Diagrams.

<p>(<i>Upper panel</i>): Flow diagram of Exp. #1 regarding times and type of treatment with pentylenetetrazole. <i>(Lower panel):</i> Schematic diagram of the distribution of proliferative (BrdU-positive) cells in the brain at day 25 after a single administration of a convulsive dose of PTZ. Note the change from a widespread distribution (<b>A</b>) to a more restricted distribution at 25 days post-seizure (<b>B</b>). In control rats, there were only a few BrdU-positive cells, often in a duplex, mitosis-like state (<b>C, D, E</b>). Many proliferative cells in PTZ-treated animals appeared to enter the brain from the circulation via leptomeningeal blood vessels (<b>F</b>, arrow points to a mitosis-like state). (<b>G</b>, <b>H</b>): Quantitation of BrdU-positive cells. One episode of convulsive seizure causes, at day 3, dramatically increased BrdU-positive cell numbers in the hippocampus (15-fold over controls; p = 0.001) (<b>G</b>) and temporal neocortex (22.5-fold over controls; p = 0.001) (<b>H</b>). Although the numbers of BrdU-positive cells decreased dramatically by day 25, their number remained, nonetheless, at relatively high levels in the hippocampus (4.8-fold; p = 0.001)(<b>G</b>) and temporal neocortex (5.6-fold; p = 0.001) (<b>H</b>) over control levels. N = 15 rats for each timepoint. <i>Abbreviations</i>: <i>Te-II</i>, temporal neocortex layer II; <i>Ent</i>, entorhinal neocortex; <i>HC</i>, hippocampus; <i>lep</i>, leptomeninx. Bars: (<b>C, D, E</b>), 20 µm; (<b>F</b>), 30 µm.</p

Localization and quantification of DCX in the rat brain during kindling development.

<p>(<b>A, B</b>) Overview of DCX staining in the ventral (<b>A,</b> arrows) and dorsal (<b>B,</b> arrows) hippocampal hilus of the kindled animals. The dorsal hippocampus of kindled animals, was highly significant (p = 0.001) enriched in DCX⁺-cells (<b>C</b>). Note that the DCX antigens were localized both in cell bodies and extensions penetrating the densely packed granule cell neurons (<b>D</b>, arrows). (<b>E-F</b>): Phenotyping of DCX-cells. After 2× PTZ some DCX⁺ positive cells (green) in the dorsal hippocampus along the hilar border with the granule cell layer had a NeuN nucleus (red) (<b>E</b>, arrows). In kindled animals some of the DCX (green)/BrdU (red) double-labeled cells had a clonal appearance (<b>F</b>, inset, 3D-image) while other DCX⁺ cells (green) sometimes displayed a fragmented BrdU-positivity (<b>F</b>, arrow). By quantitative RT-PCR there was a 3-fold increase (p = 0.01) in the relative amount of DCX transcripts in kindled animals over that of controls (<b>G</b>). Note that the number of DCX⁺ cells also is maximal when PTZ is administered every 25^th day (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039302#pone-0039302-g004" target="_blank">Fig. 4</a>H, filled circles) as opposed to every 30^th day (<b>H</b>, open circles). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer; <i>hl</i>, hilus; <i>pml</i>, polymorphic layer. Bars: (<b>A,B</b>), 200 µm; (<b>D</b>), 100 µm.</p

Number and phenotyping of BrdU-positive cells after seizure activity.

<p>Each PTZ treatment led to an accumulation of BrdU⁺ cells in the dentate gyrus of the kindled rats (9-fold, p = 0.0001; <b>A</b>). (<b>B–E</b>): 3D projections of confocal BrdU(red)/NeuN(green) double-labeled images from PTZ-treated animals. A single episode of seizure activity led to the appearance of BrdU-positive cells in the polymorphic layer that were in a mitosis-like state (<b>B</b>). Occasionally some neurons in layers II and III of the temporal neocortex also displayed BrdU⁺ cells in close apposition to neurons (<b>B</b>, insets). After 2× PTZ some BrdU-positive cells have differentiated into neurons, particularly in the granule cell layer (<b>C</b>, arrows). In addition, some BrdU-positive nuclei were detected in the walls of large blood vessels (<b>C</b>, inset). The number of double-labeled BrdU(red)/NeuN(green) increased with the number of PTZ injections and reached a maximum in the granule cells layer of kindled animals (<b>D</b>, low power; <b>E</b>, higher power). <i>Abbreviations</i>: <i>Te</i>, temporal neocortex; <i>GCL</i>, granule cell layer; <i>BV</i>, Blood Vessel.</p

Flow diagrams of the experimental design regarding times and type of treatment and EEG recording.

