Prevention and complete reversal of cyclosporine A-induced renal vasoconstriction and nephrotoxicity in the rat by fenoldopam.

The primary mechanism of cyclosporine A-induced nephrotoxicity involves renal vasoconstriction. In the present study, we have tested the effects of fenoldopam, a dopamine DA1, receptor agonist with renal vasodilator properties, on the changes in renal function induced by acute and subacute administration of cyclosporine A. In inactin-anesthetized rats, acute administration of cyclosporine A (100 mg/kg i.p.) significantly decreased paraaminohippuric acid (PAH) and inulin clearances. Fenoldopam, at a dose (10 micrograms/kg.min) which alone significantly increased PAH and inulin clearances, completely prevented the cyclosporine A-induced reductions in renal function. Similarly, subacute administration of cyclosporine A (20 mg/kg.day for 3 days) resulted in significant reductions in base-line PAH and inulin clearances which were normalized by administration of fenoldopam. These data indicate that administration of fenoldopam can both prevent and completely reverse cyclosporine A-induced renal vasoconstriction and nephrotoxicity.

Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae.

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

Divergent patterns of pulmonary blastomycosis induced by conidia and yeasts in athymic and euthymic mice.

Athymic nude (nu/nu) mice are uniformly more susceptible than euthymic nu/+ mice to lethal infection with intranasally inoculated Blastomyces dermatitidis, whether infection is initiated by yeasts or conidia. Conidial infection requires a high inoculum size; the disease produced is prolonged and disseminated. Yeasts are infective at a low inoculum size and produce a rapidly fatal pneumonia. Thymus transplantation is more protective for conidia-infected than yeast-infected nude mice, presumably because the disease course is long enough for an effect to become demonstrable. Yeast inocula multiply more rapidly in the lungs than do conidial inocula. This may relate to the greater susceptibility of conidia to heterophils evoked in the airways, and the fact that yeasts derived from conidial inocula must survive in the face of an established inflammatory reaction. When yeasts and conidia are inoculated simultaneously, the disease produced is less severe than when yeasts are inoculated alone, presumably because of a more intense and diffuse inflammatory response engendered by the conidia. Suppression of conidia-derived yeast replication is demonstrable for at least 1 wk in nu/nu mice and for 2 to 3 wk in nu/+ mice. The latter delay appears attributable to the intact immune system in nu/+ mice, and the probability that cellular immunity limits the subsequent replication of yeasts. Eventually, the immune response fails to control yeast replication, and the mice succumb. These studies provide further insights into the role of the thymus in host defense against B. dermatitidis and the basis for the differential pace of infection when mice are infected with yeasts or conidia.

In vitro antifungal susceptibility testing and measurement of levels of antifungal agents in body fluids.

Interpretation of in vitro susceptibility data for antifungal drugs is hindered by the absence of standardized test criteria. Thus, it is extremely difficult to identify a clear relation between minimal inhibitory concentrations and clinical outcome. The situation appears more readily resolvable for yeast-like than for filamentous fungi since the former are more easily quantified by standardized microbiologic techniques. Accordingly the National Committee for Clinical Laboratory Standards has initiated the process of developing standards for yeast susceptibility testing. A related issue concerns the measurement of antifungal agents in body fluids. Whereas there may be little value in measuring concentrations of amphotericin B (because of its predictable pharmacokinetics), there is value to measuring levels of flucytosine (serum concentrations may relate to bone marrow suppression and/or the development of drug resistance) and ketoconazole (which may be absorbed unpredictably from the gut). Laboratory standards for these measurements have not been established.

A Legionella-specific DNA probe detects organisms in lung tissue homogenates from intranasally inoculated mice.

We have prepared a DNA probe from internal sequences of the gene encoding the Legionella pneumophila major outer membrane protein (MOMP). Immunologic studies of the MOMP have confirmed that it possesses both genus-specific and species-specific antigenic domains, but possesses no cross-reactivity with non-Legionella species. At the DNA levels, the 3' half of the gene contains sequences that are homologous to DNA from all strains tested within the genus, whereas the 5' half of the gene has homology with L. pneumophila strains only. Homology of the gene with non-legionellae has not been detected even under low stringency conditions. To test the utility of this probe for detecting organisms in tissue, we tested crude homogenates of mouse lungs representing 1/1,000th of the total lung mass. After intranasal inoculation with 2 X 10(8) colony-forming units of L. pneumophila, mice were sacrificed at various intervals (10 mice per group). Since L. pneumophila does not produce a propagating infection in these animals, cultures of lung tissue from successive days after inoculation showed a roughly linear decline in viable L. pneumophila (total lung yield: 10(8) on Day 0, 5 X 10(7) on Day 2, 10(5) on Day 5, 10(3) on Day 9, and less than 10(2) on Day 15). By DNA dot hybridization with the MOMP probe, we detected positive signals from most animals on Days 0 and 2, suggesting a threshold sensitivity of between 50,000 and 100,000 organisms with our current methods. Advances in DNA probe technology may soon permit the rapid, specific identification of either L. pneumophila or other Legionella species in pathologic specimens.

Electron-microscopic observations of the Coccidioides immitis parasitic cycle in vivo.

BALB/c mice were inoculated by intranasal challenge with viable arthroconidia of C. immitis and in-vivo morphogenesis of the fungal pathogen was investigated by electron-microscopic examination of pulmonary lavage and cryofractured lung specimens. Samples were prepared at intervals over an 11-day period. Stages of spherule and endospore development were easily identified by scanning and thin-section electron microscopy. Details of morphogenesis of the pathogen in vivo closely resemble developmental aspects reported from in-vitro studies.

