Hypoxic "second hit" in leukocytes from trauma patients: Modulation of the immune response by histone deacetylase inhibition.

INTRODUCTION: Histone deacetylase inhibitors (HDACI), can improve survival after lethal hemorrhagic shock, and modulate the inflammatory response after hemorrhage/lipopolysaccharide (LPS). The current experiments were designed to study the effects of HDACI after hemorrhage and severe hypoxia. METHODS: Splenic leukocytes from trauma and non-trauma patients (n=4-5/group) were exposed to severe hypoxia with/without suberoylanilide hydroxamic acid (SAHA, 400 nM) for 8h. Cytokines were measured by ELISA and RT-PCR, and hypoxia inducible factor (HIF)-1a and heme oxygenase (HO)-1 by Western blot. RESULTS: After hemorrhage and hypoxia, SAHA increased IL-1b gene (4.7+/-1.2-fold) and protein expression (2.1+/-0.6-fold) in trauma splenic leukocytes. It also reduced IL-10 gene expression (0.6+/-0.2-fold), but did not alter TNFa or IL-6 levels. This unexpected pro-inflammatory response may be due to a decrease in HIF-1a and HO-1 protein levels. CONCLUSIONS: In this model of severe hypoxia, treatment with SAHA increased the inflammatory response in trauma leukocytes, possibly through inhibition of the HIF-1/HO-1 pathway. Splenic leukocytes from non-trauma patients were variably affected by SAHA. Taken in context with the known anti-inflammatory properties of HDACI after hemorrhage/LPS, these findings suggest that the immune-modulating functions of HDACI are dependent on the type and severity of both the priming injury and subsequent insult.

Identification of a novel potential biomarker in a model of hemorrhagic shock and valproic acid treatment.

BACKGROUND: The initial management of a poly-trauma patient requires evaluation for potential hemorrhage and ongoing monitoring to assess the efficacy of treatment and avoid complications related to massive blood loss. Certain serum protein levels may be altered in response to hemorrhagic shock, and may serve as useful biomarkers to guide diagnosis, prognosis, and therapeutics in traumatic hemorrhagic shock (HS). Treatment with valproic acid (VPA) has been shown to up-regulate various survival pathways and improve outcome. Here we determine whether these changes would result in altered serum biomarkers. METHODS: Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss) followed by treatment with or without VPA (300 mg/kg). Using surface enhanced laser desorption-time of flight mass spectrometry (SELDI or SELDI-TOF MS) technology, we screened serum samples obtained from five rats at different time points (baseline, post-hemorrhagic shock, and post-VPA treatment) in a lethal model of hemorrhagic shock (HS). Additionally, we used isobaric tag labeling for relative quantitation (iTRAQ) to identify potential biomarkers in the serum. Western blots were performed to validate iTRAQ-identified biomarker from independent serum samples, and to analyze protein biomarker levels in the intestine during hemorrhagic shock and treatment. RESULTS: HS and treatment with VPA affected serum levels of many proteins. One such protein with a mass spectrum around 22.7 kDa was detected in all five rats. The same serum samples subjected to iTRAQ resulted in our identification of claudin-3, a 23 kDa tight junction protein. HS elevated serum claudin-3 protein levels, which was reversed by VPA treatment in a pattern similar to the SELDI-TOF MS studies. Further validation with independent serum and intestine samples from individual rats by Western blots confirmed that HS increased the protein level of claudin-3 in serum and decreased its level in the intestine. Treatment with VPA reversed the hemorrhagic shock-induced alteration in claudin-3 to sham levels. CONCLUSIONS: HS causes an acute rise in serum claudin-3 protein levels and a concurrent decrease in intestinal claudin-3 protein expression. VPA treatment attenuates these alterations and stabilizes intestinal claudin-3 levels. Our results demonstrate for the first time that claudin-3 is a potential biomarker in HS and treatment.

Pharmacologic resuscitation: cell protective mechanisms of histone deacetylase inhibition in lethal hemorrhagic shock.

