Intrathecal delivery of protein therapeutics to the brain: A critical reassessment

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Mitochondrial DNA Variation in the Eastern Pondmussel, Ligumia nasuta (Bivalvia: Unionoida), in the Great Lakes Region

Most freshwater mussel species in the Great Lakes colonized the region from the Mississippi River basin and few appear to have colonized from Atlantic coast rivers. The Eastern Pondmussel, Ligumia nasuta, is widespread along the Atlantic coast but occurs elsewhere only in the Great Lakes, suggesting that it is one of the few Great Lakes species of Atlantic origin. Great Lakes populations are now imperiled following invasion of the lakes by dreissenid mussels. We examined patterns of diversity in the mitochondrial CO1 and ND1 genes in L. nasuta populations in the Great Lakes and in Atlantic coast rivers. Genetic diversity was low in Great Lakes populations and included only one CO1 and two ND1 haplotypes, all of which were also found in Atlantic coast populations. Genetic diversity was higher in Atlantic coast populations and included four CO1 and six ND1 haplotypes. Pairwise ФST revealed significant genetic differentiation for both genes between Atlantic coast and Great Lakes populations but not within Great Lakes populations. These results suggest that all populations of L. nasuta in the Great Lakes are derived from a single, small founder group that colonized from an Atlantic coast river. As such, Great Lakes populations may be considered a single management unit and conservation efforts based on propagation or translocation should be limited to use of Great Lakes source stock to prevent introduction of non-native haplotypes

Myocardial bridging does not predict sudden death in children with hypertrophic cardiomyopathy but is associated with more severe cardiac disease

AbstractOBJECTIVESWe sought to examine the association between systolic compression of sections of epicardial coronary vessels (myocardial bridging) with myocardial perfusion abnormalities and clinical outcome in children with hypertrophic cardiomyopathy (HCM).BACKGROUNDIt has recently been suggested that myocardial bridging is an important cause of myocardial ischemia and sudden death in children with HCM.METHODSAngiograms from 57 children with HCM were reviewed for the presence of bridging (50% or more maximum systolic arterial compression). QT interval indices, echocardiographic and cardiac catheterization findings, treadmill exercise tests, exercise thallium scintigraphy, Holter monitoring and electrophysiologic study findings were compared in children with and without bridging. The findings were also related to the presence or absence of compression of septal branches of the left anterior descending artery (LAD).RESULTSBridging was present in 23 (40%) of the children. Multiple coronary arteries were involved in four children. Bridging involved the LAD in 16 of 28 (57%) affected vessels. Myocardial perfusion abnormalities were present in 14 of 30 (47%) children without bridging and in 17 of 22 (94%) children with bridging, p = 0.002. However, bridging was associated with more severe septal hypertrophy (19 ± 8 mm vs. 28 ± 8 mm, p < 0.001), a higher septum:posterior wall thickness ratio (2.7 ± 1.2 vs. 1.8 ± 0.9, p < 0.001), and higher left ventricle (LV) outflow gradient (45 ± 37 mm Hg vs. 16 ± 28 mm Hg, p = 0.002). Compression of septal LAD branches was present in 37 (65%) of the children and was significantly associated with bridging, severity of LV hypertrophy and outflow obstruction. Multivariate analysis demonstrated that LV septal thickness and septal branch compression, and not bridging, were independent predictors of thallium perfusion abnormalities. There was a 90% power at 5% significance to detect an effect of bridging on thallium abnormalities at an odds ratio of 3. Bridging was also not associated with significantly greater symptoms, increased QT and QTc intervals and QTc dispersion, ventricular tachycardia on Holter or induced at EP study, or a worse prognosis.CONCLUSIONSBridging and compression of septal branches of the LAD are common in HCM children and are related to magnitude of LV hypertrophy. Left ventricular hypertrophy and compression of intramyocardial branches of the epicardial coronary arteries may contribute to myocardial perfusion abnormalities. Our findings suggest that bridging does not result in myocardial ischemia and may not cause arrhythmias or sudden death in HCM children

Identifying and understanding factors that affect the translation of therapies from the laboratory to patients: a study protocol

