Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Association of Diverse Staphylococcus aureus Populations with Pseudomonas aeruginosa Coinfection and Inflammation in Cystic Fibrosis Airway Infection.

Staphylococcus aureus is one of the most common pathogens isolated from the airways of cystic fibrosis (CF) patients and often persists for extended periods. There is limited knowledge about the diversity of S. aureus in CF. We hypothesized that increased diversity of S. aureus would impact CF lung disease. Therefore, we conducted a 1-year observational prospective study with 14 patients with long-term S. aureus infection. From every sputum, 40 S. aureus isolates were chosen and characterized in terms of phenotypic appearance (size, hemolysis, mucoidy, and pigmentation), important virulence traits such as nuclease activity, biofilm formation, and molecular typing by spa sequence typing. Data about coinfection with Pseudomonas aeruginosa and clinical parameters such as lung function, exacerbation, and inflammatory markers in blood (C-reactive protein [CRP], interleukin 6 [IL-6], and S100A8/9 [calprotectin]) were collected. From 58 visits of 14 patients, 2,319 S. aureus isolates were distinguished into 32 phenotypes (PTs) and 50 spa types. The Simpson diversity index (SDI) was used to calculate the phenotypic and genotypic diversity, revealing a high diversity of PTs ranging from 0.19 to 0.87 among patients, while the diversity of spa types of isolates was less pronounced. The SDI of PTs was positively associated with P. aeruginosa coinfection and inflammatory parameters, with IL-6 being the most sensitive parameter. Also, coinfection with P. aeruginosa was associated with mucoid S. aureus and S. aureus with high nuclease activity. Our analyses showed that in CF patients with long-term S. aureus airway infection, a highly diverse and dynamic S. aureus population was present and associated with P. aeruginosa coinfection and inflammation. IMPORTANCE Staphylococcus aureus can persist for extended periods in the airways of people with cystic fibrosis (CF) in spite of antibiotic therapy and high numbers of neutrophils, which fail to eradicate this pathogen. Therefore, S. aureus needs to adapt to this hostile niche. There is only limited knowledge about the diversity of S. aureus in respiratory specimens. We conducted a 1-year prospective study with 14 patients with long-term S. aureus infection and investigated 40 S. aureus isolates from every sputum in terms of phenotypic appearance, nuclease activity, biofilm formation, and molecular typing. Data about coinfection with Pseudomonas aeruginosa and clinical parameters such as lung function, exacerbation, and inflammatory markers in blood were collected. Thirty-two phenotypes (PTs) and 50 spa types were distinguished. Our analyses revealed that in CF patients with long-term S. aureus airway infection, a highly diverse and dynamic S. aureus population was associated with P. aeruginosa coinfection and inflammation.

Heart Failure Results in Inspiratory Muscle Dysfunction Irrespective of Left Ventricular Ejection Fraction.

BACKGROUND: Exercise intolerance in heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF) results from both cardiac dysfunction and skeletal muscle weakness. Respiratory muscle dysfunction with restrictive ventilation disorder may be present irrespective of left ventricular ejection fraction and might be mediated by circulating pro-inflammatory cytokines. OBJECTIVE: To determine lung and respiratory muscle function in patients with HFrEF/HFpEF and to determine its associations with exercise intolerance and markers of systemic inflammation. METHODS: Adult patients with HFrEF (n = 22, 19 male, 61 ± 14 years) and HFpEF (n = 8, 7 male, 68 ± 8 years) and 19 matched healthy control subjects underwent spirometry, measurement of maximum mouth occlusion pressures, diaphragm ultrasound, and recording of transdiaphragmatic and gastric pressures following magnetic stimulation of the phrenic nerves and the lower thoracic nerve roots. New York Heart Association (NYHA) class and 6-min walking distance (6MWD) were used to quantify exercise intolerance. Levels of circulating interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured using ELISAs. RESULTS: Compared with controls, both patient groups showed lower forced vital capacity (FVC) (p < 0.05), maximum inspiratory pressure (PImax), maximum expiratory pressure (PEmax) (p < 0.05), diaphragm thickening ratio (p = 0.01), and diaphragm strength (twitch transdiaphragmatic pressure in response to supramaximal cervical magnetic phrenic nerve stimulation) (p = 0.01). In patients with HFrEF, NYHA class and 6MWD were both inversely correlated with FVC, PImax, and PEmax. In those with HFpEF, there was an inverse correlation between amino terminal pro B-type natriuretic peptide levels and FVC (r = -0.77, p = 0.04). In all HF patients, IL-6 and TNF-α were statistically related to FVC. CONCLUSIONS: Irrespective of left ventricular ejection fraction, HF is associated with respiratory muscle dysfunction, which is associated with increased levels of circulating IL-6 and TNF-α.

Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells.

BACKGROUND AND AIMS: Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS: hGLC and hGL5 cells were treated with a fixed dose (0.1 µM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. CONCLUSIONS: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P-S1PR axis may cooperate with gonadotropins in modulating follicle development.

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice.

BACKGROUND & AIMS: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. RESULTS: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. LAY SUMMARY: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

Inspiratory muscle dysfunction and restrictive lung function impairment in congenital heart disease: Association with immune inflammatory response and exercise intolerance.

BACKGROUND: In adult patients with congenital heart disease (ACHD), both underlying disease and lung restriction contribute to exercise intolerance. In ACHD the yet incompletely understood mechanism underlying restricted ventilation may be inspiratory muscle weakness. Therefore, this study comprehensively evaluated inspiratory muscle function in ACHD and associations with systemic inflammation and the clinical severity of exercise intolerance. METHODS: 30 ACHD patients (21 men, 35 ± 12 years) and 30 healthy controls matched for age, gender and body mass index underwent spirometry, measurement of mouth occlusion pressures, and diaphragm ultrasound. Six-minute walking distance (6MWD) and New York Heart Association functional class were used to quantify exercise intolerance. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were measured using enzyme-linked immunosorbent assays. RESULTS: ACHD patients showed lower forced vital capacity (FVC), and maximum inspiratory (PImax) and expiratory (PEmax) pressures compared with controls (all p < 0.05). On ultrasound, ACHD patients showed a lower diaphragm thickening ratio (2.3 ± 0.5 vs. 2.8 ± 0.9, p < 0.01) and lower diaphragm excursion velocity during a voluntary sniff maneuver (5.7 ± 2.2 vs. 7.6 ± 2.0 cm/s, p < 0.01). Respiratory parameters, such as FVC (r = 0.53; p < 0.01) and PImax (r = 0.43; p = 0.02), correlated with 6MWD. Furthermore, amino terminal pro B-type natriuretic peptide levels were inversely correlated with FVC (r = -0.54; p < 0.01). Circulating pro-inflammatory cytokines were markedly increased, and IL-6 was correlated with 6MWD, dyspnea, and biomarkers of heart, lung and inspiratory muscle function (all p < 0.05). CONCLUSIONS: Our findings show that diaphragm dysfunction is present in ACHD and relates to restrictive ventilation disorder and exercise intolerance, possibly mediated by increased IL-6 levels.

Respiratory Muscle and Lung Function in Lung Allograft Recipients: Association with Exercise Intolerance.

