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In Norway, genetic testing is permitted from the age of 16, and females identified with pathogenic variants in the BRCA1 or BRCA2 genes (BRCA PVs) can manage their cancer risk through screening programs and/or by undergoing risk-reducing surgery from the age of 25 years'. This qualitative study aimed to explore and describe how women under the age of 25 years' experience the genetic counseling and testing process for BRCA PVs and living with an increased cancer risk. Semi-structured individual interviews were conducted with five women with BRCA PVs. Thematic cross-case data analysis with systematic text condensation was used. Three main themes were identified: (1) experience with genetic counseling and testing, (2) impact of pathogenic variants on participants' future, and (3) social and psychological support needs. Women perceive genetic counseling as a source of information and care. The decision for genetic testing was made autonomously, both in line with and conflicting with the parents' wishes. Living with genetic risk adds a layer of worry and pressure regarding future decisions. Many experienced loneliness owing to a lack of contact with other young women with BRCA PVs. The results of this study indicate the need for better support after genetic testing, such as the need for educational support groups specially designed for these young women and a meeting place with their peers. Genetic counselors need to emphasize the opportunity for follow-up counseling and give assistance to choose suitable psychologists., (© 2024 The Author(s). Journal of Genetic Counseling published by Wiley Periodicals LLC on behalf of National Society of Genetic Counselors.)

Background/objectives: Iron (Fe) is a co-factor for enzymes of the developing brain necessitating sufficient supply. We investigated the effects of administering ferric derisomaltose/Fe isomaltoside (FDI) subcutaneously to Fe-deficient (ID) pregnant rats on cerebral and hepatic concentrations of essential metals and the expression of iron-relevant genes., Methods: Pregnant rats subjected to ID were injected with FDI on the day of mating (E0), 14 days into pregnancy (E14), or the day of birth (postnatal (P0)). The efficacy was evaluated by determination of cerebral and hepatic Fe, copper (Cu), and zinc (Zn) and gene expression of ferroportin, hepcidin, and ferritin H + L in pups on P0 and as adults on P70., Results: Females fed an ID diet (5.2 mg/kg Fe) had offspring with significantly lower cerebral and hepatic Fe compared to female controls fed a standard diet (158 mg/kg Fe). Cerebral Cu increased irrespective of supplying a standard diet or administering FDI combined with the standard diet. Hepatic hepcidin mRNA was significantly lower following ID. Cerebral hepcidin mRNA was hardly detectable irrespective of iron status., Conclusions: In conclusion, administering FDI subcutaneously to ID pregnant rats on E0 normalizes fetal cerebral and hepatic Fe. When applied at later gestational ages, supplementation with additional Fe to the offspring is needed to normalize cerebral and hepatic Fe.

Background: Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood-brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA)., Methods: Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS., Results: In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression., Conclusions: VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1.

After the CLARITY-AD clinical trial results of lecanemab were interpreted as positive, and supporting the amyloid hypothesis, the drug received accelerated Food and Drug Administration approval. However, we argue that benefits of lecanemab treatment are uncertain and may yield net harm for some patients, and that the data do not support the amyloid hypothesis. We note potential biases from inclusion, unblinding, dropouts, and other issues. Given substantial adverse effects and subgroup heterogeneity, we conclude that lecanemab's efficacy is not clinically meaningful, consistent with numerous analyses suggesting that amyloid-β and its derivatives are not the main causative agents of Alzheimer's disease dementia.

The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for the delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin receptor antibodies, lowering the affinity of the targeting antibodies or implementing monovalent antibodies increase uptake by BECs and allows for further transport across the BBB. The novel demonstration of transport of targeted liposomes in post-capillary venules from blood to the brain is interesting and clearly warrants further mechanistic pursuit. The recent evidence for passing targeted nanoparticles through the BBB shows great promise for future drug delivery of biologics to the brain.

