The Hepatic Response to Severe Injury M.G. Jeschke and D.N. Herndon

(1)

M.G. Jeschke and D.N. Herndon

Introduction

After severe injury, such as thermal injury, a variable degree of liver injury is present and it is usually related to the severity of the thermal injury. Fatty changes, a very common finding, are per se reversible and their significance depends on the cause and severity of accumulation [1]. However, autopsies of burned children who died have shown that fatty liver infiltration was associated with increased bacterial translocation, liver failure, and endotoxemia, thus delineating the crucial role of the liver during the post-burn response [2 – 4]. In a recent study in 102 children, 41 females and 61 males with a total body burn size of 58 „ 2% and third degree burns in 45 „ 2%, we found that liver size and weight significantly increased during the first week post-burn (+85 „ 5%), peaked at 2 weeks post-burn (+126 „ 19%), and was increased by +89 „ 10% at discharge. At 6, 9, and 12 months the liver weight was increased by 40 – 50 % compared to predicted liver weight. In addition, liver protein synthesis was impaired for a 6-month period with a shift from constitutive hepatic proteins to acute phase proteins [5]. Liver enzymes were significantly elevated over the first 3 weeks post-burn, normalizing over time. These findings indi- cate that the hepatic acute phase response perseveres for a longer time period than previously thought [5, 6].

Immediately after the burn injury, liver damage may be associated with an increased hepatic edema formation. In an animal model, we have shown that the liver weight and the liver to body weight ratio increased significantly 2 to 7 days after burn injury when compared to controls [7]. As hepatic protein concentration was significantly decreased in the burned rats, we suggest that the liver weight gain is due to increased edema formation rather than increases in the number of hepatocytes or protein levels. An increase in edema formation may lead to cell damage, with the release of hepatic enzymes [7]. The three enzymes that achieve abnormal serum levels in hepatic diseases and during the aftermath of a severe injury are alkaline phosphatase, serum glutamic oxalacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT). Serum aspartate transaminase (AST), ala- nine transaminase (ALT) and alkaline phosphatase (ALP) are elevated between 50 to 200 % when compared with normal levels. We observed that serum AST, ALT, and ALP peaked during the first week post-burn and approached the normal range 3 – 5 weeks post-burn. If liver damage persists or sepsis occurs, enzymes stay elevated or increase again [5 – 7].

(2)

Hepatocyte Proliferation and Death

Liver damage has been associated with increased hepatocyte cell death [7]. In general, cell death occurs by two distinctly different mechanisms: Programmed cell death (apoptosis) or necrosis [8]. Apoptosis is characterized by cell shrinkage, DNA fragmentation, membrane blebbing, and phagocytosis of the apoptotic cell frag- ments by neighboring cells or extrusion into the lumen of the bowel without inflammation. This is in contrast to necrosis, which involves cellular swelling, random DNA fragmentation, lysosomal activation, membrane breakdown, and extrusion of cellular contents into the interstitium. Membrane breakdown and cellular content release induce inflammation with the migration of inflammatory cells and release of pro-inflammatory cytokines and free radicals, which leads to further tissue breakdown [8]. Pathological studies found that about 10 % to 15 % of thermally injured patients have liver necrosis at autopsy [1, 9]. The necrosis is generally focal or zonal, central or paracentral, sometimes microfocal, and related to burn shock and sepsis.

The morphological differences between apoptosis and necrosis are used to differen- tiate the two processes.

A cutaneous thermal injury induces liver cell apoptosis (Fig. 1) [7]. This increase in hepatic programmed cell death is compensated by an increase in hepatic cell proliferation, suggesting that the liver attempts to maintain homeostasis (Fig. 1).

Fig. 1. Top panel: Percent of prolif- erating cells measured by PCNA.

Lower panel: Apoptotic cells measured by TUNEL assay 1, 2, 5, and 7 days after burn, expressed as positive apoptotic hepatocytes per one thousand hepatocytes. Burned rats had significantly higher rates of hepatocyte apoptosis when compared to controls. Data presented as means „ SEM. (Burned animals n = 7 and controls n = 2 per time point). * p ‹ .05 burn vs. control.

Modified from [7] with permission

(3)

Despite the attempt to compensate for increased apoptosis by increased hepatocyte proliferation, the liver cannot regain hepatic mass and protein concentration, as we found a significant decrease in hepatic protein concentration in burned rats. It has been shown that a cutaneous burn induces small bowel epithelial cell apoptosis [10].

In the same study, the authors showed that small bowel epithelial cell proliferation was not increased, leading to a loss of mucosal cells and hence mucosal mass. Simi- lar findings were demonstrated in the heart [11 – 14]. Burn induced cardiocyte apoptosis, however, cardiocyte proliferation remained unchanged causing cardiac impairment and dysfunction [11 – 14].

The mechanisms whereby a cutaneous burn induces programmed cell death in hepatocytes are not defined. Studies suggested that, in general, hypoperfusion and ischemia-reperfusion are associated to promote apoptosis [15 – 17]. After a thermal injury it has been shown that the blood flow to the bowel decreases by nearly 60 % of baseline and stays decreased for approximately 4 hours [18]. It can be surmised that the hepatic blood flow also decreases, thus causing programmed cell death. In addition, pro-inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF) have been described as apoptotic signals [19, 20]. We have shown in our burn model that after a thermal injury serum and hepatic concentrations of pro-inflammatory cytokines, such as IL-1q , IL-6, and TNF, are increased [21–23].

