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R D Diamond

Publications and source records attributed to R D Diamond.

At least 55 records · Page 3Linked to original sources
PubMed1985

Mechanisms of resistance of Aspergillus fumigatus Conidia to killing by neutrophils in vitro.

Despite the critical role for neutrophils in host defenses against invasive aspergillosis, previous studies have established that neutrophils are unable to kill resting conidia of Aspergillus fumigatus. The mechanisms of resistance of the conidia were therefore investigated. Electron microscopy studies showed the fusion of phagosomes containing A. fumigatus conidia with lysosomes of the neutrophil. Resting conidia of A. fumigatus were then compared with those that had been preincubated in broth until swollen, but not germinated, as well as with blastospores of Candida albicans (two fungal forms that are killed by neutrophils) and zymosan particles. Despite comparable susceptibility to phagocytosis, resting conidia of A. fumigatus stimulated production of significantly less superoxide anion, hydrogen peroxide, and hypochlorous acid and induced less myeloperoxidase-dependent iodination by neutrophils than did the preincubated conidia of A. fumigatus, blastospores of C. albicans, or zymosan particles. In addition, resting conidia of A. fumigatus were relatively resistant to cell-free killing by oxidants presumed to be generated by neutrophils. Thus, resistance of resting conidia of A. fumigatus to neutrophil fungicidal mechanisms appears to be secondary to both failure of the conidia to stimulate an optimal respiratory burst as well as resistance of the conidia to neutrophil oxidants. However, the reversal of this resistance by preincubation of the conidia suggests that neutrophils still may form an important host defense against the conidia of A. fumigatus.

Aspergillus fumigatus↗
PubMed1985

A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT.

A rapid colorimetric assay for viability of fungi that uses the tetrazolium salt MTT is described. All live species of a wide range of medically important fungi that were tested reduced MTT, with resultant intracellular formation within several hours of clearly visible purple crystals of MTT-formazan. The assay proved particularly useful in measuring viability of individual hyphae; greater than 99% of live Aspergillus fumigatus and Rhizopus oryzae hyphae stained purple when incubated with MTT compared with 0% of hyphae killed by any of a variety of methods. The MTT method for measuring fungal viability closely correlated with a slide-culture technique when killing of A. fumigatus hyphae by graded concentrations of hydrogen peroxide was compared. MTT-formazan was easily extracted from the fungi by alcohol, a procedure allowing spectrophotometric quantification of fungal killing in both cell-free and cellular systems. A linear relation between inocula and MTT reduction of several fungi, including Candida albicans blastospores and A. fumigatus hyphae, was demonstrated. Human leukocytes killed 80.3% of C. albicans blastospores as measured by the MTT assay, compared with 70.5% as measured by dilutions and pour plates. Killing of A. fumigatus hyphae by human leukocytes was also quantitated by using the MTT assay.

Fungi↗
PubMed1985

Neutrophil chemotactic responses induced by fresh and swollen Rhizopus oryzae spores and Aspergillus fumigatus conidia.

With the induction of germination, Rhizopus oryzae spores and Aspergillus fumigatus conidia activate the complement system and induce neutrophil chemotaxis. In contrast, freshly isolated R. oryzae spores did not induce neutrophil migration into lung tissue of mice after intranasal inoculation. Moreover, in microchemotaxis assays neither fresh R. oryzae spores nor A. fumigatus conidia activated sera to stimulate human neutrophil chemotaxis above control migration until at least 10(7) or 10(8) spores or conidia per ml of sera were used. The increased generation of chemotactic factors by swollen spores and conidia was not due to an increased surface area, as there was decreased neutrophil chemotactic response to Rhizopus or Aspergillus hyphae when compared with swollen spores or conidia.

Animals↗
PubMed1984

In vivo bronchoalveolar macrophage defense against Rhizopus oryzae and Aspergillus fumigatus.

The ability of bronchoalveolar macrophages from normal, diabetic, and cortisone-treated mice to inhibit spore germination and kill fungal spores in vivo was investigated. The data indicated that the normal host controls different fungal infections in the lungs by different mechanisms. Prevention of mucormycosis required inhibition of fungal spore germination by alveolar macrophages. In contrast, pulmonary defense against aspergillosis depended on early killing of conidia by alveolar macrophages and not on inhibition of germination by bronchoalveolar macrophages. Bronchoalveolar macrophages in diabetic and cortisone-treated animals allowed fungal spore germination, thereby permitting infection by Rhizopus oryzae. In the cortisone-treated mouse, bronchoalveolar macrophages did not kill fungal conidia and progressive infection by Aspergillus fumigatus occurred. Fungicidal activity of bronchoalveolar macrophages was measured with a new in vivo killing assay.