BACKGROUND: We have demonstrated that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), can improve animal survival after hemorrhagic shock, and protect neurons from hypoxia-induced apoptosis. This study investigated whether VPA treatment works through the c-Jun N-terminal kinase (JNK)/Caspase-3 survival pathways. METHODS: Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss over 60 min) followed by post-shock treatment with VPA (300 mg/kg), without any additional resuscitation fluids. The experimental groups were: (1) Sham (no hemorrhage, no resuscitation), (2) no resuscitation (hemorrhage, no resuscitation), and (3) VPA treatment. The animals were sacrificed at 1, 6, or 24h (n=3/timepoint), and liver tissue was harvested. Cytosolic and nuclear proteins were isolated and analyzed for acetylated histone-H3 at lysine-9 (Ac-H3K9), total and phosphorylated JNK, and activated caspase-3 by Western blot. RESULTS: Hemorrhaged animals were in severe shock, with mean arterial pressures of 25-30 mmHg and lactic acid 7-9 mg/dL. As expected, only the VPA treated animals survived to the 6- and 24-h timepoints; none of the non-resuscitated animals survived to these time points. Treatment of hemorrhaged animals with VPA induced acetylation of histone H3K9, which peaked at 1h and returned back to normal by 24h. Hemorrhage induced phosphorylation of JNK (active form) and increased activated caspase-3 levels, representing a commitment to subsequent cell death. Treatment with VPA decreased the phospho-JNK (P=0.06) expression at 24h, without changing the total levels of JNK (P=0.89), and this correlated with attenuation of activated caspase-3 at 24h (P=0.04), compared with the non-resuscitated animals. CONCLUSION: Treatment with HDACI, induces acetylation of histone H3K9, and reduces JNK phosphorylation and subsequent caspase-3 activation. This discovery establishes for the first time that HDACI may protect cells after severe hemorrhage through modulation of the JNK/caspase-3 apoptotic pathway.

Impact of resuscitation strategies on the acetylation status of cardiac histones in a swine model of hemorrhage.

BACKGROUND: Chromatin remodeling through histone acetylation is a key control mechanism in gene transcription. We have shown previously that fluid resuscitation in rodents is coupled with highly structured post-translational modifications of cardiac histones. The current experiment was performed to validate this concept in a clinically relevant large animal model of hemorrhage and resuscitation, and to correlate the changes in histone acetylation with altered expression of immediate-early response genes. STUDY DESIGN: Yorkshire swine (n=49, 7/group, weight=40-58kg) were subjected to combined uncontrolled and controlled hemorrhage (40% of estimated blood volume) and randomly assigned to the following resuscitation groups: (1) 0.9% saline (NS), (2) racemic lactated Ringer's (dl-LR), (3) l-isomer lactated Ringer's (l-LR), (4) Ketone Ringer's (KR), (5) 6% hetastarch in saline (Hespan). KR contained an equimolar substitution of lactate with beta-hydroxybutyrate. No hemorrhage (NH) and no resuscitation (NR) groups were included as controls. Cardiac protein was used in Western blotting to analyze total protein acetylation and histone acetylation specifically. Lysine residue-specific acetylation of histone subunits H3 and H4 was further evaluated. In addition, Chromatin Immunoprecipitation (ChIP) technique was used to separate the DNA bound to acetylated histones (H3 and H4 subunits), followed by measurement of genes that are altered by hemorrhage/resuscitation, including immediate-early response genes (c-fos and c-myc), and heat shock protein (HSP) 70. RESULTS: The type of fluid used for resuscitation influenced the patterns of cardiac histone acetylation. Resuscitation with dl-LR and KR induced hyperacetylation on H3K9. KR resuscitation was also associated with increased acetylation on H3K14 and H4K5, and hypoacetylation on H3K18. The expression of genes was also fluid specific, with the largest number of changes following KR resuscitation (increased c-fos and c-myc, HSP 70 linked with H3; and increased c-myc linked with H4). Among the histone subunits studied, altered H3 acetylations were associated with the majority of changes in immediate-early gene expression. CONCLUSIONS: Acetylation status of cardiac histones, affected by hemorrhage, is further modulated by resuscitation producing a fluid-specific code that is preserved in different species. Resuscitation with KR causes histone acetylation at the largest number of lysine sites (predominately H3 subunit), and has the most pronounced impact on the transcriptional regulation of selected (immediate-early response) genes.

Prevention of hypoxia-induced neuronal apoptosis through histone deacetylase inhibition.

