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Patient and providers’ fear of fetal exposure to medications may lead to discontinuation of treatment, disease relapse, and maternal morbidity. Placental drug transporters play a critical role in fetal exposure through active transport but the majority of data are limited to the 3rd trimester, when the majority of organogenesis has already occurred. Our objective was to define gestational age (GA) dependent changes in protein activity, expression and modifications of five major placental drug transporters: SERT, P-gp, NET, BCRP and MRP3. Apical brush border membrane fractions were prepared from fresh 1st, 2nd and 3rd trimester human placentas collected following elective pregnancy termination or planned cesarean delivery. A structured maternal questionnaire was used to identify maternal drug use and exclude exposed subjects. Changes in placental transporter activity and expression relative to housekeeping proteins were quantified. There was evidence for strong developmental regulation of SERT, NET, P-gp, BCRP and MRP3. P-gp and BCRP decreased with gestation (r = −0.72, p < 0.001 and r = −0.77, p < 0.001, respectively). Total SERT increased with gestation but this increase was due to a decrease in SERT cleavage products across trimesters. Uncleaved SERT increased with GA (r = 0.89, p < 0.001) while cleaved SERT decreased with GA (r = −0.94, p < 0.001). Apical membrane NET overall did not appear to be developmentally regulated (r = −0.08, p = 0.53). Two forms of MRP3 were identified; the 50 kD form did not change across GA; the 160 kD form was steady in the 1st and 2nd trimester and increased in the 3rd trimester (r = 0.24, p = 0.02). The 50 kD form was expressed at higher levels. The observed patterns of SERT, NET P-gp, BCRP and MRP3 expression and activity may be associated with transporter activity or decreased placental permeability in the 1st trimester to transporter specific substrates including commonly used psychoactive medications such as anti-depressants, anti-psychotics, and amphetamines, while transport of nutrients and serotonin is important in the 1st trimester. Overall these observations are consistent with a strong protective effect during organogenesis. 3rd trimester estimates of fetal exposure obtained from cord blood likely significantly overestimate early fetal exposure to these medications at any fixed maternal dose.

Introduction: Extracellular vesicles derived from the fetal central nervous system (FCNSEs) can be purified from maternal serum or plasma using the protein Contactin-2/TAG1that is expressed almost exclusively by developing neurons in the hippocampus, cerebral cortex and cerebellum. We hypothesized that fetal CNSEs could be used to non-invasively detect and quantify viral mediated in-utero brain injury in the first trimester.Materials and Methods: First trimester maternal samples were collected from a human clinical population infected with primary cytomegalovirus (CMV) and a non-human primate model of Zika (ZIKV) infection. In the CMV cohort, a nested case control study was performed comparing pregnancies with and without fetal infection. Cases of fetal infection were further subdivided into those with and without adverse neurologic outcome. ZIKV samples were collected serially following maternal inoculation or saline. All ZIKV cases had histopathologic findings on necropsy. Serum was precipitated with ExoQuick solution and FCEs were isolated with biotinylated anti-Contactin-2/TAG1 antibody-streptavidin matrix immunoabsorption. FCE Synaptopodin (SYNPO) and Neurogranin (NG) protein levels were measured using standard ELISA kits and normalized to the exosome marker CD81.Results: Fetal CNSE SYNPO and NG were significantly reduced in cases of first trimester fetal CMV infection compared to those with infection limited to the mother but could not discriminate between fetal infection with and without adverse neurologic outcome. Following ZIKV inoculation, fetal CNSE SYNPO was reduced by 48 h and significantly reduced by day 4.Discussion: These data are the first to suggest that first trimester non-invasive diagnosis of fetal viral infection is possible. Fetal CNSEs have the potential to augment clinical and pre-clinical studies of perinatal viral infection. Serial sampling may be needed to discriminate between fetuses that are responding to treatment and/or recovering due to innate defenses and those that have ongoing neuronal injury. If confirmed, this technology may advance the paradigm of first trimester prenatal diagnosis and change the calculus for the cost benefit of CMV surveillance programs in pregnancy.

