7,12-Dimethylbenz[a]anthracene Induces Apoptosis in Murine Pre-B Cells through a Caspase-8 -Dependent Pathway

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) have been demonstrated to cause a variety of tumors and immunosuppressive effects. Our laboratory, and others, have demonstrated that coculture of progenitor B lymphocytes (pre-B cells) with bone marrow stromal cells and the model PAH 7,12-dimethylbenz[a]anthracene (DMBA) results in pre-B cell apoptosis. In this study we investigated the molecular events that precede apoptosis in DMBA-treated 70Z/3 cells, a pre-B cell line. Using caspase activity assays and immunoblotting techniques, we determined the temporal pattern of caspase expression in the pre-B cells. Using caspase inhibitors, we demonstrated that DMBA-mediated pre-B cell apoptosis is dependent on activation of caspase-8, whereas caspase-9 activation is essential for maximal apoptosis. We also demonstrated that DMBA activated PKR, an interferon-inducible protein kinase, in pre-B cells. PKR in turn can activate caspase-8 independently of death receptor ligation. As a result of these studies, we propose a novel PKR-dependent pathway for activation of apoptosis in DMBA-treated pre-B cells

Quantification of pharmacodynamic interactions between dexmedetomidine and midazolam in the rat

ABSTRACT The pharmacodynamic (PD) interaction between the benzodiazepine agonist midazolam and the ␣ 2 -adrenergic agonist dexmedetomidine was characterized for defined measures of anesthetic action and cardiovascular and ventilatory side effects in 33 rats. For various combinations of constant plasma concentrations of midazolam (0.1-20 g/ml) and dexmedetomidine (0.3-19 ng/ml) obtained by target-controlled infusion, the whisker reflex (WR), righting reflex (RR), startle reflex to noise (SR), tail clamp response (TC), and corneal reflex (CR) were assessed. EEG (power in 0.5-3.5-Hz frequency band), mean arterial pressure, and heart rate were recorded continuously. Blood gas values and arterial drug concentrations were determined regularly. The nature and extent of PD interaction was quantified by the model parameter synergy (SYN Ͻ 0, antagonism; SYN ϭ 0, additivity; and SYN Ͼ 0, synergy). With increasing drug concentrations WR was lost first, followed by RR, SR, TC, and CR. These effects were accompanied by an increase of the EEG measure. The drug interaction was synergistic for all stimulus-response measures and the degree of synergy increased with deeper levels of central nervous system depression (SYN was 7.3, 145, 560, 374, and 1490 for WR, RR, SR, TC, and CR, respectively). The cardiovascular side effects of dexmedetomidine, evaluated at similar PD endpoints, were reduced in the presence of midazolam. Ventilatory side effects were minor for all drug combinations. The nature and extent of the PD interactions were not reflected in the EEG measure. In clinical anesthetic practice adequate general anesthesia requires a minimum of two different classes of anesthetic drugs. A hypnotic (inhalational anesthetic, i.v. anesthetic) and an analgesic (opiate) drug are titrated to achieve adequate CNS depression. A muscle relaxant can be used to facilitate surgical procedures. This anesthetic combination is termed "balanced anesthesia" (Hug, 1990; Many variables influence the complex relationship between dosage, plasma concentration, and drug effect. To optimize the delivery of anesthetic drugs to individual patients, it is important to distinguish between pharmacokinetic (PK) and pharmacodynamic (PD) interactions. For example, Dexmedetomidine, a selective ␣ 2 -adrenergic agonist, is being studied for potential use in anesthetic practice because of its combined analgesic, sedative, hypnotic, and anxiolytic effect

Background rates of 41 adverse events of special interest for COVID-19 vaccines in 10 European healthcare databases - an ACCESS cohort study

