Biomedical subjects
D A Stevens
Publications and source records attributed to D A Stevens.
Macrophage colony-stimulating factor (M-CSF) induction of enhanced anticryptococcal activity in human monocyte-derived macrophages: synergy with fluconazole for killing.
Induction of enhanced anticryptococcal activity in human monocyte-derived macrophages (HMM) by macrophage colony-stimulating factor (M-CSF) and possible synergy with fluconazole (FCZ) for killing of Cryptococcus neoformans (CN) was studied. Fungistasis by HMM cultured in medium for 3, 5, or 7 days was minimal, 0-17%. The fungistasis of HMM cocultured with M-CSF at 1000, 5000, or 20,000 U/ml for 3, 5, or 7 days was increased significantly (P < 0.02) at all study times and by all concentrations. The optimal M-CSF concentration for HMM treatment for enhanced fungistasis was 5000 U/ml for Day 3 (84%), whereas 1000 U/ml was sufficient with more prolonged HMM culture and M-CSF treatment (Days 5-7). The enhancement by M-CSF was seen with four different donors and three patient isolates of CN. FCZ at 5 micrograms/ml was fungicidal, 28 +/- 17% (n = 8). Killing by FCZ was enhanced by HMM treated with M-CSF 5000 or 20,000 U/ml for 5 days compared to control HMM, 58% (P = 0.001) and 60% (P = 0.002) vs 48%, respectively. This was also seen with HMM cultured with 1000 U/ml M-CSF for 7 days (P < 0.05). M-CSF also induced in HMM enhancement of fungistasis by lower, fungistatic, concentrations of FCZ. These results demonstrate enhancement of anticryptococcal activity by HMM treated with M-CSF and synergy with FCZ for inhibition and killing. These findings may provide a rationale for combined treatment of FCZ and M-CSF against cryptococcosis.
Induction of novel protein synthesis by opsonized Histoplasma capsulatum ingested by murine peritoneal macrophages.
It is known that Histoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closely related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form of H. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingested H. capsulatum with [35S]-methionine labeling. Cycloheximide at 5 to 10 micrograms/ml was used to preferentially inhibit macrophage uptake of [35S]-methionine without affecting H. capsulatum uptake. Protein synthesis by H. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killed H. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingested H. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingested H. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.
A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos.
Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.
In vivo and in vitro effects of macrophage colony-stimulating factor (M-CSF) on bronchoalveolar macrophages for antihistoplasmal activity.
The in vivo and in vitro effects of M-CSF on bronchoalveolar macrophages (BAM) activity against the intracellular fungal pathogen Histoplasma capsulatum (Hc) were studied. Three days after a single subcutaneous (s.c.) dose of M-CSF (2.5 mg/kg), enhanced ex vivo antifungal activity of BAM was measured. BAM from M-CSF-treated CD-1 mice significantly (P < 0.01) inhibited the intracellular multiplication of Hc yeast cells in 20 h assays compared to BAM from control mice. This effect was not observed at days 1, 7, 11 or 21 post-treatment. A dose of 5 mg/kg s.c., but not 1 mg/kg, induced similar antifungal activity in BAM by day 3. Peritoneal macrophages (PM) from M-CSF-treated mice did not have enhanced antifungal activity at days and doses tested. BAM could also be activated for antihistoplasmal activity by M-CSF in vitro. M-CSF at 10,000 U/ml for 24 h or 5000 U/ml for 48 h induced significant (P < 0.01) inhibition of intracellular multiplication of Hc. Interferon-gamma (IFN) plus lipopolysaccharide (LPS) activated BAM and PM in vitro to inhibit intracellular multiplication of Hc (P < 0.001); the antihistoplasmal activity was completely inhibited by NG-monomethyl L-arginine (N-MMA), indicating that an L-arginine-dependent nitric oxide-producing mechanism was operative. N-MMA could not inhibit the antihistoplasmal activity of BAM or PM activated by M-CSF in vitro. The mechanism by which M-CSF-activated macrophages inhibit intracellular multiplication of Hc remains to be determined.
Safety product evaluation: six years of experience.
Safety products to prevent percutaneous injuries are rapidly being developed for health care. Decisions to purchase such products are often made by an institution's infection control committee. We evaluated 30 safety products. These products are most readily accepted when potential users perceive a need, believe they are at risk for injury, and have input into the selection process. Task forces of intended users are invaluable in determining which products to evaluate in high-risk areas. Some new devices may be unsafe if excessive manipulation or in-service education is required. New products may not be well accepted if the devices are a different size or configuration than usual or require changes in established practices or techniques. An adequate supply of new products must be available immediately when the evaluation begins or users will tend to select the older, more familiar product. Removing all similar devices except for the new product can facilitate acceptance. Frequency of use can also influence employees when trying a new product. For instance, nurses who only occasionally draw blood have different perceptions of a new blood-drawing device from those of the phlebotomy team. Key employees were designated as resource persons to complement the manufacturer's in-service education and to support transition to the new product. We recommend that decision making by infection control committees involve the individuals who will eventually directly use the new products, starting with the early phases of safety product evaluation.
