Neil C. Olson

Prostaglandin and thromboxane levels during endotoxin-induced respiratory failure in pigs.

Arterial plasma concentrations of thromboxane B2 (TxB2), prostaglandin F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) were measured during endotoxin-induced acute respiratory failure (ARF) in anesthetized 10-12 wk old pigs. A 4.5 hour (hr) infusion of endotoxin resulted in a biphasic pattern of ARF. Phase 1 (0-2 hr) was characterized by increased pulmonary artery pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient (delta A-aO2), and decreased cardiac index (CI) and lung dynamic compliance (LDC). Following a return of PVR and CI values towards baseline, a second phase (2-4.5 hr) of deteriorating function occurred and was characterized by additional increases in PVR and delta A-aO2 and decreases in CI and LDC. Baseline (i.e., 0 hr) plasma TxB2 concentrations were 241 +/- 24 pg/ml; these values peaked at 0.5 hr (3228 +/- 712 pg/ml) and declined to 1635 +/- 453 pg/ml at 4.5 hr. Plasma concentrations of PGF2 alpha slowly increased from a baseline value of 154 +/- 32 pg/ml to 2355 +/- 738 pg/ml at 4.5 hr, while PGF1 alpha values increased from 54 +/- 2 pg/ml at 0 hr to 503 +/- 172 pg/ml at 4.5 hr. Time-matched control pigs showed no changes in pulmonary hemodynamics or in plasma TxB2, PGF2 alpha or PGF1 alpha levels. These results indicate that cyclooxygenase products are increased during both phases of endotoxin-induced ARF in pigs.

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

Summary.  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.

Dimethylthiourea inhibits the inflammatory response to intravitreally-injected endotoxin.

Dimethylthiourea, a potent scavenger of toxic oxygen metabolites such as the hydroxyl radical, hypochlorous acid, and hydrogen peroxide, was tested for its ability to inhibit an experimentally induced inflammatory response. Inflammation was induced in one eye of male New Zealand white rabbits by intravitreal injection of 10 ng Escherichia coli endotoxin; the contralateral eye received an equal volume of pyrogen-free saline vehicle. Dimethylthiourea was administered intraperitoneally to these animals at 0, 300, 450 and 600 mg kg-1. At 24 h post-endotoxin injection, all vehicle-injected eyes appeared normal with the exception of a small, but significant increase in aqueous humor protein concentration in the 600 mg kg-1 dimethylthiourea group. In endotoxin-injected eyes, treatment with dimethylthiourea, especially at the highest dose, significantly reduced iridal hyperemia, aqueous humor cell number and protein and prostaglandin-E concentrations, and the ex vivo release of prostaglandin-E from the lens. The ability of dimethylthiourea to significantly inhibit the inflammatory response to intravitreally-injected endotoxin suggests that toxic oxygen metabolites may play an important role in the initiation and/or propagation of this form of acute anterior uveitis. Furthermore, the data are consistent with an important interaction between toxic oxygen and arachidonic acid metabolites.

Formation of Isoindole Derivatives of Sulfidopeptide Leukotrienes by Reaction with O-Phthalaldehyde and Separation by Reverse Phase High Performance Liquid Chromatography

Abstract A method is described for the formation of fluorescent conjugates of the sulfidopeptide leukotrienes (LTC4, LTD4, and LTE4) by reaction of the primary amine moiety of these metabolites with o-phthalaldehyde. Separation of the fluorescent derivatives was achieved by reverse-phase high performance liquid chromatography in less than 30 minutes using a convex gradient of methanol-50 mM Na Acetate-5% Tetrahydrofuran pH 5.5. Detection limits realized under the conditions described were 0.35 ng, 3.8 ng and 3.7 ng for LTC4, LTD4, and LTE4, respectively. This represents an Increased sensitivity over detection of these metabolites by ultra-violet spectroscopy. The leukotriene-OPA derivatives are fully stable for 50 minutes at 23°C and for at least 4 hours at 0°C. The method is applied to the detection of LTC4 generated by zymosan stimulated murine peritoneal macrophages.

Effect of 5-lipoxygenase and cyclooxygenase blockade on porcine hemodynamics during continous infusion of platelet-activating factor

We hypothesized that 5-lipoxygenase and cyclooxygenase products might be mediators of cardiopulmonary and systemic vascular effects induced by a 4 h continuous infusion of platelet-activating factor (PAF, 10 ng/kg/min) in anesthetized pigs. Indomethacin (cyclooxygenase inhibitor) potentiated and CGS 8515 (5-lipoxygenase inhibitor) attenuated PAF-induced increases in total peripheral resistance (TPR) from 2.5 to 4 h. However, the 5-lipoxygenase inhibitor failed to modify pulmonary vasoconstriction and hypertension caused by PAF. Except for a delay in onset (approximately 44 s) and rate of development of pulmonary hypertension during the initial 10 min of PAF infusion, the pulmonary hemodynamic changes were also not attenuated by indomethacin. On the other hand, at 4 h, the PAF-induced pulmonary hypertension and systemic vasoconstriction were completely or partially reversed, respectively, by WEB 2086 (PAF receptor antagonist). The PAF-induced increases in plasma thromboxane B2 (TXB2) were blocked by indomethacin but not by CGS 8515, and at 4 h the 5-lipoxygenase inhibitor potentiated the levels of TXB2 in pigs treated with PAF. The plasma concentrations of 6-keto-PGF1 alpha and leukotriene B4 (LTB4) were not modified by PAF or CGS 8515 + PAF. We conclude that PAF-induced increases in TPR (2.5-4 h) are potentiated by indomethacin and are dependent on 5-lipoxygenase products other than LTB4. Although the early pulmonary vascular response (< 10 min) to PAF is dependent on cyclooxygenase products, the sustained response (after 10 min) cannot be explained by either 5-lipoxygenase or cyclooxygenase products but may be mediated directly by PAF receptors.

Indomethacin and LY171883 modify porcine cardiopulmonary responses to leukotrienes

We evaluated the effects of leukotriene (LT) C4 (0.8, 1.6, 2.4 nmol/kg), LTD4 (0.2, 1.0, 2.0 nmol/kg), and LTE4 (4.6 nmol/kg) on the cardiopulmonary system in anesthetized pigs. LTC4 and LTD4 increased mean pulmonary arterial (Ppa), mean aortic (Pma), and peak tracheal (Pt) pressures and decreased cardiac index (Cl). After indomethacin (cyclooxygenase blocker) or indomethacin + LY171883 (LTD4/LTE4 receptor antagonist), the highest doses of sulfidopeptide LTs were repeated. Indomethacin attenuated the increased Ppa and Pt, but did not affect the decreased Cl or increased Pma; LY171883 blocked or greatly attenuated the residual responses. LY171883 (without indomethacin) also blocked or greatly attenuated the LT-induced increases in Ppa and Pma and the decrease in Cl. We conclude that sulfidopeptide LTs cause potent systemic and pulmonary vasoconstriction in the anesthetized pig. Moreover, approximately two-thirds of the pulmonary arterial hypertension is indirectly mediated (i.e., cyclooxygenase products), with the residual one-third possibly due to direct LT-receptor stimulation. On the other hand, systemic vasoconstriction and decreased Cl are independent of cyclooxygenase products, and thus are likely to be directly mediated by LTs. The data support an important interaction between LT receptors and release of cyclooxygenase products.

Enhanced factor VIII activity measurements using ROTEG and factor VIII-/- mice whole blood

To quantify the rate of fibrin formation after recombinant FVIII (rFVIII) administration to FVIII-deficient mouse whole blood we have employed the rotational thromboelestography (ROTEG, Pentapharm) assay system. This system demonstrated greater sensitivity than traditional assay systems and allows the analysis of clot (fibrin) formation in real time. This system uses FVIII-deficient mouse whole blood and provides a more relevant fibrin formation assay system than plasma-based systems. In contrast to ROTEG, current assays used to measure FVIIIa-dependent fibrin clot formation, such as the activated partial thromboplastin time (APTT), are less sensitive. Reasons for the decreased sensitivity are due, in part, to the absence of platelets that are necessary for optimal tenase and prothrombinase assembly. While there has always been interest in measuring low FVIIIa activity, i.e. <0.001 U mL, the technology and methodology were not available. Thromboelastography (TEG) systems have been available for decades; however, recent ROTEG technology has improved ease of use and reproducibility [1,2]. In order to evaluate the detection sensitivity of ROTEG at low levels of FVIII we examined the thromboelastographic profile of FVIII–/– mice [3] whole blood supplemented with rFVIII over a 100 000-fold concentration range. The ability to generate a clear dose– response relationship between FVIII levels of 0.00001 U mL (0.001%) to 1 U mL (100%) and the rate of fibrin formation demonstrates the enhanced sensitivity of the approach. Briefly, blood from mice was drawn from the descending vena cava under anesthesia and immediately anticoagulated with 3.8% citrate in a 1 : 9 dilution (citrate : blood). For comparison, blood from C57BL/6 mice (FVIII+/+, background strain) was used as control. Blood samples were pooled from three to five animals for analysis. Within 8 h of sampling the blood was assayed with the ROTEG system using the NATEG protocol provided by the manufacturer [1,2]. Coagulation was allowed to proceed without the exogenous addition of tissue factor. Recombinant FVIII (Refacto,Wyeth) at eachdilutionwas added in a total volumeof 3.3 lLto 330 lL of whole blood. The coagulation reaction was started by the addition of 20 lL of 200 mM CaCl2. Using this protocol the following measurements were made: clotting time (CT), the timedelay fromstart of sample run topointwhere trace is 1 mm wide; clot formation time (CFT), the time between the 1 mm wide point to the 20 mm wide point; and alpha angle, which is an indirect measurement of the rate of clot formation [1,2]. The tests were halted after the alpha angle was calculated by the software program, or 2 h, whichever happened first. Experiments consisted of drawing blood from three to five animals, pooling the blood, and adding dilutions of rFVIII. Each experiment was repeated three to four times on different days with different mice. Data are summarized as mean ± SEM of pooled blood. The effect of FVIII levels on these thromboelastographic parameters was examined with repeated 10-fold dilutions ranging from 1.0 to 0.00001 U mL final concentrations. The dose–response effect of FVIII on the rate of clot formation (alpha angle) is shown in Fig. 1A. Normal mice (FVIII+/+) had an alpha angle value of 81 ± 2, while FVIIIdeficient mice (FVIII–/–) did not generate an alpha angle over 2 h. The addition of FVIII over a range of 1.0– 0.00001 U mL elicited a clear dose–response on clot (fibrin) formation in FVIII-deficient 5,6 mouse whole blood. As the concentration of FVIII increased, the rate of clot (fibrin) formation increased in a uniform manner. Similarly, CT and CFT also exhibited a clear dose-dependency over a range of 1– 0.00001 U mL of FVIII (Fig. 1B). CT and CFT in normal (FVIII+/+) mice were 270 ± 40 and 46 ± 8 s, respectively, whereas in FVIII–/– mice the values were 2175 ± 183 and 1326 ± 104 s. FVIII, at concentrations between 0.00001 and 1 U mL, restored CT and CFT towards normal values. As the concentration of FVIII increased the CT and CFT decreased in a uniform manner. These data demonstrate that the ROTEG provides a new methodology to detect the biological activity of very low levels of FVIII in patients with hemophilia and may help to explain the wide variation of bleeding phenotype in severe hemophilia where levels are not easilymeasured below 1%or 0.01 U mL [4,5]. Correspondence: Kyle Landskroner, Bayer Healthcare, Biological Products Division, Department of Pharmacology, Research Triangle Park, NC 27709, USA. 2 3Tel.: 919 316 6027; fax: 919 316 6065; e-mail: kyle.landskroner.b@ bayer.com

