Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model.

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway.

Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

Sevoflurane Impairs Growth Cone Motility in Dissociated Murine Neurons.

BACKGROUND: Early postnatal exposure to general anesthetic agents causes a lasting impairment in learning and memory in animal models. One hypothesis to explain this finding is that exposure to anesthetic agents during critical points in neural development disrupts the formation of brain circuitry. Here, we explore the effects of sevoflurane on the neuronal growth cone, a specialization at the growing end of axons and dendrites that is responsible for the targeted growth that underlies connectivity between neurons. METHODS: Dissociated neuronal cultures were prepared from embryonic mouse neocortex. Time-lapse images of live growth cones exposed to anesthetics were taken using differential interference contrast microscopy, and the rate of change of the area of the lamellipodia and the speed of the filopodial tip were quantified as measures of motility. The involvement of the p75 neurotropin receptor (p75NTR) was tested using inhibitors applied to the media and by a coimmunoprecipitation assay. RESULTS: The rate of lamellipodial area change and filopodial tip velocity in both axonal and dendritic growth cones was significantly reduced with sevoflurane exposure between 2% and 6%. Motility could be substantially restored by treatment with Y27632 and TAT-peptide 5, which are inhibitors of Rho Kinase and p75NTR, respectively. Sevoflurane results in reduced coimmunoprecipitation of Rho-Guanosine-5'-diphosphate dissociation inhibitor after pulldown with p75NTR. CONCLUSIONS: Sevoflurane interferes with growth cone motility, which is a critical process in brain circuitry formation. Our data suggest that this may occur through an action on the p75NTR, which promotes growth inhibitory signaling by the Rho pathway.

Effects of Neonatal Hypoxic-Ischemic Injury and Hypothermic Neuroprotection on Neural Progenitor Cells in the Mouse Hippocampus.

Neonatal hypoxic-ischemic injury (HI) results in widespread cerebral encephalopathy and affects structures that are essential for neurocognitive function, such as the hippocampus. The dentate gyrus contains a reservoir of neural stem and progenitor cells (NSPCs) that are critical for postnatal development and normal adult function of the hippocampus, and may also facilitate the recovery of function after injury. Using a neonatal mouse model of mild-to-moderate HI and immunohistochemical analysis of NSPC development markers, we asked whether these cells are vulnerable to HI and how they respond to both injury and hypothermic therapy. We found that cleaved caspase-3 labeling in the subgranular zone, where NSPCs are located, is increased by more than 30-fold after HI. The population of cells positive for both proliferating cell nuclear antigen and nestin (PCNA+Nes+), which represent primarily actively proliferating NSPCs, are acutely decreased by 68% after HI. The NSPC population expressing NeuroD1, a marker for NSPCs transitioning to become fate-committed neural progenitors, was decreased by 47%. One week after HI, there was a decrease in neuroblasts and immature neurons in the dentate gyrus, as measured by doublecortin (DCX) immunolabeling, and at the same time PCNA+Nes+ cell density was increased by 71%. NSPCs expressing Tbr2, which identifies a highly proliferative intermediate neural progenitor population, increased by 107%. Hypothermia treatment after HI partially rescues both the acute decrease in PCNA+Nes+ cell density at 1 day after injury and the chronic loss of DCX immunoreactivity and reduction in NeuroD1 cell density measured at 1 week after injury. Thus, we conclude that HI causes an acute loss of dentate gyrus NSPCs, and that hypothermia partially protects NSPCs from HI.

Parthenogenetic embryonic stem cells with H19 siRNA-mediated knockdown as a potential resource for cell therapy.

Embryonic stem (ES) cells are used in cell therapy and tissue engineering due to their ability to produce different cells types. However, studies of ES cells that are derived from fertilized embryos have raised concerns about the limitations imposed by ethical and political considerations. Therefore, many studies of stem cells use the stem cells that are derived from unfertilized oocytes and adult tissue. Although parthenogenetic embryonic stem (ESP) cells also avoid ethical and political dilemmas and can be used in cell-based therapy, the ESP cells exhibit growth retardation problems. Therefore, to investigate the potential for muscle growth from genetically modified ESP cells, we established four ES cell types, including normal embryonic stem (ESN) cells, ESP cells, ESP cells that overexpress the insulin-like growth factor 2 (Igf2) gene (ESI) and ESP cells with down-regulated H19 gene expression (ESH). Using these cells, we examined the expression profiles of genes that were related to imprinting and muscle using microarrays. The gene expression patterns of ESI and ESH cells were similar and were more closely related to the ESN pattern than that of the ESP cells. Differentiated ESH cells exhibited increased expression of bone morphologic protein 4 (BMP4), which is a mesoderm marker, compared with the differentiated ESI cells. We showed that Igf2 expression was induced by H19 silencing in the ESP cells via hypermethylation of the H19 imprinting control region 1 (ICR1). Moreover, the proportion of ESH-derived chimera was slightly higher than those produced from the ESP cells. In addition, we detected increased cell proliferation in the MEF cells following H19 knock-down. These results indicate that the ESH cells may be a source of cell-based therapy for conditions such as muscular atrophy.

A highly annotated whole-genome sequence of a Korean individual.

Recent advances in sequencing technologies have initiated an era of personal genome sequences. To date, human genome sequences have been reported for individuals with ancestry in three distinct geographical regions: a Yoruba African, two individuals of northwest European origin, and a person from China. Here we provide a highly annotated, whole-genome sequence for a Korean individual, known as AK1. The genome of AK1 was determined by an exacting, combined approach that included whole-genome shotgun sequencing (27.8x coverage), targeted bacterial artificial chromosome sequencing, and high-resolution comparative genomic hybridization using custom microarrays featuring more than 24 million probes. Alignment to the NCBI reference, a composite of several ethnic clades, disclosed nearly 3.45 million single nucleotide polymorphisms (SNPs), including 10,162 non-synonymous SNPs, and 170,202 deletion or insertion polymorphisms (indels). SNP and indel densities were strongly correlated genome-wide. Applying very conservative criteria yielded highly reliable copy number variants for clinical considerations. Potential medical phenotypes were annotated for non-synonymous SNPs, coding domain indels, and structural variants. The integration of several human whole-genome sequences derived from several ethnic groups will assist in understanding genetic ancestry, migration patterns and population bottlenecks.

Higher mitochondrial DNA copy number is associated with lower prevalence of microalbuminuria.

It has been suggested that mitochondrial dysfunction contributes to the initiation and development of atherosclerosis and cardiovascular disease. We examined the association between mitochondrial DNA (mtDNA) copy number and microalbuminuria in a cross-sectional community-based study. We measured peripheral blood mtDNA copy number in 694 adults without chronic kidney disease by a real-time PCR method. The overall prevalence of microalbuminuria (defined as an albumin creatinine ratio of 30 to 299 mg/g) was 4.5%. The prevalence of microalbuminuria decreased progressively from the lower to the upper quartiles of mtDNA copy number (6.9%, 5.7%, 2.9%, and 2.3% in quartiles 1, 2, 3, and 4, respectively, P=0.017 for trend). Multiple logistic regression models showed that the quartile of mtDNA copy number was independently associated with the prevalence of microalbuminuria (P=0.01 for trend). Compared with the lowest quartile, the highest quartile had an odds ratio of 0.22 for microalbuminuria (95% confidence interval, 0.05 to 0.87; P=0.03). Higher mtDNA copy number was associated with the lower prevalence of microalbuminuria in a community-based population.