Animals↗
PubMed1984

Killing of Aspergillus fumigatus spores and Candida albicans yeast phase by the iron-hydrogen peroxide-iodide cytotoxic system: comparison with the myeloperoxidase-hydrogen peroxide-halide system.

A new fungicidal system composed of ferrous ion, H2O2, and iodide is described and compared with the myeloperoxidase-hydrogen peroxide-halide system. Both systems had similar activity against Aspergillus fumigatus spores and the Candida albicans yeast phase, but only the ferrous ion-hydrogen peroxide-iodide system was inhibited by hydroxyl radical scavengers.

Animals↗
PubMed1984

Cerebral mucormycosis in diabetic mice after intrasinus challenge.

Rhinocerebral involvement is the most common form of mucormycosis in uncontrolled diabetic patients. Streptozotocin-induced diabetic and normal mice were challenged by intrasinus inoculation of Rhizopus oryzae to determine their susceptibility to infection by this route. Ninety percent of diabetic mice and no normal mice died after inoculation with 10(6) spores. Intrasinus inoculations caused minimal direct trauma and low levels of brain inocula, but progressive cerebral infections were induced in diabetic mice.

Animals↗
PubMed1984

Specific susceptibility to mucormycosis in murine diabetes and bronchoalveolar macrophage defense against Rhizopus.

To assess the influence of diabetes mellitus in predisposing to pulmonary mucormycosis, a murine model of streptozotocin-induced diabetes was used. Intranasal inoculation of Rhizopus oryzae into diabetic mice resulted in mucormycotic infection with histopathology resembling pulmonary mucormycosis observed in humans. There was no mortality nor infection in inoculated normal mice. Diabetic mice had fatal infections caused by R. oryzae but significantly reduced mortality following inoculation with Aspergillus fumigatus. These findings reflect the specific enhanced susceptibility to mucormycosis observed in human diabetics. Normal bronchoalveolar macrophages formed part of an efficient defense against R. oryzae by inhibiting germination, the critical step in the conversion of R. oryzae to its tissue invasive phase. Bronchoalveolar macrophages inhibited spore germination in vitro and appeared to help prevent germination in vivo. In contrast, spore germination occurred in diabetic mice following intranasal inoculation. Diabetic bronchoalveolar macrophages had a decreased ability to attach to hyphae. In diabetic mice, bronchoalveolar macrophages could damage spores or hyphae of R. oryzae, but serum factors appeared to both promote spore germination and impair attachment of macrophages to spores. This murine model of diabetes mellitus provides an opportunity for evaluation of the relative importance of cell and serum-mediated host factors in the pathogenesis of mucormycosis.

Acid Phosphatase↗
PubMed1984

Changing patterns of aspergillosis infections.

Aspergillus may cause a wide range of clinical illnesses. In large part, manifestations depend on the status of underlying local and systemic host immunologic responses. Therefore, it is critical to specifically define the different types of aspergillosis. With such distinctions between allergic bronchopulmonary aspergillosis, aspergilloma, and invasive aspergillosis with vasculitis, specific host factors that predispose to the development of each of these entities are becoming recognized. Understanding of such immunologic factors better explains the occurrence of clinical forms which overlap to include more than one type of aspergillosis. In general, the different types of aspergillosis have been increasingly recognized as significant health problems, though in different subpopulations of patients. Advances in knowledge of the immunopathogenesis of aspergillosis raise prospects for more rapid diagnosis as well as for successful prevention or therapy (directed toward both the host and pathogen) of diseases caused by these fungi.

Alveolitis, Extrinsic Allergic↗
PubMed1983

Inhibition of monocyte-mediated damage to fungal hyphae by steroid hormones.

Human monocytes can damage hyphal forms of C. albicans [1], R. oryzae [2], and A. fumigatus [3]. Because corticosteroids can inhibit phagocytosis and killing of bacteria by monocytes, the effects of steroid hormones on interactions of monocytes with hyphae were studied. Monocyte-mediated hyphal damage was inhibited by 10 microM hydrocortisone; inhibitory effects of lower concentrations of hydrocortisone were less marked with Rhizopus than with Candida or Aspergillus hyphae. Addition of 10% normal human serum to hyphae and monocytes did not affect the inhibitory effects of hydrocortisone (data not shown). Comparable inhibitory effects were obtained using 0.3-1.0 microM dexamethasone (data not shown). Estrogen and progesterone also inhibited damage to Candida or Rhizopus. However, inhibition occurred at concentrations which are readily achievable in vivo by pharmacologic doses of hydrocortisone, but not by other steroid hormones. Thus, pharmacologic doses of corticosteroids might produce serum hormone concentrations which could interfere with activity of host monocytes against the tissue-invasive forms of C. albicans and A. fumigatus in vivo. However, higher pharmacologic doses of these hormones may be required to inhibit leukocyte-mediated damage to Rhizopus hyphae. In contrast, predisposition of women to superficial candidal infections cannot be explained solely by direct effects of estrogens or progesterone on leukocyte-mediated hyphal damage.

