Cardiovascular effects of pimobendan in healthy mature horses.

REASONS FOR PERFORMING STUDY: Pimobendan is an inodilator used in dogs for the management of heart failure due to myxomatous valve disease or dilated cardiomyopathy. The lack of data regarding the effects of pimobendan in horses prevents the rational use of this drug. OBJECTIVE: To determine the cardiovascular effects of pimobendan in healthy mature horses. STUDY DESIGN: Randomised experimental study. METHODS: Five horses were fasted overnight prior to receiving i.v. pimobendan (0.25 mg/kg bwt), intragastric (i.g.) pimobendan (0.25 mg/kg bwt) or i.g. placebo with a washout period of one week between each administration. Horses were instrumented for the measurement of right ventricular (RV) minimum pressure, RV maximum pressure, RV end diastolic pressure, and maximum rate of increase and decrease in RV pressure before and 0.5, 1, 2, 4, and 8 h after drug administration. Arterial blood pressure, central venous pressure, cardiac output and heart rate were measured at the same time points. Data were expressed as a maximum percentage of change over baseline values. RESULTS: There were no adverse effects associated with administration of pimobendan. The percentage increase in heart rate was significantly greater for horses given pimobendan i.g. (33 ± 4%) and i.v. (36 ± 14%) than for those given a placebo (-2 ± 7%). The percentage increase in maximum rate of increase in RV pressure (35 ± 36%) and the percentage decrease in minimum pressure (47 ± 24%) and end diastolic pressure (34 ± 13%) were significantly greater in horses given pimobendan i.v. than in those given placebo. Other variables measured were not significantly different between treatment groups. CONCLUSION: Pimobendan administered i.v. has positive chronotropic and inotropic effects in healthy mature horses and warrants further investigation for the treatment of heart failure in horses.

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses.

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.

Polymyxin B protects horses against induced endotoxaemia in vivo.

REASONS FOR PERFORMING STUDY: A safe, affordable and effective treatment for endotoxaemia in horses is needed in order to reduce the incidence of this potentially fatal condition. OBJECTIVE: To evaluate the effect of polymyxin B (PMB) on signs of experimentally-induced endotoxaemia. HYPOTHESIS: PMB ameliorates the adverse effects of endotoxaemia without causing nephrotoxicity. METHODS: Four groups of 6 healthy mature horses each received 20 ng endotoxin/kg bwt i.v. over 30 mins. Additionally, each group received one of the following i.v.; 5000 u PMB/kg bwt 30 mins before endotoxin infusion; 5000 u PMB/kg bwt 30 mins after endotoxin infusion; 1000 u PMB/kg bwt 30 mins prior to endotoxin infusion; or saline. Clinical response data and samples were collected to determine neutrophil count, serum tumour necrosis factor (TNF) activity, plasma thromboxane B2 concentration and urine gamma glutamyltranspeptidase (GGT) to creatinine ratio. RESULTS: Treatment with PMB before or after administration of endotoxin significantly reduced fever, tachycardia and serum TNF, compared to horses receiving saline. The differences in response to endotoxin were greatest between horses that received saline vs. those that received 5000 u PMB/kg bwt prior to endotoxin. Urine GGT:creatinine did not change significantly. CONCLUSIONS AND POTENTIAL RELEVANCE: This study indicates that PMB may be a safe and effective treatment of endotoxaemia, even when administered after onset. Although nephrotoxicity was not demonstrated with this model, caution should be exercised when using PMB in azotaemic patients.

Evaluation of polymyxin B in an ex vivo model of endotoxemia in horses.

