Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

A toolkit for wide-screen dynamic area of interest measurements using the Pupil Labs Core Eye Tracker.

Eye tracking measurements taken while watching a wide field screen are challenging to perform. Commercially available remote eye trackers typically do not measure more than 35 degrees in eccentricity. Analysis software was developed using the Pupil Core Eye Tracking data to analyze viewing behavior under circumstances as natural as possible, on a 1.55-m-wide screen allowing free head movements. Additionally, dynamic area of interest (AOI) analyses were performed on data of participants viewing traffic scenes. A toolkit was created including software for simple allocation of dynamic AOIs (semi-automatically and manually), measurement of parameters such as dwell times and time to first entry, and overlaying gaze and AOIs on video. Participants (n =11) were asked to look at 13 dynamic AOIs in traffic scenes from appearance to disappearance in order to validate the setup and software. Different AOI margins were explored for the included objects. The median ratio between total appearance time and dwell time was about 90% for most objects when appropriate margins were chosen. This validated open-source toolkit is readily available for researchers who want to perform dynamic AOI analyses with the Pupil Core eye tracker, especially when measurements are desired on a wide screen, in various fields such as psychology, transportation, and low vision research.

Mouse venous thrombosis upon silencing of anticoagulants depends on tissue factor and platelets, not FXII or neutrophils.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Atherothrombosis model by silencing of protein C in APOE*3-Leiden.CETP transgenic mice.

Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoe-/-) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type "0" (no lesions) to type "V" lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type "V") atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoe-/- mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs.

A synergistic DNA logic predicts genome-wide chromatin accessibility.

Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when trained with DNase-seq data for a cell type is capable of predicting expected read counts of genome-wide chromatin accessibility at every base from DNA sequence alone, with the highest accuracy at hypersensitive sites shared across cell types. We confirm that a SCM accurately predicts chromatin accessibility for thousands of synthetic DNA sequences using a novel CRISPR-based method of highly efficient site-specific DNA library integration. SCMs are directly interpretable and reveal that a logic based on local, nonspecific synergistic effects, largely among pioneer TFs, is sufficient to predict a large fraction of cellular chromatin accessibility in a wide variety of cell types.

REtrieval And cure of Chronic Hepatitis C (REACH): Results of micro-elimination in the Utrecht province.

BACKGROUND: The Netherlands is one of the six European countries considered on track to eliminate hepatitis C virus by 2030. To achieve this goal, continuous efforts have to be put into designing efficient case-finding strategies, including the retrieval of previously diagnosed hepatitis C virus-infected who are lost to follow-up. AIMS: To trace and treat all lost to follow-up hepatitis C virus patients in the Utrecht region and create an efficient retrieval strategy that can be used in future (national) retrieval initiatives. METHODS: Positive hepatitis C virus diagnostic tests (anti-hepatitis C virus IgG or hepatitis C virus-RNA) from the laboratory of all four hospitals and one central laboratory for primary care diagnostics in the province of Utrecht from 2001 to 2015 were linked to clinical records. Untreated patients with available contact information were deemed eligible for retrieval and invited for reevaluation with (virology) blood tests, fibroscan measurement and possible direct-acting antiviral therapy. MAIN RESULTS: After screening all hepatitis C virus diagnostics, 1913 chronic hepatitis C virus-infected were identified of which 14.1% (n = 269) were invited back into care. Overall, 17.4% was traced with the highest yield (28.3%) in those who lived in the Utrecht province. Through renewed patient assessments, 42 chronic hepatitis C virus infections were re-identified (76% with a history of intravenous drug use, 24% with Metavir F3-F4). Until now, 59% has either scheduled or initiated direct-acting antiviral therapy. CONCLUSION: The retrieval of previously diagnosed hepatitis C virus patients through screening of laboratory diagnostics from the past is feasible and should be pursued for further control and reduction of hepatitis C virus infection. Retrieval is most successful when performed regionally. LAY SUMMARY: To completely eliminate chronic hepatitis C virus (HCV) infection and prevent complications, undiagnosed and also previously diagnosed but lost to follow-up (LFU) HCV patients have to be brought (back) into care for therapy. Retrieval of LFU HCV patients through screening of laboratory diagnostics from the past is feasible and most successful when performed regionally.

Binding Forces of Cellulose Binding Modules on Cellulosic Nanomaterials.

In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.

The extent of microbiological testing is associated with alteration of antibiotic therapy in adults with community-acquired pneumonia.

