Killing Of Candida Albicans Research Articles

Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366, L-693,989, and L-733,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 micrograms/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology of Aspergillus fumigatus and A, flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis. Pneumocandin B0 caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.

Impairment of pulmonary macrophage function with total parenteral nutrition.

The effects of total parenteral nutrition (TPN) administration on pulmonary macrophage function and host response to gram-negative pulmonary infection were evaluated. Administration of TPN resulted in increased infectious complications in traumatized and perioperative patients, but underlying mechanisms are unclear. Twenty-six male Wistar rats underwent central vein cannulation and were randomized to isocaloric feeding of a regular chow diet (RD) plus saline infusion or TPN without chow diet for 7 days. Pulmonary alveolar macrophage (PAM phi) superoxide production, Candida albicans phagocytosis and killing, and tumor necrosis factor (TNF) production in response to endotoxin (LPS) were assessed. Mesenteric lymph nodes (MLN) were cultured. A second group of rats (n = 6/group) were inoculated intratracheally with a sublethal dose of 9 x 10(9) live Escherichia coli per animal, and the lungs were cultured quantitatively 72 hours later to assess bacterial clearance. Finally, 11 RD-fed rats and 13 TPN-fed rats received intratracheal inoculation of 1.4 x 10(10) live E. coli and were included in follow-up. Administration of TPN was associated with a significant increase in bacteria positive MLN compared with those in the RD group (p < 0.01). Pulmonary alveolar macrophage superoxide production, Candida albicans phagocytosis and killing, TNF production, and pulmonary clearance of bacteria were decreased significantly in TPN-fed rats compared with those fed a regular chow diet (p < 0.05). These pulmonary macrophage function changes were associated with a significantly higher mortality in TPN-fed rats compared with RD-fed rats after higher dose pulmonary E. coli inoculation. Defective host pulmonary antimicrobial immune responses during TPN are associated with intestinal bacterial translocation, and may explain increased infectious complications.

Engulfment and killing capabilities of neutrophils and phagocytic splenic function in persons occupationally exposed to lead

Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils as well as phagocytic splenic function from lead-exposed workers were studied. Two species of Candida were used since in individuals with myeloperoxidase deficiency neutrophils are unable to kill C. albicans, whereas C. pseudotropicalis can be effectively lysed. Phagocytosis with both antigens and phagocytic splenic function were normal in all the workers studied. However, lytic activity towards C. albicans, but not C. pseudotropicalis was impaired. This defect was observed in lead-exposed workers with blood lead levels and urinary delta-aminolevuliniv acid (ALA-U) concentrations in the “safe” (below 60 ug/dl and 6 mg/l, respectively) and toxic ranges. An impaired ability to kill C. albicans suggests that lead exposure may lead to a myeloperoxidase deficiency. With the exception of blood lead levels and ALA-U concentrations, there was no correlation between any of the other parameters examined.

Novel aspect of amphotericin B action: accumulation in human monocytes potentiates killing of phagocytosed Candida albicans.

The influence of low doses of amphotericin B on the capacity of human monocytes to kill Candida albicans was investigated. Killing rates were quantified by a novel flow cytometric assay and were found to be 37% +/- 3% (standard error of the mean) after 3 h. Preincubation of monocytes for 6 to 20 h with low concentrations of amphotericin B (0.2 microgram/ml) resulted in a markedly augmented fungicidal capacity. Enhancement of killing was 80% +/- 11% (standard error of the mean) over that by the controls. This effect did not appear to be due to amphotericin B-dependent monocyte activation; the respiratory burst and expression of human leukocyte antigen-DR were unaltered, and no stimulation of interleukin-1 beta release occurred. Cell-associated amphotericin B was extracted with acetonitrile and was quantified by scanning spectrophotometry. Amphotericin B appeared to accumulate in the cells, and intracellular concentrations attained after overnight incubation in 1 microgram of the drug per ml were estimated to be in the range of 50 fg per cell. The fact that intracellular accumulation was responsible for the enhanced fungicidal capacity of monocytes was supported by the findings that killing of Staphylococcus aureus remained normal and enhancement of killing of an amphotericin B-resistant C. albicans strain was minimal. Dramatic enhancement of monocyte fungicidal capacity probably extends to other amphotericin B-susceptible fungi and could represent a hitherto unrecognized determinant underlying the curative properties and prophylactic efficacy of this drug.

Role of interleukin-8 receptor in skin.

