Chun-Shiang Chung

Fabienne Venet

Yaping Chen

Leslie N Jones

Douglas C Wilson

Carol A Ayala
email: aayala@lifespan.org.

PhD Alfred Ayala

DEFICIENCY OF BID PROTEIN REDUCES SEPSIS-INDUCED APOPTOSIS AND INFLAMMATION, WHILE IMPROVING SEPTIC SURVIVAL

Keywords: Fas-FasL, cytokine, chemokine, Bcl-2, mice 150

Increased apoptotic cell death is believed to play a pathological role in patients with sepsis and experimental animals. Apoptosis can be induced by either a cell death receptor (extrinsic) or a mitochondrial (intrinsic) pathway. Bid, a proapoptotic member of the Bcl-2 family, is thought to mediate the cross talk between the extrinsic and intrinsic pathways of apoptosis; however, little is known about the action of Bid in the development of apoptosis and organ-specific tissue damage/cell death as seen in polymicrobial sepsis. Our results show that after the onset of sepsis, tBid (the active form of Bid) is significantly increased in mitochondrial fractions of the thymus, spleen, Peyer patches, and liver, and that Fas or FasL deficiency blocks Bid activation in various tissues after septic challenge. Increased Bid activation is correlated with increased active caspase-3, caspase-9, and apoptosis during sepsis. Bid-deficient mice exhibit significantly reduced apoptosis in the thymus, spleen, and Peyer patches compared with background mice after sepsis. Furthermore, Biddeficient mice had significantly reduced systemic and local inflammatory cytokine levels and improved survival after sepsis. These data support not only the contribution of Bid to sepsis-induced apoptosis and the onset of septic morbidity/ mortality, but also the existence of a bridge between extrinsic apoptotic signals, e.g., FasL:Fas, TNF:TNFR, and so on, and the intrinsic mitochondrial pathway via Bid-tBid activation during sepsis.

INTRODUCTION

The inability of present therapies to mitigate the devastating effects of sepsis and multiple organ failure in the critically ill patient indicates that more knowledge of the pathophysiology of sepsis is needed to develop effective interventions [START_REF] Smith | Neutrophils, host defense, and inflammation: a double-edged sword[END_REF]. Programmed cell death, or apoptosis, not only is critical for the development and homeostasis in the immune and central nervous systems of many organisms [START_REF] Thompson | Apoptosis in the pathogenesis and treatment of disease[END_REF], but also plays an important role in the pathogenesis of many diseases, such as human immunodeficiency virus immune depression, cancer, autoimmune disorders, neurodegenerative diseases, inflammatory bowel disease, and ischemic injury [START_REF] Zimmermann | The machinery of programmed cell death[END_REF]. During the past decade, growing evidence suggests that apoptosis plays a crucial role in the outcome of experimental animals and patients with sepsis. The increased apoptosis occurring during sepsis suggests a loss of immune and/or nonimmune cell populations, thereby contributing to immunosuppression and subsequent multiple organ failure [START_REF] Hotchkiss | Apoptotic cell death in patients with sepsis, shock and multiple organ dysfunction[END_REF][START_REF] Ayala | Lymphocyte apoptosis in sepsis[END_REF]. Most consistently, increased lymphoid apoptosis has been found in a variety of tissue sites after sepsis; importantly, inhibition of lymphocyte apoptosis has been shown to improve survival in animal models of sepsis (6Y9). Studies have also shown that mice lacking functional lymphocytes (i.e., RAG j/j (10) or +%T j/j mice (11) had a higher mortality when subjected to sepsis induced by cecal ligation and puncture (CLP).

There are two major apoptotic pathways in mammalian cells: cell death receptor (extrinsic) and mitochondrial (intrinsic) pathways. The extrinsic pathway is triggered by death receptors, including Fas (CD95 or APO-1), TNF receptor 1, DR3 (APO-3), TNF-related apoptosis-inducing ligand receptor 1 (DR4), and TNF-related apoptosis-inducing ligand receptor 2 (DR5). Engagement of these receptors with their ligands recruits the Fas-associated death domain, or TNF receptor 1Yassociated death domain, procaspase-8 (Fas-associated death domainYlike IL-1"Yconverting enzyme), and so on, to form a death-inducing signaling complex. This complex can activate caspase-8, which in turn cleaves and activates a signaling cascade of downstream proteases including caspase-3, caspase-6, or caspase-7, leading to apoptosis [START_REF] Thorburn | Death receptor-induced cell killing[END_REF].

The intrinsic mitochondrial pathway is regulated by B-cell CCl/lymphoma 2 (Bcl-2) family members that include both antiapoptotic (e.g., Bcl-2 and Bcl-2X L ) and proapoptotic proteins. All of the members share up to four conserved Bcl-2 homology (BH) domains 1 to 4. Proapoptotic Bcl-2 members are further divided into multiple BH domainYcontaining proteins, such as Bax and Bak, and BH-3-only proteins, such as Bid, Bim, and Bad, and so on. In response to various stress stimuli, proapoptotic members are activated and induce the permeabilization of the outer mitochondrial membrane where they provoke permeability transition and the release of apoptogenic factors including cytochrome c. Cytosolic cytochrome c binds to apoptosis proteaseYactivating factor 1, which activates procaspase-9 and subsequently initiates a downstream caspase cascade resulting in cell death [START_REF] Borner | The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions[END_REF].

