Search PubMed⌕ Search

Biomedical subjects

R D Diamond

Publications and source records attributed to R D Diamond.

At least 37 records · Page 2Linked to original sources
PubMed1989

Effect of cilofungin (LY121019), a fungal cell wall synthesis inhibitor, on interactions of Candida albicans with human neutrophils.

The effects of cilofungin, a lipopeptide antifungal agent active against several species of Candida, on human neutrophil candidacidal activity were investigated. While no increase or decrease in neutrophil killing of blastospores was observed when cilofungin was simultaneously incubated with neutrophils and yeasts, preincubation of yeasts with serum containing cilofungin resulted in approximately 90% decrease in killing of five of six strains. Attachment/phagocytosis of these strains was unchanged from controls. We conclude that cilofungin interferes with opsonization of most strains of C. albicans, resulting in a marked decrease in neutrophil candidacidal activity, a phenomenon which may be of significance in vivo in certain tissues where access to antibodies and/or complement is limited.

Antifungal Agents↗
PubMed1989

Natural inhibitor from Candida albicans blocks release of azurophil and specific granule contents by chemotactic peptide-stimulated human neutrophils.

A product released by Candida albicans hyphae which was previously determined to block the neutrophil respiratory burst also inhibited the degranulation response elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). When neutrophils were incubated with 100 micrograms of this Candida hyphal inhibitory product (CHIP) per ml and stimulated with 1,000 nM FMLP, release of the azurophil granule marker beta-glucuronidase and the specific granule marker lactoferrin was decreased to 31.2 +/- 5.7 and 35.7 +/- 5.3% of control, respectively. Inhibition was concentration dependent, with a half-maximal reduction of beta-glucuronidase and lactoferrin release being induced by 4 and 1 micrograms of CHIP per ml, respectively. In contrast to these striking alterations in response to FMLP, CHIP had no significant effect on lactoferrin release stimulated by phorbol myristate acetate. Moreover, actin polymerization, which has been suggested to be involved in regulation of degranulation, was unaffected by CHIP whether the neutrophils were stimulated by FMLP or phorbol myristate acetate. The selectivity of inhibition of neutrophil degranulation thus provides additional confirmation that CHIP is a useful agent for exploring human neutrophil activation pathways.

Actin Cytoskeleton↗
PubMed1989

Independence of neutrophil respiratory burst oxidant generation from the early cytosolic calcium response after stimulation with unopsonized Candida albicans hyphae.

We previously noted differences between neutrophil responses to unopsonized Candida albicans hyphae and responses to other particulate stimuli such as opsonized hyphae or zymosan; these differences include delayed rises in cytosolic calcium [( Ca2+]i), 1,4,5-inositol trisphosphate, and superoxide release and the total absence of early membrane depolarization. Respiratory burst stimulation is required for killing of C. albicans hyphae. Since an early rise in [Ca2+]i may act as a second messenger for burst activation by most agonists, we chelated (Ca2+)i and extracellular Ca2+ [( Ca2+)e] to compare requirements for superoxide responses to hyphae and other stimuli. Intracellular chelation, which ablated early [Ca2+]i rises, eliminated the fMet-Leu-Phe-induced respiratory burst and profoundly reduced that response to opsonized zymosan (by 96.7%), but chelation of both (Ca2+)i and (Ca2+)e only partially inhibited responses to opsonized and unopsonized hyphae (60.5 and 23.3%, respectively; the latter exceeded absolute responses evoked by opsonized zymosan, a 12-fold-more-potent stimulus for unchelated cells). Simultaneous (Ca2+)i and (Ca2+)e chelation further decreased superoxide responses to opsonized zymosan and hyphae (99.4 and 90.4%, respectively) but not to unopsonized hyphae (26.7% inhibition). Though both ingestible (zymosan) and uningestible (hyphae) opsonized particulate stimuli elicited reduced but significant respiratory bursts without early [Ca2+]i rises, the greater superoxide responses and sensitivity to chelation with opsonized zymosan suggest important differences in initiation and/or regulation of responses to these particulate stimuli. In contrast, the respiratory burst elicited by unopsonized hyphae appeared largely Ca2+ independent. If different events mediate neutrophil activation by opsonized and unopsonized hyphae, candidacidal activity in vivo may vary under divergent conditions with specific localized sites of infection.

Calcium↗
PubMed1988

Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans.

Histatins 1, 3, and 5 from human parotid secretion were isolated by gel filtration on Bio-Gel P-2 and reverse phase high performance liquid chromatography. The complete amino acid sequences of histatins determined by automated Edman degradation of the proteins, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows: (Sequence: see text). Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues, have molecular weights of 4929, 4063, and 3037, respectively, and contain 7 residues of histidine. Histatin 1 contains 1 mol of phosphate/mol of protein; histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of all three histatins are identical, and the carboxyl-terminal 7 residues of histatins 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly- (residues 23-29), in histatin 1 is absent in histatin 3; and the sequence, -Gly-Tyr-Arg- (residues 23-25), in histatin 3 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino terminal 24 residues of histatin 3. The structural data suggest that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. All three histatins exhibit the ability to kill the pathogenic yeast, Candida albicans.

Amino Acid Sequence↗
PubMed1988

A novel natural inhibitor from Candida albicans hyphae causing dissociation of the neutrophil respiratory burst response to chemotactic peptides from other post-activation events.

Previous work established that Candida albicans hyphae release several inhibitors of human neutrophil function. We now report that the crude hyphal inhibitory product (CHIP) inhibits superoxide anion (O2-) production stimulated by FMLP in a dose-related manner with an EC50 of approximately 2 micrograms/ml. CHIP also inhibited O2- production stimulated by A23187 and by opsonized zymosan, although this effect could be overcome by increasing the concentration of agonist. No inhibition of the PMA-stimulated burst was seen at any concentration of PMA tested, indicating that CHIP neither affected polymorphonuclear neutrophil viability nor quenched superoxide anion detection. A saturating dose of inhibitor had no effect on chemotaxis stimulated either by 0.1 to 100 nM FMLP or by zymosan-activated serum. Peak inositol trisphosphate levels stimulated by FMLP were not inhibited by a dose of CHIP producing maximum inhibition of FMLP-induced superoxide production. Peak changes in cytosolic free calcium levels (as measured by indo-1 fluorescence) stimulated by 50 nM or greater FMLP were unaffected by CHIP, although for subsaturating doses of FMLP a more rapid decline from peak calcium levels was seen in CHIP-exposed cells. Taken together, these data suggest that the common fungal pathogen C. albicans releases a substance that selectively impairs the neutrophil respiratory burst. It appears to do so without inhibiting the fully assembled NADPH oxidase and with minimal or no effect on events tightly coupled to FMLP-R/G protein activation, suggesting that these events may be uncoupled from activation of the burst. In addition, the absence of effect of CHIP on chemotaxis despite profound inhibition of the respiratory burst suggests these neutrophil functions may be mediated by divergent transduction pathways.

Calcium↗
PubMed1988

Temporal association of calcium mobilization, inositol trisphosphate generation, and superoxide anion release by human neutrophils activated by serum opsonized and nonopsonized particulate stimuli.

We have investigated the involvement of phospholipase C mediated polyphosphoinositide turnover in activation of polymorphonuclear leukocytes by particulate stimuli. Results showed that stimulation of leukocytes with serum opsonized zymosan (ingestible particle), serum opsonized Candida albicans hyphae (noningestible particle), or nonopsonized hyphae was followed by a transient rise in cellular inositol phosphates as has been described for neutrophil activation via the formyl peptide receptor. The kinetics of inositol trisphosphate generation paralleled both the time course of changes in cytosolic calcium concentration and the onset of superoxide anion generation, suggesting that these may be related events.

Calcium↗
PubMed1988

Induction of signal transduction in human neutrophils by Candida albicans hyphae: the role of pertussis toxin-sensitive guanosine triphosphate-binding proteins.

Nonopsonized Candida hyphae elicit from human neutrophils a transient rise in cytosolic calcium concentrations and an oxidative burst without a detectable change in membrane potential. To determine if the signal-transduction pathway used by these organisms is mediated by guanine nucleotide-binding proteins (GNPs), we examined the functional responsiveness of neutrophils pretreated with pertussis toxin (PT). In response to serum-opsonized hyphae or zymosan, the rise in cytosolic calcium, membrane depolarization, and the respiratory burst were only partially abrogated. The transient rise in calcium induced by unopsonized hyphae was, however, completely eliminated in PT-treated neutrophils. Despite total abrogation of the calcium response, PT-treated cells could still mount a respiratory burst in response to these nonopsonized hyphae. Thus, neutrophil signaling by both serum-opsonized particles and nonopsonized hyphae is only partially mediated by PT-sensitive GNPs. Furthermore, the ability of unopsonized hyphae to elicit a respiratory burst without a calcium response suggests these events are separable and confirms the versatility of these organisms as probes for investigating neutrophil activation.

Calcium↗
PubMed1988

Stimulation of neutrophil actin polymerization and degranulation by opsonized and unopsonized Candida albicans hyphae and zymosan.

We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗
PubMed1988

Synchronous bacterial and fungal septicemia. A marker for the critically ill surgical patient.

Mixed septicemia (synchronous fungal and bacterial septicemia) is an occasional, but often fatal occurrence in the critically ill patient. We reviewed 14 such cases at two hospitals. Twelve of 14 patients were in the surgical intensive care unit. Eleven patients had an average of 2.7 major surgical procedures (range 2 to 4); persistent post-operative peritoneal sepsis was common occurring in 9 patients. Bacteremia preceded mixed septicemia in 8 of 14 cases and gram negative enteric bacilli were the most common causes of bacteremia. Fungemia was due to Candida species in 13 of 14 patients and followed prolonged antibiotic therapy. The diagnosis of disseminated candidiasis was suspected during life in 13 patients and proven in six. Mixed septicemia is a marker for a distinct population of critically ill surgical patients with a high overall mortality (78% in this study). Culture of both a fungal and bacterial pathogen in a blood culture, especially if preceded by bacteremia, should alert the physician to strongly suspect disseminated fungal infection and to commence appropriate treatment. Mortality is likely to remain high unless the underlying disease states can be rapidly corrected and infection controlled.

Adult↗
PubMed1987

A rapid fluorescent assay to distinguish attached from phagocytized yeast particles.

In studies of phagocytosis and its consequences for cell activation, it is important to distinguish those particulate stimuli which are completely ingested and internalized from those which are only attached to phagocytic surfaces. Ingestion can be profoundly influenced by both the type of opsonins on the surface of the stimulus and the expression and activation of receptors on the phagocytes for these opsonins. We report a new fluorescent assay which facilitates rapid and reproducible quantitation of attached versus fully internalized live or dead yeast particles by phagocytes. The assay employs the fluorescent dye diaethanol (Uvitex 2B) which stains chitin on the cell wall of fungi and is excluded from live phagocytes. The diaethanol assay and a standard, previously published methylene blue dye exclusion assay yielded comparable results using human neutrophils or monocytes incubated with heat-killed, serum-opsonized Candida albicans. The diaethanol assay proved useful in distinguishing differences in effects of various opsonins, as human neutrophils selectively opsonized with either pooled human serum (PHS), IgG (heat-inactivated PHS) or complement (IgG-depleted PHS) completely internalized 69.5%, 91.3% and 52.5% of cell-associated zymosan particles respectively. Finally, the new assay permitted comparisons of differing macrophage populations, as resident murine peritoneal macrophages internalized only 10.6% of complement-opsonized, cell-associated zymosan particles compared with 41.7% when the macrophages were elicited with thioglycolate. The assay should prove useful to investigators studying both fungal phagocytosis and killing, as well as to those performing general studies of receptor expression, regulation and function.

Candida albicans↗
PubMed1987

Unopsonized Candida albicans hyphae stimulate a neutrophil respiratory burst and a cytosolic calcium flux without membrane depolarization.

We have defined major differences between events elicited during activation of human neutrophils by opsonized Candida hyphae (noningestible) or zymosan (ingestible) and by unopsonized Candida hyphae (also noningestible). In response to opsonized particles, a transient increase in cytosolic free calcium was initially observed, followed by depolarization in the transmembrane potential and a respiratory burst. Without opsonins, zymosan did not elicit a response from neutrophils. In contrast, unopsonized Candida hyphae elicited a transient increase in cytosolic calcium, as well as triggered a respiratory burst; they did not, however, elicit a depolarization of the neutrophil membrane. Furthermore, the rate of superoxide generation decreased after a prolonged lag period. The calcium flux was also delayed and coincided with the onset of superoxide generation. Thus, our data demonstrate that Candida albicans hyphae activate neutrophils in the absence of added serum components and initiate a neutrophil respiratory burst without an antecedent membrane depolarization.

Calcium↗
PubMed1987

Cytosolic calcium changes in individual neutrophils stimulated by opsonized and unopsonized Candida albicans hyphae.

