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D Rotrosen

Publications and source records attributed to D Rotrosen.

31 records · Page 2Linked to original sources
PubMed1989

Cytosolic calcium changes in endothelial cells induced by a protein product of human gliomas containing vascular permeability factor activity.

A vascular permeability factor (VPF) derived from serum-free conditioned medium of cultured human malignant gliomas (HG-VPF) has been described previously. The rapid kinetics of HG-VPF activity in an in vivo assay of vascular permeability suggests a direct action upon the vascular endothelial cell. To determine whether HG-VPF was capable of inducing a physiologically significant alteration in isolated endothelial cells, cytosolic calcium [Ca++]i was measured in vitro in these cells before and after their exposure to media containing this substance. This was accomplished by preloading cultured endothelial cells with a fluorescent intracellular Ca++ probe fura-2/AM. It was found that HG-VPF induced a rapid and transient elevation of [Ca++]i in normal endothelial cells derived from human umbilical vein, bovine adrenal medulla, bovine pulmonary artery, and rat brain. This effect was inhibited by chelating extracellular calcium [Ca++]e with ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetra-acetic acid (EGTA), indicating that the HG-VPF-induced response resulted from the influx of extracellular calcium. The addition of cations that act as nonspecific calcium channel blockers (Li+, Co++, Mn++, La ) completely inhibited VPF activity, further supporting the role of [Ca++]e influx. The HG-VPF activity was not, however, blocked by verapamil, a calcium antagonist that appears to be specific for voltage-gated calcium channels. Furthermore, exposure of endothelial cells to 120 mM [K+]e did not result in a calcium transient. Coincubation of endothelial cells with dexamethasone inhibited HG-VPF-induced rises in [Ca++]i, while having no effect upon cyclic nucleotide-induced changes in calcium. The present studies indicate that vascular extravasation induced by human glioma-derived VPF may be mediated by a direct action upon vascular endothelial cells. Furthermore, the observed dexamethasone-induced inhibition of this process suggests a specific cellular action for corticosteroids. This, together with previous observations that dexamethasone suppresses both the production of VPF by tumor cells in vitro and its permeability-inducing activity in vivo, may explain the efficacy of glucocorticoids in the treatment of neoplastic vasogenic brain edema. Finally, studies with a polycationic peptide (protamine) known to induce blood-brain barrier disruption in vivo revealed similar effects upon endothelial cytosolic calcium levels. As HG-VPF is a positively charged macromolecule, a common interaction between these substances and the negatively charged endothelial cell surface in the induction of permeability is suggested. Nonspecific cross-linking of charged groups of the endothelial glycocalyx and specific HG-VPF receptor binding are both valid mechanisms of HG-VPF-mediated calcium changes. Their potential relevance in the setting of microvascular permeability is discussed.

Biomechanical Phenomena↗
PubMed1988

Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors.

Chronic granulomatous diseases of childhood (CGD) are a group of disorders of phagocytic cell superoxide (O2.-) production (respiratory burst). Anion exchange chromatography separated from normal neutrophil cytosol a 47-kilodalton neutrophil cytosol factor, NCF-1, that restored activity to defective neutrophil cytosol from most patients with autosomally inherited CGD in a cell-free O2.--generating system. A 65-kilodalton factor, NCF-2, restored activity to defective neutrophil cytosol from one patient with autosomal CGD. NCF-1, NCF-2, and a third cytosol fraction, NCF-3, were inactive alone or in pairs, but together replaced unfractionated cytosol in cell-free O2.- generation. Neutrophils deficient in NCF-1, but not NCF-2, did not phosphorylate the 47-kilodalton protein. It is proposed that NCF-1, NCF-2, and NCF-3 are essential for generation of O2.- by phagocytic cells and that genetic abnormalities of these cytosol components can result in the CGD phenotype.

Blotting, Western↗
PubMed1988

Subcellular localization of Gi alpha in human neutrophils.

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.

Adenosine Diphosphate Ribose↗
PubMed1988

Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation.

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.

Arachidonic Acid↗
PubMed1987

Formyl peptide leukocyte chemoattractant uptake and release by cultured human umbilical vein endothelial cells.