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n = 12)- and endotoxin (n = 18)-treated pigs. Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 +/- 209 pg/ml), i-12-HETE (1589 +/- 517 pg/ml), and i-15-HETE (448 +/- 78 pg/ml) were significantly (P less than 0.05) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.

Increased leukotriene B4 in bronchoalveolar lavage fluid and plasma of endotoxemic pigs

We hypothesized that leukotriene B4 (LTB4) might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) and reverse phase-high performance liquid chromatography (RP-HPLC) to determine the presence of LTB4 in plasma and bronchoalveolar lavage fluid (BALF) of saline- and endotoxin-treated pigs. Endotoxin was infused at 5 micrograms/kg for 1 hour (hr) followed by 2 micrograms/kg/hr for an average of 3 hrs. Arterial plasma (collected at 0.5 hr intervals for 4 hrs) immunoreactive (i)-LTB4 was significantly increased from 2.5 to 4 hrs of endotoxemia with the peak value occurring at 3.5 hrs (i.e. 282% of baseline value). Analysis of plasma extracts using RP-HPLC revealed an ultraviolet (UV) absorbance peak (270 nm) that was coincident with authentic LTB4 standard. The levels of i-LTB4 were significantly increased in BALF recovered from endotoxemic pigs (337 +/- 71 vs 53 +/- 13 pg/ml for saline controls). Endotoxin also increased the postmortem wet/dry ratio of bloodless lung and BALF albumin concentration, indicating pulmonary edema and increased permeability of the alveolar-capillary membrane, respectively. We conclude that LTB4 is increased in plasma and BALF recovered from endotoxemic pigs and that this lipoxygenase metabolite could possibly be an important factor contributing to the pathophysiology of endotoxin-induced ARF.

Assessment of bronchoalveolar lavage fluid and plasma for sulfidopeptide leukotrienes during endotoxemia in pigs

We hypothesized that sulfidopeptide leukotrienes (LTC4, LTD4, LTE4) might be important to the pathophysiology of endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA), reverse-phase high performance liquid chromatography (RP-HPLC) and guinea pig ileum bioassay techniques to determine the presence of sulfidopeptide leukotrienes in bronchoalveolar lavage fluid (BALF) and plasma of saline (n = 12)- and endotoxin (n = 12)-treated pigs. Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for 3.5 hrs, caused pulmonary hypertension, a biphasic increase in systemic and pulmonary vascular resistances, hypoxemia, bronchoconstriction and hemoconcentration. The levels of immunoreactive sulfidopeptide leukotrienes were not significantly increased in BALF recovered from endotoxemic pigs. Arterial plasma samples (collected at 0.5 hr intervals for 4.5 hrs) were below the detectable limits of the RIA. During RP-HPLC, ethanol extracted BALF failed to show an ultraviolet (UV) absorbance peak (280 nm) that was coincident with authentic standards. Concentrated BALF samples and BALF eluate fractions (collected at a retention time consistent with authentic LTC4) failed to cause a sustained contraction of guinea pig ileum. We conclude that sulfidopeptide leukotrienes are not increased in BALF or plasma recovered from endotoxemic pigs and that these lipoxygenase metabolites might not be important factors contributing to the pathophysiology of endotoxin-induced ARF. An alternate explanation is that the sulfidopeptide leukotrienes are rapidly metabolized so as to be undetectable by the methods employed.