An in vivo neuroimmune organoid model to study human microglia phenotypes.

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, we developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Our data show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, we demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly.

Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior.

The dentate gyrus of the mammalian hippocampus continuously generates new neurons during adulthood. These adult-born neurons become functionally active and are thought to contribute to learning and memory, especially during their maturation phase, when they have extraordinary plasticity. In this Review, we discuss the molecular machinery involved in the generation of new neurons from a pool of adult neural stem cells and their integration into functional hippocampal circuits. We also summarize the potential functions of these newborn neurons in the adult brain, their contribution to behavior, and their relevance to disease.

Cellular complexity in brain organoids: Current progress and unsolved issues.

Brain organoids are three-dimensional neural aggregates derived from pluripotent stem cells through self-organization and recapitulate architectural and cellular aspects of certain brain regions. Brain organoids are currently a highly exciting area of research that includes the study of human brain development, function, and dysfunction in unprecedented ways. In this Review, we discuss recent discoveries related to the generation of brain organoids that resemble diverse brain regions. We provide an overview of the strategies to complement these primarily neuroectodermal models with cell types of non-neuronal origin, such as vasculature and immune cells. Recent transplantation approaches aiming to achieve higher cellular complexity and long-term survival of these models will then be discussed. We conclude by highlighting unresolved key questions and future directions in this exciting area of human brain organogenesis.

Modified thromboelastometric tests provide improved sensitivity and specificity to direct oral anticoagulants compared to standard thromboelastometric tests in-vitro.

BACKGROUND: The detection of direct oral anticoagulants (DOACs) is still challenging but important in emergency patients. We recently demonstrated that modified thromboelastometry can detect rivaroxaban and dabigatran. Data on the detection rates of modified compared to standard thromboelastometric tests of apixaban and edoxaban, are missing. The aim of this in-vitro dose-effect-study was to add data on these DOACs and to evaluate thromboelastometric tests in-vitro using data of both studies. METHODS: The study was approved by the Ludwig-Maximilians-University ethics committee (No 17-525-2). Written informed consent was obtained from all individuals. Blood samples of healthy volunteers and samples of 10 volunteers for each DOAC were used. Blood samples were spiked with six different concentrations of edoxaban and apixaban (0ng/ml; 31.25ng/ml; 62.5ng/ml; 125ng/ml; 250 ng/ml; 500ng/ml). Modified tests (low-tissue-factor test TFTEM and ecarin-based test ECATEM) as well as standard tests (e.g. FIBTEM) analyzing extrinsic pathway of coagulation were used. Receiver operating characteristics analyzes were performed as well as regression analyzes. RESULTS: TFTEM CT correlated well with anti-Xa levels of apixaban and edoxaban (apixaban: r2 = 0.8064 p < 0.0001; edoxaban: r2 = 0.8603; p < 0.0001). The detection of direct FXa inhibitors (> 30 ng/mL) was successful with FIBTEM CT with a sensitivity and specificity of 81% and 90%, respectively. As expected, ECATEM CT was not prolonged by direct FXa-inhibitors due to its specificity for direct thrombin inhibitors. Again, TFTEM CT provided the highest sensitivity and specificity for the detection of direct FXa inhibitors with 96% and 95%, respectively. ECATEM test showed 100% sensitivity and 100% specificity for the detection of dabigatran. CONCLUSIONS: Our study presents modified thromboelastometric tests with improved detection of even low DOAC concentrations > 30 ng/mL, including apixaban in-vitro. The study thus complements the previously published data on dabigatran and rivaroxaban. Validation studies must confirm the results due to the explanatory design of this study.

Point-of-care detection and differentiation of anticoagulant therapy - development of thromboelastometry-guided decision-making support algorithms.

