Intrathymic LLMC present lipid droplets and vesicles of different electrondensities and autolipophagossome-like structures.

<p><b>A:</b> Upper panel shows intrathymic LLMC in middle-aged mouse. Semi-thin section fixed with osmium tetroxide. LLMC stained in brownish yellow. Lower panel shows TEM higher magnification of LLMC (false colored yellow) seen in the upper panel containing vesicles of different electrondensities. <b>B:</b> Left panel shows TEM photomicrograph of cytoplasmic lysosomes (indicated by black arrowheads) with lipofuscin and mixed content next to the LLMC nucleus (indicated by a black asterisk). Selected area with autolipophagosome-like multimembrane structure inside a lipid droplet is shown in higher magnification in the inset on the bottom left corner. Middle panel shows vesicles and lipid droplets of LLMC presenting electron-dense membranes typical of autolipophagosome formation. Small panels correspond to higher magnification of selected areas in the middle panel. White arrowheads indicate double membranes of autophagossomes with mixed content in lipid droplets. Right panel shows cytoplasmic lipofuscin granules and distinct electron-dense lipid droplets surrounded by a multi-laminar structure (indicated by a black arrowhead) in a LLMC. <b>C:</b> intrathymic LLMC (white arrow) positively labeled for Beclin-1 (green) and PLIN (red) by immunofluorescence. Cell nuclei labeled with Hoechst (blue). ld: lipid droplets. Scale bars in A, upper panel: 15μm, lower panel: 4μm; B left panel: 1μm, inset: 0.5μm; in B middle panel: 0.5μm; in B small panels: 0.1μm; in B right panel: 0.2μm; C: 15 μm.</p

Pericyte-like LLMC possibly interacting with lymphocytes and endothelial cells in the aging thymus.

<p><b>A:</b> Semi-thin thymic section of middle-aged mice fixed with osmium tetroxide and stained with toluidine blue shows pericyte (green arrowhead) and pericyte-like LLMC (red arrowhead) possibly interacting with endothelial cells. <b>B:</b> TEM photomicrograph shows pericyte-like LLMC (false colored yellow) surrounding an endothelial cell and possibly interacting with various lymphocytes. White asterisk indicates elongated nucleus of a migrating lymphocyte. <b>C-E:</b> Higher magnifications of regions seen in B. Black arrowheads indicate close apposition between membranes of LLMC and thymic lymphocytes. In E, LLMC and endothelial cell share basal membrane. Asterisks indicate area of extracellular matrix that forms basal membrane <b>F:</b> PV-LLMC (false colored yellow) surrounding an endothelial cell and possibly interacting with lymphocytes in an old thymus. <b>G:</b> Higher magnification of selected area in F. Black arrowhead shows possible membrane contact between LLMC and lymphocyte. BV: blood vessel; P: pericyte. Scale bars in A: 15μm; B: 5μm; C, D: 1μm; and E: 2μm; F: 2μm and G: 400nm.</p

Distribution, morphology and density analysis of LLMC in the aging thymus.

<p>Semi-thin thymic section of middle-aged mouse shows LLMC with brownish yellow color due to fixation with osmium-tetroxide. <b>A:</b> intrathymic LLMC presenting fusiform (black arrowhead), globular (yellow arrowhead) or stellate (white arrowhead) morphologies in the thymic parenchyma. <b>B:</b> LLMC adjacent to the thymic septa with fusiform (black arrowheads) and globular (yellow arrowhead) morphologies. <b>C:</b> Fusiform or Stellate pericyte-like LLMC (red arrowhead) visualized around vessels in the thymic parenchyma, adjacent to endothelial cells. <b>D</b>: Schematic representation showing distribution and morphologies of LLMC in the aged thymus. <b>E, F</b> and <b>G</b>: Oil Red O lipid staining of thymic cryosections from mice with 3, 12 and 24 months of age, respectively. ad: adipocyte; Ca: capsule. Scale bars in A: 20μm; B and C: 25μm; D, E, F: 30μm; <b>H</b>: Distribution and density of LLMC in the whole area of 5μm thymic tissue sections from mice with 3, 12 or 24 months of age (n = 4). C/S represents SC-LLMC adjacent to capsule or septa; V represents PV-Perivascular LLMC; and P represents P-LLMC, deeper in the parenchyma. <b>I</b>: Total lymphocyte number in the thymus of mice with 3, 12 or 24 months of age (n = 4, per each age group). Results are shown as mean ± SEM. *p<0.05; **p<0.005; ***p<0.0005.</p

Iron chelation decreases BDNF expression in human neuroblastoma cells.