Interleukin-8 (IL-8) is a potent chemotactic and proinflammatory cytokine, produced in the skin by a variety of cells in response to inflammatory stimuli. Recent studies suggest that in addition to its potent actions on leukocytes, IL-8 exerts a direct influence on several functions of human epidermal cells such as chemotaxis, Candida albicans killing activity or proliferation. The effects of IL-8 are mediated by binding to two types of specific high-affinity receptors which contain seven transmembrane domains typical of guanine nucleotide binding protein (G protein)-coupled receptors. In the skin, a broad spectrum of cells such as neutrophils, T lymphocytes, mast cells, dermal macrophages, endothelial cells and keratinocytes possess binding sites for IL-8. Recently, increased expression of epidermal IL-8 receptors has been observed in psoriasis an inflammatory and hyperproliferative skin disease. Since the effects of IL-8 may be modulated at the receptor level, the pharmacological manipulation of the IL-8 receptor may prove an important target for the therapy of skin diseases with increased IL-8 levels.

Recombinant Interferon- Enhances Resistance to Acute Disseminated Candida albicans Infection in Mice

The effect of recombinant rat interferon-gamma (rIFN-gamma) on acute disseminated Candida albicans infection in mice was investigated. Outgrowth of C. albicans in kidneys, spleen, and liver of mice treated with one intravenous (iv) dose of rIFN-gamma before iv injection of 5 x 10(5) cfu of C. albicans was significantly lower than in controls over 7 days. rIFN-gamma was protective when given 1 day before, simultaneously with, or 1-3 days after infection but not when given 3 days before. In mice pretreated with hydrocortisone acetate, rIFN-gamma significantly reduced the outgrowth only when 10(3) cfu of C. albicans was injected. Injection of rIFN-gamma did not reduce the outgrowth of C. albicans in cyclophosphamide-pretreated mice and significantly increased the capacity of peripheral blood and exudate peritoneal granulocytes to kill C. albicans in vitro. Thus, rIFN-gamma enhances host resistance against acute disseminated C. albicans infection in mice through activation of polymorphonuclear leukocytes.

Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin.

Candida albicans was found to be highly susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by enzymatic cleavage of bovine lactoferrin. Effective concentrations of the peptide varied within the range of 18 to 150 micrograms/ml depending on the strain and the culture medium used. Its effect was lethal, causing a rapid loss of colony-forming capability. 14C-labeled lactoferricin B bound to C. albicans and the rate of binding appeared to be consistent with the rate of killing induced by the peptide. The extent of binding was diminished in the presence of Mg2+ or Ca2+ ions which acted to reduce its anticandidal effectiveness. Binding occurred optimally at pH 6.0 and killing was maximal near the same pH. Such evidence suggests the lethal effect of lactoferricin B results from its direct interaction with the cell surface. Cells exposed to lactoferricin B exhibited profound ultrastructural damage which appeared to reflect its induction of an autolytic response. These findings suggest that active peptides of lactoferrin could potentially contribute to the host defense against C. albicans.

Isolation and partial characterization of glycolipid fractions from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18) that inhibit activated macrophages

Glycolipid fractions from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18) inhibited the killing of Candida albicans by activated bovine peripheral-blood-derived macrophages. Fractions were derived by using the matrix solid-phase dispersion technique, which is a new method of simultaneous lysis and partial fractionation of components of bacterial cells. Further purification of active fractions was performed by concanavalin A affinity chromatography, centrifugal filtration, and differing solvent solubility. Three different fractions were isolated and partially characterized. Two of these fractions have characteristics typical of glycolipids, and the third fraction has characteristics compatible with a peptidoglycolipid. This peptidoglycolipid fraction has been purified and named MIF-A3.

The development of a radiometric assay for monocyte phagocytosis and killing

Monocyte and macrophage phagocytosis and killing has an important role in host defence against infectious microorganisms. Established methods for investigating these activities in vitro are time-consuming, expensive and prone to subjective error. An in vitro assay has been developed which measures monocyte phagocytosis and killing of Candida albicans. In this system, monocyte function is assessed by a radiometric assay based on the incorporation of [3H]uridine into viable yeast cells. This technique allows the simultaneous but independent measurement of the average number of yeast cells ingested by monocytes and the percentage of the ingested organisms killed within a defined incubation period. These tests can be carried out on a single sample of peripheral blood, are relatively easy to perform, and provide objective results within 24 h of receipt of the specimen. This method gave consistent results when monocyte-enriched harvests were obtained by four different separation techniques. This assay could prove a valuable tool for the investigation of monocyte function in individuals with recurrent infections to determine the contribution this makes to their clinical condition. It may also be used to investigate in vitro the influence of cytokines and other chemical agents on monocyte function.

