Mean scores of homocysteine levels for all patients, restenosis group, and comparative group

<p><b>Copyright information:</b></p><p>Taken from "Hyperhomocysteinemia and recurrent carotid stenosis"</p><p>http://www.biomedcentral.com/1471-2261/8/1</p><p>BMC Cardiovascular Disorders 2008;8():1-1.</p><p>Published online 17 Jan 2008</p><p>PMCID:PMC2245907.</p><p></p

Number of patients with carotid restenosis (CR) according to occurrence in months

<p><b>Copyright information:</b></p><p>Taken from "Hyperhomocysteinemia and recurrent carotid stenosis"</p><p>http://www.biomedcentral.com/1471-2261/8/1</p><p>BMC Cardiovascular Disorders 2008;8():1-1.</p><p>Published online 17 Jan 2008</p><p>PMCID:PMC2245907.</p><p></p

FACS quantification of infiltrating leukocytes in GF/IL 17 mice and controls relative to all CD45 positive cells.

<p>FACS quantification of infiltrating leukocytes in GF/IL 17 mice and controls relative to all CD45 positive cells.</p

GF/IL17 mice express IL-17A mRNA and protein without major histological defects or leukocyte infiltration.

<p><b>(A)</b> Relative expression of <i>Il17a</i> mRNA in GF/IL17 mice is equally distributed between forebrain, hindbrain and spinal cord. In comparison to disease models in WT mice (peak EAE, West Nile Virus encephalitis, experimental cerebral Malaria) CNS expression levels of <i>Il17a</i> in otherwise untreated GF/IL17 mice exceed levels of all tested disease models irrespective of the cellular source. <b>(B)</b><i>Il17a</i> mRNA expression from GF/IL17 primary astrocytes and WT controls was quantified using real time PCR. <b>(C)</b> Astrocyte secretion of IL-17A protein was confirmed using ELISA. Supernatants of GF/IL17 and WT control primary astrocytes were analyzed after 12 hours of culture. In supernatant of WT control astrocytes IL-17A protein was not detectable. <b>(D</b>–<b>U)</b> Routine histological characterization of mice excluded major tissue damage or leukocyte infiltration. Representative areas of hippocampus <b>(D</b>–<b>I)</b> , cerebellum <b>(J</b>–<b>O)</b>, or cortex <b>(P</b>–<b>U)</b> are shown in WT controls <b>(D, F, H, J, L, N, P, R, T)</b> and GF/IL17 transgenic mice <b>(E, G, I, K, M, O, Q, S, U)</b> by HE <b>(D, E, J, K, P, Q)</b>, LFB <b>(F, G, L, M)</b>, anti murine NeuN mAb <b>(H, I, T, U)</b>, or anti murine PLP mAb <b>(N, O, R, S)</b> staining (age 9 month).</p

Transgenic IL-17A acts synergistically with other inflammatory stimuli and potentiates LPS induced microgial activation.

<p>GF/IL17 transgenic animals and littermate controls between 2 and 3 month were injected twice with 50 µg LPS i.p. in 24 hours or treated with mock injections of PBS. (<b>A</b>) Quantitative rt-PCR revealed a strong upregulation of the expression of inflammatory cytokines <i>Tnf</i>, <i>Il1b</i>, and <i>Ccl2</i> by LPS treatment whereas <i>Il6</i> was not induced following endotoxemia. This effect was markedly pronounced in GF/IL17 transgenic animals (<i>Tnf</i>: p < 0,01; <i>Il1b</i>: p < 0,05). Furthermore <i>Ccl2</i> expression was strikingly upregulated in some of the LPS treated GF/IL17 mice compared with LPS treated wild-type controls but due to the high interindividual variance not considered as significant. (<b>B</b>) Representative flow cytometry profiles from mock- or LPS-treated GF/IL17 and WT mice. LPS treatment induced a population of CD45^high/CD11b⁺ activated microglia in both WT and GF/IL17 mice. Chronic IL-17A stimulation strikingly augmented this effect, respectively. The numbers above the indicated gate show the mean percentages of FSC/SSC gated populations. (<b>C</b>) For the statistical analysis of infiltrating cell numbers a ratio between CD45^high/CD11b⁺ activated microglia and CD45^dim/CD11b⁺ resting microglia was calculated for each individual mouse.GF/IL17 transgenic animals exhibited a significantly elevated ratio of CD45^high/CD11b⁺ activated to CD45^dim/CD11b⁺ microglia after LPS treatment, respectively (p < 0,05). (<b>D</b>) Lectin immunohistochemistry revealed a pronounced accumulation of activated microglia (arrows) in the periventricular regions (asterisk: choroid plexus) after LPS treatment in GF/IL17 mice. (<b>E</b>) Intracellular staining of TNF-α after LPS treatment. Only CD45 positive microglia and monocytes/macrophages are stained by anti TNF-α antibody (left histogram) whereas GLAST positive astrocytes are negative for TNF-α (right histogram). In GF/IL17 transgenic mice (light gray) compared with wildtype mice (dark gray) CD45 positive microglia and monocytes/macrophages exhibit a stronger intracellular TNF-α staining after LPS treatment.</p