BACKGROUND: We have recently discovered that administration of valproic acid (VPA), a histone deacetylase inhibitor, enhances nuclear histone acetylation and improves survival after lethal hemorrhage in rats. In the present study, neurons were subjected to severe hypoxic condition in vitro to test whether VPA would prevent hypoxia-induced apoptosis, and to explore the possible mechanisms. METHODS: Primary hippocampal and cortical cultures dissociated from E18 rat embryos were plated in quadruplicate at a density of 2 x 10/well in neurobasal medium supplemented with B-27 on glass cover-slips coated with poly-l-lysine. On the 10th day after plating, cells were incubated in a hypoxia chamber (0.5% O2, 10% CO2, 89.5% N2) at 37 degrees C for 6 hour and 16 hour in the presence or absence of VPA (1 mmol/L). The cells were then fixed, stained with antiactivated caspase-3 and antiacetyl histone H3 lysine 9 (Ac H3 K9) antibodies and visualized under confocal microscope. The caspase-3 positive cells were counted as apoptotic. Ratio of the apoptotic to total cells stained with 4',6-diamidino-2-phenylindole was determined. Numerical data were subjected to t test analysis. p < 0.05 was considered statistically significant. Western blot was performed to determine the level of acetylation of nuclear factor-kappa B (NF-kappaB) and phospho-JNK (c-Jun N-terminal kinase) in cells treated with or without VPA. Luciferase report assay was employed to analyze the activation of NF-kappaB after the cells were transfected with NF-kBLuc with or without VPA treatment. RESULTS: Exposure of neurons to VPA prevented apoptotic cell death under hypoxic conditions (20% apoptosis). In contrast, about 95% cells underwent apoptosis at the same level of hypoxia. VPA treatment induced acetylation of histone H3 K9 and NF-kappaB lysine 310. NF-kappaB was activated at the same time as the protein acetylation. Moreover, JNK phosphorylation was inhibited after the cells were treated with VPA under hypoxia condition. CONCLUSION: VPA enhances acetylation of histone 3 at lysine 9 and NF-kappaB at 310, induces NF-kappaB activation, reduces JNK activation, and protects the neurons from hypoxia-induced apoptosis in vitro.

Surviving blood loss without fluid resuscitation.

BACKGROUND: Patients with massive blood loss often die before delivery of definitive care, especially in austere environments. Strategies that can maintain life during evacuation and transport to higher levels of care may be lifesaving. We have previously shown that administration of histone deacetylase inhibitors (HDACI) enhance gene transcription through specific modifications of DNA-associated histone proteins. Furthermore, it protects against organ damage when given before hemorrhage. The current experiment was done to test whether administration of HDACI after lethal hemorrhage, without fluid resuscitation, would improve outcome by creating a pro-survival phenotype. METHODS: Seventy-two male Wistar-Kyoto rats (n = 12 per group) were subjected to 60% blood volume loss for 1 hour (40% arterial bleed for 10 minutes and 20% venous bleed for 50 minutes). After hemorrhage, animals were randomized to receive one of two HDACI: (1) valproic acid (VPA, 300 mg/kg in 0.25 mL saline), or (2) suberoyanilide hydroxamic acid (SAHA, 7.5 mg/kg in 0.25 mL saline). Control groups included (3) no hemorrhage (Sham), (4) no resuscitation (NR), (5) 0.9% saline resuscitation, 3 times the volume of shed blood (NS), and (6) vehicle control, 0.25 mL 0.9% saline (VEH). Hemodynamic data were recorded continuously, and physiologic parameters were measured serially. Survival for 3 hours was the primary endpoint for this experiment. RESULTS: Nonresuscitated shock (NR group) was highly lethal and only 25% of the animals survived for 3 hours. Administration of HDACI after hemorrhage (without fluid resuscitation) significantly improved survival (75% and 83% in VPA and SAHA groups, respectively, p < 0.05 vs. NR). Survival was 40%, 100%, and 100% in the VEH, Sham, and NS resuscitation groups, respectively. CONCLUSIONS: This study demonstrates that post-shock administration of HDACI can significantly improve early survival in a highly lethal model of hemorrhagic shock, even in the absence of conventional fluid resuscitation. This approach may be especially relevant for austere environments where fluids are in limited supply, such as a battlefield.

Putting life on hold-for how long? Profound hypothermic cardiopulmonary bypass in a Swine model of complex vascular injuries.

