A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes

AbstractA newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases

Pulmonary vasoconstrictor action of KCNQ potassium channel blockers

KCNQ channels have been widely studied in the nervous system, heart and inner ear, where they have important physiological functions. Recent reports indicate that KCNQ channels may also be expressed in portal vein where they are suggested to influence spontaneous contractile activity. The biophysical properties of K+ currents mediated by KCNQ channels resemble a current underlying the resting K+ conductance and resting potential of pulmonary artery smooth muscle cells. We therefore investigated a possible role of KCNQ channels in regulating the function of pulmonary arteries by determining the ability of the selective KCNQ channel blockers, linopirdine and XE991, to promote pulmonary vasoconstriction. Linopirdine and XE991 both contracted rat and mouse pulmonary arteries but had little effect on mesenteric arteries. In each case the maximum contraction was almost as large as the response to 50 mM K+. Linopirdine had an EC50 of around 1 μM and XE991 was almost 10-fold more potent. Neither removal of the endothelium nor exposure to phentolamine or α,β-methylene ATP, to block α1-adrenoceptors or P2X receptors, respectively, affected the contraction. Contraction was abolished in Ca2+-free solution and in the presence of 1 μM nifedipine or 10 μM levcromakalim

25-Hydroxyvitamin D and pre-clinical alterations in inflammatory and hemostatic markers: a cross sectional analysis in the 1958 British Birth Cohort

BACKGROUND: Vitamin D deficiency has been suggested as a cardiovascular risk factor, but little is known about underlying mechanisms or associations with inflammatory or hemostatic markers. Our aim was to investigate the association between 25-hydroxyvitamin D [25(OH)D, a measure for vitamin D status] concentrations with pre-clinical variations in markers of inflammation and hemostasis. METHODOLOGY/PRINCIPAL FINDINGS: Serum concentrations of 25(OH)D, C-reactive protein (CRP), fibrinogen, D-dimer, tissue plasminogen activator (tPA) antigen, and von Willebrand factor (vWF) were measured in a large population based study of British whites (aged 45 y). Participants for the current investigation were restricted to individuals free of drug treated cardiovascular disease (n = 6538). Adjusted for sex and month, 25(OH)D was inversely associated with all outcomes (p or =75 nmol/l compared to < 25 nmol/l. D-dimer concentrations were lower for participants with 25(OH)D 50-90 nmol/l compared to others (quadratic term p = 0.01). We also examined seasonal variation in hemostatic and inflammatory markers, and evaluated 25(OH)D contribution to the observed patterns using mediation models. TPA concentrations varied by season (p = 0.02), and much of this pattern was related to fluctuations in 25(OH)D concentrations (p < or =0.001). Some evidence of a seasonal variation was observed also for fibrinogen, D-dimer and vWF (p < 0.05 for all), with 25(OH)D mediating some of the pattern for fibrinogen and D-dimer, but not vWF. CONCLUSIONS: Current vitamin D status was associated with tPA concentrations, and to a lesser degree with fibrinogen and D-dimer, suggesting that vitamin D status/intake may be important for maintaining antithrombotic homeostasi

Characterizing the pathotype of neonatal meningitis causing <i>Escherichia coli</i> (NMEC)

Background Neonatal meningitis-causing Escherichia coli (NMEC) is the predominant Gram-negative bacterial pathogen associated with meningitis in newborn infants. High levels of heterogeneity and diversity have been observed in the repertoire of virulence traits and other characteristics among strains of NMEC making it difficult to define the NMEC pathotype. The objective of the present study was to identify genotypic and phenotypic characteristics of NMEC that can be used to distinguish them from commensal E. coli. Methods A total of 53 isolates of NMEC obtained from neonates with meningitis and 48 isolates of fecal E. coli obtained from healthy individuals (HFEC) were comparatively evaluated using five phenotypic (serotyping, serum bactericidal assay, biofilm assay, antimicorbial susceptibility testing, and in vitro cell invasion assay) and three genotypic (phylogrouping, virulence genotyping, and pulsed-field gel electrophoresis) methods. Results A majority (67.92 %) of NMEC belonged to B2 phylogenetic group whereas 59 % of HFEC belonged to groups A and D. Serotyping revealed that the most common O and H types present in NMEC tested were O1 (15 %), O8 (11.3 %), O18 (13.2 %), and H7 (25.3 %). In contrast, none of the HFEC tested belonged to O1 or O18 serogroups. The most common serogroup identified in HFEC was O8 (6.25 %). The virulence genotyping reflected that more than 70 % of NMEC carried kpsII, K1, neuC, iucC, sitA, and vat genes with only less than 27 % of HFEC possessing these genes. All NMEC and 79 % of HFEC tested were able to invade human cerebral microvascular endothelial cells. No statistically significant difference was observed in the serum resistance phenotype between NMEC and HFEC. The NMEC strains demonstrated a greater ability to form biofilms in Luria Bertani broth medium than did HFEC (79.2 % vs 39.9 %). Conclusion The results of our study demonstrated that virulence genotyping and phylogrouping may assist in defining the potential NMEC pathotype

Design and baseline characteristics of a prospective cohort study for determinants of osteoporotic fracture in community-dwelling elderly Japanese men: the Fujiwara-kyo Osteoporosis Risk in Men (FORMEN) Study

