Preliminary analysis of early outcomes of a prospective, randomized trial of complete steroid avoidance in liver transplantation.

Steroids are a mainstay in liver transplantation for induction and maintenance immunosuppression but are associated with significant adverse effects. While prior studies have successfully limited the use of steroids, whether complete steroid avoidance will improve outcomes remains unclear. To further evaluate the need for steroids, consenting patients who underwent liver transplantation between June 2002 and May 2004 were entered into a prospective, randomized trial to receive either standard therapy (tacrolimus, mycophenolate mofetil, steroid induction/maintenance) or complete steroid avoidance (standard therapy without steroid induction/maintenance). Clinically suspected rejection was confirmed by biopsy and treated with pulse steroid therapy. Outcomes were compared on an intention to treat basis. Of the 72 patients enrolled, 36 (50%) were randomized to the steroid avoidance group with a mean follow up of 412 +/- 41 days. Donor and recipient characteristics were similar between groups. The steroid avoidance group was more likely to have significant infections (52% vs 28%, P = .03). There was a trend toward an increased rate of acute rejection (25% vs 14%, P = .23). Twelve of 36 recipients (33%) enrolled in the steroid avoidance group later received steroids. The incidence of recurrent hepatitis C was similar between groups. The 1-year patient (90% vs 83%, P = .44) and graft survivals (90% vs 81%, P = .27) were similar between groups. These data suggest complete steroid avoidance in liver transplantation results in acceptable patient and graft survival. However, the potential long-term benefits of steroid avoidance, including a decrease in severity of recurrent hepatitis C, remain under investigation.

The combination of growth hormone with hepatocyte growth factor alters the acute phase response.

Recombinant human growth hormone (rhGH) and hepatocyte growth factor (HGF) have both been shown to individually modulate hepatic acute phase reactant proteins and cytokine expression following trauma through different pathways. Recombinant hGH has also been shown to decrease serum and hepatic HGF concentrations after a thermal injury. We hypothesized that the combination of rhGH plus HGF improves the burn-induced acute phase response. Fifty-six male Sprague-Dawley rats received a 60% TBSA third-degree scald burn and were randomly divided to receive either rhGH (2.5 mg/kg/day sc.) plus HGF (200 microg/kg i.v. every 12 h) or placebo (saline). Rats were sacrificed on post-burn days 1, 2, 5, or 7 and serum constitutive and acute phase proteins, TNF-alpha, IL-1beta, IL-6 and liver total protein measured. Hepatic cytokine gene expression, triglyceride concentration, and hepatocyte proliferation were also measured. In rats receiving rhGH/HGF, serum albumin increased on days 5 and 7 and transferrin on day 7 after burn compared to placebo (P<0.05). Haptoglobin decreased 5 days after burn compared to placebo (P<0.05). RhGH/HGF increased serum TNF-alpha on day 2 after burn, while it decreased serum IL-1beta on day 1 after burn compared with placebo (P<0.05). RhGH/HGF had no effect on hepatic cytokine gene expression compared with placebo. Liver total protein content and hepatocyte proliferation increased on days 1, 2, 5, and 7 after burn with rhGH/HGF treatment (P<0.05). These findings indicate that rhGH in combination with HGF exert additive effects on constitutive hepatic proteins and partial inhibitory effects on acute phase protein and cytokine expression. RhGH/HGF has a strong mitogenic effect on hepatocytes.

Cutaneous burn increases apoptosis in the gut epithelium of mice.