Fluconazole in the treatment of chronic pulmonary and nonmeningeal disseminated coccidioidomycosis. NIAID Mycoses Study Group.
PURPOSE: To determine the efficacy and safety of fluconazole as treatment for coccidioidomycosis. PATIENTS AND METHODS: This was a multicenter, open-label, single-arm study. Of 78 patients enrolled, 22 had soft-tissue, 42 had chronic pulmonary, and 14 had skeletal coccidioidomycosis. Forty-nine had at least one concomitant disease, 7 of whom had HIV infection. Patients were given oral fluconazole 200 mg/d. Nonresponders were increased to 400 mg/d. Treatment courses were long: a mean of 323 +/- 230 days at 200 mg and 433 +/- 178 days at 400 mg. Predefined assessment of disease-related abnormalities was performed at the time of enrollment and repeated at least every 4 months. A satisfactory response was defined as any reduction of baseline abnormality by month 4 and at least 51% reduction by month 8. RESULTS: Among 75 evaluable patients, a satisfactory response was observed in 12 (86%) of the 14 patients with skeletal, 22 (55%) of the 40 patients with chronic pulmonary, and 16 (76%) of the 21 patients with soft-tissue disease. Five patients (7%) required modification of treatment due to toxicity. Forty-one patients who responded were followed off drug. Fifteen (37%) of them experienced reactivation of infection. CONCLUSION: Fluconazole 200 or 400 mg/d is well tolerated and a moderately effective treatment for chronic pulmonary or nonmeningeal disseminated coccidioidomycosis. The relapse rate following therapy is high. Treatment trials with higher doses appear warranted. The relative efficacy of fluconazole versus other azoles or amphotericin B remains unknown.
High concentrations of beta-adrenergic agonists inhibit DNA binding of glucocorticoids in human lung in vitro.
Explore the source record for details and available documents.
Percutaneous injury analysis: consistent categorization, effective reduction methods, and future strategies.
OBJECTIVE: To report the results of an 8-year analysis of percutaneous injuries (PI), to describe interventions to decrease these injuries, and to discuss future prevention strategies. DESIGN: Using consistent methods, 881 percutaneous injury reports were reviewed, categorized, and analyzed from 1986 through 1993. SETTING: A 620-bed acute-care county teaching hospital located in San Jose, California, that is affiliated with Stanford University Medical School, Palo Alto, California. PARTICIPANTS: Employees of Santa Clara Valley Medical Center who reported percutaneous injuries from 1986 through 1993. INTERVENTIONS: Placement of needle disposal containers in all patient care areas, 1987; education, 1987 to present; communication of percutaneous injury analyses to all departments, 1988 to present; and safety product evaluation and purchases, 1991 to present. RESULTS: The total number of PI decreased by 65% (P = .0007) from 1986 through 1993. Recapping injuries decreased from 1986 through 1993 by 88% (P < .0002); interventions that included convenient placement of needle disposal containers and consistent annual education may have contributed to this decrease. Injuries from manipulating intravenous lines or heparin locks decreased in 1992 (P < .03) after purchase of a needleless system for intravenous lines. Injuries from improper disposal or from abrupt patient movement did not decrease significantly over the 8-year period. CONCLUSIONS: This institution has conducted percutaneous injury analysis for 8 years, utilizing consistent reviewers and categorization methods. Successful interventions have reduced recapping injuries, injuries from manipulating intravenous lines/heparin locks, and the overall numbers of PI. The categories of "Improper Disposal" and "Patient Moved Abruptly" present challenges for future reductions, as well as the recently identified problem of staff not using available safety devices or using them improperly.
Enhanced sensitivity to androstenone following regular exposure to pemenone.