<p>After Exp. #2 (<b>A</b>) the proportion of rats that achieved full kindling status reached 80% (<b>C</b>; p = 0.001). By comparison, 31% of rats administered a subconvulsive dose of PTZ at all times (Exp. #3)(<b>B</b>) reached full kindling status after two injections and up to 81% of animals reached full kindling status after the fourth treatment (<b>C;</b> p = 0.001). (<b>D</b>–<b>F</b>): A subconvulsive PTZ treatment elicited intermittent non-ictal events that are typically dependent on the behavioral state of the animal (active or passive wakefulness)(<b>D</b>). After the second PTZ injection, the animals usually demonstrated mild multifocal body jerks (<b>E</b>). After the third PTZ injection, the animals showed typical seizure activity associated with motor arrest (<b>F</b>).</p

L-NAME treatment increased neurogenesis and seizure susceptibility.

<p>Daily treatment with L-NAME for 24 days (<b>A</b>) resulted in a significant increase in the number of fully seizing animals after the second PTZ administration on day 25 (<b>B</b>). In control animals, nestin immunoreactivity was detected in capillary walls (<b>C</b>). By quantitative RT-PCR there was a 7.8-fold (p = 0.001) in nestin mRNA levels at day 3 post-seizure (<b>D</b>). L-NAME-treated animals had significantly more (2.7-fold; p = 0.001) nestin mRNA than did animals treated with PTZ alone at day 25 (<b>D</b>) whereas kindled animals did not show an increased level of nestin mRNA (<b>D</b>). At the tissue level, nestin immunoreactivity at day3 in PTZ-treated animals was confined to radial glia-like cells in the inner molecular layer of the dentate gyrus, the polymorphic layer and, interestingly, to the CA2 region (arrow) (<b>E</b>, and inset). After 25 days, the nestin-like immunoreactivity was restricted to the polymorphic layer (<b>F</b> and inset). <i>Abbreviations</i>: <i>DG</i>, dentate gyrus; <i>pml</i>, polymorphic layer; <i>CA2</i>, hippocampal region.</p

L-NAME treatment increased doublecortin levels in the rat hippocampus.

<p>L-NAME treatment increased DCX immunoreactivity on Western blots (<b>A</b>) by 2.2-fold (p = 0.02) at day 50 post-seizure (<b>B</b>). By immunohistochemistry, numerous DCX-positive cells were detected in the subgranular zone of the dorsal hippocampus by day 50 post-seizure (<b>C</b>). Quantitatively, L-NAME elicited significant increases (1.5-fold, p = 0.01) in the number of DCX-positive cells (<b>D</b>). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer.</p

The donation status of the liver allograft influences the cytokine production in culture.

<p>Cytokines in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-α (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-ells. Numbers indicate epatocellular injury iα (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC un-stimulated and stimulated HMC. Mann Whitney U or Kruskal Wallis test was performed. ns = non-significant, * p<0.05, **p<0.01, ***p<0.001.</p

Clinical parameters of recipients.

<p>Clinical parameters of recipients.</p

Intrahepatic T-cells respond to HMGB1 <i>in vitro</i>.

<p>(A, B) Biopsies obtained from liver allografts before (left) and one-hour after (right) reperfusion. (A) Haematoxylin and eosin staining of a representative biopsy from DBD or DCD livers showing hepatocellular necrosis (plain arrow) and neutrophil aggregation (arrowhead) in post-reperfusion samples; (B) Immuno-histochemical staining for HMGB1 of the biopsies shown in (A) reveals an increased proportion of hepatocytes showing nuclear expression of HMGB1 in post-reperfusion samples compared to the pre-reperfusion counterparts but no cytoplasmic expression; (C) Production of IFN-γ from T-cells of a representative donor co-cultured with syngeneic DC at the ratio of 20:1 for 7 days. Percentage of IFN-γ-producing cells gated on CD8+CD3+ T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml; (D) Proliferative response of T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml. 1 = negative, 3 = positive control and 2 = HMGB1; (E) Quantitative analysis of IFN-γ concentration in supernatants of cultures. NT = negative control, S = aCD3/CD28 stimulation, HMGB1 = cultures stimulated in presence of 100 or 10ng/ml of HMGB1. Depiction of IFN-γ concentration in pg/ml (left) and ratio of increase reported to the negative control (right). Kruskal Wallis test was used.</p

CD107a expression in liver isolated CD8+ T-cells.

<p>(A) In vitro CD107a expression on liver (DBD and DCD) CD8+ T-cells was analysed after a co-culture with K562 cell line. Mann-Whitney U or Spearman correlation test was used (B) Gating of liver mononuclear cells to analyse CD107a expression on CD8+ T-cells. ns = not significant, * p<0.05.</p