BACKGROUND: Rapid induction of profound hypothermia for emergency preservation and resuscitation can improve survival from uncontrolled lethal hemorrhage in large animal models. We have previously demonstrated that profound hypothermia (10 degrees C) must be induced rapidly (2 degrees C/min) and reversed gradually (0.5 degrees C/min) for best results. However, the maximum duration of hypothermic arrest in a clinically relevant trauma model remains unknown. METHODS: Uncontrolled lethal hemorrhage was induced in 22 swine by creating an iliac artery and vein injury, followed 30 minutes later (simulating transport time) by laceration of the descending thoracic aorta. Through a thoracotomy approach, a catheter was placed in the aorta, and cold organ preservation solution was infused using a roller pump to rapidly induce profound hypothermia (10 degrees C) which was maintained with low-flow cardiopulmonary bypass. Vascular injuries were repaired during the asanguinous hypothermic low flow period. Profound hypothermia was maintained (n = 10-12 per group) for either 60 minutes or 120 minutes. After repair of injuries, animals were rewarmed (0.5 degrees C/min) and resuscitated on cardiopulmonary bypass, and whole blood was infused during this period. Animals were monitored for 4 weeks for neurologic deficits, organ dysfunction, and postoperative complications. RESULTS: The 4-week survival rates in 60- and 120-minute groups were 92% and 50%, respectively (p < 0.05). The surviving animals were neurologically intact and had no long-term organ dysfunction, except for one animal in the 120-minute group. The animals subjected to 120 minutes of hypothermia had significantly worse lactic acidosis, displayed markedly slower recovery, and had significantly higher rates of postoperative complications, including late deaths because of infections. CONCLUSION: In a model of lethal injuries, rapid induction of profound hypothermia can prevent death. Profound hypothermia decreases but does not abolish metabolism. With current methods, the upper limit of hypothermic arrest in the setting of uncontrolled hemorrhage is 60 minutes.

Environmental impact of accelerated clinical care in a high-volume center.

BACKGROUND: "Fast-track" surgery, involving multimodal care, improves efficiency and short-term outcomes in patients undergoing bowel resection. The sustainability of the benefits and the "drag" effect on non-participating surgeons through changed nursing and resident practice is undetermined. METHODS: 297 consecutive elective colon resections (DRG149) within three study periods (pre-change, immediate post-change, long-term post-change) were retrospectively reviewed. Two surgeons began to "fast-track" their patients in 1999 independently from the other surgeons in the hospital. Surgeons were grouped into "fast-track surgeons," "high-volume surgeons," (>/=10 cases per year) and "low-volume surgeons," (<10 cases per year). Comparisons of duration of stay (DOS), readmission rates, and mortality were made for each of three time periods and surgeon groups. Trends were also compared with unrelated hospital non-colectomy control groups (uncomplicated craniotomy DRG 001 and pancreatic surgery DRG 192) and to a colectomy control group from a statewide database (DRG 149). RESULTS: Mean DOS for colon resection significantly decreased among the "fast-track" surgeons and among all the other surgeons in the hospital, from 6.3 +/- 0.3 days, down to 3.7 +/- 0.1 days. We found no significant difference in mortality or readmission rates between the study periods. 15% of the cases were performed laparoscopically, and the improvements in outcome were independent of surgical technique. Control group analyses demonstrated that the environmental impact on outcome was independent of hospital-wide or regional improvement efforts. CONCLUSIONS: Implementation of a new practice pattern in a shared environment leads to improved outcomes for patients of other surgeons within the same environment. Dissemination and cross-pollination of new methods through resident, nurse, and case manager practice pattern modification creates a favorable force for change and this impact is sustained over a 3-year period.

Histone deacetylase as therapeutic target in a rodent model of hemorrhagic shock: effect of different resuscitation strategies on lung and liver.

BACKGROUND: DNA transcription is regulated in part by acetylation of nuclear histones, controlled by 2 groups of enzymes: histone deacetylases (HDAC) and histone acetyl transferases (HAT). We have shown previously that hemorrhage and resuscitation are associated with HDAC/HAT imbalance, which influences the acetylation status of cardiac histones. The goals of this study were to determine whether: (1) resuscitation after hemorrhage affects histone acetylation in a fluid- and organ-specific fashion; and (2) administration of HDAC inhibitors influences histone acetylation and subsequent gene expression. METHODS: In the first experiment, rats (n = 6/group) were subjected to volume-controlled hemorrhage and resuscitated with: (1) racemic lactated Ringer's (DL-LR); (2) L-lactated Ringer's (L-LR); (3) 7.5% hypertonic saline (HTS); (4) ketone Ringer's (KR); or (5) pyruvate Ringer's (PR). Control groups included: (6) no hemorrhage (Sham); and (7) hemorrhage with no resuscitation (NR). In the second experiment (n = 5/group), 3 HDAC inhibitors, valproic acid (VPA), trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), were added to normal saline and used as fluid for resuscitation. At the end of resuscitation, lung and liver tissues were subjected to subcellular protein fractionation and Western blotting to analyze histone acetylation. In addition, cDNA microarrays and RT-PCR were used to measure expression of selected genes. RESULTS: Hemorrhage did not change the level of histone acetylation in lungs, whereas resuscitation predominantly hyperacetylated histones. An analysis of histone acetylation on 10 lysine sites showed that L-LR, HTS, and KR resuscitation caused the largest number of changes (7, 6, and 6 respectively). SAHA hyperacetylated 7 sites in liver and affected expression of 57 genes (44 up, 13 down). CONCLUSIONS: Resuscitation with various fluids, as well as infusion of pharmacologic HDAC inhibitors affects histone acetylation in a fluid- and organ-specific fashion, even when administered post-insult for a limited period of time. Uniquely affected genes are associated with metabolism, cellular growth, proliferation, differentiation, transformation, and cellular signaling.