Brain homeostasis depends on the existence of the blood-brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB's tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens-1, VE-cadherin and β-catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho-ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild-type and Sort1-/- brain endothelial cells can pave the way for a better understanding of sortilin's role in the healthy and diseased BBB., (© 2021 Federation of European Biochemical Societies.)

Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain.

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor angioarchitecture in large three-dimensional datasets from optically cleared samples. This methodology provides a precise and comprehensive analysis of extravasation in brain tumors and allows for correlation of extravasation patterns with specific features of the heterogeneous brain tumor vasculature.

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain., Competing Interests: Conflict of interest disclosure The authors declare no competing interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity and the rapid development of chemoresistance. In the brain, this is further hampered by the blood-brain barrier that reduces passive diffusion of drugs to a minimum. Tumors grow invasively and form new blood vessels, also in brain tissue where remodeling of pre-existing vasculature is substantial. The cancer-associated vessels in the brain are considered leaky and thus could facilitate the transport of chemotherapeutic agents. Yet, brain tumors are extremely difficult to treat, and, in this review, we will address how different aspects of the vasculature in brain tumors contribute to this.

Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter- and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

Exosomes were first described as waste carriers implicated in reticulocyte maturation but has during the past decade been associated with many other cellular functions. The biogenesis of exosomes has been extensively studied and several protein machineries have been identified to dictate their production and release. The newly discovered branches of the autophagy system implicate secretion of waste in endosomal-derived vesicles as is thought for exosome release. Many of the proteins that have been identified as responsible for the formation and release of these vesicles are the same as those identified in exosome biogenesis. In this Perspective, we discuss the possibility of exosomes being a part of the autophagy machinery and the consequences this could have on interpretation of exosome functions.

Obtaining efficient drug delivery to the brain remains the biggest challenge for the development of therapeutics to treat diseases of the central nervous system. The main obstacle is the blood-brain barrier (BBB), which impedes the entrance of most molecules present in the systemic circulation, especially large molecule drugs and nanomedicines. To overcome this obstacle, targeting strategies binding to nutrient receptors present at the luminal membrane of the BBB are frequently employed. Amongst the numerous potential targets at the BBB, the transferrin receptor (TfR) remains the most common target used to ensure sufficient drug delivery to the brain. In this review, we provide a full account on the use of the TfR as a target for brain drug delivery by describing the function of the TfR in the BBB, the historical background of its use in drug delivery, and the most recent evidence suggesting TfR-targeted medicines to be efficient for brain drug delivery with a clear clinical potential., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The idea of using extracellular vesicles (EVs) for targeted drug delivery was first introduced in 2011 and has since then gained increasing attention as promising new candidates in the field. Targeting EVs to areas of disease can be achieved through a complex process of designing and inserting a targeting ligand to the surface of the EVs. Although this can be obtained via chemical conjugation, the most important strategy has been to transfect or modulate the EV-producing cell to endow the EVs with the desired targeting capabilities. However, since EVs are harvested from biological sources, their composition is highly heterogeneous, which makes it difficult to control the purity and quality of the resulting EV-based drug delivery vehicles. In this review, we present a detailed account of EVs in targeted drug delivery based on a systematic literature search. We discuss the potential advantages of EVs compared to synthetic lipid-based nanocarriers, and the methodological and biological limitations associated with their use as targeted drug delivery vehicles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 10 3 antibodies/μm 2 ), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over- or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10 10 EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery., Competing Interests: Competing interests: All authors have declared no conflicting interest in relation to the theme and contents of the manuscript. The anti-TfRA, anti-TfRD, and anti-TfRA/BACE1 antibodies were obtained from Genentech, Inc. via a Material Transfer Agreement (MTA A15783). Genentech, Inc. had no influence on study design, experimental procedures, data analysis, or preparation of the manuscript.

