David W. A. Beno

Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells.

The reactive oxygen intermediate H2O2 can function as a signaling molecule to activate gene expression. In this study, we demonstrate that oxidant stress induced by tumor necrosis factor α (TNFα) or H2O2 differentially regulates intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) gene expression in endothelial and epithelial cells. Northern blot analysis revealed that TNFα induced both ICAM-1 and IL-8 expression in either the A549 lung epithelial cell line or the human microvessel endothelial cell line (HMEC-1). In contrast, H2O2 selectively induced only ICAM-1 in HMEC-1 and only IL-8 in A549. This cell type-specific pattern of IL-8 expression was also observed in several other endothelial and epithelial cells. TNFα induced greater IL-8 gene expression as compared with H2O2, but the kinetics of induction were similar. The induction of epithelial IL-8 message was accompanied by a corresponding increase in functional IL-8 protein secretion as determined by a neutrophil motility assay. The increased neutrophil motility stimulated by conditioned media from H2O2- or TNFα-exposed A549 cells was completely inhibited by an anti-IL-8 antibody. TNFα and H2O2 also induced a differential pattern of CC chemokine expression in A549. While TNFα induced both RANTES and MCP-1, H2O2 induced only MCP-1. These data suggest that epithelial cells under oxidant stress contribute to the inflammatory cytokine network by selective production of IL-8, MCP-1, and RANTES, which may critically influence the site-specific recruitment of leukocyte subsets.

THE EFFECT OF ANESTHESIA AND SURGERY ON CYP3A ACTIVITY IN RATS

The purpose of this investigation was to examine the effects of surgery and anesthesia on in vivo CYP3A activity and portal venous blood flow. Midazolam, a CYP3A probe for both rats and humans, was administered orally (2.7 mg), intravenously (0.57 mg), or via the portal vein (0.57 mg) to rats 4 h after anesthesia with ketamine/xylazine and surgery for placement of indwelling vascular and duodenal catheters and 3 days after surgery (chronic). The systemic clearance of midazolam was 51 ± 4 ml/min/kg in the chronic animals, and this was significantly decreased (29 ± 1 ml/min/kg, P = 0.024) in acute rats studied 4 to 6 h after anesthesia and surgery. The hepatic availability (FH), directly determined from the aortic and hepatic venous concentration gradient, was significantly higher in the acute animals (0.57 ± 0.05) compared with the chronic animals (0.33 ± 0.07, P = 0.001). Hepatic availability was determined using a classical approach in which FH was calculated from the area under the plasma concentration versus time curve ratio after portal venous or intravenous administration. FH was higher in the acute rats (0.48) compared with the chronic animals (0.27 ± 0.03). Portal venous blood flow was significantly lower in the acute animals (5.0 ± 0.4 ml/min/100 g body weight) compared with the chronic animals (9.1 ± 0.9 ml/min/100 g body weight, P = 0.015). The effect of surgery and anesthesia was confirmed using the indicator dye dilution method after infusion of [14C]polyethylene glycol 4000 into the superior mesenteric artery. Our data suggest that anesthesia and surgery decreases both hepatic CYP3A activity and hepatic blood flow in rats. Studies performed in rats within 3 days of surgery and anesthesia are conducted under nonphysiologic conditions and therefore provide inaccurate assessment of drug disposition, in particular, clearance and bioavailability.

Splenectomy ablates endotoxin-induced IFNγ response in rats

ABSTRACTThe mechanism of liver injury in endotoxemia is unclear. Previous studies have shown that splenectomy protects the liver from endotoxin-induced injury. The purpose of this study was to determine the relationship of TNFα and IFNγ release and endotoxin-induced liver injury in splenectomized an

Chronic Staphylococcal enterotoxin B and lipopolysaccharide induce a bimodal pattern of hepatic dysfunction and injury.