BACKGROUND: In lung transplant recipients (LTRs), restrictive ventilation disorder may be present due to respiratory muscle dysfunction that may reduce exercise capacity. This might be mediated by pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). OBJECTIVE: We investigated lung respiratory muscle function as well as circulating pro-inflammatory cytokines and exercise capacity in LTRs. METHODS: Fifteen LTRs (6 female, age 56 ± 14 years, 63 ± 45 months post-transplantation) and 15 healthy controls matched for age, sex, and body mass index underwent spirometry, measurement of mouth occlusion pressures, diaphragm ultrasound, and recording of twitch transdiaphragmatic (twPdi) and gastric pressures (twPgas) following magnetic stimulation of the phrenic nerves and the lower thoracic nerve roots. Exercise capacity was quantified using the 6-min walking distance (6MWD). Plasma IL-6 and TNF-α were measured using enzyme-linked immunosorbent assays. RESULTS: Compared with controls, patients had lower values for forced vital capacity (FVC; 81 ± 30 vs.109 ± 18% predicted, p = 0.01), maximum expiratory pressure (100 ± 21 vs.127 ± 17 cm H2O, p = 0.04), diaphragm thickening ratio (2.2 ± 0.4 vs. 3.0 ± 1.1, p = 0.01), and twPdi (10.4 ± 3.5 vs. 17.6 ± 6.7 cm H2O, p = 0.01). In LTRs, elevation of TNF-α was related to lung function (13 ± 3 vs. 11 ± 2 pg/mL in patients with FVC ≤80 vs. >80% predicted; p < 0.05), and lung function (forced expiratory volume after 1 s) was closely associated with diaphragm thickening ratio (r = 0.81; p < 0.01) and 6MWD (r = 0.63; p = 0.02). CONCLUSION: There is marked restrictive ventilation disorder and respiratory muscle weakness in LTRs, especially inspiratory muscle weakness with diaphragm dysfunction. Lung function impairment relates to elevated levels of circulating TNF-α and diaphragm dysfunction and is associated with exercise intolerance.

Enhanced expression of the sphingosine-1-phosphate-receptor-3 causes acute myelogenous leukemia in mice.

Acute myeloid leukemia (AML) carries a 10-100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.

Elevating Endogenous Sphingosine-1-Phosphate (S1P) Levels Improves Endothelial Function and Ameliorates Atherosclerosis in Low Density Lipoprotein Receptor-Deficient (LDL-R-/-) Mice.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high-density lipoprotein (HDL) exerting several atheroprotective effects in vitro. However, the few studies addressing anti-atherogenic effects of S1P in vivo have led to disparate results. We here examined atherosclerosis development in low-density lipoprotein receptor (LDL-R)-deficient (LDL-R-/-) mice with elevated endogenous S1P levels. METHODS AND RESULTS: Sub-lethally irradiated LDL-R-/- mice were transplanted with bone marrow deficient in sphingosine kinase 2 (SphK2), which led to the elevation of S1P concentrations in erythrocytes, plasma and HDL by approximately 1.5- to 2.0-fold in SphK2-/-/LDL-R-/- mice. Afterwards, mice were fed a Western diet for 14 weeks. Elevation of endogenous S1P significantly reduced atherosclerotic lesion formation by approximately half without affecting the plasma lipid profile. Furthermore, the macrophage content of atherosclerotic lesions and lipopolysaccharide-induced monocyte recruitment to the peritoneal cavity were reduced in SphK2-/-/LDL-R-/- mice. Studies using intra-vital microscopy revealed that endogenous S1P lowered leukocyte adhesion to capillary wall and decreased endothelial permeability to fluorescently labelled LDL. Moreover, SphK2-/-/LDL-R-/- mice displayed decreased levels of vascular cell adhesion molecule 1 in atherosclerotic lesions and in plasma. Studies in vitro demonstrated reduced monocyte adhesion and transport across an endothelial layer exposed to increasing S1P concentrations, murine plasma enriched in S1P or plasma obtained from SphK2-deficient animals. In addition, decreased permeability to fluorescence-labelled dextran beads or LDL was observed in S1P-treated endothelial cells. CONCLUSION: We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R-/- mice that are mediated by favourable modulation of endothelial function.

Discordant prostate specific antigen test results despite WHO assay standardization.

