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Departments of
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Medicine and
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The few studies performed in this area have been hampered by concerns about the purity of the melanin preparations used and the difficulty in applying standard serologic techniques to this compound. In anterior chamber uveitis models, melanin injected into the anterior chamber of laboratory animals resulted in the development of a brisk inflammatory response (8, 10). However, further purification of the putative melanin preparations by boiling in HCl inactivated the proinflammatory properties of the substance, suggesting that the effects were due to contaminating proteins (9). Melanins extracted from skin have also been used to elicit an immune response; however, the preparations used in this work contained cellular debris (11), and it is unclear whether the response was to melanin or the associated material (12). More recently, two groups have shown that precursors to melanin found in the sera of patients with melanoma can be immunogenic, and mAbs reactive with these compounds have been generated (13, 14). Hence, there is circumstantial evidence that melanin can be immunogenic, but this has not been rigorously established.
We re-examined the question of whether natural melanins are immunogenic using melanin isolated from Cryptococcus neoformans, a human pathogenic fungus responsible for life-threatening meningoencephalitis in 6 to 8% of patients with AIDS (15). C. neoformans produces melanin when grown in medium with phenolic substrates (16), and we have developed techniques for isolating the melanin (17). The synthesis of melanin is catalyzed by a phenol oxidase (laccase) (18), and the production of melanin is associated with virulence (19, 20, 21), possibly by quenching oxidants produced by immune effector cells and interfering with leukocyte phagocytosis (21, 22). Although the actual structure of melanin is unknown, we hypothesized that the pigment has a complex structure that includes one or more antigenic determinants. We demonstrate that fungal melanin can elicit an Ab response and describe serologic techniques for the study of melanin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanin was isolated from heavily pigmented C. neoformans strain 24067 cells as previously described (17). Briefly, C. neoformans cells were grown in defined media (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, and 3 µM thiamine) and 1 mM phenolic substrate (L-dopa or (-)epinephrine; Sigma, Cleveland, OH) for 10 days for melanin production. The melanized cells were collected and washed once in 1 M sorbitol and 0.1 M sodium citrate (pH 5.0). Novozyme 234 (Biopacific, Emeryville, CA) was added at a concentration of 10 mg/ml, and the suspension was incubated for 1 h at 30°C. The protoplasts were collected and suspended in 4 M guanidinium isothiocyanate for 30 min at room temperature. Cell debris were then collected and suspended in 6 M HCl at 100°C for 1 h. This procedure completely dissolves nonmelanized cells. However, for melanized cells, a black pellet remains, consisting of melanin "ghosts." The pellet was dialyzed exhaustively against dH2O. Electron spin resonance spectroscopy has previously shown that this residual material is melanin (17).
Immunization
Five- to six-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were given an i.p. injection of purified C. neoformans L-dopa melanin in PBS emulsified with CFA (Sigma) at a 1/1 (v/v) dilution. Two immunizing doses were evaluated: 100 and 300 µg/mouse. Booster injections, at the same dosage as the initial injection (100 or 300 µg), were given using a suspension of melanin in PBS without adjuvant at 6 and 8 wk. The mice were bled before immunization and at 2, 4, 7, and 9 wk.
In a different set of experiments, 6- to 8-wk-old BALB/c (The Jackson Laboratory), C57BL/6 (National Cancer Institute, Rockville, MD), and BALB/c T cell-deficient (nude) mice (The Jackson Laboratory) were given the 300-µg dose every 2 wk. The first dose was administered with CFA (1/1, v/v), and subsequent injections used IFA (1/1, v/v). The mice were bled before immunization and at 12 wk. Five mice of each strain were immunized.
Sera
The sera were stored at -20°C before use. To evaluate IgG and IgA titers, the sera were treated with ß-ME (1/1, v/v), 0.15 M ß-ME in Tris-buffered saline (TBS;4 25 mM Tris, 126 mM NaCl, and 2.6 mM KCl, pH 7.2), and incubated for 1 h at 37°C to reduce the multimeric form of IgM (23). ß-ME reduction removes the potential competitive masking of low affinity IgG subtypes or IgA by high avidity multimeric IgM. The titer of IgM was determined using sera not treated with ß-ME.