BACKGROUND: DOAC detection is challenging in emergency situations. Here, we demonstrated recently, that modified thromboelastometric tests can reliably detect and differentiate dabigatran and rivaroxaban. However, whether all DOACs can be detected and differentiated to other coagulopathies is unclear. Therefore, we now tested the hypothesis that a decision tree-based thromboelastometry algorithm enables detection and differentiation of all direct Xa-inhibitors (DXaIs), the direct thrombin inhibitor (DTI) dabigatran, as well as vitamin K antagonists (VKA) and dilutional coagulopathy (DIL) with high accuracy. METHODS: Following ethics committee approval (No 17-525-4), and registration by the German clinical trials database we conducted a prospective observational trial including 50 anticoagulated patients (n = 10 of either DOAC/VKA) and 20 healthy volunteers. Blood was drawn independent of last intake of coagulation inhibitor. Healthy volunteers served as controls and their blood was diluted to simulate a 50% dilution in vitro. Standard (extrinsic coagulation assay, fibrinogen assay, etc.) and modified thromboelastometric tests (ecarin assay and extrinsic coagulation assay with low tissue factor) were performed. Statistical analyzes included a decision tree analyzes, with depiction of accuracy, sensitivity and specificity, as well as receiver-operating-characteristics (ROC) curve analysis including optimal cut-off values (Youden-Index). RESULTS: First, standard thromboelastometric tests allow a good differentiation between DOACs and VKA, DIL and controls, however they fail to differentiate DXaIs, DTIs and VKAs reliably resulting in an overall accuracy of 78%. Second, adding modified thromboelastometric tests, 9/10 DTI and 28/30 DXaI patients were detected, resulting in an overall accuracy of 94%. Complex decision trees even increased overall accuracy to 98%. ROC curve analyses confirm the decision-tree-based results showing high sensitivity and specificity for detection and differentiation of DTI, DXaIs, VKA, DIL, and controls. CONCLUSIONS: Decision tree-based machine-learning algorithms using standard and modified thromboelastometric tests allow reliable detection of DTI and DXaIs, and differentiation to VKA, DIL and controls. TRIAL REGISTRATION: Clinical trial number: German clinical trials database ID: DRKS00015704 .

Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder.

Bipolar disorder is a complex neuropsychiatric disorder that is characterized by intermittent episodes of mania and depression; without treatment, 15% of patients commit suicide. Hence, it has been ranked by the World Health Organization as a top disorder of morbidity and lost productivity. Previous neuropathological studies have revealed a series of alterations in the brains of patients with bipolar disorder or animal models, such as reduced glial cell number in the prefrontal cortex of patients, upregulated activities of the protein kinase A and C pathways and changes in neurotransmission. However, the roles and causation of these changes in bipolar disorder have been too complex to exactly determine the pathology of the disease. Furthermore, although some patients show remarkable improvement with lithium treatment for yet unknown reasons, others are refractory to lithium treatment. Therefore, developing an accurate and powerful biological model for bipolar disorder has been a challenge. The introduction of induced pluripotent stem-cell (iPSC) technology has provided a new approach. Here we have developed an iPSC model for human bipolar disorder and investigated the cellular phenotypes of hippocampal dentate gyrus-like neurons derived from iPSCs of patients with bipolar disorder. Guided by RNA sequencing expression profiling, we have detected mitochondrial abnormalities in young neurons from patients with bipolar disorder by using mitochondrial assays; in addition, using both patch-clamp recording and somatic Ca(2+) imaging, we have observed hyperactive action-potential firing. This hyperexcitability phenotype of young neurons in bipolar disorder was selectively reversed by lithium treatment only in neurons derived from patients who also responded to lithium treatment. Therefore, hyperexcitability is one early endophenotype of bipolar disorder, and our model of iPSCs in this disease might be useful in developing new therapies and drugs aimed at its clinical treatment.

Functional testing of tranexamic acid effects in patients undergoing elective orthopaedic surgery.

