Evaluation of human cytokine production and effects of pharmacological agents in a heterologous system in vivo.

The ability of human monocytes adoptively transferred into the peritoneal cavity of BALB/c mice to produce tumor necrosis factor-alpha (TNF) and interleukin 1 beta (IL-1) was studied. Human monocytes were isolated from fresh, heparinized blood obtained by venipuncture. BALB/c mice were administered 2-10 x 10(6) cells and challenged with lipopolysaccharide intraperitoneally. 2 h later, they were killed and a peritoneal washout was obtained. The washouts were assayed for TNF and, in some cases, IL-1 content using a species specific enzyme-linked immunosorbant assay (ELISA). This model allowed for the simultaneous evaluation of the production of mouse and human inflammatory cytokines. Significant levels of both human and mouse TNF were seen as early as 60 min after challenge. Peak levels for both were seen at 120 min post administration of LPS. Both human and mouse TNF concentrations declined at the 2 h time point. The phosphodiesterase type 4 inhibitor, R-rolipram was found to inhibit both human and mouse TNF production while SB CSAID, novel kinase inhibitor SB 203580 inhibited human IL-1 and TNF as well as mouse TNF. This model was reliable, reproducible and allowed evaluation of pharmacological agents for their effect on human cytokine production in a heterologous setting in vivo.

Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 3. Bis[tetrahydroisoquinoline]s.

7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline (SK&F 64139) is a potent inhibitor of phenylethanolamine N-methyltransferase (IC50 = 10 muM) that may have therapeutic utility in man. A series of related compounds in which two 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline molecules have been bridged from nitrogen to nitrogen by an unbranched alkyl chain have been prepared and have demonstrated potent inhibitory properties (0.08 to 2 muM). In contrast simple substitution on the nitrogen of 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline with a variety of substituents gives compounds with greatly diminished inhibitory potencies (IC50 = 2 to greater than 100 muM) relative to SK&F 64139. Kinetic studies with a C6 analogue have shown that it is competitive with respect to phenylethanolamine and uncompetitive with respect to S-adenosylmethionine. The increased potency of some of the bis analogues relative to that seen with the tetrahydroisoquinolines having larger alkyl groups on nitrogen suggests that several of the bis compounds show supplemented or cooperative binding to the enzyme, presumably as a result of the second tetrahydroisoquinoline moiety.

Tetrahydrothiadiazoloisoquinolines: synthesis and inhibition of phenylethanolamine-N-methyltransferase.

A series of 7,8-fused heterocyclic tetrahydroisoquinolines were synthesized and tested as inhibitors of rabbit adrenal phenylethanolamine-N-methyltransferase (PNMT). 6,7,8,9-Tetrahydro[1,2,3]thiadiazolo[5,4-h]isoquinoline 5 (SK&F 86607) was found to be a potent inhibitor of PNMT with an IC50 similar to that of 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (1, SK&F 64139). The isomeric tetrahydro[1,2,3]thiadiazolo[4,5-h]- and tetrahydro[1,2,5]thiadiazolo[3,4-h]isoquinolines, 13 and 20, were also potent PNMT inhibitors. However, substitution of Cl at position 5 (17) resulted in loss of potency similar to the loss observed in the 5-chloro analogue of 1. The 1,2,5 isomer 20 showed only a small drop in activity at 10(-6) M. All of the thiadiazoles were more potent than the 7,8-benzo-fused analogue 36. Fusion of other five-membered heterocyclic ring systems at the 7,8-position, e.g. triazole 22 and imidazoles 24 and 26, resulted in a decrease of PNMT inhibition. The alpha-adrenoreceptor affinities of 1 and 5 were also compared.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Pharmacology of the pyrroloimidazole, SK&F 105809--II. Antiinflammatory activity and inhibition of mediator production in vivo.

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid.

SK&F 105809 [2-(4- methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2- a] imidazole] was determined to be a prodrug for the sulfide metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole] which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration (IC50) of 3 microM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The IC50 was 100 microM in the absence of peroxidase activity, whereas an IC50 of 3 microM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production with IC50 values of 0.1 and 2 microM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited (IC50 2 microM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B2 and LTB4 production ex vivo with a half-maximal dose (ED50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an IC50 of greater than 200 microM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB4 or leukotriene D4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

ARDS-like lung injury produced by endotoxin in platelet-activating factor-primed rats.

