Thromboelastography measurements of whole blood from factor VIII-deficient mice supplemented with rFVIII.

The rotational thromboelastography (ROTEG) assay system allows the real-time analysis of clot formation (fibrin formation) in a whole-blood assay format. The ROTEG system provides significant advantages over the current plasma-based assay systems as it includes the important interactions between cellular and plasmatic coagulation factors. We have employed the ROTEG system to characterize clot formation dynamics in factor VIII- deficient mouse whole blood and examined the ability of recombinant FVIII (rFVIII) supplementation to restore the normal phenotype. The ability to generate a clear dose-response relationship by adding rFVIII to FVIII-deficient murine whole blood (FVIII-/-) demonstrates the feasibility of this approach. A dose-response from 1 U to 0.00001 U mL(-1) demonstrates the enhanced sensitivity of the ROTEG system. Further characterization of this experimental approach may provide a potential tool for comparing the activity of FVIII concentrates and/or evaluating FVIII mutants.

Plasminogen did not affect lung function in surfactant-treated preterm lambs.

Preterm infants with respiratory distress syndrome develop fibrin-rich hyaline membranes within the alveoli and have depressed fibrinolytic activity, which is thought to be due to a relative deficiency of plasminogen. Local fibrin deposition inhibits surfactant function and amplifies inflammation. We hypothesized that plasminogen administration to surfactant-treated preterm lambs would prevent fibrin-rich hyaline membrane formation, resulting in the amelioration of lung pathology and improved lung function. We randomly treated preterm lambs (gestational age 127-129 days) with either 16 mg of lysine-plasminogen (n = 10) or saline (n = 10), and ventilated them for 5 h. There were no significant differences in physiologic measurements of lung function (ventilation efficiency index, oxygenation index, dynamic compliance, quasi-static pressure volume curve), measures of lung injury (alveolar wash protein content and (125)I-albumin recovery) or surfactant pool size. The degree and extent of bronchiolar erosion and hyaline membrane formation were similar in the two groups. Plasminogen administration did not improve lung function or prevent hyaline membrane formation in surfactant-treated lambs.

A murine skeletal muscle ischemia-reperfusion injury model: differential pathology in BALB/c and DBA/2N mice.

Ischemia-reperfusion injuries can occur with diseases such as myocardial infarction and stroke and during surgical procedures such as organ transplantation and correction of aortic aneurysms. We developed a murine model to mimic abdominal aortic aneurysm repair with cross-clamping of the aorta distal to the renal artery. After model development, we compared the normal complement BALB/c mouse with the C5-deficient DBA/2N mouse. To assess quantitative differences, we measured neuromuscular function up to 72 h after ischemia with a subjective clinical scoring system, as well as plasma chemistries, hematology, and histopathology. There were significant increases in clinical scores and creatine phosphokinase, lactate dehydrogenase, and muscle histopathology scores in BALB/c mice compared with those in DBA/2N mice and sham-surgery mice. Muscle histopathology scores of the cranial tibialis and quadriceps correlated well with clinical signs, creatine phosphokinase, and lactate dehydrogenase, and indicated the greatest pathology in these muscle groups. We developed a murine model of skeletal muscle ischemia-reperfusion injury that can utilize the benefits of murine genetic and transgenic models to assess therapeutic principles of this model. Additionally, we have shown a significant reduction in clinical signs, plasma muscle enzyme concentrations, and muscle pathology in the C5-deficient DBA/2N mouse in this model.

Monoclonal antibody to tumor necrosis factor-alpha attenuates plasma interleukin-6 levels in porcine gram-negative sepsis.

