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*
Department of Microbiology and
Cell and Molecular Biology Program, School of Medicine, University of Nevada, Reno, NV 89557; and
Division of Infectious Diseases,
§
Department of Medicine, 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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-1,3-linked mannose backbone that is O-acetylated and
substituted with single side chains of xylose and glucuronic acid. The
degree of xylose substitution and O-acetylation can vary,
producing four primary serotypes (A–D) and eight chemotypes
(6, 7, 8, 9). Vaccination with a tetanus toxoid conjugate of GXM confers protection in a murine model of cryptococcosis (10). Similarly, administration of mAbs prolongs survival and decreases fungal tissue burden in experimentally infected mice (11, 12, 13). Effector mechanisms reported for anti-GXM Abs include clearance of soluble GXM by reticuloendothelial cells (14), enhanced granuloma formation (15), Ab-dependent cell-mediated cytotoxicity (16), and increased nitric oxide production by macrophages (17). However, the most important function of anti-GXM Abs is likely to be opsonization. The availability of mAbs has enabled a detailed evaluation of the biological activities of anti-GXM Abs that was not possible with polyclonal Abs. Several studies found that protective efficacy is influenced by mAb quantity (11), isotype (18, 19), and epitope specificity (12, 20).
Although the role of isotype in the biological function of an Ab is well established, the contribution of epitope specificity to protective efficacy is less well understood, particularly in the context of anticapsular Abs. Recent studies of passive immunization in experimental cryptococcosis identified protective and nonprotective anti-GXM Abs. The Abs were of the same isotype but differed in their epitope specificity (12, 20). Similarly, we found the epitope specificity of anti-GXM mAbs to be a critical variable in complement activation by encapsulated cryptococci (21). Depending on the epitope specificity of the Ab, addition of an anti-GXM mAb to the milieu 1) produces early C3 deposition via the classical pathway, 2) has no effect on C3 deposition via the classical pathway, 3) suppresses C3 binding by the alternative pathway, or 4) has no effect on the alternative pathway.
In the course of preparing cells for our studies of Ab-mediated deposition of C3 in the cryptococcal capsule, we examined the capsular reaction produced by several anti-GXM mAbs. Abs with different epitope specificities produced strikingly different capsular reactions when viewed by differential interference contrast (DIC) microscopy. The goals of the present study were to 1) clearly establish the role of epitope specificity of a mAb or epitope expression by a yeast cell in producing a given capsular reaction, 2) determine the molecular basis for different capsular reactions, and 3) identify any correlation between a given capsular reaction and Ab effector functions such as complement activation and the protective potential of an Ab. To our knowledge, our results are the first demonstration of two distinct capsular reactions and provide the first molecular explanation for the capsular reaction. Further, our results show an unequivocal association between the type of capsular reaction and biological activities of Abs that are important in host resistance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serotype A strains CN6, 388, and 62066 and serotype D strains 34875, 127, and 9375 were used throughout. Strain 388 was obtained from K. J. Kwon-Chung (National Institutes of Health, Bethesda, MD); all others were provided by R. Cherniak (Georgia State University, Atlanta, GA). The GXM chemotyping scheme described by Cherniak et al. (6) places serotype A strains CN6 and 62066 in GXM chemotype 5 and the three serotype D strains in chemotype 1. The chemotype of strain 388 has not been determined. Yeast cells used in all experiments were incubated for 4 days at 37°C on a synthetic medium (22) in 5% CO2 and killed by an overnight incubation with 1.0% formaldehyde. These conditions produce yeast cells with large capsules (23). The cells were then washed with sterile 0.01 M phosphate-buffered 150 mM saline (pH 7.3; PBS) and stored at 4°C.
