Selective Inhibition of SIRT2 Improves Outcomes in a Lethal Septic Model.

BACKGROUND: Seven isoforms of histone deacetylase Class III have been reported - Sirtuin (SIRT) 1-7. We recently demonstrated that EX-527, an inhibitor of SIRT1, reduces mortality in a mouse model of lethal-cecal-ligationand- puncture (CLP)-induced septic shock. Our present study was aimed at determining whether selective inhibition of SIRT2, with AGK2, would decrease animal death and attenuate the inflammatory response in a septic model. METHODS: Experiment I: C57BL/6J mice were intraperitoneally given either AGK2 (82 mg/kg) in dimethyl sulfoxide (DMSO) or DMSO alone, and 2 h later subjected to CLP. Survival was monitored for 240 hours. Experiment II: mice treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2, with sham mice (operating but without any treatment) serving as controls. Peritoneal fluid and peripheral blood were examined at 24 and 48 hours for cytokine production. Samples of blood at 48 h were also allocated to assess coagulability using Thrombelastography (TEG). Morphological changes of bone marrow were evaluated from long bones (femurs and tibias) with hematoxylin and eosin (H&E) staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: cytokines in supernatant of the cultured normal primary splenocytes were measured after the cells were stimulated by lipopolysaccharide and treated with or without AGK2 (10 µM) for 6 hours. RESULTS: AGK2 significantly reduced mortality and decreased levels of cytokines in blood (TNF-α: 298.3±24.6 vs 26.8±2.8 pg/ml, p=0.0034; IL-6: 633.4±82.8 vs 232.6±133.0 pg/ml, p=0.0344) and peritoneal fluid (IL-6: 704.8±67.7 vs 391.4±98.5 pg/ml, p=0.033) compared to vehicle control. Also, AGK2 suppressed the TNF-α and IL-6 production in the cultured splenocytes (TNF-α: 68.1±6.4 vs 23.9±2.8 pg/ml, p=0.0009; IL-6: 73.1±4.2 vs 49.6±3.0 pg/ml; p=0.0051). The TEG data showed that the mice subjected to CLP displayed prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function, and clot rigidity. AGK2 treatment was associated with dramatic improvements in fibrin cross-linkage and clot formation times, without a significant impact on the clot initiation parameters or platelet function. Additionally, AGK2 significantly attenuated the bone marrow atrophy (58.3±6.5 vs 30.0±8.2%, p=0.0262). CONCLUSION: Selective inhibition of SIRT2 significantly improves survival, and attenuates sepsis-associated "cytokine storm", coagulopathy, and bone marrow atrophy in a mouse model of lethal septic shock.

Histone cleavage as a mechanism for epigenetic regulation: current insights and perspectives.

Discovered over a century ago, histones constitute one of the oldest families of proteins and have been remarkably conserved throughout eukaryotic evolution. However, only for the past 30 years have histones demonstrated that their influence extends far beyond packaging DNA. To create the various chromatin structures that are necessary for DNA function in higher eukaryotes, histones undergo posttranslational modifications. While many such modifications are well documented, others, such as histone tail cleavage are less understood. Recent studies have discovered several proteases that cleave histones and have suggested roles for clipped histones in stem cell differentiation and aging in addition to infection and inflammation; the underlying mechanisms, however, are uncertain. One histone class in particular, histone H3, has received outstanding interest due to its numerous N-terminal modification sites and prevalence in regulating homeostatic processes. Here, with special consideration of H3, we will discuss the novel findings regarding histone proteolytic cleavage as well as their significance in the studies of immunology and epigenetics.

Translational barriers and opportunities for emergency preservation and resuscitation in severe injuries.