We, therefore, suggest that two possible mechanisms, decreased splanchnic blood- flow and elevation of pro-inflammatory cytokines, are involved in increased hepatocyte apoptosis by initiating intracellular signaling mechanisms. Signals that may be involved encompass many signals that play an important role during the acute phase response.

A pathophysiologic association with apoptosis is mitochondrial impairment and dysfunction. After severe stress, mitochondrial function and structure is impaired as shown in patients with systemic inflammatory response syndrome (SIRS), sepsis, or after burn injury [24 – 27]. Our group recently showed that post-burn mitochondrial state-3 respiration and the respiratory control index were significantly attenuated in muscle and liver. In addition, we found that calcium dependent onset of mitochondrial permeability transition (MPT) was markedly accelerated indicating severe mitochondrial structure damage post-burn (Fig. 2). Hepatocyte apoptosis was significantly increased along with increased caspases-3 and -9 and decreased Bcl-2 concentration post-burn [28]. We concluded that a burn induces hepatocyte apoptosis

Fig. 2. Calcium dependent mito- chondrial permeability transition (MPT). Burn injury induces damage to the mitochondrial membrane, which leads to a significant accelerated onset of MPT.

(4)

via caspases-3 and -9 and causes a significant impairment to mitochondrial function and structure. We suggest that mitochondrial dysfunction and increased hepatocyte apoptosis could be the underlying mechanism for the marked hepatomegaly observed after a severe burn.

Bile Formation

Bile secretion is an active process, relatively independent of total liver blood flow, except in conditions of shock. Bile is formed at two sites: a) the canalicular membrane of the hepatocyte, and b) the bile ductules or ducts. Total unstimulated bile flow in a 70 kg man has been estimated to be 0.41 to 0.43 ml/min. Eighty percent of the total daily production of bile (approximately 1500 ml) is secreted by hepatocytes and 20 % is secreted by the bile duct epithelial cells. In trauma and sepsis, intrahepatic cholestasis occurs frequently and appears to be an important pathophysiologic factor, occurring without demonstrable extrahepatic obstruction. This phenomenon has been described in association with a number of processes, such as hypoxia, drug toxicity, or total parenteral nutrition [29]. The mechanisms of intrahepatic cholestasis seem to be associated with an impairment of basolateral and canicular hepatocyte transport of bile acids and organic anions [30, 31]. This is most likely due to decreased transporter protein and RNA expression thus leading to increased bile.

Intrahepatic cholestasis, which is one of the prime manifestations of hepatocellular injury, was present in 26 % of patients in a clinical study [9]. All of these cases were concurrent with sepsis. The cellular damage observed in sepsis is more likely the result of decreased hepatic blood flow than of direct cellular damage [32]. We have recently shown that a burn causes a significant decrease in bile acid output (Fig. 3) [33]. As bile acids are contributors to hepatic regeneration it seems likely that decreased bile acid output represents another factor for hepatocyte damage post- burn [34].

Fig. 3. Bile acid output of thermally injured animals in comparison to control rats. Bile was collected over consecutive 10 min periods. When comparing the corresponding periods, bile acid secretion was significantly reduced in the burned group. *p‹ 0.001 burn vs. control; **p‹ 0.01 burn vs. control. From [33] with permission. Copyright 2006 The Endocrine Society

(5)

Hepatic Acute Phase Response

The acute phase response is a cascade of events initiated to prevent tissue damage and to activate repair processes (Fig. 4) [35]. The acute phase response is initiated by activated phagocytic cells, fibroblasts, and endothelial cells which release pro- inflammatory cytokines leading to the systemic phase of the acute phase response.

The systemic reaction affects the hypothalamus which leads to fever, the pituitary- adrenal axis so as to release steroid hormones, the liver which causes the synthesis and secretion of acute phase proteins, the bone marrow which promulgates further hemopoietic responses, and the immune system which allows activation of the retic- uloendothelial system and the stimulation of lymphocytes. However, a crucial step in this cascade of reactions involves the interaction between the site of injury and the liver, which is the principle organ responsible for producing acute phase proteins and modulating the systemic inflammatory response [35].

After major trauma, such as a severe burn, hepatic protein synthesis shifts from hepatic constitutive proteins, such as albumin, pre-albumin, transferrin, and retinol- binding protein to acute phase proteins [35 – 37]. Acute phase proteins are divided into type I acute phase proteins, such as haptoglobin and [1-acid glycoprotein, mediated by IL-1-like cytokines (IL-1[ / q , TNF) and type II acute phase proteins, such as [2-macroglobulin and fibrinogen, which are mediated by IL-6-like cytokines (IL-6, IL-11) [35 – 37]. We recently evaluated the inflammatory-cytokine expression profile after a severe burn and found that pro-inflammatory mediators, namely IL-6, IL-8, monocyte chemoattractant protein (MCP)-1 and TNF, increase 2 – 100 fold immediately post-burn. The inflammatory cascade decreases over time and by 5 – 6 weeks most of the cytokines approach normal levels [38].