Previous experiments suggest the critical central role of the neutrophil (PMN) respiratory burst in the prevention and containment of disseminated candidiasis. A rise in cytosolic free calcium concentrations ([Ca2+]i) has been documented as an early event after PMN stimulation which is involved in the subsequent genesis of microbicidal and inflammatory respiratory burst products. [Ca2+]i were therefore determined in individual PMN, loaded with the fluorescent calcium probe fura-2 as they attached to and spread over serum-opsonized or unopsonized Candida albicans hyphae, particles that are too big to be completely ingested. After contact between hyphae and PMN, the PMN rapidly spread over hyphal surfaces. Although both opsonized and unopsonized hyphae stimulated similar magnitudes of peak median increases in PMN [Ca2+]i, the kinetics of responses differed; median [Ca2+]i peaked within 1 min after contact with opsonized hyphae versus 4 min after contact with unopsonized hyphae. Moreover, a detectable calcium transient did not invariably follow contact and spreading of each individual PMN over a hyphal surface. In contrast to patterns seen after stimulation of PMN with opsonized zymosan, in which [Ca2+]i is greatest in the periphagosomal region, there was a more uniform distribution throughout the cytoplasm in PMN stimulated with the noningestable hyphae. These alterations in the early patterns and timing of PMN stimulation may reflect analogous differences in subsequent events which control the efficiency and specificity of microbicidal responses to uningestible hyphae and which also determine whether host tissues are damaged by the generation of toxic PMN activation products.

Benzofurans↗
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

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor.

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.

Actins↗
PubMed1986

The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion.

The neutral histidine-rich polypeptide (HRP) from human parotid secretion was isolated by ion-exchange and gel-filtration chromatography. The complete amino acid sequence determined by automated Edman degradation of the protein, tryptic and Staphylococcus aureus V8 protease peptides, and digestion with carboxypeptidase A is: (Formula: see text) where Pse represents phosphoserine. The polypeptide contains 38 residues and has Mr 4929. The charged amino acids predominate with 7 histidine, 4 arginine, 3 lysine, 3 aspartic acid, 3 glutamic acid residues, and 1 phosphoserine. Assuming minimal charge contributions from histidine and one negative charge from phosphoserine at pH 7, the net charge of HRP is balanced by an equal contribution of basic and acidic residues. Furthermore, the distribution of hydrophilic and hydrophobic residues along the polypeptide chain indicates that there is no structural polarity. The polypeptide lacks threonine, alanine, valine, cysteine, methionine, and isoleucine. HRP did not display sequence similarity with any protein sequence in the National Biomedical Research Foundation Data Bank. HRP is an active inhibitor of hydroxyapatite crystal growth from solutions supersaturated with respect to calcium phosphate salts and therefore must play a role in the stabilization of mineral-solute interactions in oral fluid. In addition, HRP is a potent inhibitor of Candida albicans germination and therefore may be a significant component of the antimicrobial host defense system in the oral cavity.

Amino Acid Sequence↗
PubMed1986

In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages.

The fungicidal activity of six rabbit neutrophil cationic peptides (NP) against resting (dormant) spores, preincubated (swollen) spores, and hyphae of Aspergillus fumigatus and Rhizopus oryzae was examined. Whereas resting spores of both fungi were totally resistant to killing by up to 500 micrograms of NP/ml, preincubated spores and hyphae were sensitive, under optimal conditions, to less than 1 microgram /ml. NP-1 and NP-2, also found in rabbit bronchoalveolar macrophages, were the most active of the peptides. Activity was inhibited with increasing ionic strength, calcium but not magnesium, incubation at 4 C, and the chitin fragments chitobiose and chitotriose. 125I-labeled NP-1 bound to all three forms of A. fumigatus. Finally, killing of A. fumigatus conidia by rabbit bronchoalveolar macrophages was faster and greater if the conidia were first preincubated in RPMI 1640 medium. Thus, although NP did not kill the resting A. fumigatus conidia, a role for cationic peptides in macrophage-mediated killing is not precluded, as fungicidal activity of both isolated cationic peptides and intact bronchoalveolar macrophages appeared to occur only after the conidia became activated.

Animals↗
PubMed1986

Comparative activities of UK-49,858 and amphotericin B against Blastomyces dermatitidis infections in mice.

UK-49,858, a new antifungal triazole derivative, was compared with amphotericin B in the treatment of pulmonary infections by Blastomyces dermatitidis in male BALB/cByJ mice. Administration of UK-49,858 in daily doses of 25 or 50 mg/kg for 21 days gave 30 and 100% survival rates, respectively. These results compared with 100% mortality in infected controls and 100% survival among mice treated with amphotericin B. UK-49,858 did not eradicate the fungus from the lungs of surviving animals, while amphotericin B effected sterilization of the lungs in 66% of the survivors.

Amphotericin B↗