ObjectiveTo determine the effect of chronic exposure to endotoxin (lipopolysaccharide) and Staphylococcal enterotoxin B on hepatic injury and function. DesignProspective, controlled trial. SettingResearch laboratory in a university hospital. SubjectsMale Sprague-Dawley rats weighing 325–350 g with chronic vascular and bile catheters. InterventionsChronically catheterized rats were treated daily with saline, 50 &mgr;g/kg Staphylococcal enterotoxin B alone, 1000 &mgr;g/kg lipopolysaccharide alone, 1000 &mgr;g/kg lipopolysaccharide with 50 &mgr;g/kg Staphylococcal enterotoxin B, or 100 &mgr;g/kg lipopolysaccharide with 50 &mgr;g/kg Staphylococcal enterotoxin B for 10 days. Serum and biliary measures of hepatic injury and dysfunction were measured before and then 6 hrs and 1, 2, 3, 7, and 10 days after the start of treatment. The animals were killed at 10 days and the livers examined histologically. Measurements and Main ResultsMean rates of bile flow, biliary indocyanine green excretion, and bile acid flux were significantly decreased immediately after treatment (6 hr, 1 and 2 days) and then at 10 days. Increases in biliary and serum &ggr;-glutamyltransferase and serum bile acids also occurred in a similar bimodal pattern. Animals treated with lipopolysaccharide or Staphylococcal enterotoxin B alone became tolerant and did not develop the bimodal pattern of hepatic dysfunction. Histologic examination of the liver at 10 days revealed periportal inflammation and fibrosis. ConclusionsThe combination of lipopolysaccharide and Staphylococcal enterotoxin B leads to late liver injury, whereas either toxin alone does not. These data may explain the frequent development of liver dysfunction in patients exposed to multiple bacterial toxins such as in sepsis, multiple-system organ failure, and other diseases with altered intestinal permeability.

Differential induction of hepatic dysfunction after intraportal and intravenous challenge with endotoxin and Staphylococcal enterotoxin B.

We have previously shown that systemic infusion of the bacterial toxins Staphylococcal enterotoxin B (SEB) and endotoxin (LPS) induces hepatic dysfunction as measured by decreased biliary indocyanine green (ICG) excretion. In this study, we compare the effects of these bacterial toxins after infusion into the portal and systemic circulation and directly measure biliary bile acid excretion as a measure of cholestasis. We hypothesized that bacterial toxins infused into the portal vein would induce greater hepatic dysfunction than toxins infused into the systemic circulation. Using a chronically catheterized rat model, biliary bile acid excretion was directly measured after infusion of LPS at 10 and 100 &mgr;g/kg with and without 50 &mgr;g/kg SEB into the portal vein (IPV) or inferior vena cava (IV) at baseline, and at 6 and 24 h. We found that when LPS was infused alone, only IPV administration caused a significant decrease in bile acid excretion at 6 h. There was no change in bile acid excretion after IV administration of LPS. In contrast, when the combination of LPS and SEB was infused, both IV and IPV administration significantly decreased bile acid excretion at 6 and 24 h. At 6 h post-LPS and -SEB administration, the decrease in bile acid excretion was significantly greater after IPV than IV administration. There was no site-specific difference in IFN-&ggr; release after infusion of toxins. However, peak TNF&agr; release was decreased in IPV-infused rats [10 &mgr;g/kg (P < 0.05) or 100 &mgr;g/kg (P = ns) LPS with SEB] compared with the same doses in IV-infused rats. These data question the role of systemic TNF-&agr; and IFN-&ggr; in regulating hepatic dysfunction and suggest a differential functional response of the liver to systemic and gut-derived septic events. This study also further explains the frequent development of liver dysfunction in patients with sepsis, multisystem organ failure, and other diseases with altered intestinal permeability.

Nonstressed rat model of acute endotoxemia that unmasks the endotoxin-induced TNF-α response