Although these two apoptotic pathways can act independently, it seems that there is cross talk between the extrinsic and intrinsic pathways. It has been proposed that FasL-FasYdriven B-extrinsic-[ or B-intrinsic-[ apoptosis depends on whether it takes place in a type I or type II cell [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. Type I cells (e.g., B lymphoblasts) seem to activate large amounts of caspase-8 by the death-inducing signaling complex and directly activate downstream caspase-3 and/or other proteases resulting in apoptosis. These cells bypass the mitochondria in Fas-mediated apoptosis, which cannot be blocked by Bcl-2 overexpression [START_REF] Strasser | Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis[END_REF]. Alternatively, type II cells (e.g., Jurkat T cells or hepatocytes) seem to produce less active caspase-8, and the mitochondria are needed to trigger downstream caspase activation for apoptosis, which can be inhibited by Bcl-2 or Bcl-X L [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. The nature of the transition of apoptotic signals from death receptors to the mitochondria became clear when Bid protein was discovered. Upon the engagement of death receptors and their ligands, caspase-8 is activated and cleaves cytoplasmic Bid into truncated Bid (tBid), which then translocates to the mitochondria where it promotes Bax and Bak activation and the subsequent intrinsic death pathway [START_REF] Li | Cleavage of BID by Caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF]. Studies have shown that hepatocytes lacking Bid are resistant to Fas or TNF-!Yinduced cell death [START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF][START_REF] Guicciardi | Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alphaYinduced hepatocyte apoptosis[END_REF]. Furthermore, wild-type mice treated with Fas agonist antibody (clone Jo2) exhibited massive hepatocyte apoptosis and severe liver injury; a response not observed in bid-deficient mice [START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF]. Several studies, including ours, have demonstrated that immune cell apoptosis is increased in a variety of tissue sites during sepsis (4, 19Y21), and that organ damage and mortality associated with sepsis in mouse models are at least in part caused by the activation of the Fas-FasL signaling pathway [START_REF] Chung | Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis[END_REF]. Our previous studies demonstrated that the inhibition of FasL-Fas signaling caused by FasL gene deficiency, delayed (12 h post-CLP) treatment with FasL-binding protein (FasFP), or siRNA silencing of Fas not only protected some immune cell populations from apoptosis, but also reduced the morbidity and mortality seen in sepsis [START_REF] Wesche | In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice[END_REF][START_REF] Chung | Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis[END_REF][START_REF] Chung | Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity[END_REF]. However, the basis for the cross talk between Fas-mediated and mitochondrial-driven apoptosis during sepsis remains largely unclear. Although much is known about the action of Bid in various cell lines or death receptorYinduced mortality/liver injury models, its role in immune versus nonimmune cell apoptosis as seen in sepsis is less well characterized. Therefore, in the current study, we examined the impact of Bid protein activation on sepsis-induced mortality, apoptosis, and inflammation.

MATERIALS AND METHODS

Animals

The studies described here were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Rhode Island Hospital Committee on Animal Use and Care. Bid-deficient mice (Bid j/j , which is generated on a C57BL/6 background) were a gift from the late Dr Stanley Korsmeyer [START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF]. Fas receptor (lpr, B6.MRL-Tnfrsf6lpr/J)Y or FasL (gld, B6Smn.C3-Faslgld/J)deficient and the background control C57BL/6 mice were purchased from Jackson's laboratories (Bar Harbor, Me). Mice were age (8Y10 weeks) and sex (male) matched for all experiments.

Sepsis model of CLP

Polymicrobial sepsis was induced in mice with the CLP model as previously described [START_REF] Chung | Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?[END_REF]. Briefly, mice were anesthetized with isoflurane (Abbott Laboratory, Chicago, Ill); their abdomen was shaved and scrubbed with Betadine. A midline incision (1.0Y1.5 cm) was made below the diaphragm to expose the cecum. The cecum was ligated, punctured twice with a 22gauge needle, and gently compressed to extrude a small amount of fecal content through the punctures. The cecum was returned to the abdomen, and the incision was closed in layers with 6-0 Ethilon suture (Ethicon, Inc, Somerville, NJ). The animals then were resuscitated with 1.0 mL of Ringer's lactate solution by s.c. injection. The sham controls were subjected to the same surgical procedure, i.e., laparotomy and cecal isolation, but the cecum was neither ligated nor punctured. For survival studies, C57BL/6 and Bid j/j mice were subjected to CLP, and survival was monitored for 10 days.

Sample preparation

Mice were killed, and the thymus, spleen, Peyer patches, liver, and lung were collected at various time points. Thymocytes and splenocytes were obtained by gently grinding the organs between frosted glass slides according to the methods previously described [START_REF] Chung | SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis[END_REF]. Contaminating red blood cells were lysed with a hypotonic solution. Cells were then centrifuged, counted (by trypan blue exclusion), and either lysed in lysis buffer (50 mM Tris-HCl, pH7.5, 5 mM EDTA, 150 mM NaCl, 10 mM sodium phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 0.5% Triton X-100, and protease inhibitor cocktail) (Sigma, St Louis, Mo) or used for other analyses. Peyer patches, liver, and lung samples were homogenized in lysis buffer using a PowerGen 125 homogenizer (Fisher Scientific, Pittsburgh, Pa) and centrifuged. Protein content of cell/tissue lysate was determined using the BIO-RAD protein assay reagents (Hercules, Calif ).

Preparation of mitochondrial and cytosolic fractions

Mitochondria were isolated using the Mitochondrial Fractionation kit according to the manufacturer's instruction (Active Motif, Carlsbad, Calif ) [START_REF] Chung | SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis[END_REF]. In brief, cells/tissues were collected as previously described, washed with phosphate buffered saline, resuspended in cytosolic buffer, incubated, and homogenized in a Dounce homogenizer (Wheaton, Millville, NJ) 30 to 50 strokes on ice. Nuclei and unbroken cells were pelleted by centrifugation at 800g for 20 min at 4-C. The supernatant was centrifuged again at 16,000g for 20 min at 4-C to separate the supernatant (cytosolic fraction) and mitochondrial pellet, which was then washed and resuspended in lysis buffer. Protein content of cytosolic and mitochondrial fractions was measured as previously described.

