PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor.

PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.

Activation of rat macrophages by Betafectin PGG-glucan requires cross-linking of membrane receptors distinct from complement receptor three (CR3).

PGG-glucan (Betafectin) is a soluble, highly purified yeast (1,3)-beta-glucan with broad anti-infective and immunomodulatory activities. These studies evaluated the ability of PGG-glucan to directly elicit O2- and tumor necrosis factor alpha (TNF-alpha) production by rat leukocytes in vitro. Particulate beta-glucan stimulated O2- production by the rat NR8383 alveolar macrophage cell line and resident rat peritoneal macrophages, but soluble PGG-glucan did not. In contrast, presentation of PGG-glucan to cells after covalent immobilization to a plastic surface caused a direct stimulation of O2- and TNF-alpha production. The O2- response of rat leukocytes to immobilized PGG-glucan was inhibited by soluble PGG-glucan, indicating that cellular responses to both immobilized and soluble PGG-glucan occur via common cell surface receptors. Because complement receptor type three (CR3) has been proposed as a beta-glucan receptor on human leukocytes, NR8383 cells were evaluated for the presence of CR3. Indirect immunofluorescence and flow cytometric analysis showed that despite being responsive to both particulate and immobilized beta-glucans, NR8383 cells expressed no detectable CR3. These results indicate that the beta-glucan receptors on NR8383 cells are not CR3 and suggest that physical presentation plays an important role in inducing pro-inflammatory leukocyte responses to PGG-glucan.

Transesophageal echocardiographic assessment of systolic mitral leaflet displacement among patients with mitral valve prolapse.

Though qualitative transthoracic echocardiographic criteria for abnormal systolic leaflet motion are widely accepted as diagnostic characteristics of mitral valve prolapse, transesophageal echocardiographic criteria have not been evaluated against such a standard. Because transesophageal imaging planes are not identical to transthoracic imaging planes, validation of transesophageal echocardiographic criteria for mitral valve prolapse is needed. Eleven patients with mitral valve prolapse (based on physical findings and transthoracic echocardiographic criteria) and 11 healthy persons underwent prospective transesophageal echocardiography in two orthogonal imaging planes. Measurements of maximal leaflet displacement superior to the annular hinge points and mitral prolapse area subtended by the displaced mitral leaflets and the chord connecting the annular hinge points were performed in triplicate and averaged by a blinded observer. Though maximal systolic leaflet displacement was greater among patients with mitral valve prolapse than healthy subjects for both the transesophageal four-chamber (0.66+/-0.39 cm versus 0.05+/-0.11 cm, p < 0.001) and two chamber views (0.57+/-0.44 cm versus 0.20+/-0.25 cm, p < 0.04), no unique value differentiated patients with from those without mitral valve prolapse. Mitral prolapse area was greater for patients with mitral valve prolapse than for healthy subjects in both transesophageal four-chamber (1.23+/-1.18 cm2 versus 0.03+/-0.06 cm2, p < 0.02) and two-chamber views (1.73+/-1.65 cm2 versus 0.21+/-0.31 cm2, p < 0.02). Whereas a mitral prolapse area of 0.20 cm2 uniquely differentiated patients with from those without mitral valve prolapse in the four-chamber view, data overlap prevented determination of a similar diagnostic criterion for the two-chamber view. The difficulty in defining quantitative transesophageal echocardiographic criteria for mitral valve prolapse based on leaflet displacement alone suggested that the simple qualitative observation of leaflet displacement above the annular hinge points should not be used as a defining morphologic criterion for mitral valve prolapse.

Neuprex XOMA Corp.

XOMA is developing Neuprex, an injectable recombinant protein fragment (rBPI-21) of the naturally-occurring bactericidal/permeability increasing (BPI) protein, for serious infections and infectious complications of trauma and surgery. Neuprex is the most advanced compound from XOMA's BPI platform. Neuprex is planning to commence a clinical trial for at least one additional indication in 1998 [293193]. Meningococcemia The lead indication for Neuprex is meningococcemia, for which phase III trials are expected to be completed in the second half of 1998 [275644]. In June 1998, Neuprex received Orphan Drug designation from the FDA for the treatment of severe meningococcal disease. XOMA is preparing components of a BLA to be submitted to the FDA as soon as the ongoing phase III trial is complete [290059]. In an open-label pilot study in 26 patients, completed in 1996, the mortality rate was 3.8% (one death) compared with 22% (12 deaths in 55 patients) of patients treated at the same medical centers from the previous two years [264164]. A 200-patient phase III trial, testing Neuprex in children with severe meningococcemia, is being carried out in the UK and North America. An independent Data Safety Monitoring Board recommended continuation of the trial following an interim analysis of the data [264164]. Post hepatectomy A phase II trial in partial hepatectomy patients has stopped enrollment at 35 patients (due to the slow rate; XOMA intends to make progress in other hepatic studies). Data from the first interim safety analysis, on twelve patients (low-dose group), suggested that patients receiving Neuprex spent less time on respirator, in the ICU and in the hospital [282257]. Antibiotic-resistant infections In vitro studies have shown that rBPI-21 can make resistant bacteria more antibiotic-susceptible and enhance the potency of a broad range of traditional antibiotics. Hence, XOMA has commenced studies to examine Neuprex in situations where infections may be resistant to antibiotics. A 21-patient open-label trial of Neuprex as an adjuvant to antibiotics in the treatment of severe intra-abdominal infections has been completed. The results showed a consistent, dose-related improvement in patient outcome as measured by time to improvement and time to fever-free status, but few resistant bacterial strains were found in this study and none that were resistant to all three antibiotics used in the study [282257]. In November 1997, XOMA began a clinical trial evaluating Neuprex as an adjunctive therapy with conventional antibiotics in bacterial lung infections in cystic fibrosis (CF) patients. A second study site was added in January 1998. The pilot study will enroll up to 24 CF patients hospitalized for acute bacterial exacerbations [264398]. Post-hemorrhagic trauma The company is initiating a phase III clinical trial to evaluate Neuprex as a treatment to prevent infections and infectious complications in patients suffering from hemorrhagic trauma [268444]. A 401-patient, phase II hemorrhagic trauma trial showed a positive overall benefit in patients receiving Neuprex versus placebo [282257]. A follow-on study in 169 patients confirmed the original dosing regimen [282257]. Bactericidal/permeability increasing protein platform BPI is a human host-defense protein discovered in 1978 by Drs Peter Elsbach & Gerrold Weiss, at New York University (NYU) Medical School. BPI occurs in neutrophils. XOMA scientists have discovered three active areas (functional domains) within the N-terminal fragment of human BPI (the amino-terminal 199 amino acid fragment). Many peptides, derived from these domains, have been identified and characterized. Five classes of bioactivity have been shown: antifungal, antibacterial, endotoxin-neutralizing, heparinneutralizing and anti-angiogenic [282257]. XOMA has succeeded in producing recombinant versions of BPI proteins but has also modified them to fine-tune their function and improve their pharmacodynamic profile [162407,183566, 184024]. XOMA is New York University's exclusive licensee for patents relating to human therapeutic and diagnostic uses of rBPI. XOMA has also built a broad patent position related to its own work with BPI [282257]. In addition, in July 1998, XOMA licensed all of Incyte's BPI-related intellectual property, adding seven patents to XOMA's portfolio, which now includes worldwide rights to all existing BPI patents [293193].

MAK-195F Knoll AG.

BASF is developing the monoclonal antibody MAK-195F, a tumor necrosis factor (TNF)-alpha antagonist, for the potential treatment of septic shock. It is in phase III clinical trials in the US and Europe [198973]. Results from the trials were expected between late 1997 and early 1998 [200089], but none have yet been reported. Launch of the drug was expected in 2000 [277817]. Results from a phase II clinical trial on 122 patients in Germany did not show prolonged survival in patients with septic shock but did indicate lowered levels of IL-6. The treatment was well-tolerated [222840]. A patent disclosing similar human antibodies that bind to TNF was published by BASF in August 1997 (WO-09729131).

PGG-Glucan activates NF-kappaB-like and NF-IL-6-like transcription factor complexes in a murine monocytic cell line.

PGG-Glucan (Betafectin) is a novel soluble beta-glucan immunomodulator that enhances leukocyte microbicidal activities without inducing inflammatory cytokines. Although several different receptors for soluble and particulate beta-glucans have been described, the signal transduction pathway(s) used by soluble beta-glucans have not been elucidated. We report that in a murine monocytic cell line (BMC2.3) PGG-Glucan activates nuclear factor-kappaB (NF-kappaB)-like and NF-interleukin-6 (IL-6)-like transcription factors. Electrophoretic mobility shift assays showed that PGG-Glucan activation of the factors is time- and concentration-dependent. The NF-kappaB-like complex includes subunit p65 (rel-A) as one of its components, but apparently not p50 (kappaB1), p52 (kappaB2), p68 (rel-B), or p75 (C-rel) family members. The NF-IL-6-like complex contains subunit C/EBP-beta (NF-IL-6alpha) as one of its components, but apparently not C/EBP-alpha or C/EBP-delta (NF-IL-6beta). As expected, lipopolysaccharide (LPS) activated p65/p50 NF-kappaB and C/EBP-beta NF-IL-6 complexes, increased the nuclear titer of p65 and p50 antigens, and increased cytokine (IL-1beta, tumor necrosis factor alpha) mRNA production. In contrast, PGG-Glucan increased the nuclear titer of p65, but apparently not p50, and did not induce cytokine mRNA production. These data demonstrate that PGG-Glucan utilizes signal transduction pathways different from those used by LPS. The data suggest that activation of the PGG-Glucan-stimulated factors is not sufficient to stimulate cytokine mRNA transcription.

Transesophageal echocardiographic assessment of the contribution of intrinsic tissue thickness to the appearance of a thick mitral valve in patients with mitral valve prolapse.

OBJECTIVES: This prospective, blinded transesophageal echocardiographic study was performed to determine the relative contributions of leaflet redundancy and overlap versus intrinsic tissue thickening as mechanisms for the apparent increase in diastolic thickness of the mitral valve. BACKGROUND: Increased diastolic thickness of the mitral valve has been identified as an echocardiographic feature that predicts subsequent adverse sequelae in patients with mitral valve prolapse (MVP). METHODS: Eleven patients with clinical and transthoracic echocardiographic evidence of MVP and 11 age-matched control subjects underwent protocol transesophageal echocardiography to image the mitral valve in two orthogonal planes and to measure its thickness in systole and diastole. RESULTS: Maximal diastolic width of the slack, unloaded anterior leaflet was significantly greater in patients with MVP than in control subjects (mean +/- SD: 0.64 +/- 0.20 cm vs. 0.30 +/- 0.04 cm, p < 0.001). Similarly, diastolic posterior leaflet width was greater in patients with MVP (0.67 +/- 0.39 cm vs. 0.31 +/- 0.06 cm, p < 0.01). In contrast, minimal systolic width of the distended pressure-loaded mitral valve was not significantly different between patients with MVP and control subjects for either the anterior (0.22 +/- 0.05 cm vs. 0.20 +/- 0.04 cm, p = NS) or the posterior (0.25 +/- 0.07 cm vs. 0.24 +/- 0.05 cm, p = NS) leaflets. The percent change in leaflet width from diastole to systole (% delta W), an index of the contribution of dynamic factors (e.g., leaflet redundancy and overlap) to the apparent increase in diastolic leaflet thickness, was significantly greater in patients with MVP than in control subjects for both the anterior (% delta W 62 +/- 13% vs. 34 +/- 16%, p < 0.001) and the posterior (% delta W 54 +/- 19% vs. 22 +/- 21%, p < 0.005) leaflets. CONCLUSIONS: The apparent increase in diastolic mitral leaflet thickness in patients with MVP versus control subjects is largely attributable to dynamic factors such as leaflet redundancy, overlap and deformation. During diastole, when the mitral leaflets are slack and unstressed, the leaflets appear markedly thickened in patients with MVP. In contrast, during systole, when developed intraventricular pressure distends the leaflets, causing them to stretch and balloon into the left atrium, the intrinsic tissue thickness is much less than that measured in diastole. These findings have important implications for the morphologic criteria used to diagnose MVP and the potential pathophysiologic mechanisms for adverse sequelae in this syndrome.

Expression and functional characterization of recombinant human vascular cell adhesion molecule-1 (VCAM1) synthesized by baculovirus-infected insect cells.

Vascular cell adhesion molecule-1 (VCAM1) is a cell surface glycoprotein produced by the vascular endothelium, as well as on macrophage-like and dendritic cell types, in response to certain inflammatory stimuli. VCAM1 interacts with the integrin VLA4 present on mononuclear leukocytes. We have isolated the cDNA for VCAM1 using RT-PCR by screening a cDNA library from IL-1 beta-activated human endothelial cells. To obtain large quantities of VCAM1 for structural and functional studies, we have produced this protein in insect cells using a baculovirus expression system. Insect cells infected with recombinant virus synthesized human VCAM1 at levels exceeding 3% of total cellular protein following 72 h postinfection. VCAM1-expressing insect cells were shown to bind specifically to a variety of VLA4 expressing cell lines (Jurkat, THP-1, U937). Thus, recombinant VCAM1 protein produced in the baculovirus expression system was localized to the cell surface and was biologically active. Large-scale availability of this adhesion protein should enhance efforts toward the discovery of new antiadhesive (anti-inflammatory and antiatherogenic) therapeutics.

Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq.

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.

Identification and immunolocalization of phospholipase C in bovine rod outer segments.

Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphate. Approximately 60-70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of approximately 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.

Persistent cardioprotection by azapropazone in a canine model of coronary artery thrombosis and thrombolysis.

Activated neutrophils and possibly xanthine oxidase-derived free radicals are believed to be mediators of ischemia and reperfusion-induced myocardial damage. We studied the cardioprotective effect of the neutrophil stabilizer and xanthine oxidase inhibitor azapropazone in dogs subjected to thrombotic occlusion of the left anterior descending coronary artery (LAD), induced by intracoronary introduction of a copper coil, followed 60 min later by thrombolytic treatment with intracoronary streptokinase and 4-day reperfusion; we then determined infarct size by triphenyltetrazolium stain. Azapropazone [100 mg/kg intravenously (i.v.) followed by a 24-h i.v. infusion of 10 mg/kg/h, n = 8] or vehicle (n = 10) treatments were started immediately before the streptokinase infusion. Steady-state plasma levels of azapropazone ranged from 97 to 163 micrograms/ml during the infusion. Myocardial blood flow and underperfused area at risk were determined using radiolabeled microspheres. Results were as follows (mean +/- SEM): area at risk (percentage of left ventricle) azapropazone 22.7 +/- 3.16 and vehicle 21.8 +/- 4.13; infarct size (percentage of area at risk), azapropazone 45.1 +/- 11.8 and vehicle 75.7 +/- 10.6, p less than 0.03; collateral blood flow (ml/min/g), azapropazone 0.27 +/- 0.02 and vehicle 0.23 +/- 0.02; total ischemic period (min), azapropazone 106 +/- 5.9 and vehicle 91.5 +/- 4.9. Azapropazone had no effects on heart rate (HR), blood pressure (BP), or rate/pressure product (RPP). These dta show that azapropazone limits infarct size in a canine model of coronary thrombosis and long-term reperfusion and that this cardioprotection is independent of cardiovascular parameters.

Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis.

Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids.

Cellular and biochemical characterization of the anti-inflammatory effects of DuP 654 in the arachidonic acid murine skin inflammation model.

The possible utility of DuP 654, a potent 5-lipoxygenase inhibitor, for treating human inflammatory skin disease was investigated in murine skin treated with 1.0 mg arachidonic acid (AA). When DuP 654 was applied to murine skin treated with AA, it inhibited the resulting inflammation and influx of cells. High performance liquid chromatography and radioimmunoassay analysis of lipid extracts from AA-treated ears indicated that the influx of polymorphonuclear leukocytes (PMN) was temporally preceded by an appearance of significant amounts of 5-HETE (6.7 +/- 1.4 ng/ear) and Leukotriene B4 LTB4 0.92 +/- 0.2 ng/ear) when compared with extracts of untreated ears (5-HETE, 02 +/- 0.3 ng/ear; LTB4, less than 0.1 ng/ear). The levels of the 5-lipoxygenase products were reduced by treatment with 10 micrograms/ear DUP 654. Lipid extracts from AA-treated ears contain chemotactic activity for human PMN and this chemotactic activity in the AA-treated ears could be reduced but not eliminated by immunosorption with anti-LTB4 antibodies coupled to protein A-agarose. The appearance of the chemotactic activity was inhibited by DuP 654. Taken together, these data suggest that DuP 654 may have utility in human inflammatory skin disease.

Extracellular phospholipase A2 activity in two in vivo models of inflammation.

Two "in vivo" models of inflammation have been used to investigate the role of phospholipases A2 (PLA2) in inflammation. These models are casein-induced peritonitis in the rat and zymosan-induced peritonitis in the mouse. The extracellular PLA2 activities from peritoneal lavage fluid in these two models are similar: they are calcium dependent and have broad neutral pH optima. However, the relationship between extracellular PLA2 activity and cell influx in these models are not identical. In zymosan peritonitis, PLA2 activity preceded peak cell influx, reaching a maximum within 15 min after zymosan injection, while cell influx peaked by 8 hr. In casein-induced peritonitis, the PLA2 activity peaked at 24 hr, while cell influx continued through 48 hr. The origins of the PLA2 activities in both models remain unclear; one potential source is the plasma. Understanding the role of extracellular PLA2 activity in "in vivo" models, and investigating specific inhibitors in these models may aid in our understanding of the role of extracellular PLA2 in diseases such as rheumatoid arthritis, endotoxin shock and pancreatitis.