BACKGROUND: Gut mucosal homeostasis depends on a balance between cell proliferation and cell death. After cutaneous burn injury, gut mucosal weight has been shown to decrease. This decrease in weight was paradoxically associated with an increase in gut proliferative factors. For mucosal weight to decrease in the presence of increased proliferation, there must be an even greater increase in cell death. We postulate that cutaneous burn injury causes an increase in gut epithelial cell death primarily by apoptosis. STUDY DESIGN: We produced a 30% full-thickness scald burn in the dorsum of anesthetized male C57BL6 mice and collected the proximal small bowel at 12, 24, 36, 48, and 60 hours after injury. Sham burned animals served as controls. Apoptosis and proliferation were measured by immunohistochemical assays (terminal deoxyuridine nick-end labeling for apoptosis and proliferative cell nuclear antigen assay for proliferation). Apoptosis was also measured by ELISA for cytoplasmic histone-associated DNA fragments. Mucosal height was determined on histologic sections. The two groups were compared at each time point using Wilcoxon two-sample test and t-test with Bonferroni's correction where appropriate. RESULTS: The percentage of apoptotic cells (number of cells stained by terminal deoxyuridine nick-end labeling per 100 villus cells) was significantly higher at 12, 24, and 48 hours after injury. This increase was corroborated by an increase in the ELISA at 12 hours. Proliferation as measured by immunostaining for proliferative cell nuclear antigen significantly increased at 12, 24, 48, and 60 hours. Mucosal height as a gross measure of mucosal atrophy was not different between the groups. CONCLUSIONS: We have shown an increase in apoptosis coupled with an increase in proliferation after a burn injury. These results imply an increase in cell turnover in the gut epithelial cells after a burn. Impaired bowel function has been demonstrated repeatedly after burn injury, and this increase in cell turnover may be related.

Characteristics associated with liver graft failure: the concept of a donor risk index.

Transplant physicians and candidates have become increasingly aware that donor characteristics significantly impact liver transplantation outcomes. Although the qualitative effect of individual donor variables are understood, the quantitative risk associated with combinations of characteristics are unclear. Using national data from 1998 to 2002, we developed a quantitative donor risk index. Cox regression models identified seven donor characteristics that independently predicted significantly increased risk of graft failure. Donor age over 40 years (and particularly over 60 years), donation after cardiac death (DCD), and split/partial grafts were strongly associated with graft failure, while African-American race, less height, cerebrovascular accident and 'other' causes of brain death were more modestly but still significantly associated with graft failure. Grafts with an increased donor risk index have been preferentially transplanted into older candidates (>50 years of age) with moderate disease severity (nonstatus 1 with lower model for end-stage liver disease (MELD) scores) and without hepatitis C. Quantitative assessment of the risk of donor liver graft failure using a donor risk index is useful to inform the process of organ acceptance.

Burn and starvation increase programmed cell death in small bowel epithelial cells.

Maintenance of gut mucosal homeostasis depends on a balance between cell proliferation and cell death. Gut mucosal integrity is impaired after severe burn and during starvation. We determined the effect of burn, starvation, and the combination of both on small bowel epithelial apoptosis and proliferation. Fifty adult male Fischer 344 rats (260-300 g) received a 60% full-thickness scald burn and were randomly divided into fed and starved groups. Small intestine was taken at 12, 24, and 48 hr after injury. All animals in the 12-hr group were starved while recovering from anesthesia. Apoptosis was quantified by immunohistochemical staining (TUNEL) and mucosal proliferation was determined by bromodeoxyuridine (BrdU) incorporation. The apoptotic index was higher in burned rats compared to controls at 12 hr after burn; both these groups were starved (P < 0.05). At 24 and 48 hr after burn, apoptosis was highest in the starved groups, with no additional effects of burn (P < 0.05). Mucosal epithelial cell proliferation was not different between groups at any time point. In conclusion, burn and starvation both increase apoptosis in the small bowel mucosa; however, these effects are not additive. Apoptosis could be attenuated by enteral feeding, which delineates the importance of early enteral feeding initiation after injury to maintain mucosal integrity.

Recombinant human growth hormone (rhGH) downregulates hepatocyte growth factor (HGF) in burns.