The aim of this study was to explore the relationship between the extent of microbiological testing and the frequency of antibiotic alteration in adults hospitalised with community-acquired pneumonia (CAP). We retrospectively studied 283 immunocompetent patients hospitalised with CAP. Information on microbiological testing and prescribed antibiotics was obtained. Patients were grouped according to the number of different microbiological tests performed within the first 2 days of admission (0-5 tests). Alteration rates were compared between these groups. Antimicrobial alteration was defined as a switch at day 3 of hospital stay to (1) a narrower spectrum antibiotics, or (2) a different class of antibiotics, or (3) a switch from dual therapy to monotherapy (4) or discontinuation of antibiotic treatment because the indication for antibiotic treatment did no longer exist. For each additional test performed, a stepwise increase in percentage of patients with altered antibiotic regimen ranging from 0 to 59% (p = 0.001) was found. Multivariate logistic regression analyses showed that performing PCR assay for atypical pathogens was most strongly associated with any alteration of antibiotic treatment (OR 2.6 (95% CI 1.4-4.9)) and with changes in atypical coverage specifically (OR 3.1 (95% CI 1.6-6.0). The extent of microbiological testing was positively associated with antibiotic alteration in adults hospitalised with CAP. Antibiotic treatment was most likely to be altered in patients in whom PCR assay for atypical pathogens was performed.

The effects of habituation and adding a rest-frame on experienced simulator sickness in an advanced mobility scooter driving simulator.

The aim of this article is to investigate the effect of a physical rest-frame, habituation and age on simulator sickness in an advanced mobility scooter driving simulator. Twenty-six young and 34 older adults completed a total of 12 drives in an advanced mobility scooter driving simulator over two visits. A 2x2 crossover design was used to measure the effect of a rest frame that was added to the driving simulator on either the first or second visit. The Simulator Sickness Questionnaire was used to measure simulator sickness symptoms. A significant decrease in simulator sickness was observed between the first and the second visit. Older adults reported more severe simulator sickness symptoms compared to younger participants. No effect of rest-frame could be found. Habituation appears to be the most effective method to reduce simulator sickness in an advanced mobility scooter driving simulator. More research is needed to investigate simulator sickness in patient groups. Practitioner summary: Experiencing simulator sickness is a major problem across all types of simulators. The present experiment investigated the effect of a rest-frame, habituation and age on developing simulator sickness symptoms in an advanced mobility scooter driving simulator. Habituation appeared to be the most effective method to reduce simulator sickness.

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice.

Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology.

Silencing of Anticoagulant Protein C Evokes Low-Incident but Spontaneous Atherothrombosis in Apolipoprotein E-Deficient Mice-Brief Report.

OBJECTIVE: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype. APPROACH AND RESULTS: On lowering of plasma protein C levels with small interfering RNA (siProc) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as siProc-treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in siProc mice with thrombi had a similar composition and size as plaques in siProc mice without thrombi. Seven out of 25 siProc mice featured clots in the left atrium of the heart. CONCLUSIONS: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.

Group A Streptococcal Meningitis With the M1UK Variant in the Netherlands.

This study reports an epidemiological assessment of laboratory-confirmed group A streptococcal meningitis cases in the Netherlands using more than 40 years of national bacteriological surveillance data.

Oligonucleotides targeting coagulation factor mRNAs: use in thrombosis and hemophilia research and therapy.

Small interfering (si) RNAs and antisense oligonucleotides (ASOs; here for simplicity reasons, both referred to as oligonucleotides) are small synthetic RNA or DNA molecules with a sequence complementary to a (pre)mRNA. Although the basic mechanisms of action between siRNAs and ASO are distinct, a sequence-specific interaction of the both oligonucleotides with the target (pre)mRNA alters the target's fate, which includes highly effective sequence-specific blockade of translation and consequently depletion of the corresponding protein. For a number of years, these oligonucleotides have been used as a tool in biological research to study gene function in vitro. More recently, safe and specific delivery of these oligonucleotides to the liver of mammals has been achieved and optimized. This not only allowed their use for in vivo gene studies in physiology and disease, but also opened the opportunity for the development of a new generation of RNA-specific drugs for therapeutic purposes. In 2013, the first oligonucleotide product targeting RNA from the hepatic cholesterol pathway was approved. For blood coagulation, a large portion of key proteins are produced in the liver, and thereby siRNAs and ASOs can also be used as appropriate tools to target these proteins in vivo. In this review, we describe the first use of oligonucleotides for this purpose from zebrafish to primates. As the use of oligonucleotides allows avoidance of early lethality associated with full deficiency of several coagulation factors, it has proved to be of value for studying these proteins in physiology and disease. Currently, oligonucleotides are tested as therapeutics, with the ultimate goal to beneficially modulate the hemostatic balance in thrombosis and hemophilia patients. We discuss both the preclinical and clinical studies of a number of siRNAs and ASOs with the potential to be introduced as drugs for prophylactic and/or treatment of thrombosis or hemophilia. We conclude that for the coagulation field, oligonucleotides are of value for research purposes, and now the moment has come to fulfill their promise as therapeutics.

Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production.

Mice deficient in the anticoagulants antithrombin (Serpinc1) or protein C (Proc) display premature death due to thrombosis-related coagulopathy, thereby precluding their use in gene function studies and thrombosis models. We used RNA interference to silence Serpinc1 and/or Proc in normal adult mice. The severe coagulopathy that followed combined "knockdown" of these genes is reported. Two days after siRNA injection, thrombi (occlusive) were observed in vessels (large and medium-sized) in multiple tissues, and hemorrhages were prominent in the ocular, mandibular, and maxillary areas. Tissue fibrin deposition and reduction of plasma fibrinogen accompanied this phenotype. The coagulopathy was prevented by dabigatran etexilate treatment. Silencing of Serpinc1 alone yielded a comparable but milder phenotype with later onset. The phenotype was absent when Proc was targeted alone. We conclude that RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes in vivo and provides a novel, controlled mouse model for spontaneous venous thrombosis.

P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

Difficulties in Daily Life Reported by Patients With Homonymous Visual Field Defects.

BACKGROUND: Homonymous visual field defects (HVFD) are common after postchiasmatic acquired brain injury and may have a significant impact on independent living and participation in society. Vision-related difficulties experienced in daily life are usually assessed using questionnaires. The current study 1) links the content of 3 of these questionnaires to the International Classification of Functioning, Disability and Health (ICF) and 2) provides analyses of vision-related difficulties reported by patients with HVFD and minimal comorbidities. METHODS: Fifty-four patients with homonymous hemianopia or quadrantanopia were asked about difficulties experienced in daily life because of their HVFD. This was performed during a structured interview including 3 standardized questionnaires: National Eye Institute Visual Functioning Questionnaire, Independent Mobility Questionnaire, and Cerebral Visual Disorders Questionnaire. The reported difficulties were linked to the ICF according to the ICF linking rules. Main outcome measures were presence or absence of experienced difficulties. RESULTS: The ICF linking procedure resulted in a classification table that can be used in future studies of vision-related difficulties. Besides well-known difficulties related to reading, orientation, and mobility, a high proportion of patients with HVFD reported problems that previously have not been documented in the literature, such as impaired light sensitivity, color vision, and perception of depth. CONCLUSIONS: A systematic inventory of difficulties experienced in daily life by patients with HVFD was performed using the ICF. These findings have implications for future study, assessment and rehabilitation of patients with HVFD.

Mesh Or Patch for Hernia on Epigastric and Umbilical Sites (MORPHEUS trial): study protocol for a multi-centre patient blinded randomized controlled trial.

BACKGROUND: Evidence is accumulating that, similar to other ventral hernias, umbilical and epigastric hernias must be mesh repaired. The difficulties involved in mesh placement and in mesh-related complications could be the reason many small abdominal hernias are still primary closed. In laparoscopic repair, a mesh is placed intraperitoneally, while the most common procedure is open surgery is pre-peritoneal mesh placement. A recently developed alternative method is the so-called patch repair, in this approach a mesh can be placed intraperitoneally through open surgery. In theory, such patches are particularly suitable for small hernias due to a reduction in the required dissection. This simple procedure is described in several studies. It is still unclear whether this new approach is associated with an equal risk of recurrence and complications compared with pre-peritoneal meshes. The material of the patch is in direct contact with intra-abdominal organs, it is unknown if this leads to more complications. On the other hand, the smaller dissection in the pre-peritoneal plane may lead to a reduction in wound complications. METHODS/DESIGN: 346 patients suffering from an umbilical or epigastric hernia will be included in a multi-centre patient-blinded trial, comparing mesh repair with patch repair. Randomisation will take place for the two operation techniques. The two devices investigated are a flat pre-peritoneal mesh and a Proceed Ventral Patch®. Stratification will occur per centre. Post-operative evaluation will take place after 1, 3, 12 and 24 months. The number of complications requiring treatment is the primary endpoint. Secondary endpoints are Verbal Descriptor Scale (VDS) pain score and VDS cosmetic score, operation duration, recurrence and costs. An intention to treat analysis will be performed. DISCUSSION: This trial is one of the first in its kind, to compare different mesh devices in a randomized controlled setting. The results will help to evaluate mesh repair for epigastric an umbilical hernia, and find a surgical method that minimizes the complication rate. TRIAL REGISTRATION: Netherlands Trail Registration (NTR) www.trialregister.nl 2010 NTR2514 NL33995.060.10.

