Low thrombin generation in postmenopausal women using estetrol.

OBJECTIVE: Estetrol (E4) represents a novel estrogen of interest to relieve vasomotor symptoms. E4 activates the nuclear estrogen receptor α (ERα) but antagonizes the estradiol ERα-dependent membrane-initiated steroid signaling pathway. The distinct pharmacological properties of E4 could explain its low impact on hemostasis. This study aimed to assess the effect of E4 on coagulation in postmenopausal women, using the thrombin generation assay (TGA). METHODS: Data were collected from a multicenter, randomized, placebo-controlled, dose-finding study in postmenopausal women (NCT02834312). Oral E4 (2.5 mg, n = 42; 5 mg, n = 29; 10 mg, n = 34; or 15 mg, n = 32) or placebo (n = 31) was administered daily for 12 weeks. Thrombograms and TGA parameters were extracted for each subject at baseline and after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, all treatment groups showed a mean thrombogram (±95% confidence interval [CI] of the mean) within the reference ranges, that is, the 2.5th-97.5th percentile of all baseline thrombograms (n = 168), as well as for TGA parameters. CONCLUSIONS: The intake of E4 15 mg for 12 weeks led to significant but not clinically relevant changes compared to baseline as the mean values (±95% CI of the mean) remained within reference ranges, demonstrating a neutral profile of this estrogen on hemostasis.

CSF and venous blood flow from childhood to adulthood studied by real-time phase-contrast MRI.

PURPOSE: In vivo measurements of CSF and venous flow using real-time phase-contrast (RT-PC) MRI facilitate new insights into the dynamics and physiology of both fluid systems. In clinical practice, however, use of RT-PC MRI is still limited. Because many forms of hydrocephalus manifest in infancy and childhood, it is a prerequisite to investigate normal flow parameters during this period to assess pathologies of CSF circulation. This study aims to establish reference values of CSF and venous flow in healthy subjects using RT-PC MRI and to determine their age dependency. METHODS: RT-PC MRI was performed in 44 healthy volunteers (20 females, age 5-40 years). CSF flow was quantified at the aqueduct (Aqd), cervical (C3) and lumbar (L3) spinal levels. Venous flow measurements comprised epidural veins, internal jugular veins and inferior vena cava. Parameters analyzed were peak velocity, net flow, pulsatility, and area of region of interest (ROI). STATISTICAL TESTS: linear regression, student's t-test and analysis of variance (ANOVA). RESULTS: In adults volunteers, no significant changes in flow parameters were observed. In contrast, pediatric subjects exhibited a significant age-dependent decrease of CSF net flow and pulsatility in Aqd, C3 and L3. Several venous flow parameters decreased significantly over age at C3 and changed more variably at L3. CONCLUSION: Flow parameters varies depending on anatomical location and age. We established changes of brain and spinal fluid dynamics over an age range from 5-40 years. The application of RT-PC MRI in clinical care may improve our understanding of CSF flow pathology in individual patients.

Impact of estetrol (E4) on hemostasis, metabolism and bone turnover in postmenopausal women.

OBJECTIVE: This study aimed to determine the effects of estetrol (E4) on hemostasis, lipids, carbohydrate metabolism and bone turnover in postmenopausal women. METHODS: This study was a multicenter, randomized, double-blind placebo-controlled phase 2 trial. Participants (n = 180, age 43-64 years) received E4 2.5 mg, 5 mg, 10 mg and 15 mg or placebo once daily for 12 weeks. Changes from baseline at week 12 were evaluated versus placebo for hemostasis parameters, sex hormone binding globulin (SHBG), lipids, carbohydrate metabolism and bone markers. RESULTS: Changes for hemostasis parameters were minimal with a small increase only in the normalized activated protein C sensitivity ratio in the E4 15 mg group versus placebo. SHBG increased in the E4 5 mg, 10 mg and 15 mg groups versus placebo. High-density lipoprotein cholesterol increased in all E4 groups; changes were not consistent for other lipids. Significant decreases versus placebo were seen for insulin resistance (E4 10 mg group), hemoglobin A1c (E4 15 mg group) and type 1 collagen C-terminal telopeptide (E4 10 mg and 15 mg groups). Small decreases in osteocalcin in the E4 5 mg, 10 mg and 15 mg groups were significant versus the increase observed in placebo. CONCLUSION: E4 had limited impact on hemostasis and potentially beneficial effects on lipids, carbohydrate metabolism and bone turnover.