Previous investigators have demonstrated that the tumor necrosis factor-α (TNF-α) response to endotoxin is inhibited by exogenous corticosterone or catecholamines both in vitro and in vivo, whereas others have reported that surgical and nonsurgical stress increase the endogenous concentrations of these stress-induced hormones. We hypothesized that elevated endogenous stress hormones resultant from experimental protocols attenuated the endotoxin-induced TNF-α response. We used a chronically catheterized rat model to demonstrate that the endotoxin-induced TNF-α response is 10- to 50-fold greater in nonstressed (NS) rats compared with either surgical-stressed (SS, laparotomy) or nonsurgical-stressed (NSS, tail vein injection) models. Compared with the NS group, the SS and NSS groups demonstrated significantly lower mean peak TNF-α responses at 2 mg/kg and 6 μg/kg endotoxin [NS 111.8 ± 6.5 ng/ml and 64.3 ± 5.9 ng/ml, respectively, vs. SS 3.9 ± 1.1 ng/ml ( P < 0.01) and 1.3 ± 0.5 ng/ml ( P < 0.01) or NSS 5.2 ± 3.2 ng/ml ( P < 0.01) at 6 μg/kg]. Similarly, baseline concentrations of corticosterone and catecholamines were significantly lower in the NSS group [84.5 ± 16.5 ng/ml and 199.8 ± 26.2 pg/ml, respectively, vs. SS group 257.2 ± 35.7 ng/ml ( P< 0.01) and 467.5 ± 52.2 pg/ml ( P < 0.01) or NS group 168.6 ± 14.4 ng/ml ( P < 0.01) and 1,109.9 ± 140.7 pg/ml ( P < 0.01)]. These findings suggest that the surgical and nonsurgical stress inherent in experimental protocols increases baseline stress hormones, masking the endotoxin-induced TNF-α response. Subsequent studies of endotoxic shock should control for the effects of protocol-induced stress and should measure and report baseline concentrations of corticosterone and catecholamines.Previous investigators have demonstrated that the tumor necrosis factor-alpha (TNF-alpha) response to endotoxin is inhibited by exogenous corticosterone or catecholamines both in vitro and in vivo, whereas others have reported that surgical and nonsurgical stress increase the endogenous concentrations of these stress-induced hormones. We hypothesized that elevated endogenous stress hormones resultant from experimental protocols attenuated the endotoxin-induced TNF-alpha response. We used a chronically catheterized rat model to demonstrate that the endotoxin-induced TNF-alpha response is 10- to 50-fold greater in nonstressed (NS) rats compared with either surgical-stressed (SS, laparotomy) or nonsurgical-stressed (NSS, tail vein injection) models. Compared with the NS group, the SS and NSS groups demonstrated significantly lower mean peak TNF-alpha responses at 2 mg/kg and 6 micrograms/kg endotoxin [NS 111.8 +/- 6.5 ng/ml and 64.3 +/- 5.9 ng/ml, respectively, vs. SS 3.9 +/- 1.1 ng/ml (P < 0.01) and 1.3 +/- 0.5 ng/ml (P < 0.01) or NSS 5.2 +/- 3.2 ng/ml (P < 0.01) at 6 micrograms/kg]. Similarly, baseline concentrations of corticosterone and catecholamines were significantly lower in the NSS group [84.5 +/- 16.5 ng/ml and 199.8 +/- 26.2 pg/ml, respectively, vs. SS group 257. 2 +/- 35.7 ng/ml (P < 0.01) and 467.5 +/- 52.2 pg/ml (P < 0.01) or NS group 168.6 +/- 14.4 ng/ml (P < 0.01) and 1,109.9 +/- 140.7 pg/ml (P < 0.01)]. These findings suggest that the surgical and nonsurgical stress inherent in experimental protocols increases baseline stress hormones, masking the endotoxin-induced TNF-alpha response. Subsequent studies of endotoxic shock should control for the effects of protocol-induced stress and should measure and report baseline concentrations of corticosterone and catecholamines.

Ursodeoxycholic acid ameliorates ibuprofen-induced enteropathy in the rat

Background The objective of the study was to determine whether ursodeoxycholic acid (Ursodiol) is protective against ibuprofen (IBU)-induced enteropathy. Methods Using the chronically catheterized rat model, IBU (60 mg/kg body weight per day) was infused via the gastric catheter twice daily. Pancreatic enzyme (PE; 10,000 U lipase/kg body weight per day) and Ursodiol (10 mg/kg body weight per day) in two doses were infused via the duodenal catheter. Rats were assigned to one of six treatment groups and were administered treatment for 20 days: control, IBU, PE, IBU + PE, IBU + Ursodiol, and IBU + PE + Ursodiol. The entire jejunum, ileum, cecum, and colon were available for histologic analysis using previously described techniques. Results Addition of Ursodiol to high-dose IBU and normal doses of PE showed a significant reduction in the percentage of rats with ulcers (P < 0.05), total number of serositis events (P < 0.01), total number of severe ulcers (P < 0.001), and an absence of ulcers in the large intestine. Conclusions Ursodiol, the drug of choice for the treatment of cystic fibrosis liver disease, may offer a safe method of using high-dose IBU in these patients by ameliorating the enteropathy.

The effect of surgical stress on the endotoxin-induced interferon-γ response

: Most animal studies of cytokine release during sepsis or endotoxemia have used models in which studies are performed during or immediately after surgical stress. In a previous study, we showed that surgical stress as measured by elevated endogenous corticosterone concentrations attenuated the endotoxin-induced tumor necrosis factor alpha (TNFalpha) response. To determine whether surgical stress attenuates the endotoxin-induced interferon-gamma (IFN-gamma) response, chronically catheterized male Sprague-Dawley rats were treated with endotoxin 10 microg/kg immediately after surgery for catheter placement (surgical stress group, SS group) or at least 4 days postoperative (nonstressed group, NS group). We found that peak endotoxin-induced IFN-gamma responses were similar in the SS and NS groups (2094 +/- 315 pg/mL vs. 1863 +/- 307 pg/mL). Baseline corticosterone concentrations were significantly elevated in the SS group compared to the NS group (273.8 +/- 15.2 ng/mL vs. 30.0 +/- 8.5 ng/mL, P < 0.001). Peak TNFalpha concentrations were significantly reduced in the SS group compared to the NS group (5.2 +/- 1.9 ng/mL vs. 69.9 +/- 10.3 ng/mL, P = 0.0002). While peak serum TNFalpha concentrations were inversely related to baseline corticosterone concentrations, there was no correlation between peak IFN-gamma concentrations and baseline corticosterone concentrations or between TNFalpha and IFN-gamma concentrations. We conclude that surgical stress associated with elevated concentrations of endogenous corticosterone does not attenuate the endotoxin-induced IFN-gamma response despite an attenuation of the endotoxin-induced TNFalpha response. Because the effect of stress on different cytokines is varied, studies of sepsis and endotoxemia must account for the effects of experimentally-induced stress on cytokine responses.