Profound hypothermic cardiopulmonary bypass facilitates survival without a high complication rate in a swine model of complex vascular, splenic, and colon injuries.

BACKGROUND: Induction of a profound hypothermia for emergency preservation and resuscitation in severe hemorrhagic shock can improve survival from lethal injuries, but the impact of hypothermia on bleeding and infectious complications has not been completely determined. STUDY DESIGN: Uncontrolled hemorrhage was induced in 26 swine (95 to 135 lbs) by creating an iliac artery and vein injury, and 30 minutes later, by lacerating the descending thoracic aorta. Through a left thoracotomy approach, profound total body hypothermia (10 degrees C) was induced (2 degrees C/min) by infusing cold organ preservation solution into the aorta. The experimental groups were: vascular injuries alone (group 1, n=10), vascular and colon injuries (group 2, n=8), and vascular, colon, and splenic injuries (group 3, n=8). All injuries were repaired during 60 minutes of low-flow cardiopulmonary bypass (CPB) with hemodilution and profound hypothermia; then the animals were slowly rewarmed (0.5 degrees C/min) back to normothermia. Survivors were monitored for 6 weeks for postoperative bleeding, neurologic deficits, cognitive function (learning new skills), organ dysfunction, and septic complications. RESULTS: Six-week survival rates were 90% in group 1, 87.5% in group 2, and 75% in group 3 (p > 0.05). One animal in each group died from acute cardiac failure during the early postoperative phase. Splenic salvage was possible in all animals, and none required complete splenectomy for hemorrhage control. All surviving animals were neurologically intact, displayed normal learning capacity, and had no longterm organ dysfunction. None of the animals had postoperative hemorrhage or experienced septic complications. One animal in group 3 died on the ninth postoperative day because of bowel obstruction (volvulus). CONCLUSIONS: Induction of profound hypothermia can preserve the viability of key organs during repair of lethal injuries. This strategy can be used even in the presence of solid organ and bowel injuries to improve survival, without any considerable increase in postoperative complication rates.

Differential effect of resuscitation on Toll-like receptors in a model of hemorrhagic shock without a septic challenge.

UNLABELLED: It has been shown that the inflammatory response and cellular damage after hemorrhagic shock are influenced by resuscitation strategies. Toll-like receptors (TLRs) play an important role in signal transduction in inflammatory conditions. However, alterations in TLR expression following hemorrhagic shock and resuscitation have not been well documented. This study was conducted to measure the impact of different resuscitation strategies on TLR expression and downstream signaling in key organs. METHODS: Sprague Dawley rats (n=38) were subjected to a severe volume-controlled hemorrhage protocol. After 75 min of shock, they were resuscitated over 45 min as follows: (1) lactated Ringer's (LR, 81 ml/kg), (2) ketone Ringer's (KR, 81 ml/kg), (3) 7.5% hypertonic saline (HTS, 9.7 ml/kg), (4) 6% hetastarch (HEX, 27 ml/kg), (5) pyruvate Ringer's (PR, 81 ml/kg). Sham hemorrhage (NH) and no resuscitation (NR) groups served as controls. The KR and PR solutions were identical to LR except for equimolar substitution of racemic lactate with beta hydroxybutyrate and sodium pyruvate, respectively. At the end of resuscitation, the expression of TLRs (types 1-10), and cytokines (IL-10, IL-1beta and TNF-alpha) were measured in the lung and spleen using RT-PCR. Levels of phosphorylated and total IkB-alpha and NF-kappaB were detected by Western blotting. The systemic and lung protein levels of TNF-alpha were measured using ELISA and immunohistochemistry. RESULTS: Expression of TLRs in the lung was affected more than in the spleen by hemorrhagic shock and resuscitation. In the lung, hemorrhage increased TLR-2, -3 and -6 (but not TLR-4) mRNA expression, with an up-regulation of the ratio of phosphor-NF-kappaBp65 and total NF-kappaBp65, NF-kappaBp65 activation, and enhanced systemic and tissue TNF-alpha protein levels. Post-resuscitation, TLR mRNA profile and subsequent downstream proteins in the lung and spleen were affected by the choice of resuscitation strategy. CONCLUSIONS: Hemorrhagic shock activates TLR signaling in lung, but not the spleen, probably through an up-regulation of TLR gene expression, and activation of NF-kappaB pathway. Resuscitation modulates this response in a fluid- and tissue-specific fashion.