Recent observations support an active role for the vascular endothelial cell in the induction and evolution of the inflammatory response. Since prior studies suggested that cultured bovine endothelial cells express high affinity binding sites for the neutrophil chemotactic oligopeptide formyl methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), we sought to further characterize the interaction between formyl peptide chemoattractants and human vascular endothelial cells. Cultured human umbilical vein endothelial cells and peripheral blood neutrophils specifically bound f-Met-Leu-[3H]Phe, whereas specific binding to cultured fibroblasts, smooth muscle, and epithelial cells was negligible. Endothelial cells expressed 3.6 +/- 0.7 X 10(5) binding sites/cell with a Kd of 210 +/- 31 nM. Although the hexapeptide formyl norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (f-Nle-Leu-Phe-Nle-Tyr-Lys) and the tetrapeptide f-Met-Leu-Phe-Lys completed with f-Met-Leu-[3H]Phe for binding to endothelial cells, specific binding of 125I-f-Nl-Leu-Phe-Tyr-Lys or f-Met-Leu-Phe-Lys-fluorescein to endothelial cells was not observed, suggesting that steric constraints on formyl peptide binding differ between endothelial cells and leukocytes. At 37 degrees C, cell-associated f-Met-Leu-[3H]Phe greatly exceeded that bound at 0 degrees C and was incorporated predominantly into a nondisplaceable compartment. Release of f-Met-Leu-[3H]Phe or radioactive breakdown products from this compartment was time- and temperature-dependent with a t1/2 of approximately equal to 20 min at 37 degrees C. Resolution of the radioactive products released from f-Met-Leu-[3H]Phe-loaded endothelial cells by thin layer chromatography indicated that greater than or equal to 57% of the released material co-migrated with intact f-Met-Leu-[3H]Phe. Degradative release was blocked by agents that interfere with lysosomal acidification. The radioactive material released from f-Met-Leu-[3H]Phe-loaded endothelial cells bound specifically to neutrophils. This binding was inhibited 50.2 +/- 6.4% by a greater than or equal to 10(3)-fold excess of nonradioactive f-Met-Leu-Phe whereas binding of authentic f-Met-Leu-[3H]Phe was inhibited 89.4 +/- 3.0%. Supernatant obtained from f-Met-Leu-[3H]Phe-loaded endothelial cells elicited a rise in neutrophil cytosolic free calcium ([Ca2+]i) measured by quin2 fluorescence. The change in neutrophil [Ca2+]i depended on ligand binding to the neutrophil formyl peptide receptor since endothelial supernatants were devoid of activity in the presence of the f-Met-Leu-Phe antagonist, tert-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1987

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae.

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51Cr release from radiolabeled monolayers. From these studies, we conclude that neutrophils are capable of killing Candida hyphae selectively within human vascular endothelial cell monolayers and may have protective rather than detrimental effects on endothelial cell integrity.

Candida albicans↗
PubMed1986

Expression of specific binding sites on Candida with functional and antigenic characteristics of human complement receptors.

Receptors for C3 degradation fragments (CR1, CR2, and CR3) are present on many human cells including phagocytes and lymphoid cells and may be critical in the attachment of invading microorganisms. In these studies Candida were found to mimic the human CR by binding erythrocytes coated with specific human C3 fragments. Yeast forms of Candida species were adhered to glass slides and were allowed to germinate. Sheep erythrocytes (E) were coated with IgM (EA) and human complement components to prepare EA, EAC14, EAC3b, EAC3bi, and EAC3d. These test cells were then examined for adherence to the organism. Antibodies to human CR1, CR2, and CR3 were used to evaluate their potential for blocking adherence of the test erythrocytes to Candida. Fluorescein-labeled antibodies to human complement receptors were also used to characterize the binding sites. EAC3bi and EAC3d, but not E, EA, or EAC14, bound extensively to the germ tubes and pseudohyphae of Candida albicans and C. stellatoidea. EAC3b bound infrequently. Other Candida species, generally considered less pathogenic, bound significantly fewer specific test erythrocytes than C. albicans. Monoclonal antibodies to human CR1 and CR3 (3D9, 1B4, C511, 2B6, anti-B2, Mo1, and anti-Mac-1), in general, did not block adherence of test erythrocytes. Blocking of adherence of EAC3bi and EAC3d test erythrocytes coated with small quantities of C3 fragments occurred with high concentrations of monoclonal (anti-CR2) HB-5 and polyclonal (anti-CR2) anti-GP 140. Immunofluorescence studies demonstrated binding of Mo-1 to the germinated forms of the organism, whereas binding of the other antibodies was not seen. These studies suggest a surface constituent on the organism similar to CR on human cells. Additional studies are necessary to further define the molecular nature of the binding site. The ability of organisms to mimic human CR may be more generalized than previously known and may serve as a mechanism for modification of the inflammatory and immune response.