The signal cascade of cytokines is the following: The cytokines bind to their receptors and activate intracellular signals by tyrosine phosphorylation, for the type I acute phase response c-Jun/c-fos, hepatic nuclear factor-kappa B (NF-κB) or the CCAAT/enhancer-binding-proteins (C/EBPs) [39 – 42]. The intracellular signal cascade for type II has been shown to be a tyrosine phosphorylation and activation of intracellular tyrosine kinases (JAKs), latent cytoplasmic transcription factors, STAT1, STAT3, and STAT5 (signal transducer and activator of transcription),

Fig. 4. Scheme of the hepatic acute phase response.

(6)

or mitogen-activated protein [40, 43, 44]. These signals activate transcription, translation, and expression of acute phase proteins. IL-6, in particular, has been speculated to be the main mediating cytokine. IL-6 activates glycoprotein 130 and the JAK-kinases (JAK-1) leading to activation of STAT1 and 3 translocating to the nucleus. The intranuclear genes for acute phase proteins are turned on.

In contrast to acute phase proteins, constitutive hepatic proteins are downregu- lated [35, 37, 45, 46]. After a thermal injury, albumin and transferrin decrease by 50 – 70 % below normal levels [5, 6]. Studies have shown that two mechanisms are responsible for the decrease in constitutive hepatic proteins. First, the liver re-priori- tizes its protein synthesis from constitutive hepatic proteins to acute phase proteins [35]. This has been shown in many studies in which the mRNA synthesis for constitutive hepatic proteins is decreased. The other mechanism for decreased constitutive hepatic protein concentration is the capillary leakage and the loss of these proteins into the massive extravascular space and burn wound. Albumin and transferrin, however, have important physiologic functions as they serve as transporter proteins and contribute to osmotic pressure and plasma pH [45, 46]. The downregulation of albumin and transferrin after trauma has been described as potentially harmful and the synthesis of these proteins has been used as a predictor of mortality, nutritional status, and severity of stress, and as an indicator of improved recovery [39, 46 – 48].

The question whether albumin substitution in patients with hypoalbuminemia is beneficial or detrimental represents a current study focus.

The aim of the acute phase response is to protect the body from further damage, and this aim will be achieved when all elements of the acute phase response coalesce in a balanced fashion. However, a prolonged increase in pro-inflammatory cytokines and acute phase proteins has been shown to be associated with a hypercatabolic state, increased risk of sepsis, multiple organ failure (MOF), morbidity, and mortality [39, 47, 48]. Therefore, an important therapeutic approach to improve survival after trauma may be the modulation of the acute phase response by decreasing acute phase proteins and pro-inflammatory cytokines and increasing constitutive hepatic proteins [49]. The use of antibodies against pro-inflammatory cytokines such as TNF, IL-1q , or their receptors showed promising results in-vitro and in animal models by increasing survival rates in septicemia [50 – 53]. However, when these approaches entered clinical trials it became evident that these promising animal data could not be repeated in humans. New approaches encompass the attenuation or blockade of high mobility group box 1 (HMGB1) a late mediator responsible for lethality in the state of septicemia [54 – 57], macrophage inhibitory factor (MIF) [58 – 62], receptor for advanced glycation end products (RAGE) [63 – 66], or other danger signals (e.g., damage-associated molecular pattern molecules [DAMPs]) [67].

We and others have chosen a different approach. We hypothesized that endogenous hormones can alter inflammation and the acute phase response. Over the last decade, we determined the effects of growth hormone, hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I), ICF-I in combination with its principle binding protein-3 (IGF-I/BP-3), and insulin on the hepatic acute phase response and cytokine expression as a physiological approach to attenuate the pro-inflammatory cascade.

(7)

Possible Treatments to Alter the Acute Phase and Inflammatory Response

Recombinant Human Growth Hormone

Recombinant human growth hormone (rhGH) modulates the acute phase response by affecting pro-inflammatory, IL-1-like cytokine expression followed by decreased type I acute phase proteins and increasing constitutive hepatic proteins [23, 68].

No effect on IL-6-like cytokines and type II acute phase proteins could be demonstrated. RhGH administration increased endogenous albumin levels, reducing the amount of required exogenous substitution to maintain normal serum albumin levels. Similar to acute phase proteins, the mechanisms by which rhGH increases endogenous albumin concentrations are unknown; however, rhGH may exert this effect through activation of C/EBPq [69]. Another side effect of rhGH that has been recently delineated is an increase in hepatic triglyceride concentration and development of a fatty liver [23, 70, 71]. RhGH administration over 10 days increased hepatic triglyceride concentration by nearly 50 % in burned rats [23].