Turbulence in surgical suction heads as detected by MRI.

BACKGROUND: Blood loss is common during surgical procedures, especially in open cardiac surgery. Allogenic blood transfusion is associated with increased morbidity and mortality. Blood conservation programs in cardiac surgery recommend re-transfusion of shed blood directly or after processing, as this decreases transfusion rates of allogenic blood. But aspiration of blood from the wound area is often associated with increased hemolysis, due to flow induced forces, mainly through development of turbulence. METHODS: We evaluated magnetic resonance imaging (MRI) as a qualitative tool for detection of turbulence. MRI is sensitive to flow; this study uses velocity-compensated T1-weighted 3D MRI for turbulence detection in four geometrically different cardiotomy suction heads under comparable flow conditions (0-1250 mL/min). RESULTS: Our standard control suction head Model A showed pronounced signs of turbulence at all flow rates measured, while turbulence was only detectable in our modified Models 1-3 at higher flow rates (Models 1 and 3) or not at all (Model 2). CONCLUSIONS: The comparison of flow performance of surgical suction heads with different geometries via acceleration-sensitized 3D MRI revealed significant differences in turbulence development between our standard control Model A and the modified alternatives (Models 1-3). As flow conditions during measurement have been comparable, the specific geometry of the respective suction heads must have been the main factor responsible. The underlying mechanisms and causative factors can only be speculated about, but as other investigations have shown, hemolytic activity is positively associated with degree of turbulence. The turbulence data measured in this study correlate with data from other investigations about hemolysis induced by surgical suction heads. The experimental MRI technique used showed added value for further elucidating the underlying physical phenomena causing blood damage due to non-physiological flow.

Velocity vector reconstruction for real-time phase-contrast MRI with radial Maxwell correction.

PURPOSE: To develop an auto-calibrated image reconstruction for highly accelerated multi-directional phase-contrast (PC) MRI that compensates for (1) reconstruction instabilities occurring for phase differences near ± π and (2) phase errors by concomitant magnetic fields that differ for individual radial spokes. THEORY AND METHODS: A model-based image reconstruction for real-time PC MRI based on nonlinear inversion is extended to multi-directional flow by exploiting multiple flow-encodings for the estimation of velocity vectors. An initial smoothing constraint during iterative optimization is introduced to resolve the ambiguity of the solution space by penalizing phase wraps. Maxwell terms are considered as part of the signal model on a line-by-line basis to address phase errors by concomitant magnetic fields. The reconstruction methods are evaluated using simulated data and cross-sectional imaging of a rotating-disc, as well as in vivo for the aortic arch and cervical spinal canal at 3T. RESULTS: Real-time three-directional velocity mapping in the aortic arch is achieved at 1.8 × 1.8 × 6 mm3 spatial and 60 ms temporal resolution. Artificial phase wraps are avoided in all cases using the smoothness constraint. Inter-spoke differences of concomitant magnetic fields are effectively compensated for by the model-based image reconstruction with integrated radial Maxwell correction. CONCLUSION: Velocity vector reconstructions based on nonlinear inversion allow for high degrees of radial data undersampling paving the way for multi-directional PC MRI in real time. Whether a spoke-wise treatment of Maxwell terms is required or a computationally cheaper frame-wise approach depends on the individual application.

Estetrol-Drospirenone combination oral contraceptive: a clinical study of contraceptive efficacy, bleeding pattern and safety in Europe and Russia.

