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Biomedical subjects

R A Blackwood

Publications and source records attributed to R A Blackwood.

At least 19 recordsLinked to original sources
PubMed2001

Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils.

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca(2+) concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca(2+) mobilization, whereas mAbs against the beta(2) integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca(2+) signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.

Carbohydrate Metabolism↗
PubMed2001

Phospholipase A(2)-mediated fusion of neutrophil-derived membranes is augmented by phosphatidic acid.

Phosphatidic acid (PA), the product of phospholipase D (PLD) metabolism, is not only an important second messenger in neutrophil signal transduction but PA generation increases membrane fusogenicity. Following neutrophil stimulation, PA formation can be detected in azurophil, specific, and plasma membrane vesicle subcellular fractions, suggesting a potential role for PA formation in granule-plasma membrane fusion. Neutrophil stimulation also activates phospholipase A(2) (PLA(2)) and the release of arachidonic acid. In vitro fusion of plasma membrane vesicles and specific granules with complex liposomes were dependent on PLA(2) (<10 microM Ca(2+)) while the presence of PA in the liposomes augmented the effects of PLA(2). Azurophil granules were extremely resistant to fusion (no fusion at 12 mM Ca(2+) even in the presence of PLA(2)). However, in the presence of both PA and PLA(2) fusion could be detected at <5 microM Ca(2+), suggesting a direct role for phospholipid metabolism in neutrophil degranulation.

Cell Degranulation↗
PubMed2001

Disseminated vaccine strain varicella as the acquired immunodeficiency syndrome-defining illness in a previously undiagnosed child.

The Food and Drug Administration licensed a live-virus varicella vaccine (Varivax; Merck & Co Inc, West Point, PA) in March 1995. Prelicensure adverse events were minimal; however, since licensure and increased vaccine use, rare previously undetected risks have arisen. Presented here is the clinical course of a previously undiagnosed, human immunodeficiency virus-infected boy who developed dissemination of the vaccine strain of varicella zoster after immunization. chickenpox, human immunodeficiency virus, pneumonia, encephalopathy, varicella vaccine, adverse events, dissemination.

Chickenpox↗
PubMed2000

Clustering of urokinase receptors (uPAR; CD87) induces proinflammatory signaling in human polymorphonuclear neutrophils.

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 +/- 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of beta-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.

Calcium↗
PubMed1999

Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes.

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.

Benzoquinones↗
PubMed1998

Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol.

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule-plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+-dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil.

Annexin A1↗
PubMed1997

Carvedilol versus verapamil in chronic stable angina: a multicentre trial.

OBJECTIVE: In a multicentre, double-blind, parallel group study, the anti-anginal and the anti-ischaemic efficacy of 12 weeks of therapy with the vasodilating beta-adrenoceptor-blocker carvedilol 25 mg b.i.d. was compared with verapamil 120 mg t.i.d. METHODS: During a 2-week placebo run-in period, patients were required to have two treadmill exercise tests (modified Bruce Protocol) differing by not more than 15% with regard to total exercise time (TET). Of 313 patients enrolled, 248 were randomized and 212 completed the study according to the protocol. RESULTS: The primary variable TET was analysed using the Cox Proportional Hazards Model to take into account censored values due to the patient stopping the exercise test for reasons other than angina. Forty-three per cent of patients allocated to carvedilol and 36% to verapamil did not stop with angina at the final visit. There was no difference in the TET between the groups, the risk ratio being 1.14 in favour of carvedilol (90% CI 0.85-1.52). TET increased from 378 s at baseline to 436 s at the final visit in the carvedilol group and from 386 to 438 s in the verapamil group. Results for time to angina and time to 1 mm ST-segment depression were similar. Compared to verapamil, carvedilol significantly reduced HR, systolic BP and rate pressure product at peak exercise. Analysis of 48 h Holter monitor data showed a greater reduction of HR and PVCs with carvedilol. Lown grading improved in both groups. Adverse events were reported by 48% (3.2% serious adverse events) of patients taking carvedilol and 58% (5.7% serious adverse events) taking verapamil. CONCLUSION: Carvedilol is at least as effective as verapamil in the management of chronic stable angina and demonstrated a favourable adverse event profile.

Adrenergic beta-Antagonists↗
PubMed1997

Pulsed-field gel electrophoresis genomic fingerprinting of hospital Escherichia coli bacteraemia isolates.

Pulsed-field gel electrophoresis (PFGE), because of the increased sensitivity it affords over other methods of bacterial genotyping, represents a potentially powerful tool for the characterisation of isolates from hospital infections. Genomic fingerprinting by PFGE was applied to all clinical isolates of Escherichia coli obtained from blood during a 6-month period (78 isolates, 58 patients) at the University of Michigan Medical Center. The rare-restriction patterns of these isolates, in contrast to those of isolates from the E. coli reference collection (ECOR), were not randomly distributed through the E. coli species. Four related clusters, which represented c. 21% of the blood isolates, were identified. Two of these genotypic clusters were also clustered temporally, their members all being isolated within the same 2-week period, while the other two clusters spanned the study period. These observations indicate in-hospital endemic vectors or the occurrence of specialised E. coli lineages that are capable of invading the bloodstream and exploiting in-hospital vectors, or both.