Western blot analysis

The presence of Bid, tBid, active caspase-3, and active caspase-9 in samples was assessed by Western blot as previously described [START_REF] Chung | SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis[END_REF]. In brief, samples were boiled, separated on 16% denaturing polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (Novex, San Diego, Calif ). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 and incubated with antimouse Bid and tBid (MAB860, R & D System, Minneapolis, Minn)Yactive caspase-3 (which detects cleaved caspase-3 p17 and p19, but does not recognize full-length caspase-3 or other cleaved caspases) (9664S, Cell Signaling Technology, Danvers, Mass) or Yactive caspase-9 (9509, Cell Signaling) antibodies overnight at 4-C. Membranes were washed and incubated with horseradish peroxidaseYconjugated secondary antibody. After washing, proteins were visualized by enhanced chemiluminescence detection and densitometrically assessed by Alpha-Innotech image analyzer. VDAC1 (Novus Biologicals, Littleton, Colo) was used as a mitochondrial loading control, and "-Actin (Abcam, Cambridge, Mass) was used as a cytosolic loading control.

Assessment of apoptosis

Flow cytometric analysis-Thymocytes and splenocytes were stained with propidium iodide ([PI] 0.1% NP-40, 0.1% sodium citrate, and 50 mg/mL PI; Sigma) or terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) reagent (In Situ Cell Death Detection Kit, Fluorescin, Roche Applied Science, Indianapolis, Ind) and analyzed by BD FACSArray Bioanalyzer [START_REF] Chung | SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis[END_REF].

Microscopic examination of hematoxylin and eosin, TUNEL, or active caspase-3 tissue section staining-The thymus, spleen, lung, liver, and Peyer patches were excised and fixed in 10% buffered formaldehyde, and paraffinembedded tissue sections were prepared. Hematoxylin and eosin (H & E) staining was performed by Core Research Laboratories at Rhode Island Hospital. The TUNEL staining was performed according to the manufacturer's instructions (Roche). Active caspase-3 was detected by immunohistochemical staining as previously described [START_REF] Perl | Silencing of Fas-but not caspase-8 in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis[END_REF]. In brief, paraffin-embedded tissue slides were dewaxed, rehydrated, and incubated with 3% H 2 O 2 in phosphate buffered saline to block endogenous peroxidase followed by nonspecific blocking with 5% normal goat serum. After blocking, the slides were incubated with the antiYactive caspase-3 antibody, washed, incubated with a biotinylated goat-antirabbit antibody (BD Biosciences, San Diego, Calif ), followed by a streptavidin-biotin peroxidase (Sigma). Chromogenic peroxidase substrate, metal-enhanced diaminobenzidine (Pierce, Rockford, Ill), was added to visualize active caspase-3 staining. Methyl green was used to counterstain nuclei. The slides were examined under a light or fluorescent microscope for evidence of apoptosis by blinded observers. Image processing and analysis for TUNEL and caspase-3 staining was performed using iVision (BioVision Technologies, Exton, Pa). Positive staining was expressed as percent area stained over total area (% area stained).

Transmission electron microscopy-The thymus, spleen, and liver were excised, submerged in half-strength Karnovsky fixative in 0.15 M sodium cacodylate buffer at 4-C, and immediately prepared into 1-mm 3 blocks and allowed to fix overnight. After fixation, the tissue was washed and postfixed in 1% osmium tetroxide (OsO 4 ) for 1 h at 4-C. After postfixation, samples were rinsed and dehydrated in a graded series of acetone and embedded in Spurr epoxy resin. Semithin sections (1 2m) were prepared using a Reichert Ultracut S microtome, stained with methylene blueYazure II and evaluated for areas of interest. Ultrathin sections (50Y60 nm) were then prepared, retrieved onto 300-mesh copper grids, and contrasted with uranyl acetate and lead citrate. Sections were examined using a Morgagni 268 transmission electron microscope, and images were collected with an AMT Advantage 542 CCD camera system.

Quantification of cytokine/chemokine levels in plasma or peritoneal fluids and liver enzyme ALT in plasma

Blood was collected in heparinized syringes by cardiac puncture, and plasma was obtained by centrifugation and stored at j70-C until analyzed. Peritoneal fluids were obtained from mice by lavage, clarified by centrifugation, and stored at j70-C as previously described [START_REF] Chung | Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity[END_REF]. Mouse TNF-!, monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-10 levels were determined in plasma or peritoneal fluids using cytometric bead array technique (BD Cytometric Bead Array Mouse Inflammation Kit, BD Biosciences) according to the manufacturer's instructions [START_REF] Perl | Silencing of Fas-but not caspase-8 in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis[END_REF] dilution and incubated for 2 h at room temperature in the dark. Samples were washed with wash buffer and centrifuged at 200g at room temperature for 5 min. Supernatant was discarded; samples were resuspended in wash buffer and analyzed on a BD FACSArray bioanalyzer. Plasma levels of alanine aminotransferase (ALT), as an index of liver injury, were determined using an assay kit (Pointe Scientific, Inc, Canton, Mich) according to the manufacturer's instructions.

Presentation of data and statistical analysis

The results are presented as a mean T SEM for each group. Mann-Whitney U test was used to analyze the comparison between two groups. For data comparing more than two groups, a one-way ANOVA was executed followed by a Student-Newman-Keuls test to determine the differences. The survival data were compared using the log-rank survival analysis. P e 0.05 was considered statistically significant.