OBJECTIVE We have developed novel methods for isolating fetal central nervous system (CNS)-derived extracellular vesicles (FCEs) from maternal plasma as a non-invasive platform for testing aspects of fetal neurodevelopment in early pregnancy. We investigate the hypothesis that levels of defined sets of functional proteins in FCEs can be used to detect abnormalities in fetal neuronal and glial proliferation, differentiation, and survival. METHOD Maternal plasma was obtained between 10 and 19 weeks from women with current heavy EtOH exposure and matched controls. FCE levels of synaptophysin, synaptotagmin, synaptopodin, and neurogranin were quantified normalized to the exosome marker CD81. Quantitative RT-PCR was performed with specific primers for miR-9. RESULTS FCE cargo protein levels of synaptophysin, synaptotagmin, synaptopodin, and neurogranin were all significantly reduced in pregnancies exposed to current heavy EtOH use (P

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.

Introduction Novel plant DING proteins (full-length 38 kDa p38SJ, and 27 kDa p27SJ) exhibit phosphatase activity and modulate HIV-1 gene transcription. Previously, we demonstrated that DING regulates HIV-1 gene transcription by dephosphorylation and inactivation of CTD RNA polymerase II, the major elongating factor of HIV-1 Long Terminal Repeats (LTR). Because the transcription of HIV-1 is controlled by several viral and cellular factors, including p65/p50 subunits of NF-κB, we hypothesized that DING phosphatase can also affect the phosphorylation and activity of p65 NF-κB, in addition to C-terminal Domain (CTD) of RNA Polymerase II (RNAPII), to suppress HIV-1 gene transcription and inhibit HIV-1 infection. Methods Here, we describe the inhibition of HIV-1 infection and the p65/p50 NF-κB phosphorylation by DING protein, analyzed by ELISA and northern-blot assays, western-blot assays, cell fractionation, and promoter-reporter assays in DING-expressing cells, using a pTet-on inducible system. Results Results from HIV-1 infection assays demonstrate a strong inhibition of HIV-1 and HIV-LTR RNA expression by DING protein, determined by p24 ELISA and by northern blot assay. Results from the western blot assays and cell fractionation assays show that there is an increase in the level of hypo-phosphorylated form of p65 NF-κB in DING-expressing cells. Both fractions of p65/p50, nuclear or cytoplasmic, are affected by DING phosphatase, but more cytoplasmic accumulation of p65 NF-κB was found in the presence of DING, suggesting that subsequent activation and nuclear import of active NF-κB is affected by DING. The major portion of nuclear p65 was dephosphorylated in DING-expressing cells. The promoter-reporter assay demonstrated that DING-mediated dephosphorylation and dysregulation of NF-κB p65 lead to the suppression of its binding to HIV-1 LTR, and resulted in the inhibition of p65-mediated activation of LTR transcription. Mapping of the region within LTR that was affected by DING revealed that both, NF-κB and CTD RNA Polymerase II binding sites were important, and cooperativity of these cellular factors was diminished by DING. In addition, mapping of the region within DING-p38SJ that affected LTR transcription, revealed that phosphate-binding domain is essential for this inhibitory activity. Conclusion We have demonstrated the effect of DING phosphatases on HIV-1 infection, phosphorylation of p65 NF-κB, and transcription of HIV-1 LTR. Our studies suggest that one possible mechanism by which DING can regulate the expression of HIV-1 LTR can be through dysregulation of the transcription factor NF-κB p65 by preventing its phosphorylation and translocation to the nucleus and binding to the HIV-1 LTR, an action that could contribute to the utility of DING p38SJ as an antiviral agent. Importantly, DING not only inhibits HIV-1 LTR gene transcription in the presence of increased p65 NF-κB, but also suppresses HIV-1 infection. DING protein improved inhibitory effects of the known anti-retroviral drugs, Tenofovir (TFV) and Emtricitabine (FTS) on HIV-1, since in the combination with these drugs; the suppression of HIV-1 by DNG was significantly higher when it was in combination with these drugs, compared to controls or cases without DING. Thus, our data support the use of neuroprotective DING proteins as novel therapeutic antiviral drugs that suppress HIV-1 LTR transcription by interfering with the function of NF-κB.

