Vehicle-treated Control Animals Research Articles

Cardiomyocyte-Specific Overexpression of Activated Yes-Associated Protein Modified-RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration.

The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system (YAP5SACM-SMRTs or GFPCM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via invitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SACM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SACM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFPCM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFPCM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SACM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SACM-SMRTs appeared to promote cardiomyocyte proliferation. The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

The glutamate receptor antagonist ifenprodil inhibits hepatitis E virus infection.

Infection with hepatitis E virus (HEV) represents a global problem, with over 20 million people infected annually. No specific antiviral drugs are available for treating HEV infection, necessitating the development of novel targeted therapeutics. Here, we report that the N-methyl-D-aspartate receptor (NMDAR) antagonist ifenprodil, a clinically approved drug used to treat idiopathic pulmonary fibrosis (IPF), is an HEV inhibitor in liver-derived cells. In vitro investigation demonstrates that ifenprodil suppresses viral protein expression in a dose-dependent manner in human hepatoma cells by inhibiting early stages of viral infection. We also found that ifenprodil modulates host cell intrinsic biological processes distinct from virus-induced innate immunity, inhibiting HEV RNA accumulation in primary human hepatocytes. Finally, the inhibitory effect of ifenprodil in vivo was also tested in rabbits challenged with the HEV-3ra CHN-BJ-R14 strain. Fecal virus shedding was below the limit of detection in two animals for both ribavirin-treated and ifenprodil-treated rabbits compared to vehicle-treated control animals. Our data demonstrate that ifenprodil is an effective anti-HEV compound with potential as a therapeutic candidate for the treatment of HEV infection.

Normotensive placental extracellular vesicles provide long-term protection against hypertension and cardiovascular disease

BackgroundWomen with normotensive pregnancy are at a reduced risk of developing cardiovascular disease postpartum compared to those who experience hypertensive conditions during pregnancy. However, the underlying mechanisms remain poorly understood. During normotensive pregnancy, vast numbers of placental extracellular vesicles are released into the maternal circulation, which protect endothelial cells from activation and alter maternal vascular tone. We hypothesized that placental extracellular vesicles play a mechanistic role in lowering risk of cardiovascular disease following normotensive pregnancy. ObjectiveThis study aimed to investigate the long-term effects of placental extracellular vesicles derived from normotensive term placentae on the cardiovascular system and explore the mechanisms underlying their biological effects. Study DesignSpontaneously hypertensive rats were injected with placental EVs from normotensive term pregnancies (2 mg /kg each time, n=8) or vehicle control (n=9) at 3 months of age. Blood pressure and cardiac function were regularly monitored from 3 months to 15 months of age. The response of mesenteric resistance arteries to vasoactive substances was investigated to evaluate vascular function. Cardiac remodelling, small artery remodelling, and renal function were investigated to comprehensively assess the impact of placental extracellular vesicles on cardiovascular and renal health. ResultsCompared to vehicle-treated control animals, rats treated with normotensive placental extracellular vesicles exhibited a significantly lower increase in blood pressure and improved cardiac function. Furthermore, the vasodilator response to the endothelium-dependent agonist acetylcholine was significantly enhanced in the normotensive placental extracellular vesicle-treated spontaneously hypertensive rats compared to the control. Moreover, treatment with placental extracellular vesicles reduced wall thickening of small renal vessels and attenuated renal fibrosis. ConclusionPlacental extracellular vesicles from normotensive term pregnancies have long-lasting protective effects reducing hypertension and mitigating cardiovascular damage in vivo.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Differential expression of glycogen synthase kinase 3α and 3β isomers in brain cortex of mice following high doses of glucose

Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase. We investigated the effects of Subcutaneous (SC) glucose administration on the expression of glycogen synthase kinase 3 (GKS-3) isomers (α and β) genes in the cerebral cortex of mice with the aim of determining the possible mechanism(s) involved in mitochondrial dysfunction induced by hyperglycemia. Adult male BALB/c mice were treated with 12 gm/kg glucose solution SC once daily for 3 days. Ultrastructure study, histopathological analysis, and Real-time PCR investigations were carried out on the cerebral cortex from glucose treated mice and from vehicle-treated control animals. We observed significant ultrastructural damage of mitochondria in the cerebral cortex of mice received high doses of glucose. Histopathological alterations in the cortex of these animals were also detected. A significant increased of GSK-3α gene expression and decreased expressions of GKS-3β gene in high glucose treated animals were noticed. The hyperglycemia-induced ultrastructural changes may occur via modulation of gene expression of GSK-3 isomers, and we hypothesize this as an early etiopathological factor in hyperglycemia-related neurodegenerative diseases (NDD). It considered the first study describing "modulation of expression of GSK-3 isomer genes" as a possible mechanism of hyperglycemia-induced mitochondrial dysfunction.