INTRODUCTION: Hepatocyte growth factor (HGF) and recombinant human growth hormone (rhGH) have both been shown to increase albumin serum concentrations after major injury. However, the effect of rhGH on HGF production after injury is unknown. We postulated that rhGH effects constitutive protein concentrations by inducing HGF expression. METHODS: In order to determine a dose response, 20 male Sprague-Dawley rats received three different concentrations of rhGH 1, 2.5, and 5 mg/kg and a saline treatment. Rats were sacrificed 10 days after burn and serum albumin and HGF plasma concentrations were measured. Eighty male Sprague-Dawley rats received a 60% TBSA third-degree scald burn injury and were randomly divided into three groups, control, burn plus saline treatment, or burn plus rhGH treatment (2.5 mg/kg SQ qD). Rats were sacrificed 2, 5, 7, and 14 days after burn, and serum albumin, plasma, and hepatic tissue HGF concentrations were determined (ELISA, Institute of Immunology, Tokyo, Japan). RESULTS: At 10 days postburn serum albumin levels were significantly increased with rhGH 2.5 mg/kg treatment (P < 0.05); however, HGF plasma concentrations were significantly decreased with a dose of 5 mg/kg of rhGH compared to control and rhGH 1.0 mg/kg (P < 0.05). Serum albumin concentrations decreased immediately after burn and remained low until at least the 14th day after injury. RhGH-treated animals had higher levels of albumin on Day 7 after burn (P < 0.05). Plasma HGF levels decreased immediately after burn, but increased after the second day postburn. Beginning on the 5th day after injury, HGF levels in non-rhGH-treated rats were significantly higher compared to those in rhGH-treated rats (P < 0.05). Hepatic tissue HGF concentrations were higher in non-rhGH-treated rats compared to rhGH treated animals 7 days after burn (P < 0.01). CONCLUSION: Although rhGH treatment improves constitutive protein synthesis, rhGH decreases HGF concentration in a dose-dependent manner. The improvements in constitutive protein concentrations do not occur via a HGF dependent pathway.

Hepatocyte growth factor modulates the hepatic acute-phase response in thermally injured rats.

OBJECTIVE: Hepatocyte growth factor (HGF) has been shown to modulate the acute-phase response in vitro. The specific in vivo role of HGF in this multifactorial response, however, remains unknown. This study examines the effects of exogenous HGF on the acute-phase response in thermally injured rats. DESIGN: Prospective, randomized, laboratory study. SETTINGS: Shriners Hospital for Children and University of Texas Medical Branch laboratories. SUBJECTS: Fifty-six male Sprague-Dawley rats (weight range, 300-325 g). INTERVENTION: Animals received a 60% total body surface area third-degree scald burn and were randomly divided to receive either 400 microg/kg/day i.v. HGF or saline (control). MEASUREMENTS AND MAIN RESULTS: Serum acute-phase proteins, cytokines, and insulin-like growth factor (IGF)-I concentrations, as well as liver weight, protein and triglyceride content, IGF-I concentrations, and cytokine gene expression were measured 1, 2, 5, or 7 days after burn. Serum albumin was increased on days 2, 5, and 7 after burn, and transferrin was increased on day 7 after burn in HGF-treated rats compared with controls (p<.05). HGF increased alpha2-macroglobulin concentrations on postburn days 2, 5, and 7 compared with controls (p<.05). Serum interleukin-6 and tumor necrosis factor-alpha were significantly higher within 2 days of burn in rats treated with HGF (p<.05). HGF increased the hepatic gene expression of tumor necrosis factor-alpha compared with controls (p<.05). Serum IGF-I decreased in rats receiving HGF 1, 2, and 5 days after burn, whereas liver IGF-I concentrations were higher on days 1 and 7 after burn compared with controls (p<.05). Hepatic protein concentrations were higher in the HGF group compared with controls on postburn days 1, 2, and 7, with a concomitant increase in total liver weight (p<.05). HGF exerted a strong mitogenic effect on hepatocytes 1 and 2 days after thermal injury compared with controls (p<.05). CONCLUSIONS: These findings suggest that HGF modulates the acute-phase response in vivo after burn and causes changes in liver morphology.

Recombinant human growth hormone alters acute phase reactant proteins, cytokine expression, and liver morphology in burned rats.