The field of extracellular vesicle (EV)-based drug delivery systems has evolved significantly through the recent years, and numerous studies suggest that these endogenous nanoparticles can function as efficient drug delivery vehicles in a variety of diseases. Many characteristics of these EV-based drug delivery vehicles suggest them to be superior at residing in the systemic circulation and possibly at mediating therapeutic effects compared to synthetic drug delivery vehicles, e.g. liposomes. In this Commentary, we discuss how some currently published head-to-head comparisons of EVs versus liposomes are weakened by the inadequate choice of liposomal formulation, and encourage researchers to implement better controls to show any potential superiority of EVs over other synthetic nanoparticles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain.

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

In the recent years, the possibility of utilizing extracellular vesicles for drug delivery purposes has been investigated in various models, suggesting that these vesicles may have such potential. In addition to the choice of donor cell type for vesicle production, a major obstacle still exists with respect of loading the extracellular vesicles efficiently with the drug of choice. One of the proposed solutions to this problem has been drug loading by electroporation, where small pores are created in the membrane of the extracellular vesicles, hereby allowing for free diffusion of the drug compound into the interior of the vesicle. We investigated the utility of adipose-derived stem cells (ASCs) as an efficient exosome donor cell type with a particular focus on the treatment of glioblastoma multiforme (GBM). In addition, we evaluated electroporation-induced effects on the ASC exosomes with respect to their endogenous potential of stimulating GBM proliferation, and morphological changes to single and multiple ASC exosomes. We found that electroporation does not change the endogenous stimulatory capacity of ASC exosomes on GBM cell proliferation, but mediates adverse morphological changes including aggregation of the exosomes. In order to address this issue, we have successfully optimized the use of a trehalose-containing buffer system as a way of maintaining the structural integrity of the exosomes.

The potential therapeutic utility of extracellular vesicles (EVs) has spawned an interest into a scalable production, where the quantity and purity of EV samples is sufficient for clinical applications. EVs can be isolated using several different protocols; however, these isolation protocols and the subsequent methods of quantifying the resulting EV yield have not been sufficiently standardized. Therefore, the possibility of comparing different studies with respect to these parameters is limited. In this review, we have presented factors that might influence the yield and function of EVs from cell culture supernatants. The methods of isolation, downstream quantification, and culture conditions of the EV producing cells have been discussed. In order to examine the inter-study coherency of EV yields, 259 studies were initially screened, and 46 studies were included for extensive downstream analysis of EV yields where information pertaining to the isolation protocols and quantification methods was obtained from each study. Several other factors influencing yield were compared, such as cell type producing EVs, cell confluence level, and cell stimulation. In conclusion, various factors may impact the resulting EV yield, including technical aspects such as EV isolation and quantification procedures, and biological aspects such as cell type and culture conditions. The reflections presented in this review might aid in future standardization of the workflow in EV research.

An unmet need exists for therapeutic compounds to traverse the brain capillary endothelial cells that denote the blood-brain barrier (BBB) to deliver effective treatment to the diseased brain. The use of nanoparticle technology for targeted delivery to the brain implies that targeted liposomes encapsulating a drug of interest will undergo receptor-mediated uptake and transport through the BBB with a subsequent unfolding of the liposomal content inside the brain, hence revealing drug release to adjacent drug-demanding neurons. As transferrin receptors (TfRs) are present on brain capillary endothelial, but not on endothelial cells elsewhere in the body, the use of TfR-targeted liposomes - colloidal particulates with a phospholipid bilayer membrane - remains the most relevant strategy to obtain efficient drug delivery to the brain. However, many studies have failed to provide sufficient quantitative data to proof passage of the BBB and significant appearance of drugs inside the brain parenchyma. Here, we critically evaluate the current evidence on the use of TfR-targeted liposomes for brain drug delivery based on a thorough investigation of all available studies within this research field. We focus on issues with respect to experimental design and data analysis that may provide an explanation to conflicting reports, and we discuss possible explanations for the current lack of sufficient transcytosis across the BBB for implementation in the design of TfR-targeted liposomes. We finally provide a list of suggestions for strategies to obtain substantial uptake and transport of drug carriers at the BBB with a concomitant transport of therapeutics into the brain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This review presents the present-day literature on the anatomy and physiological mechanisms of the blood-brain barrier and the problematic of cerebral drug delivery in relation to malignant brain tumors. First step in treatment of malignant brain tumors is resection, but there is a high risk of single remnant infiltrative tumor cells in the outer zone of the brain tumor. These infiltrative single-cells will be supplied by capillaries with an intact BBB as opposed to the partly leaky BBB found in the tumor tissue before resection. Even though BBB penetrance of a chemotherapeutic agent is considered irrelevant though the limited success rate for chemotherapeutic treatability of GBM tumors indicate otherwise. Therefore drug delivery strategies to cerebral cancer after resection should be tailored to being able to both penetrate the intact BBB and target the cancer cells. In this review the intact bloodbrain barrier and cerebral cancer with main focus on glioblastoma multiforme (GBM) is introduced. The GBM induced formation of a blood-tumor barrier and the consequences hereof is described and discussed with emphasis on the impact these changes of the BBB has on drug delivery to GBM. The most commonly used drug carriers for drug delivery to GBM is described and the current drug delivery strategies for glioblastoma multiforme including possible routes through the BBB and epitopes, which can be targeted on the GBM cells is outlined. Overall, this review aims to address targeted drug delivery in GBM treatment when taking the differing permeability of the BBB into consideration.