17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics.

To facilitate the development of broad-spectrum retina neuroprotectants that can be delivered through topical dosage forms, this proteomics study focused on analyzing target engagements through the identification of functional protein networks impacted after delivery of 17β-estradiol in eye drops. Specifically, the retinae of ovariectomized Brown Norway rats treated with daily eye drops of 17β-estradiol for three weeks were compared to those of vehicle-treated ovariectomized control animals. We searched the acquired raw data against a composite protein sequence database by using Mascot, as well as employed label-free quantification to detect changes in protein abundances. Our investigation using rigorous validation criteria revealed 331 estrogen-regulated proteins in the rat retina (158 were up-regulated, while 173 were down-regulated by 17β-estradiol delivered in eye drops). Comprehensive pathway analyses indicate that these proteins are relevant overall to nervous system development and function, tissue development, organ development, as well as visual system development and function. We also present 18 protein networks with associated canonical pathways showing the effects of treatments for the detailed analyses of target engagements regarding potential application of estrogens as topically delivered broad-spectrum retina neuroprotectants. Profound impact on crystallins is discussed as one of the plausible neuroprotective mechanisms.

The impact of silencing feed-forward parvalbumin-expressing inhibitory interneurons in the cortico-thalamocortical network on seizure generation and behaviour

Feed-forward inhibition (FFI) is an essential mechanism within the brain, to regulate neuronal firing and prevent runaway excitation. In the cortico-thalamocortical (CTC) network, fast spiking parvalbumin-expressing (PV+) inhibitory interneurons regulate the firing of pyramidal cells in the cortex and relay neurons in the thalamus. PV+ interneuron dysfunction has been implicated in several neurological disorders, including epilepsy. Previously, we demonstrated that loss of excitatory AMPA-receptors, specifically at synapses on PV+ interneurons in CTC feedforward microcircuits, occurs in the stargazer mouse model of absence epilepsy. These mice present with absence seizures characterized by spike and wave discharges (SWDs) on electroencephalogram (EEG) and concomitant behavioural arrest, similar to childhood absence epilepsy. The aim of the current study was to investigate the impact of loss of FFI within the CTC on absence seizure generation and behaviour using new Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology. We crossed PV-Cre mice with Cre-dependent hM4Di DREADD strains of mice, which allowed Cre-recombinase-mediated restricted expression of inhibitory Gi-DREADDs in PV+ interneurons. We then tested the impact of global and focal (within the CTC network) silencing of PV+ interneurons. CNO mediated silencing of all PV+ interneurons by intraperitoneal injection caused the impairment of motor control, decreased locomotion and increased anxiety in a dose-dependent manner. Such silencing generated pathological oscillations similar to absence-like seizures. Focal silencing of PV+ interneurons within cortical or thalamic feedforward microcircuits, induced SWD-like oscillations and associated behavioural arrest. Epileptiform activity on EEG appeared significantly sooner after focal injection compared to peripheral injection of CNO. However, the mean duration of each oscillatory burst and spike frequency was similar, irrespective of mode of CNO delivery. No significant changes were observed in vehicle-treated or non-DREADD wild-type control animals. These data suggest that dysfunctional feed-forward inhibition in CTC microcircuits may be an important target for future therapy strategies for some patients with absence seizures. Additionally, silencing of PV+ interneurons in other brain regions may contribute to anxiety related neurological and psychiatric disorders.

Dezocine Alleviates Morphine-Induced Dependence in Rats.