The responses elicited by olfactory stimuli may be modified throughout an organism's life. For example, Wysocki et al. found that regular presentations of 5a-androst-16-en-3-one (androstenone) to anosmic subjects resulted in a graded increase in the perceived intensity of this substance in about half of their subjects (Wysocki et al., 1989). The increased sensitivity they observed appeared to be specific to the exposed odorant and was presumed to occur only in anosmic subjects. Here, we continue our explorations of the individual differences in olfactory capabilities among subjects initially classified by their ability to detect and identify the odor of the diastereoisomeric ketone, cis-4-(4'-t-butylcyclo-hexyl)-4-methyl-2-pentanone (pemenone) which shares with androstenone a pronounced urine-sweaty odor. We asked if regular pemenone exposure enhances the sensitivity of human subjects to pemenone, androstenone, isovaleric acid, or phenylethyl alcohol and, if shifts in threshold occurred, were they specific to particular odorants and classes of subject? Detection thresholds for the four substances were determined before and after 7-8 weeks of regular, biweekly, exposure to pemenone (n = 18 subjects) or a control substance (22 subjects). Significant decreases in threshold were seen in the experimental group, relative to the control group, for androstenone, but not for the other compounds evaluated. Neither gender nor initial pemenone osmicity significantly affected the frequency of subjects with enhanced sensitivity. These findings show that a subject's sensitivity to one odorant can be enhanced by exposure to another, chemically distinct compound.(ABSTRACT TRUNCATED AT 250 WORDS)
Synergy of fluconazole with human monocytes or monocyte-derived macrophages for killing of Candida species.
Possible synergy between fluconazole and monocyte-derived macrophages (MDM) for significant killing of Candida species and between fluconazole and monocytes for increased candidacidal activity were studied. Human serum in test medium (RPMI 1640 with 10% fresh autologous human serum) was highly fungistatic for Candida species. Fluconazole in test medium, even at high concentrations, was not fungicidal. Monocytes alone were fungicidal for all Candida species and synergized with fluconazole for increased killing of a fluconazole-sensitive and a fluconazole-resistant Candida albicans isolate. MDM obtained by culturing for 3 or 5 days were not significantly fungicidal for C. albicans but could synergize with fungistatic fluconazole for significant killing. MDM alone killed Candida krusei and Candida glabrata but did not synergize with fluconazole for significantly increased killing. These results, which demonstrate synergy of fluconazole and mononuclear phagocytes for killing, help explain the in vivo efficacy of fluconazole against candidiasis.
Killing of Histoplasma capsulatum by macrophage colony stimulating factor-treated human monocyte-derived macrophages: role for reactive oxygen intermediates.
The interaction of human macrophages with the yeast-form of Histoplasma capsulatum was studied. The use of culture and a short-term assay period instead of microscopy gave direct evidence of the fungicidal activity of human macrophages. The present study reports the novel finding of fungicidal activity of macrophages derived from monocytes in the presence of macrophage colony-stimulating-factor (MCSF). The induction of fungicidal activity by this cytokine was dose dependent. MCSF at 10,000 U/ml was optimal with 73(SD3)% killing. Inhibition of macrophage killing by superoxide dismutase (SOD), but not catalase (CAT) or N-monomethyl-L-arginine (NMMA), established the role of the superoxide anion in the killing mechanism. The fungistatic activity of MCSF-derived human macrophages in a 24-h assay was also dose dependent and was not inhibited by SOD, CAT or NMMA. MCSF at 10,000 U/ml produced optimal macrophage fungistatic activity, 34.6(SD4)%.
Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum.
The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs. Little is known about the interaction of BAM and Hc, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis. A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-activated BAM. Fungistatic activity of BAM was determined with a 24-h assay. A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used. Activated BAM killed Hc (reduced inoculum CFU by 25 +/- 12%; n = 4). The fungicidal activity of BAM was abrogated by 0.2 mM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase. In fungistatic assays activated BAM inhibited multiplication of Hc by 61 +/- 4% (n = 3) compared with cocultures with control BAM. However, Hc multiplied 100% more in control BAM cocultures than in medium alone. Data indicated that this was due to advantages that Hc has in the intracellular environment. Only NMMA inhibited fungistatic activity of activated BAM. In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained. In conclusion, activated BAM and PM kill yeast cells of Hc by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway.
Different components in human serum inhibit multiplication of Cryptococcus neoformans and enhance fluconazole activity.
The inhibitory effect of human serum on the multiplication of Cryptococcus neoformans and the interaction with fluconazole were studied. Compared with cryptococcal multiplication in RPMI 1640 medium alone, 5% human serum in medium inhibited multiplication by 76% +/- 6% (n = 8). The inhibitory effect of human serum was donor independent, [corrected] heat stable (56 degrees C, 30 min), and not due to albumin or globulin. Bovine and murine sera were not inhibitory at that concentration. A fungistatic concentration of fluconazole (5.0 micrograms/ml) in medium plus 5% human serum resulted in 40% +/- 5% (n = 8) killing (reduction of inoculum CFU) in a 24-h assay. Bovine or murine sera did not have the enhancing effect, and this human serum activity was heat stable and donor independent. At 2.5 micrograms of fluconazole per ml, fungistasis by fluconazole plus human serum was significantly greater than with either alone. Higher serum concentrations [corrected] potentiated fluconazole more. At higher fluconazole concentrations (e.g., 20 micrograms/ml) fluconazole alone could kill, but serum potentiated this. A fluconazole-resistant isolate (MIC, 100 micrograms/ml) was not killed by fluconazole (5.0 micrograms/ml) in 5% human serum, but human serum potentiated the partial fluconazole inhibition. When human serum was dialyzed (molecular weight cutoff, 6,000 to 8,000) against phosphate-buffered saline, it lost the ability to synergize with fluconazole for killing Cryptococcus organisms but not the capacity to inhibit multiplication. Filtration of serum suggested the filtrate with a molecular weight of < 10,000 could interact synergistically with fluconazole for killing but could not inhibit cryptococcal multiplication. These findings indicate that human serum has two components, one (macromolecular) with a unique ability to inhibit C. neoformans and a low-molecular-weight component that enhances fluconazole anticryptococcal activity.