The mechanisms have been discussed in clinical studies, where the authors speculated that rhGH increased peripheral lipolysis and due to a lack of transporter proteins (low density lipoprotein [LDL], high density lipoprotein [HDL]) triglycerides accumulate in the liver [70, 71]. We demonstrated in pediatric burn patients that rhGH increased free fatty acid concentration when compared to placebo, indicating that rhGH stimulates peripheral lipolysis and subsequently free fatty acid concentration [68]. Given the fact that the acute phase response is a contributor to mortality after trauma, rhGH administration appears not to cause an increase in mortality in severely burned children as described by Takala et al. in trauma and septic patients [72], as rhGH does not cause an increased and prolonged acute phase response.

Hepatocyte Growth Factor

Administration of hepatocyte growth factor (HGF) stimulates constitutive hepatic proteins after burn injury in-vivo [73]. In fact, serum transferrin reached normal levels 7 days after injury with HGF treatment, whereas in saline-treated animals, serum transferrin remained low. Serum albumin levels decreased; however, begin- ning at day 2 after burn, HGF attenuated this drop in serum albumin. The exact mechanisms by which HGF stimulates constitutive hepatic proteins are unknown;

however, HGF is capable of stimulating the synthesis of C/EBPq , which regulates constitutive hepatic proteins [74]. In contrast to recent in vitro studies, where the authors demonstrated that HGF decreased acute phase proteins, we showed in vivo that HGF increased serum [2-macroglobulin (type II acute phase protein), with no effect on [1-acid glycoprotein and haptoglobin (type I acute phase proteins) [75, 76].

Type II acute phase proteins are mediated through IL-6 like cytokines, including cytokines such as IL-6 and IL-11 [35]. IL-6, secreted by Kupffer cells in the liver, is capable of regulating the synthesis of transcription factors that have response elements in the 5’-flanking region of the HGF gene that may be potentially utilized in inducing HGF gene expression at the transcriptional level [77, 78]. Therefore, IL-6 appears likely to substantially and quickly upregulate HGF mRNA and HGF mRNA receptor expression [79]. However, the interaction between HGF and IL-6 in vitro has been shown to be complex and controversial [75, 80]. In our study, we demonstrated that administration of rhHGF stimulated serum IL-6, along with an increase

(8)

in its dependent type II acute phase proteins, serum [2-macroglobulin and TNF [73]. HGF has been shown to have some beneficial effects and to be a potential therapeutic agent; however, more studies need to be done before this growth factor can be applied in patients.

Insulin-Like Growth Factor-I in Combination with its Principle Binding Protein-3

Insulin-like growth factor-I is a 7.7 kDa single chain polypeptide of 70 amino acids with sequence homology to pro-insulin [81]. In the body, 95 – 99 % of IGF-I is bound and transported with one of its six binding proteins (IGFBPs) 1 – 6 [81]. The major- ity of IGF-I is bound to IGFBP-3. Administration of the IGF-I/BP-3 complex as a therapeutic agent provides several advantages over the administration of IGF-I alone, because when IGF-I is already bound to IGFBP-3, it rapidly transforms into a ternary complex, which confers decreased serum clearance and allows the delivery of significantly larger amounts of IGF-I without inducing hypoglycemia and electro- lyte imbalances. In general, IGF-I has been shown to improve cell recovery, wound healing, peripheral muscle protein synthesis, gut and immune function after thermal injury [82, 83]. Recent evidence suggests that IGF-I is instrumental in the early phases of liver regeneration after trauma and modulates the hepatic acute phase response in burned rats [22, 84]. In thermally injured children, rhIGF-I in combination with its principle binding protein modulates the hepatic acute phase response by decreasing the pro-inflammatory cytokines, IL-1q and TNF, followed by a decrease in type I acute phase proteins. IGF-I/BP-3 had no effect on IL-6 and type II acute phase proteins. Decreases in acute phase protein and pro-inflammatory cytokine synthesis were associated with increases in constitutive hepatic protein synthesis [85, 86]. Attenuating the hepatic acute phase response with IGF-I/BP-3 modulated the hypermetabolic response, which may prevent MOF and improve clinical outcome after a thermal injury without any detectable adverse side effects. The data shown would make IGF-I/BP-3 an ideal therapeutic agent; however, recently our group found that IGF-I/BP-3 increased the risk of peripheral neuropathies, thus limiting the use of this agent [unpublished observations].

Insulin

In severely burned rodents, insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein and fat, while insulin decreased the hepatic inflammatory response signal cascade by decreasing hepatic pro-inflammatory cytokine mRNA and proteins, IL-1q and TNF, at pre-translational levels [21, 28]. Insulin increased hepatic cytokine mRNA and protein expression of IL-2 and IL-10 at a pre-translational level when compared with controls. In addition, insulin affected hepatic signal transcription factors and attenuated inflammation at a molecular level. Insulin increased hepatocyte proliferation along with Bcl-2 concentration, while decreasing hepatocyte apoptosis along with decreased concentrations of caspase-3 and -9, thus improving liver morphology (Fig. 5) [21, 28]. From this animal study, we concluded that insulin attenuates the inflammatory response by decreasing the pro-inflammatory and increasing the anti-inflammatory cascade, thus, restoring hepatic homeostasis. This effect was not only limited to the post- burn response but also to other post-stress models [87, 88]. We showed that insulin administration during endotoxemia (lipopolysaccharide [LPS] administration) improved hepatic protein synthesis, inflammation, acute phase protein synthesis,