<p><b>A.</b> SH-SY5Y cells were treated with either vehicle or 100 µM deferoxamine (DFO) for 24 hours. RT-PCR measures of BDNF transcripts show a decrease in total BDNF transcripts (tBDNF, variants 1–14, 16–18 (*p<0.01, n = 4–5). <b>B.</b> Iron chelation did not cause changes in cell survival as assessed by trypan blue exclusion 24 hours post-treatment (n = 5). <b>C.</b> SH-SY5Y cells were transfected with luciferase reporters for the promoter regions of BDNF I–IV and were then treated with 100 µM deferoxamine for 24 hours. No significant differences were observed between control and treated cells (n = 3–4).</p

Heterogeneity of intrathymic LLMC phenotypes.

<p><b>A</b>: Left panel: Preadipocytes immunostained for PPARγ2 in the thymic parenchyma close to the capsule and blood vessels. Small panels show representative photomicrographs of intrathymic lipid-laden preadipocytes immunolabeled for PPARγ2 (green) or PLIN (red); merged images in the right panel. <b>B:</b> Left panel shows thymic brown adipocytes expressing UCP-1 near blood vessels in the parenchyma. Small panels show representative photomicrographs of intrathymic brown adipocytes immunolabeled for UCP1 (green) or PLIN (red); merged images in the right panel <b>C:</b> intrathymic macrophagic LLMC expressing PLIN (red) and Iba 1 (green). <b>D:</b> Pericytic LLMC (white arrowheads) expressing PLIN (red) and NG2 (green) adjacent to the capsule. Insets show higher magnification of perilipin and NG2 staining around the lipid droplets of the pericytic LLMC. <b>E</b>: myofibroblastic LLMC stained with antibody for PLIN (red) and αSMA (green) surrounding a blood vessel. <b>F:</b> Thymic epithelial cells positively stained with antibody anti-Pan cytokeratin (CK, green) did not express PLIN (red). Nuclei stained with DAPI or Hoechst dye (blue). BV: blood vessels, Ca: capsule, S: septae. Scale bars in A, left panels: 15μm; right panel: 7 μm in B, left panels: 25μm; right panel: 5μm; in C: 10μm; in D: 20μm, inset: 10μm; in E: 15μm; in F: 30μm.</p

Intrathymic LLMC possibly interact with lymphocytes, epithelial and endothelial cells.

<p><b>A:</b> Representative photomicrograph of fusiform SC-LLMC (black arrowhead); and globular (yellow arrowhead) or stellate (white arrowhead) P-LLMC in middle-aged mouse thymus section. Tissue fixation with osmium tetroxide reveals lipid-droplets in brownish yellow color. <b>B:</b> TEM photomicrographs show P-LLMC (false colored yellow), adjacent to subcapsular epithelial cells (SCE) (false colored blue), possibly interacting with lymphocytes in the thymic parenchyma. Selected traced area show SCE and LLMC in the limitation between parenchyma and capsule region (Ca); <b>C:</b> Higher magnification of the selected area in panel B shows a LLMC prolongation (false colored yellow) in the basal membrane between the two layers of SCE (false colored blue), in the limit of the thymic parenchyma. <b>D</b>: Semi-thin thymus tissue sections fixed with osmium tetroxide and counterstained with toluidine blue reveal globular P-LLMC (black asterisk) surrounding four lymphocytes. <b>E:</b> TEM photomicrograph showing a lymphocyte surrounded by the P-LLMC in Panel D. Selected traced area shows limiting areas between lymphocytes and LLMC (black asterisk in the nucleus); <b>F</b>: Higher magnification of the selected area in panel E shows the nucleus (white asterisk) and plasma membrane of a lymphocyte (black arrowheads) surrounded by the LLMC with a more electron-lucent cytoplasm. <b>G:</b> A LLMC (false colored yellow) embraces a lymphocyte with a telopode-like prolongation and possibly establishes membrane contact with another lymphocyte in the old thymus. A plasma cell (PL) is located adjacent to an intrathymic LLMC. <b>H:</b> Higher magnification of selected area in G shows possible membrane contact between LLMC and lymphocyte. Black arrowhead indicates close apposition between cell membranes; <b>I:</b> A P-LLMC (false colored yellow) possibly establishing contact with a lymphocyte. Inset on the bottom left corner demonstrates higher magnification of selected area showing close apposition between the cell membranes of LLMC and lymphocyte; <b>J:</b> P-LLMC (false colored yellow) showing a telopode-like prolongation embracing thymic lymphocytes. Telocyte podom-like structure is indicated (black arrowhead). <b>K:</b> TEM photomicrograph showing P-LLMC (false colored yellow) surrounding lymphocytes close to thymic epithelial cell (false colored blue) around the perivascular space (PVS); <b>L:</b> TEM photomicrograph showing LLMC (false colored yellow) in the PVS surrounded by epithelial cells (false colored blue). BV, blood vessel. Ca: capsule; PL: plasma cell; PVS: Perivascular space. Scale bars in A: 20 μm; B, G, J, L: 5 μm; C, E, K: 2 μm; D: 15 μm; F,H: 0.5 μm; I: 1μm and inset in I: 0.2 μm.</p

