workers examined in 2002-2005 and 1 year after exposure termination in 2007. The arithmetic mean endotoxin exposure was 5800-11,000 EU/m(3) among the high exposure group and 390 EU/m(3) in the low exposure group. Assessment of lung function included spirometry and gas diffusion in 2003, 2004 and 2007. Rhinometry was performed in 2004 and 2007. Blood analysis included leukocyte cell count and measurement of the acute phase proteins: C-reactive protein, interleukin-6, eosinophilic cationic protein, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1, chemoattractant protein RANTES, platelet-derived growth factor BB, fibrinogen and D-dimer. RESULTS: In the low exposure group, but not in the high exposure group, there were significant improvements in both forced vital capacity (FVC) (290 ml) and forced expiratory volume in 1 s (FEV(1)) (180-210 ml) (p=0.004-0.03) 1 year after the end of exposure. The number of leukocytes and eosinophilic cationic protein and D-dimer levels increased significantly with increasing endotoxin exposures and decreased significantly 1 year after exposure termination. Changes in acute phase proteins suggested exposure-related tolerance. CONCLUSIONS: An inflammatory tendency during an exposure period of 4 years seems to reverse 1 year after cessation of exposure to endotoxins from a single species. Lung function improved significantly among workers exposed to low levels of endotoxin but not among the highly exposed workers.

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.

Striking decrease in the total precursor B-cell compartment during early childhood as evidenced by flow cytometry and gene expression changes.

The number of circulating B-cells in peripheral blood plateaus between 2 and 24 months of age, and thereafter declines gradually. How this reflects the kinetics of the precursor B-cell pool in the bone marrow is of clinical interest, but has not been studied thoroughly in humans. The authors analyzed bone marrow (n = 37) from healthy children and adults (flow cytometry) searching for age-related changes in the total precursor B-cell compartment. In an age-matched cohort (n = 25) they examined age-related global gene expression changes (Affymetrix) in unsorted bone marrow with special reference to the recombination activating gene 1, RAG1. Subsequently, they searched the entire gene set for transcripts correlating to the RAG1 profile to discover other known and possibly new precursor B-cell related transcripts. Both methods disclosed a marked, transient increase of total precursor B-cells at 6-20 months, followed by a rapid decrease confined to the first 2 years. The decline thereafter was considerably slower, but continued until adulthood. The relative composition of total precursor B-cells, however, did not change significantly with age. The authors identified 54 genes that were highly correlated to the RAG1 profile (r >or= .9, p < 1 x 10(-8)). Of these 54 genes, 15 were characteristically B-lineage associated like CD19, CD79, VPREB, EBF1, and PAX5; the remaining 39 previously not described as distinctively B-lineage related. The marked, transient increase in precursor B-cells and RAG1 transcriptional activity is not reflected by a similar peak in B-cells in peripheral blood, whereas the sustained plateau concurs in time.

Using global gene expression patterns to characterize Annexin V positive and negative human monocytes in culture.

OBJECTIVE: To investigate the early apoptosis that may be detected by Annexin V binding to phosphatidylserine and propidium iodide (PI) exclusion in human monocytes. When studying monocytes in culture, less than 40 % of these cells survive after 7 days. MATERIAL AND METHODS: In the first 4 h, 24 % of monocytes in culture develop into Annexin V(+)/PI(-) cells. Human monocytes were investigated at 0 h and sorted into Annexin V(+) and Annexin V(-) by FACS after 4 h. Gene expression was examined by microarray analyses. RESULTS: At 4 h, Annexin V(+) monocytes versus Annexin V(-) cells showed 1220 differentially expressed genes. Ingenuity Pathway Analysis disclosed 153 genes related to cell death. Among these were caspase activators, caspase 6, Apaf 2 and FAS, as well as the autophagy gene ATG5. In addition, examination of the most up-regulated or down-regulated genes among the 1220 revealed genes involved in other biological processes, as well as genes not yet annotated. These included the non-annotated genes LOC28480 (fold change: 82) and 225767-at (fold change: 68) and the transcription factor SOX 4 (fold change: 24). conclusions: We suggest that apoptosis in cultured monocytes, as evidenced by Annexin V(+), operates through genes well known in apoptosis, but that the process also involves additional genes not commonly associated with apoptosis.

Identification of genes particularly sensitive to lipopolysaccharide (LPS) in human monocytes induced by wild-type versus LPS-deficient Neisseria meningitidis strains.

