Endothelial function and the risk for endothelial dysfunction differ between males and females. Besides the action of estrogen, sex chromosome gene expression and programming effects also provoke ...this sexual dimorphism. MicroRNAs (miRNAs) have emerged as regulators of endothelial cell function and dysfunction. We here hypothesized distinct miRNA expression patterns in male versus female human endothelial cells that contribute to the functional differences. We used our well-established model of fetal endothelial cells isolated from placenta (fpEC) and analyzed sexual dimorphic miRNA expression and potentially affected biological functions. Next-generation miRNA sequencing of fpEC isolated after pregnancies with male and female neonates identified sex-dependent miRNA expression patterns. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath and mirSystem softwares, and suggested differences in barrier function and actin organization. The identified pathways were further investigated by monolayer impedance measurements (ECIS) and analysis of F-actin organization (Phalloidin). Nine miRNAs were differentially expressed in fpEC of male versus female neonates. Functional pathways most significantly regulated by these miRNAs included 'Adherens junction', 'ECM receptor interaction' and 'Focal adhesion'. These pathways control monolayer barrier function and may be paralleled by altered cytoskeletal organization. In fact, monolayer impedance was higher in fpEC of male progeny, and F-actin staining revealed more pronounced peripheral stress fibers in male versus female fpEC. Our data highlight that endothelial cell function differs between males and females already in utero, and that altered miRNAs are associated with sex dependent differences in barrier function and actin organization.
Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media ...specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.
Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to ...evaluate the performance of the recently introduced cobas® HCV GT assay (Roche) and to compare it to two comparator assays.
The cobas® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE® HCV 5'NC Genotyping Kit (Siemens) and the VERSANT® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included.
When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample.
The cobas® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.
To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we ...performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.
•Loss-of-function mutations in KIF5A are a cause of amyotrophic lateral sclerosis•ALS-associated KIF5A mutations are distinct from HSP and CMT mutations in KIF5A•Identification of KIF5A highlights the role of cytoskeleton in ALS pathogenesis
Using a large-scale genome-wide association study and exome sequencing, we identified KIF5A as a novel gene associated with ALS. Our data broaden the phenotype resulting from mutations in KIF5A and highlight the importance of cytoskeletal defects in the pathogenesis of ALS.
C3 glomerulopathy and idiopathic Ig-associated membranoproliferative GN are kidney diseases characterized by abnormal glomerular complement C3 deposition. These conditions are heterogeneous in ...outcome, but approximately 50% of patients develop kidney failure within 10 years.
To improve identification of patients with poor prognosis, we performed a detailed analysis of percutaneous kidney biopsies in a large cohort of patients. Using a validated histologic scoring system, we analyzed 156 native diagnostic kidney biopsies from a retrospective cohort of 123 patients with C3 glomerulopathy and 33 patients with Ig-associated membranoproliferative GN. We used linear regression, survival analysis, and Cox proportional hazards models to assess the relationship between histologic and clinical parameters with outcome.
Frequent biopsy features were mesangial expansion and hypercellularity, glomerular basement membrane double contours, and endocapillary hypercellularity. Multivariable analysis showed negative associations between eGFR and crescents, interstitial inflammation, and interstitial fibrosis/tubular atrophy. Proteinuria positively associated with endocapillary hypercellularity and glomerular basement membrane double contours. Analysis of second native biopsies did not demonstrate associations between immunosuppression treatment and improvement in histology. Using a composite outcome, risk of progression to kidney failure associated with eGFR and proteinuria at the time of biopsy, cellular/fibrocellular crescents, segmental sclerosis, and interstitial fibrosis/tubular atrophy scores.
Our detailed assessment of kidney biopsy data indicated that cellular/fibrocellular crescents and interstitial fibrosis/tubular atrophy scores were significant determinants of deterioration in kidney function.
Solid cancers that metastasize to the lungs represent a major therapeutic challenge. Current treatment paradigms for lung metastases consist of radiation therapy, chemotherapies, and surgical ...resection, but there is no single treatment or combination that is effective for all tumor types. To address this, oncolytic myxoma virus (MYXV) engineered to express human tumor necrosis factor (vMyx-hTNF) was tested after systemic administration in an immunocompetent mouse K7M2-Luc lung metastatic osteosarcoma model. Virus therapy efficacy against pre-seeded lung metastases was assessed after systemic infusion of either naked virus or ex vivo-loaded autologous bone marrow leukocytes or peripheral blood mononuclear cells (PBMCs). Results of this study showed that the PBMC pre-loaded strategy was the most effective at reducing tumor burden and increasing median survival time, but sequential intravenous multi-dosing with naked virus was comparably effective to a single infusion of PBMC-loaded virus. PBMC-loaded vMyx-hTNF also potentially synergized very effectively with immune checkpoint inhibitors anti-PD-1, anti-PD-L1, and anti-cytotoxic T lymphocyte associated protein 4 (CTLA-4). Finally, in addition to the pro-immune stimulation caused by unarmed MYXV, the TNF transgene of vMyx-hTNF further induced the unique expression of numerous additional cytokines associated with the innate and adaptive immune responses in this model. We conclude that systemic ex vivo virotherapy with TNF-α-armed MYXV represents a new potential strategy against lung metastatic cancers like osteosarcoma and can potentially act synergistically with established checkpoint immunotherapies.
