A human co-culture cell model incorporating microglia supports glioblastoma growth and migration, and confers resistance to cytotoxics.

Despite the importance of the tumor microenvironment in regulating tumor progression, few in vitro models have been developed to understand the effects of non-neoplastic cells and extracellular matrix (ECM) on drug resistance in glioblastoma (GBM) cells. Using CellTrace-labeled human GBM and microglial (MG) cells, we established a 2D co-culture including various ratios of the two cell types. Viability, proliferation, migration, and drug response assays were carried out to assess the role of MG. A 3D model was then established using a hyaluronic acid-gelatin hydrogel to culture a mixture of GBM and MG and evaluate drug resistance. A contact co-culture of fluorescently labeled GBM and MG demonstrated that MG cells modestly promoted tumor cell proliferation (17%-30% increase) and greater migration of GBM cells (>1.5-fold increase). Notably, the presence of MG elicited drug resistance even when in a low ratio (10%-20%) relative to co-cultured tumor cells. The protective effect of MG on GBM was greater in the 3D model (>100% survival of GBM when challenged with cytotoxics). This new 3D human model demonstrated the influence of non-neoplastic cells and matrix on chemoresistance of GBM cells to three agents with different mechanisms of action suggesting that such sophisticated in vitro approaches may facilitate improved preclinical testing.

Combinatorial Intracellular Delivery Screening of Anticancer Drugs.

Conventional drug solubilization strategies limit the understanding of the full potential of poorly water-soluble drugs during drug screening. Here, we propose a screening approach in which poorly water-soluble drugs are entrapped in poly(2-(methacryloyloxyethyl phosphorylcholine)-poly(2-(diisopropylaminoethyl methacryate) (PMPC-PDPA) polymersomes (POs) to enhance drug solubility and facilitate intracellular delivery. By using a human pediatric glioma cell model, we demonstrated that PMPC-PDPA POs mediated intracellular delivery of cytotoxic and epigenetic drugs by receptor-mediated endocytosis. Additionally, when delivered in combination, drug-loaded PMPC-PDPA POs triggered both an enhanced drug efficacy and synergy compared to that of a conventional combinatorial screening. Hence, our comprehensive synergy analysis illustrates that our screening methodology, in which PMPC-PDPA POs are used for intracellular codelivery of drugs, allows us to identify potent synergistic profiles of anticancer drugs.

Predicting mortality in patients admitted to the intensive care unit after open vascular surgery.

PURPOSES: Vascular surgery (VS) has a higher perioperative mortality than other types of surgery. We compared different scores for predicting mortality in patients admitted to the intensive care unit (ICU) after open VS. METHODS: Patients admitted to the ICU after open VS from 2006 to 2013 were included. We calculated the Acute Physiology and Chronic Health Evaluation (APACHE), Simplified Acute Physiology Score (SAPS), Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity (POSSUM) and Preoperative Score to Predict Postoperative Mortality (POSPOM). We performed multivariate logistic regression to assess independent factors with the calculation of odds ratios (ORs) and 95% confidence intervals (CIs). We tested the predictive ability of the scores using the area under the receiver operating characteristics curve (AUROC). RESULTS: A total of 833 consecutive patients were included. Hospital mortality was 5.1% (1.3% after intermediate-risk and 8.4% after high-risk surgery). In the multivariate analysis, the age (OR 1.04, 95% CI 1.01-1.08, p = 0.013), smoking status (OR 2.46, 95% CI 1.16-5.21, p = 0.019), surgery risk (OR 2.92, 95% CI 1.05-8.08, p = 0.040), serum sodium level (OR 1.17, 95% CI 1.10-1.26, p < 0.001), urea (OR 1.01, 95% CI 1.01-1.02, p = 0.001) and leukocyte count (OR 1.05, 95% CI 1.01-1.10, p = 0.009) at admission were considered independent predictors. Hematocrit (0.86, 95% CI 0.80-0.93, p < 0.001) was considered an independent protective factor. The AUROC of our model was 0.860, compared to SAPS (0.752), APACHE (0.774), POSPOM (0.798) and POSSUM (0.829). CONCLUSION: The observed mortality was within the predicted range (1-5% after intermediate-risk and > 5% after high-risk surgery). POSSUM and POSPOM had slightly better predictive capacity than SAPS or APACHE.