(9)

Fig. 5. Burn caused a significant increase in hepatocyte apoptosis compared to normal. Insulin administra- tion decreased hepatocyte apoptosis at all time points. *p‹ 0.05 insulin vs control. Data presented as mean „ SEM with n=7 for each group and each time point. Insulin significantly increased hepatocyte proliferation on days 1, 5, and 7 compared to controls. Modified from [28] with permission

and homeostasis. Bioluminescence showed that insulin improved hepatic glucose metabolism and glycolysis (Fig. 6). Gene chip analysis revealed a strong anti-inflammatory effect of insulin on inflammatory mediators. In order to confirm our animal data, we conducted a human study [89]. Insulin administration decreased pro- inflammatory cytokines and proteins, while increasing constitutive-hepatic proteins.

Burned children receiving insulin required significantly less albumin substitution to maintain normal levels compared to controls. Insulin decreased free fatty acids and serum triglycerides when compared to controls.

In conclusion, several studies have shown that insulin attenuates the inflammatory response by decreasing the pro-inflammatory, and increasing the anti-inflammatory cascade, thus, restoring systemic homeostasis, which has been shown to be critical for organ function and survival in critically ill patients. Insulin appears to be

(10)

normal

LPS

LPS+insulin ATP

normal

LPS

LPS+insulin Glucose

normal

LPS

LPS+insulin Lactate

Fig. 6 (Legend see p. 661)

(11)

˜

Fig. 6. Bioluminescence of the liver. ATP was present in normal animals. Endotoxemia caused decreased levels of intracellular hepatic ATP. There were no significant differences between LPS and LPS+insulin. Intrahepatic glucose levels were low. LPS caused an increase in intrahepatic glucose levels, which were decreased to normal concentrations with insulin administration. Rats receiving LPS+insulin had normal hepatic glucose levels, p‹ 0.05. Endoto- xemia caused a significant increase in lactate, whereas animals receiving LPS+insulin had normal lactate levels, p‹ 0.05. Color interpretation from highest to lowest concentration: red8orange8yellow8green8light blue8dark blue. From [88] with permission from the European Association for the Study of the Liver

a safe and effective drug to affect hepatic dysfunction. Moreover, as tight euglycemic control has been shown to be advantageous, studies in this area are warranted.

Propranolol

Finally, we would like to mention propranolol, a non-selective q1/q2blocker. Beta blockade results in a decrease in urinary nitrogen loss, with decreased peripheral lipolysis and whole body urea production [90], decreased resting energy expenditure, and improved skeletal muscle protein kinetics [91]. Furthermore, propranolol pre- served fat-free mass when compared to controls [91]. Propranolol also decreases hepatic fat storage by limiting fatty acid delivery in severely burned pediatric patients [92]. In addition, we showed that propranolol decreased peripheral lipolysis and improved insulin responsiveness [93]. Recently, we further showed that propranolol has a profound effect on fat infiltration of the liver by reversing hepatomegaly [92]. We propose that propranolol reduces hepatomegaly by inhibiting lipolysis and reducing liver blood flow, and, in turn, delivery of fatty acids to the liver. The effect of propranolol on the hepatic acute phase response, systemic inflammatory reaction, and immune system is being examined in ongoing clinical studies at our institute.

Conclusion

The liver plays a crucial role in the aftermath of a thermal injury. The synthesis of constitutive hepatic proteins, acute phase proteins, cytokines, and other mediators makes it a determining factor for survival. For a long time, hepatic dysfunction was tolerated without any treatment option. In the field of burns, it appears that new treatment options will be available to successfully attenuate the hypermetabolic hepatic acute phase response. A new approach for improving hepatic function may be the use of anabolic, anti-inflammatory agents; however, there is currently no effective treatment for hepatic dysfunction.

References

1. Linares HA (1988) Autopsy findings in burned children. In: Carvajal HF, Parks DH (eds) Burns in Children: Pediatric Burn Management. Year Book Medical Pub, Chicago

2. Barret JP, Jeschke MG, Herndon DN (2001) Fatty infiltration of the liver in severely burned pediatric patients: autopsy findings and clinical implications. J Trauma 51:736 – 739

3. Barrow RE, Hawkins HK, Aarsland A, et al (2005) Identification of factors contributing to hepatomegaly in severely burned children. Shock 24:523 – 528

4. Barrow RE, Mlcak R, Barrow LN, Hawkins HK (2004) Increased liver weights in severely burned children: comparison of ultrasound and autopsy measurements. Burns 30:565 – 568 5. Jeschke MG, Mlcak RP, Herndon DN (2007) Morphologic changes of the liver after a severe

thermal injury. Shock (in press)

(12)

6. Jeschke MG, Barrow RE, Herndon DN (2004) Extended hypermetabolic response of the liver in severely burned pediatric patients. Arch Surg 139:641 – 647

7. Jeschke MG, Low JF, Spies M, et al (2001) Cell proliferation, apoptosis, NF-kappaB expression, enzyme, protein, and weight changes in livers of burned rats. Am J Physiol Gastrointest Liver Physiol 280:G1314 – 1320