BACKGROUND: Hypothermia is commonly used for organ and tissue preservation in multiple clinical settings, but its role in the management of injured patients remains controversial. There is no doubt that temperature modulation is a powerful tool, and hypothermia has been shown to protect cells during ischaemia and reperfusion, decrease organ damage and improve survival. Yet hypothermia is a double-edged sword: unless carefully managed, its induction can be associated with a number of complications. METHODS: A literature review was performed to include important papers that address the impact of hypothermia on key biological processes, and explore the potential therapeutic role of hypothermia in trauma/haemorrhage models. RESULTS: No clinical studies have been conducted to test the therapeutic benefits of hypothermia in injured patients. However, numerous well designed animal studies support this concept. Despite excellent preclinical data, there are several potential barriers to translating hypothermia into clinical practice. CONCLUSION: Therapeutic hypothermia is a promising life-saving strategy. Appropriate patient selection requires a thorough understanding of how temperature modulation affects various biological mechanisms.

[The war on (too many) fluids. New horizons in combat medicine]. / Krieg und Flüssigkeitsmanagement. Neue Horizonte.

In the coming years, our approach to the bleeding patient will have to change radically. The inevitable knowledge from the wars in Iraq and Afghanistan permits the prediction that conventional crystalloids will sooner or later disappear from volume replacement therapy. The dogma that fluids must always be given will be abandoned, to be replaced by the practice of careful and goal-directed resuscitation. In the near future, we would rely on designer fluids and sophisticated pharmacological agents to deliver personalized resuscitation based upon the specific needs of the individual patient.

An update on fluid resuscitation.

Hemorrhagic shock is the leading cause of death in civilian and military trauma. Effective hemorrhage control and better resuscitation strategies have the potential of saving lives. However, if not performed properly, resuscitation can actually exacerbate cellular injury caused by hemorrhagic shock, and the type of fluid used for resuscitation plays an important role in this injury pattern. It is logical to prevent this cellular injury through wiser resuscitation strategies than attempting immunomodulation after the damage has already occurred. It is important to recognize that unlike numerous other variables, resuscitation is completely under our control. We decide who, when and how should get resuscitated. This paper summarizes data from a number of studies to illustrate the differential effects of commonly used resuscitation fluids on cellular injury, and how these relate to clinical practice. In addition, some novel resuscitation strategies are described that may become clinically available in the near future.

Design of preservation solutions for universal tissue preservation in vivo: demonstration of efficacy in preclinical models of profound hypothermic cardiac arrest.

The design of new solutions for the universal preservation of tissues is a quest that would facilitate multiple-organ harvesting from organ donors since current preservation solutions do not provide optimum preservation for all organs. In contrast, a new approach to bloodless surgery using hypothermic blood substitution (HBS) to protect the whole body during profound hypothermic circulatory arrest (clinical suspended animation) has focused on the development of a hybrid solution design with the objective of providing universal tissue preservation. In this study, a porcine model of uncontrolled lethal hemorrhage was employed. A combination of two new solutions, maintenance and purge, was used in a cardiopulmonary bypass (CPB) technique to affect profound hypothermia and prolonged cardiac arrest (60 min), with resuscitation after surgical repair of the vascular deficit induced to affect exsanguination. After rewarming and recovery, pigs were monitored for 6 weeks for neurological deficits, cognitive function (learning new skills), and organ dysfunction. All the normothermic control animals died (n = 10), whereas 90% (9 of 10) in the HBS group survived (P < .05). Moreover, all of the survivors were neurologically intact, displayed normal learning and memory capability, and had no long-term organ dysfunction. Histology of brains after 6 weeks revealed no ischemic damage in marked contrast to control animals, which all showed diffuse ischemic damage. The demonstrated efficacy of these synthetic, acellular HBS solutions for protection of all the tissues in the body during clinical suspended animation justifies their consideration for multiple-organ harvesting from cadaveric and living donors.

Resuscitation-induced pulmonary apoptosis and intracellular adhesion molecule-1 expression in rats are attenuated by the use of Ketone Ringer's solution.