Opioid dependence is a major public health issue without optimal therapeutics. This study investigates the potential therapeutic effect of dezocine, a nonaddictive opioid, in opioid dependence in rat models. Dezocine was administered intraperitoneally to a morphine-dependent rat model to investigate its effect on withdrawal and conditioned place preference (CPP). Effect of dezocine on morphine withdrawal syndrome and CPP was analyzed using 2-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Buprenorphine and vehicle solution containing 20% (v/v) dimethyl sulfoxide were used for positive and negative control, respectively. The astrocytes activation in nucleus accumbens was assessed by immunofluorescence assay of glial fibrillary acidic protein. Effect of dezocine and buprenorphine on the internalization of κ opioid receptor (KOR) was investigated using Neuro2A expressing KOR fused to red fluorescent protein tdTomato (KOR-tdT). Buprenorphine and dezocine were screened against 44 G-protein-coupled receptors, ion channels, and transporter proteins using radioligand-binding assay to compare the molecular targets. The mean withdrawal score was reduced in rats treated with 1.25 mg·kg dezocine compared to vehicle-treated control animals starting from the day 1 (mean difference: 7.8; 95% confidence interval [CI], 6.35-9.25; P < .0001 by 2-way ANOVA). Significance was observed at all treatment days, including day 7 (mean difference: 2.13; 95% CI, 0.68-3.58; P < .001 by 2-way ANOVA). Furthermore, dezocine inhibited the reinstatement of morphine-induced CPP (mean difference: 314; 95% CI, 197.9-430.1; P < .0001 by 2-way ANOVA) compared to the control group. Chronic morphine administration induced astrocytes activation in nucleus accumbens, which was attenuated by dezocine. Dezocine blocked the agonist-induced KOR internalization in vitro, 1 of the mechanisms involved in the downstream signaling and development of opioid dependence. Dezocine had affinity to norepinephrine and serotonin transporters and sigma-1 receptor, whereas buprenorphine showed no activity against these targets. Dezocine could potentially be used to alleviate opioid dependence. Due to the unique molecular target profile different from buprenorphine, it might have important value in studying the mechanisms of morphine dependence and developing novel therapeutic approaches.

A Reduction in B, T, and Natural Killer Cells Expressing CD38 by TAK-079 Inhibits the Induction and Progression of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.

Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC\u2013MS and ion mobility enabled HILIC\u2013MS

IntroductionAs large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening.ObjectivesTo develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system.MethodsA rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation.ResultsThe rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database.ConclusionA rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites.

Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation

High-risk cornea transplant recipients represent a patient population with significant un-met medical need for more effective therapies to prevent immunological graft rejection due to heightened anti-donor immune response. In this study, a rat model of pre-existing anti-donor immunity was developed in which corneal allografts were rejected earlier than in non-pre-sensitized recipients. In this model, third-party (non-donor, non-recipient strain) allogeneic mesenchymal stromal cells (allo-MSC) were administered intravenously 7 and 1 days prior to transplantation. Rejection-free graft survival to 30 days post-transplant improved from 0 to 63.6% in MSC-treated compared to vehicle-treated control animals (p = < 0.0001). Pre-sensitized animals that received third-party allo-MSC prior to transplantation had significantly higher proportions of CD45+CD11b+ B220+ monocytes in the lungs 24 h after the second MSC injection and significantly higher proportions of CD4+ FoxP3+ regulatory T cells in the graft-draining lymph nodes at the average day of rejection of control animals. In in vitro experiments, third-party allo-MSC polarized primary lung-derived CD11b/c+ myeloid cells to a more anti-inflammatory phenotype, as determined by cytokine profile and conferred them with the capacity to suppress T cell activation via prostaglandin E2 and TGFβ1. In experiments designed to further validate the clinical potential of the protocol, thawed cryopreserved, third-party allo-MSC were shown to be similarly potent at prolonging rejection-free corneal allograft survival as their freshly-cultured counterparts in the pre-sensitized high-risk model. Furthermore, thawed cryopreserved third-party allo-MSC could be co-administered with mycophenolate mofetil without adversely affecting their immunomodulatory function. In conclusion, a clinically-relevant protocol consisting of two intravenous infusions of third-party allo-MSC during the week prior to transplantation, exerts a potent anti-rejection effect in a pre-sensitized rat model of high-risk corneal allo-transplantation. This immune regulatory effect is likely to be mediated in the immediate post-transplant period through the promotion, by allo-MSC, of alternatively-activated macrophages in the lung and, later, by enhanced regulatory T-cell numbers.

The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients and type I interferon (IFN) has been evaluated in a few infected transplantation patients in vivo. However, no effective and specific treatments against HEV infections are currently available.In this study, we evaluated the natural compound silvestrol, isolated from the plant Aglaia foveolata, and known for its specific inhibition of the DEAD-box RNA helicase eIF4A in state-of-the-art HEV experimental model systems. Silvestrol blocked HEV replication of different subgenomic replicons in a dose-dependent manner at low nanomolar concentrations and acted additive to ribavirin (RBV). In addition, HEV p6-based full length replication and production of infectious particles was reduced in the presence of silvestrol. A pangenotypic effect of the compound was further demonstrated with primary isolates from four different human genotypes in HEV infection experiments of hepatocyte-like cells derived from human embryonic and induced pluripotent stem cells. In vivo, HEV RNA levels rapidly declined in the feces of treated mice while no effect was observed in the vehicle treated control animals. In conclusion, silvestrol could be identified as pangenotypic HEV replication inhibitor in vitro with additive effect to RBV and further demonstrated high potency in vivo. The compound therefore may be considered in future treatment strategies of chronic hepatitis E in immunocompromised patients.

Astrocytic N-Myc Downstream-regulated Gene-2 Is Involved in Nuclear Transcription Factor κB-mediated Inflammation Induced by Global Cerebral Ischemia.

Inflammation is a key element in the pathophysiology of cerebral ischemia. This study investigated the role of N-Myc downstream-regulated gene-2 in nuclear transcription factor κB-mediated inflammation in ischemia models. Mice (n = 6 to 12) with or without nuclear transcription factor κB inhibitor pyrrolidinedithiocarbamate pretreatment were subjected to global cerebral ischemia for 20 min. Pure astrocyte cultures or astrocyte-neuron cocultures (n = 6) with or without pyrrolidinedithiocarbamate pretreatment were exposed to oxygen-glucose deprivation for 4 h or 2 h. Astrocytic nuclear transcription factor κB and N-Myc downstream-regulated gene-2 expression, proinflammatory cytokine secretion, neuronal apoptosis and survival, and memory function were analyzed at different time points after reperfusion or reoxygenation. Proinflammatory cytokine secretion was also studied in lentivirus-transfected astrocyte lines after reoxygenation. Astrocytic nuclear transcription factor κB and N-Myc downstream-regulated gene-2 expression and proinflammatory cytokine secretion increased after reperfusion or reoxygenation. Pyrrolidinedithiocarbamate pretreatment significantly reduced N-Myc downstream-regulated gene-2 expression and proinflammatory cytokine secretion in vivo and in vitro, reduced neuronal apoptosis induced by global cerebral ischemia/reperfusion (from 65 ± 4% to 47 ± 4%, P = 0.0375) and oxygen-glucose deprivation/reoxygenation (from 45.6 ± 0.2% to 22.0 ± 4.0%, P < 0.001), and improved memory function in comparison to vehicle-treated control animals subjected to global cerebral ischemia/reperfusion. N-Myc downstream-regulated gene-2 lentiviral knockdown reduced the oxygen-glucose deprivation-induced secretion of proinflammatory cytokines. Astrocytic N-Myc downstream-regulated gene-2 is up-regulated after cerebral ischemia and is involved in nuclear transcription factor κB-mediated inflammation. Pyrrolidinedithiocarbamate alleviates ischemia-induced neuronal injury and hippocampal-dependent cognitive impairment by inhibiting increases in N-Myc downstream-regulated gene-2 expression and N-Myc downstream-regulated gene-2-mediated inflammation.

Protective Role of Methanol Stem Bark Extract of Myriantus arboreus P. Beauv in Anthracene-Induced Nephrotoxicity in Laboratory Rats