This study examined the kinetics of IL-6 release into the systemic circulation in a porcine model of bacterial sepsis induced by infusion of live Pseudomonas aeruginosa. Three groups of animals were studied. Group I (n = 12) animals received a 1 hr infusion of live P. aeruginosa. Group II (n = 6) animals received monoclonal antibody to tumor necrosis factor-alpha (TNF-alpha) (15 mg/kg) prior to induction of sepsis. Group III (n = 7) animals received sterile saline only. TNF-alpha and interleukin-6 (IL-6) levels rose sharply, in group I following pseudomonas infusion. Following a peak at 120 min after the bacterial infusion (4.8 +/- 0.7 U/ml at 120 min vs 0.4 +/- 0.2 U/ml at 0 min), TNF-alpha levels subsequently declined prior to the end of the experiment. In contrast, IL-6 levels rose sharply, subsequent to TNF-alpha, peaked at 180 min, and remained significantly elevated throughout the study period (5.3 +/- 0.9 ng/ml vs 0.05 +/- 0.01 ng/ml, 0 min). In animals pretreated with monoclonal antibody to TNF-alpha, no increase in TNF-alpha activity was detected at any time during the period of study. IL-6 levels in antibody-treated animals, although greatly attenuated, still rose significantly above baseline (2.02 +/- 0.8 ng/ml at 180 min vs 0.05 +/- 0.01 ng/ml at 0 min) and above levels in control animals. We conclude that although TNF-alpha plays an important role in synthesis and release of IL-6, there is a TNF-alpha-independent pathway for release of IL-6 in sepsis.

Effects of interleukin-2 on the pulmonary microvasculature in anesthetized sheep.

Interleukin-2 (IL-2) is reputed to cause pulmonary microvascular injury. We studied the pulmonary and splanchnic microcirculation of anesthetized sheep after one dose (1.8 x 10(6) IU/kg) of IL-2 (n = 9) and after six doses (1.8 x 10(6) IU.kg-1.dose-1) of IL-2 over 3 days (n = 9). Seven control sheep received only 5% dextrose diluent. We measured hemodynamics and lymph dynamics in anesthetized sheep after the final dose of IL-2 or diluent. After one dose of IL-2, caudal mediastinal node (mainly pulmonary) lymph flow was stable, whereas thoracic duct lymph flow increased from a baseline of 54 +/- 6 to 124 +/- 22 ml/h. After 3 days of IL-2, the caudal mediastinal node lymph flow increased from 7.7 +/- 5.5 to 19.0 +/- 14.8 ml/h 5-6 h after the final dose of IL-2, and thoracic duct lymph flow increased from 84 +/- 43 to 143 +/- 42 ml/h. The lymph-to-plasma protein concentration ratio increased after IL-2 for thoracic duct but not for caudal mediastinal node lymph. The equilibration rate of 125I-albumin from plasma to caudal mediastinal node lymph did not change, whereas plasma-to-thoracic duct lymph equilibration was faster after both one dose and 3 days of IL-2. Positron emission tomography showed no increase in the pulmonary transcapillary escape rate for 68Ga-labeled transferrin or in extravascular lung water (n = 4). We conclude that IL-2 at doses two to three times those used clinically does not significantly injure the pulmonary microcirculation of sheep.

Combined ibuprofen and monoclonal antibody to tumor necrosis factor-alpha attenuate hemodynamic dysfunction and sepsis-induced acute lung injury.

A number of key mediators are implicated in the pathophysiology of sepsis. In previous studies of a septic porcine model, ibuprofen pretreatment prevented the early but not the late rise in pulmonary vascular resistance index (PVRI) and the early but not the late fall in arterial PO2 (PaO2), whereas monoclonal antibody to tumor necrosis factor alpha (anti-TNF alpha) prevented the late but not the early rise in PVRI and the late but not the early fall in PaO2. This study examined the impact of pretreatment with combined ibuprofen and anti-TNF-alpha on the course of sepsis and acute lung injury (ALI) in pigs. Three groups were studied for 5 hours. Groups I (n = 9) and II (n = 5) received a 1-hour infusion of Pseudomonas aeruginosa. Group II received ibuprofen (12.5 mg/kg) and anti-TNF-alpha (5 mg/kg) before P. aeruginosa, and a further bolus of ibuprofen at 120 minutes. Group III (n = 11) received sterile saline. Group I demonstrated a significant (p < 0.05) rise in plasma TNF-alpha that was abolished in group II. The SVRI in group II did not change significantly from baseline through the study and the SVRI rose sharply in group I following onset of the infusion of P. aeruginosa, as did PVRI. There was no significant change in PVRI from baseline in group II, except for the final 60 minutes; PVRI in group II was significantly less than in group I throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of monoclonal antibody against tumor necrosis factor alpha in an endotoxemic baboon model.

