Richard E. Dean

Small-molecule selectin inhibitor protects against liver inflammatory response after ischemia and reperfusion

BACKGROUND The selectin family of adhesion molecules plays a key role in the neutrophil-mediated injury observed after ischemia and reperfusion. In our study, we investigated the effects of TBC-1269, a novel small-molecule, nonoligosaccharide inhibitor of P-, E-, and L-selectin binding, in the liver inflammatory response after 90 minutes of warm ischemia. STUDY DESIGN Total liver ischemia was produced in Sprague-Dawley rats for 90 minutes using an extracorporeal portosystemic shunt. The animals were divided into five groups including: the sham (group 1), ischemic control (group 2) receiving only the vehicle, and the treated groups receiving TBC-1269 at a dose of 25 mg/kg at different times of administration: 15 minutes before reperfusion (group 3), at reperfusion (group 4), and 15 minutes after reperfusion (group 5). The following indices were analyzed: 7-day survival, liver injury tests, liver tissue myeloperoxidase as an index of neutrophil infiltration, and liver histology. RESULTS TBC-1269 treated groups experienced a significant increase in survival compared with controls. Best overall survival, 70%, was observed when TBC-1269 (Texas Biotechnology Corporation, Houston, TX) was administered 15 minutes before reperfusion (p < 0.05). This group also showed a marked decrease (p < 0.05) in liver enzyme levels at 6 hours after reperfusion. Neutrophil migration was also significantly ameliorated (81%), as reflected by decreased myeloperoxidase levels. We observed improved histologic damage scores in the treated group compared with controls (p < 0.05). CONCLUSIONS A small-molecule selectin inhibitor (TBC-1269) had a protective effect in livers subjected to 90 minutes of warm hepatic ischemia and 6 hours of reperfusion by decreasing neutrophil infiltration, migration and subsequent tissue damage. The best protective effect was achieved when the compound was administered 15 minutes before reperfusion. These findings offer a new therapeutic alternative for protection against ischemia and reperfusion injury.

Mechanism of the beneficial effects of ATP-MgCl2 following trauma-hemorrhage and resuscitation: Downregulation of inflammatory cytokine (TNF, IL-6) release

Although ATP-MgCl2 improves hepatocellular function in a nonheparinized model of trauma-hemorrhage and crystalloid resuscitation, it remains unknown whether the beneficial effects of this agent are due to downregulation of the release of the inflammatory cytokines, tumor necrosis factor (TNF), and interleukin-6 (IL-6) under those conditions. To study this, rats underwent a 5-cm laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximum bleedout volume was returned in the form of Ringers lactate (RL). The animals were then resuscitated with four times the volume of shed blood with RL over 60 min. ATP-MgCl2 (50 mumoles/kg body weight each) or an equivalent volume of normal saline was infused intravenously for 95 min. This infusion was started during the last 15 min of RL resuscitation. Plasma levels of TNF and IL-6 were measured at 1.5 hr after the completion of resuscitation by cytokine-dependent cellular assays. Hepatic blood flow was determined by in vivo indocyanine green clearance (corrected by hepatic extraction ratio for indocyanine green), radioactive microspheres, and [3H]-galactose clearance techniques. The results indicate that the levels of circulating TNF and IL-6 increased significantly in the hemorrhaged-resuscitated animals. ATP-MgCl2 treatment, however, markedly decreased the synthesis and/or release of these cytokines to levels similar to the sham group. The markedly decreased hepatic blood flow (as determined by three different methods) and hepatic extraction ratio for indocyanine green were also restored by ATP-MgCl2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adequate crystalloid resuscitation restores but fails to maintain the active hepatocellular function following hemorrhagic shock

Studies have shown that active hepatocellular function is depressed early after trauma-hemorrhage and persists despite resuscitation with two or three times (x) the volume of maximum bleedout (MB) with lactated Ringers solution (LR). However, it is not known if a larger volume of fluid resuscitation corrects this dysfunction. To study this, rats were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the MB volume was returned in the form of LR, and then resuscitated with 4x or 5x the volume of MB with LR. Three doses of indocyanine green (ICG) were given intravenously and [ICG] measured in vivo using an in-vivo hemoreflectometer. The initial velocity of the clearance of ICG was calculated. Maximal velocity of the clearance (Vmax: the number of functional ICG receptors) and kinetic constant (Km: the efficiency of the active transport) were determined from the Lineweaver-Burk plot. Vmax decreased during hemorrhage, was restored to control levels at 0-4 hours after resuscitation, but decreased at 4-8 hours after resuscitation despite restoration of cardiac output following resuscitation with 5x LR. This could be the result of increased TNF release. The Km also decreased during hemorrhage, but increased at 0-1.5 hours and remained at control levels even 4-8 hours after resuscitation. Thus the failure of Vmax to remain at control levels following adequate fluid resuscitation may form the basis of cellular dysfunction and multiple organ failure after severe hemorrhagic shock.