ELISA
The amount of melanin used per well was determined by selecting the concentration of cryptococcus-derived melanin resulting in the best signal to noise ratio. Polystyrene 96-well ELISA plates (Corning Glass Works, Corning, NY) were coated with serially diluted melanin suspensions from 1 x 107 to 2.5 x 103 particles of L-dopa melanin/ml dH2O. Gentle rocking at room temperature overnight evenly distributed and dried the particles in the wells. The plates were baked for 30 min at 80°C to fix the melanin. Blocking for nonspecific binding was performed with 2% BSA in dH2O overnight and then with 5% powdered milk in dH2O for 1 h, both at room temperature. Three washes with 250 µl of 0.1% Tween-20 in TBS were performed between each incubation. Following blocking, 50 µl of TBS or a 1/200 (v/v) dilution of 9 wk sera heated at 37°C for 1 h with ß-ME was applied in duplicate and serially diluted in 1% BSA. Alkaline phosphatase-labeled goat anti-mouse (AP-GAM) IgG (Fisher Biotech, Fisher Scientific, Orangeburg, NY) was then incubated on the plate at 37°C for 1 h. Ab binding was detected by developing the plates with p-nitrophenyl phosphate substrate (Sigma). OD was measured at 405 nm with a Ceres 900HDi (Bio-Tek Instruments, Winooski, VT). The signal to noise ratio was calculated by dividing the average of the readings from the wells with sera by those from wells without sera.
Using plates prepared with 5 x 105 melanin particles/well, sera were applied, serially diluted, and incubated at 37°C for 1 h. AP-GAM IgG1, IgG2A, IgG2B, IgG3, IgA, or IgM (Fisher Biotech) were incubated on the plate at 37°C for 1 h. The reactions were developed with p-nitrophenyl phosphate substrate. The absorbance at 405 nm was measured, and the titer was arbitrarily defined as the lowest dilution that gave an absorbance reading equal to or greater than twice the background. Background absorbances were in the range of 0.04 to 0.06.
Plates were also prepared with L-dopa- and (-)epinephrine-derived cryptococcal melanin, synthetic melanin (Sigma), and chemically treated melanin (see below). The melanins were placed in duplicate on the same plate. The ELISA protocol described above was followed using ß-ME-treated preimmune or 9 wk sera.
Inhibition ELISA
Suspensions of purified cryptococcal melanin particles were blocked with 2% BSA for 1 h at room temperature and then incubated with a 1/200 (v/v) dilution of sera in 2% BSA for 1 h at room temperature. The melanin particles were pelleted by centrifugation at 5000 rpm for 5 min, and the supernatants were plated in duplicate into ELISA plates prepared with 5 x 105 melanin particles/well. The plates were incubated for 1 h at 37°C. AP-GAM IgG and IgM (Fisher Biotech) were incubated on the plate at 37°C for 1 h. The reactions were developed with p-nitrophenyl phosphate substrate and measured at an OD of 405 nm.
Immunofluorescence (IF)
Ten microliters of a suspension containing 106 L-dopa cryptococcus-derived melanin particles/ml in dH2O or 105 melanized or nonmelanized C. neoformans cells grown for 10 days were dried on poly-L-lysine-coated slides (Sigma). Blocking for nonspecific binding was performed with 10% goat sera in 1% BSA for 1 h at room temperature. IF of the cells was tested using preimmune and 9 wk sera. Sera from BALB/c mice was applied at a dilution of 1/100 (v/v) in 1% BSA to the slide and incubated in a moisture chamber for 1 h at room temperature. After washing off excess Ab, the slide was covered with 50 µl of a 1/100 dilution of FITC-conjugated GAM IgG and IgM (Fisher) in 1% BSA for 1 h at room temperature. The slide was washed to eliminate unbound FITC reagent, and a coverslip was placed using a mounting solution (50% glycerol, 50% PBS, and 0.1 M N-propyl gallate). IF was observed using a Zeiss Axiophot UV microscope at a magnification of x1000.