BACKGROUND: In May 2020, the ACCESS (The vACCine covid-19 monitoring readinESS) project was launched to prepare real-world monitoring of COVID-19 vaccines. Within this project, this study aimed to generate background incidence rates of 41 adverse events of special interest (AESI) to contextualize potential safety signals detected following administration of COVID-19 vaccines. METHODS: A dynamic cohort study was conducted using a distributed data network of 10 healthcare databases from 7 European countries (Italy, Spain, Denmark, The Netherlands, Germany, France and United Kingdom) over the period 2017 to 2020. A common protocol (EUPAS37273), common data model, and common analytics programs were applied for syntactic, semantic and analytical harmonization. Incidence rates (IR) for each AESI and each database were calculated by age and sex by dividing the number of incident cases by the total person-time at risk. Age-standardized rates were pooled using random effect models according to the provenance of the events. FINDINGS: A total number of 63,456,074 individuals were included in the study, contributing to 211.7 million person-years. A clear age pattern was observed for most AESIs, rates also varied by provenance of disease diagnosis (primary care, specialist care). Thrombosis with thrombocytopenia rates were extremely low ranging from 0.06 to 4.53/100,000 person-years for cerebral venous sinus thrombosis (CVST) with thrombocytopenia (TP) and mixed venous and arterial thrombosis with TP, respectively. INTERPRETATION: Given the nature of the AESIs and the setting (general practitioners or hospital-based databases or both), background rates from databases that show the highest level of completeness (primary care and specialist care) should be preferred, others can be used for sensitivity. The study was designed to ensure representativeness to the European population and generalizability of the background incidence rates. FUNDING: The project has received support from the European Medicines Agency under the Framework service contract nr EMA/2018/28/PE

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Determination of platinum surface contamination in veterinary and human oncology centres using inductively coupled plasma mass spectrometry

The objective of this study was to determine the surface contamination with platinum-containing antineoplastic drugs in veterinary and human oncology centres. Inductively coupled plasma mass spectrometry was used to measure platinum levels in surface samples. In veterinary and human oncology centres, 46.3 and 68.9% of the sampled surfaces demonstrated platinum contamination, respectively. Highest platinum levels were found in the preparation rooms (44.6 pg cm-2) in veterinary centres, while maximal levels in human centres were found in oncology patient-only toilets (725 pg cm-2). Transference of platinum by workers outside areas where antineoplastic drugs were handled was observed in veterinary and human oncology centres. In conclusion, only low levels of platinum contamination attributable to carboplatin were found in the sampled veterinary oncology centres. However, dispersion of platinum outside areas where antineoplastic drugs were handled was detected in veterinary and human oncology centres. Consequently, not only personnel, but also others may be exposed to platinum

Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood spots in the quantification of miltefosine in dried blood samples

Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly endemic in resource-poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We developed and validated an LC-MS/MS method for the quantification of miltefosine with this novel sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within ±10.8%), precision (CV%≤11.9%), recovery, carry-over and matrix effect. VAMS samples were stable for at least one month at room temperature and 37°C. The impact of haematocrit on assay accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A clinical validation will be required to investigate whether VAMS is an appropriate and cost-effective alternative sampling method to conventional DBS sampling

The risk of new fragility fractures in patients with chronic kidney disease and hip fracture-a population-based cohort study in the UK

Chronic kidney disease (CKD) is a risk factor for fractures. However, in hip fracture patients, CKD G3-G5 was associated with a higher mortality risk and not associated with a higher risk of subsequent non-hip fractures compared to eGFR > 60 ml/min. The higher mortality risk may, as competing risk, explain our findings. INTRODUCTION: Chronic kidney disease (CKD) is a known risk factor for fragility fractures. Patients aged 50+ with a recent fragility fracture have an increased risk of subsequent fractures. Our aim was to evaluate the association between CKD stages G3-G5 versus estimated glomerular filtration rate (eGFR) > 60 ml/min and the risk of a new non-hip fracture or fragility fracture in patients with a first hip fracture. METHODS: Population-based cohort study using the UK general practices in the Clinical Practice Research Datalink. Associations between CKD stage and first subsequent fracture were determined using Cox proportional hazard analyses to estimate hazard ratios (HRs). To explore the potential competing risk of mortality, cause-specific (cs) HRs for mortality were estimated. RESULTS: CKD G3-G5 was associated with a lower risk of any subsequent non-hip fracture (HR: 0.90, 95%CI: 0.83-0.97), but not with the risk of subsequent major non-hip fragility fracture. CKD G3-G5 was associated with a higher mortality risk (cs-HR: 1.05, 95%CI: 1.01-1.09). Mortality risk was 1.5- to 3-fold higher in patients with CKD G4 (cs-HR: 1.50, 95%CI: 1.38-1.62) and G5 (cs-HR: 2.93, 95%CI: 2.48-3.46) compared to eGFR > 60 ml/min. CONCLUSIONS: The risk of a subsequent major non-hip fragility fractures following hip fracture was not increased in patients with CKD G3-G5 compared to eGFR > 60 ml/min. Mortality risk was higher in both hip fracture and non-hip fracture patients with CKD G4 and G5. The higher mortality risk may, as competing risk, explain our main finding of no increased or even decreased subsequent fracture risk after a hip fracture in patients with CKD G3-G5

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism