Insanity as a Defense to the Civil Fraud Penalty

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of proinflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-. signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression

Efficient and innocuous delivery of small interfering RNA to microglia using an amphiphilic dendrimer nanovector.

Aim: Alterations of microglia, the brain-resident macrophages, are associated with numerous brain pathologies. Genetic manipulation of microglia in diseases using small interfering RNA (siRNA) is hampered by the lack of safe and efficient siRNA delivery methods. We assessed the amphiphilic dendrimer (AD) for functional siRNA delivery and gene knockdown in primary microglia. Materials & methods: We characterized the ability of AD to form nanoparticles with siRNA, and studied their size, surface potential, cell uptake and gene silencing in rodent microglia. Results: AD effectively delivered siRNA to primary microglia and decreased target gene and protein expression, leading to transcriptomic changes without affecting basal microglial functions. Conclusion: The dendrimer AD promises to be an innocuous carrier for siRNA delivery into microglia

Characteristics of the Alternative Phenotype of Microglia/Macrophages and its Modulation in Experimental Gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas

Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3

Stearoyl-CoA Desaturase Regulates Angiogenesis and Energy Metabolism in Ischemic Cardiomyocytes

New blood vessel formation is a key component of the cardiac repair process after myocardial infarction (MI). Hypoxia following MI is a major driver of angiogenesis in the myocardium. Hypoxia-inducible factor 1α (HIF1α) is the key regulator of proangiogenic signaling. The present study found that stearoyl-CoA desaturase (SCD) significantly contributed to the induction of angiogenesis in the hypoxic myocardium independently of HIF1α expression. The pharmacological inhibition of SCD activity in HL-1 cardiomyocytes and SCD knockout in an animal model disturbed the expression and secretion of proangiogenic factors including vascular endothelial growth factor-A, proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor α, monocyte chemoattractant protein-1, and Rantes), metalloproteinase-9, and platelet-derived growth factor in ischemic cardiomyocytes. These disturbances affected the proangiogenic potential of ischemic cardiomyocytes after SCD depletion. Together with the most abundant SCD1 isoform, the heart-specific SCD4 isoform emerged as an important regulator of new blood vessel formation in the murine post-MI myocardium. We also provide evidence that SCD shapes energy metabolism of the ischemic heart by maintaining the shift from fatty acids to glucose as the substrate that is used for adenosine triphosphate production. Furthermore, we propose that the regulation of the proangiogenic properties of hypoxic cardiomyocytes by key modulators of metabolic signaling such as adenosine monophosphate kinase, protein kinase B (AKT), and peroxisome-proliferator-activated receptor-γ coactivator 1α/peroxisome proliferator-activated receptor α depends on SCD to some extent. Thus, our results reveal a novel mechanism that links SCD to cardiac repair processes after MI

Quantification of selected M1/M2 phenotype-associated gene expression in CD11b⁺ cells isolated from naïve and tumor-bearing mice.

<p>Gene expression was analyzed by real-time PCR and the results are presented as fold changes of CD11b⁺ cells isolated from tumor brains versus those from naïve brain tissue. Numbers corresponding to the significantly changed genes (t-test generated p-value<0.05) are marked in bold; NA - not available.</p

Influx of microglia/macrophages into the tumor is blocked by CsA.

<p>A. Representative confocal images of Iba1 staining in intact brain tissue, tumor-bearing brain slices from mice treated with PBS or CsA. Scale bar = 20 µm. B–C. Quantification of microglia and blood-derived macrophages in naïve, tumor-bearing and CsA-treated mice (4 per group). Each bar represents the mean ± SEM. ^***<i>p</i><0.001, ^**<i>p</i><0.01 tumor-bearing versus naïve mice; ^##<i>p</i><0.01, CsA-treated versus PBS-treated tumor-bearing mice.</p

Alterations of gene expression in infiltrating microglia/macrophages and intracranial gliomas are modulated by CsA.

<p>A. Gene expression in magnetically sorted CD11b⁺ cells from tumor-bearing and naïve brains was determined by qPCR. Expression of five genes was significantly altered in CD11b⁺ cells: <i>arg-1 (p = 0.000003)</i>; <i>cxcl14 (p = 0.0001)</i>; <i>ifn-β1 (p = 0.0002)</i>; <i>cox-2 (p = 0.000002)</i>; <i>mt1-mmp (p = 0.00002)</i>; n = 5 animals per group; ^*<i>p</i><0.05, ^**<i>p</i><0.01. The middle line represents the median value. Lower ΔC_T are consistent with higher gene expression. B. Quantification of arginase activity in brain tissue extracts from naïve and tumor-bearing mice treated either with PBS or CsA. Results represent the mean ± SEM of 4–5 mice; ^*<i>p</i><0.05, tumor-bearing versus tumor-free hemispheres; ^#<i>p</i><0.05, CsA (10 mg/kg, 8th) versus PBS-treated, tumor-bearing mice. C. MMP-2 activity in proteins extracts from the brains of naïve (N1–5) and tumor-bearing mice (T1–5) determined by gel zymography. Active MMP-2 detected as a prominent band at 62 kDa. D. Quantification of MMP-2 activity using the cleavage of fluorescent DQ-gelatin substrate; means ± SEM of 4–6 mice; ^**<i>p</i><0.01, tumor-bearing versus naïve brain extracts; ^###<i>p</i><0.001, ^#<i>p</i><0.05, CsA- versus PBS-treated tumor-bearing mice.</p

Accumulation and activation of microglia/macrophages in experimental glioma.

<p>A. Representative confocal images of brain sections 15 days after implantation of pEGFP-N1 GL261 cells into the striatum of C57BL/6 mice. Note the infiltration and morphological transformation of glioma-infiltrating Iba1⁺ cells. Scale bar: left image – 1000 µm, right image – 20 µm. B. Contralateral and ipsilateral hemisphere from tumor-bearing brain 15 days after injection of pEGFP-N1 GL261 cells. Images showed merged Iba1 and EGFP fluorescence. Scale bar = 750 µm. C. Microglia/macrophages were separated using a magnetic-bead-conjugated anti-CD11b antibody and stained with CD45 PerCP-Cy5.5 and CD11b PE prior to FACS acquisition. Propidium iodide staining was performed to eliminate necrotic/apoptotic cells (Gate R3, R4) and viable cells were gated (Gate R1; <b>B1</b>, Gate R2; <b>B2</b>). D. Efficiency of CD11b-positive cells separation in the negative fraction (CD11b-negative cells) from each sample was controlled. E. Representative dot plots for microglia (Gate R4, CD11b⁺/CD45^low) and macrophages (Gate R5, CD11b⁺/CD45^high) from tumor-bearing hemispheres. F. Kinetics of microglia/macrophage influx into tumor tissue. CD11b⁺ cells separated from the brains of naïve, sham operated and tumor-bearing mice at day 3, 8 or 15 after implantation (n = 4/group) were sorted according to CD45 expression. Each bar represents the mean ± SEM; ^***<i>p</i><0.001, ^*<i>p</i><0.05 tumor-bearing mice at 8th day versus naïve mice; ^##<i>p</i><0.01 tumor-bearing mice at day 15 versus day 8.</p

Cytokine profile in glioma-bearing brains.

<p>A. The levels of ten pro/anti-inflammatory cytokines were determined by flow cytometry in protein extracts isolated from hemispheres of naïve, LPS-injected and tumor-bearing mice. The results show the means ± SEM (n = 5 per group); ^#<i>p</i><0.05 significant change between LPS-treated v. naïve mice; ^*<i>p</i><0.05 significant difference between tumor-bearing v. naïve mice. B. Elevated levels of IL-10 and GM-CSF detected in tumors are reduced by CsA treatment. Each dot represents an individual animal; a horizontal line represents a mean of each group; ^*<i>p</i><0.05; ^**<i>p</i><0.01. C. Expression of IL-10 in glioma-infiltrating microglia and macrophages. Left panel: expression of IL-10 on sorted CD11b⁺ cells (50,000 cells) determined by flow cytometry with the anti-IL-10 antibody conjugated to Alexa Fluor647. Representative histograms of IL-10 detection in microglia (light grey) and macrophages (dark grey) cells from naïve (a) and tumor-bearing (b) brains. Right panel: quantification of microglia/macrophages expressing IL-10 in naïve and tumor-bearing brains (means ± SEM, 3 animals per group); significant increase of IL-10-positive microglia (** <i>p</i><0.01) and macrophages (^###<i>p</i><0.001) in tumor-bearing brains. D. Quantification of <i>gm-csf</i> and <i>m-csf</i> expression in GL261 glioma cells and non-transformed astrocytes (means ± SD from 3 experiments). E. Quantitative evaluation of <i>gm-csf</i> mRNA expression in GL261 glioma cells exposed to 0.1 and 1 µM CsA for 24 hours (means ± SD from two experiments with three or four replicates per condition) compared to untreated control cells. Statistical analysis was done by Student <i>t</i> test, ** <i>p</i><0.01.</p