Primary insertion of a silastic spring-loaded silo for gastroschisis.

Gastroschisis is traditionally managed by emergency primary closure, with a temporary silo reserved for large defects unable to be closed primarily. We recently have begun primary Silastic (Dow Coming, Midland, MI) spring-loaded silo (SLS) closure followed by elective closure and report our preliminary experience. A total of 15 infants (weight range, 2.1-13.5 kg) at 2 different institutions were treated by SC by 3 different surgeons between 1998 and 2002. A 3-, 4-, or 5-cm (ring diameter) silo was used depending on size of abdominal wall defect. Elective closure was performed in the operating room or at the bedside. Surgical parameters assessed included success of SLS, peak inspiratory pressures (PIPs) pre- and post-SLS closure, total time of staged closure with SLS, time to full feedings, and intra- and postoperative complications. Fifteen of 15 infants were successfully treated by SLS closure followed by elective closure. Two of 15 (13.3%) experienced temporary dislodgement of the silo prior to permanent closure. In both cases, the silo was safely reinserted at the bedside. Comparison of PIP values measured at various stages of SLS closure revealed no significant difference (P > 0.05). Mean times to final fascial closure (3.7 days) and full enteral feedings (22 days) were similar to historical controls obtained from the surgical literature. In 1 case where there was associated intestinal atresia, SLS closure was effective in permitting concomitant elective closure and re-establishment of bowel continuity. All children are alive and well at the time of this report. SLS closure permits safe, gentle, and gradual reduction of the exposed viscera leading to successful permanent abdominal wall closure. Respiratory embarrassment and hemodynamic instability associated with emergent (primary) closure of large abdominal wall defects can thus be avoided.

Histone deacetylase inhibitors prevent apoptosis following lethal hemorrhagic shock in rodent kidney cells.

BACKGROUND: We have previously demonstrated that treatment with histone deacetylase inhibitors (HDACI), such as valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), can improve survival after hemorrhagic shock in animal models. Hemorrhage results in hypoacetylation of proteins which is reversed by HDACI. These agents are known to acetylate insulin receptor substrate-I (IRS-I), which in turn activates the Akt survival pathway. This study investigated whether HDACI exert their beneficial effects through the Akt survival pathway. METHODS: Wistar-Kyoto rats (N=21) underwent hemorrhage (60% blood loss) and were randomized into 3 groups; no resuscitation (NR), and treatment with VPA or SAHA. Kidneys were harvested at 1, 6, and 24h after HDACI treatment and analyzed for acetylated histone 3 at lysine 9 residue (Ac-H3K9), phosphorylated Akt (phospho-Akt), BAD and Bcl-2 proteins. RESULTS: Hemorrhaged animals were in severe shock, with mean arterial pressures of 25-30mmHg and lactic acid 7-9mg/ml. Only animals treated with VPA and SAHA survived to the 6- and 24-h timepoints. Treatment with HDACI produced a biologic effect on rat kidney cells inducing acetylation of histone H3K9, which peaked after 1h of treatment, and was statistically significant in the VPA group (p=0.01) compared to NR. Phospho-Akt protein increased in the VPA group with a reciprocal decrease in the pro-apoptotic BAD protein in both groups which was statistically significant in the VPA group after 1h (p=0.007) and 24h (p=0.006) of treatment and in the SAHA group after 24h of treatment (p=0.028). Anti-apoptotic Bcl-2 protein markedly increased after 6 (p=0.04) and 24h (p=0.014) of VPA treatment. Bcl-2 also increased in the SAHA group, but failed to reach statistical significance. CONCLUSION: Treatment with HDACI increases phosphorylation of Akt with a subsequent decrease in the pro-apoptotic BAD protein. The above mechanism facilitates the action of anti-apoptotic protein Bcl-2. HDACI protect kidney cells subjected to hemorrhagic shock in rodents through the Akt survival pathway.

Candida infection and colonization among non-trauma emergency surgery patients.