BACKGROUND: The effects of exogenous recombinant human growth hormone (rhGH) on hepatic acute phase reactant proteins, cytokine expression, and liver morphology were studied in thermally injured rats to define whether rhGH alters the acute phase response. MATERIALS AND METHODS: Sprague-Dawley rats (56 males) receiving a 60% TBSA third-degree scald burn were randomly divided into two groups to receive either 2.5 mg/kg/day sc rhGH or saline. Rats were sacrificed on Postburn Days 1, 2, 5, and 7. Serum acute phase reactant proteins and cytokines TNF-alpha, IL-1alpha, IL-1beta, and IL-6 were measured. Hepatocyte proliferation, hepatic cytokine gene expression, and liver protein concentrations were determined. RESULTS: Recombinant hGH increased serum albumin on Days 5 and 7 after burn (P < 0.05). Serum haptoglobin and alpha1-acid glycoprotein levels decreased at 2, 5, and 7 days after burn compared to saline (P < 0.05). In rats treated with rhGH, serum IL-1beta decreased 1 day postburn, while serum TNF-alpha increased 5 days after burn compared to saline (P < 0.05). Serum IL-6 and IL-1alpha did not change. Hepatic RNA levels for TNF-alpha were significantly elevated on Day 1 postburn compared to saline (P < 0. 05). Hepatic protein content increased on Days 2, 5, and 7 postburn compared to saline (P < 0.05). Hepatocyte proliferation in rhGH-treated rats increased on Day 5 after burn (P < 0.05). CONCLUSION: Data indicate that rhGH alters the hepatic acute phase response by decreasing type I acute phase proteins and modulating IL-1-like cytokine expression. These changes are associated with increased hepatocyte mitosis and serum and total liver protein concentrations.

Anabolic effects of insulin-like growth factor in combination with insulin-like growth factor binding protein-3 in severely burned adults.

BACKGROUND: The purpose of this study was to determine the anabolic effects of recombinant human insulin-like growth factor-I (rhIGF-1) complexed with its principal binding protein IGF-1 binding protein-3 (IGFBP-3) in severely burned adults. METHODS: Ten burned adults were studied consecutively after receiving saline (pretreatment), then rhIGF-1/IGFBP-3 (treatment) for 5 days. Doses were 1, 2, and 4 mg/kg per day. Glucose, electrolytes, hormones, and leg muscle protein metabolism were determined. Nine other studies were performed on similarly injured adults at comparable times to the treatment studies to control for time effects. RESULTS: Serum IGF-1 and IGFBP-3 levels increased with all doses, but no incremental increases were found. Leg protein balance improved with rhIGF-1/IGFBP-3, which was associated with an increase in muscle protein fractional synthetic rate. These effects were independent of time. All patients were euglycemic without electrolyte imbalances. CONCLUSION: Net protein synthesis in the isolated leg of severely burned adults improved with rhIGF-1/IGFBP-3 without development of glucose abnormalities.

Increased small bowel epithelial turnover in interleukin-1 receptor knockout mice.

OBJECTIVE: To determine whether interleukin-1 (IL-1) affects the cellular homeostasis of small bowel mucosa, the authors studied apoptosis and proliferation in small bowel epithelium in two groups of C57 mice: an IL-1 receptor knockout group, and a control wild-type group. SUMMARY BACKGROUND DATA: Gut mucosal integrity is maintained by a balance of cell proliferation and cell death. Recent reports suggest that IL-1, a proinflammatory cytokine, increases cell death by apoptosis in some epithelial cells. METHODS: Twenty-four male C57BL6 IL-1 receptor (type I) knockout mice were killed, and small bowel was removed for study. Twenty-four wild-type mice (C57-BL6) served as controls. Body weights, bowel length, and mucosal morphology were examined for phenotypic differences. Apoptosis was quantified by terminal deoxyuridine nick-end labeling (TUNEL) immunohistochemical staining and cellular proliferation by proliferation cell nuclear antigen staining. Whole mucosal protein was analyzed for nuclear factor-kappaB expression. Groups were analyzed by t test. RESULTS: The absence of IL-1 type I receptor in knockout mice was verified by reverse transcriptase-polymerase chain reaction. IL-1 receptor null mice were larger than wild-type controls, with a longer small bowel; however, the index of small bowel length to total body weight did not differ between groups. The percentage of apoptotic cells was higher in IL-1 receptor null mice than in wild-type mice; the proliferation index also increased. Mucosal height and other measures of mucosal morphology were not different. Genotypic absence of IL-1 receptors was associated with decreased expression of nuclear factor-kappaB in whole mucosal protein extracts. CONCLUSIONS: Both apoptosis and proliferation increased in gut epithelial cells of mice without IL-1 receptors, suggesting increased cell turnover with no change in net balance. This model represents an opportunity to examine potential mechanisms of gut epithelial turnover in vivo, under both normal conditions and in conditions of mucosal proliferation and atrophy.