8. Steller H (1995) Mechanisms and genes of cellular suicide. Science 267:1445 – 1449

9. Teplitz C (1996) The pathology of burns and the fundamentals of burn wound sepsis. In:

Herndon D (ed) Total Burn Care, 1^stedn. WB Saunders, Philadelphia, pp 45 – 50

10. Wolf SE, Ikeda H, Matin S, et al (1999) Cutaneous burn increases apoptosis in the gut epithelium of mice. J Am Coll Surg 188:10 – 16

11. Carlson DL, Lightfoot E Jr, Bryant DD, et al (2002) Burn plasma mediates cardiac myocyte apoptosis via endotoxin. Am J Physiol Heart Circ Physiol 282:H1907 – 1914

12. Carlson DL, Willis MS, White DJ, Horton JW, Giroir BP (2005) Tumor necrosis factor-alpha- induced caspase activation mediates endotoxin-related cardiac dysfunction. Crit Care Med 33:1021 – 1028

13. Horton JW, Maass DL, Ballard-Croft C (2005) Rho-associated kinase modulates myocardial inflammatory cytokine responses. Shock 24:53 – 58

14. Lightfoot E Jr, Horton JW, Maass DL, White DJ, McFarland RD, Lipsky PE (1999) Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis. Shock 11:29 – 34

15. Baron P, Traber LD, Traber DL, et al (1994) Gut failure and translocation following burn and sepsis. J Surg Res 57:197 – 204

16. Ikeda H, Suzuki Y, Suzuki M, et al (1998) Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium. Gut 42:530 – 537 17. Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY (1998) Programmed cell death induced by

ischemia-reperfusion in rat intestinal mucosa. Am J Physiol 274:G270 – 276

18. Ramzy PI, Wolf SE, Irtun O, Hart DW, Thompson JC, Herndon DN (2000) Gut epithelial apoptosis after severe burn: effects of gut hypoperfusion. J Am Coll Surg 190:281 – 287 19. Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr (1993) Tumor necrosis factor and interleu-

kin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol 13:3301 – 3310

20. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE (1997) Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Am J Pathol 151:891 – 896

21. Jeschke MG, Einspanier R, Klein D, Jauch KW (2002) Insulin attenuates the systemic inflammatory response to thermal trauma. Mol Med 8:443 – 450

22. Jeschke MG, Herndon DN, Vita R, Traber DL, Jauch KW, Barrow RE (2001) IGF-I/BP-3 administration preserves hepatic homeostasis after thermal injury which is associated with increases in no and hepatic NF-kappa B. Shock 16:373 – 379

23. Jeschke MG, Herndon DN, Wolf SE, et al (1999) Recombinant human growth hormone alters acute phase reactant proteins, cytokine expression, and liver morphology in burned rats. J Surg Res 83:122 – 129

24. Brealey D, Karyampudi S, Jacques TS, et al (2004) Mitochondrial dysfunction in a long-term rodent model of sepsis and organ failure. Am J Physiol Regul Integr Comp Physiol 286:R491 – 497

25. Brealey D, Singer M (2003) Mitochondrial Dysfunction in Sepsis. Curr Infect Dis Rep 5:

365 – 371

26. Davies NA, Cooper CE, Stidwill R, Singer M (2003) Inhibition of mitochondrial respiration during early stage sepsis. Adv Exp Med Biol 530:725 – 736

27. Singer M, Brealey D (1999) Mitochondrial dysfunction in sepsis. Biochem Soc Symp 66:

149 – 166

28. Klein D, Schubert T, Horch RE, Jauch KW, Jeschke MG (2004) Insulin treatment improves hepatic morphology and function through modulation of hepatic signals after severe trauma.

Ann Surg 240:340 – 349

29. Cano N, Gerolami A (1983) Intrahepatic cholestasis during total parenteral nutrition. Lancet 1:985

30. Bolder U, Jeschke MG, Landmann L, et al (2006) Heat stress enhances recovery of hepatocyte bile acid and organic anion transporters in endotoxemic rats by multiple mechanisms. Cell Stress Chaperones 11:89 – 100

(13)

31. Bolder U, Ton-Nu HT, Schteingart CD, Frick E, Hofmann AF (1997) Hepatocyte transport of bile acids and organic anions in endotoxemic rats: impaired uptake and secretion. Gastroen- terology 112:214 – 225

32. Hurd T, Lysz T, Dikdan G, McGee J, Rush BF Jr, Machiedo GW (1988) Hepatic cellular dysfunction in sepsis: an ischemic phenomenon? Curr Surg 45:114 – 116

33. Jeschke MG, Bolder U, Chung DH, et al (2007) Gut mucosal homeostasis and cellular mediators after severe Thermal trauma, the effect of insulin-like growth factor-I in combination with insulin-like growth factor binding protein-3. Endocrinology 148:354 – 362

34. Huang W, Ma K, Zhang J, et al (2006) Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration. Science 312:233 – 236