Pre-Clinical Drug Testing in 2D and 3D Human In Vitro Models of Glioblastoma Incorporating Non-Neoplastic Astrocytes: Tunneling Nano Tubules and Mitochondrial Transfer Modulates Cell Behavior and Therapeutic Respons.

The role of astrocytes in the glioblastoma (GBM) microenvironment is poorly understood; particularly with regard to cell invasion and drug resistance. To assess this role of astrocytes in GBMs we established an all human 2D co-culture model and a 3D hyaluronic acid-gelatin based hydrogel model (HyStem™-HP) with different ratios of GBM cells to astrocytes. A contact co-culture of fluorescently labelled GBM cells and astrocytes showed that the latter promotes tumour growth and migration of GBM cells. Notably, the presence of non-neoplastic astrocytes in direct contact, even in low amounts in co-culture, elicited drug resistance in GBM. Recent studies showed that non-neoplastic cells can transfer mitochondria along tunneling nanotubes (TNT) and rescue damaged target cancer cells. In these studies, we explored TNT formation and mitochondrial transfer using 2D and 3D in vitro co-culture models of GBM and astrocytes. TNT formation occurs in glial fibrillary acidic protein (GFAP) positive "reactive" astrocytes after 48 h co-culture and the increase of TNT formations was greater in 3D hyaluronic acid-gelatin based hydrogel models. This study shows that human astrocytes in the tumour microenvironment, both in 2D and 3D in vitro co-culture models, could form TNT connections with GBM cells. We postulate that the association on TNT delivery non-neoplastic mitochondria via a TNT connection may be related to GBM drug response as well as proliferation and migration.

Assessment of main complications of regional anesthesia recorded in an acute pain unit in a tertiary care university hospital: a retrospective cohort.

BACKGROUND: Regional anesthesia has been increasingly used. Despite its low number of complications, they are associated with relevant morbidity. This study aims to evaluate the incidence of complications after neuraxial block and peripheral nerve block. METHODS: A retrospective cohort study was conducted, and data related to patients submitted to neuraxial block and peripheral nerve block at a tertiary university hospital from January 1, 2011 to December 31, 2017 were analyzed. RESULTS: From 10,838 patients referred to Acute Pain Unit, 1093(10.1%) had side effects or complications: 1039 (11.4%) submitted to neuraxial block and 54 (5.2%) to peripheral nerve block. The most common side effects after neuraxial block were sensory (48.5%) or motor deficits (11.8%), nausea or vomiting (17.5%) and pruritus (8.0%); The most common complications: 3 (0.03%) subcutaneous cell tissue hematoma, 3 (0.03%) epidural abscesses and 1 (0.01%) arachnoiditis. 204 of these patients presented sensory or motor deficits at hospital discharge and needed follow-up. Permanent peripheral nerve injury after neuraxial block had an incidence of 7.7:10,000 (0.08%). The most common side effects after peripheral nerve block were sensory deficits (52%) and 21 patients maintained follow-up due to symptoms persistence after hospital discharge. CONCLUSION: Although we found similar incidences of side effects or even lower than those described, major complications after neuraxial block had a higher incidence, particularly epidural abscesses. Despite this, other serious complications, such as spinal hematoma and permanent peripheral nerve injury, are still rare.

Syndapin-2 mediated transcytosis of amyloid-ß across the blood-brain barrier.

A deficient transport of amyloid-ß across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-ß efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-ß transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-ß clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-ß expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-ß clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-ß build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-ß assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-ß across the blood-brain barrier.

The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis.