Introduction Mitochondrial dysregulation is a key event in HIV-1 infection. Recent studies have suggested that age-related neurodegenerative disorders are associated with increased mitochondrial DNA (mtDNA) damage. As accelerated ageing was found in HIV-1 patients, we hypothesized that HIV-1 infection or HIV-1 proteins can lead to mtDNA damage. Unrepaired mtDNA impairs mitochondrial function, which can lead to oxidative stress and cell death. Investigations of mechanisms of mtDNA damage are limited by the lack of available human models. Methods We compared mtDNA or nDNA (nuclear DNA) damage in human cortical neurons and PBMC cells. Primary neuronal cultures were incubated with conditioned media from HIV-1 infected PBMC, or HIV-1 viral proteins Tat or Vpr. Total genomic DNA (nuclear and mtDNA) was isolated using the QIAamp Kit. Nuclear and mtDNA were amplified using the long q-PCR/Gene Amp XL Kit. Real-Time RT-PCR using mitochondrial energy metabolism array was performed to assess mitochondrial energy metabolism markers. Superoxide dismutase (SOD) activity in neuronal cells was measured by the OxiSelect SOD Activity Assay. Reactive oxygen species (ROS) were determined by the confocal microscopy. ATP levels were analyzed using ATP determination biochemical assay. Mitochondrial, cytoplasmic and nuclear proteins were studied by quantitative western-blot assay. Results We show that both treatment of neuronal cells with HIV-1 conditioned media, or infection of PBMC with HIV-1 increase mtDNA damage in cells. mtDNA damage was also seen in neuronal cells, incubated with HIV-1 proteins, Tat and Vpr. Next, we confirmed that mtDNA damage was also increased in neuronal cells transfected by Tat expressing plasmids. We showed that mtDNA was not damaged in neuronal cells following treatment with heat inactivated HIV-1 or Tat protein. Further, we demonstrated that HIV-1 or Tat caused more mtDNA damage compared to nuclear DNA damage in neuronal cells. Finally, we showed that Tat dysregulates RNA expression of several genes regulating mitochondrial energy metabolism, suggesting involvement of Tat in mitochondrial bioenergetics in human neurons. Finally, our hypothesis was confirmed by qWestern analysis of mitochondrial and apoptotic proteins demonstrating the accumulation of apoptotic Bax and Bad proteins in mitochondrial fraction of Tat-treated neuronal cells, suggesting toxic effects of Tat on mitochondrial survival. Conclusion We showed an increase of mtDNA damage in primary neurons, treated with HIV-1 proteins and in PBMC, infected with HIV-1. Increased mtDNA damage can lead to neurodegeneration, and cause neuronal apoptosis. Our system presents a suitable model to study mtDNA changes during HIV-1 infection.

The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O 2 •- ) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O 2 •- levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ m was lower, mitochondrial O 2 •- levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2 •- levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.

Maternal opioid use disorder is common, resulting in significant neonatal morbidity and cost. Currently, it is not possible to predict which opioid-exposed newborns will require pharmacotherapy for neonatal abstinence syndrome. Further, little is known regarding the effects of maternal opioid use disorder on the developing human brain. We hypothesized that novel methodologies utilizing fetal central nervous system-derived extracellular vesicles isolated from maternal blood can address these gaps in knowledge. Plasma from opioid users and controls between 9 and 21 weeks was precipitated and extracellular vesicles were isolated. Mu opioid and cannabinoid receptor levels were quantified. Label free proteomics studies and unbiased small RNA next generation sequencing was performed in paired fetal brain tissue. Maternal opioid use disorder increased mu opioid receptor protein levels in extracellular vesicles independent of opioid equivalent dose. Moreover, cannabinoid receptor levels in extracellular vesicles were upregulated with opioid exposure indicating cross talk with endocannabinoids. Maternal opioid use disorder was associated with significant changes in extracellular vesicle protein cargo and fetal brain micro RNA expression, especially in male fetuses. Many of the altered cargo molecules and micro RNAs identified are associated with adverse clinical neurodevelopmental outcomes. Our data suggest that assays relying on extracellular vesicles isolated from maternal blood extracellular vesicles may provide information regarding fetal response to opioids in the setting of maternal opioid use disorder. Prospective clinical studies are needed to evaluate the association between extracellular vesicle biomarkers, risk of neonatal abstinence syndrome, and neurodevelopmental outcomes.