LLMC may interact with mast cells in the thymus.

<p><b>A:</b> Oil Red O⁺ LLMC (white arrowheads) possibly interacting with toluidine blue-stained metachromatic purple mast cells in the thymus from middle-aged mice. <b>B:</b> TEM photomicrograph shows intrathymic LLMC contacting mast cell (MC). Scale bars in A: 15 μm; and B: 0.5 μm. <b>C:</b> Graphic representation of intrathymic mast cells density in mice with 3, 12 or 24 months of age (n = 4). Results are shown as mean ± SEM. * p<0.05; ** p<0.005.</p

CpKO mice have decreased concentrations of iron in the cerebral cortex and striatum.

<p>Atomic absorption measures of cortical and striatal biopsy punches show significant decreases in iron concentrations in CpKO compared to wild type mice in both the cerebral cortex ( n = 18; *p<0.02) and the striatum (n = 7; *p<0.03).</p

The expression of BDNF is reduced in the cerebral cortex and striatum of ceruloplasmin-deficient mice.

<p><b>A.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2 and 4 in the cortex of the CpKO mouse (*p<0.04, n = 5). <b>B.</b> RT-PCR measures of the 4 BDNF transcripts show a decrease in BDNF transcripts 1, 2, 3 and 4 in the striatum of the CpKO mouse (*p<0.05, n = 5). <b>C.</b> Examples of immunoblots of BDNF protein levels in the cortex and striatum (top panel) and densitometry results showing significant decreases BDNF protein levels (normalized to actin level) using Image J software. (*p<0.03; n = 4 WT and 3 CpKO mice).</p

Rapamycin blocks CXCL12 induced migration and actin polymerization of T cells.

<p>(A) Primary human T cells were labeled with calcein (5 µM) for 1 h in media and washed. Pretreatment of labeled cells was done with or without rapamaycin (100 ng/ml) for 1 h. Cells (1×10⁵ in 100 µl) were placed in the upper wells of 24-well transwell migration chambers with 5 µm pores (Corning, Corning, NY). In the lower wells, either medium alone or CXCL12 (100 ng/ml) was added to a total volume of 600 µl, and the chambers were incubated for 2 hours at 37°C in 5% CO2 incubator. Triplicate well determinations were performed for each treatment. The level of fluorescence of cells migrating across the chamber was assessed using a microfluorimeter. * indicates the value is statistically significant at p<0.05 level (n = 4). PMI indicates ‘percent migration index’. (B) Primary human T cells were treated with CXCL12 for 30 minutes in the presence or absence of pretreated rapamycin. Confocal microscopy was done according to the procedure mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a> to monitor actin polymerization. Red color indicates the actin polymerization. Representative images are shown from each treatment group. (C) Effect of rapamycin and KU-0063794 (KU) on CXCL12-induced cell migration, and effect of MIP3β and TARC in the presence or absence of rapamycin on the migration of resting T cells were performed as described in panel A. (D) Migration assay for CEM cells was performed similarly as primary T cells described above. (E) Dose response curve for rapamycin effect.</p