35. Moshage H (1997) Cytokines and the hepatic acute phase response. J Pathol 181:257 – 266 36. Baumann H, Gauldie J (1994) The acute phase response. Immunol Today 15:74 – 80 37. Hiyama DT, von Allmen D, Rosenblum L, Ogle CK, Hasselgren PO, Fischer JE (1991) Synthe-

sis of albumin and acute-phase proteins in perfused liver after burn injury in rats. J Burn Care Rehabil 12:1 – 6

38. Finnerty CC, Herndon DN, Przkora R, et al (2006) Cytokine expression profile over time in severely burned pediatric patients. Shock 26:13 – 19

39. De Maio A, Mooney ML, Matesic LE, Paidas CN, Reeves RH (1998) Genetic component in the inflammatory response induced by bacterial lipopolysaccharide. Shock 10:319 – 323

40. Kishimoto T, Taga T, Akira S (1994) Cytokine signal transduction. Cell 76:253 – 262 41. Gilpin DA, Hsieh CC, Kuninger DT, Herndon DN, Papaconstantinou J (1996) Effect of thermal

injury on the expression of transcription factors that regulate acute phase response genes: the response of C/EBP alpha, C/EBP beta, and C/EBP delta to thermal injury. Surgery 119:674 – 683 42. Alam T, An MR, Papaconstantinou J (1992) Differential expression of three C/EBP isoforms

in multiple tissues during the acute phase response. J Biol Chem 267:5021 – 5024

43. Siebenlist U, Franzoso G, Brown K (1994) Structure, regulation and function of NF-kappa B.

Annu Rev Cell Biol 10:405 – 455

44. Yao J, Mackman N, Edgington TS, Fan ST (1997) Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors. J Biol Chem 272:17795 – 17801

45. Fey G, Gauldie J (1990) The Acute Phase Response of the Liver in Inflammation. W.B. Saun- ders, Philadelphia

46. Rothschild MA, Oratz M, Schreiber SS (1988) Serum albumin. Hepatology 8:385 – 401 47. Livingston DH, Mosenthal AC, Deitch EA (1995) Sepsis and multiple organ dysfunction syn-

drome: a clinical-mechanistic overview. New Horiz 3:257 – 266

48. Selzman CH, Shames BD, Miller SA, et al (1998) Therapeutic implications of interleukin-10 in surgical disease. Shock 10:309 – 318

49. Yin MJ, Yamamoto Y, Gaynor RB (1998) The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396:77 – 80

50. Tracey KJ, Fong Y, Hesse DG, et al (1987) Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662 – 664

51. Tracey KJ, Lowry SF, Fahey TJ 3rd, et al (1987) Cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog. Surg Gynecol Obstet 164:415 – 422 52. Alexander HR, Doherty GM, Buresh CM, Venzon DJ, Norton JA (1991) A recombinant

human receptor antagonist to interleukin 1 improves survival after lethal endotoxemia in mice. J Exp Med 173:1029 – 1032

53. Pruitt JH, Copeland EM, 3rd, Moldawer LL (1995) Interleukin-1 and interleukin-1 antago- nism in sepsis, systemic inflammatory response syndrome, and septic shock. Shock 3:

235 – 251

54. Czura CJ, Yang H, Tracey KJ (2003) High mobility group box-1 as a therapeutic target down- stream of tumor necrosis factor. J Infect Dis 187 (Suppl 2):S391 – 396

55. Lotze MT, Tracey KJ (2005) High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 5:331 – 342

56. Wang H, Bloom O, Zhang M, et al (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science 285:248 – 251

57. Wang H, Yang H, Czura CJ, Sama AE, Tracey KJ (2001) HMGB1 as a late mediator of lethal systemic inflammation. Am J Respir Crit Care Med 164:1768 – 1773

(14)

58. Benigni F, Atsumi T, Calandra T, et al (2000) The proinflammatory mediator macrophage migration inhibitory factor induces glucose catabolism in muscle. J Clin Invest 106:1291 – 1300 59. Calandra T (2003) Macrophage migration inhibitory factor and host innate immune

responses to microbes. Scand J Infect Dis 35:573 – 576

60. Calandra T, Echtenacher B, Roy DL, et al (2000) Protection from septic shock by neutraliza- tion of macrophage migration inhibitory factor. Nat Med 6:164 – 170

61. Calandra T, Froidevaux C, Martin C, Roger T (2003) Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis. J Infect Dis 187 (Suppl 2):S385 – 390

62. Roger T, David J, Glauser MP, Calandra T (2001) MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature 414:920 – 924

63. Bierhaus A, Humpert PM, Morcos M, et al (2005) Understanding RAGE, the receptor for advanced glycation end products. J Mol Med 83:876 – 886

64. Peyroux J, Sternberg M (2006) Advanced glycation endproducts (AGEs): pharmacological inhibition in diabetes. Pathol Biol (Paris) 54:405 – 419

65. Rong LL, Gooch C, Szabolcs M, et al (2005) RAGE: a journey from the complications of diabetes to disorders of the nervous system – striking a fine balance between injury and repair.