Impact of allele-selective silencing of von Willebrand factor in mice based on a single nucleotide allelic difference in von Willebrand factor.

INTRODUCTION: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact. MATERIALS AND METHODS: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding. RESULTS: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times. CONCLUSIONS: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.

Appropriateness of empirical treatment and outcome in bacteremia caused by extended-spectrum-ß-lactamase-producing bacteria.

We studied clinical characteristics, appropriateness of initial antibiotic treatment, and other factors associated with day 30 mortality in patients with bacteremia caused by extended-spectrum-ß-lactamase (ESBL)-producing bacteria in eight Dutch hospitals. Retrospectively, information was collected from 232 consecutive patients with ESBL bacteremia (due to Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae) between 2008 and 2010. In this cohort (median age of 65 years; 24 patients were <18 years of age), many had comorbidities, such as malignancy (34%) or recurrent urinary tract infection (UTI) (15%). One hundred forty episodes (60%) were nosocomial, 54 (23%) were otherwise health care associated, and 38 (16%) were community acquired. The most frequent sources of infection were UTI (42%) and intra-abdominal infection (28%). Appropriate therapy within 24 h after bacteremia onset was prescribed to 37% of all patients and to 54% of known ESBL carriers. The day 30 mortality rate was 20%. In a multivariable analysis, a Charlson comorbidity index of ≥ 3, an age of ≥ 75 years, intensive care unit (ICU) stay at bacteremia onset, a non-UTI bacteremia source, and presentation with severe sepsis, but not inappropriate therapy within <24 h (adjusted odds ratio [OR], 1.53; 95% confidence interval [CI], 0.68 to 3.45), were associated with day 30 mortality. Further assessment of confounding and a stratified analysis for patients with UTI and non-UTI origins of infection did not reveal a statistically significant effect of inappropriate therapy on day 30 mortality, and these results were insensitive to the possible misclassification of patients who had received ß-lactam-ß-lactamase inhibitor combinations or ceftazidime as initial treatment. In conclusion, ESBL bacteremia occurs mostly in patients with comorbidities requiring frequent hospitalization, and 84% of episodes were health care associated. Factors other than inappropriate therapy within <24 h determined day 30 mortality.

Effects of two weeks of metformin treatment on whole-body glycocalyx barrier properties in db/db mice.

BACKGROUND: The anti-diabetic drug metformin has been demonstrated to exert a protective effect against vascular complications in diabetes independent of its glucose lowering action. Since the endothelial glycocalyx has been indicated to have important vasculoprotective properties and to be vulnerable to degradation by hyperglycemic conditions, we evaluated in the current study the effect of short-term metformin treatment on whole-body glycocalyx barrier properties in a mouse model of non-insulin dependent diabetes mellitus (db/db mouse). METHODS: Glycocalyx barrier properties were measured in an acute experiment in three groups of mice: 1) db/db mice without treatment serving as controls, 2) db/db mice which received metformin for two weeks in the drinking water serving as experimental group, and 3) C57Bl/6 mice serving as reference group. Animals were put under anesthesia (ketamine, medetomidine, and atropine) and carotid artery blood pressure was continuously monitored. To probe the glycocalyx a mixture of the tracers FITC-labeled 70 kDa dextrans (Dex70) or fluorescein-labeled red blood cells (RBCs) versus Texas Red-labeled 40 kDa dextrans (Dex40) was infused and blood samples subsequently collected for 30 min to determine the initial vascular distribution volume and clearance of these tracers. Urine was collected and dry-to-wet weight of heart and kidney were determined after the experiment. Group differences were tested using unpaired t-tests. RESULTS: Metformin treatment did not affect body weight, fasting blood glucose and arterial blood pressure. Compared to C57Bl/6 mice, db/db mice showed a diminished initial exclusion and increased vascular clearance of Dex70 versus Dex40 (P < 0.05), and both were improved by the metformin treatment (P < 0.05). While urine production was higher in the db/db mice compared to C57Bl/6 (P < 0.05), heart and kidney of the metformin treated animals showed comparable dry-to-wet weights compared to the C57Bl/6 mice. CONCLUSIONS: Two weeks of metformin in the drinking water is associated with an improvement in glycocalyx barrier properties in db/db mice, as evidence by an enhanced exclusion and retention of 70 kDa dextrans in the vasculature. In addition, metformin improved hydration of heart and kidney. Previous reported cardiovascular benefits of metformin may well involve an improvement of the endothelial glycocalyx.