Muscle protein catabolism after severe burn: effects of IGF-1/IGFBP-3 treatment.

OBJECTIVE: To determine the effects of recombinant human insulin-like growth factor-1 (IGF-1) complexed with its principal binding protein, IGFBP-3, on skeletal muscle metabolism in severely burned children. SUMMARY BACKGROUND DATA: Severe burns are associated with a persistent hypermetabolic response characterized by hyperdynamic circulation and severe muscle catabolism and wasting. Previous studies showed that nutritional support and pharmacologic intervention with anabolic agents such as growth hormone and insulin abrogated muscle wasting and improved net protein synthesis in the severely burned. The use of these agents, however, has several adverse side effects. A new combination of IGF-1 and IGFBP-3 is now available for clinical study. METHODS: Twenty-nine severely burned children were prospectively studied before and after treatment with 0.5, 1, 2, or 4 mg/kg/day IGF-1/IGFBP-3 to determine net balance of protein across the leg, muscle protein fractional synthetic rates, and glucose metabolism. Another group was studied in a similar fashion without IGF-1/IGFBP-3 treatment as time controls. RESULTS: Seventeen of 29 children were catabolic before starting treatment. The infusion of 1.0 mg/kg/day IGF-1/IGFBP-3 increased serum IGF-1, which did not further increase with 2.0 and 4.0 mg/kg/day. IGF-1/IGFBP-3 treatment at 1 to 4 mg/ kg/day improved net protein balance and increased muscle protein fractional synthetic rates. This effect was more pronounced in catabolic children. IGF-1/IGFBP-3 did not affect glucose uptake across the leg or change substrate utilization. CONCLUSIONS: IGF-1/IGFBP-3 at doses of 1 to 4 mg/kg/day attenuates catabolism in catabolic burned children with negligible clinical side effects.

Efficacy of a high-carbohydrate diet in catabolic illness.

OBJECTIVE: To determine within the setting of isocaloric, isonitrogenous enteral diets whether a diet that supplies most of its calories from fat or carbohydrate would be most beneficial at limiting muscle protein wasting in catabolic illness. DESIGN: Prospective, randomized, crossover trial. SETTING: Academic pediatric burn unit in tertiary medical center. PATIENTS: Fourteen severely burned (>40% total body surface area) children underwent systemic metabolic and cross-leg muscle protein kinetic studies. INTERVENTIONS: All were treated clinically in a similar manner, including early excision and grafting, antimicrobial therapy, and isocaloric, isonitrogenous enteral nutritional support. Subjects randomly received either a high-carbohydrate enteral diet (3% fat, 82% carbohydrate, 15% protein), or a high-fat enteral diet (44% fat, 42% carbohydrates, 14% protein) for 1 week and then crossed over to the other diet for a second week. MEASUREMENTS AND MAIN RESULTS: On day 5 of each diet, muscle protein kinetics were determined from femoral arterial and venous blood samples during a primed-constant d5-phenylalanine infusion. Indirect calorimetry was used to determine systemic resting energy expenditure and respiratory quotient. The seven boys and seven girls were 7.1 +/- 1.1 (mean +/- sem) years old and suffered burns over 65 +/- 4% of their bodies, with 52 +/- 6% being third-degree burns. Muscle protein degradation markedly decreased (p <.01) with administration of the high-carbohydrate diet. Protein synthesis was unaltered. Endogenous insulin concentrations increased during the high-carbohydrate feeding period. No differences in energy expenditure were seen between study diets. CONCLUSIONS: In severely burned pediatric patients, enteral nutrition supplied predominantly as carbohydrate rather than fat improves the net balance of skeletal muscle protein across the leg. This is attributable to decreased protein breakdown, suggesting a protein-sparing effect of high-carbohydrate feedings.