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

Despite advances in our knowledge about glioblastoma multiforme (GBM) pathology, clinical challenges still lie ahead with respect to treatment in GBM due to high prevalence, poor prognosis, and frequent tumor relapse. The implication of microRNAs (miRNAs) in GBM is a rapidly expanding field of research with the aim to develop more targeted molecular therapies. This review aims to present a comprehensive overview of all the available literature, evaluating miRNA signatures as a function of prognosis and survival in GBM. The results are presented with a focus on studies derived from clinical data in databases and independent tissue cohorts where smaller samples sizes were investigated. Here, miRNA associated to longer survival (protective) and miRNA with shorter survival (risk-associated) have been identified and their signatures based on different prognostic attributes are described. Finally, miRNAs associated with disease progression or survival in several studies are identified and functionally described. These miRNAs may be valuable for future determination of patient prognosis and could possibly serve as targets for miRNA-based therapies, which hold a great potential in the treatment of this severe malignant disease.

The increasing interest in identifying molecular biomarkers to determine patient prognosis in glioblastoma multiforme (GBM) has resulted in several microRNA (miRNA)-based signatures able to predict progression-free and overall survival. However, the coherency between these signatures is small, and correlations to clinicopathological features other than survival are seldom seen. The aim of this study was to identify any significant relationship between miRNA signatures and clinicopathological data by combining pathological features with miRNA and mRNA analysis in fourteen GBM patients. In total, 161 miRNAs were shown to cluster the GBM tumor samples into long- and short-term-surviving patients. Many of these miRNAs were associated with differential expression in GBM, including a number of miRNAs shown to confer risk or protection with respect to clinical outcome and to modulate the mesenchymal mode of migration and invasion. An inverse relationship between miR-125b and nestin expression was identified and correlated with overall survival in GBM patients, eloquently illustrating how clinicopathological findings and molecular profiling may be a relevant combination to predict patient outcome. The intriguing finding that many of the differentially expressed miRNAs contained exosome-packaging motifs in their mature sequences suggests that we must expand our view to encompass the complex intercellular communication in order to identify molecular prognostic biomarkers and to increase our knowledge in the field of GBM pathogenesis.