INTRODUCTION: Total PSA (tPSA) and free PSA (fPSA) are the most commonly used biomarkers for early detection of prostate cancer. Despite standardization efforts, many available PSA assays may still produce discordant results. In the present study, we compared four PSA assays calibrated to the WHO standards 96/670 and 96/668 for tPSA and fPSA, respectively. METHODS: Within the scope of the Prostate Cancer Early Detection Study Based on a ''Baseline'' PSA Value in Young Men (PROBASE), we tested tPSA and fPSA in serum samples from 50 patients in the four different PROBASE sites using four WHO-calibrated assays from Roche (Elecsys, Cobas), Beckman-Coulter (Access-II) and Siemens (ADVIA Centaur). The comparison was performed using the Passing-Bablok regression method. RESULTS: Compared to Access, the median tPSA levels for Centaur, Elecsys, and Cobas were +3%, +11%-20%, and +17%-23%, respectively, while for median fPSA levels the differences for Centaur, Elecsys, and Cobas were +49%, +29%-31%, and +22%, respectively. DISCUSSION: Despite all investigated assays being WHO-calibrated, the Elecsys and Cobas tPSA assays produced considerably higher results than the Access and Centaur assays. Differences in fPSA-recovery between all investigated assays were even more pronounced. When applying the tPSA cutoff of 3.1 µg/L recommended for WHO-calibrated assays, the use of higher calibrated assays may lead to unnecessary prostate biopsies. Conversely, if the historical threshold of 4 µg/L is applied when using WHO-calibrated assays, it could lead to falsely omitted prostate biopsies.

The Simultaneous measurement of serum testosterone and 5α-dihydrotestosterone by gas chromatography-mass spectrometry (GC-MS).

BACKGROUND: Simultaneous measurement of testosterone (T) and 5α-dihydrotestosterone (DHT) is important for diagnosing androgen deficiency states and hyperandrogenism in males and females, respectively. However, immunoassays used for T and DHT determination suffer from inadequate specificity and sensitivity, while tandem mass spectrometry is expensive and demanding in use. METHODS AND RESULTS: We developed a selective gas chromatography-mass spectrometry (GC-MS) method for parallel T and DHT measurement. The assay showed a linear response up to 46.5nmol/L, intra- and interassay imprecision and inaccuracy <15% and recoveries in spiked samples >90% for both analytes. The limit of quantitation was 0.117nmol/L for T and 0.168nmol/L for DHT. Comparison with immunoassays revealed good agreement for T in males, but a bias in favour of immunoassays at low concentrations for T in females and DHT in both sexes. We established reference ranges for T and DHT and suggest interval partitioning for T according to age in men and menstrual cycle in women. Assay validation in a clinical setting suggests that measuring DHT or T/DHT ratio may help identify patients with polycystic ovary syndrome. CONCLUSION: We developed a selective, simple and inexpensive GC-MS method for parallel measurement of T and DHT with potential use in the clinical laboratory.

First trimester TTR-RBP4-ROH complex and angiogenic factors in the prediction of small for gestational age infant's outcome.

PURPOSE: To study the role of the TTR-RBP4-ROH complex components (transthyretin, serum retinol binding protein, retinol) and of angiogenic factors PlGF (placental growth factor) and sFlt-1 (soluble fms-like tyrosine kinase-1) in pregnancies complicated by small for gestational age infants (SGA). METHODS: Case control study conducted on maternal serum collected between 11 + 0 to 13 + 6 weeks of gestation. TTR, RBP4, ROH, PlGF and sFlt-1 were measured in SGA patients (birth weight <10%) who delivered at term (n = 37) and before 37 weeks of gestation (n = 17) and in a matched control group with uneventful pregnancies (n = 37). RESULTS: We found decreased RBP4 in SGA patients that delivered fetuses <3% and in fetuses delivered after the 37 weeks of gestation compared to controls [1.50 (95% CI 1.40-1.75) vs 1.62 (95% CI 1.47-1.98), p < 0.05]. Further, we found lower PlGF and sFlt-1 concentrations in SGA that delivered before 37 weeks of gestation compared to controls (respectively, PIGF and sFlt-1: 39.7 pg/ml (95% CI 32.3-66.3) vs 62.9 pg/ml (95% CI 45.2-78.4) and 906 pg/ml (95% CI 727-1626) vs 1610 pg/ml (95% CI 1088-212), p < 0.05). CONCLUSIONS: First trimester maternal serum RBP4 and angiogenic factors PlGF and sFlt-1 can differently predict the timing of delivery of pregnancies complicated by SGA fetuses.

Determination of serum cholestane-3ß,5α,6ß-triol by gas chromatography-mass spectrometry for identification of Niemann-Pick type C (NPC) disease.