Fungi↗
PubMed1983

Mechanisms of destruction of Aspergillus fumigatus hyphae mediated by human monocytes.

Normal monocytes attached to and destroyed Aspergillus hyphae by morphologic and metabolic criteria. Inhibition by anaerobiosis, azide, cyanide, halide-free conditions, catalase, histidine, and tryptophan suggested mediation of hyphal damage primarily through the myeloperoxidase system. However, myeloperoxidase-independent oxidative or nonoxidative mechanisms appeared active in hyphal damage by monocytes from patients with myeloperoxidase deficiency or chronic granulomatous disease (CGD). Moreover, hyphae were damaged by lysates and granule-enriched fractions of monocytes from patients with CGD, whereas comparable fractions of normal monocytes were active only with added halide and H2O2. Hyphal damage by both whole monocytes and granule-enriched fractions from patients with CGD was inhibited by polyanions, a result suggesting that cationic proteins may be involved. Hyphal damage by normal monocytes or monocytes from patients with CGD was inhibited by cells that lacked antihyphal activity (chlorpromazine-treated normal neutrophils or neutrophils from patients with CGD, respectively). This effect may predispose patients with CGD to chronic, invasive aspergillosis.

Aspergillosis↗
PubMed1982

Monocyte-mediated damage to Rhizopus oryzae hyphae in vitro.

Clinicopathological correlations from human cases and experimental animal studies suggest that neutrophils are critical components of the host response to mucormycosis but that other cellular defense mechanisms appear to be important as well. Since our previous studies demonstrated that Rhizopus oryzae hyphae which are too large to be ingested completely can be damaged and probably killed by human neutrophils, we studied the antihyphal activity of human monocytes. As with neutrophils, light and electron microscopic studies indicated that monocytes attached to hyphae and appeared to destroy them in the absence of serum. As judged by our previously described assay for the leukocyte-induced inhibition of [14C]uracil uptake by hyphae, quantitative damage to hyphae by monocytes was 40.8 +/- 2.2% in 54 experiments. Neither attachment to nor damage of hyphae by monocytes was augmented by the presence of 10% human serum. As with neutrophils, monocyte-mediated damage of R. oryzae was significantly decreased by some inhibitors of oxidative metabolism and scavengers of the potentially microbicidal oxidative leukocyte products, which included 10(-4)M sodium azide, 10 (-3) M sodium cyanide, catalase, 10(-3) M histidine, 10(-3) M tryptophan, and 10(-4) M 1,4-diazobicyclo[2.2.2]octane but not superoxide dismutase, 1.4 X 10(-2) M dimethyl sulfoxide, and 4.0 X 10(-1) M mannitol. Moreover, monocytes from three patients with chronic granulomatous disease failed to damage hyphae at all. In contrast to our previous data for neutrophils, polyanions (10(-5) M polyaspartic or polyglutamic acid) did not inhibit monocyte-mediated hyphal damage. Thus, monocytes can damage and probably kill R. oryzae hyphae by oxidative mechanisms and so may be involved in host defense mechanisms against mucormycosis.

Azides↗
PubMed1982

Chemotaxis of human neutrophils and monocytes induced by Cryptococcus neoformans.

Chemotaxis of human neutrophils and monocytes was stimulated by sera activated with greater than or equal to 1.25 x 10(6) Cryptococcus neoformans. Leukocytes from five renal transplant recipients had depressed chemotactic responses to C. neoformans-activated sera when compared with normal subjects (P less than 0.05). Concentrations of cryptococcal capsular polysaccharide less than 1 mg/ml failed to generate chemotactic factors from sera.

Chemotactic Factors↗
PubMed1982

Damage to Aspergillus fumigatus and Rhizopus oryzae hyphae by oxidative and nonoxidative microbicidal products of human neutrophils in vitro.