Within the brain, endothelial cells lining the blood vessels meticulously coordinate the transport of nutrients, energy metabolites and other macromolecules essential in maintaining an appropriate activity of the brain. While small molecules are pumped across specialised molecular transporters, large macromolecular cargos are shuttled from one side to the other through membrane-bound carriers formed by endocytosis on one side, trafficked to the other side and released by exocytosis. Such a process is collectively known as transcytosis. The brain endothelium is recognised to possess an intricate vesicular endosomal network that mediates the transcellular transport of cargos from blood-to-brain and brain-to-blood. However, mounting evidence suggests that brain endothelial cells (BECs) employ a more direct route via tubular carriers for a fast and efficient transport from the blood to the brain. Here, we compile the mechanism of transcytosis in BECs, in which we highlight intracellular trafficking mediated by tubulation, and emphasise the possible role in transcytosis of the Bin/Amphiphysin/Rvs (BAR) proteins and glycocalyx (GC)-a layer of sugars covering BECs, in transcytosis. Both BAR proteins and the GC are intrinsically associated with cell membranes and involved in the modulation and shaping of these membranes. Hence, we aim to summarise the machinery involved in transcytosis in BECs and highlight an uncovered role of BAR proteins and the GC at the brain endothelium.

Exploring Poly(Ethylene Glycol)-Poly(Trimethylene Carbonate) Nanoparticles as Carriers of Hydrophobic Drugs to Modulate Osteoblastic Activity.

Current treatment options for bone-related disorders rely on a systemic administration of therapeutic agents that possess low solubility and intracellular bioavailability, as well as a high pharmacokinetic variability, which in turn lead to major off-target side effects. Hence, there is an unmet need of developing drug delivery systems that can improve the clinical efficacy of such therapeutic agents. Nanoparticle delivery systems might serve as promising carriers of hydrophobic molecules. Here, we propose 2 nanoparticle-based delivery systems based on monomethoxy poly(ethylene glycol)-poly(trimethyl carbonate) (mPEG-PTMC) and poly(lactide-co-glycolide) for the intracellular controlled release of a small hydrophobic drug (dexamethasone) to osteoblast cells in vitro. mPEG-PTMC self-assembles into stable nanoparticles in the absence of surfactant and shows a greater entrapment capacity of dexamethasone, while assuring bioactivity in MC3T3-E1 and bone marrow stromal cells cultured under apoptotic and osteogenic conditions, respectively. The mPEG-PTMC nanoparticles represent a potential vector for the intracellular delivery of hydrophobic drugs in the framework of bone-related diseases.

Designing peptide nanoparticles for efficient brain delivery.

The targeted delivery of therapeutic compounds to the brain is arguably the most significant open problem in drug delivery today. Nanoparticles (NPs) based on peptides and designed using the emerging principles of molecular engineering show enormous promise in overcoming many of the barriers to brain delivery faced by NPs made of more traditional materials. However, shortcomings in our understanding of peptide self-assembly and blood-brain barrier (BBB) transport mechanisms pose significant obstacles to progress in this area. In this review, we discuss recent work in engineering peptide nanocarriers for the delivery of therapeutic compounds to the brain: from synthesis, to self-assembly, to in vivo studies, as well as discussing in detail the biological hurdles that a nanoparticle must overcome to reach the brain.

Incidence, predictors and validation of risk scores to predict postoperative mortality after noncardiac vascular surgery, a prospective cohort study.