OBJECTIVES: To assess the contraceptive efficacy, bleeding pattern and safety of a combined oral contraceptive containing estetrol (E4) 15 mg and drospirenone (DRSP) 3 mg. DESIGN: Multicenter, open-label, phase 3 trial. SETTING: Sixty-nine sites in Europe and Russia. POPULATION: Sexually active women aged 18-50 years with regular menstrual cycles and body mass index ≤35 kg/m2 . METHODS: E4/DRSP was administered in a 24 active/4 placebo regimen for up to 13 cycles. Visits were scheduled during Cycles 2, 4, 7 and 10 and after completing treatment during which adverse events (AEs) were collected. Participants recorded medication intake, vaginal bleeding/spotting, use of other contraceptive methods and sexual intercourse on a daily diary. MAIN OUTCOME MEASURES: Pearl Index (PI) for women 18-35 years (overall and method-failure), bleeding pattern and AEs. RESULTS: A total of 1553 women aged 18-50 years, including 1353 from 18 to 35 years old, received the study medication. PI was 0.47 pregnancies/100 woman-years (95% CI 0.15-1.11); method failure PI was 0.29 pregnancies/100 woman-years (95% CI 0.06-0.83). Scheduled bleeding/spotting occurred in 91.9-94.4% of women over Cycles 1 to 12 and lasted a median of 4-5 days per cycle. The percentage of women with unscheduled bleeding/spotting episodes decreased from 23.5% in Cycle 1 to <16% from Cycle 6 onwards. The most common AEs were headache (7.7%), metrorrhagia (5.5%), vaginal haemorrhage (4.8%) and acne (4.2%). One treatment-related serious AE was reported, a lower extremity venous thromboembolism. One-hundred and forty-one (9.1%) women discontinued study participation because of treatment-related adverse events. CONCLUSION: E4/DRSP provides effective contraception, a predictable bleeding pattern and a favourable safety profile. TWEETABLE ABSTRACT: A phase 3 trial with E4/DRSP shows high contraceptive efficacy, a predictable bleeding pattern and favourable safety profile.

Treatment of early-stage mycosis fungoides: results from the PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) study.

BACKGROUND: The PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) study is a prospective analysis of an international database. Here we examine front-line treatments and quality of life (QoL) in patients with newly diagnosed mycosis fungoides (MF). OBJECTIVES: To identify (i) differences in first-line approaches according to tumour-nodes-metastasis-blood (TNMB) staging; (ii) parameters related to a first-line systemic approach and (iii) response rates and QoL measures. METHODS: In total, 395 newly diagnosed patients with early-stage MF (stage IA-IIA) were recruited from 41 centres in 17 countries between 1 January 2015 and 31 December 2018 following central clinicopathological review. RESULTS: The most common first-line therapy was skin-directed therapy (SDT) (322 cases, 81·5%), while a smaller percentage (44 cases, 11·1%) received systemic therapy. Expectant observation was used in 7·3%. In univariate analysis, the use of systemic therapy was significantly associated with higher clinical stage (IA, 6%; IB, 14%; IIA, 20%; IA-IB vs. IIA, P < 0·001), presence of plaques (T1a/T2a, 5%; T1b/T2b, 17%; P < 0·001), higher modified Severity Weighted Assessment Tool (> 10, 15%; ≤ 10, 7%; P = 0·01) and folliculotropic MF (FMF) (24% vs. 12%, P = 0·001). Multivariate analysis demonstrated significant associations with the presence of plaques (T1b/T2b vs. T1a/T2a, odds ratio 3·07) and FMF (odds ratio 2·83). The overall response rate (ORR) to first-line SDT was 73%, while the ORR to first-line systemic treatments was lower (57%) (P = 0·027). Health-related QoL improved significantly both in patients with responsive disease and in those with stable disease. CONCLUSIONS: Disease characteristics such as presence of plaques and FMF influence physician treatment choices, and SDT was superior to systemic therapy even in patients with such disease characteristics. Consequently, future treatment guidelines for early-stage MF need to address these issues.

Dynamic water/fat separation and B0 inhomogeneity mapping-joint estimation using undersampled triple-echo multi-spoke radial FLASH.

PURPOSE: To achieve dynamic water/fat separation and B0 field inhomogeneity mapping via model-based reconstructions of undersampled triple-echo multi-spoke radial FLASH acquisitions. METHODS: This work introduces an undersampled triple-echo multi-spoke radial FLASH sequence, which uses (i) complementary radial spokes per echo train for faster spatial encoding, (ii) asymmetric echoes for flexible and nonuniform echo spacing, and (iii) a golden angle increment across frames for optimal k-space coverage. Joint estimation of water, fat, B0 inhomogeneity, and coil sensitivity maps from undersampled triple-echo data poses a nonlinear and non-convex inverse problem which is solved by a model-based reconstruction with suitable regularization. The developed methods are validated using phantom experiments with different degrees of undersampling. Real-time MRI studies of the knee, liver, and heart are conducted without prospective gating or retrospective data sorting at temporal resolutions of 70, 158, and 40 ms, respectively. RESULTS: Up to 18-fold undersampling is achieved in this work. Even in the presence of rapid physiological motion, large B0 field inhomogeneities, and phase wrapping, the model-based reconstruction yields reliably separated water/fat maps in conjunction with spatially smooth inhomogeneity maps. CONCLUSIONS: The combination of a triple-echo acquisition and joint reconstruction technique provides a practical solution to time-resolved and motion robust water/fat separation at high spatial and temporal resolution.