Tranexamic acid (TXA) can reduce blood loss and transfusion rates in orthopaedic surgery. In this regard, a new viscoelastometric test (TPA-test, ClotPro), enables the monitoring of TXA effects. This prospective observational study evaluated and correlated TXA plasma concentrations (cTXA) following intravenous and oral administration in patients undergoing elective orthopaedic surgery with lysis variables of TPA-test. Blood samples of 42 patients were evaluated before TXA application and 2, 6, 12, 24 and 48 h afterwards. TPA-test was used to determine lysis time (LT) as well as maximum lysis (ML) and cTXA was measured using Ultra-High-Performance-Liquid-Chromatography/Mass-Spectrometry. Data are presented as median (min-max). LTTPA-test and MLTPA-test correlated with cTXA (r = 0.9456/r = 0.5362; p < 0.0001). 2 h after intravenous TXA administration all samples showed complete lysis inhibition (LTTPA-test prolongation: T1: 217 s (161-529) vs. T2: 4500 s (4500-4500);p < 0.0001), whereas after oral application high intraindividual variability was observed as some samples showed only moderate changes in LTTPA-test (T1: 236 s (180-360) vs. T2: 4500 s (460-4500); p < 0.0001). Nevertheless, statistically LTTPA-test did not differ between groups. MLTPA-test differed 2 h after application (i.v.: 9.0% (5-14) vs. oral: 31% (8-97); p = 0.0081). In 17/21 samples after oral and 0/21 samples after intravenous administration cTXA was < 10 µg ml-1 2 h after application. TPA-test correlated with cTXA. MLTPA-test differed between intravenous and oral application 2 h after application. Most patients with oral application had TXA plasma concentration < 10 µg ml-1. The duration of action did not differ between intravenous and oral application. Additional studies evaluating clinical outcomes and side-effects based on individualized TXA prophylaxis/therapy are required.

Coagulation under Mild Hypothermia Assessed by Thromboelastometry.

INTRODUCTION: While previous studies have shown a significant impact of extreme hypo- and hyperthermia on coagulation, effects of much more frequently occurring perioperative mild hypothermia are largely unknown. This study therefore aimed to analyze the effects of mild hypothermia using rotational thromboelastometry in vitro. MATERIALS AND METHODS: Twelve healthy volunteers were included in this study. Standard thromboelastometric tests (EXTEM, INTEM, FIBTEM) were used to evaluate coagulation in vitro at 39, 37, 35.5, 35, and 33°C. Beyond standard thromboelastometric tests, we also evaluated the effects of mild hypothermia on the TPA-test (ClotPro, Enicor GmbH, Munich, Germany), a new test which aims to detect fibrinolytic capacity by adding tissue plasminogen activator to the sample. Data are presented as the median with 25/75th percentiles. RESULTS: Extrinsically activated coagulation (measured by EXTEM) showed a significant increase in clot formation time (CFT; 37°C: 90 s [81/105] vs. 35°C: 109 s [99/126]; p = 0.0002), while maximum clot firmness (MCF) was not significantly reduced. Intrinsically activated coagulation (measured by INTEM) also showed a significant increase in CFT (37°C: 80 s [72/88] vs. 35°C: 94 s [86/109]; p = 0.0002) without significant effects on MCF. Mild hypothermia significantly increased both the lysis onset time (136 s [132/151; 37°C] vs. 162 s [141/228; 35°C], p = 0.0223) and lysis time (208 s [184/297; 37°C] vs. 249 s [215/358; 35°C]; p = 0.0259). CONCLUSION: This demonstrates that even under mild hypothermia coagulation is significantly altered in vitro. Perioperative temperature monitoring and management are greatly important and can help to prevent mild hypothermia and its adverse effects. Further investigation and in vivo testing of coagulation under mild hypothermia is needed.

Neutrophil extracellular trap formation and nuclease activity in septic patients.

BACKGROUND: There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well. METHODS: Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 µg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis. RESULTS: In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers. CONCLUSIONS: Unstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. TRIAL REGISTRATION: DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.

Comparison of Two Different Fibrinogen Concentrates in an in vitro Model of Dilutional Coagulopathy.