RESULTS

Bid is activated/cleaved and tBid is translocated from the cytosol to the mitochondria after the onset of sepsis It has been clearly documented that upon activation, cytosolic full-length Bid is cleaved into tBid by active caspase-8 [START_REF] Li | Cleavage of BID by Caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF]. tBid translocates to the mitochondrial membrane and activates the caspase-mediated apoptotic cascade. To verify that there was activation of Bid after the onset of sepsis, we initially did a time course using thymocytes and splenocytes. C57BL/6 mice were subjected to sham or CLP procedures, and cells were harvested 4, 24, and 48 h postsurgery for assessment of Bid (cytosolic fractions) and tBid (mitochondrial fractions) by immunoblotting. At 4 h post-CLP, tBid was detected in splenocytes (no difference was observed between sham and septic mice), but not in thymocytes. tBid expression peaked at 24 h and was barely detectable at 48 h after CLP in both sites (Fig. 1A). Based on these data, 24 h was chosen for assessment in most of the subsequent experiments. We observed that in C57BL/6 mice, polymicrobial sepsis induced an increase in activation of Bid and translocation of tBid from the cytoplasm to mitochondria in Peyer patches and liver as well as the thymocytes and splenocytes, but not in the lung, when compared with sham controls at 24 h post-CLP (Fig. 1B). Because Bid has been identified as a critical protein in connecting the death receptor (Fas, TNF receptor 1, etc) and mitochondrial death pathways, and as our laboratory has previously shown that the signaling through the Fas/FasL pathway contributes to septic morbidity/mortality [START_REF] Wesche | In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice[END_REF][START_REF] Chung | Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis[END_REF][START_REF] Chung | Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity[END_REF], we investigated the effects of Fas-mediated pathway on Bid activation after septic challenge. Bid and tBid were determined by immunoblotting of cells/tissues taken from Fas j/j (lpr) or FasL j/j (gld) mice 24 h after surgery. Interestingly, it seems that when the Fas-FasL pathway is disrupted through Fas or FasL deficiency, the activation of Bid is differentially affected in various tissues taken from lpr or gld mice (Fig. 1B). Our results show that not only was Bid activation after sepsis diminished in thymocytes, splenocytes, and the livers of lpr mice as compared with C57BL/6 CLP mice, but this activation in lpr sham animals was also reduced as compared with C57BL/6 sham mice. In gld mice, septic insult did not lead to an increase in Bid activation/translocation in all cells/tissues tested as seen in C57BL/6 CLP mice. However, unlike lpr mice, the basal mitochondrial levels of tBid in gld sham and CLP animals were generally comparable to C57BL/6 shams, with the exception of the liver where tBid activation in both gld sham and CLP mice was comparable to that of C57BL/6 CLP mice (Fig. 1B).

Bid deficiency reduces septic mortality

To determine whether deficiency of proapoptotic Bid protein could provide protection against septic mortality, C57BL/6 and Bid j/j mice were subjected to CLP, and their survival was monitored for 10 days (Fig. 2). The survival rate for the C57BL/6 background mice steadily declined during FIG. 2. Bid deficiency improved survival after sepsis. C57BL/6 background and Bid-deficient mice were subjected to CLP, and 10-day survival was recorded. Bid j/j mice showed an improvement in survival when compared with C57BL/6 background mice, and the difference was statistically significant (P G 0.05, log-rank survival analysis; n = 13Y17 mice per group). the first 7 days to approximately 30%. This was significantly different from the Bid j/j mice, which exhibited slower mortality that resulted in a survival rate of approximately 78% from day 4 until day 10.

Bid deficiency reduces sepsis-induced apoptosis in different cells/tissues

To compare the extent of sepsis-induced apoptosis between C57BL/6 and Bid j/j mice, several methods were used. Flow cytometric assessment of apoptotic DNA fragmentation was performed using the DNA-binding agent propidium iodide and TUNEL staining. A significant increase in apoptosis of thymocytes and splenocytes was observed in both the septic C57BL/6 and Bid j/j mice when compared with their respective shams at 24 h post-CLP (Fig. 3). However, the extent of apoptosis in cells taken from septic Bid j/j mice was significantly lower than that from septic C57BL/6 mice. In addition to DNA analysis, increased active capsase-3 was detected by Western blot analysis, verifying the increased apoptosis in these cells. The results also show that Bid deficiency reduced caspase-3 activation in the spleen, thymus, and Peyer patches 24 h after sepsis (Fig. 4A). In addition, we examined the kinetics of caspase-3 activation in thymocytes and splenocytes at 4 and 48 h post-CLP in the C57BL/6 mice. Figure 4B shows an increase in active caspase-3 in thymocytes but no changes in splenocytes when compared with shams 4 h after sepsis. Similar to Bid activation, active caspase-3 levels peaked at 24 h and diminished to lower than sham controls 48 h after CLP. These results suggest that the kinetics of caspase-3 activation is slightly different between thymocytes and splenocytes during sepsis; however, both cell types showed a peak of caspase-3 activation at 24 h post-CLP. Because caspase-9 is the central initiator caspase of the intrinsic death pathway, we also determined whether the activation of caspase-9 was affected by Bid deficiency after sepsis. Immunoblot results demonstrated that although active caspase-9 was increased 24 h after sepsis in C57BL/6 background mice, no such FIG. 4. Active caspase-3 levels in Bid j/j mice were reduced after sepsis. A, thymocytes, splenocytes, and Peyer patches were collected from C57BL/6 background or Bid j/j mice 24 h after subjected to sham or CLP procedures. Active caspase-3 (p17/p19) was determined by Western blot analyses (left panels) and semiquantitated by densitometry, expressed as integrated density (IDT) values of tBid relative to IDT values of "-actin (right panels). B, Kinetics of caspase-3 activation in C57BL/6 mice after CLP. *P G 0.05 vs. respective sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 4 to 8 mice per group. FIG. 5. Deficiency of Bid reduced active caspase-9 levels in thymocytes and splenocytes after sepsis. Thymocytes, splenocytes, and the liver were harvested from C57BL/6 background or Bid j/j mice 24 h after subjected to sham or CLP procedures. Activate caspase-9 was determined by Western blot analyses (left panels) and semiquantitated by densitometry, expressed as integrated density (IDT) values of tBid relative to IDT values of "-actin (right panels). *P G 0.05 vs. respective sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 4 to 8 mice per group. FIG. 6. Representative H & E staining of tissue sections for detection of apoptosis by morphology examined under light microscopy. C57BL/6 background or Bid j/j mice were subjected to sham or CLP procedures, and 24 h later, paraffin-embedded tissue sections were prepared. Sham animals (C57BL/6 and Bid j/j ) display normal histology of thymus (A), spleen (D), and Peyer patches (G). Although septic C57BL/6 mice exhibited typical apoptotic morphology (arrows) in all three tissues (B, thymus; E, spleen; H, Peyer patches) when compared with sham-operated mice, Bid-deficient animals showed fewer apoptotic cells in these tissues after sepsis (C, thymus; F, spleen; I, Peyer patches). Original magnification Â400.

increase was observed in the spleen and thymus of septic Bid j/j mice (Fig. 5). Alternatively, caspase-9 was equally activated in the septic livers of both C57BL/6 and Bid j/j mice (Fig. 5).