BACKGROUND: Resuscitation with Lactated Ringer's solution after hemorrhagic shock in rats has been shown to cause early cellular injury in the lung. We hypothesized that the use of energy substrates, such as ketone bodies, in the resuscitation fluids would protect against this injury. As markers of cellular injury we measured the induction of apoptotic cell death and the expression of Intracellular Adhesion Molecule-1 (ICAM-1). STUDY DESIGN: Male Sprague Dawley rats (n = 35) under inhaled isoflurane anesthesia had placement of femoral arterial and venous catheters. A three-stage hemorrhage model was used for this experiment. There was an initial hemorrhage of 27 mL/kg for 10 minutes. During the next 75 minutes another 8 mL/kg of blood was withdrawn at a steady rate. The resuscitation fluids were then infused for 45 minutes during which the third continuous hemorrhage of 8 mL/kg was performed. The animals were randomized to five groups: 1) sham hemorrhage (n = 6); 2) sham resuscitation (n = 7); 3) Lactated Ringer's resuscitation, three times the volume of shed blood (n = 8); 4) Ketone Ringer's (containing 28 mEq/L of beta-hydroxybutyrate) resuscitation, three times the volume of shed blood (n = 7); and 5) plasma resuscitation, volume equal to shed blood (n = 7). The animals were sacrificed 1 hour after resuscitation and lungs were harvested. Western blot technique was used for the determination of proapoptotic protein (bax), antiapoptotic protein (bcl-2), apoptotic fragments of poly ADP-ribose polymerase, and ICAM-1. Sections of lung were also subjected to immunostaining using antibodies to bax and ICAM-1 proteins (reported as number of positive cells/mm2). RESULTS: Lactated Ringer's resuscitation caused a significant increase in pulmonary apoptosis and ICAM-1 expression compared with the sham hemorrhage group. Animals resuscitated with Ketone Ringer's solution and plasma did not show this injury pattern. CONCLUSIONS: Substitution of lactate with ketone bodies in the resuscitation fluid attenuates the expression of cellular injury markers in the lung.

E- and P-selectin expression depends on the resuscitation fluid used in hemorrhaged rats.

BACKGROUND: E- and P-selectins are adhesion molecules that effect neutrophil-mediated reperfusion injury. Our hypothesis was that the expression of E- and P-selectins is dependent on the type of fluid used for resuscitation and that lactated Ringer's (LR) solution would result in an early upregulation of these molecules. METHODS: Male Sprague-Dawley rats (n = 36) were subjected to a 27 ml/kg hemorrhage over 5 min followed by a 1-h shock period and 1-h of resuscitation. The animals were randomized into the following resuscitation groups: (1) sham; (2) hemorrhage, no resuscitation; (3) whole blood (27 ml/kg); (4) 3:1 lactated Ringer's (81 ml/kg); (5) sham hemorrhage, infusion of lactated Ringer's (81 ml/kg); (6) 7. 5% hypertonic saline (9.7 ml/kg). Immediately after resuscitation, the spleen and lung were harvested for measurement of E- and P-selectin mRNA expression with reverse transcriptase- polymerase chain reaction (RT-PCR), and protein expression with immunostaining. RESULTS: LR resuscitation and LR infusion without prior hemorrhage significantly increased the E- and P-selectin mRNA in the lung and spleen. Immunostaining demonstrated that the adhesion molecule expression was mainly located in perivascular/peribronchial areas in the lung, and the marginal and trabecular areas in the spleen. Pulmonary edema and inflammatory cell infiltration were observed only in the animals that were hemorrhaged and resuscitated with LR. No resuscitation and resuscitation with whole blood caused no significant increase in selectin expression. CONCLUSION: LR resuscitation and LR infusion without hemorrhage are associated with early increased expression of E- and P-selectin molecules in the lung and spleen.

Xenogeneic patch closure of the small intestine: a novel approach to fistula management.

Previous work from our laboratory demonstrated the feasibility of utilizing placental-derived collagen tissue matrix (CTM) as a bowel wall substitute. We reasoned that this technique would also be suitable in managing intestinal fistulae. To test this hypothesis, we created a chronic cecal fistula in rats and randomly managed some with primary repair and others with CTM replacement. Leak rates, mortality, bursting pressures and histologic scores were similar, suggesting that a chronic fistula can be successfully managed with either a CTM or primary repair.