Real-time multi-directional flow MRI using model-based reconstructions of undersampled radial FLASH - A feasibility study.

The purpose of this work was to develop an acquisition and reconstruction technique for two- and three-directional (2d and 3d) phase-contrast flow MRI in real time. A previous real-time MRI technique for one-directional (1d) through-plane flow was extended to 2d and 3d flow MRI by introducing in-plane flow sensitivity. The method employs highly undersampled radial FLASH sequences with sequential acquisitions of two or three flow-encoding datasets and one flow-compensated dataset. Echo times are minimized by merging the waveforms of flow-encoding and radial imaging gradients. For each velocity direction individually, model-based reconstructions by regularized nonlinear inversion jointly estimate an anatomical image, a set of coil sensitivities and a phase-contrast velocity map directly. The reconstructions take advantage of a dynamic phase reference obtained by interpolating consecutive flow-compensated acquisitions. Validations include pulsatile flow phantoms as well as in vivo studies of the human aorta at 3 T. The proposed method offers cross-sectional 2d and 3d flow MRI of the human aortic arch at 53 and 67 ms resolution, respectively, without ECG synchronization and during free breathing. The in-plane resolution was 1.5 × 1.5 mm2 and the slice thickness 6 mm. In conclusion, real-time multi-directional flow MRI offers new opportunities to study complex human blood flow without the risk of combining differential phase (i.e., velocity) information from multiple heartbeats as for ECG-gated data. The method would benefit from a further reduction of acquisition time and accelerated computing to allow for extended clinical trials.

Differentiation of ICOS+ and ICOS- recent thymic emigrant regulatory T cells (RTE T regs) during normal pregnancy, pre-eclampsia and HELLP syndrome.

Two different subsets of naturally occurring regulatory T cells (nTregs), defined by their expression of the inducible co-stimulatory (ICOS) molecule, are produced by the human thymus. To examine the differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) recent thymic emigrant (RTE) T regs during normal pregnancy and in the presence of pre-eclampsia or haemolysis elevated liver enzymes low platelet (HELLP)-syndrome, we used six-colour flow cytometric analysis to determine the changes in the composition of the ICOS(+) and ICOS(-) T reg pools with CD45RA(+) CD31(+) RTE T regs, CD45RA(+) CD31(-) mature naive (MN) T regs, CD45RA(-) CD31(+) and CD45RA(-) CD31(-) memory Tregs. With the beginning of pregnancy until term, we observed a strong differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE, but not CD45RA(+) CD31(-) MN T regs, into CD45RA(-) CD31(-) memory T regs. At the end of pregnancy, the onset of spontaneous term labour was associated with a significant breakdown of ICOS(+) CD45RA(-) CD31(-) memory T regs. However, in the presence of pre-eclampsia, there was a significantly increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs into CD45RA(-) CD31(+) memory T regs, wherein the lacking differentiation into CD45RA(-) CD31(-) memory T regs was partially replaced by the increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(-) MN Tregs into CD45RA(-) CD31(-) memory T regs. In patients with HELLP syndrome, this alternatively increased differentiation of CD45RA(-) CD31(-) MN T regs seemed to be exaggerated, and presumably restored the suppressive activity of magnetically isolated ICOS(+) and ICOS(-) T regs, which were shown to be significantly less suppressive in pre-eclampsia patients, but not in HELLP syndrome patients. Hence, our findings propose that the regular differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs ensures a healthy pregnancy course, while their disturbed differentiation is associated with the occurrence of pre-eclampsia and HELLP syndrome.

Outbreak of listerosis due to imported cooked ham, Switzerland 2011.