To further confirm that the results observed in the isolated cells/tissues was representative of in vivo changes at the tissue level, we sought morphological evidence of apoptosis in the thymus, spleen, Peyer patch, and liver using light and electron microscopy. Figures 6 and7 illustrate typical light microscopic pictures of H & EYstained tissue sections and electron micrographs, respectively. A marked increase in apoptotic cells, characterized by cell shrinkage, pyknosis, and deformation of the cell membrane, was seen in the thymus (Figs. 6B and 7B for C57BL/6, and 6C and 7C for Bid j/j ), spleen (Figs. 6E and7E for C57BL/6, and 6F and 7F for Bid j/j Fig. 6, H [C57BL/6] and I [Bid j/j ]) taken from C57BL/6 and Bid j/j mice at 24 h after sepsis as compared with shams (note: because there was no difference between C57BL/6 and Bid j/j shams, one set of sham was used as representative in Figures 6, 7, 8, and9). However, these types of morphological changes were much less evident in septic Bid j/j mice (Figs. 6, C, F, andI, and7, C and F) compared with septic C57BL/6 mice (Figs. 6, B, E, andH, and7, B andE). Interestingly, in the liver, the electron micrographs indicated relatively normal mitochondrial morphology (see insets) and minimum signs of overt apoptosis after sepsis in either mouse strain (Fig. 7, H [C57BL/6] and J [Bid j/j ]) compared with their respective shams (Fig. 7, G [C57BL/6] and I [Bid j/j ]). The most apparent finding was a loss of glycogen stores and a substantial increase in lipid-like vesicles (which may be related to sepsis-induced steatosis or fatty change often seen in patients) encountered in the hepatocytes of both septic C57BL/6 and Bid j/j mice. Of note, although less obvious compared with the septic mouse hepatocytes, the Bid j/j sham mice seem to have more of these lipidlike droplets than the background shams (Fig. 7, G andI). This could be associated with the unexpected role of tBid in transfer and recycling of mitochondrial phospholipids [START_REF] Esposti | Bid, a widely expressed proapoptotic protein of Bcl-2 family, displays lipid transfer activity[END_REF] and in the regulation of lipid "-oxidation [START_REF] Giordano | tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1[END_REF].

Immunohistochemical staining for active caspase-3 (Fig. 8) and TUNEL (Fig. 9) were also performed to further assess the extent of apoptosis. We observed results consistent with the earlier morphological assessment. A substantial increase in active caspase-3 (thymus, spleen, and Peyer patches) and TUNEL (thymus and spleen) staining were observed in both C57BL/6 (Figs. 8, B, E, andH, and9, B andE) and Bid j/j (Figs. 8,C, F, and I, and 9, C and F) mice 24 h after the induction of sepsis as compared with shams (Figs. 8, A, D, andG, and9, A andD). However, Bid j/j mice had a reduction in sepsis-induced apoptosis in the tissues tested. As seen with H & E staining, we did not observe obvious changes in active caspase-3 or TUNEL staining in the liver (data not shown). In addition, although we found that sepsis induced a significant elevation in plasma levels of the liver enzyme ALT in C57BL/6 and Bid j/j mice when compared with their respective shams, ALT levels were not different between the septic mice of both strains (C57BL/6 sham, 8.7 T 2.3 IU/mL; C57BL/6 CLP, 35.6 T 6.7 IU/mL; Bid j/j sham, 10.6 T 2.8 IU/mL; Bid j/j CLP, 45.7 T 16.1 IU/ mL). This suggests that other mechanisms (necrotic death and/or autophagy) could contribute to cell death in the liver in our model of sepsis.

Bid deficiency reduces sepsis-induced systemic and local proinflammatory and anti-inflammatory cytokines/chemokines

Plasma cytokines/chemokines were measured in sham and septic mice 12 and 24 h after surgery. Plasma levels of TNF-!, MCP-1, IL-10, and IL-6 were markedly increased in C57BL/6 mice 12 (Fig. 10, A andB) and 24 h (Fig. 10, C andD) after the onset of sepsis compared with sham mice. However, Bid j/j FIG. 7. Representative electron micrographs of tissue sections for morphological detection of apoptosis. C57BL/6 background or Bid j/j mice were subjected to sham or CLP procedures, and 24 h later, tissue sections were prepared for electron microscopy. Sham animals (C57BL/6 and Bid j/j ) display normal histology of thymus (A) and spleen (D). Although septic C57BL/6 mice exhibited a typical cluster of apoptotic cells in these tissues (B, thymus; E, spleen) when compared with sham-operated mice, Bid-deficient animals showed fewer apoptotic cells after sepsis (C, thymus; F, spleen). Original magnification Â4,400. In the liver, the electron micrographs showed relatively normal mitochondria (inset, original magnification Â5,540) and minimum signs of apoptosis after sepsis in either C57BL/6 (H) or Bid j/j (J) mice compared with their shams (G, C57BL/6; I, Bid j/j ). However, Bid j/j mice seem to have more of the lipid-like droplets (arrows) (I, sham; J, CLP). Original magnification Â2,800.

mice did not respond to septic challenge as vigorously as C57BL/6 mice. At 12 h after CLP, although plasma levels of TNF-!, MCP-1, and IL-6 (but not IL-10) in Bid j/j septic mice were significantly elevated compared with their shams, IL-6 and IL-10 levels were markedly lower and TNF-! and MCP-1 were slightly lower than those in C57BL/6 septic mice. By 24 h after CLP, all four cytokine levels in plasma of Bid j/j mice were significantly lower compared with C57BL/6 mice. Likewise, Bid deficiency reduced local cytokine/chemokine production in the peritoneal cavity of septic mice. MCP-1 and IL-10 levels at 12 h (Fig. 10, E andF) and TNF-!, MCP-1, and IL-10 levels at 24 h (Fig. 10, G andH) in peritoneal fluids from Bid j/j septic mice were significantly lower than those in septic C57BL/6 mice.