Introduction Alterations of white matter integrity and subsequent white matter structural deficits are consistent findings in Fetal Alcohol Syndrome (FAS), but knowledge regarding the molecular mechanisms underlying these abnormalities is incomplete. Experimental rodent models of FAS have shown dysregulation of cytokine expression leading to apoptosis of oligodendrocyte precursor cells (OPCs) and altered oligodendrocyte (OL) differentiation, but whether this is representative of human FAS pathogenesis has not been determined. Methods Fetal brain tissue (12.2-21.4 weeks gestation) from subjects undergoing elective termination of pregnancy was collected according to an IRB-approved protocol. Ethanol (EtOH) exposure status was classified based on a detailed face-to-face questionnaire adapted from the National Institute on Alcohol Abuse and Alcoholism Prenatal Alcohol and Sudden Infant Death Syndrome and Stillbirth (PASS) study. Twenty EtOH-exposed fetuses were compared with 20 gestational age matched controls. Cytokine and OPC marker mRNA expression was quantified by Real-Time Polymerase chain reaction (qRT-PCR). Patterns of protein expression of OPC markers and active Capase-3 were studied by Fluorescence Activated Cell Sorting (FACS). Results EtOH exposure was associated with reduced markers of cell viability, OPC differentiation, and OL maturation, while early OL differentiation markers were unchanged or increased. Expression of mRNAs for proteins specific to more mature forms of OL lineage (platelet-derived growth factor α (PDGFRα) and myelin basic protein (MBP) was lower in the EtOH group than in controls. Expression of the multifunctional growth and differentiation-promoting growth factor IGF-1, which is essential for normal development, also was reduced. Reductions were not observed for markers of early stages of OL differentiation, including Nuclear transcription factor NK-2 homeobox locus 2 (Nkx2.2). Expression of mRNAs for the proinflammatory cytokine, tumor necrosis factor-α (TNFα), and several proinflammatory chemokines was higher in the EtOH group compared to controls, including: Growth regulated protein alpha/chemokine (C-X-C motif) ligand 1 (GRO-α/CXCL1), Interleukin 8/chemokine (C-X-C motif) ligand 8 (IL8/CXCL8), Chemokine (C-X-C motif) ligand 6/Granulocyte chemotactic protein 2 (CXCL16/GCP2), epithelial-derived neutrophil-activating protein 78/chemokine (C-X-C motif) ligand 5 (ENA-78/CXCL5), monocyte chemoattractant protein-1 (MCP-1). EtOH exposure also was associated with an increase in the proportion of cells expressing markers of early stage OPCs, such as A2B5 and NG2. Finally, apoptosis (measured by caspase-3 activation) was increased substantially in the EtOH group compared to controls. Conclusion Prenatal EtOH exposure is associated with excessive OL apoptosis and/or delayed OL maturation in human fetal brain. This is accompanied by markedly dysregulated expression of several chemokines and cytokines, in a pattern predictive of increased OL cytotoxicity and reduced OL differentiation. These findings are consistent with findings in animal models of FAS.

Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associate with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy.

Objective Neuronal exosomes purified from peripheral blood samples have been proposed as diagnostic tool in the setting of acute brain injury but never tested clinically. We hypothesized that exosome protein biomarkers would change over time following acute hypoxic brain injury and would predict response to therapy. Methods Synaptopodin (SYNPO), an actin‐associated protein present in postsynaptic spines, was evaluated as a potential biomarker as well as: synaptophysin, neuron‐specific enolase, and mitochondrial cytochrome c oxidase. A secondary analysis was performed on neonatal samples collected at 8, 10, and 14 h after the initiation of therapeutic‐controlled hypothermia for acute hypoxic–ischemic encephalopathy (n = 14). Neuronal exosomes were purified from serum and protein levels were quantified using standard ELISA methods. The primary study outcomes were length of stay (LOS), discharge on seizure medication (DCMED), and composite neuroimaging score (NIS). Results The slope of change in neuronal exosome SYNPO between 8 and 14 h appeared to be the most promising biomarker for all three clinical study outcomes. SYNPO was highly correlated with LOS (−0.91, P < 0.001). SYNPO increased in 6/8 without DCMED and was worse or neutral in 5/5 with DCMED (P = 0.02). All four neonates with an abnormal NIS had neutral or decreasing SYNPO (P = 0.055). Other candidate biomarkers were not associated with outcomes. Interpretation This report provides the first clinical evidence that neural exosomes turn over rapidly enough in the peripheral circulation to be used as a “troponin‐like” test following acute brain injury. Optimal sampling and biomarkers likely vary with type of brain injury.