Antibodies, Monoclonal↗
PubMed1986

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers.

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1-selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F-actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.

Actins↗
PubMed1985

Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration.

To elucidate the pathogenesis of hematogenous Candida infections, we developed an in vitro model of Candida adherence to and penetration of human endothelial cells. We enhanced or inhibited adherence in order to probe mechanisms of attachment. Adherence of Candida albicans showed a linear relation to Candida inoculum (range, 10(2)-10(5) cfu, r = .99, P less than .01) and exceeded that of less virulent Candida species and that of Saccharomyces cerevisiae (P less than .01). Candida immune serum blocked attachment (greater than 95% inhibition; P less than .001), however, this activity was abolished by immunoprecipitation of immune serum with C. albicans mannan (P less than .001) and was unaffected by immunoprecipitation with S. cerevisiae mannan or by adsorption with particulate chitin. Adherence was diminished by exposing C. albicans to heat (greater than 99% inhibition; P less than .01), UV light (98% inhibition; P less than .01), or sodium periodate (greater than 72% inhibition; P less than .01). An extract from heat-exposed C. albicans blocked adherence (greater than 51% inhibition; P less than .001). Transmission electron microscopy demonstrated that viable or killed Candida organisms were attached to endothelial cells, were enveloped by membrane processes from the endothelial cell surface, and were incorporated into the endothelial cells within phagosomes. Cytochalasin B blocked incorporation without blocking surface attachment.

Adhesiveness↗
PubMed1982

Detection of fungemia obscured by concomitant bacteremia: in vitro and in vivo studies.

Our recent clinical experience suggested that bacteremia may interfere with the detection of concomitant fungemia when standard blood culture methods are used. To determine the extent to which bacteria may interfere with fungal isolation from blood cultures, an in vitro model simulating blood cultures taken during concomitant fungemia and bacteremia was created. Each of six bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) was combined with each of three pathogenic yeasts (Candida albicans, Candida tropicalis, and Torulopsis glabrata) in vented blood culture bottles containing enriched brain heart infusion broth and fresh normal human blood. Blood culture bottles were analyzed at 1, 2, and 7 days of incubation. Gram strains and subcultures onto chocolate and MacConkey agars failed to detect fungi in 37.0, 66.7, and 100% of samples, respectively. However, subcultures onto Sabouraud dextrose agar failed in only 13% of the samples (occurring only with P. aeruginosa). In a rabbit model of concomitant fungemia with C. albicans and bacteremia with P. aeruginosa, no yeasts were recovered from blood cultures despite 100% detection of P. aeruginosa. Therefore, the usual microbiological techniques may be inadequate to detect fungemia when concomitant bacteremia is present.

Animals↗
PubMedDate not supplied

Adherence of Candida species to host tissues and plastic surfaces.

Adherence of Candida species to host tissues and nonbiologic materials has been studied in vivo and in vitro. Attachment of Candida albicans to mucosal cells, fibrin-platelet matrices, vascular endothelial cells, and plastic materials has been examined to elucidate early events in the pathogenesis of mucosal colonization and infection, candidal endocarditis, tissue invasion from the intravascular space, and infection of prosthetic devices. Adherence of C. albicans and Candida tropicalis exceeds that of less virulent Candida species, and germinated C. albicans cells adhere to host tissues more readily than do yeast-phase organisms. The adhesin of Candida that mediates attachment has yet to be characterized at the molecular level; however, on the basis of competitive inhibition by crude and purified cell wall products, blocking by antibody and lectin, and controlled degradation of the cell surface of Candida, it appears that mannans and mannoproteins are important constituents of the adhesin. The methods currently used to assay adherence of Candida all have limitations, and an approach to resolving these limitations is one of several areas that warrant further investigation.

Animals↗