DISCUSSION

Many studies, including experimental animal and clinical studies, have demonstrated that apoptotic cell death via either the death receptor or mitochondrial pathway plays a critical role in sepsis (4, 19Y21). However, the function of BH3-only proapoptotic protein Bid, which is thought to be a key protein connecting the extrinsic to the intrinsic cell death pathway, in sepsis-induced apoptosis has not been clearly defined. In the current study, we examined the contribution of Bid protein FIG. 8. Representative immunohistochemical staining of active caspase-3 in tissue sections. C57BL/6 background or Bid j/j mice were subjected to sham or CLP procedures, and 24 h later, paraffin-embedded tissue sections were prepared and stained with antiYcaspase-3. Sham animals display no or slight staining of active caspase-3 in the thymus (A), spleen (D), and Peyer patches (G). Although septic C57BL/6 mice exhibited extensive active caspase-3 staining (arrows) in all three tissues (B, thymus; E, spleen; H, Peyer patches) when compared with sham-operated mice, Bid-deficient animals showed less staining in these tissues after sepsis (C, thymus; F, spleen; I, Peyer patches). Original magnification Â400. Quantification of the images was processed and analyzed using iVision software. Positive staining was defined through thresholding, the resulting images were analyzed, and data were expressed as percent area stained over total area (% area stained). *P G 0.05 vs. sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 3 per group.

to apoptosis in several tissues, its impact on mortality, and its effect on inflammation in response to a polymicrobial septic challenge (CLP) in mice.

Although much is known about the action of Bid in various cell lines and acute death receptorYinduced mortality/liver injury [START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF][START_REF] Guicciardi | Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alphaYinduced hepatocyte apoptosis[END_REF][START_REF] Chen | Bid-independent mitochondrial activation in tumor necrosis factor alpha-induced apoptosis and liver injury[END_REF], its role in sepsis is less well characterized. A recent clinical study demonstrated that gene expression of proapoptotic Bcl-2 family members, including Bim and Bid, was upregulated, and antiapoptotic members were downregulated in the peripheral blood from patients with severe sepsis [START_REF] Stuber | Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis[END_REF]. However, because of the nature of such observational clinical data, whether Bid gene expression in these patient samples contributed to sepsis pathology could not be determined.

To investigate the involvement of Bid in the increased apoptosis as seen in sepsis, we initially compared the changes in Bid activation in several organs taken from sham or septic C57BL/6 mice at 4, 24, and 48 h after procedures. Our results demonstrated that there is a significant increase in Bid cleavage/activation (tBid) and translocation of tBid to mitochondria of cells within the thymus, spleen, Peyer patches, and liver 24 h after the onset of polymicrobial sepsis induced by CLP. Because Bid is a unique BH3-only member and is thought to be required for the connection of extrinsic cell death initiated by the Fas-FasL pathway and the activation of the intrinsic mitochondrial pathway, we chose to use lpr (Fas-deficient) or gld (FasL-deficient) mice to examine the effects of Fas-FasL signaling on Bid activation during sepsis. We found that inhibition of the Fas-FasL pathway did affect Bid activation during sepsis. The activation of Bid was completely blocked after septic insult, as indicated by its translocation to the mitochondria in all tissues assessed from sham or CLP lpr mice when compared with C57BL/6 wildtype mice. However, the degree of Bid activation in gld mice seemed to be somewhat more tissue dependent. Although Bid activation caused by CLP was still blunted in the thymus, spleen, and Peyer patches taken from gld mice, we observed higher basal levels, equivalent to C57BL/6 sham level, of tBid in both sham and CLP mice. Interestingly, Bid activation in the liver of both gld sham and CLP mice was as high as septic C57BL/6 mice. These results suggest that FasL-independent pathways of basal Bid activation may exist in the liver. There are, however, other possible explanations: (a) in the absence of having a functional FasL protein, compensatory mechanisms involving other death receptor ligands are upregulated to account for this higher basal mitochondrial level of tBid; or (b) it is also possible that besides caspase-8, there are other proteases activated during sepsis including caspase-2, caspase-3, caspase-10, or granzyme B, which may serve to cleave/ activate Bid as well [START_REF] Yin | Bid, a BH3-only multi-functional molecule, is at the cross road of life and death[END_REF].