We recently reported that the combined administration of lipopolysaccharide (LPS) and platelet-activating factor (PAF) in rats, at doses that are completely devoid of any effect when given alone, caused lung injury characterized by neutrophil adhesion to lung capillaries and postcapillary venules, neutrophil accumulation in the lung parenchyma, platelet-fibrin deposits in postcapillary venules, and pulmonary edema. A marked increase in lung myeloperoxidase activity and an elevation of serum tumor necrosis factor-alpha and thromboxane B2, along with leukopenia and thrombocytopenia, were also noticed. The present study aimed to examine whether repeated LPS-PAF stimulus can cause progressive lung injury reminiscent of adult respiratory distress syndrome (ARDS). A second LPS-PAF challenge, 4 h (n = 11) after the original challenge, induced mortality (69% at 24 h, P < 0.01) and some of the pathological changes seen in clinical ARDS, including severe pulmonary edema, alveolar proteinaceous exudates, monocytic infiltration, and a further increase in lung myeloperoxidase activity (700%, P < 0.01). Repeated LPS-PAF dosing also resulted in sustained increased serum tumor necrosis factor-alpha levels (1,610 +/- 470 pg/ml, P < 0.01) and further exacerbation of the leukopenia (-68 +/- 6%, P < 0.01) and thrombocytopenia (-65 +/- 8%, P < 0.01). These data suggest that repeated LPS-PAF actions are sufficient to elicit pathophysiology of ARDS-like lung injury.

Reduction of myocardial reperfusion injury with human soluble complement receptor type 1 (BRL 55730).

This study was designed to evaluate the cardioprotective effects of a solubilized human complement receptor, sCR1, in the rat subjected to myocardial infarction. Following coronary artery occlusion for 0.5 h and reperfusion for 24 h (MI/R group), myocardial infarct size (determined by planimetric analysis) was 18.3 +/- 2.1% of the left ventricle (n = 16), while myeloperoxidase activity (a biochemical marker of neutrophil activation) was increased from 0.94 +/- 0.09 U/g tissue in the sham occluded + vehicle group to 2.96 +/- 0.17 U/g tissue in the MI/R + vehicle treated group (P < 0.01). Injection of sCR1 (5 mg/kg i.v., 5 min prior to coronary artery occlusion) produced plasma concentrations of 154 +/- 4 microgram/ml 1 min prior to coronary artery occlusion, and concentrations of 86 +/- 2 and 58 +/- 3 micrograms/ml at 40 min and 125 min after dosing (n = 6). sCR1 reduced myocardial infarct size to 11.3 +/- 2.2% of the left ventricle, and attenuated the increase in myeloperoxidase activity to 2.11 +/- 0.20 U/g tissue (n = 18; P < 0.01, compared to the MI/R + vehicle group). Administration of sCR1 5 min prior to reperfusion afforded a 25.3% non-significant reduction in myocardial injury. These results suggest a beneficial effect of sCR1 in myocardial ischemia/reperfusion injury by reducing the infiltration of neutrophils and attenuating the extent of myocardial injury.

Induction of plasma exudation and inflammatory cell infiltration by leukotriene C4 and leukotriene B4 in mouse peritonitis.

Leukotriene induction of the fluid and cellular phases of the inflammatory response in the mouse was evaluated. Intraperitoneal injection of leukotriene C4 (LTC4 250 ng) led to dye extravasation but not polymorphonuclear leukocyte (PMN) infiltration, whereas injection of leukotriene B4 (LTB4 250 ng), led to PMN infiltration but not dye extravasation. The injection of both leukotrienes did not result in synergy. LTC4 did not appear to induce significant release or formation of chemotactic mediators, but the dye extravasation induced by LTC4 was inhibited by the vasoactive amine antagonist cyproheptadine and not by the eicosanoid inhibitors phenidone or naproxen. The response was markedly inhibited by the cytokine and eicosanoid inhibitors SK&F 86002 and SK&F 104493. PMN infiltration induced by LTB4 was not inhibited by SK&F 86002 or phenidone but was abrogated by colchicine treatment. LTB4 in this model did not appear to cause release or formation of vasoactive mediators. These leukotrienes appeared to be independent, complementary, and sufficient to mount a complete inflammatory response in the mouse.

Differentiation in vivo of classical non-steroidal antiinflammatory drugs from cytokine suppressive antiinflammatory drugs and other pharmacological classes using mouse tumour necrosis factor alpha production.

The stimulation of tumour necrosis factor alpha (TNF alpha) production by lipopolysaccharide (LPS) has been widely used, both in vitro and in vivo, to examine the biochemistry and pharmacology of inflammatory cytokine production. It appears that classical nonsteroidal antiinflammatory drugs (NSAIDs) (prostaglandin H synthase 1 (PGHS-1) inhibitors) do not inhibit but instead stimulate cytokine production. In the current study, the authors utilized LPS-induced TNF alpha production in the Balb/c mouse to evaluate the activity of a classical NSAID, a mixed inhibitor, and SmithKline Beecham cytokine suppressive antiinflammatory drugs (CSAID). The results corroborated the stimulation of TNF alpha production by NSAIDs (indomethacin, naproxen, ibuprofen) and indicated that the stimulation rank-ordered with the potency of inhibition of PGHS-1. Neither acetaminophen nor nabumetone was found to stimulate TNF alpha production significantly. Tenidap, a compound reported to inhibit 5-lipoxygenase, cyclooxygenase and cytokine production, also stimulated TNF alpha production while the 5-lipoxygenase inhibitor, phenidone, was inactive. The CSAID (exemplified by SK&F 86002, SK&F 105809 and SK&F 104351), strongly inhibited TNF alpha production in this model system (ED50s of 32, 48, and 34 mg/kg p.o., respectively). These results clearly differentiate CSAID from the other compounds tested and suggest that CSAID are relatively weak inhibitors of PGHS 1 while being potent inhibitors of inflammatory cytokine production.

Effect of kallidin, substance P, and other basic polypeptides on the production of respiratory macromolecules.

When canine tracheal explants were incubated in culture medium 199 in the presence of D-glucosamine labeled with carbon-14 for 24 hours, a significant amount of radioactivity was found in the secreted macromolecules. When kallidin was present in the culture medium, the amount of radioactivity associated with a portion of these macromolecules was increased. A met-lys-bradykinin derivative had a similar effect, but bradykinin did not. When hexadimethrine, an inhibitor of kinin formation, was present in the culture medium, the amount of radioactivity in the macro-molecular fraction was decreased. Substance P and the structurally related polypeptides, physalaemin and eledoisin, also enhanced the production of tracheal macromolecules; they were several-fold more active than kallidin. The effect of polypeptides on the activities of glycosyltransferases was also investigated. One of the enzymes present in a microsomal fraction prepared from the mucosal lining of canine trachea was uridine diphosphate (UDP)-galactose:mucin galactosyltransferase, which required a 25 mM concentration of maganese ions to be present in the assay mixture to obtain maximal enzymatic activity. When the concentration of manganese ions was decreased to 2.5 mM, there was less than one third of the maximal enzymatic activity, but full activity could be restored by the addition of kallidin. Several other basic polypeptides had a similar effect on the enzymatic activity. Kallidin had little or no effect on the activities of several other glycosyltransferases. The results suggest that basic polypeptides may be important in controlling the synthesis and/or release of respiratory glycoproteins.

Effect of inhibitors of eicosanoid metabolism in murine collagen-induced arthritis.

The dual inhibitors of arachidonic acid metabolism, Smith Kline & French (SK&F) 86002, SK&F 104351, and phenidone; the corticosteroid, dexamethasone; and the selective cyclooxygenase inhibitors, ibuprofen, indomethacin, naproxen, and piroxicam were evaluated for their antiarthritic potency in the murine, collagen-induced arthritis model. The ability of these compounds to alter the severity of arthritic lesions and to reduce serum levels of the acute-phase reactant, serum amyloid P component (SAP) were monitored. Serum concentrations of SAP were found to correlate strongly (r = 0.985) with disease severity at day 35 postimmunization. Treatment with SK&F 86002, SK&F 104351, phenidone, or dexamethasone significantly reduced disease severity, as judged by clinical score (55%, 72%, 41%, and 45% inhibition, respectively) and SAP levels (62%, 94%, 52%, and 94% inhibition, respectively) in arthritic mice. This profile of activity was not shared by the selective cyclooxygenase inhibitors, which did not uniformly inhibit disease activity by both parameters. The results suggest that dual inhibitors of 5-lipoxygenase and cyclooxygenase may prove more effective than selective cyclooxygenase inhibitors as anti-arthritic agents.

Assessment of myeloperoxidase activity in whole rat kidney.

A method to quantitate myeloperoxidase (MPO) activity from rat whole kidney is described. Polymorphonuclear leukocyte (PMN) infiltration into tissue is a hallmark of acute inflammation. Historically, the degree of inflammation has been quantified by the identification and enumeration of PMNs histologically or by some other means. More recently, the enzyme activity of MPO, a marker enzyme for PMN, and freshly emigrated monocytes in many inflamed tissues has replaced these methods. The kidney, however, has been identified as a tissue from which MPO cannot be measured. Indeed, kidney homogenized by a standard extraction procedure was devoid of MPO activity. We modified the established methodology so that kidney was homogenized in 5 mM potassium phosphate buffer (PB) first and then centrifuged at 30,000 g for 30 min at 4 degrees C prior to extraction. The resulting 30,000 g pellets expressed MPO activity after suspending them in 50 mM PB containing 0.5% hexadecyltrimethylammoniumbromide (HTAB). Interference in the assay was observed with supernatants from control and inflamed kidney, which appeared to be due to kidney-derived material forming a complex with HTAB. After washing the pellets twice, we noted that their extracts exhibited greater activity, and interference from supernatants was abolished. Using this method, we observed that acutely inflamed kidneys from rats treated with sheep nephrotoxic immunoglobulin G (IgG) had significantly elevated MPO activity over kidneys from control rats. Thus, the described technique allows for the routine assay of MPO in kidney tissue.

Cardioprotective effects of the vasodilator/beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat.

The purpose of this study was to evaluate the cardioprotective effects of carvedilol, a beta-adrenergic blocker and vasodilator, in two models of ischemic myocardial damage in the rat. Following coronary artery occlusion for 0.5 h and reperfusion for 24 h (MI/R group), left ventricular (LV) injury was determined by planimetric analysis of triphenyltetrazolium chloride-stained tissue, and polymorphonuclear leukocyte infiltration was assessed by measuring myeloperoxidase (MPO) activity. In the vehicle-treated MI/R group, infarct size was 14.2 +/- 1.3% of the LV (n = 16), and MPO activity was increased to 2.8 +/- 0.7 from 0.14 +/- 0.03 U/g tissue in the vehicle-treated sham-occluded group (p less than 0.01). Carvedilol (1 mg/kg i.v., 15 min prior to coronary artery occlusion and at 3.5 h following reperfusion) reduced myocardial infarct size to 7.5 +/- 1.2% of the LV (n = 14; p less than 0.01) and attenuated the increase in MPO activity to 1.4 +/- 0.4 U/g tissue (p less than 0.05). A lower dose of carvedilol (i.e. 0.3 mg/kg i.v.) did not limit myocardial infarct size or the increase in MPO activity. In a model of permanent coronary artery occlusion, 24-hour survival was reduced from 85% in sham-occluded animals (n = 38) to 44% in the vehicle-treated MI group (n = 84; p less than 0.01). In comparison to the vehicle-treated MI group, carvedilol (0.3 mg/kg i.v., 15 min prior to coronary artery occlusion and 1 mg/kg 4 h after occlusion) improved survival by 55% (n = 64; p less than 0.05, compared to the vehicle-treated MI group), whereas the same dose of propranolol (n = 42) had no significant effect on survival. These results indicate that carvedilol reduces myocardial ischemia/reperfusion injury, and significantly improves survival in a permanent coronary artery occlusion model of myocardial infarction.

Method for quantification of myocardial infarction and inflammatory cell infiltration in rat cardiac tissue.

A method to quantitate both creatine phosphokinase (CPK) and myeloperoxidase (MPO) activity from the same cardiac tissue homogenate preparation is described. Depletion of CPK specific activity is used to quantitate myocardial infarct size, while MPO activity is utilized as a marker for polymorphonuclear leukocyte and monocyte infiltration into inflammatory sites. However, the standard assay systems are not compatible, necessitating the use of different groups of animals for these two parameters. This leads to an increase in cost, effort, and variability. The described method utilized a standard CPK methodology. It was found that interference in the MPO assay was likely caused by 2-mercaptoethanol present in the homogenate buffer (IC50 = 90 microM). Washing of the 30 K X g pellet followed by rehomogenization restored the MPO activity. Negligible MPO activity was found in the original supernatant or washes. Through the use of this technique, MPO activity was measured in the hearts of myocardial infarcted animals. The results indicated that MPO activity generated from CPK homogenate pellets compared favorably to the activity seen using standard methodology homogenates. The procedure described thus allowed the simultaneous determination of myocardial CPK specific activity and MPO activity, resulting in decreased animal usage and potentially less variability.

Temporal relation between neutrophil accumulation and myocardial reperfusion injury.

Infiltration of polymorphonuclear leukocytes (PMN) is associated with the progression of myocardial infarction and reperfusion injury. However, little is known about the time course of cellular infiltration. To investigate this issue, rats were subjected to 30 min of coronary artery occlusion followed by reperfusion for less than or equal to 96 h. Myocardial injury was determined by measuring the depletion of myocardial creatine phosphokinase activity, and PMN infiltration was assessed by measuring myeloperoxidase (MPO) activity. MPO activity increased from 0.7 U/g tissue in non-operated animals, to a peak of 6.7 +/- 0.8 and 6.4 +/- 1.4 U/g at 6 and 24 h after coronary artery reperfusion, respectively. MPO activity decreased to 3.3 +/- 0.8 U/g at 48 h and 1.1 +/- 0.4 U/g at 96 h, suggesting diminished PMN accumulation. Histological examination confirmed the accumulation and resolution of PMN over the 96-h period. At 24 h, there was a significant linear correlation between infarct size and MPO activity, whereas at 96 h no relationship was found. These data indicate that PMN infiltration occurs early in response to reperfusion injury and persists for only 24 h after initiation of reperfusion. These findings suggest that attempts to moderate inflammatory cell responses to myocardial injury should be administered early after coronary artery reperfusion to limit the accumulation of potentially deleterious inflammatory cells.

Reduction of myocardial damage and polymorphonuclear leukocyte accumulation following coronary artery occlusion and reperfusion by the thromboxane receptor antagonist BM 13.505.

This study was designed to assess the effect of the thromboxane receptor antagonist, BM 13.505, in limiting myocardial damage and polymorphonuclear leukocyte accumulation in rats subjected to coronary artery occlusion for 30 min with reperfusion for 24 h (MI/R). Myocardial injury and polymorphonuclear leukocyte infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.24 +/- 0.33 U/mg protein in sham MI/R-vehicle-treated animals (n = 18), and were significantly decreased to 6.51 +/- 0.44 U/mg protein in MI/R-vehicle animals (n = 22). Myocardial MPO values were 2.4 +/- 0.5 U/g LVFW in sham MI/R animals, and significantly increased to 10.9 +/- 1.3 U/g LVFW in MI/R-vehicle animals. Administration of BM 13.505 (30 mg/kg, i.p.) 1 min prior to coronary occlusion resulted in CPK values of 7.83 +/- 0.45 U/mg protein and MPO levels of 6.1 +/- 0.9 U/g LVFW (p less than 0.05, compared to the MI/R-vehicle group). The survival rate in the MI/R-BM 13.505 group was 74 and 65% at 2 and 24 h, respectively, and was not different from the MI/R-vehicle group. There were no significant differences in mean arterial blood pressure or heart rate between the MI/R-vehicle and MI/R-BM 13.505 groups, indicating that changes in myocardial oxygen demand do not explain the protective effects. A lower dose did not reduce myocardial injury, indicating that the effects of BM 13.505 were dose dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor-alpha mediates endotoxin-induced lung injury in platelet activating factor-primed rats.

We have reported recently that lipopolysaccharide endotoxin and platelet activating factor cooperate in priming relationships to elicit lung microvascular injury. Lung injury was associated with elevated serum levels of tumor necrosis factor-alpha (TNF alpha) and histological findings highly reminiscent of the adult respiratory distress syndrome. The present study was designed to examine the role of TNF alpha in lipopolysaccharide/platelet activating factor-induced lung injury by utilizing a highly specific monoclonal antibody which block TNF alpha actions (anti-TNF alpha monoclonal antibody). Pretreatment with anti-TNF alpha monoclonal antibody (2.5-25 mg/kg i.v., n = 5-9) dose-dependently prevented the lipopolysaccharide/platelet activating factor-induced histopathological changes, lung edema (P < .01), lung myeloperoxidase activity (P < .01), elevation of neutrophil count in bronchoalveolar lavage fluid (P < .01) and increased serum thromboxane B2 (P < .01). Indomethacin (6 mg/kg i.v., n = 5) failed to modify the lung injury despite complete inhibition of thromboxane B2 formation (P < .05). These data suggest that TNF alpha might play a key role in initiation of the early inflammatory changes which lead to adult respiratory distress syndrome.