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How to deliver and specific arming of oncolytic viruses are two of the biggest questions currently facing the field of oncolytic virotherapy. In this study, leukocytes are used to deliver TNF-α-armed myxoma virus to lung metastatic osteosarcoma tumors. The efficacy of these modalities is shown alone and in combination with immune checkpoint inhibitors.
Background/aim:
Cost-efficient methods are essential for successful participant recruitment in clinical trials. Patient portal messages are an emerging means of recruiting potentially eligible ...patients into trials. We assessed the response rate and complaint rate from direct-to-patient, targeted recruitment through patient portals of an electronic medical record for a clinical trial, and compared response rates by differences in message content.
Methods:
The Study to Understand Fall Reduction and Vitamin D in You (STURDY) trial is a National Institutes of Health–sponsored, community-based study of vitamin D supplementation for fall prevention in older adults conducted at Johns Hopkins. Potential participants were identified using the Epic electronic medical record at the Johns Hopkins Health System based on age (≥70 years), ZIP code (30-mile radius of study site), and prior activation of a patient portal account. We prepared a shorter message and a longer message. Both had basic information about study participation, but the longer message also contained information about the significance of the study and a personal invitation from the STURDY principal investigator. The Hopkins Institutional Review Board did not require prior consent from the patient or their providers. We calculated the response rate and tracked the number of complaints and requests for removal from future messages. We also determined response rate according to message content.
Results:
Of the 5.5 million individuals receiving care at the Johns Hopkins Health System, a sample of 6896 met our inclusion criteria and were sent one patient portal recruitment message between 6 April 2017 and 3 August 2017. Assessment of enrollment by this method ended on 1 December 2017. There were 116 patients who expressed interest in the study (response rate: 1.7%). Twelve (0.2%) recipients were randomized. There were two complaints (0.03%) and one request to unsubscribe from future recruitment messages (0.01%). Response rate was higher with the longer message than the shorter message (2.1% vs 1.2%; p = 0.005).
Conclusion:
Patient portal messages inviting seniors to participate in a randomized controlled trial resulted in a response rate similar to commercial email marketing and resulted in very few complaints or opt-out requests. Furthermore, a longer message with more content enhanced response rate. Recruitment through patient portals might be an effective strategy to enroll trial participants.
BACKGROUND:The association between elevated levels of triglycerides and insulin may be weaker in African American women (AAW) than in women of other groups, leading to under diagnosis of the ...metabolic syndrome (MetS) in AAW when using the National Cholesterol Education Program (NCEP) criteria, as those criteria do not include a marker of insulin resistance, using elevated triglycerides to provide an indirect indication of insulin resistance.
OBJECTIVES:To determine the degree of agreement between two definitions for the MetS, that described by the NCEP and the NCEP criteria with the addition of a marker of insulin resistance in a sample of AAW.
METHOD:This non experimental pilot study took place in the General Clinical Research Center of a major medical center. Thirty-three AAW 19-45 years of age were screened using the NCEP criteria for MetS, additional markers of insulin resistance, and a 2-hour Oral Glucose Tolerance Test.
FINDINGS:Six (18%) women were classified as having the MetS using the NCEP criteria. When one of three markers for insulin resistance (hyperinsulinemia, acanthosis nigricans, or Homeostatic Model Assessment Insulin Resistance) was added to the criteria, 15 (45.5%) to 19 (59.5%) of the women were then identified as having MetS.
DISCUSSION:As identified in the literature, the prevalence of cardiovascular disease risk in AAW may be underestimated based on the sole use of the NCEP criteria. Further, because there is some evidence that insulin resistance develops before many other indicators, the addition of a marker of insulin resistance may assist in earlier identification of AAW at high risk for cardiovascular disease.
Building upon a growing literature that resists the pathologizing effects of developmentalist and comparative framings, this fascinating collection of case studies pushes the frontiers of scholarship ...on African urbanism through detailed and nuanced ethnographic analyses of life in a diverse set of cities across the continent. These contributions explore a range of innovative institutions, discourses, and material practices through which claims to citizenship are enacted and contested by a diverse array of actors. They treat cities as sites of experimentation, privileging the ordinary, daily, under-the-radar negotiations through which emergent reconfigurations of citizenship are being continually forged. In doing so, they provide a more culturally informed perspective on African politics and society.
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