Agglutination
L-dopa melanin particles purified from C. neoformans were incubated in 1% BSA overnight to block nonspecific protein binding. Starting with a 1/20 (v/v) dilution of BALB/c serum, fivefold dilutions of serum in 1% BSA (50 µl/well) were performed in 96-well polystyrene ELISA plates, and 105 L-dopa melanin particles in 1% BSA were added to each well. After incubation at 37°C for 1 h, agglutination was assessed by examining the wells for particle aggregation with a microscope (x400 magnification). The agglutination titer was defined as the highest dilution of sera at which agglutination was observed.
Chemical treatment of melanin
In addition to the standard cryptococcal melanin purification protocol, samples were prepared for ELISA, analysis of carbon/nitrogen ratios, and scanning electron microscopy subsequent to guanidinium incubation (time zero) and after boiling in 6 M HCl (0.5, 1, 2, 4, or 6 h). Two samples were prepared using boiling 6 M NaOH for 1 h, one after guanidinium treatment only and one following 1 h of boiling in HCl. Samples were also prepared for scanning electron microscopy using NaOCl at concentrations of 0.01% (1 mM), 0.1% (10 mM), or 0.5% (50 mM) for 2 h at room temperature. Following treatment in the respective chemical, the pellets were washed three times in dH2O and dialyzed as described above.
Carbon/nitrogen ratios
Quantitative Technologies (Whitehouse, NJ) performed the quantitative analysis of the carbon/nitrogen ratios in the cryptococcal melanin samples. Using a 2400 Perkin-Elmer CHN elemental analyzer (Norwalk, CT), the samples were converted to gases such as CO2, H2O, and N2 by combustion. The gases were separated under steady state conditions and measured as a function of thermal conductivity.
Scanning electron microscopy
Chemically treated cryptococcal melanin particles were fixed overnight in a 4% glutaraldehyde solution in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, and 1.4 mM KH2PO4). The particles were then transferred onto polylysine-coated coverslips and dehydrated by incubation in graded ethanol. The samples were mounted with gold-palladium and viewed with a JEOL (Tokyo, Japan) JAM-6400 electron microscope (17).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the Ab response to melanin, we modified conventional
serologic methods to study Ab binding to melanin. Our standard melanin
preparations from C. neoformans are comprised of 5.3 ±
1.1 µm (n = 20, by scanning electron
microscopy) hollow particles (17) that are highly negatively charged
(24) and are the melanin ghosts of melanized yeast cells. Development
of an ELISA required attachment of melanin particles to a solid phase
support. Essential steps for the successful ELISA were the use of heat
to bind the melanin particles firmly to polystyrene microtiter plates
and the extensive use of blocking reagents to reduce nonspecific
binding of Ab to melanin. In this regard, nonspecific binding of Igs to
melanin has been described (10), and thorough blocking can prevent it.
Analysis of the ELISA signal to noise ratio using sera from immunized
mice revealed that deposition of 5 x 105 purified
cryptococcal melanin particles/well produced optimal results (Fig. 1
). Preincubation of sera with melanin
particles decreased the binding of serum Ab to melanin plates (Fig. 2).
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The mouse Ab response to fungal melanin
Mice injected with either 100 or 300 µg of purified cryptococcal
melanin in CFA i.p. produced an Ab response to melanin. Serum Ab was
detected by ELISA, melanin particle agglutination, and IF. The Ab
response to the 300-µg injection was significantly greater than that
to the 100-µg dose as measured by ELISA and agglutination. Figure 3 compares the injection doses by
agglutination. IF studies revealed that sera from vaccinated mice
produced annular fluorescence, suggesting that Ab binding occurred
diffusely on the surface of the melanin particle.
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shows the Ab response as measured by
ELISA for the various isotypes. All mice made IgM and IgG, but no IgA
was detected. Among IgG subclasses, IgG2b was the dominant isotype.
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Sera were examined for reactivity with whole melanized and
nonmelanized cryptococcal cells by IF. No fluorescence was seen with
preimmune sera or nonmelanized cells (data not shown). Reactivity was
observed using hyperimmune sera with melanized cells (Fig. 5). The fluorescent pattern was annular
and localized to the cell wall where melanin is deposited. Reactivity
was significantly better using cells immobilized on the slide than with
cells in suspension.