Exosomes denote a class of secreted nanoparticles defined by size, surface protein and lipid composition, and the ability to carry RNA and proteins. They are important mediators of intercellular communication and regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest them to be important both for diagnostic and therapeutic purposes, prompting the idea of using exosomes as drug delivery vehicles, especially for gene therapy. This review covers the current status of evidence presented in the field of exosome-based drug delivery systems. Components for successful exosome-based drug delivery, such as choice of donor cell, therapeutic cargo, use of targeting peptide, loading method and administration route are highlighted and discussed with a general focus pertaining to the results obtained in models of different cancer types. In addition, completed and on-going clinical trials are described, evaluating exosome-based therapies for the treatment of different cancer types. Due to their endogenous origin, exosome-based drug delivery systems may have advantages in the treatment of cancer, but their design needs further refinement to justify their usage on the clinical scale., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Neurodegenerative disorders are characterized by the presence of inflammation in areas with neuronal cell death and a regional increase in iron that exceeds what occurs during normal aging. The inflammatory process accompanying the neuronal degeneration involves glial cells of the central nervous system (CNS) and monocytes of the circulation that migrate into the CNS while transforming into phagocytic macrophages. This review outlines the possible mechanisms responsible for deposition of iron in neurodegenerative disorders with a main emphasis on how iron-containing monocytes may migrate into the CNS, transform into macrophages, and die out subsequently to their phagocytosis of damaged and dying neuronal cells. The dying macrophages may in turn release their iron, which enters the pool of labile iron to catalytically promote formation of free-radical-mediated stress and oxidative damage to adjacent cells, including neurons. Healthy neurons may also chronically acquire iron from the extracellular space as another principle mechanism for oxidative stress-mediated damage. Pharmacological handling of monocyte migration into the CNS combined with chelators that neutralize the effects of extracellular iron occurring due to the release from dying macrophages as well as intraneuronal chelation may denote good possibilities for reducing the deleterious consequences of iron deposition in the CNS.

Glioblastoma multiforme (GBM) is an incurable form of brain cancer with a very poor prognosis. Because of its highly invasive nature, it is impossible to remove all tumor cells during surgical resection, making relapse inevitable. Further research into the regulatory mechanism underpinning GBM pathogenesis is therefore warranted, and over the past decade, there has been an increased focus on the functional role of microRNA (miRNA). This systematic review aims to present a comprehensive overview of all the available literature on the expression profiles and function of miRNA in GBM. Here, we have reviewed 163 papers and identified 253 upregulated, 95 downregulated, and 17 disputed miRNAs with respect to expression levels; 85 % of these miRNAs have not yet been functionally characterized. A focus in this study has been 26 interesting miRNAs involved in the mesenchymal mode of migration and invasion, demonstrating the importance of miRNAs in the context of the cellular niche. Both oncogenic and tumor-suppressive miRNAs were found to affect target genes involved in cell migration, cytoskeletal rearrangement, invasiveness, and angiogenesis. Clearly, the distinct functional properties of these miRNAs need further investigation and might hold a great potential in future molecular therapies targeting GBM.

Background: The prevalence of type 2 diabetes is increasing, but the exact prevalence of the disease and its accompanying late complications are unknown. In the Anglo-Danish-Dutch study of Intensive Treatment in People with Screen-detected Diabetes in Primary Care (ADDITION study), patients with hitherto undiagnosed type 2 diabetes are identified using a stepwise screening strategy in selected general practices. This article reports the occurrence of diabetic retinopathy in this population., Methods: In Arhus and Copenhagen counties, a total of 12,708 of the persons invited by mail were screened for diabetes mellitus. Consequently, 763 persons with type 2 diabetes were identified; 670 of these (335 from each of the two centres) underwent a general physical examination (including measurement of blood pressure and HbA1c) and an ophthalmological examination (including measurement of visual acuity and fundus photography). Retinopathy was graded from the photographs by counting all retinopathy lesions., Results: Forty-five (6.8%) of the examined patients had any retinopathy, of which the majority was minimal. No patients had severe non-proliferative or proliferative diabetic retinopathy. There was no significant difference between age, sex and visual acuity among patients with and without retinopathy. However, the patients with retinopathy had significantly higher HbA1c and systolic and diastolic blood pressure than the patients without retinopathy., Conclusion: Patients with screen-detected diabetes have a low prevalence of diabetic retinopathy and no vision-threatening lesions. Screening for diabetic retinopathy should be focused on those patients who have already been diagnosed with type 2 diabetes during routine clinical practice.