Transgenic CNS expression of IL-17A induces glial activation

<p>. <b>(A)</b> IHC for GFAP in the hippocampus of WT and GF/IL17 transgenic mice at 9 month. GFAP-staining revealed a strong astrocytic activation by morphological criteria in GF/IL17 mice. <b>(B)</b> Astrocytosis was confirmed by anti-GFAP immunoblotting. Whole brain lysates were analyzed by immunoblotting for the presence of GFAP. Anti-tubulin immunoblotting served as internal loading control on the same membrane. <b>(C)</b> Densitometric quantifications (arbitrary densitometry units) from immunoblots of B after normalization by tubulin densitometry units obtained from the same immunoblot. (*p < 0.05). <b>(D)</b> Tomato-lectin-staining in the hippocampus revealed an activated microglial morphology in GF/IL17A transgenic animals characterized by rounded cell bodies and microglial clustering (open arrows). In addition Lectin staining displayed prominent microvasculature in GF/IL17 mice compared with WT controls (closed arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057307#pone-0057307-g003" target="_blank">Figure 3</a> for vascular pathology). <b>(E)</b> IHC of frozen brain sections for Iba1 (red), CD68 (green) and Dapi (blue). GF/IL17 mice showed a strong immunoreactivity for the activation marker CD68 in Iba1 stained microglia (white arrows indicating colocalisation of the lysosomal markes CD68 and Iba1, age: 9 month). (F) Representative flow cytometric analysis of surface marker expression from freshly isolated microglia in GF/IL17 mice (red) and WT littermate controls (blue). Dashed histogram: isotype control. Histograms were gated on microglial population according to forward/side scatter profile. GF/IL17 mice displayed similar surface expression levels for CD45 compared with WT whereas CD11b expression was upregulated in GF/IL17 mice compared with WT. (G) Statistical analysis of mean fluorescence intensity of freshly isolated microglia in GF/IL17 mice (red) and WT littermate controls (blue). Comparable expression levels of CD45 in GF/IL17 and WT mice whereas CD11b expression levels were significantly upregulated in GF/IL17 mice compared with WT controls (*p < 0.05).</p

Astrocytic expression of IL-17A induces a vascular pathology with capillary calcifications, microvascular rarefaction and thickening of endothelial layer and basement membrane of vessel walls without disturbing blood-brain barrier integrity.

<p>GFAP immunohistochemistry (<b>A–B</b>), lectin (<b>C</b>) and Alizarin red S staining (<b>D</b>) of the thalamus in WT mice (<b>A</b>) and GF/IL17 transgenic animals (<b>C–D</b>) at the age of 09 month. Microvessels surrounded by GFAP positive astrocytic endfeet and laminin stained endothelia are filled with hematoxillin positive material (white arrows). Around calcified microvessels astrocytes display an activated morphology. Deposits are labelled orange/red in Alizarin red S staining (<b>D</b>) confirming vascular calcifications. Transmission electron microscopy of capillaries in the corpus callosum of (<b>E</b>) wild-type mice and (<b>F</b>) GF/IL17 transgenic mice at the age between 10 and 12 month exhibit morphological criteria of microangiopathy: the endothelial cell layer <i>(Endo)</i> appears enlarged compared with WT. Basement membrane <i>(red-colorored)</i> surrounding the vascular endothelium appears heavily thickened in TG animals. GF/IL17 mice display numerous duplications of the basement membrane spanning the perivascular space harbouring pericytes <i>(green colored)</i>. Inside capillaries erythrocytes <i>(Ery)</i> are detectable. Scale bar represents 1 µm. (<b>G</b>) Measurement of basement membrane thickness revealed a significant diameter increase in GF/IL17 mice (p < 0,05) (<b>H</b>) Confocal microscopy of 50 µm sections labelled with anti-laminin displayed a dense microvascular network in the white matter of WT animals. (<b>I</b>) Rarefaction of microvasculature in corresponding areas in GF/IL17 mice. Furthermore arterioles appear thickened. (Age: 10–12 month of both transgenic and wild-type mice) (<b>J</b>) To examine blood-brain barrier integrity Evans blue dye (EBD) extravasation into tissue was quantified. Levels of tissue EBD in brains and spinal cord are equal in WT and GF/IL17 mice. Liver tissue served as positive controls.</p

Regulation of inflammation related genes in GF/IL17 mice relative to littermate controls (arbitrary units).

<p>Regulation of inflammation related genes in GF/IL17 mice relative to littermate controls (arbitrary units).</p

sj-docx-1-eso-10.1177_23969873231202363 – Supplemental material for Endovascular treatment of cerebral sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia

Supplemental material, sj-docx-1-eso-10.1177_23969873231202363 for Endovascular treatment of cerebral sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia by Johannes Weller, Katarzyna Krzywicka, Anita van de Munckhof, Franziska Dorn, Katharina Althaus, Felix J Bode, Monica Bandettini di Poggio, Brian Buck, Timothy Kleinig, Charlotte Cordonnier, Vanessa Dizonno, Jiangang Duan, Ahmed Elkady, Beng Lim Alvin Chew, Carlos Garcia-Esperon, Thalia S Field, Catherine Legault, Mar Morin Martin, Dominik Michalski, Johann Pelz, Silvia Schoenenberger, Simon Nagel, Marco Petruzzellis, Nicolas Raposo, Mona Skjelland, Domenico Sergio Zimatore, Sanjith Aaron, Mayte Sanchez van Kammen, Diana Aguiar de Sousa, Erik Lindgren, Katarina Jood, Adrian Scutelnic, Mirjam R Heldner, Sven Poli, Antonio Arauz, Adriana B Conforto, Jukka Putaala, Turgut Tatlisumak, Marcel Arnold, Jonathan M Coutinho, Albrecht Günther, Julian Zimmermann and José M Ferro in European Stroke Journal</p