Background: Myriantus arboreus P. Beauv (Cecropiaceae) is used in traditional medicine for management of systemic pathologies. Objective: This study was conducted to investigate the protective role of the methanol stem bark extract of M. arboreus (MEMA) in anthracene-induced nephrotoxicity in laboratory rats. Materials and Methods: Forty rats were randomly divided into five groups of eight rats each comprising Group 1: 10 mL/kg of 5% Tween 20 as vehicle treated control; Group 2: 50 mg/kg anthracene (ANTH) as positive control; Group 3: 100 mg/kg; and Group 4: 200 mg/kg MEMA respectively. Groups 3 and 4 were administered the extract for 7 days followed by co-administration with 50 mg/kg ANTH p.o. for 21 days. Animals in Group 5 were administered only the extract at 200 mg/kg for 21 days p.o. Results: Creatinine was significantly (p&lt;0.01) increased on exposure to ANTH. However, creatinine was significantly (p&lt;0.01) reduced in rats co-administered MEMA and ANTH. Renal tissues showed a significant (p&lt;0.001) reduction in reduced glutathione (GSH) and superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in ANTH exposed rats compared to MEMA and ANTH exposed. Catalase (CAT) was significantly (p&lt;0.05) increased in animals administered only MEMA compared to ANTH and vehicle treated control animals. Malondialdehyde (MDA) was significantly (p&lt;0.01) increased on exposure to ANTH but was significantly reduced on treatment with 200 mg/kg MEMA. On histopathological assessment, ANTH exposed rats presented with section of the kidney showing massive renal necrosis. However, there was a remodeling of renal architecture observed as normal renal architecture with minimal renal damage on co-administration with MEMA. Conclusion: The extract significantly reduced the anthracene-induced increase in creatinine level and also modulated the architectural damage of the kidney caused by anthracene. The improvements in the antioxidant indices are suggestive renoprotective mechanisms of the extract.

Inhibition of ERG Activity in Patient-derived Prostate Cancer Xenografts by YK-4-279.

The aim of the current study was to determine the effects of the ERG small-molecule inhibitor YK-4-279 on ERG+ prostate cancer patient-derived xenografts (PDX). ERG activity was blocked using YK-4-279 in three subcutaneously-implanted ERG+ (LuCaP 23.1, 86.2 and 35) and one ERG- (LuCaP 96) PDX. Treated animals tumor volume (TV), body weight (BW) and serum prostate-specific antigen (PSA) were compared to vehicle-treated control animals. Gene expression, proliferation, apoptosis, microvessel density and ERG expression were also assessed. Administration of YK-4-279 decreased TV (p=0.026), proliferation (p=0.0038) and PSA (p=0.022) in Severe Combined Immunodeficiency (SCID) mice bearing LuCaP 23.1 tumors. LuCaP 86.2, LuCaP 35 and LuCaP 96 showed no significant changes in TV, or PSA. Mineralocorticoid receptor (MR) and MR-direct target genes were up-regulated in treatment-resistant LuCaP 86.2 and LuCaP 35 PDX. YK-4-279 decreased ERG+ LuCaP 23.1 tumor growth, but not LuCaP 86.2 and LuCaP 35 ERG+ tumor growth.

Endothelial colony-forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFC) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFC may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia-reperfusion (I/R). Rat pulmonary microvascular endothelial cells (PMVEC) with high proliferative potential were compared with pulmonary artery endothelial cells (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 h of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of ICAM-1 and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

Interaction Effect of Social Isolation and High Dose Corticosteroid on Neurogenesis and Emotional Behavior.

Hypercortisolemia is one of the clinical features found in depressed patients. This clinical feature has been mimicked in animal studies via application of exogenous corticosterone (CORT). Previous studies suggested that CORT can induce behavioral disturbance in anxious-depressive like behavior, which is associated with suppressed neurogenesis. Hippocampal neurogenesis plays an important role in adult cognitive and behavioral regulation. Its suppression may thus lead to neuropsychiatric disorders. Similar to the effects of CORT on the animals’ depression-like behaviors and neurogenesis, social deprivation has been regarded as one factor that predicts poor prognosis in depression. Furthermore, social isolation is regarded as a stressor to social animals including experimental rodents. Hence, this study aims to examine if social isolation would induce further emotional or anxiety-like behavior disturbance and suppress neurogenesis in an experimental model that was repeatedly treated with CORT. Sprague-Dawley rats were used in this study to determine the effects of different housing conditions, either social isolated or group housing, in vehicle-treated control and CORT-treated animals. Forced swimming test (FST), open field test (OFT) and social interaction test (SIT) were used to assess depression-like, anxiety-like and social behaviors respectively. Immunohistochemistry was performed to quantify the number of proliferative cells and immature neurons in the hippocampus, while dendritic maturation of immature neurons was analyzed by Sholl analysis. Social isolation reduced latency to immobility in FST. Furthermore, social isolation could significantly reduce the ratio of doublecortin and bromodeoxyuridine (BrdU) positive cells of the neurogenesis assay under CORT-treated condition. The current findings suggested that the behavioral and neurological effect of social isolation is dependent on the condition of hypercortisolemia. Furthermore, social isolation may possibly augment the signs and symptoms of depressed patients with potential alteration in neurogenesis.