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Since we immunized with a melanin preparation derived from
melanized C. neoformans cells, we were concerned that the Ab
response could have been directed toward nonmelanin substances in the
particle that remain attached despite acid digestion. To investigate
this possibility, we conducted additional acid and/or base digestions
on melanized cells for varying lengths of time and collected the
particles to determine carbon/nitrogen ratios, shape of the particles
by SEM, and measurements of reactivity with immune sera. Boiling
melanin particles in HCl for 1 to 4 h did not significantly alter
their carbon/nitrogen ratio (Table I).
SEM revealed that prolonged hot acid treatment resulted in the collapse
of many of the spheres, and 6 h of heating significantly altered
the shapes and surfaces of the particles (Fig. 6). Similar results were observed when
particles were boiled in concentrated NaOH with or without first
exposing them to HCl. Immune serum was more reactive with melanin
digested in HCl for 1 h than with melanin preparations made only
with Novozyme 234 and detergent. There was no significant loss of
reactivity observed after 2 h of HCl digestion, but longer times
of boiling in HCl and/or NaOH resulted in a significant reduction in
their ability to bind Ab (Fig. 7). Hence,
the epitopes recognized by sera from mice injected with melanin are
resistant to acid hydrolysis, but prolonged exposure to concentrated
acid or base appears to destroy the epitope.
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To determine whether Ab elicited in response to
L-dopa C. neoformans melanin particles
recognized other melanins, the reactivity of immune sera with
epinephrine, C. neoformans melanin, and synthetic melanin
was studied by ELISA. In addition to binding the yeast-derived melanin,
serum from mice injected with L-dopa melanin reacts, albeit
to a lesser extent, with both epinephrine and synthetic melanin (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of melanin to induce an Ab response is an interesting
finding considering its unusual biochemical features. Melanin is a
poorly characterized macromolecule that is a stable free radical (25, 26). Melanins are insoluble in all biologic fluids, and there are no
known enzymes that degrade them. For example, the melanin used in this
experiment is a preparation that consists of large (
5 µm)
particles resistant to hydrolysis in concentrated HCl acid. In general,
Ab responses are made to molecules that are processed by the immune
system (i.e., proteins) or to soluble polymers with repeating epitopes
(i.e., polysaccharides).
Our experiments with BALB/c nude mice demonstrate that melanin can act
as a T cell-independent (TI) Ag. TI Ag can induce an immune response by
Ag directly cross-linking Ig receptors on B cell surfaces (27). In
addition, interactions between TI Ag and cytokines, complement, or NK
cells have been reported to trigger B cell Ab production (28). Melanin
is presumed to be a large m.w. polymer that may contain repeating
epitopes. This description fits one of the distinct features of TI Ag.
In addition, melanin particles persist for long periods in vivo (12),
and TI Ag are considered to have long half-lives (29). Hillman and
associates developed a polymer containing a repeating peptide sequence
that stimulated TI Ab production (30). In results similar to ours,
Hillman showed that isotype switching occurred in the immune response
to their polymer in BALB/c nude mice. Isotype switching in nude mice
may occur with the assistance of small numbers of T cells found in nude
mice (31) and/or by cytokines, such as the secretion of IFN-
by NK
cells (32).
The melanins have historically been notoriously difficult compounds to study. The finding that Abs can be elicited to melanin antigenic determinants suggests that it may be possible to develop serologic reagents for the study of melanins. The demonstration that Abs raised to L-dopa C. neoformans melanin react with epinephrine C. neoformans melanin and synthetic melanin suggests that all three melanins contain shared antigenic structures. The finding that Abs raised against L-dopa melanin bind significantly less strongly to epinephrine melanin and synthetic melanin suggests that these melanins differ in antigenic structure, epitope density, and/or epitope accessibility.