The development of a radiometric assay for monocyte phagocytosis and killing

Monocyte and macrophage phagocytosis and killing has an important role in host defence against infectious microorganisms. Established methods for investigating these activities in vitro are time-consuming, expensive and prone to subjective error. An in vitro assay has been developed which measures monocyte phagocytosis and killing of Candida albicans. In this system, monocyte function is assessed by a radiometric assay based on the incorporation of [3H]uridine into viable yeast cells. This technique allows the simultaneous but independent measurement of the average number of yeast cells ingested by monocytes and the percentage of the ingested organisms killed within a defined incubation period. These tests can be carried out on a single sample of peripheral blood, are relatively easy to perform, and provide objective results within 24 h of receipt of the specimen. This method gave consistent results when monocyte-enriched harvests were obtained by four different separation techniques. This assay could prove a valuable tool for the investigation of monocyte function in individuals with recurrent infections to determine the contribution this makes to their clinical condition. It may also be used to investigate in vitro the influence of cytokines and other chemical agents on monocyte function.

Effects of miocamycin and erythromycin on polymorphonuclear cell function.

Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Candida albicans and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significantly enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration.

Effect of lectins on hepatic clearance and killing of Candida albicans by the isolated perfused mouse liver.

The isolated perfused mouse liver model was used to study the effects of various lectins on hepatic trapping and killing of Candida albicans. After mouse livers were washed with 20 to 30 ml of perfusion buffer, 10(6) C. albicans CFU were infused into the livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicated that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. When included in both preperfusion and postperfusion buffers (0.2 mg of lectin per ml), Ulex europeaus lectin (binding specificity for fucose) decreased hepatic trapping of C. albicans by 37% and eluted trapped C. albicans from the liver only when included in postperfusion buffer. By comparison, treatment of C. albicans with U. europeaus lectin before infusion had no effect on the trapping or killing of yeast cells. When Lens culinaris lectin (binding specificity for mannose) was included in the perfusion buffers, hepatic killing of C. albicans increased by 16% with no significant effect on hepatic killing when yeast cells were treated with L. culinaris lectin before infusion. Forty to 55% of the infused C. albicans were killed when concanavalin A (binding specificities for mannose and glucose), Glycine max (binding specificity for N-acetylgalactosamine), or Arachis hypogea (binding specificity for galactose) lectin was included in the perfusion buffer or when yeast cells were treated with these lectins before their infusion. When C. albicans was treated with concanavalin A at a concentration of less than 0.02 mg/ml, hepatic killing of yeast cells was not significantly increased. The data suggest that a fucose-containing receptor on the surface of either sinusoidal endothelial cells or Kupffer cells is involved in the trapping of C. albicans by the perfused mouse liver. Moreover, lectins with binding specificities for mannose, N-acetylgalactosamine, and galactose increased hepatic killing of C. albicans.

Fungicidal effect of human lactoferrin against Candida albicans

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37°C. Fe 3+ -saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37°, 42°C). The presence of 1 mM d-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the antifungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.

Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model

The isolated perfused mouse liver model was used to study the effect of Arg-Gly-Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD-treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells.

Effect of cilofungin on phagocytosis and intracellular killing of Candida albicans by human neutrophils.

The effect of a beta glucan synthase inhibitor, cilofungin, amphotericin B and 5-fluorocytosine on opsonization, phagocytosis and intracellular killing of Candida albicans blastospores by glass-adherent human neutrophils was determined by microscopy and dye exclusion staining. Pretreatment of blastospores for 2, 4, 6 and 18 h with 1.25 mg/l cilofungin resulted in 24-28% of neutrophils ingesting compared to 24% with no drug present. There was no significant difference in the phagocytic index with increased incubation time. Percentage ingestion and phagocytic index values were not significantly different when blastospores were pretreated for 2 h in the presence of 3, 7, 15, 31 and 62 mg/l cilofungin. Comparable concentrations of amphotericin B and 5-fluorocytosine gave similar results. In the absence of cilofungin, intracellular killing after 2 h was 54%. In the presence of 1 and 10 mg/l cilofungin, 85-90% of intracellular blastospores were killed. Therapeutic levels of amphotericin B or 5-fluorocytosine did not enhance intracellular killing. These data demonstrate the potentiation of intracellular killing of Candida albicans by neutrophils in the presence of cilofungin.

Histochemical and Immunochemical Study of the Fate of Candida albicans Inside Human Neutrophil Phagolysosomes

To further define the ultrastructural events associated with the killing of Candida albicans by human neutrophils, four methods were used: (1) the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of vicinal-glycol-containing complex carbohydrates; (2) the localization of thermostable immunodeterminants of the yeast cell wall, mannans or mannoproteins, using monospecific antibodies and a protein A-gold complex (monAb-gold); (3) the localization of mannose residues with concanavalin A labeled with gold particles (Con A-gold); (4) the localization of chitin oligomers using wheat germ agglutinin and ovomucoid labeled with gold particles (WGA-gold). The mannan-rich cell wall layers were progressively lost as shown by altered PA-TCH-SP reactivity and a diffuse pattern of staining with Con A-gold and monAb-gold. The de novo appearance of conspicuous amounts of glycogen-like particles near the plasmalemma and in the cell wall was interpreted as evidence of a reparative process of the yeast cell wall. Chitin was seemingly unaltered and readily demonstrated by the WGA-gold in the wall remnants of ghost cells.