BACKGROUND: Noncardiac vascular surgery (VS) patients have comorbidities that increase the risk of death after surgery. Assessing that risk is important to allocate the necessary resources and improve quality of care. We aimed to evaluate the incidence and predictors of 30-day post-operative mortality (POM) after VS and compare the performance of existing risk scores. MATERIALS AND METHODS: Prospective cohort study including consecutive patients submitted to elective VS at a tertiary university hospital. We collected patients' demographics/perioperative data and calculated Surgical Apgar, age-adjusted Charlson Comorbidity Index (CCI), Vascular-Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity (V-POSSUM) and Preoperative Score to Predict Postoperative Mortality (POSPOM). We performed multivariate logistic regression to assess independent factors with Odds Ratio (OR) and 95% confidence interval (CI) calculation and Cox-regression for time-to-event analysis. We tested the predictive ability of the scores using the area under ROC curve (AUROC). RESULTS: POM was 6.2% (n = 19/306), not different from expected by V-POSSUM (6.5%) or POSPOM (5.6%). Post-operative myocardial infarction (MI) and acute kidney injury (AKI) were associated with higher POM (OR 4.8, p = 0.011 and OR 5.4, p = 0.001, respectively). On multivariate analysis, Chronic kidney disease (CKD) (OR 4.0, p = 0.021), Age (OR 1.1, p = 0.002), Peripheral arterial disease (PAD) (OR 8.0, p = 0.006), intra-operative red blood cells (RBC) Transfusion (OR 1.9, p < 0.001) and Atrial fibrillation (OR 8.4, p = 0.002) were considered independent predictors of POM (CAPTA score). The AUROC of our model was 0.882, better V-POSSUM (0.858), POSPOM (0.784), CCI (0.732) or Surgical Apgar (0.649). CONCLUSION: Observed POM was similar to predicted by V-POSSUM or POSPOM. Age, PAD, CKD, atrial fibrillation and intraoperative RBC transfusion were independent risk factors for POM. Score V-POSSUM performed better than POSPOM, CCI or Surgical Apgar.

On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias.

The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor-related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.

Major Cardiac Events in Patients Admitted to Intensive Care After Vascular Noncardiac Surgery: A Retrospective Cohort.

Introduction. Patients proposed to vascular noncardiac surgery (VS) have several comorbidities associated with major adverse cardiac events (MACE). We evaluated incidence, predictors, and outcomes, and compared different scores to predict MACE after VS. Methods. We included all patients admitted from 2006 to 2013. Perioperative MACE included cardiac arrhythmias, myocardial infarction (MI), cardiogenic pulmonary edema (CPE), acute heart failure (AHF), and cardiac arrest (CA). Lee Revised Cardiac Risk Index (RCRI), Vascular Quality Initiative (VQI-CRI), Vascular Study Group of New England (VSG-CRI), and South African Vascular Surgical (SAVS-CRI) Cardiac Risk Indexes were calculated and analyzed. We performed multivariate logistic regression to assess independent predictors with calculation of odds ratio (OR) and 95% confidence interval (CI). To reduce overfitting, we used leave-one-out cross-validation approach. The Predictive ability of scores was tested using area under receiver operating characteristic curve (AUROC). Results. A total of 928 patients were included. We observed 81 MACE (28 MI, 22 arrhythmias, 10 CPE, 9 AHF, 12 CA) in 60 patients (6.5%): 3.3% in intermediate-risk surgery and 9.8% in high-risk surgery. Previous history of coronary artery disease (OR = 3.2, CI = 1.8-5.7), atrial fibrillation (OR = 5.1, CI = 2.4-11.0), insulin-treated diabetes mellitus (OR = 3.26, CI = 1.51-7.06), mechanical ventilation (OR = 2.75, CI = 1.41-4.63), and heart rate (OR = 1.02, CI = 1.01-1.03) at admission were considered independent risk factors in multivariate analysis. The AUROC of our model was 0.79, compared with RCRI (0.66), VSG-CRI (0.69), VQI-CRI (0.71), and SAVS-CRI (0.73). Conclusions. Observed MACE were within predicted range (1% to 5% after intermediate-risk surgery and >5% after high-risk surgery). SAVS-CRI and VQI-CRI had slightly better predictive capacity than VSG-CRI or RCRI.

PAMAM dendrimers: blood-brain barrier transport and neuronal uptake after focal brain ischemia.