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3ß,5α,6ß-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography-mass spectrometry (GC-MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC-MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03-200ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03ng/mL and 0.01ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5-10-fold increase in C-triol levels. We developed a quick and sensitive GC-MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing.

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression.

BACKGROUND AND AIMS: Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. METHODS: Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. RESULTS: Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. CONCLUSIONS: HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects.

Factors Associated with Worse Lung Function in Cystic Fibrosis Patients with Persistent Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an important pathogen in cystic fibrosis (CF). However, it is not clear which factors are associated with worse lung function in patients with persistent S. aureus airway cultures. Our main hypothesis was that patients with high S. aureus density in their respiratory specimens would more likely experience worsening of their lung disease than patients with low bacterial loads. METHODS: Therefore, we conducted an observational prospective longitudinal multi-center study and assessed the association between lung function and S. aureus bacterial density in respiratory samples, co-infection with other CF-pathogens, nasal S. aureus carriage, clinical status, antibiotic therapy, IL-6- and IgG-levels against S. aureus virulence factors. RESULTS: 195 patients from 17 centers were followed; each patient had an average of 7 visits. Data were analyzed using descriptive statistics and generalized linear mixed models. Our main hypothesis was only supported for patients providing throat specimens indicating that patients with higher density experienced a steeper lung function decline (p<0.001). Patients with exacerbations (n = 60), S. aureus small-colony variants (SCVs, n = 84) and co-infection with Stenotrophomonas maltophilia (n = 44) had worse lung function (p = 0.0068; p = 0.0011; p = 0.0103). Patients with SCVs were older (p = 0.0066) and more often treated with trimethoprim/sulfamethoxazole (p = 0.0078). IL-6 levels positively correlated with decreased lung function (p<0.001), S. aureus density in sputa (p = 0.0016), SCVs (p = 0.0209), exacerbations (p = 0.0041) and co-infections with S. maltophilia (p = 0.0195) or A. fumigatus (p = 0.0496). CONCLUSIONS: In CF-patients with chronic S. aureus cultures, independent risk factors for worse lung function are high bacterial density in throat cultures, exacerbations, elevated IL-6 levels, presence of S. aureus SCVs and co-infection with S. maltophilia. TRIAL REGISTRATION: ClinicalTrials.gov NCT00669760.

Six months of daily treatment with vardenafil improves parameters of endothelial inflammation and of hypogonadism in male patients with type 2 diabetes and erectile dysfunction: a randomized, double-blind, prospective trial.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, characterized by a reduction of nitric oxide (NO)-mediated relaxation. Phosphodiesterase type 5 inhibitors (PDE5i) improve NO levels. The aim of the study was to investigate whether long-term, chronic treatment with the PDE5i vardenafil improves systemic endothelial function in diabetic men. DESIGN: A prospective, investigator-initiated, randomized, placebo-controlled, double-blind, clinical trial was conducted. METHODS: In total, 54 male patients affected by T2DM, diagnosed within the last 5 years, and erectile dysfunction were enrolled, regardless of testosterone levels. In all, 26 and 28 patients were assigned to verum and placebo groups respectively. The study consisted of an enrollment phase, a treatment phase (24 weeks) (vardenafil/placebo 10  mg twice in a day) and a follow-up phase (24 weeks). Parameters evaluated were as follows: International Index of Erectile Function 15 (IIEF-15), flow-mediated dilation (FMD), serum interleukin 6 (IL6), endothelin 1 (ET-1), gonadotropins and testosterone (measured by liquid chromatography/tandem mass spectrometry). RESULTS: IIEF-15 erectile function improved during the treatment (P<0.001). At the end of the treatment both FMD (P=0.040) and IL6 (P=0.019) significantly improved. FMD correlated with serum testosterone levels (R(2)=0.299; P<0.001). Testosterone increased significantly under vardenafil treatment and returned in the eugonadal range only in hypogonadal men (n=13), without changes in gonadotropins. Chronic vardenafil treatment did not result in relevant side effects. CONCLUSION: This is the first double-blind, placebo-controlled clinical trial designed to evaluate the effects of chronic treatment of vardenafil on endothelial health-related parameters and sexual hormones in patients affected by a chronic disease. Chronically administered vardenafil is effective and improves endothelial parameters in T2DM patient. Moreover, chronic vardenafil therapy improves hypogonadism in diabetic, hypogonadal men.