Mechanism of immunosuppression following hemorrhage: Defective antigen presentation by macrophages

The mechanism by which simple hemorrhage profoundly impairs the proliferative response of T lymphocytes to mitogen and alloantigen, produces a defect in interleukin-2 generation, and increases the susceptibility to sepsis remains unknown. Since antigen presentation (AP) by the macrophage (M phi) plays a critical role in the antigen-specific activation of T-helper cells and lymphokine production, we investigated whether the function of the M phi as an AP cell is altered following hemorrhage. C3H/HEJ mice were bled to a mean BP of 35 mm Hg, maintained at that level for 1 hr, and then resuscitated. There was no mortality with this model. Control mice were not bled but otherwise treated identically. Immediately after resuscitation the mice were sacrificed and peritoneal M phi (PM phi) as well as splenic adherent cells (SAC) were harvested. AP function was tested by coculturing different numbers of PM phi and SAC with D10.G4.1 cells (2 x 10(4) cells/well) in the presence of conalbumin (300 micrograms/ml). This T-helper cell clone proliferates upon recognition of conalbumin in the context of Iak (a M phi surface membrane glycoprotein), thus directly reflecting M phi AP capability. After 72 hr of incubation, the cultures were pulsed with [3H]thymidine and harvested. D10.G4.1 proliferations induced via AP by PM phi and SAC from hemorrhaged-resuscitated mice were 29 and 24% of control, respectively (P less than 0.05). Thus, we conclude that AP by M phi following hemorrhage is defective despite adequate resuscitation, a mechanism which could explain the state of immunosuppression and enhanced susceptibility to sepsis.

Prostaglandin E2 depresses antigen-presenting cell function of peritoneal macrophages.

Eicosanoids play a prominent role in trauma. Such mediators of inflammation negatively influence cell-mediated immunity (CMI). There is, however, no information available on the effect of eicosanoids on a critical event in CMI, i.e., antigen-presenting (AP) cell function of macrophages (M luminal diameter), a cellular process responsible for the activation of T and B lymphocytes. The aim of this study, therefore, was to examine the effect of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) on AP cell function of the peritoneal M luminal diameter. To study this, a T-helper-cell clone, D10.G4.1 was employed. This cell clone proliferates in the presence of Iak (Class II glycoprotein, MAC product) bearing M luminal diameter and specific antigen (conalbumin A) thus directly reflecting the AP capability of the M luminal diameter. Peritoneal M luminal diameter were harvested from B10.BR mice (H2k) and their AP was tested in vitro by incubating varying numbers of M luminal diameter with 2 X 10(4) D10.G4.1 cells/well and conalbumin (400 micrograms/ml) in the presence and absence of different concentrations of PGE2 or TXB2. Cultures were incubated for 72 hr, pulsed with [3H]-thymidine, and harvested. At concentrations of 10, 30, and 100 nM of PGE2, D10.G4.1 proliferations were 38, 35, and 20% of control, respectively (P less than 0.05 compared to control). TXB2 added at the above-mentioned concentrations did not suppress the proliferative response of D10. Thus, PGE2 but not TXB2 has a potent immunosuppressive effect on AP of peritoneal M luminal diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Exogenous and Endogenous Nitric Oxide but Not iNOS Inhibition Improves Function and Survival of Ischemically Injured Livers

The role of nitric oxide (NO) in liver ischemia/reperfusion I/R) injury remains controversial and few works have shed more information regarding the effect of exogenous (EX) and/or endogenous NO (EN) under conditions of I/R of the liver. We investigated the role of exogenous and endogenous NO and inducible nitric oxide synthase (iNOS) inhibition in liver function, neutrophil infiltration, and animal survival after liver I/R. Sprague-Dawley rats were subjected to total hepatic ischemia for 90 min using an extracorporeal porto-systemic shunt. The animals were divided into five groups, including the sham porto-systemic shunt with no ischemia, the control ischemic group, the L-arginine-treated group, the sodium nitroprusside (SNP or NaNP)-treated group, and the L- N 6 -(1-iminoethyl) lysine hydrochloride (L-NIL) (selective iNOS inhibitor)-treated group. The animal survival was followed for 7 days. Liver injury tests, tissue myeloperoxidase (MPO), and histology were analyzed at 6 h postreperfusion. L-Arginine- and sodium nitroprusside-treated groups demonstrated significant improvement in 7 days survival in comparison to the control (20%) ( p < .05). The best overall survival was obtained with SNP (70%), followed by survival in the L-arginine treated group (60%). The iNOS inhibitor group (40%) did not show any statistical significance when compared to the control group ( p > .05). Liver injury tests and histology scores in the SNP- and L-arginine-treated groups showed significant improvement when compared to the control group ( p < .01 and p < .05, respectively). The iNOS group demonstrated only a slight improvement in these parameters. The liver MPO (as a measurement of neutrophil migration into the liver parenchyma) was significantly decreased only in the SNP and L-arginine groups ( p < .05) but not in the iNOS group ( p > .5). We conclude that NO exogenous donors and substrates for the endogenous pathway are beneficial for the liver after severe I/R and could be important therapeutic targets to prevent damage following this phenomenon.