The effects of high-dose ibuprofen and pancreatic enzymes on the intestine of the rat.

BACKGROUND High-dose ibuprofen therapy limits the progression of lung disease in patients with cystic fibrosis. However, ibuprofen increases intestinal permeability, which potentiates intestinal damage caused by high-dose pancreatic enzyme treatment, as was shown in a previous study by this group. In the present study, the combined effects of ibuprofen and pancreatic enzyme treatment on the intestine and liver were examined. METHODS Using a chronically catheterized rat model, high-dose ibuprofen (60 mg/kg x day in two doses), with or without pancreatic enzyme treatment was infused into gastric and duodenal catheters, respectively, for 20 days. Six groups were studied: control group; ibuprofen treatment alone; pancreatic enzyme treatment alone (two groups: normal dose, 10,000 U lipase/kg x day and high dose, 40,000 U lipase/kg x day); and ibuprofen combined with pancreatic enzyme (two groups: ibuprofen with high-dose pancreatic enzyme and ibuprofen and low-dose pancreatic enzyme). After treatment, rats were autopsied, and complete histologic analyses of the entire intestine and liver were performed. RESULTS Ibuprofen caused mild ulceration of the small intestine in 50% of rats. Pancreatic enzyme treatment alone did not induce ulceration of the intestine. The combination of pancreatic enzyme and ibuprofen treatment increased the severity of the ulcers in the small intestine but not the number of ulcers or the percentage of rats affected. Ibuprofen treatment alone did not cause ulcers in the large intestine, but with the addition of pancreatic enzymes, ulceration and fibrosis were present. CONCLUSIONS Ibuprofen at doses used to limit progression of cystic fibrosis lung disease caused enteropathy in 50% of rats. There was synergism between ibuprofen and pancreatic enzyme treatment in the production of severe ulcers. Ulcers in the cecum and colon were increased with combined ibuprofen and pancreatic enzyme treatment compared with incidence in control animals.

Hepatic Extraction of Lactic Acid Remains Intact After Endotoxemia 236

We previously showed that endotoxin-induced lactic acidemia is initially caused by a decrease in clearance of lactic acid (L) and not by an increase in production (Pediatr Res 41:1997, 38A). Previous investigators have shown that hepatocellular function is impaired early in the course of sepsis (Wang et. al. Shock 3:1995). Because the liver is a major site of L metabolism, we hypothesized that impaired hepatic extraction of L would contribute to endotoxin-induced lactic acidemia. To test this hypothesis, we measured simultaneous hepatic venous (HV), portal venous (PV) and aortic (A) lactic acid concentrations after 2 mg/kg LPS was infused into the IVC in chronically catheterized rats (n=3). Blood L concentrations (nmol/ml) were analyzed using an enzymatic assay. The extraction of L by the liver, non-hepatic splanchnic organs, and the entire splanchnic system was determined from the hepatic venous-portal venous (HV-PV), portal venous-aortic (PV-A), and hepatic venous-aortic (HV-A) concentration gradients, respectively. Values in table are mean (SD). Aortic L concentration increased 4.5 fold peaking at 90 min post LPS infusion. The positive L(PV-A) remained unchanged indicating that the non-hepatic splanchnic organs are net producers of L but do not contribute to the increase in L after LPS. The increasingly negative L(HV-PV) after LPS indicates that the liver retains the ability to extract L from the PV after LPS. The increasingly negative L(HV-A) indicates that the splanchnic system as a whole is a net clearer of L after LPS. We conclude that the ability of the liver to extract lactate is not impaired by LPS. However, the net hepatic flux of L is related both to the concentration gradient of L across the liver and blood flow through the liver. Since L(HV-PV) becomes increasingly negative, we speculate that the decreased net extraction of lactate after LPS may be related to a decrease in hepatic blood flow.