Tumor necrosis factor alpha (TNF) has been described as a primary mediator of the pathophysiology associated with bacterial sepsis/endotoxemia. We tested the efficacy and possible mechanisms of protection of a monoclonal antibody against TNF (TNF Mab) in a low mortality (29%), endotoxemic baboon model. A number of parameters were monitored at days 0, 1, 2 and 5-7 after challenge with 2 mg E. coli endotoxin/kg. TNF Mab pretreatment (15 mg/kg) prevented the increase in detectable serum TNF and the early perturbations in cardiovascular function which occurred in the control group. Early metabolic dysfunction was delayed in the TNF MAb group and was attenuated thereafter. Dysfunction of the kidney, liver, and coagulation systems and the increased IL-6 levels were significantly attenuated in the TNF MAb group; neutrophil activation was not affected by TNF MAb. No deaths occurred in the TNF MAb group. These results support the hypothesis that TNF plays a central role in the pathophysiology of endotoxic shock.

Monoclonal antibody to tumor necrosis factor alpha attenuates cardiopulmonary dysfunction in porcine gram-negative sepsis.

Tumor necrosis factor (TNF) is implicated in the pathophysiology of gram-negative sepsis. This study examined physiologic and biochemical effects of pretreatment with an anti-TNF alpha monoclonal antibody immediately before the onset of sepsis. Three groups of anesthetized ventilated pigs were studied for 300 minutes. Groups 1 (n = 12) and 2 (n = 6) received a 1-hour infusion of live Pseudomonas aeruginosa. Group 2 was pretreated with anti-TNF alpha monoclonal antibody (15 mg/kg). Group 3 (n = 8) received intravenous sterile saline. Group 1 exhibited a significant rise in plasma TNF activity, which was abolished in group 2. Cardiac index was reduced in both groups 1 and 2 in the first hour but recovered in group 2 (3.3 +/- 0.4 l/min per square meter at 300 minutes in group 2 vs 1.3 +/- 0.2 L/min per square meter in group 1). Metabolic acidosis was attenuated (arterial pH, 7.39 +/- 0.01 in group 2 vs 7.16 +/- 0.03 at 300 minutes in group 1). Increased extravascular lung water was also attenuated (5.9 +/- 0.7 in group 2 vs 13.2 +/- 1.5 mL/kg at 300 minutes in group 1). However, pulmonary hypertension and hypoxemia, which are known cyclooxygenase effects, were not affected. In the early phase of the study, plasma thromboxane B2 levels were elevated in both groups 1 and 2. We conclude that anti-TNF alpha monoclonal antibody offered significant protection against the effects of sepsis, but that other mediators may be responsible for the early changes seen in this model.

Effects of recombinant human interleukin-2 and excipient infusion on plasma levels of prostaglandins and thromboxane B2 in sheep.

Systemic administration of recombinant human interleukin-2 (rIL-2) is used for treating some patients with advanced cancer. This therapy is limited by severe adverse reactions of cardiovascular and gastrointestinal origin. The effects of rIL-2 treatment on plasma levels of some prostanoids were examined in sheep in order to elucidate the mechanism of these adverse reactions. A total of 8 adult female Suffolk sheep were used. rIL-2 (0.1 mg/kg) was infused over a 30-min period in 4 sheep. Four different sheep in the control group received an excipient, which consisted of 5% mannitol and sodium dodecyl sulfate (SDS) (187 micrograms SDS/mg rIL-2), in the same way. Plasma levels of prostaglandin (PG) F2 alpha in rIL-2-treated animals showed significant increases from 1.5 to 6 h over the excipient treated animals with a maximal increase of 138% of the pooled zero time control value (p less than or equal to 0.01) at 6 h. The pooled zero time control plasma PGF2 alpha concentration was 443.0 +/- 45.9 pg/ml. Plasma levels of 6-keto-PGF1 alpha in rIL-2-treated animals showed a significant increase (p less than or equal to 0.02) over the excipient treated animals at 0.5 h but the value was only 103% of the pooled zero time control. Plasma levels of 6-keto-PGF1 alpha in excipient-treated animals showed a maximal increase of 156% of the pooled zero time control value (55.6 +/- 8.9 pg/ml) at 5 h and it was significantly higher than the rIL-2-treated sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid pretreatment reduces interleukin-2 toxicity in sheep.