Effects of GTP gamma S and fMet-Leu-Phe on rat PMN phospholipase C (PLC) activity.

The PLC activities associated with the cytosol and plasma membrane subcellular fractions of rat PMN were tested for sensitivity to stimulation by GTP gamma S and/or fMet-Leu-Phe. PMN plasma membrane PLC was stimulated 10-20% by 50 microM GTP gamma S (p less than 0.02). fMet-Leu-Phe alone had no effect on the plasma membrane PLC activity, but addition of fMet-Leu-Phe and GTP gamma S together stimulated PLC activity by 20-30% (p less than 0.05). Neither GTP gamma S nor fMet-Leu-Phe had any significant effect on the cytosolic PLC activity. These data demonstrate the presence of a PLC activity in rat PMN plasma membranes that is sensitive to stimulation by GTP gamma S and fMet-Leu-Phe.

Biochemical and pharmacologic characterization of a phosphatidylinositol-specific phospholipase C in rat neutrophils.

The phosphatidylinositol (PI)-specific phospholipase C (PLC) activity contained in sonicates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15-20 min at 37 degrees C and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5-6.0, is enhanced 1.5-3-fold by the addition of deoxycholate, and is Ca++ dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 +/- 85 microM and a Vmax of 34.3 +/- 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 +/- 66 microM and a Vmax of 14.3 +/- 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values less than or equal to 100 microM) inhibitors of the PMN PLC.

Effect of azapropazone and allopurinol on myocardial infarct size in rats.

The effect of the xanthine oxidase inhibitor allopurinol and the non-steroidal antiinflammatory agent azapropazone on infarct size in rats, subjected to 48 h of occlusion of the left anterior descending coronary artery, were studied. Allopurinol (50 mg/kg i.p., twice daily from 24 h before to 48 h after LAD occlusion) and azapropazone (100 mg/kg i.p twice daily from 24 h before to 48 h after LAD occlusion) significantly reduced infarct size when compared to saline-treated rats. These data point towards involvement of xanthine oxidase derived free radicals in evolving myocardial infarction in rats; beneficial effect of azapropazone in this model may be related to the drug's ability to inhibit xanthine oxidase as well as various key neutrophil functions.

Acute lung inflammation in rats induced by phorbol myristate acetate (PMA).

Intratracheal administration of PMA produces acute lung injury in part due to the generation of O2-derived free radicals. This study evaluated the role of the antioxidant enzyme superoxide dismutase (SOD) in PMA-induced lung injury in the rat. PMA was instilled into rats intratracheally (20-60 micrograms/kg), and the lungs were lavaged 4 hr later. Total number of cells recovered from lavage after PMA treatment was not different from the total number recovered from controls; lavagable PMNs increased in a dose-dependent manner. Albumin in lavage fluid (an index of lung vascular permeability) was significantly increased at 60 micrograms/kg PMA. SOD (10,000 U) + PMA (60 micrograms/kg) reduced the albumin level but significantly increased both total number of cells and number of PMNs recovered from lavage fluid. To investigate the possibility that SOD decreases the ability of PMNs to adhere, PMN aggregation was measured in vitro. The results indicated that 10,000 U SOD can inhibit PMA-induced aggregation by 50%. In contrast, aggregation to other stimuli (e.g., fMet-Leu-Phe, A23187) was unaffected by SOD. We conclude SOD prevents PMA-induced lung permeability and diminishes PMN adherence.

Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug.

Azapropazone at concentrations of 0.1 to 1 mM inhibited by 30-70% rat neutrophil migration, aggregation, and degranulation in response to the synthetic chemotactic peptide fMet-Leu-Phe. Binding studies using fNle-Leu-[3H]Phe, a radiolabeled analog of fMet-Leu-Phe, showed that azapropazone did not inhibit these responses by interfering with fMet-Leu-Phe binding. Azapropazone also decreased both the apparent rate of production and maximal levels of superoxide anion (O2-) generated by cells stimulated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA). The concentrations of azapropazone that inhibit these neutrophil responses in vitro approximate those previously found in vivo after administration of therapeutic doses of drug to rats or humans. Taken together, the data suggest that the efficacy of azapropazone in gouty arthritis may be partly due to its ability to inhibit key neutrophil functional responses in vivo.