From 24 April to 31 July 2011, nine cases of listeriosis were registered in the cantons of Aargau, Basel-Land and Zurich, Switzerland. In six of the cases, infection with Listeria monocytogenes was laboratory confirmed, while three remained suspected cases. The suspected cases were family members of confirmed cases with identical or similar symptoms. All confirmed cases were infected with a L. monocytogenes strain belonging to serovar 1/2a: all had an indistinguishable pulsotype by pulsed-field gel electrophoresis (PFGE). The same strain was detected in samples of cooked ham that were on sale from a particular retailer. Two samples of ham tested contained 470 and 4,800 colony-forming units (CFU) L. monocytogenes per gram respectively. Data of shopper cards from two confirmed cases could be evaluated: both cases had purchased the contaminated ham. The outbreak initiated a product recall and alert actions at national and European level, through the Rapid Alert System for Food and Feed (RASFF). Following the RASFF alert, the company producing the contaminated ham was inspected by the responsible authorities. Their investigations showed that the ham was not contaminated in the production plant, but in the premises of a company to which slicing and packing was outsourced.

Maximum velocity projections within 30 seconds: a combination of real-time three-directional flow MRI and cross-sectional volume coverage.

This work is a proof-of-concept realization of a novel technique for rapid volumetric acquisition, reconstruction, and visualization of three-directional (3dir) flow velocities. The technique combines real-time 3dir phase-contrast (PC) flow magnetic resonance imaging (MRI) with real-time cross-sectional volume coverage. It offers a rapid examination without dependence on electrocardiography (ECG) or respiratory gating during a continuous image acquisition at up to 16 fps. Real-time flow MRI utilizes pronounced radial undersampling and a model-based nonlinear inverse reconstruction. Volume coverage is achieved by automatically advancing the slice position of each PC acquisition by a small percentage of the slice thickness. Post-processing involves the calculation of maximum intensity projections along the slice dimension resulting in six direction-selective velocity maps and a maximum speed map. Preliminary applications to healthy subjects at 3 T comprise mapping of the carotid arteries and cranial vessels at 1.0 mm in-plane resolution within 30 s as well as of the aortic arch at 1.6 mm resolution within 20 s. In conclusion, the proposed method for rapid mapping of 3dir flow velocities offers a quick assessment of the vasculature either to provide a first clinical survey or to plan for more detailed studies.

Whole-body magnetic resonance imaging in two minutes: cross-sectional real-time coverage of multiple volumes.

This work describes a novel technique for rapid and motion-robust whole-body magnetic resonance imaging (MRI). The method employs highly undersampled radial fast low angle shot (FLASH) sequences to cover large volumes by cross-sectional real-time MRI with automatic slice advancement after each frame. The slice shift typically amounts to a fraction of the slice thickness (e.g., 10% to 50%) in order to generate a successive series of partially overlapping sections. Joint reconstructions of these serial images and their respective coil sensitivity maps rely on nonlinear inversion (NLINV) with regularization to the image and sensitivity maps of a preceding frame. The procedure exploits the spatial similarity of neighboring sections. Whole-body scanning is accomplished by measuring multiple volumes at predefined locations, i.e., at fixed table positions, in combination with intermediate automatic movements of the patient table. Individual volumes may take advantage of different field-of-views, image orientations, spatial and temporal resolutions as well as contrasts. Preliminary proof-of-principle applications to healthy subjects at 3 T without cardiac gating and during free breathing yield high-quality anatomic images with acquisition times of less than 100 ms. Spin-density and T1 contrasts are obtained by spoiled FLASH sequences, while T2-type (i.e., T2/T1) contrast results from refocused FLASH sequences that generate a steady state free precession (SSFP) free induction decay (FID) signal. Total measuring times excluding vendor-controlled adjustment procedures are less than two minutes for a 100 cm scan that, for example, covers the body from head to thigh by three optimized volumes and more than 1,300 images. In conclusion, after demonstrating technical feasibility the proposed method awaits clinical trials.

FLASHlight MRI in real time-a step towards Star Trek medicine.

This work describes a dynamic magnetic resonance imaging (MRI) technique for local scanning of the human body with use of a handheld receive coil or coil array. Real-time MRI is based on highly undersampled radial gradient-echo sequences with joint reconstructions of serial images and coil sensitivity maps by regularized nonlinear inversion (NLINV). For this proof-of-concept study, a fixed slice position and field-of-view (FOV) were predefined from the operating console, while a local receive coil (array) is moved across the body-for the sake of simplicity by the subject itself. Experimental realizations with a conventional 3 T magnet comprise dynamic anatomic imaging of the head, thorax and abdomen of healthy volunteers. Typically, the image resolution was 0.75 to 1.5 mm with 3 to 6 mm section thickness and acquisition times of 33 to 100 ms per frame. However, spatiotemporal resolutions and contrasts are highly variable and may be adjusted to clinical needs. In summary, the proposed FLASHlight MRI method provides a robust acquisition and reconstruction basis for future diagnostic strategies that mimic the usage of ultrasound. Necessary extensions for this vision require remote control of all sequence parameters by a person at the scanner as well as the design of more flexible gradients and magnets.