Head-To-Head Assessment of Diagnostic Performance of Testosterone Immunoassays in Patients With Polycystic Ovary Syndrome.

BACKGROUND: Determination of plasma testosterone is critical for the proper diagnosis of polycystic ovary syndrome (PCOS), but the interpretation of biochemical tests is hampered by inadequate specificity and precision of available immunoassays. We here compared the diagnostic performance of three testosterone immunoassays (Advia Centaur, Immulite 2000 XPi, Cobas e411) in PCOS patients using receiver operator characteristics curve analysis. METHODS AND RESULTS: Plasma levels of testosterone, androstendione, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, estradiol, progesterone, steroid hormone binding globulin, luteinizing hormone, and follicular stimulating hormone were determined in 188 patients with PCOS and 202 controls. Free testosterone (fT) levels and free androgen index (FAI) were calculated. Testosterone levels measured on Advia Centaur, Immulite 2000 XPi, and Cobas e411 showed clear linear relationship to each other. Testosterone measured with Advia Centaur showed discriminatory performance superior to Immulite 2000 XPi and Cobas e411. Calculation of fT or FAI improved the performance of Advia Centaur and Immulite 2000 XPi, which nevertheless performed better than Cobas e411. The performance of other parameters was inferior to that of testosterone, fT, and FAI. CONCLUSION: Present study documents striking differences between testosterone immunoassays with respect to their capacity to identify PCOS patients and favors the use of calculated parameters reflecting active testosterone in plasma.

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR-/-) Mice.

OBJECTIVE: Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive. METHODS AND RESULTS: We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR-/-/LDLR-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR-/--macrophages to TNFα-stimulated endothelial cells was decreased in vitro accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. Ex vivo incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells in vitro. CONCLUSION: uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.

Gadolinium chloride modulates bradykinin-induced pulmonary vasoconstriction and hypoxic pulmonary vasoconstriction during polymicrobial abdominal sepsis in rats.

BACKGROUND: Macrophages importantly contribute to sepsis-induced lung injury. As their impact on pulmonary endothelial injury and dysregulation of hypoxic pulmonary vasoconstriction (HPV) remains unclear, we assessed pulmonary endothelial dysfunction and HPV by macrophage inhibition via gadolinium chloride (GC) pre-treatment in rats with peritonitis (cecal ligation and puncture [CLP]). METHODS: The following four study groups were made: Group I: SHAM and group II: SHAM + GC (pre-treatment with NaCl 0.9% or GC 14 mg/kg body weight (b.w.) intravenously 24 hours prior to sham laparotomy); group III: CLP and group IV: CLP + GC (pre-treatment with NaCl 0.9% or GC 14 mg/kg b.w. 24 hours prior to induction of peritonitis). Exhaled nitric oxide (exNO), bradykinin-induced pulmonary vasoconstriction (=surrogate marker of endothelial dysfunction) and HPV were investigated in isolated and perfused lungs (n = 40). Using the same protocol wet to dry lung weight ratio and myeloperoxidase (MPO) activity were investigated in separate rats (n = 28). In additional rats (n = 12) of groups III and IV nitrite levels in alveolar macrophages (AM) were measured. RESULTS: In sepsis, GC pre-treatment significantly attenuated exNO levels, AM-derived nitrite levels, lung MPO activity, and restored blunted HPV, but severely enhanced endothelial dysfunction in healthy and septic animals. CONCLUSION: Macrophages exhibit a controversial role in sepsis-induced lung injury. The GC-induced restoration of inflammation parameters to sham levels is clearly limited by the negative impact on CLP-induced endothelial injury in this setting. The exact link between the GC-associated modulation of the NO pathway demonstrated and septic lung injury needs to be determined in future studies.