BACKGROUND: Candida is a significant pathogen among critically ill patients. However, candidiasis among non-trauma emergency surgery (NTES) patients has not been previously investigated. Herein we describe the incidence of both colonization and infection from Candida and risk factors for invasive disease in this population. METHODS: For this retrospective single center study we included all NTES patients with ICU stay ≥4 days from May 1(st), 2002 to April 30(th), 2007. Patients were divided into 3 non-overlapping groups: 1) patients with Candida-infection, 2) patients with Candida colonization and 3) patients with negative Candida cultures. Groups were compared by univariate and multivariate analyses to identify significant risk factors for invasive candidiasis. RESULTS: Of all 289 eligible patients, 63 (21.7%) fulfilled the criteria for Candida infection and 110 (38%) were included in the Candida colonization group. Interestingly, from the 63 patients with invasive candidiasis, 25 (39.7%) were infected by a non-albicans species. Upon multivariate analyses, ventilator-associated pneumonia (VAP) (Odds Ratio [OR]: 2.34; 95%, Confidence Interval [CI]: 1.213-4.533, p = 0.0112), bacteremia (OR: 4.778; 95%CI: 1.519-15.029, p = 0.0075) and surgical complications (OR: 3.903; 95%CI: 1.335-11.412, p = 0.0129) were independent risk factors for the development of Candida infection. Candida infection and colonization were both found to correlate with approximately $40,000-100,000 mean additional costs). Interestingly, candidemia was associated with 63% all-cause mortality. For all other forms of candidiasis, mortality was not significantly different among groups. CONCLUSION: We found that Candida infection is alarmingly high among NTES patients with prolonged intensive care unit (ICU) stay. Surgical complications and bacterial infections (VAP and bacteraemia) were significantly correlated with the development of candidiasis. Candidiasis reached a rate of 21.7/100 discharges, which is significantly higher than most established high-risk populations for candidiasis. Future studies should review the need for antifungal prophylaxis on this population.

Fulminant Clostridium difficile colitis: patterns of care and predictors of mortality.

HYPOTHESIS: There exist predictors of mortality and the need for colectomy among patients with fulminant Clostridium difficile colitis. DESIGN: Retrospective study. SETTING: Academic tertiary referral center. PATIENTS: We reviewed the records of 4796 inpatients diagnosed as having C difficile colitis from January 1, 1996, to December 31, 2007, and identified 199 (4.1%) with fulminant C difficile colitis, as defined by the need for colectomy or admission to the intensive care unit for C difficile colitis. MAIN OUTCOME MEASURES: Risk of inpatient mortality was determined by multivariate analysis according to clinical predictors, colectomy, and medical team. RESULTS: The inhospital mortality rate for fulminant C difficile colitis was 34.7%. Independent predictors of mortality included the following: (1) age of 70 years or older, (2) severe leukocytosis or leukopenia (white blood cell count, >or=35 000/microL or <4000/microL) or bandemia (neutrophil bands, >or=10%), and (3) cardiorespiratory failure (intubation or vasopressors). When all 3 factors were present, the mortality rate was 57.1%; when all 3 were absent, the mortality rate was 0%. Patients who underwent colectomy had a trend toward decreased mortality rates (odds ratio, 0.49; 95% confidence interval, 0.21-1.1; P = .08). Among patients admitted primarily for fulminant C difficile colitis, care in the surgical department compared with the nonsurgical department resulted in a higher rate of operation (85.1% vs 11.2%; P < .001) and lower mortality rates (12.8% vs 39.3%; P = .001). Patients admitted directly to the surgical department had a shorter mean (SD) interval from admission to operation (0 vs 1.7 [2.8] days; P = .001). CONCLUSIONS: Despite awareness and treatment, fulminant C difficile colitis remains a highly lethal disease. Reliable predictors of mortality exist and should be used to prompt aggressive surgical intervention. Survival rates are higher in patients who were cared for by surgical vs nonsurgical departments, possibly because of more frequent and earlier operations.

Protective effect of suberoylanilide hydroxamic acid against LPS-induced septic shock in rodents.