Melanin immune sera were demonstrated to bind to melanized C. neoformans cells, but not to nonmelanized cells. Accessibility of Ab to cell wall structures suggests that Ab responses to cell wall Ag during the course of infection may be biologically relevant. Although functional studies were not pursued here, this observation provides information about melanin in the cell wall. Previous studies using EM have suggested that melanin is deposited primarily in the inner part of the cell wall (21). The finding that Abs can bind to melanized cryptococcal cells indicates that either some Abs can diffuse into the cell wall or that melanin is present throughout the cell wall, including its surface. The biologic function of Abs to fungal melanin is unknown, but the fact that melanin is accessible to Ab binding raises the possibility that melanin-binding Ab could mediate biologic effects in C. neoformans infection.
The studies described here were made possible by the modification of several serologic techniques to compensate for the unusual properties of melanin. Melanins are charged hydrophobic particles that can aggregate, attach poorly to solid supports, and bind proteins nonspecifically. The melanin ELISA succeeded only after determining that drying and heating of melanin suspensions resulted in the solid attachment of the melanin particles to the 96-well polystyrene plates. Extensive blocking with 2% BSA and 5% powdered milk was essential to prevent nonspecific binding. The optimal signal to background absorbance ratio was obtained with 5 x 105 melanin particles/well. At higher concentrations of melanin, the background increased. The ELISA is sensitive to Ag concentration, but proved to be an excellent system for detecting Abs to melanin. To our knowledge this is the first successful ELISA using naturally occurring melanin for the detection of melanin-binding Ab. By extensive blocking we were also able to modify two classic serologic techniques, agglutination and indirect IF, to confirm the ELISA results.
In summary, we demonstrate that melanin can elicit an Ab response in mice, a finding that establishes melanin as an immunologically active molecule that is recognized by the immune system. The mechanism by which melanin stimulates the production of an Ab response is unknown, but has similarities to TI Ag. The development of serologic techniques for the measurement of melanin-binding Abs may be useful for serologic studies in diseases involving melanin-containing cells and infections with pathogens that produce melanin. Furthermore, the existence of melanin-binding Abs indicates that polyclonal and monoclonal Ab reagents can be generated for further study of the enigmatic melanin compounds.
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Acknowledgments |
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Footnotes |
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2 The data in this paper are from a thesis to be submitted by Angel Luis Rosas in partial fulfillment of the requirements for the degree of doctor of philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University (Bronx, NY).
3 Address correspondence and reprint requests to Dr. A. Casadevall, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. E-mail address:
4 Abbreviations used in this paper: TBS, Tris-buffered saline; AP-GAM, alkaline phosphatase-labeled goat anti-mouse; IF, immunofluorescence; TI, T cell-independent.
Received for publication December 8, 1997. Accepted for publication February 10, 1998.
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References |
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: a model for T cell-independent class switching in response to T cell-independent type 2 antigens. J. Exp. Med. 175:1367.This article has been cited by other articles:
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  P. Mandal, U. Banerjee, A. Casadevall, and J. D. Nosanchuk Dual Infections with Pigmented and Albino Strains of Cryptococcus neoformans in Patients with or without Human Immunodeficiency Virus Infection in India J. Clin. Microbiol., September 1, 2005; 43(9): 4766 - 4772. [Abstract] [Full Text] [PDF]
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 A. J. Mednick, J. D. Nosanchuk, and A. Casadevall Melanization of Cryptococcus neoformans Affects Lung Inflammatory Responses during Cryptococcal Infection Infect. Immun., April 1, 2005; 73(4): 2012 - 2019. [Abstract] [Full Text] [PDF]