OBJECTIVE: To evaluate effects of polymyxin B sulfate (PMB) on response of horses to endotoxin, using an ex vivo model. ANIMALS: 8 healthy horses. PROCEDURE: In a crossover design, 3 doses of PMB (100, 1,000, and 10,000 U/kg of body weight) and physiologic saline solution (control) were evaluated. Prior to and for 24 hours after administration of PMB, blood samples were collected into heparinized tubes for use in 2 assays. For the endotoxin-induced tumor necrosis factor (TNF) assay, blood samples were incubated (37 C for 4 h) with 1 ng of Escherichia coli or Salmonella Typhimurium endotoxin/ml of blood. Plasma was harvested and assayed. For the residual endotoxin activity assay, plasma was collected into sterile endotoxin-free borosilicate tubes, diluted 1:10 with pyrogen-free water, and incubated for 10 minutes at 70 C. Escherichia coli endotoxin (0.1 or 1 ng/ml of plasma) was added to the thawed samples prior to performing the limulus ameobocyte lysate assay. Serum creatinine concentrations were monitored for 1 week. RESULTS: Compared with baseline values, PMB caused a significant dose- and time-dependent decrease in endotoxin-induced TNF activity. Compared with baseline values, residual endotoxin activity was significantly reduced after administration of 10,000 U of PMB/kg. Compared with baseline values, 1,000 and 5,000 U of PMB/kg should inhibit 75% of endotoxin-induced TNF activity for 3 and 12 hours, respectively. Serum creatinine concentrations remained within the reference range. CONCLUSION AND CLINICAL RELEVANCE: Results of the study suggest that PMB is a safe, effective inhibitor of endotoxin-induced inflammation in healthy horses.

Effect of intravenous infusion of omega-3 and omega-6 lipid emulsions on equine monocyte fatty acid composition and inflammatory mediator production in vitro.

The effect of intravenous administration of lipid emulsions enriched with omega-3 (n3) and omega-6 (n6) fatty acids on equine monocyte phospholipid fatty acid composition and the synthesis of inflammatory mediators in vitro was evaluated. In a randomized crossover design, horses were infused intravenously with 20% lipid emulsions containing n3 or n6 fatty acids. Monocytes were isolated from the horses before and 0 h, 8 h, 24 h, and 7 days after lipid infusion. Monocyte fatty acid analysis demonstrated incorporation of the parenteral n3 and n6 fatty acids in monocyte phospholipids immediately after infusion, with changes in the fatty acid composition persisting for up to 7 days after infusion. In vitro production of the inflammatory mediators thromboxane B2/thromboxane B3 (TXB(2/3)) and tumor necrosis factor-alpha (TNFalpha) by peripheral blood monocytes was diminished by n3 lipid infusion and was unchanged or increased by n6 lipid infusion. The results of this study demonstrate that short-term infusions of n3 and n6 fatty acid-enriched lipid emulsions alter the fatty acid composition of equine monocyte phospholipids and modify the inflammatory response of these cells in vitro. These results also support further investigation into the use of parenteral n3 fatty acids as part of the supportive therapy of patients with multiple organ dysfunction (MODS) or systemic inflammatory response syndrome (SIRS).

Tumor necrosis factor and interleukin-6 activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease.

The purpose of this study was to evaluate the diagnostic and prognostic significance of tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6) activities and endotoxin concentration in blood and peritoneal fluid of 155 adult horses with acute abdominal disease (colic). Samples also were obtained from 20 healthy adult horses. Blood and peritoneal fluid supernatant TNF and IL-6 activities and endotoxin concentration were significantly greater in horses with colic, compared with healthy horses. In horses with colic, the peritoneal fluid endotoxin concentration and TNF and IL-6 activities were significantly greater than those in blood. Within the colic group, peritoneal fluid IL-6 activity was the analyte that was most frequently increased. Blood and peritoneal fluid supernatant TNF and IL-6 activities were significantly greater when endotoxin was detected in the same sample. Blood and peritoneal fluid IL-6 activity was significantly greater in horses with inflammatory or strangulating lesions, compared with horses having nonstrangulating or noninflammatory lesions. Compared with all other data categories, diagnostic accuracy for nonsurvival was greatest (80%) when blood IL-6 activity exceeded 60 units/mL. The results of this study indicate that endotoxin was present in the peritoneal cavity of at least one third of horses with any acute disease of the abdomen. In horses presented for colic, blood or peritoneal fluid IL-6 activity was more useful than either TNF activity or endotoxin concentration for distinguishing lesion type. Although diagnostic accuracy for the prediction of nonsurvival was good for all of the analytes, negative values were more useful in the prediction of a favorable outcome than were abnormally increased values in the prediction of mortality.

Effect of tumor necrosis factor antibody given to horses during early experimentally induced endotoxemia.