Efficacy of the triazole D0870 in a murine model of systemic histoplasmosis.
The efficacy of D0870 was studied in a murine model of systemic histoplasmosis and was compared with that of fluconazole. All regimens of D0870 (1 or 10 mg/kg of body weight given daily or 1, 10, or 100 mg/kg given every other day) and 100 mg of fluconazole per kg given daily provided complete protection from lethality. Comparison of the number of viable Histoplasma capsulatum yeasts remaining in the spleen or liver indicated that D0870 was superior to fluconazole in clearing and curing infection. D0870 was 10- to 100-fold more efficacious than fluconazole in the treatment of experimental histoplasmosis.
Activities of the triazole SCH 51048 against Coccidioides immitis in vitro and in vivo.
The activity of the novel triazole SCH 51048 was tested against Coccidioides immitis. SCH 51048 inhibited C. immitis in vitro; MICs for 13 isolates ranged from < or = 0.39 to 0.78 micrograms/ml, and minimum fungicidal concentrations ranged from < or = 0.39 to 1.6 micrograms/ml. In vivo, no mice treated with SCH 51048 at 2 to 50 mg/kg of body weight or 100 mg of fluconazole or itraconazole per kg died of systemic coccidioidomycosis, whereas 60 to 100% of the control mice died. SCH 51048 given at 25 or 50 mg/kg was curative, whereas fluconazole or itraconazole given at 100 mg/kg was not curative. Pharmacokinetic studies showed peak levels in serum of > 14 micrograms/ml, with an estimated half-life of > 12 h. SCH 51048 was 5- to 50-fold or more superior to fluconazole or itraconazole.
Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice.
We have previously determined the relative virulence of isolates of Saccharomyces cerevisiae on the basis of differences in proliferation and resistance to clearance in CD-1 mice. These infections were not fatal. To further characterize S. cerevisiae pathogenesis, we studied a virulent clinical isolate, YJM128, and an avirulent nonclinical isolate, Y55, in C5-deficient mice. DBA/2N mice were infected intravenously with YJM128 or Y55, and temporal burdens of yeast cells in various organs were determined. After infection with 10(7) CFU, Y55 increased by 13-fold and YJM128 increased by 20-fold in the brain from day 0 to 3. In addition, YJM128 increased by 4-fold in the kidneys, whereas Y55 decreased by 16-fold. Both isolates declined in number in other organs. In all studies, 90% of mice infected with 10(7) CFU of YJM128 died between days 2 and 7, whereas no mice infected with equivalent numbers of Y55 died. No mice died after infection with 10(6) CFU of Y55 or YJM128. The importance of C5 was confirmed by studies using B10.D2/oSnJ (C5-) mice and their congenic C5+ counterparts. Again, the C5- mice were most susceptible to infection with S. cerevisiae, with 63% infected with YJM128 dying by day 7; no C5+ mice died. No Y55-infected mice died, and mean burdens in the brain at day 14 were sevenfold lower in C5+ mice than in C5- mice. Seven of 10 other S. cerevisiae isolates were also more virulent in DBA/2N than CD-1 mice, causing > or = 40% mortality. These data indicate that C5 is a critical factor in host resistance against S. cerevisiae infections and further confirm the pathogenic potential of some isolates of S. cerevisiae.
Genotypic analysis of pseudoepidemic due to contamination of Hanks' balanced salt solution with Candida parapsilosis.
A cluster of isolates of Candida parapsilosis recovered from clinical specimens was demonstrated, by both classical and molecular epidemiological techniques, to have resulted from contamination in the laboratory. The source of the pseudoepidemic was a repeatedly utilized contaminated container of Hanks' balanced salt solution used in specimen processing. The patterns of restriction fragment length polymorphisms of DNA extracted from the clinical and environmental isolates were identical to each other but composed a newly identified unique C. parapsilosis DNA type.