Our previous studies established that human neutrophils could damage and probably kill hyphae of Aspergillus fumigatus and Rhizopus oryzae in vitro, primarily by oxygen-dependent mechanisms active at the cell surface. These studies were extended, again quantitating hyphal damage by reduction in uptake of (14)C-labeled uracil or glutamine. Neither A. fumigatus nor R. oryzae hyphae were damaged by neutrophils from patients with chronic granulomatous disease, confirming the importance of oxidative mechanisms in damage to hyphae. In contrast, neutrophils from one patient with hereditary myeloperoxidase deficiency damaged R. oryzae but not A. fumigatus hyphae. Cell-free, in vitro systems were then used to help determine the relative importance of several potentially fungicidal products of neutrophils. Both A. fumigatus and R. oryzae hyphae were damaged by the myeloperoxidase-hydrogen peroxide-halide system either with reagent hydrogen peroxide or enzymatic systems for generating hydrogen peroxide (glucose oxidase with glucose, or xanthine oxidase with either hypoxanthine or acetaldehyde). Iodide with or without chloride supported the reaction, but damage was less with chloride alone as the halide cofactor. Hydrogen peroxide alone damaged hyphae only in concentrations >/=1 mM, but 0.01 mM hypochlorous acid, a potential product of the myeloperoxidase system, significantly damaged R. oryzae hyphae (a 1 mM concentration was required for significant damage to A. fumigatus hyphae). Damage to hyphae by the myeloperoxidase system was inhibited by azide, cyanide, catalase, histidine, and tryptophan, but not by superoxide dismutase, dimethyl sulfoxide, or mannitol. Photoactivation of the dye rose bengal resulted in hyphal damage which was inhibited by histidine, tryptophan, and 1,4-diazobicyclo(2,2,2)octane. Lysates of neutrophils or separated neutrophil granules did not affect A. fumigatus hyphae, but did damage R. oryzae hyphae. Similarly, three preparations of cationic proteins purified from human neutrophil granules were more active in damaging R. oryzae than A. fumigatus hyphae. This damage, as with the separated granules and whole cell lysates, was inhibited by the polyanion heparin. Damage to R. oryzae hyphae by neutrophil cationic proteins was enhanced by activity of the complete myeloperoxidase system or by hydrogen peroxide alone in subinhibitory concentrations. These data support the importance of oxidative products in general and the myeloperoxidase system in particular in damage to hyphae by neutrophils. Cationic proteins may also contribute significantly to neutrophil-mediated damage to R. oryzae hyphae.

Aspergillus fumigatus↗
PubMed1982

Generation of chemotactic factors by Rhizopus oryzae in the presence and absence of serum: relationship to hyphal damage mediated by human neutrophils and effects of hyperglycemia and ketoacidosis.

As our previous studies had shown that human neutrophils could kill Rhizopus oryzae hyphae in vitro, interactions of these hyphae with neutrophils and serum were further explored. Heated or fresh normal human sera suppressed hyphal metabolic activity as determined by [14C]uracil uptake, but severe ketoacidosis (8 X 10(-3) M beta-hydroxybutyric acid plus 2 X 10(-3) M acetoacetic acid at pH 7.0) negated this effect. Hyperglycemia (500 mg/dl) and severe ketoacidosis did not affect damage to hyphae by human neutrophils. Hyphae generated factors from sera which induced comparable chemotactic responses by neutrophils obtained from both normal and diabetic subjects, using a leading front assay performed in modified Boyden chambers. Zymosan-stimulated neutrophil chemotaxis was marginally depressed only by the combined elevation of both glucose (500 mg/dl) and ketoacids (10(-2)M) irrespective of pH (7.0 to 7.4), but not by any of these factors alone. Protein-containing supernatants from live or killed hyphae were chemotactic in the absence of serum based upon "checkerboard" assays varying the concentrations of hyphal supernatants above and below filters in the Boyden chambers. The supernatant-induced chemotactic response by neutrophils from diabetic subjects was minimally less than that of normal neutrophils (P less than 0.05). These findings indicate that R. oryzae hyphae can generate chemotactic factors which might prove to influence the inflammatory response to infections in vivo, and that severe hyperglycemia and ketoacidosis might affect interaction between the host and invading hyphae in mucormycosis.

Chemotactic Factors↗
PubMed1981

Monocyte-mediated serum-independent damage to hyphal and pseudohyphal forms of Candida albicans in vitro.