OBJECTIVE: To test efficacy of murine monoclonal, rabbit polyclonal recombinant equine or human tumor necrosis factor-alpha (rETNF or rHTNF, respectively) antibodies to inhibit native equine tumor necrosis factor (TNF) activity. ANIMALS: 8 and 18 healthy adult horses for parts 1 and 2 of the study, respectively. PROCEDURES: In part 1, supernates from endotoxin-activated peritoneal macrophages were incubated with various dilutions of each rETNF antibody and subsequently tested for TNF activity. Serum was also obtained from a horse 1 hour after infusion with 20 ng of endotoxin/kg of body weight and was incubated with various dilutions of rabbit polyclonal rHTNF antibody. In part 2, 20 ng of endotoxin/kg was infused in horses during a 30-minute period. Fifteen minutes after the endotoxin infusion was initiated, 1 of 3 preparations was infused: 0.1 mg of rabbit polyclonal (rHTNF antibody/kg, 0.1 mg of human IgG/kg, or 500 ml of 5% dextrose. Clinical and hematologic data were collected for 24 hours. RESULTS: Compared with the monoclonal antibody, the rabbit polyclonal rETNF antibody was more effective in inhibiting TNF activity. The 50% effective doses of the murine monoclonal rETNF, rabbit polyclonal rETNF, and rabbit rHTNF antibodies were 1.8, 0.8, and 0.6 micrograms of antibody/ml, respectively. In part 2, endotoxin infusion resulted in significant alternations in all variables; however, differences among treatment groups were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Although murine monoclonal and rabbit polyclonal rETNF or rHTNF antibodies are capable of inhibiting native equine TNF activity in vitro, when given after initiation of endotoxemia, administration of 0.1 mg of rabbit polyclonal rHTNF/kg does not alter the response to infusion of endotoxin.

Hemostatic and fibrinolytic indices in neonatal foals with presumed septicemia.

Thirteen coagulation tests evaluating hemostatic and fibrinolytic indices and serum cytokine and plasma endotoxin concentrations were obtained in 34 foals with a positive sepsis score (septic group) and 46 age-matched healthy foals. Compared to healthy foals, the prothrombin, activated partial thromboplastin, and whole blood recalcification times were significantly longer in septic foals. The fibrinogen and fibrin degradation products concentrations, percent plasminogen, alpha-2 antiplasmin, and plasminogen activator inhibitor activities, and tumor necrosis factor and interleukin-6 activities were greater in septic foals. Protein C antigen and antithrombin III activity were significantly lower in septic foals. Blood cultures were positive for growth and endotoxin was detected in 19 of 29 and 15 of 30 septic foals, respectively. In septicemic foals with detectable endotoxin in the plasma, the prothrombin and activated partial thromboplastin times were significantly longer and the plasminogen and antithrombin III activities were significantly less than in septic foals in which endotoxin was not detected. Twenty-three of the 34 septic foals did not survive. Septic foals that did not survive were most likely to have a positive blood culture in which a gram-negative organism was isolated. Histopathologic evidence of hemorrhage was evident in 11 foals at postmortem examination and thrombosis was identified in 2 foals. The prothrombin time was significantly longer in foals that had multisite hemorrhage at postmortem examination. The results of this study indicate that clinically relevant alternations in hemostatic and fibrinolytic indices occur in neonatal foals with septicemia and that derangements can be correlated with the presence of endotoxin in plasma. Derangements in hemostatic or fibrinolytic indices were helpful in identification of septic foals with increased risk of coagulopathy, but were not helpful in predicting hemorrhage as compared to thrombus formation. Survival of septicemic foals was correlated with gram-negative bacteremia, but not with the presence of endotoxin or coagulopathy.

Effect of pentoxifylline, flunixin meglumine, and their combination on a model of endotoxemia in horses.

OBJECTIVE: To compare effects of a single dose of pentoxifylline (PTX), flunixin meglumine (FM), and their combination (FM/PTX) in a model of equine endotoxemia. ANIMALS: 24 healthy horses, aged 2 to 15 years. PROCEDURE: 4 groups (n = 6/group) received 30 ng of Escherichia coli O55:B5 endotoxin/kg of body weight, i.v., over 30 minutes, and 1 of the following preparations 15 minutes before and 8 hours after endotoxin infusion: FM, 1.1 mg/kg; PTX, 8 mg/kg; FM/PTX, 1.1 mg of FM and 8 mg of PTX/kg; and saline solution bolus (ENDO). Clinical and hematologic variables were measured over 24 hours. RESULTS: Compared with ENDO, FM given before endotoxin significantly reduced TxB2, and 6-keto-PGF1 concentrations, pulse, rectal temperature, and attitude score. Pentoxifylline given before endotoxin resulted in significantly higher 6-keto-PGF1 concentration at 1.5 hours and significantly lower PAI-1 activity at 12 hours. Tumor necrosis factor and IL-6 activities in horses given PTX alone were not significantly different from values in those given the saline bolus. FM/PTX induced effects similar to those of FM alone on endotoxin-induced changes in temperature and TxB2 concentration, and 6-keto-PGF1 concentration was significantly lower than that in horses of the ENDO group at 1 hour. In horses of the FM group, 6-keto-PGF1 concentration was significantly lower than that in horses of the ENDO group, from 0.5 hour to 2 hours. Horses of the FM and FM/PTX groups had significantly higher IL-6 activity at 1.5 and 2 hours than did horses of the PTX and ENDO groups; those of the FM and FM/PTX groups had significantly lower WBC count than did those of the PTX and ENDO groups. CONCLUSIONS: FM/PTX may help offset deleterious hemodynamic effects of endotoxin more effectively than does either FM or PTX alone.

Effects of pentoxifylline infusion on response of horses to in vivo challenge exposure with endotoxin.

OBJECTIVE: To evaluate the effect of pentoxifylline on response of horses to in vivo challenge exposure with endotoxin. ANIMALS: 24 healthy horses in 3 treatment groups: pentoxifylline, endotoxin, or endotoxin and pentoxifylline. PROCEDURE: Horses of the pentoxifylline group were given a bolus of pentoxifylline (7.5 mg/kg of body weight, i.v.), followed by an infusion (3 mg/kg/h) over 3 hours, and those of the endotoxin group were given 20 ng of endotoxin/kg i.v. over 30 minutes. Those of the combination group were given both of the aforementioned compounds; pentoxifylline was administered immediately after endotoxin. Clinical (rectal temperature, heart and respiratory rates, blood pressure) and hematologic (WBC count; whole blood recalcification time; plasma fibrinogen, thromboxane B2, and 6-keto-prostaglandin F1 alpha concentrations; plasma plasminogen activator inhibitor activity; and serum tumor necrosis factor and interleukin 6 activities) variables were evaluated over 24 hours. RESULTS: Compared with baseline values, there were no significant changes in any variable over time in the horses receiving only pentoxifylline, with the exception of a significant increase in WBC count. Rectal temperature, heart rate, mean blood pressure, WBC count, whole blood recalcification time, fibrinogen concentration, plasminogen activator inhibitor activity, tumor necrosis factor and interleukin 6 activities, and plasma thromboxane B2 concentration changed significantly over time in horses of the endotoxin and endotoxin-pentoxifylline combination groups. Respiratory rate and plasma 6-keto-prostaglandin F1 alpha concentration changed significantly over time only in horses of the endotoxin group. Compared with values for the endotoxin group, rectal temperature and respiratory rate were significantly lower, and whole blood recalcification time was longer for the endotoxin/pentoxifylline group. CONCLUSION: Beneficial effects of pentoxifylline are limited when it is administered i.v. to horses after in vivo challenge exposure with endotoxin.

The effects of pentoxifylline infusion on plasma 6-keto-prostaglandin F1 alpha and ex vivo endotoxin-induced tumour necrosis factor activity in horses.

Pentoxifylline (7.5 mg/kg) was bolused intravenously to eight healthy horses and was immediately followed by infusion (1.5 mg/kg/h) for 3 h. Clinical parameters were recorded and blood samples were collected for 24 h. Plasma was separated and concentrations of pentoxifylline, its reduced metabolite I, and 6-keto-prostaglandin F1 alpha were determined. Heparinized whole blood was also incubated ex vivo with 1 ng Escherichi coli endotoxin/mL blood for 6 h before determination of plasma tumour necrosis factor activity. The peak plasma concentrations of pentoxifylline and metabolite I occurred at 15 min after bolus injection and were 9.2 +/- 1.4 and 7.8 +/- 4.3 micrograms/mL, respectively. The half-life of elimination (t1/2 beta) of pentoxifylline was 1.44 h and volume of distribution (Vdarea) was 0.94 L/kg. The mean plasma concentration of 6-keto-prostaglandin F1 alpha increased over time, with a significant increase occurring 30 min after the bolus administration. Ex vivo plasma endotoxin-induced tumour necrosis factor activity was significantly decreased at 1.5 and 3 h of infusion. These results indicate that infusion of pentoxifylline will increase 6-keto-prostaglandin F1 alpha and significantly suppress endotoxin-induced tumour necrosis factor activity in horses during the period of infusion.

Endotoxin induced expression of tumour necrosis factor, tissue factor and plasminogen activator inhibitor activity by peritoneal macrophages.

Peritoneal fluid was collected aseptically from 30 healthy adult horses and 115 horses with acute gastrointestinal disease and supernatant was separated from cells by centrifugation followed by freezing until assayed for endotoxin and tumour necrosis factor activity. Peritoneal macrophages obtained from healthy horses were incubated in vitro for 3, 6, 12 or 24 h in the absence (media control) or presence of Escherichia coli 055:B5 endotoxin (final concentrations of 1, 10, 100 or 1000 ng/ml). Macrophages obtained from horses with acute gastrointestinal disease were incubated for 12 h in the absence (media control) or presence of 100 ng endotoxin/ml. At the conclusion of the incubation, macrophage supernatants were collected and frozen at -70 degrees C until analysed for tumour necrosis factor activity. Macrophage membranes were lysed and frozen at -70 degrees C until assayed for tissue factor and plasminogen activator inhibitor type 2 activity. Compared to cells incubated with media, incubation of macrophages, obtained from healthy horses, with endotoxin significantly increased tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity. These increases were dependent on the endotoxin concentration and the duration of incubation. Compared to cells incubated with media alone, incubation of macrophages, obtained from horses with acute gastrointestinal disease with endotoxin, significantly increased tumour necrosis factor and tissue factor activity. Endotoxin induced tumour necrosis factor activity in vitro was significantly less for macrophages from horses with acute gastrointestinal disease, as compared to that produced by similarly treated cells obtained from healthy horses. For those horses with acute gastrointestinal disease, macrophages obtained from horses with either endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant had significantly less endotoxin induced tumour necrosis factor in vitro, as compared to similarly treated cells obtained from horses without endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant. The results of this study indicate that exposure of equine peritoneal macrophages to endotoxin results in a significant increase in tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity. After in vitro exposure to endotoxin, there is significant down-regulation of inflammatory mediator production by peritoneal macrophages obtained from endotoxaemic horses. These results suggest that these macrophages may exhibit early endotoxin tolerance.

Effect of free and vesicle-bound cysteine proteinases of Porphyromonas gingivalis on plasma clot formation: implications for bleeding tendency at periodontitis sites.

Infection by Porphyromonas gingivalis is strongly associated with adult periodontitis, with proteinases from this bacterium now considered to be important virulence factors. In order to investigate possible pathological functions of these enzymes, we examined the effect of both free and vesicle-bound forms of the two major cysteine proteinases (gingipains) of P. gingivalis on plasma clot formation by using thrombin time (TT) measurements. Both Lys-gingipain (gingipain-K) and Arg-gingipain (gingipain-R) prolonged plasma TT in a dose- and time-dependent manner, and this was also found with vesicles which are the biological carriers of P. gingivalis proteinases. The increase in plasma TT by vesicles could be completely reversed by treatment with nonspecific cysteine proteinase inhibitors but only partially by compounds selective for either gingipain-K or gingipain-R. Preincubation of vesicles with a gingipain-K-specific inhibitor (z-FK-ck) reduced plasma TT more than a gingipain-R-specific inhibitor (leupeptin), suggesting that under physiological conditions gingipain-K was more effective in fibrinogen destruction. Each purified enzyme also markedly increased fibrinogen TT, gingipain-R being fourfold more potent than gingipain-K. However, in plasma, gingipain-R was ineffective because of the inhibitory effect of albumin. These results imply that cysteine proteinases, especially gingipain-K, abrogate the clotting potential of fibrinogen and, therefore, may contribute to the bleeding tendency and to persistent inflammation in periodontitis sites infected with P. gingivalis.

Fibrinolytic activity in plasma from horses with gastrointestinal diseases: changes associated with diagnosis, surgery, and outcome.

Plasma fibrinolytic activity was evaluated over 5 consecutive days in 59 horses admitted to the Large Animal Teaching Hospital with acute gastrointestinal diseases. Only horses hospitalized for at least 5 days were included in the study. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) were quantitated using standard chromogenic activity assays. Statistical analyses were performed using analysis of variance; differences were considered significant when P < or = .05. Activity of PAI-1, the primary endogenous inhibitor of fibrinolysis, was significantly increased on hospital days 2, 4, and 5 in horses that died, when compared with those that were discharged from the hospital. Plasma PAI-1 activity was not different at admission, but was significantly increased on hospital days 2 and 3 in horses that underwent surgery, when compared with those that did not, suggesting an acute phase response to surgical intervention. Horses with strangulating intestinal lesions had significantly increased PAI-1 activity on day 3, while PAI-1 activity was significantly greater in horses with inflammatory conditions at the time of admission, when compared with horses with strangulating or nonstrangulating/noninflammatory lesions. Among all horses, PAI-1 activity was significantly higher and tPA activity was significantly lower on day 2 when compared with other hospital days. These results suggest that fibrinolysis is inhibited early in the course of inflammatory gastrointestinal diseases and in response to surgery. In addition, among all horses, the prognosis for survival was poor for those with persistently increased PAI-1 activity, reflecting treatment failure and the loss of hemostatic regulation.

Pentoxifylline inhibits mediator synthesis in an equine in vitro whole blood model of endotoxemia.

Whole blood from 10 healthy horses was aseptically collected into heparin or citrate anticoagulant and incubated in vitro for 6 hr in the absence (saline control) or presence of 1 ng endotoxin/ml blood. Pentoxifylline (0.1, 1, 10, or 100 micrograms/ml blood) was added 1 hr before, at the same time, or 1 hr after endotoxin. As compared to saline controls, pentoxifylline alone had no effect on mediator production, with the exception of significantly increasing 6-ketoprostaglandin F1 alpha concentration. Pentoxifylline inhibited endotoxin-induced increases in tumor necrosis factor (TNF) and interleukin-6 (IL-6) activity in a dose-related fashion, whether added before, at the same time, or after endotoxin. Pentoxifylline significantly inhibited tissue factor activity, but only when added before endotoxin. Pentoxifylline had no effect on endotoxin-induced 6-keto-prostaglandin F1 alpha production, but significantly inhibited thromboxane B2 (TxB2) production. The results of this study indicate that pentoxifylline, at blood concentrations consistent with those achieved in vivo, has effects that may be beneficial in the treatment of endotoxemia.

Comparison of the effects of ketoprofen and flunixin meglumine on the in vitro response of equine peripheral blood monocytes to bacterial endotoxin.

The purpose of this study was to investigate the in vitro effects of flunixin meglumine, a cyclo-oxygenase inhibitor, and ketoprofen, a reported cyclo-oxygenase and lipoxygenase inhibitor, on the synthesis of cyclo-oxygenase end-products thromboxane B2 and prostaglandin E2, lipoxygenase derived 12-hydroxyeicosatetraenoic acid, tumor necrosis factor and tissue factor. Six adult horses were each randomly administered flunixin meglumine (1.1 mg/kg) or ketoprofen (2.2 mg/kg) intravenously every 12 hours with the drug treatments separated by two weeks. Blood samples were obtained prior to initiating treatment, the last day of treatment and for two consecutive days after the termination of treatment for measurement of serum concentrations of thromboxane B2 as well as isolation of peripheral blood monocytes. Quantitation of unstimulated, endotoxin- and calcium ionophore-induced synthesis of thromboxane B2, prostaglandin E2, 12-hydroxyeicosatetraenoic acid, tumor necrosis factor and tissue factor by peripheral blood monocytes was performed in vitro. Both flunixin meglumine and ketoprofen significantly decreased serum concentrations of thromboxane B2 demonstrating in vivo cyclo-oxygenase inhibition. There were no significant differences between drug treatment groups in the in vitro production of thromboxane B2, prostaglandin E2, 12-hydroxy-eicosatetraenoic acid, tumor necrosis factor or tissue factor. This study does not identify significant differences between the effects of flunixin meglumine and ketoprofen.