Recombinant interleukin-2 (rIL-2) has shown promise in the treatment of patients with advanced cancer. However, toxicity of this therapy remains a major problem with its use in some patients. In this study we examined whether steroids could reduce the adverse cardiopulmonary effects of rIL-2. Seven sheep were surgically prepared with vascular catheters and lung lymph fistulas. Each sheep received either a single dose of rIL-2 (100 micrograms/kg) or rIL-2 plus methylprednisolone (30 mg/kg) followed by the reverse treatment 1 week later. Lung lymph flow increased markedly after rIL-2 with a peak QL of 140% +/- 30% (above baseline). Steroid pretreatment significantly reduced this lymph flow increase with peak lung lymph flow being only 40% +/- 16% (p less than 0.004). The lymph/plasma protein ratio tended to increase after rIL-2, but these changes were not statistically significant. After rIL-2, cardiac output, heart rate, core temperature, and mean pulmonary artery pressure increased (p less than 0.05), whereas systemic vascular resistance and arterial PO2 decreased (p less than 0.05). These changes did not occur with steroid pretreatment. The results of this study demonstrate that steroids reduced the adverse cardiopulmonary effects of rIL-2. We believe that rIL-2 induces activation of arachidonic acid metabolism, which leads to the production of multiple inflammatory mediators that cause increased microvascular permeability and the adverse cardiopulmonary effects of rIL-2.

In vivo biology of recombinant interleukin-2 infusion in sheep: cardiopulmonary manifestations of an intravascular immune-inflammatory response.

We have described the physiologic changes that occur in the conscious sheep during five days of dosing with rIL-2. Major effects noted were a decrease in systemic vascular resistance (vasodilation) accompanied by an increase in cardiac output (blood flow) and heart rate and a mild increase in pulmonary vascular resistance (vasoconstriction). Body temperature increased and may have contributed to the peripheral vasodilation. Lung microvascular fluid and protein flux increased following rIL-2 administration, as evidenced by an increase in lung lymph flow and protein clearance, and circulating levels of leukocytes decreased. These phenomena were temporally related. Since the composite biologic response that occurred following rIL-2 administration was characterized by changes in vascular caliber and blood flow, increased vascular permeability, and margination of leukocytes, we propose that the response be viewed as an intravascular inflammatory reaction.

Fat emulsion catabolism in vitro and in vivo--sex related differences.

The removal rates of an intravenously administered 10% fat emulsion (Intralipid) from plasma in male and female conscious rats are described. The plasma concentration of fat emulsion particles at various time intervals following a bolus administration (0.2 g/kg) was measured by nephelometry. At the dose employed, the removal of fat emulsion from the plasma followed first order kinetics, ie, a constant fraction was removed from the plasma per unit of time, K2 (%/min). Females exhibited a significantly greater fractional removal rate (K2) than comparably aged males (21.0 +/- 1.0 vs 15.0 +/- 1.4, p less than 0.05). Postheparin lipoprotein lipase, measured using fat emulsion as substrate, also was significantly greater in female rats compared with males. Our results demonstrate that, in rats, fat emulsion (Intralipid) is catabolized more rapidly in females than in males and a greater lipoprotein lipase activity in female rats may be the causative factor.

Protection of ischemic myocardium: comparison of effects of propranolol, bevantolol and N-dimethyl propranolol on infarct size following coronary artery occlusion in anesthetized dogs.