Background: The process of translating preclinical findings into a clinical setting takes decades. Previous studies have suggested that only 5-10% of the most promising preclinical studies are successfully translated into viable clinical applications. The underlying determinants of this low success rate (e.g. poor experimental design, suboptimal animal models, poor reporting) have not been examined in an empirical manner. Our study aims to determine the contemporary success rate of preclinical-to-clinical translation, and subsequently determine if an association between preclinical study design and translational success/failure exists. Methods: Established systematic review methodology will be used with regards to the literature search, article screening and study selection process. Preclinical, basic science studies published in high impact basic science journals between 1995 and 2015 will be included. Included studies will focus on publicly available interventions with potential clinical promise. The primary outcome will be successful clinical translation of promising therapies - defined as the conduct of at least one Phase II trial (or greater) with a positive finding. A case-control study will then be performed to evaluate the association between elements of preclinical study design and reporting and the likelihood of successful translation. Discussion: This study will provide a comprehensive analysis of the therapeutic translation from the laboratory bench to the bedside. Importantly, any association between factors of study design and the success of translation will be identified. These findings may inform future research teams attempting preclinical-to-clinical translation. Results will be disseminated to identified knowledge users that fund/support preclinical research

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach

Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123

P-glycoprotein (P-gp) expression at the blood-brain barrier prevents unwanted blood-borne toxins and signalling molecules from entering the brain. Primary and immortalised human brain endothelial cells (BECs) represent two suitable options for studying P-gp function in vitro. The limited supply of primary human BECs and their instability over passage number makes this choice unattractive for medium/high throughput studies. The aim of this study was to further characterise the expression of P-gp by an immortalised human BEC line, hCMEC/D3, in order to evaluate their use as an in vitro human blood-brain barrier model. P-gp expression was stable over a high passage number (up to passage 38) and was polarised on the apical plasma membrane, consistent with human BECs in vivo. In addition, hCMEC/D3 cell P-gp expression was comparable, albeit slightly lower to that observed in primary isolated human BECs although P-gp function was similar in both cell lines. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 (rh123) from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells also displayed polarised P-gp transport, since both tariquidar and vinblasine selectively increased the apical-to-basolateral permeability of hCMEC/D3 cells to rh123. The results presented here demonstrate that hCMEC/D3 cells are a suitable model to investigate substrate specificity of P-gp in BECs of human origin

Mitochondria, Energetics, Epigenetics, and Cellular Responses to Stress

Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria–nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation. Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function. Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25–26 March 2013. Here, we summarize key points and ideas emerging from this meeting. Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways. Conclusions: Understanding mitochondria–cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors. Citation: Shaughnessy DT, McAllister K, Worth L, Haugen AC, Meyer JN, Domann FE, Van Houten B, Mostoslavsky R, Bultman SJ, Baccarelli AA, Begley TJ, Sobol RW, Hirschey MD, Ideker T, Santos JH, Copeland WC, Tice RR, Balshaw DM, Tyson FL. 2014. Mitochondria, energetics, epigenetics, and cellular responses to stress. Environ Health Perspect 122:1271–1278; http://dx.doi.org/10.1289/ehp.140841

Transcriptional control of the multi-drug transporter ABCB1 by transcription factor Sp3 in different human tissues

The ATP-binding cassette (ABC) transporter ABCB1, encoded by the multidrug resistance gene MDR1, is expressed on brain microvascular endothelium and several types of epithelium, but not on endothelia outside the CNS. It is an essential component of the blood-brain barrier. The aim of this study was to identify cell-specific controls on the transcription of MDR1 in human brain endothelium. Reporter assays identified a region of 500bp around the transcription start site that was optimally active in brain endothelium. Chromatin immunoprecipitation identified Sp3 and TFIID associated with this region and EMSA (electrophoretic mobility shift assays) confirmed that Sp3 binds preferentially to an Sp-target site (GC-box) on the MDR1 promoter in brain endothelium. This result contrasts with findings in other cell types and with the colon carcinoma line Caco-2, in which Sp1 preferentially associates with the MDR1 promoter. Differences in MDR1 transcriptional control between brain endothelium and Caco-2 could not be explained by the relative abundance of Sp1:Sp3 nor by the ratio of Sp3 variants, because activating variants of Sp3 were present in both cell types. However differential binding of other transcription factors was also detected in two additional upstream regions of the MDR1 promoter. Identification of cell-specific controls on the transcription of MDR1 indicates that it may be possible to modulate multi-drug resistance on tumours, while leaving the blood brain barrier intact