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 (10(6)), LPS-deficient N. meningitidis (10(6) and 10(8)), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (10(6)) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 10(6) LPS-deficient N. meningitidis organisms. However, 10(8) 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 10(6) wild-type N. meningitidis and 10(8) 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 10(6) wild-type N. meningitidis but not by 10(8) LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-beta), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-beta may activate the type I IFN signaling pathway, and an unknown number of IFN-beta-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 10(6) wild-type N. meningitidis organisms than when stimulated by 10(8) 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.

The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference.

BACKGROUND: Pseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (ABCA17P). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents. RESULTS: Systematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene ABCC6, ABCC6P1 and ABCC6P2, are transcribed. ABCC6P1 and ABCC6 possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene ABCC6P1 resulted in a significant reduction of ABCC6 mRNA expression levels. CONCLUSION: The human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of ABCC6 mRNA levels by targeted suppression of the expression of its pseudogene ABCC6P1 provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.

Does granulocyte colony-stimulating factor ameliorate the proinflammatory response in human meningococcal septic shock?

OBJECTIVE: To test the hypothesis that granulocyte colony-stimulating factor acts cooperatively with interleukin-10 in down-regulating monocyte function in severe meningococcal septic shock. 1) We quantified the plasma levels of granulocyte colony-stimulating factor, interleukin-10, Neisseria meningitidis lipopolysaccharide and the number of N. meningitidis DNA copies in 28 patients with systemic meningococcal disease. 2) We studied the inhibitory effect of recombinant human granulocyte colony-stimulating factor on normal human monocytes stimulated with purified meningococcal lipopolysaccaride. 3) We monitored the inhibitory effects of endogenously produced granulocyte colony-stimulating factor and interleukin-10 in meningococcal shock plasmas on monocytes. DESIGN: Comparative, experimental study. SETTING: University Hospital and laboratory. SUBJECTS: Twenty-eight patients with systemic meningococcal disease, 13 with persistent shock, 7 died, and 15 without shock. MEASUREMENTS AND MAIN RESULTS: The median levels of granulocyte colony-stimulating factor in shock and nonshock patients were 1.7 x 10(6) and 8.1 x 10(2) pg/mL; interleukin-10, 2.1 x 10(4) and 4 x 10(1) pg/mL; number of N. meningitidis DNA copies, 2.9 x 10(7) and <10(3)/mL; and lipopolysaccharide, 105 and <0.04 endotoxin units/mL, respectively. The plasma levels of granulocyte colony-stimulating factor were reduced by 50% within 4 to 6 hrs after initiation of antibiotic treatment. In model experiments with lipopolysaccharide-stimulated human monocytes, recombinant human granulocyte colony-stimulating factor and interleukin-10 reduced the release of tumor necrosis factor-alpha by mean 30% and 92%, respectively. When plasmas from three shock patients were depleted of native granulocyte colony-stimulating factor or interleukin-10 by immunoprecipitation, no increase in tumor necrosis factor-alpha release occurred after removal of granulocyte colony-stimulating factor, whereas removal of interleukin-10 increased the tumor necrosis factor-alpha release eight-fold. CONCLUSIONS: Although granulocyte colony-stimulating factor in plasma increases by five orders of magnitude in patients with meningococcal shock, the anti-inflammatory effect on patients' monocytes is uncertain.

Quantitation of serum free light chains in combination with protein electrophoresis and clinical information for diagnosing multiple myeloma in a general hospital population.

BACKGROUND: Serum free light chain (SFLC) measurements have recently come into use as an aid for diagnosing monoclonal gammopathy. We evaluated SFLC measurements in combination with serum protein electrophoresis (SPE) and clinical information for diagnosing multiple myeloma (MM) in a hospital population. METHODS: We measured SFLCs in 3818 sera received for SPE over a 1-year period when patient symptoms or biochemical findings suggested myeloma-related tissue damage (n = 1067). We reviewed SPE and SFLC results from 489 patients together with their final diagnoses obtained from the hospital information technology department. RESULTS: SFLC measurement, combined with SPE and clinical information, allowed identification of 95% of patients (38 of 40) with previously undiagnosed MM, macroglobulinemia, or primary amyloidosis. Additionally, we identified 45 patients with monoclonal gammopathy of undetermined significance (MGUS) and 4 with plasmacytoma. Of patients followed at our hospital in whom SFLCs were not measured, only 1 patient was diagnosed with MM. This patient had anemia and was mistakenly not tested for SFLCs. An abnormal kappa/lambda ratio was found in 26 of 29 patients with MM but also in 36 of 203 patients with renal impairment, polyclonal immunoresponse, or other nonhematological diagnoses. None of the 203 patients with nonhematological disease had a kappa/lambda ratio <0.05 or >10. CONCLUSIONS: The combined use of SPE, SFLC measurements, and clinical criteria allows MM to be efficiently diagnosed or excluded based on serum measurements only.