The relative extent of myocardial infarction produced by occlusion of the left anterior descendens coronary artery in anesthetized dogs was determined under control conditions or following treatment (4 h after ligation) with propranolol (1 mg/kg), bevantolol (3 mg/kg) or N-dimethyl propranolol (10 mg/kg). Doses of drugs were selected to provide similar reductions in heart rate, aortic blood pressure and contractility. Infarct size was estimated indirectly from levels of plasma creatine phosphokinase and measured histochemically by nitroblue tetrazolium stain. A significant ( p < 0.001) correlation between methods was found. Propranolol and bevantolol (beta adrenergic antagonists) produced a significant (p < 0.05) reduction (approximately 50% decrease) in infarct size, measured at 8 h following induction of ischemia, while N-dimemthyl propranolol (no beta antagonist activity) produced no effect. While all three agents produced similar hemodynamic actions and thereby reduced primary determinants of myocardial oxygen demand, only the beta blockers were able to afford protection of ischemic myocardium.

Effect of propranolol and nitroglycerin plus methoxamine on transmural creatine kinase activity after acute coronary occlusion.

Transmural creatine kinase activity was determined 5 hours after acute occlusion of the left anterior descending coronary artery in 27 open chest anesthetized dogs. In seven dogs, propranolol, 2 mg/kg, was given intravenously over a 10 minute period 10 minutes after occlusion. In 10 dogs, nitroglycerin, 300 microgram/min, was infused intravenously for 1 hour 10 minutes after occlusion. Methoxamine, 300 to 500 microgram, was administered to return blood pressure and heart rate to prenitroglycerin levels. In untreated dogs, there was a distinct transmural gradient of creatine kinase activity in the ischemic region from subepicardium to subendocardium: nonischemic subepicardium 1,187 +/- 50 international units (IU)/g versus ischemic subepicardium 1,054 +/- 46 IU/g and nonischemic subendocardium 1,170 +/- 53 IU/g versus ischemic subendocard;um 766 +/- 42 IU/g, respectively. Administration of propranolol did not affect the transmural creatine kinase gradient after 5 hours of occlusion. In contrast, nitroglycerin plus methoxamine significantly (P less than 0.05) decreased subendocardial creatine kinase depletion after 5 hours of occlusion (776 +/- 42 versus 978 +/- 47 IU/g). These findings demonstrate the unique capability of nitroglycerin plus methoxamine to protect the subendocardium during ischemic insult.

Transmural triglycerides in acute myocardial ischaemia.

The effect of coronary artery occlusion on endogenous triglycerides of left ventricular subepicardium and subendocardium was studied in the open-chest anaesthetised dog. Under control conditions, the subepicardium was found to have a greater concentration of triglycerides than the subendocardium. Thirty minutes after acute coronary artery occlusion there was a decrease followed by a steady increase at 60, 120, and 240 min in subepicardial triglycerides of the ischaemic region. No change in triglycerides in the subendocardium of normal or ischaemic regions was observed. The initial decrease of subepicardial triglycerides in the ischaemic region was blocked by administration of propranolol or bevantolol (CI-775; a specific beta 1 antagonist) given 30 min before occlusion. It is concluded that the effect of coronary artery ligation on transmural endogenous triglycerides is biphasic with an initial period of increased mobilisation followed by a period of increased deposition.

Effect of propranolol on enzymatic and histochemical estimates of infarct size in experimental myocardial infarction.

Acute myocardial infarction was produced in anesthetized dogs by ligation of the left anterior descendens coronary artery. Propranolol (2 mg/kg i.v.) administered 4 hours post ligation was examined for its ability to reduce infarct size estimated by histochemical and enzymatic methods. There was a signficant correlation between these two methods in their estimation of infarct size. Treatment with propranolol significantly decreased infarct size estimated with both methods. It is concluded that some portion of the myocardium can be protected against infarction by pharmacologic intervention as late as 4 hours after the onset of coronary artery occlusion.