INTRODUCTION: Fibrinogen concentrates are widely used to restore clot stability in situations of bleeding. Fibrinogen preparations are produced using different production methods, resulting in different compounds. Thus, different preparations might have a distinct impact on blood coagulation. We tested the effect of fibrinogen concentrates Haemocomplettan® (CSL Behring, Marburg, Germany) and fibryga® (Octapharma GmbH, Langenfeld, Germany) on the impairments induced by 60% dilutional coagulopathy in vitro. MATERIALS AND METHODS: The influence of the fibrinogen concentrates fibryga® and Haemocomplettan® on colloid (gelatine, hydroxyethyl starch [HES], albumin)-induced or crystalloid (Ringer's acetate)-induced dilutional coagulopathy was analysed using rotational thromboelastometry (ROTEM®) and standard laboratory tests. The following experimental conditions were analysed in vitro: whole blood, 60% dilution (40% blood and 60% diluent) ± 50 or 100 mg/kg-1 fibryga® or Haemocomplettan®, respectively. RESULTS: Dilution with either diluent resulted in prolonged clotting time (CT) in an extrinsic activated test (CTEXTEM) and decreased maximum clot firmness (MCFFIBTEM) as expressed, e.g., by gelatine: (59.5 s [62/54.8] vs. 95 s [102.8/86.8]; p < 0.001 and 14 mm [16/10.5] vs. 3 mm [4-3]; p < 0.001). Substitution after 60% dilution with HES resulted in no difference between the preparations, except for shorter thrombin time with fibryga® (14 s [15/14] vs. 18 s [18.8/17]; p = 0.0093; low dose). CTEXTEM was higher with Haemocomplettan® in a gelatine-induced dilution (51 s [54.5/47.5] vs. 63 s [71/60.3]; p = 0.0202; low dose) whereas thrombin time was lower with fibryga® (19.5 s [20.8/19] vs. 27 s [29/25.3]; p = 0.0017). In dilution with albumin, differences in CTEXTEM (69 s [76.5/66] vs. 56 s [57/53.3]; p = 0.0114; low dose) and thrombin time (18 s [18/17] vs. 24.5 s [25.8/24]; p = 0.0202; low dose) were seen. In dilution with crystalloid solution, again differences in CTEXTEM (53.5 s [57.8/53] vs. 45 s [47/43]; p = 0.035; low dose) and thrombin time (17 s [17/16] vs. 23.5 s [24/23]; p = 0.0014; low dose) were seen. Fibrinogen levels were more increased by high-dose substitution of both preparations. CONCLUSION: Based on this data it can be stated that both fibryga® and Haemocomplettan® had the same performance in our in vitro model except for CTEXTEM and thrombin time.

Hypoxia Inducible Factor-2 Alpha and Prolinhydroxylase 2 Polymorphisms in Patients with Acute Respiratory Distress Syndrome (ARDS).

Hypoxia-inducible-factor-2α (HIF-2α) and HIF-2 degrading prolyl-hydroxylases (PHD) are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS). Specifically, functionally active genetic variants of HIF-2α (single nucleotide polymorphism (SNP) [ch2:46441523(hg18)]) and PHD2 (C/T; SNP rs516651 and T/C; SNP rs480902) are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs' prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2α or PHD2 genes are (1) common, and (2) independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay) and HIF-2α-polymorphism (restriction digest + agarose-gel visualization), and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2α CC-carriers (CG: 0.38%; GG: 0%), 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%; CC: 78.8%), and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%; CC: 52.4%). PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09-10.22; p = 0.034) within 30-days, whereas the other SNPs had no significant impact (p = ns). The homozygous HIF-2α GG-genotype was not present in our Caucasian ARDS cohort; however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS.

The Wnt adaptor protein ATP6AP2 regulates multiple stages of adult hippocampal neurogenesis.

In the mammalian hippocampus, canonical Wnt signals provided by the microenvironment regulate the differentiation of adult neural stem cells (NSCs) toward the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways and the role of Wnt/Planar cell polarity (PCP) signaling in adult neurogenesis remains unknown. Using in vitro assays on differentiating adult NSCs, we identified a transition of Wnt signaling responsiveness from Wnt/ß-catenin to Wnt/PCP signaling. In mice, retroviral knockdown strategies against ATP6AP2, a recently discovered core protein involved in both signaling pathways, revealed that its dual role is critical for granule cell fate and morphogenesis. We were able to confirm its dual role in neurogenic Wnt signaling in vitro for both canonical Wnt signaling in proliferating adult NSCs and non-canonical Wnt signaling in differentiating neuroblasts. Although LRP6 appeared to be critical for granule cell fate determination, in vivo knockdown of PCP core proteins FZD3 and CELSR1-3 revealed severe maturational defects without changing the identity of newborn granule cells. Furthermore, we found that CELSR1-3 control distinctive aspects of PCP-mediated granule cell morphogenesis with CELSR1 regulating the direction of dendrite initiation sites and CELSR2/3 controlling radial migration and dendritic patterning. The data presented here characterize distinctive roles for Wnt/ß-catenin signaling in granule cell fate determination and for Wnt/PCP signaling in controlling the morphological maturation of differentiating neuroblasts.

REST Regulates Non-Cell-Autonomous Neuronal Differentiation and Maturation of Neural Progenitor Cells via Secretogranin II.

RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non-cell-autonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non-cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non-cell-autonomously via SCG2. SIGNIFICANCE STATEMENT: Our results reveal that REST regulates differentiation and maturation of neural progenitor cells in vitro by orchestrating both cell-intrinsic and non-cell-autonomous factors and that Scg2 is a major secretory target of REST with a differentiation-enhancing activity in a paracrine manner. In vivo, REST depletion causes accelerated differentiation of newborn neurons at the expense of spine defects, suggesting a potential role for REST in the timing of the maturation of granule neurons.

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

BACKGROUND: Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. METHODS: Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. RESULTS: Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. CONCLUSIONS: The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Hypoxia inducible factor-1 alpha and prolinhydroxlase 2 polymorphisms in patients with severe sepsis: a prospective observational trial.

BACKGROUND: Hypoxia-inducible-factor-1α (HIF-1α) and HIF-1 degrading prolyl-hydroxylases (PHD) are key regulators of the hypoxic-inflammatory response. Functionally active genetic variants in the HIF-1α (C/T; Single Nucleotide Polymorphism (SNP) rs11549465) and the PHD2 gene (EGLN1; C/T; SNP rs516651 and T/C; SNP rs480902) are associated with altered HIF-1α mRNA nuclear translocation and an altered adaptation to hypoxia. Furthermore, the HIF system is important in surviving inflammatory disorders and sepsis. Thus, we tested the hypotheses, that SNPs in the HIF-1α or PHD2 genes are (1) common in Caucasians, with 2) the HIF-1α genetic variant being associated with an altered HIF-1α mRNA expression; and 3) independent risk factors for 30-day mortality in severe sepsis. METHODS: After ethics approval, 128 septic patients (Caucasian descent) were included prospectively within 24 h after first diagnosing sepsis. Patients characteristics and severity of illness (simplified acute physiology score II), genotypes (Taqman assay), and their influence on leukocyte HIF-1α-mRNA-expression (Real-Time PCR) and 30-day mortality were determined. RESULTS: Frequencies were 0.8 % for homozygous HIF-1α TT-carriers (CT 17.6 %; CC 81.6 %), 2.5 % for homozygous PHD2 SNP rs516651 TT-allele carriers (CT 17.5 % and CC 80 %), and 9.4 % for homozygous PHD2 SNP rs480902 TT-allele carriers (CT 34.4 % and CC 56.3 %). While HIF-1α T-allele carriers had a borderline decrease in HIF-1α-mRNA-expression (p = 0.06) neither HIF-1α nor PHD2 SNPs were (independent) risk factors for 30-day mortality. CONCLUSIONS: Genetic variants in HIF-1α and PHD2 genes exist in Caucasians but do not appear to alter 30-day mortality in sepsis.

Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation.

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

LDL attenuates VEGF-induced angiogenesis via mechanisms involving VEGFR2 internalization and degradation following endosome-trans-Golgi network trafficking.

Considerable efforts have been made to amplify angiogenesis under conditions of hypoxia and ischemia by vascular endothelial growth factor (VEGF) delivery, so far with limited success. Ischemic vascular diseases are often associated with hypercholesterolemia. To elucidate whether the exposure to blood lipids influences VEGF responses of microvessels, we characterized effects of low density lipoprotein (LDL) exposure on the proliferation, migration and tube formation of human umbilical vein endothelial cells. By examining the expression, phosphorylation and downstream signals of VEGF's receptor VEGFR2, we characterized mechanisms controlling angiogenic responses following LDL exposure. LDL attenuated endothelial proliferation, migration and tube formation in a dose-dependent way. Reduced abundance of VEGFR2 and VEGFR1 were noticed in LDL-exposed endothelial cells. In subcellular localization studies that we combined with pharmacological experiments, we showed that the loss of VEGFR2 resulted from its internalization and degradation, the latter of which required syntaxin-16-dependent endosome-trans-Golgi network trafficking. As a consequence, VEGFR2 phosphorylation and downstream signals -specifically Akt and ERK1/2 phosphorylation- were attenuated in response to VEGF treatment. VEGF only partly reversed the effects of LDL on angiogenesis under conditions of normoxia and hypoxia. Our results suggest that angiogenic responses to VEGF are compromised in hypercholesterolemia as a consequence of endosomal VEGFR2 degradation.