ATP-MgCl2 restores the depressed hepatocellular function and hepatic blood flow following hemorrhage and resuscitation.

Although ATP-MgCl2 produces a myriad of beneficial effects following organ ischemia and simple hemorrhagic shock in animal models which involved heparinization and/or blood resuscitation, it is not known whether ATP-MgCl2 has any salutary effect on the depressed active hepatocellular function (AHF) and hepatic microvascular blood flow (HMBF) in a nonheparinized model of trauma and severe hemorrhage in the absence of blood resuscitation. To determine this, rats underwent a midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum shed blood volume was returned in the form of Ringers lactate (RL). The animals were then resuscitated with four times the volume of shed blood with RL. ATP-MgCl2, 50 mumoles/kg body weight (BW) each or an equivalent volume of normal saline, was infused intravenously for 95 min during and following crystalloid resuscitation. At 1.5 and 4 hr after resuscitation, AHF (Vmax, maximal velocity of indocyanine green clearance; Km, efficiency of the active transport process) was determined without blood sampling by using an in vivo indocyanine green clearance technique. HMBF was measured with laser Doppler flowmetry. Results indicate that Vmax, Km, and HMBF decreased significantly at 1.5-4 hr after hemorrhage and resuscitation. ATP-MgCl2 infusion restored the depressed Vmax, Km, and HMBF and prevented the occurrence of hepatic edema. The restoration of AHF with ATP-MgCl2 treatment may be due to its direct salutary effect on the active indocyanine green transport process and/or due to improvement in hepatic microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)

P-Selectin Blockade Is Beneficial after Uncontrolled Hemorrhagic Shock

OBJECTIVES The selectins play an important role in the neutrophil-mediated injury after hemorrhagic shock and resuscitation. The aim of this study was to investigate the effect of P-selectin blockade after HS and resuscitation. METHODS Forty-eight Sprague-Dawley rats were subjected to controlled combined with uncontrolled HS and resuscitation. Rats were divided into three groups (n = 16/group): (1) sham, no HS; (2) control, HS + resuscitation + drug vehicle; (3) treated, HS + anti-P-selectin monoclonal antibody. Transaminase levels to measure hepatocellular injury, liver myeloperoxidase to assess neutrophil infiltration, and histology were analyzed and compared between groups. Survival was followed for 3 days and was compared statistically. RESULTS Survival significantly increased from 30% in the control group to 70% in the treated group. Hepatocellular and structural injury as well as neutrophil infiltration were significantly decreased in treated animals. CONCLUSION Blockade of P-selectin resulted in decreased hepatocellular injury and increased survival in our model of uncontrolled HS. Selectins may be important therapeutic targets for blockade in the treatment of HS.

Plasma Vitamin and Mineral Status in Home Parenteral Nutrition Patients

Home parenteral nutrition (HPN) provides long-term nutritional support for persons whose absorptive capacity is compromised by a variety of intestinal malabsorption problems. However, the presence of vitamin and mineral deficiency syndromes that normally would not have time to develop in the hospitalized patient receiving total parenteral nutrition has been reported in patients receiving HPN. This study entails a longitudinal survey of plasma concentrations of vitamins A, E, and 1,25-dihydroxyvitamin D, as well as the minerals zinc, copper, and selenium, in patients receiving HPN. Plasma samples from eight patients who had been on HPN for 1-92 months before the study began were obtained once a month over a 12-month period. The blood was drawn immediately before their evening infusion of TPN in order to approximate fasting plasma nutrient concentrations. Patient values were compared to fasting control values and to published norms. Values for vitamin A, 1,25-dihydroxyvitamin D, and zinc all were within the normal range, and there was no evidence of metabolic bone disease. Plasma vitamin E and copper concentrations exceeded the normal range for most of the 12-month period. Of all of the nutrients studied, only plasma selenium concentrations were consistently in the low-normal to below-normal range. Selenium levels in patients on HPN should be monitored regularly, and supplementation may be necessary if clinical conditions warrant.

Feeling the stress: perceptions of burnout among general surgery program directors.

PURPOSE To document the types and levels of stress experienced by general surgery program directors as they fulfill their education and administrative responsibilities. METHODS This study consisted of a 3-part survey that incorporated 2 established instruments to help determine the presence of burnout in program directors. A personal projects analysis was used to help identify the tasks most relevant to the role of program director as well as to evaluate their perceptions of these tasks. The Maslach Burnout Inventory (MBI) was used to measure the degree of burnout among program directors. Demographic data were gathered to develop a picture of the background of the program directors and how they spent their time. RESULTS A total of 71.8% of program directors responded. Of all tasks, teaching received the highest ratings for importance, enjoyment, and control, as well as the lowest ratings for stress. Emotional exhaustion was the most notable aspect of burnout in program directors on the MBI. Program directors scoring high in burnout were younger, had been in their current position fewer years, and had fewer years overall as a program director. CONCLUSIONS Burnout is more related to age and experience of program director than to features of the program itself.