Drug delivery to the central nervous system is restricted by the blood-brain barrier (BBB). However, with the onset of stroke, the BBB becomes leaky, providing a window of opportunity to passively target the brain. Here, cationic poly(amido amine) (PAMAM) dendrimers of different generations were functionalized with poly(ethylene glycol) (PEG) to reduce cytotoxicity and prolong blood circulation half-life, aiming for a safe in vivo drug delivery system in a stroke scenario. Rhodamine B isothiocyanate (RITC) was covalently tethered to the dendrimer backbone and used as a small surrogate drug as well as for tracking purposes. The biocompatibility of PAMAM was markedly increased by PEGylation as a function of dendrimer generation and degree of functionalization. The PEGylated RITC-modified dendrimers did not affect the integrity of an in vitro BBB model. Additionally, the functionalized dendrimers remained safe when in contact with the bEnd.3 cells and rat primary astrocytes composing the in vitro BBB model after hypoxia induced by oxygen-glucose deprivation. Modification with PEG also decreased the interaction and uptake by endothelial cells of PAMAM, indicating that the transport across a leaky BBB due to focal brain ischemia would be facilitated. Next, the functionalized dendrimers were tested in contact with red blood cells showing no haemolysis for the PEGylated PAMAM, in contrast to the unmodified dendrimer. Interestingly, the PEG-modified dendrimers reduced blood clotting, which may be an added beneficial function in the context of stroke. The optimized PAMAM formulation was intravenously administered in mice after inducing permanent focal brain ischemia. Twenty-four hours after administration, dendrimers could be detected in the brain, including in neurons of the ischemic cortex. Our results suggest that the proposed formulation has the potential for becoming a successful delivery vector for therapeutic application to the injured brain after stroke reaching the ischemic neurons.

Assessment of main complications of regional anesthesia recorded in an acute pain unit in a tertiary care university hospital: a retrospective cohort

Abstract Background Regional anesthesia has been increasingly used. Despite its low number of complications, they are associated with relevant morbidity. This study aims to evaluate the incidence of complications after neuraxial block and peripheral nerve block. Methods A retrospective cohort study was conducted, and data related to patients submitted to neuraxial block and peripheral nerve block at a tertiary university hospital from January 1, 2011 to December 31, 2017 were analyzed. Results From 10,838 patients referred to Acute Pain Unit, 1093(10.1%) had side effects or complications: 1039 (11.4%) submitted to neuraxial block and 54 (5.2%) to peripheral nerve block. The most common side effects after neuraxial block were sensory (48.5%) or motor deficits (11.8%), nausea or vomiting (17.5%) and pruritus (8.0%); The most common complications: 3 (0.03%) subcutaneous cell tissue hematoma, 3 (0.03%) epidural abscesses and 1 (0.01%) arachnoiditis. 204 of these patients presented sensory or motor deficits at hospital discharge and needed follow-up. Permanent peripheral nerve injury after neuraxial block had an incidence of 7.7:10,000 (0.08%). The most common side effects after peripheral nerve block were sensory deficits (52%) and 21 patients maintained follow-up due to symptoms persistence after hospital discharge. Conclusion Although we found similar incidences of side effects or even lower than those described, major complications after neuraxial block had a higher incidence, particularly epidural abscesses. Despite this, other serious complications, such as spinal hematoma and permanent peripheral nerve injury, are still rare.

Peptide Self-Assemblies for Drug Delivery.

Peptide amphiphiles (PAs) are novel engineered biomaterials able to self-assemble into supramolecular systems that have shown significant promise in drug delivery across the cell membane and across challenging biological barriers showing promise in the field of brain diseases, regenerative medicine and cancer. PAs are amino-acid block co-polymers, with a peptide backbone composed usually of 8-30 amino acids, a hydrophilic block formed by polar amino acids, a hydrophobic block which usually entails either non-polar or aromatic amino acids and alkyl, acyl or aryl lipidic tails and in some cases a spacer or a conjugated targeting moiety. Finely tuning the balance between the hydrophilic and hydrophobic blocks results in a range of supramolecular structures that are usually stabilised by hydrophobic, electrostatic, ß-sheet hydrogen bonds and π-π stacking interactions. In an aqueous environment, the final size, shape and interfacial curvature of the PA is a result of the complex interplay of all these interactions. Lanreotide is the first PA to be licensed for the treatment of acromegaly and neuroendocrine tumours as a hydrogel administered subcutaneously, while a number of other PAs are undergoing preclinical development. This review discusses PAs architecture fundamentals that govern their self-assembly into supramolecular systems for applications in drug delivery.