Bacteremia↗
PubMed1997

Phospholipase D activity facilitates Ca2+-induced aggregation and fusion of complex liposomes.

Phospholipase D (PLD) activation in stimulated neutrophils results in the conversion of membrane phosphatidylcholine (PC) to phosphatidic acid (PA). This change in membrane phospholipid composition has two potentially positive effects on degranulation. It 1) replaces a nonfusogenic phospholipid with a fusogenic one and 2) increases the potential for interactions between membranes and the annexins. Modeling neutrophil degranulation, we examined the effect of PLD (Streptomyces chromofuscus) hydrolysis on the aggregation and fusion of liposomes in the presence and absence of annexin I. We found that PLD-mediated conversion of PC to PA lowered the [Ca2+] required for fusion. Annexin I increased the rate of fusion in the presence of PA, although it did not lower threshold [Ca2+], which remained above the physiological range. However, after hydrolysis by PLD, annexin I lowered the [Ca2+] required for aggregation by almost three orders of magnitude, to near physiological concentrations. These studies indicate that the activation of PLD and the production of PA may play a role in annexin-mediated membrane-membrane apposition.

Annexin A1↗
PubMed1996

Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils.

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide ('DPX') to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 microM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Calcium↗
PubMed1996

PLA2 promotes fusion between PMN-specific granules and complex liposomes.

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude. Furthermore, although lysophospholipids inhibited fusion, the incorporation of arachidonic acid into liposome membranes overcame the inhibitory effects of the lysophospholipids. Thus with PLA2 and annexins we were able to obtain fusion of complex liposomes at concentrations of Ca2+ that are close to physiological. Our data suggest that the activation of PLA2 and the generation of arachidonic acid may be the major fusion-promoting event mediating neutrophil degranulation.

Arachidonic Acid↗
PubMed1996

Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor.

During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP). We observed reciprocal inhibition with respect to chemotaxis. We demonstrated that homologous desensitization, with respect to the mobilization of intracellular calcium stores, lasted approximately 15 min. Heterologous desensitization between the fMLP receptor and the C5a receptor was reciprocal; either stimulant would diminish the cells' response to stimulation by the other for approximately 3-5 min. However, we observed a unidirectional heterologous desensitization of the IL-8 receptor by both the fMLP and the C5a receptor. This unidirectional heterologous desensitization was observed with respect to both calcium mobilization and arachidonic acid production (i.e., prestimulation of the IL-8 receptor had no effect on subsequent stimulation by either fMLP or C5a).

Antigens, CD↗
PubMed1996

Colchicine inhibits arachidonate release and 5-lipoxygenase action in alveolar macrophages.

Although colchicine is known to inhibit leukotriene synthesis in neutrophils, its effect on other aspects of arachidonic acid (AA) metabolism as well as its mechanism of action are unknown. To address these questions, we investigated the effects of colchicine on resident rat alveolar macrophages (AM), cells that generate a variety of lipoxygenase and cyclooxygenase products after stimulation. Pretreatment of AM with 10 microM colchicine for 1 h dramatically inhibited the synthesis of all 5-lipoxygenase (5-LO) metabolites from endogenous AA in ionophore A-23187-stimulated cells. In addition, colchicine inhibited the total release of AA as well as prostanoids to a lesser extent. Similar effects were observed with the other microtubule-disruptive agents nocodazole and vinblastine, and 5-LO product formation stimulated by the particulate agonist zymosan was inhibited as well. A selective inhibitory effect of colchicine on the 5-LO pathway was demonstrated by monitoring the synthesis of 5-LO products from exogenously supplied AA. Cell-free enzyme assays showed that this effect was not through a direct inhibition of the 5-LO enzyme. Moreover, colchicine did not affect the translocation of 5-LO to the nuclear envelope. We next evaluated the effect of colchicine on the levels of the two 5-LO cofactors, ATP and Ca2+. Although colchicine did not affect ATP levels, it did abrogate the ionophore-induced increase in intracellular Ca2+ concentration; the inhibitory effect of colchicine on 5-LO metabolism in AM was partially overcome by stimulation with higher doses of A-23187. We conclude that microtubular disruption inhibits agonist-induced increase in intracellular Ca2+ concentration, with multiple consequences for AA metabolism. These include a reduction in the liberation of AA from membrane phospholipids as well as the selective inhibition of processing of AA by 5-LO.

Adenosine Triphosphate↗
PubMed1995

Effect of calcium on phenothiazine inhibition of neutrophil degranulation.

The phenothiazines are known to be potent inhibitors of calmodulin and have been used as probes for examining calmodulin-dependent cellular functions. We report here that the characteristics of phenothiazine inhibition of exocytosis in neutrophils more closely resemble their interaction with the annexins in vitro. Ca(2+)-dependent aggregation of liposomes mediated by either annexin I or annexin II was inhibited by the phenothiazines. Inhibition of liposome aggregation was not caused by interference with the binding of annexins to phospholipids. Rather, the phenothiazines increased the concentration of Ca2+ required for aggregation. Likewise, in neutrophils permeabilized with streptolysin O, inhibition of degranulation by phenothiazines could be overcome by increasing [Ca2+]. These results suggest that inhibition by phenothiazines of neutrophil degranulation is secondary to the ability of these compounds to inhibit membrane-membrane contact promoted by the annexins.

Annexins↗