As a pathological index of sepsis, apoptosis has been observed in septic patients and experimental animals (4, 19Y21). Using the CLP model of sepsis, we found that deletion of the Bid gene significantly reduced apoptosis in the spleen, thymus, and Peyer patches, but this was less evident in the lung and liver. From our past experiences, sepsis produced by CLP had minimal effects on lung function [START_REF] Ayala | Shock-induced PMN mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency[END_REF]; therefore, we did not anticipate finding substantial changes in lung tissue apoptosis 24 h after CLP. However, other studies have indicated that Bid deficiency ameliorated lung injury and provided early protection from mortality in a mouse model of endotoxemia [START_REF] Wang | The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death[END_REF], suggesting a role for Bid in lung cell apoptosis. Unfortunately, although endotoxic shock is an excellent model of acute inflammatory and/or toxic shock, it is not considered FIG. 9. Representative immunohistochemical TUNEL staining in tissue sections. C57BL/6 background or Bid j/j mice were subjected to sham or CLP procedures, and 24 h later, paraffin-embedded tissue sections were prepared and stained with TUNEL. Sham animals display no or slight staining of TUNEL in thymus (A) and spleen (D). Although septic C57BL/6 mice exhibited extensive typical apoptotic morphology in tissues (B, thymus; E, spleen) when compared with sham-operated mice, Bid-deficient animals showed less TUNEL staining in these tissues after sepsis (C, thymus; F, spleen). Original magnification Â400. Quantification of the images was processed and analyzed using iVision software. Positive staining was defined through thresholding, the resulting images were analyzed, and data were expressed as percent area stained over total area (% area stained). *P G 0.05 vs. sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 3 per group. by many to be an adequate model of sepsis as the levels of endotoxin used are substantially higher than those encountered in CLP or in septic patients [see review in [START_REF] Ayala | Lymphocyte apoptosis in sepsis[END_REF]]. With respect to the liver, neither background nor Bid j/j mice had obvious evidence of apoptosis histologically (light or electron microscopy) after sepsis, although tBid translocation to the mitochondria was increased in the livers of septic background C57BL/6 mice. In fact, the livers taken from septic Bid j/j mice exhibited similar levels of active caspase-9 and more lipid-like droplets in electron micrographs when compared with septic C57BL/6 mice. The reason for this is not clear; however, this is possibly caused by the activation of diverse downstream signaling pathways. It seems that Bid is essential for hepatocyte apoptosis. Deletion of Bid has been shown to result in an effective blockade of Fas-mediated hepatocyte apoptosis, liver damage, and mortality using an agonist anti-Fas antibody (Jo2) treatment in vivo [START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF]. Nearly 90% of wildtype mice die within 4 h in this acute model of liver injury. However, the same laboratory also demonstrated that although Bid-deficient mice were significantly more resistant to TNF-!Ymediated (an alternative death receptor activator) liver injury and lethality, it only temporarily delayed hepatocyte cell death, liver injury, and mortality (8 h as compared with 4 h) [START_REF] Chen | Bid-independent mitochondrial activation in tumor necrosis factor alpha-induced apoptosis and liver injury[END_REF]. Hence, sepsis induced by CLP, which is a complex stimulus, affects most of the organs and activates numerous biological responses and mediators that might affect the apparent actions of Bid on apoptosis as it occurs in various tissues/ organs. We noted that after CLP, Bid deficiency only partially suppressed apoptosis in various tissues. This is not unexpected as Bid-independent mitochondrial activation can also be in-duced by other mechanisms including reactive oxygen species accumulation, c-Jun N-terminal kinase activation [START_REF] De Smaele | Induction of gadd45" by NF-.B downregulates pro-apoptotic JNK signaling[END_REF] and reactive oxygen speciesYinduced mitochondrial permeability transition [START_REF] Zhao | Bid activates multiple mitochondrial apoptotic mechanisms in primary hepatocytes after death receptor engagement[END_REF]. Unlike the anti-FasYinduced liver injury model, Bid deficiency seems to have less effect on the liver in our model of sepsis induced by CLP. Despite the lack of morphological evidence of obvious apoptotic cell death in the liver after sepsis in both mouse strains, we observed elevated plasma levels of ALT, an index of liver injury, in both septic C57BL/6 and Bid j/j mice. This suggests that other cell death mechanisms (necrotic death and/or autophagy) may be involved in sepsis-induced liver injury. Conversely, the decrease in sepsis-induced apoptosis in Bid j/j mice was evident mainly in lymphoid organs such as thymus, spleen, and Peyer patches, and less overt in nonlymphoid tissues such as the lung or liver. Interestingly, previous studies from our laboratory have documented that sepsis-induced thymocyte apoptosis is steroid driven and is not dependent on endotoxin, TNF-!, or Fas pathways [START_REF] Ayala | Does Fas ligand or endotoxin contribute to thymic apoptosis during polymicrobial sepsis?[END_REF]. We believe that the observation of reduced thymus apoptosis in septic Bid j/j mice is likely not related to Fas activation. Indeed, studies have shown that glucocorticoid, which increases during stress/injury, can induce the cleavage/ activation of Bid in cultured lymphocytes [START_REF] Planey | Inhibition of glucocorticoid-induced apoptosis in 697 pre-B lymphocytes by the mineralocorticoid receptor N-terminal domain[END_REF] and eosinophils [START_REF] Segal | Bid activation during induction of extrinsic and intrinsic apoptosis in eosinophils[END_REF]. tBid can then activate the mitochondrial death pathway and induce steroid-mediated apoptosis in leukocytes.

As previously mentioned, many animal studies have shown that overexpression of antiapoptotic or deletion/knockdown of proapoptotic genes can improve septic survival. Our previous studies also indicate that blocking Fas-FasL signaling using Fas fusion protein or Fas siRNA reduced septic mortality FIG. 10. Cytokine/chemokine levels in plasma (circulation) and peritoneal cavity (local). C57BL/6 background or Bid j/j mice were subjected to sham or CLP procedures; 12 and 24 h later, plasma and peritoneal fluid were collected for measurement of cytokine/chemokine concentration (pg/mL) by cytometric bead array. Plasma levels of TNF-!, MCP-1, IL-10, and IL-6 were significantly increased in septic C57BL/6 mice at 12 (A, B) and 24 (C, D) h compared with their shams. Although TNF-!, MCP-1, and IL-6 at 12 h and MCP-1 and IL-6 at 24 h in septic Bid j/j mice were also increased compared with shams, these plasma cytokine/chemokine levels were significantly decreased compared with C57BL/6 mice at 24 h and to a lesser extent at 12 h after CLP. Bid deficiency also reduced local cytokine/chemokine production in the peritoneal cavity after CLP. The MCP-1 and IL-10 levels at 12 h (E, F) and TNF-!, MCP-1, and IL-10 levels at 24 h (G, H) in peritoneal fluids from Bid j/j septic mice were significantly lower than those in septic C57BL/6 mice. *P G 0.05 vs. respective sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 6 to 8 mice per group. [START_REF] Wesche | In vivo delivery of caspase 8 or Fas siRNA improves the survival of septic mice[END_REF][START_REF] Chung | Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis[END_REF][START_REF] Chung | Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity[END_REF]. In agreement with these studies, reduced apoptosis is associated with an improved survival rate in Bid j/j mice subjected to CLP, indicating that Bid protein plays an important role in sepsis-induced apoptosis and mortality. This is supported by a recent report by Chang et al. [START_REF] Chang | Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis[END_REF], in which an increased survival rate was observed in Bid j/j mice after CLP.