Ethanol induces neuronal cell injury and death by dysregulating several signaling events that are controlled, in part, by activation of MAPK/ERK1/2 and/or inactivation of its corresponding phosphatase, PP1. Recently, we have purified a novel protein of 38 kDa in size, p38SJ, from a callus culture of Hypericum perforatum, which belongs to an emerging DINGG family of proteins with phosphate binding activity. Here, we show that treatment of neuronal cells with p38SJ protects cells against injury induced by exposure to ethanol. Furthermore, pre-treatment of neuronal cells with p38SJ diminishes the level of the pro-apoptotic protein Bax and some events associated with apoptosis such as caspase 3 cleavage. In addition, by inducing stress, alcohol can elevate production of reactive oxygen species (ROS) that leads to a decrease in the activity of superoxide dismutase (SOD). Our results showed that p38SJ restores the activity of SOD in the ethanol treated neuronal cells. These observations provide a novel biological tool for developing new approaches for preventing neuronal cell death induced by ethanol and possibly treatment of neurological disorders associated with alcohol abuse. J. Cell. Physiol. 221: 499–504, 2009. © 2009 Wiley-Liss, Inc.

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.

JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.

The nucleic acid-binding protein Puralpha is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Puralpha also regulates homologous recombination-directed DNA repair (HRR).Cells lacking Puralpha showed enhanced sensitivity to cisplatin as evaluated by assays for cell viability and cell clonogenicity. This was seen both in Puralpha-negative MEFs and in human glioblastoma cells treated with siRNA directed against Puralpha. MEFs lacking Puralpha also showed enhanced H2AX phosphorylation in response to cisplatin. Repair of a reporter plasmid that had been treated with cisplatin was decreased in a reactivation assay using Puralpha-negative MEFs and the capacity of nuclear extracts from Puralpha-negative MEFs to perform non-homologous end-joining in vitro was also impaired.We investigated the effects of the DNA damage-inducing cancer chemotherapeutic agent cisplatin on mouse embryo fibroblasts (MEFs) from PURA(-/-) knockout mice that lack Puralpha.Puralpha has a role in the cellular response to cisplatin-induced DNA damage and may provide new therapeutic modalities for cisplatin-resistant tumors.

Objective The purpose of this study was to develop an animal model for intrapartum inflammation at term to investigate the interactions between maternal and fetal inflammatory responses and adverse neurologic outcome. Study Design Lipopolysaccharide (160, 320, or 640 μg/kg) was administered intraperitoneally to day 20 term-pregnant Sprague Dawley rat dams 2, 4, and 6 hours before sample collection. Maternal outcomes included dam core temperature and plasma interleukin 6 (IL-6). Fetal outcomes included plasma IL-6, brain IL-6 messenger RNA expression, and brain IL-6 protein expression. Primary cortical cell cultures were prepared to examine neuronal morphologic condition. Neurite counts were obtained with the use of automated Sholl analysis. Results Maternal plasma IL-6 levels peaked 2 hours after lipopolysaccharide stimulus and rapidly resolved, except for an observed low level persistence at 6 hours with 640 μg/kg. Fetal plasma and placental IL-6 expression also peaked rapidly but only persisted in placental samples. Fetal brain IL-6 RNA and protein expression was significantly higher than control litters at 6 hours after the exposure to both 320 μg/kg ( P ≤ .05) and 640 μg/kg ( P ≤ .01). Cortical cells from fetuses that were exposed for 6 hours to maternal systemic inflammation showed reduced neurite number and neurite length ( P Conclusion Our results demonstrate that fetal brain injury follows isolated systemic maternal inflammation and that fetal brain inflammation lags after maternal stimulus, which creates a potential 4-hour clinical window for therapeutic intervention.