Kaplan–Meier analysis of cumulative survival.

<p>Kaplan–Meier analysis of cumulative survival.</p

Risk of ventilator-associated conditions: VAC using Cox proportional hazard model (Stepwise Variable Selection) (n = 303).

<p>Risk of ventilator-associated conditions: VAC using Cox proportional hazard model (Stepwise Variable Selection) (n = 303).</p

Patient disposition chart.

<p>CDC, Centers for Disease Control and Prevention; ECMO, extracorporeal membrane oxygenation; ICU, intensive-care unit; MV, mechanical ventilation; PCPS, percutaneous cardiopulmonary support; NPPV, noninvasive positive pressure ventilation; VAC, ventilator-associated conditions</p

Baseline demographics and outcome.

<p>Baseline demographics and outcome.</p

Ventilator-associated conditions univariate analysis.

<p>Ventilator-associated conditions univariate analysis.</p

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

<div><p>Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II <i>in vitro</i>. Irbesartan inhibited not only their <i>in vitro</i> chemotaxis but also <i>in vivo</i> migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.</p></div

Experimental design.

<p>(A) Clones from a CD34^–c-kit⁺Sca-1⁺lineage^– (CD34^–KSL) cell or a total of 2×10⁶ bone marrow-total nucleated cells (BM-TNCs) isolated from enhanced green fluorescent protein (EGFP)-transgenic mice were transplanted into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, mice were intraperitoneally injected with CCl₄ or olive oil twice a week for 12 weeks. (B) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. Ly6C⁺ monocytes, peripheral blood (PB)-TNCs, or a PB-Ly6C⁺ cell-depleted population isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (C) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. They were fed chow containing irbesartan or normal chow for 2 weeks. Ly6C⁺ monocytes isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (D) C57BL/6J-Ly5.1 mice that received 2×10⁶ EGFP⁺ BM-TNCs were fed chow containing irbesartan or normal chow for 6 weeks. In another group of mice, RS504393 or vehicle was subcutaneously administered once a day for 6 weeks. One week after the initiation of irbesartan or RS504393 treatment, CCl₄ treatment was started and continued for 5 weeks.</p

Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies

<div><p>Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these “semaphorin-mimicking” antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved “C” shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies.</p></div

Effect of irbesartan on hematopoietic lineage cell infiltration into the CCl₄-injured pancreas.

<p>EGFP⁺ BM-TNC-transplanted mice were fed a normal chow diet or an irbesartan-containing diet and treated with CCl₄ for 6 weeks. (A) Numbers of EGFP⁺F4/80⁺ monocytic cells, EGFP⁺Ly6G⁺ neutrophils, EGFP⁺B220⁺ B cells, and EGFP⁺CD3ε⁺ T cells in the pancreas of both groups. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed a normal chow. (B) Panels show EGFP as green, CCR2 as red, F4/80 as blue, and the merged images of EGFP, CCR2, F4/80, and DIC images. White triangles, the yellow triangle, and the yellow asterisk indicate EGFP⁺F4/80⁺CCR2⁺ cells, an EGFP⁺F4/80⁺CCR2^– cell, and an EGFP⁺F4/80⁻CCR2⁺ cell, respectively. Scale bars, 30 µm. (C). Numbers of EGFP⁺F4/80⁺CCR2⁺ cells and EGFP⁺F4/80⁺CCR2^– cells in the pancreas of both groups. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed a normal chow. HPF, high power field.</p

Visualization of the mouse PlxnA1_1-6-Fab complex using negative-stain EM.

<p>Purified PlxnA1_1-6 protein alone (A) or in complex with Fab fragments derived from PXB361b (B), PXB693 (C), and PXB727 (D) were separated by size exclusion chromatography on a Superdex 200 column. The upper subpanels show the overlaid chromatograms of PlxnA1_1-6 protein (blue line), PlxnA1_1-6 mixed with Fab (red line), and Fab alone (gray dotted line). SDS-PAGE gel profiles of the collected fractions (thick horizontal bars) are shown in the <i>insets</i>. The peak fraction (asterisk) was subjected to negative-stain EM and image analysis. The molecular size of the collected peak calculated using the positions of standard marker proteins is indicated in the chromatogram, together with the theoretical value in parenthesis. The lower subpanels present a gallery of 20 class averages obtained from ~1,000 selected particles. The number of individual particles represented by each average is shown at the bottom left corner in either white (clockwise) or yellow (counter-clockwise) to indicate their tail curvature orientation. Bar: 100 Å.</p