Warfarin dose and INR related to genotypes of CYP2C9 and VKORC1 in patients with myocardial infarction.

BACKGROUND: Warfarin treatment has a narrow therapeutic range, requiring meticulous monitoring and dosage titration. Individual dosage requirement has recently partly been explained by genetic variation of the warfarin metabolizing enzyme CYP2C9 and the Vitamin K-activating enzyme VKORC1. In the WARIS-II study, comparing three different antithrombotic regimens after myocardial infarction, warfarin treatment reduced thrombotic events, but was associated with more frequent bleeding than use of acetylsalisylic acid (ASA) alone. AIMS: The primary aim of the present study was to investigate the relation between genotypes of CYP2C9 and VKORC1 and warfarin maintenance dose in myocardial infarction. The secondary aim was to relate the genotypes to international normalized ratio (INR). METHODS: Genotyping was performed in 212 myocardial infarction patients from the WARIS-II study by robotic isolation of DNA from EDTA whole blood (MagNa Pure LC) before PCR amplification (LightCycler) and melting point analysis. RESULTS: The 420 C>T substitution of CYP2C9*2, the 1075 A>C substitution of CYP2C9*3 and the 1173 C>T substitution of VKORC1 had minor allele frequencies of, 11.3%, 5.7% and 36.6% respectively. Warfarin weekly dose varied between 17 mg and 74 mg among the patients. INR did not vary between genotypes. Warfarin dosage requirement was significantly associated with CYP2C9 and VKORC1 genotypes, treatment group and age. The VKORC1 genotype contributed 24.5% to the interindividual variation in warfarin dosage, whereas the combined CYP2C9 genotypes were only responsible for 7.2% of the dose variation. CONCLUSION: CYP2C9 and VKORC1 genotype frequencies in myocardial infarction patients appear similar to other patient groups and have similar impact on warfarin maintenance dose.

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.

Fibrinogen and fibrin induce synthesis of proinflammatory cytokines from isolated peripheral blood mononuclear cells.

Fibrinogen in plasma includes three main fractions; high-molecular-weight (HMW)-fibrinogen, low-molecular-weight (LMW)-fibrinogen, and very-low-molecular-weight (LMW')-fibrinogen. During acute-phase conditions, plasma fibrinogen levels and the HMW-/LMW-fibrinogen ratio increase rapidly due to increased synthesis of HMW-fibrinogen. The consequences of elevated plasma fibrinogen levels and local deposition of fibrin in inflammatory tissues observed during acute-phase conditions are not clear. We wanted to investigate proinflammatory effects of fibrinogen and fibrin on peripheral blood mononuclear cells (PBMC) as reflected by altered mRNA expression and synthesis of the proinflammatory cytokines IL-6, TNF-alpha and IL-1 beta, and to explore the significance of altered HMW-/LMW-fibrinogen ratio. PBMC were isolated from whole blood using Lymphoprep. HMW-fibrinogen was separated from unfractioned fibrinogen by ammonium sulphate precipitation. Cells were incubated with unfractioned fibrinogen, HMW-fibrinogen or fibrin. Cytokine levels in cell lysates were determined using ELISA assays. Real-time PCR was used for mRNA quantification. We found that fibrinogen significantly increased mRNA levels, and induced synthesis of the proinflammatory cytokines IL-6 and TNF-alpha in PBMC in a dose dependent manner. Median (25, 75 percentile) IL-6 and TNF-alpha concentrations were 12 (5, 40) pg/ml and 16 (0,61) pg/ml, respectively. Median mRNA quantity was increased 12.3- (6.6, 48.6) and 1.7- (1.5, 6.5) fold for IL-6 and TNF-alpha compared to controls. The stimulatory effect of unfractioned fibrinogen was not significantly different from HMW-fibrinogen. Fibrinogen and fibrin were equally effective in promoting cytokine synthesis from PBMC. The results support that fibrin and fibrinogen may actively modulate the inflammatory process by inducing synthesis of proinflammatory cytokines from PBMC.

[ABCA-transporters: regulators of cellular lipid transport]. / ABCA-transportører - regulatorer av den cellulaere lipidtransporten.