Deep breathing couples CSF and venous flow dynamics.

Venous system pathologies have increasingly been linked to clinically relevant disorders of CSF circulation whereas the exact coupling mechanisms still remain unknown. In this work, flow dynamics of both systems were studied using real-time phase-contrast flow MRI in 16 healthy subjects during normal and forced breathing. Flow evaluations in the aqueduct, at cervical level C3 and lumbar level L3 for both the CSF and venous fluid systems reveal temporal modulations by forced respiration. During normal breathing cardiac-related flow modulations prevailed, while forced breathing shifted the dominant frequency of both CSF and venous flow spectra towards the respiratory component and prompted a correlation between CSF and venous flow in the large vessels. The average of flow magnitude of CSF was increased during forced breathing at all spinal and intracranial positions. Venous flow in the large vessels of the upper body decreased and in the lower body increased during forced breathing. Deep respiration couples interdependent venous and brain fluid flow-most likely mediated by intrathoracic and intraabdominal pressure changes. Further insights into the driving forces of CSF and venous circulation and their correlation will facilitate our understanding how the venous system links to intracranial pressure regulation and of related forms of hydrocephalus.

Spinal CSF flow in response to forced thoracic and abdominal respiration.

BACKGROUND: Respiration-induced pressure changes represent a powerful driving force of CSF dynamics as previously demonstrated using flow-sensitive real-time magnetic resonance imaging (MRI). The purpose of the present study was to elucidate the sensitivity of CSF flow along the spinal canal to forced thoracic versus abdominal respiration. METHODS: Eighteen subjects without known illness were studied using real-time phase-contrast flow MRI at 3 T in the aqueduct and along the spinal canal at levels C3, Th1, Th8 and L3. Subjects performed a protocol of forced breathing comprising four cycles of 2.5 s inspiration and 2.5 s expiration. RESULTS: The quantitative results for spinal CSF flow rates and volumes confirm previous findings of an upward movement during forced inspiration and reversed downward flow during subsequent exhalation-for both breathing types. However, the effects were more pronounced for abdominal than for thoracic breathing, in particular at spinal levels Th8 and L3. In general, CSF net flow volumes were very similar for both breathing conditions pointing upwards in all locations. CONCLUSIONS: Spinal CSF dynamics are sensitive to varying respiratory performances. The different CSF flow volumes in response to deep thoracic versus abdominal breathing reflect instantaneous adjustments of intrathoracic and intraabdominal pressure, respectively. Real-time MRI access to CSF flow in response to defined respiration patterns will be of clinical importance for patients with disturbed CSF circulation like hydrocephalus, pseudotumor cerebri and others.

Reduction of brain metastases in plasminogen activator inhibitor-1-deficient mice with transgenic ocular tumors.

Plasminogen activator inhibitor-1 is known to play a paradoxical positive role in tumor angiogenesis, but its contribution to metastatic spread remains unclear. We studied the impact of plasminogen activator inhibitor (PAI)-1 deficiency in a transgenic mouse model of ocular tumors originating from retinal epithelial cells and leading to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not affect primary tumor growth or vascularization, but was associated with a smaller number of brain metastases. Brain metastases were found to be differentially distributed between the two genotypes. PAI-1-deficient mice displayed mostly secondary foci expanding from local optic nerve infiltration, whereas wild-type animals displayed more disseminated nodules in the scissura and meningeal spaces. SuperArray GEarray analyses aimed at detecting molecules potentially compensating for PAI-1 deficiency demonstrated an increase in fibroblast growth factor-1 (FGF-1) gene expression in primary tumors, which was confirmed by reverse transcription-polymerase chain reaction and western blotting. Our data provide the first evidence of a key role for PAI-1 in a spontaneous model of metastasis and suggest that angiogenic factors, such as FGF-1, may be important for primary tumor growth and may compensate for the absence of PAI-1. They identify PAI-1 and FGF-1 as important targets for combined antitumor strategies.

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation.

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.