Human peripheral blood monocytes attached to Candida albicans hyphae in the absence of serum and damaged the hyphae without completely ingesting them. Attachment and damage was not augmented by the addition of serum. Damage to hyphae was quantitated by a previously developed metabolic assay that measured leukocyte-induced reduction in uptake of [(14)C]cytosine by the hyphae. Use of cells from patients with hereditary disorders of leukocyte function, chronic granulomatous disease, and myeloperoxidase deficiency indicated that myeloperoxidase-independent and nonoxidative mechanisms could sometimes damage hyphae where oxidative mechanisms were impaired. Damage to hyphae by normal monocytes was inhibited by concentrations of sodium azide and sodium cyanide that primarily affect myeloperoxidase activity, as well as by halide-free conditions, catalase, and putative antagonists of hypochlorous acid or singlet oxygen. Iodination of hyphae, a myeloperoxidase and hydrogen peroxide-dependent process of monocytes, was similarly inhibited by sodium azide, sodium cyanide, and catalase. Under anaerobic conditions, damage to hyphae was reduced by 64.0-68.4%. In contrast, inhibitors of potential nonoxidative antifungal mechanisms, iron salts to saturate iron chelators, and polyanionic amino acid polymers to neutralize cationic proteins did not block damage to hyphae by monocytes. Preparations rich in lysosomal granules from fractionated normal monocytes also did not damage hyphae. Overall, it appeared that oxidative mechanisms were most important for damage to hyphae by normal monocytes. Electron microscopy confirmed that Candida hyphae were damaged and probably killed by monocytes, but monocytes appeared to sustain significant damage in the process. In the absence of serum, monocyte cell membranes became closely approximated to Candida cell walls. It appeared that some Candida could escape this partial engulfment, as they were seen floating free with vesicular trilaminar membrane remnants covering hyphal surfaces. In general, monocytes appeared to be damaged by interactions with Candida hyphae more than neutrophils had been in previous studies.

Candida albicans↗
PubMed1980

Damage to Candida albicans hyphae and pseudohyphae by the myeloperoxidase system and oxidative products of neutrophil metabolism in vitro.

In previous studies, we noted that Candida hyphae and pseudohyphae could be damaged and probably killed by neutrophils, primarily by oxygen-dependent nonphagocytic mechanisms. In extending these studies, amount of damage to hyphae again was measured by inhibition of [(14)C]cytosine uptake. Neutrophils from only one of four patients with chronic granulomatous disease damaged hyphae at all, and neutrophils from this single patient damaged hyphae far less efficiently than simultaneously tested neutrophils from normal control subjects. Neutrophils from neither of two subjects with hereditary myeloperoxidase deficiency damaged the hyphae. This confirmed the importance of oxidative mechanisms in general and myeloperoxidase-mediated systems in particular in damaging Candida hyphae. Several potentially fungicidal oxidative intermediates are produced by metabolic pathways of normal neutrophils, but their relative toxicity for Candida hyphae was previously unknown. To help determine this, cell-free in vitro systems were used to generate these potentially microbicidal products. Myeloperoxidase with hydrogen peroxide, iodide, and chloride resulted in 91.2% damage to hyphal inocula in 11 experiments. There was less damage when either chloride or iodide was omitted, and no damage when myeloperoxidase was omitted or inactivated by heating. Azide, cyanide, and catalase (but not heated catalase) inhibited the damage. Systems for generation of hydrogen peroxide could replace reagent hydrogen peroxide in the myeloperoxidase system. These included glucose oxidase, in the presence of glucose, and xanthine oxidase, in the presence of either hypoxanthine or acetaldehyde. In the presence of myeloperoxidase and a halide, the toxicity of the xanthine oxidase system was not inhibited by superoxide dismutase and, under some conditions, was marginally increased by this enzyme. This suggested that superoxide radical did not damage hyphae directly but served primarily as an intermediate in the production of hydrogen peroxide. The possible damage to hyphae by singlet oxygen was examined using photoactivation of rose bengal. This dye damaged hyphae in the presence of light and oxygen. The effect was almost completely inhibited by putative quenchers of singlet oxygen: histidine, tryptophan, and 1,4-diazobicyclo[2.2.2]octane. These agents also inhibited damage to hyphae by myeloperoxidase, halide, and either hydrogen peroxide or a peroxide source (xanthine oxidase plus acetaldehyde). Myeloperoxidase-mediated damage to hyphae was also inhibited by dimethyl sulfoxide, an antioxidant and scavenger of the hydroxyl radical. These data support the involvement of oxidative mechanisms and the myeloperoxidase-H(2)O(2)-halide system, in particular in damaging hyphae in vitro and perhaps in vivo as well.

Candida albicans↗