BACKGROUND: The transport of lipids, which is orchestrated by a multitude of molecular factors, is a key feature of the physiology of living cells. A new group of transporter protein, the A-subclass of ATP-binding cassette (ABC) transporters, was recently discovered. ABCA-transporters play pivotal roles in cellular lipid transport and their discovery has brought important new insights into the molecular basis of this process. This review article presents the biology of ABCA-transport proteins and their implication for clinical medicine. MATERIAL AND METHODS: Literature retrieved from Pubmed, including own research results, formed the basis for the article. RESULTS AND INTERPRETATION: Mutations in ABCA-transporter genes have been shown to result in hereditary diseases involving major physiologicical processes in the cardiovascular, respiratory, visual and integumentary systems. Accumulated evidence suggests that ABCA-transporters play critical roles in the pathogenesis of complex multifactorial disorders with a high incidence; such as atherosclerosis, age-related macula degeneration and Alzheimer's disease.

Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study.

Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

The human ortholog of the rodent testis-specific ABC transporter Abca17 is a ubiquitously expressed pseudogene (ABCA17P) and shares a common 5' end with ABCA3.

BACKGROUND: During the past years, we and others discovered a series of human ATP-binding cassette (ABC) transporters, now referred to as ABC A-subfamily transporters. Recently, a novel testis-specific ABC A transporter, Abca17, has been cloned in rodent. In this study, we report the identification and characterization of the human ortholog of rodent Abca17. RESULTS: The novel human ABC A-transporter gene on chromosome 16p13.3 is ubiquitously expressed with highest expression in glandular tissues and the heart. The new ABC transporter gene exhibits striking nucleotide sequence homology with the recently cloned mouse (58%) and rat Abca17 (51%), respectively, and is located in the syntenic region of mouse Abca17 indicating that it represents the human ortholog of rodent Abca17. However, unlike in the mouse, the full-length ABCA17 transcript (4.3 kb) contains numerous mutations that preclude its translation into a bona fide ABC transporter protein strongly suggesting that the human ABCA17 gene is a transcribed pseudogene (ABCA17P). We identified numerous alternative ABCA17P splice variants which are transcribed from two distinct transcription initiation sites. Genomic analysis revealed that ABCA17P borders on another ABC A-subfamily transporter - the lung surfactant deficiency gene ABCA3. Surprisingly, we found that both genes overlap at their first exons and are transcribed from opposite strands. This genomic colocalization and the observation that the ABCA17P and ABCA3 genes share significant homologies in several exons (up to 98%) suggest that both genes have evolved by gene duplication. CONCLUSION: Our results demonstrate that ABCA17P and ABCA3 form a complex of overlapping genes in the human genome from which both non-coding and protein-coding ABC A-transporter RNAs are expressed. The fact that both genes overlap at their 5' ends suggests interdependencies in their regulation and may have important implications for the functional analysis of the disease gene ABCA3. Moreover, this is the first demonstration of the expression of a pseudogene and its parent gene from a common overlapping DNA region in the human genome.

Flow cytometry-sorted non-viable endotoxin-treated human monocytes are strongly procoagulant.

Monocytes/macrophages are important in disease states such as gram-negative sepsis and coronary artery disease. Following exposure to lipopolysaccharide (LPS), monocytes express tissue factor (TF), the main initiator of blood coagulation. We previously demonstrated that human monocytes treated with high concentrations of LPS, or with LPS and calcium ionophore, displayed higher TF activity than monocytes treated with only low concentrations of LPS, even though the monocytes under all conditions expressed similar amounts of cell surface TF antigen. Such restrainedTF activity is often referred to as encryption and its release as de-encryption. We also observed that the increase in TF activity, de-encryption, coincided with an increase in cell surface phosphatidylserine (PS) representing apoptosis and necrosis. In the present work, we separated LPS and LPS and calcium ionophore-treated human monocytes into two populations, one of mainly viable, PS negative cells, and one of mainly non-viable, PS positive cells, by sorting flow-cytometry. We observed that non-viable cells expressed considerably less TF antigen than viable cells. Despite this, non-viable cells were clearly more procoagulant than viable cells in two different coagulation assays. Procoagulant activity was dependent on both TF and PS. We consider the higher content of externalized PS in non-viable monocytes as the major reason for the stronger procoagulant activity of these cells. Thus, TF de-encryption appears largely to occur on PS positive, non-viable cells under these conditions. This supports the important role of PS in coagulation, and it suggests that PS expression signifying cell death, may be clinically relevant.

Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis.

Tissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.