Influence of fungal surface components on the interaction of Coccidioides immitis with polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMNs) possess phagocytic and fungicidal activity against Coccidioides immitis that declines during maturation from arthroconidia to round cells, is lost throughout spherule maturation, and returns when endospores are released from ruptured spherules. Studies of PMN chemiluminescence, iodination, and degranulation give similar results. Phagocytosis of forms other than spherules is strain dependent and enhanced by immune serum. The absence of adequate PMN-spherule interaction may be attributed to the production of an extracellular fibrillar matrix, glycoprotein in composition, that restricts intimate PMN-spherule contact. When the spherule ruptures, PMNs enter to phagocytose endospores that are themselves invested by a matrix derived from the inner spherule wall. The immunochemical relationship between the outer matrix and the inner matrix remains to be discovered. Nevertheless, presence of the outer matrix may help to explain the long-standing histopathologic observation that PMNs fail to attack spherules until they release their endospores.

Factors governing adherence of Candida species to plastic surfaces.

The ability of Candida albicans and Candida spp. to adhere to inert polymeric surfaces may allow these organisms direct ingress into the human host. Biophysical characterization of this adherence shows that the forces responsible for such adherence are attractive London-van der Waals forces (or hydrophobic forces) and electrostatic forces. The hydrophobic affinity of yeasts was determined by (i) a water-hydrocarbon two-phase assay and by (ii) measurement of the contact angle (theta) of a liquid droplet on a monolayer of yeast cells. The hydrophobicity of the yeasts correlated with the tendency of yeasts to adhere to polystyrene and was reduced in the presence of Tween 20. The adherence of yeasts to polymers of increasing hydrophobicity (determined by the contact angle method) was directly proportional to theta. Yeast surface charges were altered by selectively blocking amino and carboxyl groups. The more positively charged yeasts adhered in greater numbers. Increasing the molarity of NaCl increased yeast adherence. These forces probably contribute to the negative cooperativity (determined by Scatchard and Hill plot) that characterizes the adherence of yeasts to polymers.

Antibacterial activities of antineoplastic agents.

Fourteen antineoplastic agents were examined for in vitro antibacterial activity against 101 aerobic and anaerobic bacterial isolates representing indigenous human microflora and selected opportunistic pathogens. Only 5-fluorouracil, mitomycin, and etoposide demonstrated inhibitory effects at achievable plasma concentrations, while the remaining drugs lacked appreciable antibacterial activities.

Introduced leishmaniasis on Taiwan.

Leishmaniasis is not known to be indigenous to Taiwan but a number of imported cases of visceral as well as post-kala-azar dermal leishmaniasis have been seen. Only two autochthonous cases of cutaneous-subcutaneous diseases have been documented in aborigines but no cases of visceral leishmaniasis have been reported. Although a significant number of imported cases of leishmaniasis have been seen, the disease has apparently not been established on the island.

Simplified bioassay method for measurement of flucytosine or ketoconazole.

A simple agar-well diffusion bioassay suitable for measurement of flucytosine or ketoconazole was developed by using Candida pseudotropicalis ATCC 46764 as the assay organism. A test medium composed of (per liter) 7 g of Trypticase peptone, 7 g of YNB (yeast-nitrogen base), 15 g of glucose, and 15 g of agar was seeded with an inoculum which had been grown to no. 2 McFarland turbidity after 4 to 6 h in YNB-glucose broth. Determinations of flucytosine or ketoconazole were performed without necessity of heating or diluting of serum samples to alleviate amphotericin B interference. A linear relationship between zone diameters and log10 concentration of the drugs was observed over the pharmacologically relevant ranges of 25 to 160 micrograms/ml for flucytosine and 0.5 to 20 micrograms/ml for ketoconazole. The mean coefficient of variability for samples measured on 5 separate days was 2.4% for flucytosin and 4.0% for ketoconazole. This assay represents a significant improvement over previous bioassay methods in that a single test system may be used for measurement of either flucytosine or ketoconazole, no serum dilution or pretreatment is required, inoculum preparation is accomplished entirely on the day of the assay, and sharp, clearly defined zones of inhibition are obtained with both drugs.

Intracellular and extracellular defenses of human phagocytes against Blastomyces dermatitidis conidia and yeasts.

The lesions of blastomycosis are characterized by both suppuration and granuloma formation, but the relative roles of human neutrophils, monocytes, and macrophages against Blastomyces dermatitidis are poorly defined. Our studies reveal that B. dermatitidis yeasts are generally too large to be ingested by polymorphonuclear neutrophils (PMNs), and are killed predominantly by external PMN attachment and degranulation, whereas conidia are first ingested, then killed. PMN function is maximal in the presence of serum, divalent cations, and complement, and killing is more efficient for conidia (approximately 50%) than for yeasts (approximately 20%). PMNs that have degranulated, but remain attached to yeasts, block access by contiguous PMNs. When degranulated PMNs are removed, allowing access by fresh PMNs, there is a further increment in yeast killing. Both conidia and yeasts are killed by predominantly oxidative PMN mechanisms, with conidia being greater activators of the respiratory burst, and proportionately more influenced by oxidative inhibitors. Peripheral blood monocytes can kill conidia (approximately 35%), but are feebly active against yeasts (approximately 5%). Monocyte-derived macrophages kill approximately 90% of conidia and 40% of yeasts. The dramatic susceptibility of conidia, the infective particles of B. dermatitidis, to nonspecific phagocytic host defenses may help to explain the relative rarity of blastomycosis as a clinical problem. The presence of PMNs in lesions of blastomycosis may indicate an active, although limited, role of these cells in host defense against B. dermatitidis yeasts.