Restor Neurol Neurosci 23:355 – 365

66. Wendt T, Harja E, Bucciarelli L, et al (2006) RAGE modulates vascular inflammation and atherosclerosis in a murine model of type 2 diabetes. Atherosclerosis 185:70 – 77

67. Foell D, Wittkowski H, Vogl T, Roth J (2007) S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 81:28 – 37

68. Jeschke MG, Barrow RE, Herndon DN (2000) Recombinant human growth hormone treatment in pediatric burn patients and its role during the hepatic acute phase response. Crit Care Med 28:1578 – 1584

69. Schwander JC, Hauri C, Zapf J, Froesch ER (1983) Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status. Endocrinology 113:297 – 305

70. Aarsland A, Chinkes D, Wolfe RR (1996) Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man. J Clin Invest 98:2008 – 2017

71. Aarsland A, Chinkes D, Wolfe RR, et al (1996) Beta-blockade lowers peripheral lipolysis in burn patients receiving growth hormone. Rate of hepatic very low density lipoprotein triglyceride secretion remains unchanged. Ann Surg 223:777 – 789

72. Takala J, Ruokonen E, Webster NR, et al (1999) Increased mortality associated with growth hormone treatment in critically ill adults. N Engl J Med 341:785 – 792

73. Jeschke MG, Herndon DN, Wolf SE, et al (2000) Hepatocyte growth factor modulates the hepatic acute-phase response in thermally injured rats. Crit Care Med 28:504 – 510

74. Seth A, Gonzalez FA, Gupta S, Raden DL, Davis RJ (1992) Signal transduction within the nucleus by mitogen-activated protein kinase. J Biol Chem 267:24796 – 24804

75. Guillen MI, Gomez-Lechon MJ, Nakamura T, Castell JV (1996) The hepatocyte growth factor regulates the synthesis of acute-phase proteins in human hepatocytes: divergent effect on interleukin-6-stimulated genes. Hepatology 23:1345 – 1352

76. Pierzchalski P, Nakamura T, Takehara T, Koj A (1992) Modulation of acute phase protein synthesis in cultured rat hepatocytes by human recombinant hepatocyte growth factor. Growth Factors 7:161 – 165

77. Michalopoulos GK, DeFrances MC (1997) Liver regeneration. Science 276:60 – 66

78. Michalopoulos GK, Appasamy R (1993) Metabolism of HGF-SF and its role in liver regeneration. Exs 65:275 – 283

79. Zarnegar R (1995) Regulation of HGF and HGFR gene expression. EXS 74:33 – 49

80. Ohira H, Miyata M, Kuroda M, et al (1996) Interleukin-6 induces proliferation of rat hepatocytes in vivo. J Hepatol 25:941 – 947

81. Humbel RE (1990) Insulin-like growth factors I and II. Eur J Biochem 190:445 – 462 82. Huang KF, Chung DH, Herndon DN (1993) Insulin-like growth factor 1 (IGF-1) reduces gut

atrophy and bacterial translocation after severe burn injury. Arch Surg 128:47 – 53

83. Strock LL, Singh H, Abdullah A, Miller JA, Herndon DN (1990) The effect of insulin-like growth factor I on postburn hypermetabolism. Surgery 108:161 – 164

(15)

84. Jeschke MG, Herndon DN, Barrow RE (2000) Insulin-like growth factor I in combination with insulin-like growth factor binding protein 3 affects the hepatic acute phase response and hepatic morphology in thermally injured rats. Ann Surg 231:408 – 416

85. Jeschke MG, Barrow RE, Herndon DN (2000) Insulinlike growth factor I plus insulinlike growth factor binding protein 3 attenuates the proinflammatory acute phase response in severely burned children. Ann Surg 231:246 – 252

86. Jeschke MG, Barrow RE, Suzuki F, Rai J, Benjamin D, Herndon DN (2002) IGF-I/IGFBP-3 equilibrates ratios of pro- to anti-inflammatory cytokines, which are predictors for organ function in severely burned pediatric patients. Mol Med 8:238 – 246

87. Jeschke MG, Klein D, Bolder U, Einspanier R (2004) Insulin attenuates the systemic inflammatory response in endotoxemic rats. Endocrinology 145:4084 – 4093

88. Jeschke MG, Rensing H, Klein D, et al (2005) Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats. J Hepatol 42:870 – 879 89. Jeschke MG, Klein D, Herndon DN (2004) Insulin treatment improves the systemic inflamma-

tory reaction to severe trauma. Ann Surg 239:553 – 560

90. Herndon DN, Nguyen TT, Wolfe RR, et al (1994) Lipolysis in burned patients is stimulated by the beta 2-receptor for catecholamines. Arch Surg 129:1301 – 1304

91. Herndon DN, Hart DW, Wolf SE, Chinkes DL, Wolfe RR (2001) Reversal of catabolism by beta-blockade after severe burns. N Engl J Med 345:1223 – 1229

92. Barrow RE, Wolfe RR, Dasu MR, Barrow LN, Herndon DN (2006) The use of beta-adrenergic blockade in preventing trauma-induced hepatomegaly. Ann Surg 243:115 – 120

93. Morio B, Irtun O, Herndon DN, Wolfe RR (2002) Propranolol decreases splanchnic triacylgly- cerol storage in burn patients receiving a high-carbohydrate diet. Ann Surg 236:218 – 225