Septic mortality is thought to be closely related to the development of a systemic inflammatory response. Dysregulation of the proinflammatory and anti-inflammatory cytokine balance has been postulated to play an important role in the outcome of sepsis [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. In this regard, our present data indicates that Bid deficiency reduced inflammation not only systemically, but also locally. Although plasma levels of cytokine/chemokine (TNF-!, IL-6, IL-10, and MCP-1) were significantly elevated in C57BL/6 mice at 12 and 24 h after sepsis, Bid deficiency markedly reduced these circulating cytokine/chemokine levels especially at 24 h post-CLP. Similarly, these cytokine/chemokine levels in the local peritoneal cavity were also significantly reduced in Bid j/j mice after insult, with the exception of IL-6. However, the sources and mechanisms behind the release of these mediators and how Bid protein regulates the inhibition by Bid j/j are not clear.

In addition to its initially characterized proapoptotic role, Bid protein may also be involved in other cellular functions. Studies have indicated that Bid may play a role in promoting hepatocarcinogenesis [START_REF] Bai | Deletion of bid impedes cell proliferation and hepatic carcinogenesis[END_REF] and DNA damageYinduced or replicative stressYinduced apoptosis or cell cycle arrest [START_REF] Zinkel | A role for proapoptotic BID in the DNA-damage response[END_REF], although Kaufmann et al. [START_REF] Kaufmann | The BH3-only protein Bid is dispensable for DNA damageY and replicative stressYinduced apoptosis or cellcycle arrest[END_REF] have reported that Bid protein does not function in the DNA damage response. Bid has also been shown to be required for maintenance of myeloid homeostasis and suppression of myeloid tumorigenesis [START_REF] Zinkel | Proapoptotic BID is required for myeloid homeostasis and tumor suppression[END_REF]. Moreover, Bid seems to be essential for the resolution of inflammation in a mouse model of arthritis, as Bid deficiency prolonged joint inflammation and delayed the resolution of arthritis [START_REF] Scatizzi | Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis[END_REF]. The significance of these divergent functions of Bid remains open to debate and needs more investigation to broaden our understanding of this protein.

In summary, our findings indicate that sepsis induces Bid activation and translocation of tBid from the cytoplasm to the mitochondria. Bid deficiency rescues mice from septic mortality, and this is associated with reduced apoptosis in lymphoid organs as well as the suppression of local and systemic cytokine/chemokine levels during sepsis. BH3 domain-only proteins can regulate Bcl-2 proteins by neutralizing (depressing) antiapoptotic proteins, activating proapoptotic proteins, or directly binding and activating Bax/Bak to induce apoptosis [START_REF] Baskin-Bey | Death by association: BH3 domain-only proteins and liver injury[END_REF]. These BH3-only proteins seem to be critical to mediating the cell death program, and thus understanding their function will be important to identify how the induction of apoptosis contributes to septic mortality, which may provide insight into a novel therapeutic target for management of this condition.

  FIG. 1. Sepsis-induced changes in Bid activation and tBid translocation from cytosol to mitochondria that the activation was differentially affected by blockade of Fas-FasL signaling. A, C57BL/6 mice were subjected to sham or CLP. Thymocytes and splenocytes were harvested at 4, 24, and 48 h after surgery. The extent of total Bid (p22) in cytosolic fractions and tBid (p15) protein in the mitochondrial fractions were determined by Western blot analyses. B, C57BL/6 background, gld or lpr mice were subjected to sham or CLP, and 24 h later, thymocytes, splenocytes, liver, and Peyer patches were harvested. The extent of total Bid and tBid were determined by Western blot analyses (left panels) and semiquantitated by densitometry and expressed as integrated density (IDT) values of tBid relative to IDT values of VDAC1 (right panels). *P G 0.05 vs. respective sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 4 to 8 mice per group. NT indicates not tested.

FIG. 3 .

 3 FIG.3. Bid deficiency reduced sepsis-induced apoptosis in thymocytes and splenocytes. Cells were isolated from C57BL/6 background or Bid j/j mice 24 h after subjected to sham or CLP procedures. Apoptosis was determined by TUNEL assay or PI staining. A decreased apoptosis in thymocytes (A) and splenocytes (B) taken from Bid j/j mice after sepsis was observed when compared with C57BL/6 background mice. *P G 0.05 vs. respective sham; # P G 0.05 vs. C57BL/6 CLP. One-way ANOVA and a Student-Newman-Keuls test, mean T SEM; n = 4 to 8 mice per group.

SHOCK VOL. 34, NO. 2 CHUNG ET AL.

SHOCK VOL. 34, NO. 2 CHUNG ET AL.

SHOCK VOL. 34, NO. 2 CHUNG ET AL.

Copyright © 2010 by the Shock Society. Unauthorized reproduction of this article is prohibited. Downloaded from http://journals.lww.com/shockjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0h CywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 04/11/2023

SHOCK VOL. 34, NO. 2 CHUNG ET AL.

ACKNOWLEDGMENTS

The authors thank Mr Paul Monfils and Ms Virginia Hovanesian, Core Research Laboratories, Rhode Island Hospital, for assistance with histology and imagine analysis.

This study was supported by the National Institutes of Health project (grant no. GM53209-10). .1097/SHK.0b013e3181cf70fb


Dina Okhrimchuk

Pierre Hurtevent

Marc Andre Gonze

Anne Probst

Marie Simon Cornu

Marie Simon

Study of the long-term behaviour of 137cs atmospheric fallouts in french forests

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

D

  . Okhrimchuk 1 *, P. Hurtevent 1 , M.-A. Gonze 1 , A. Probst 2 , M. Simon Cornu 1 Study of the long-term behaviour of 137 Cs atmospheric fallouts in French forests 1 Institut de radioprotection et de sûreté nucléaire (IRSN), Centre de Cadarache, 13115 Saint Paul lez Durance, France 2 EcoLab, Université de Toulouse, CNRS, 31400 Toulouse, France

Fig. 1 :FreshFig. 3 :

 13 Fig.1: TREE4 model structure