Elizabeth A. Sailhamer

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-&agr; in vivo (circulation) and in vitro (culture medium), increased the neutrophilic cell population in the spleen, enhanced the expression of TNF-&agr; and IL-1&bgr; 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.

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 1st, 2002 to April 30th, 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

Cell protective mechanism of valproic acid in lethal hemorrhagic shock

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 thatCandidainfection 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.

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, 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.

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

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.

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

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.

Identification of a Novel Potential Biomarker in a Model of Hemorrhagic Shock and Valproic Acid Treatment

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.

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

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.

Operative time is a poor surrogate for the learning curve in laparoscopic colorectal surgery

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.