This study investigates the role of endogenous platelet-activating factor (PAF) in the production of nitric oxide (NO) by constitutive and inducible isoforms of NO synthase (NOS) in endotoxin shock. In anesthetized rats, 3 hours of endotoxemia resulted in a fall in mean arterial blood pressure (MAP) from 127±5 (control) to 61±7 mm Hg and a reduction of the pressor responses to norepinephrine (NE, 1 jug* kg-') from 33±3 (control) to 17±2 mm Hg. Endotoxemia for 3 hours also resulted in a significant reduction in the contractile effects of NE (10-8 to 10-6 mol/L) in thoracic aortas ex vivo. This hyporeactivity to NE was due to an enhanced formation of NO, for it was restored by the NOS inhibitor macrophages and vascular smooth muscle cells, resulting in the formation of large quantities of NO.'14"5 Production of NO in response to LPS is the principal mediator of hypotension, peripheral vasodilatation, and vascular hyporeactivity to vasoconstrictor agents in endotoxemia. '6-22 The aims of the present study were to investigate whether (1) the beneficial hemodynamic effects of the PAF receptor antagonist WEB 2086 in endotoxemia involve inhibition of NO synthesis by constitutive and/or inducible isoform of NOS and (2) PAF stimulates the formation of NO by activation and/or induction of NOS in vivo. Materials and Methods Hemodynamic MeasurementsMale Wistar rats (260 to 320 g, Glaxo Laboratories Ltd, Greenford, Middlesex, UK) were anesthetized with thiopentone sodium (Trapanal, 3% solution, 120 mg* kg-l IP). The trachea was cannulated to facilitate respiration, and rectal temperature was maintained at 37°C using a homeothermic blanket (BioSciences, Sheerness, Kent, UK). The right carotid artery was cannulated and connected to a pressure transducer by guest on

An enhanced formation of nitric oxide (NO) due to induction of a calcium-independent (inducible) NO synthase (iNOS) contributes importantly to the cardiovascular failure caused by bacterial endotoxin. Repeated challenges with small doses of endotoxin result in tolerance to both peripheral vascular failure and death caused by subsequent injection of a higher dose of endotoxin. Here we investigate whether tolerance to endotoxin is associated with a lack of induction of iNOS in vivo and whether endogenous glucocorticoids play a role in the development of endotoxin tolerance. In anesthetized rats, i.v. administration of Escherichia coli endotoxin [lipopolysaccharide (LPS); 2 mg-kg-'] resulted In a prolonged decrease in mean arterial blood pressure (MAP) and hyporeactivity to the contractfle responses elicited by norepinephrine (NE; 10 nM) in aortic rings ex vivo. Hyporeactivity to NE was partially reversed by NG-nitro-L-argine methyl ester (0.3 mM) in vitro, suggesting that an enhanced formation of NO contributes to this hyporeactivity. There was a substantial increase in the activity of iNOS in the lung 3 h after i.v. injection of LPS (0.2 ± 0.1 to 6.6 ± 0.6 pmolnmgl -min-'; n = 5; P < 0.01). Rats injected i.p. with LPS (0.5 mg-kg-') for 4 consecutive days became tolerant to an i.v. injection of LPS (2 mg-kg-') in that both hypotension and vascular hyporeactivity to NE were signicantly attenuated. Moreover, in these endotoxin-tolerant rats, the induction of iNOS by LPS in the lung was attenuated by 63% ± 6%. Injection of LPS caused a 9-fold increase in plasma corticosterone (CCS) levels within 2 h and CCS levels remained snificantly elevated 6 and 24 h after LPS. Animals rendered tolerant to endotoxin by administration of a low dose of LPS (0.5 mg kg-', i.p.) for 4 days still had a 6-fold increase in plasma CCS levels 24 h after the last iqjection of LPS. When endotoxin-tolerant rats were treated with the glucocorticoid receptor antagonist RU 486 (50 mg kg-1, p.o. 3 h prior to LPS), there was a restoration of the effects of LPS (2 mg-kg- ', i.v.) in causing hypotension, vascular hyporeactivity to NE, and iNOS induction in the lung. However, in control rats RU 486 enhanced neither the decrease in MAP nor the induction of iNOS in response to LPS (2 mg-kg', i.v.). Thus, cardiovascular tolerance to endotoxin is accompanied and explained by reduced induction of iNOS in vivo due to the elevation of endogenous glucocorticoid levels.Endotoxin (or bacterial lipopolysaccharide; LPS), a component of the outer membrane ofGram-negative bacteria, is the prime initiator of septic shock. Despite the considerable progress made in understanding the pathophysiology of septic shock, therapy is still very limited and the mortality of patients with established shock remains high (1).Tolerance to endotoxin develops after repeated administration of small doses to animals and is characterized by the reduced effect of subsequent challenge with a high-doseThe publication costs of this article were defrayed in part by page charge p...

The pineal secretory product melatonin was found to exert protective effects in septic shock. In a host infected by bacterial lipopolysaccharide (LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNF-alpha) is rapidly increased, suggesting that TNF-alpha is associated with the etiology of endotoxic shock. Recent reports show that the expression of NO synthase (NOS) II and the production of superoxide anion (O2*-) also contribute to the pathophysiology of septic shock. In the present study we demonstrate that melatonin prevents circulatory failure in rats with endotoxemia and improves survival in mice treated with a lethal dose of LPS. The beneficial hemodynamic effects of melatonin in the endotoxemic animal appear to be associated with the inhibition of (i) the release of TNF-alpha in plasma, (ii) the expression of NOS II in liver, and (iii) the production of O2*- in aortae. In addition, the infiltration of polymorphonuclear neutrophils into the liver from the surviving LPS mice treated with melatonin was reduced. Thus, our results support the clinical use of melatonin in endotoxemia.

The pathogenesis of multiple organ dysfunction syndrome (MODS) in septic shock is complicated and not fully understood. Some studies show that an overproduction of nitric oxide (NO) leads to the refractory hypotension and multiple organ failure, while other studies suggest that free radicals, e.g. superoxide (O2−), contribute to the detrimental effect on vascular responsiveness and tissue/organ damage. Thus, this study was performed on the Wistar rat by using cecal ligation and puncture (CLP) to induce septic shock‐associated MODS. We evaluated the effect of an antioxidant melatonin in CLP‐induced septic rats and demonstrated that melatonin (3 mg/kg, i.v. at 3, 6, 12 hr after CLP) significantly (a) attenuated hyporeactivity to norepinephrine and delayed hypotension, (b) reduced plasma index of hepatic and renal dysfunction, (c) diminished plasma NO and interleukin‐1β (IL‐1β) concentrations as well as aortic O2− levels, (d) reduced marked infiltration of polymorphonuclear neutrophils (PMNs) in the lung and liver tissues, and (e) promoted the survival rate at 18 hr to twofold compared with the CLP alone group. The current study underlined the inhibition of plasma NO and IL‐1β as well as aortic O2− production and the reduction of PMN infiltration may lead to the amelioration of MODS, which may contribute to the beneficial effect of antioxidants (e.g. melatonin in this study) in conscious rats with peritonitis‐induced lethality. Thus, the antioxidant could be a novel agent for the treatment of septic animals or patients in the early stage.

There is good evidence that endotoxemia, sepsis, and septic shock are associated with the generation and release of reactive oxygen species (ROS) such as superoxide anion (O2), indicating that oxygen-derived free radicals play an important role in the pathogenesis of sepsis/shock. Studies on the application of free oxygen radical scavengers to limit the damage to tissues and organs have been recently attempted. A stable piperidine nitroxide of low molecular weight (Tempol) can permeate biological membranes and scavenge O2 in vitro and in vivo. Thus, we investigated effects of Tempol on the circulatory failure and multiple organ injuries caused by a clinically relevant polymicrobial sepsis model in the rat-cecal ligation and puncture (CLP). CLP not only successfully induced circulatory failure but also substantially increased plasma concentrations of glutamate-oxalate-transferase and glutamate-pyruvate-transferase (indicators of liver injury), creatinine and blood urea nitrogen (indicators of kidney injury), and decreased base excess in arterial blood in the late stage, indicating the development of multiple organ injury in this study. These were also confirmed by a histologic examination showing that the CLP-induced sepsis accompanied increase of polymorphonuclear neutrophil (PMN) infiltration in the lung and sequestration in the liver. Our results demonstrated that Tempol not only ameliorated the deterioration of hemodynamic changes and renal and liver injuries but also attenuated PMN infiltration in the lung and sequestration in the liver (histology). In addition, Tempol improved the survival in CLP-induced septic rats. Moreover, Tempol reduced the plasma NO. and interleukin-1beta and organ O2 levels in CLP-treated rats. In conclusion, Tempol prevented circulatory failure and attenuated organ dysfunction/injury as well as decreased the mortality rate in CLP-treated animals. These beneficial effects of Tempol may be attributed to inhibition of ROS formation (e.g., NO. and O2), suggesting antioxidant (e.g., Tempol) is a potential therapeutic agent in the treatment of intraperitoneal septic shock.

Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cellimpermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment. Experimental Design:We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays. Results: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular h-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong h-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts. Conclusions: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.

A watch and wait policy avoids the morbidity associated with radical surgery and preserves oncologic outcomes in our retrospective study from a single institute. It could be considered a therapeutic option in patients with locally advanced rectal cancer following chemoradiotherapy with a complete clinical response.

Dairy cows are highly susceptible to ketosis after parturition. In the present study, we evaluated the expression of fatty acid β-oxidation-related enzymes in the liver of ketotic (n=6) and nonketotic (n=6) cows. Serum levels of nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and glucose were determined by using standard biochemical techniques. The mRNA abundance and protein content of acyl-CoA synthetase long-chain (ACSL), carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), acyl-CoA dehydrogenase long chain (ACADL), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), and acetyl-CoA carboxylase (ACC) were evaluated by real-time PCR and ELISA. We found that serum glucose levels were lower in ketotic cows than in nonketotic cows, but serum BHBA and NEFA concentrations were higher. Messenger RNA and protein levels of ACSL were significantly higher in livers of ketotic cows than those in nonketotic cows. In contrast, mRNA levels of CPT I and mRNA and protein levels of CPT II, ACADL, HMGCS, and ACC were decreased in the liver of ketotic cows. Serum NEFA concentration positively correlated with ACSL protein levels and negatively correlated with protein levels of CPT II, HMGCS, ACADL, and ACC. In addition, serum BHBA concentration negatively correlated with protein levels of CPT II, HMGCS, and ACADL. Overall, fatty acid β-oxidation capability was altered in the liver of ketotic compared with nonketotic cows. Furthermore, high serum NEFA and BHBA concentrations play key roles in affecting pathways of fatty acid metabolism in the liver.