<p>Abstract</p> <p>Background</p> <p>Osteoporosis and osteoporotic fracture in men are significant public health problems in an aging society. However, information on male osteoporosis remains impressively lacking, especially for Asians. We designed the Fujiwara-kyo Osteoporosis Risk in Men (FORMEN) study as an ancillary study of a cohort study, the Fujiwara-kyo study, to determine risk factors for osteoporotic fractures in Japanese men.</p> <p>Methods/Design</p> <p><it><b>Design</b></it>: A community-based single-centre prospective cohort study with at least a 5-year follow-up</p> <p><it><b>Subjects</b></it>: All the male participants of the Fujiwara-kyo study who were living in the four cities studied, aged 65 years and older, and able to walk without aid from others.</p> <p><it><b>Primary outcome</b></it>: Incidence of osteoporotic fractures including vertebral and clinical non-vertebral fractures.</p> <p><it><b>Additional outcomes</b></it>: Change in bone mineral density (BMD), change in hip geometry, onset of receiving benefits from Long-term Care Insurance (LCI), health-related quality of life, and mortality.</p> <p><it><b>Baseline measurements</b></it>: BMD at the lumbar spine (LS) and hip (TH), hip geometry, vertebral deformity assessment, bone turnover markers, physical and cognitive performance, various medical and lifestyle factors, and geriatric psychosocial measures confirmed by interviews based on self-administrated questionnaires.</p> <p><it><b>Outcome surveillance</b></it>: Annual mail surveys and a follow-up survey at the fifth year comprising similar items to the baseline study will be used to determine the outcomes. Receipt of benefits from LCI and mortality will be obtained from the city governments.</p> <p><it><b>Current status</b></it>: The baseline study was conducted for 2174 eligible men, and 2012 completed the study and were eligible for follow-up. Prevalence rates of osteoporosis (BMD 2.5 SD or more below the young adult mean (YAM)) and low BMD (BMD 1 SD or more below YAM) in at least one of LS and TH were calculated to be 4.4% and 41.8%, respectively. The proportion of men with low BMD only at TH showed a significant increasing trend with aging (p < 0.0001) while that only at LS showed a decreasing trend (p = 0.0386). The prevalence rate of osteoporosis was underestimated when diagnosed using only BMD at LS. Other baseline measurements were successfully obtained.</p> <p>Discussion</p> <p>FORMEN baseline study was performed as designed and the FORMEN cohort study was successfully launched.</p

Intramyocardial Transplantation of Undifferentiated Rat Induced Pluripotent Stem Cells Causes Tumorigenesis in the Heart

BACKGROUND: Induced pluripotent stem cells (iPSCs) are a novel candidate for use in cardiac stem cell therapy. However, their intrinsic tumorigenicity requires further investigation prior to use in a clinical setting. In this study we investigated whether undifferentiated iPSCs are tumorigenic after intramyocardial transplantation into immunocompetent allogeneic recipients. METHODOLOGY/PRINCIPAL FINDINGS: We transplanted 2 × 10(4), 2 × 10(5), or 2 × 10(6) cells from the established rat iPSC line M13 intramyocardially into intact or infarcted hearts of immunocompetent allogeneic rats. Transplant duration was 2, 4, or 6 weeks. Histological examination with hematoxylin-eosin staining confirmed that undifferentiated rat iPSCs could generate heterogeneous tumors in both intracardiac and extracardiac sites. Furthermore, tumor incidence was independent of cell dose, transplant duration, and the presence or absence of myocardial infarction. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease

Search for a Technicolor omega_T Particle in Events with a Photon and a b-quark Jet at CDF

If the Technicolor omega_T particle exists, a likely decay mode is omega_T -> gamma pi_T, followed by pi_T -> bb-bar, yielding the signature gamma bb-bar. We have searched 85 pb^-1 of data collected by the CDF experiment at the Fermilab Tevatron for events with a photon and two jets, where one of the jets must contain a secondary vertex implying the presence of a b quark. We find no excess of events above standard model expectations. We express the result of an exclusion region in the M_omega_T - M_pi_T mass plane.Comment: 14 pages, 2 figures. Available from the CDF server (PS with figs): http://www-cdf.fnal.gov/physics/pub98/cdf4674_omega_t_prl_4.ps FERMILAB-PUB-98/321-

Measurement of the B0 anti-B0 oscillation frequency using l- D*+ pairs and lepton flavor tags

The oscillation frequency Delta-md of B0 anti-B0 mixing is measured using the partially reconstructed semileptonic decay anti-B0 -> l- nubar D*+ X. The data sample was collected with the CDF detector at the Fermilab Tevatron collider during 1992 - 1995 by triggering on the existence of two lepton candidates in an event, and corresponds to about 110 pb-1 of pbar p collisions at sqrt(s) = 1.8 TeV. We estimate the proper decay time of the anti-B0 meson from the measured decay length and reconstructed momentum of the l- D*+ system. The charge of the lepton in the final state identifies the flavor of the anti-B0 meson at its decay. The second lepton in the event is used to infer the flavor of the anti-B0 meson at production. We measure the oscillation frequency to be Delta-md = 0.516 +/- 0.099 +0.029 -0.035 ps-1, where the first uncertainty is statistical and the second is systematic.Comment: 30 pages, 7 figures. Submitted to Physical Review