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 S. Youngchim, R. J. Hay, and A. J. Hamilton Melanization of Penicillium marneffei in vitro and in vivo Microbiology, January 1, 2005; 151(1): 291 - 299. [Abstract] [Full Text] [PDF]
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 E. Dadachova, J. D. Nosanchuk, L. Shi, A. D. Schweitzer, A. Frenkel, J. S. Nosanchuk, and A. Casadevall Dead cells in melanoma tumors provide abundant antigen for targeted delivery of ionizing radiation by a mAb to melanin PNAS, October 12, 2004; 101(41): 14865 - 14870. [Abstract] [Full Text] [PDF]
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D. S. Alviano, A. J. Franzen, L. R. Travassos, C. Holandino, S. Rozental, R. Ejzemberg, C. S. Alviano, and M. L. Rodrigues Melanin from Fonsecaea pedrosoi Induces Production of Human Antifungal Antibodies and Enhances the Antimicrobial Efficacy of Phagocytes Infect. Immun., January 1, 2004; 72(1): 229 - 237. [Abstract] [Full Text] [PDF]
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 D. van Duin, A. Casadevall, and J. D. Nosanchuk Melanization of Cryptococcus neoformans and Histoplasma capsulatum Reduces Their Susceptibilities to Amphotericin B and Caspofungin Antimicrob. Agents Chemother., November 1, 2002; 46(11): 3394 - 3400. [Abstract] [Full Text] [PDF]
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J. D. Nosanchuk, B. L. Gomez, S. Youngchim, S. Diez, P. Aisen, R. M. Zancope-Oliveira, A. Restrepo, A. Casadevall, and A. J. Hamilton Histoplasma capsulatum Synthesizes Melanin-Like Pigments In Vitro and during Mammalian Infection Infect. Immun., September 1, 2002; 70(9): 5124 - 5131. [Abstract] [Full Text] [PDF]
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 A. L. Rosas, R. S. MacGill, J. D. Nosanchuk, T. R. Kozel, and A. Casadevall Activation of the Alternative Complement Pathway by Fungal Melanins Clin. Vaccine Immunol., January 1, 2002; 9(1): 144 - 148. [Abstract] [Full Text] [PDF]
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W. J. Dai, A. Hemphill, A. Waldvogel, K. Ingold, P. Deplazes, H. Mossmann, and B. Gottstein Major Carbohydrate Antigen of Echinococcus multilocularis Induces an Immunoglobulin G Response Independent of {alpha}{beta}+ CD4+ T Cells Infect. Immun., October 1, 2001; 69(10): 6074 - 6083. [Abstract] [Full Text] [PDF]
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B. L. Gomez, J. D. Nosanchuk, S. Diez, S. Youngchim, P. Aisen, L. E. Cano, A. Restrepo, A. Casadevall, and A. J. Hamilton Detection of Melanin-Like Pigments in the Dimorphic Fungal Pathogen Paracoccidioides brasiliensis In Vitro and during Infection Infect. Immun., September 1, 2001; 69(9): 5760 - 5767. [Abstract] [Full Text] [PDF]
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 C. A. D'Souza, J. A. Alspaugh, C. Yue, T. Harashima, G. M. Cox, J. R. Perfect, and J. Heitman Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans Mol. Cell. Biol., May 1, 2001; 21(9): 3179 - 3191. [Abstract] [Full Text]
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 C. Luberto, D. L. Toffaletti, E. A. Wills, S. C. Tucker, A. Casadevall, J. R. Perfect, Y. A. Hannun, and M. Del Poeta Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans Genes & Dev., January 15, 2001; 15(2): 201 - 212. [Abstract] [Full Text]
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M. L. Rodrigues, L. R. Travassos, K. R. Miranda, A. J. Franzen, S. Rozental, W. de Souza, C. S. Alviano, and E. Barreto-Bergter Human Antibodies against a Purified Glucosylceramide from Cryptococcus neoformans Inhibit Cell Budding and Fungal Growth Infect. Immun., December 1, 2000; 68(12): 7049 - 7060. [Abstract] [Full Text] [PDF]
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A. L. Rosas, J. D. Nosanchuk, M. Feldmesser, G. M. Cox, H. C. McDade, and A. Casadevall Synthesis of Polymerized Melanin by Cryptococcus neoformans in Infected Rodents Infect. Immun., May 1, 2000; 68(5): 2845 - 2853. [Abstract] [Full Text] [PDF]
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J. D. Nosanchuk, J. Rudolph, A. L. Rosas, and A. Casadevall Evidence That Cryptococcus neoformans Is Melanized in Pigeon Excreta: Implications for Pathogenesis Infect. Immun., October 1, 1999; 67(10): 5477 - 5479. [Abstract] [Full Text] [PDF]
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J. D. Nosanchuk, P. Valadon, M. Feldmesser, and A. Casadevall Melanization of Cryptococcus neoformans in Murine Infection Mol. Cell. Biol., January 1, 1999; 19(1): 745 - 750. [Abstract] [Full Text] [PDF]
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