Saphenous vein graft infection: a fatal complication of postoperative mediastinitis.

Infection and erosion of the saphenous vein graft with mediastinal hemorrhage is a rare but highly lethal complication of cardiac surgery. This is associated with a mortality rate of 50%. We present a patient who died during the postoperative period due to this complication.

Can pleural effusions cause cardiac tamponade?

Pleural effusion(s) can increase the pressure of an otherwise insignificant pericardial effusion to a degree that can result in cardiac tamponade. The case histories presented here illustrate the importance of recognizing this phenomenon and altering our treatment algorithm to drain the pleural effusions instead of the pericardial collections.

Polyethylene glycol-superoxide dismutase inhibits lipid peroxidation in hepatic ischemia/reperfusion injury.

BACKGROUND: Hepatic injury after ischemia/reperfusion is attributed to the development of oxygen free radical (OFR)-mediated lipid peroxidation--a process that can be measured through its byproducts, specifically malondialdehyde. The use of free radical scavengers can offer significant protection against OFR-induced liver injury. We hypothesize that a new potent OFR scavenger, polyethylene glycol-superoxide dismutase (PEG-SOD), can inhibit OFR-mediated lipid peroxidation in hepatic ischemia/reperfusion injury. METHODS: Twelve male Sprague-Dawley rats (300-350 g) were subjected to occlusion of the left and middle hepatic arteries and portal veins for 90 min, followed by 120 min reperfusion. PEG-SOD (5000 units/kg) was given intravenously before vascular occlusion and again immediately upon reperfusion to six rats. Normal saline was given to the remaining six rats to be used as a control group. The right hepatic lobe (used as internal control) and left hepatic lobe were harvested separately and tissue malondialdehyde was measured. RESULTS: A marked increase in lipid peroxide was found in the normal saline group after 2 h reperfusion. Treatment with PEG-SOD prevented the rise in tissue malondialdehyde. The mean difference in the malondialdehyde between the left and right hepatic lobes were 13.20 +/- 6.35 and 1.70 +/- 3.65 nmol/g in the normal saline (control) and PEG-SOD groups, respectively. This difference was found to be statistically significant (P < 0.005) using Student's t-test. CONCLUSIONS: PEG-SOD can effectively attenuate hepatic ischemia/reperfusion injury by inhibiting OFR-mediated lipid peroxidation.

Acquired immunity to TNF: effects on intestinal wound healing.

Increased concentrations of tumor necrosis factor (TNF) damage normal tissue and produce a shock-like syndrome--changes that can be prevented with anti-body-specific antisera. These findings suggest that TNF-stimulated immunity should protect normal tissue and promote wound healing. To test this hypothesis, 30 Fischer 344 rats (150-200 g) were serially immunized against TNF (20 micrograms/kg). Convalescent sera assayed (micro-ELISA) for circulating antibodies revealed titers (2.54 +/- 0.08 au) significantly higher (P < 0.00001) in immunized animals than in nonimmunized controls (0.11 +/- 0.06 au). Following this, 10 immunized (Group I), 10 nonimmunized (Group II), and 10 control rats underwent partial cecectomy with primary anastomosis. Animals from Groups I and II received TNF (25 micrograms/kg) while controls received saline intravenously on Postoperative Days 1, 3, and 5. Animals were then sacrificed to determine: (1) hydroxyproline content of the anastomosis, (2) mitochondrial respiratory control ratio, and (3) pyruvate dehydrogenase activity of the muscle. We found that (1) exposure to increased concentrations of TNF (Group II) depresses (P < 0.01) biologic markers of wound healing and (2) acquired immunity to TNF (Group I) eliminates this response. In conclusion, acquired immunity to TNF protects the healing intestinal anastomosis from the effects of exposure to increased levels of TNF.