In vitro study of contact-mediated killing of Candida albicans hyphae by activated murine peritoneal macrophages in a serum-free medium

Activated peritoneal macrophages obtained from Listeria-immune mice were demonstrated to kill nonphagocytosable Candida albicans hyphae by contact-mediated mechanisms in a serum-free synthetic medium. The actual killing of hyphae was confirmed by a microculture technique utilizing the dimorphic nature of the fungus. The most efficient candidacidal activity was demonstrated by the macrophages obtained from mice first immunized with live Listeria monocytogenes and then elicited with heat-killed L. monocytogenes cells. Resident macrophages from control mice showed only low candidacidal activity against C. albicans hyphae and yeast cells. Direct physical contact appeared to be required for macrophages to efficiently kill oversized C. albicans hyphae. Efficient in vitro killing of hyphae also required relatively high effector/target cell ratios (50 or higher). The contact-mediated candidacidal activity of activated macrophages was not significantly abrogated by oxygen-radical scavengers, suggesting the involvement of oxygen-independent mechanisms. These results suggest that the enhanced nonspecific immunity to candidiasis seen in Listeria-immune hosts can be attributed, at least in part, to activated fungicidal macrophages. The ability of macrophages to detect and destroy both yeast and hyphal C. albicans cells is clearly an important element of the host defense against candidiasis.

Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry

We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii) relative percentage of yeast cells that become associated with phagocytes, and (iii) percentage of killing of C. albicans. The first two parameters are obtained through the additional use of a phycoerythrin-conjugated monoclonal antibody that selectively labels monocytes and polymorphonuclear granulocytes in peripheral blood. Killing is assessed by solubilizing blood cells with deoxycholate to liberate yeast cells from the phagocytes. Viable yeast cells retain carboxyfluorescein, but nonviable cells lose the fluorescent marker; thus, the reduction in number of fluorescent particles directly reflects phagocytic killing. Results obtained by the present method correlated excellently with parallel enumerations by colony counting. Test results with seven healthy individuals revealed a marked dissociation between the process of opsonophagocytosis, which was essentially complete after 20 min at 37 degrees C, and killing rates, which were 48% +/- 11% and 63% +/- 9% (standard deviation) after 1 and 2 h, respectively, when yeast cell-to-phagocyte ratios were in the range of 0.5:1 to 2:1. The described assay is unrivaled in simplicity, rapidity, and reproducibility and generates results for a large number of samples within hours.

Intracellular killing of Candida albicans by human neutrophils is potentiated by exposure to combinations of amphotericin B and 5‐fluorocytosine

Combination treatment with amphotericin B and 5-fluorocytosine is synergistic and has become clinically useful in the treatment of various forms of systemic candidosis. The synergy between these two compounds may be explained in part by their combined effect on the interaction between fungal cells and host phagocytes. Pretreatment of Candida albicans for 2 h with either amphotericin B or 5-fluorocytosine or the two agents in combination did not inhibit or enhance phagocytosis by glass-adherent human neutrophils (P greater than 0.05). Intracellular killing of pretreated yeast cells was not influenced by antifungals alone (P greater than 0.05), but pretreatment of C. albicans with 5.0 mg l-1 amphotericin B + 10 l-1 5-fluorocytosine or 5.0 mg l-1 amphotericin B + 50 mg l-1 5-fluorocytosine significantly enhanced the ability of neutrophils to kill the number of viable yeast cells intracellularly (P less than 0.001).

Mechanisms of host defense against Candida species. II. Biochemical basis for the killing of Candida by mononuclear phagocytes.

We studied the biochemical basis of candidacidal activity by comparing the killing of Candida albicans, a serious pathogen, and Candida parapsilosis, a low-grade pathogen, by human monocytes (Mo) and monocyte-derived macrophages. Mo killed C. parapsilosis significantly better than C. albicans. The two species triggered the respiratory burst and release of myeloperoxidase (MPO) and beta-glucuronidase in Mo to an equivalent extent. In contrast to Mo, macrophages killed both species to an equivalent extent. Mo exhibited a greater candida-stimulated respiratory burst than did monocyte-derived macrophages, and the respiratory burst was required for the killing of both species. C. parapsilosis was killed much more easily than C. albicans by exposure to low concentrations of hypochlorite or monochloramine, MPO-dependent oxidants released by Mo but not macrophages, which lack MPO. With six different Candida strains there was a significant correlation between killing by Mo and susceptibility to hypochlorite (r = 0.926) or monochloramine (r = 0.981) (p less than 0.01 for each). Species differences in resistance to killing by Mo may be related to differences in sensitivity to MPO-derived oxidants, and the ability of C. albicans to resist the effects of these oxidants may be a virulence factor associated with this species.