The Anticryptococcal Factor of Blood Serum

Szilagyi, George; Reiss, Frederick; Smith, J. C.

doi:10.1038/jid.1966.47

Cited by 24 publications

(8 citation statements)

References 6 publications

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“…Our studies have shown the significant fungistatic effect of human serum on C neoformans, which is consistent with previous reports [2][3][4]. We found that the serum frozen and thawed up to 10 times inhibited growth as well as fresh frozen human serum.…”

Section: Discussionsupporting

confidence: 95%

“…This suggests that apotransferrin and iron may not be the major factors responsible for the fungistatic activity of human serum. The role of an iron binding factor in growth inhibition of C. neoformans by normal human serum has been previously studied by Szilagyi and Reiss [4]. Five #g of iron used in their study gave inconsistent results and they concluded that in addition to the iron binding factor in serum, other factors may be involved in the inhibitory activity of serum.…”

Section: Discussionmentioning

confidence: 94%

“…In early studies, the fungistatic effect of human serum against Cryptococcus neoformans has been described [2,3], and several attempts have been made to characterize the inhibitory factor(s) responsible [2][3][4][5]. Candida aIbicans, also an opportunistic fungus, has shown similar phenomena and, on investigation, transferrin and iron were found to be the major factors involved [6].…”

Section: Introductionmentioning

confidence: 98%

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Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

1996

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Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl3 on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 degrees C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 microM, of FeCl3 at both apotransferrin concentrations. By contrast, addition of FeCl3 to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl3. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.

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Section: Discussionsupporting

confidence: 95%

Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 98%

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Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

1996

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“…Early attempts to identify an inhibitory component(s) were limited by the technology available (12,18). Here a sequential method was used for characterization of the anticryptococcal activity.…”

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confidence: 99%

Characterization of an Anticryptococcal Protein Isolated from Human Serum

et al. 2000

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Human serum at low concentrations inhibits the growth of Cryptococcus neoformans in vitro. Fractionation of serum yielded a purified inhibitory protein with a molecular mass of ϳ81.8 kDa, a pI of ϳ6.2, and an amino acid sequence that matched that of human transferrin. The inhibitory activity and that of apotransferrin and 5% human serum were reversed by 10 M freshly prepared FeCl 3 .Human serum inhibits the growth of Cryptococcus neoformans (1, 11). Early attempts to identify an inhibitory component(s) were limited by the technology available (12,18). Here a sequential method was used for characterization of the anticryptococcal activity.C. neoformans isolates CDC 9759 and 46545 were grown on Sabouraud's dextrose agar or blood agar plates at 35°C for 48 h. Yeast cells were washed in saline and suspended in RPMI 1640 (GIBCO, Grand Island, N.Y.) with or without human serum fractions and dispensed, 0.1 ml per microtest plate well, in sets of quadruplicate cultures. In other experiments, C. neoformans was cultured in iron-free human transferrin (siderophilin) (Sigma Chemical Company, St. Louis, Mo.).Cultures were incubated at 37°C in 5% CO 2 -95% air for 24 h, harvested, and washed with sterile distilled water. Dilutions of harvested material were plated on blood agar plates, CFU were enumerated after 48 h at 35°C. Percent inhibition was calculated by the formula [1 Ϫ (experimental CFU/control CFU)] ϫ 100. CFU from RPMI 1640 cultures constituted the control. This definition measures the sum of events during incubation where some cells may be static, some multiply, and others die.Serum from healthy donors and human AB serum (GIBCO) were stored at Ϫ80°C.For anion-exchange gel chromatography, DEAE-Sephacel (Pharmacia LKB, Uppsala, Sweden) columns (2.5 by 6.5 cm and 3 by 12 cm) were prepared. Four milliliters of serum was applied to the smaller column, and 12 ml was applied to the larger. Columns were eluted sequentially with Tris-HCl (0.1 M, pH 8.5) buffer and then 0.1, 0.2, and 0.3 M NaCl in buffer. Five-milliliter fractions were collected, and the protein concentration of fractions was estimated by absorbance at 280 nm. Fractions that formed a protein peak were pooled, lyophilized, dialyzed against distilled water, and filter sterilized.Sephadex G-200 (Pharmacia, Piscataway, N.J.) columns (2.5 by 90 cm) were used for molecular sieve chromatography. Samples were eluted from the column with phosphate-buffered saline diluted 1:10. Fractions were processed as described above.A precast Bis-Tris polyacrylamide minigel electrophoresis system, which runs at neutral pH, 7.0 (NuPAGE; NOVEX, San Diego, Calif.), was used for one-dimensional gel electrophoresis. Electrophoresis was done on 4 to 12% gels using a 3-(N-morpholino)propanesulfonic acid-sodium dodecyl sulfate buffer in an Xcell-II apparatus. Proteins were fixed, stained with Coomassie blue, photographed with a digital camera, and printed with a graphic printer.NOVEX isoelectric focusing (IEF) gels, pH 3 to 7, nondenaturing (no urea), containing 5% polyacrylamide and 2%...

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“…Free iron is important for growth of Candida albicans in serum and has also been suggested for C. neoformans. At pH 7.4, transferrin removes free iron, making it unavailable, whereas at a pH of less than 6 transferrin is unable to bind iron and it is available (3,9). We tested the possible importance of transferrin by comparing growth in serum with and without added iron.…”

Section: Resultsmentioning

confidence: 99%

Treatment of Experimental Murine Cryptococcosis: a Comparison of Miconazole and Amphotericin B

Graybill

Mitchell

Levine³

1978

Antimicrob Agents Chemother

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Miconazole was compared with amphotericin B in the treatment of murine cryptococcosis. Both subcutaneous and intraperitoneal administration of miconazole produced serum levels higher than the minimum inhibitory concentration for the challenge strain. However, maximal tolerable doses of miconazole gave no increase in survival. When combined with amphotericin B, miconazole demonstrated neither additive nor antagonistic effects on survival. Spleen and brain counts of cryptococci were not lowered by miconazole; also, miconazole did not alter the effect of amphotericin B on reducing tissue counts. In vitro studies confirmed that the strain of Cryptococcus neoformans was quite susceptible to both miconazole and amphotericin B. However, miconazole had a delayed onset of antifungal activity. This was apparent even at miconazole levels 20 times greater than the minimum inhibitory concentration. Also, the antifungal activity of miconazole was markedly inhibited by serum. Delayed antifungal activity and serum inhibition may limit the in vivo effectiveness of miconazole in murine cryptococcosis.

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The Anticryptococcal Factor of Blood Serum

Cited by 24 publications

References 6 publications

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Characterization of an Anticryptococcal Protein Isolated from Human Serum

Treatment of Experimental Murine Cryptococcosis: a Comparison of Miconazole and Amphotericin B

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