We have recently found that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improves survival in a lethal model of hemorrhagic shock in rats. The purpose of the present study was to determine whether SAHA treatment would prevent LPS-induced septic shock and improve the survival in a murine model. C57BL/6J mice were randomly divided into two groups. Experimental mice were given intraperitoneal SAHA (50 mg/kg) in vehicle dimethyl sulfoxide fluid (n = 10). The control mice (n = 10) received vehicle dimethyl sulfoxide only. They were injected with LPS (20 mg/kg, i.p.) 2 h later, and the animals from the treatment group were given a second dose of SAHA. Survival was monitored during the next 7 days. In a parallel study, mice treated with or without SAHA were subjected to LPS insult while normal (sham) mice serviced as controls. 1) Lungs were harvested at 3 and 48 h for analysis of gene expression and pathologic changes, respectively; 2) spleens were isolated for analysis of neutrophilic cell population. In addition, RAW264.7 mouse macrophages were cultured to assess the effects of SAHA on LPS-induced inflammation in vitro. All mice in the control group that were subjected to LPS challenge died in less than 48 h. However, SAHA-treated animals displayed a significantly higher 1-week survival rate (87.5%) compared with the control group (0%). Moreover, LPS insult decreased the acetylation of histone proteins (H2A, H2B, and H3), elevated the levels of TNF-alpha in vivo (circulation) and in vitro (culture medium), increased the neutrophilic cell population in the spleen, enhanced the expression of TNF-alpha and IL-1beta genes in lung tissue, and augmented the pulmonary neutrophil infiltration. In contrast, SAHA treatment markedly attenuated all of these LPS-induced alterations. We report for the first time that administration of SAHA (50 mg/kg) significantly attenuates a variety of inflammatory markers and improves long-term survival after a lethal LPS insult.

Acetylation: a novel method for modulation of the immune response following trauma/hemorrhage and inflammatory second hit in animals and humans.

BACKGROUND: Hemorrhage induces an imbalance in histone acetyl transferase/histone deacetylase (HAT/HDAC) ratio. Correction of this imbalance with histone deacetylase inhibitors (HDACI) improves survival. We aimed to identify whether this was due to modulation of the post-shock immune response. METHODS: We established a "two-hit" model in which rats (n=11; 5-6/group) and humans (n=10; 5/group) sustained trauma/hemorrhage, followed by exposure of splenic leukocytes to lipopolysaccharide (LPS, 10 ng/mL) for 8 or 24 hours. The leukocytes were treated with: No treatment, SAHA (suberoylanilide hydroxamic acid, HDACI, 400 nM), or Garcinol (HAT inhibitor, 20 microM). RESULTS: Hemorrhage in the animals produced severe shock and a pro-inflammatory state. SAHA reduced TNFa secretion in the hemorrhaged leukocytes after LPS "second-hit" (34.0%, P = .003), whereas it increased transcript levels of TNFa and IL-1b (2.1+/-0.3 and 5.1+/- 2.2 fold respectively, P < .05). Leukocytes from trauma patients displayed 2 distinct responses to SAHA after LPS "second-hit," with markedly increased or decreased cytokine levels. CONCLUSIONS: SAHA normalizes TNFa levels following hemorrhage and LPS "second hit" in the rats, whereas trauma patients respond to SAHA in 2 distinct patterns, with either marked attenuation or exaggeration of inflammatory cytokines. Cytokine levels were independent of gene expression, implicating acetylation of non-nuclear proteins as the dominant regulatory mechanism.

Cell protective mechanism of valproic acid in lethal hemorrhagic shock.

BACKGROUND: We have demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve animal survival after hemorrhagic shock and protect neurons from hypoxia-induced apoptosis. This study investigated whether VPA treatment works through the beta-catenin survival pathways. METHODS: Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss) followed by treatment with or without VPA (300 mg/kg). Brains were harvested after 1, 6, and 24 hours and analyzed for acetylated histone-H3 at lysine-9 (Ac-H3K9), acetylated and total beta-catenin, and Bcl-2 by Western blot. In addition, primary neurons dissociated from E18 rat embryos were exposed to hypoxia (0.5% O(2)) for 16 hours with or without VPA (1 mmol/L) and analyzed using confocal microscopy. RESULTS: After treatment of hemorrhaged animals with VPA, acetylated beta-catenin was found in both the cytosol and nucleus, along with Ac-H3K9. Bcl-2 transcript increased after 1 hour followed by an increase in Bcl-2 protein at 6 hours. Confocal imaging demonstrated that after VPA treatment, beta-catenin translocated into the nucleus and colocalized with Ac-H3K9. CONCLUSION: VPA treatment acetylates H3K9 and beta-catenin and enhances translocation of beta-catenin into the nucleus, where it colocalizes with Ac-H3K9 and stimulates the transcription of survival gene bcl-2. This finding suggests that VPA protects cells after severe insult through the beta-catenin survival pathway.