mAbs and Ig fragments
mAbs 3C2, 471, 339, 1255, and 302 are IgG1. Several properties
of the Abs are summarized in Table I
;
detailed descriptions are provided in previous reports
(24, 25, 26, 27). All IgG mAbs were purified from mouse ascites
using caprylic acid precipitation of proteins other than IgG followed
by GXM immunoaffinity and protein A affinity chromatography alone or in
combination (28). Concentrations of the above Abs were
determined by UV spectroscopy using OD280 = 1.35
for 1 mg IgG/ml and OD280 = 1.2 for IgM. mAbs
12A1, 2D10, 13F1, and 21D2 are IgM Abs with protective efficacies in a
murine model of cryptococcosis that are well characterized. mAbs 12A1
and 2D10 are protective; mAbs 13F1 and 21D2 are not protective
(12, 20). Ab concentrations of mAbs 12A1, 2D10, 13F1, and
21D2 were determined by ELISA relative to isotype-matched standards of
known concentration. All IgM mAbs were in the pentameric form.
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Evaluation of Ab binding by DIC microscopy
C. neoformans cells (2 x 104) were mixed with each mAb (50 µg/ml PBS, unless otherwise noted) in a 50-µl reaction volume and incubated for 5 min at 37°C, transferred to microscope slides, and immediately examined. Capsular reactions were assessed by DIC microscopy using a Nikon Eclipse E800 microscope with a x100 oil immersion objective.
Evaluation of capsular binding by direct immunofluorescence
mAbs 3C2 and 302 were labeled with Texas Red (TR) or FITC using labeling kits from Molecular Probes (Eugene, OR). These mAbs were added singly or sequentially (50 µg/ml each) to C. neoformans CN6 cells (2 x 104 cells) and incubated for 5 min at 37°C. The cells were then washed once with PBS; the final cell pellet was resuspended in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA) and transferred to microscope slides.
Location of the FITC- or TR-labeled Abs in the capsule was determined by epifluorescence microscopy using a Nikon Eclipse E800 microscope with a x100 oil immersion objective. Images from both DIC and immunofluorescence microscopy were acquired using a Photonic Science (Millham, U.K.) integrating CCD camera and Image Pro Plus 3.0 image analysis software (Media Cybernetics, Silver Spring, MD). Fluorescence images were digitally deconvolved with MicroTome version 4.0 (VayTek, Fairfield, IA).
Kinetics of C3 binding
Normal human serum (NHS) used for complement activation studies was obtained from the peripheral blood of 10–12 healthy adult volunteers, pooled, and stored at -80°C. C3 was purified from frozen human plasma by previously described methods (29). C3 was labeled with 125I by the IodoGen procedure (30).
Kinetics for activation and binding of C3 to cryptococcal cells were determined as previously described (21) Briefly, C. neoformans cells (4.0 x 105) were incubated in a 1.0-ml reaction mixture containing 1) 40% NHS, 2) 125I-labeled C3 sufficient to produce a specific activity of 50,000 cpm/µg total C3, and 3) various amounts of anti-GXM mAbs. GVB2+ (sodium Veronal (5 mM)-buffered saline (142 mM), pH 7.3, with 0.1% gelatin, 0.15 mM CaCl2, and 1 mM MgCl2) was the buffer in experiments where C3 deposition via the classical pathway was determined. GVB-Mg-EGTA (sodium Veronal-buffered saline with 0.1% gelatin, 10 mM EGTA, and 10 mM MgCl2) was the buffer if C3 deposition via only the alternative pathway was assessed. Chelation of Ca2+ with EGTA selectively inhibits the classical pathway (31, 32). Mixtures containing NHS, 125I-C3, mAb, and the appropriate buffer were prewarmed for 5 min at 37°C, and the reaction was initiated by addition of cryptococcal cells. At various times, duplicate samples (30 µl) were withdrawn and added to 150 µl of stop solution (PBS containing 0.1% SDS and 20 mM EDTA). C. neoformans cells were washed with PBS containing 0.1% SDS using Millipore (Bedford, MA) MABX-N12 filter plates fitted with BV 1.2-µm pore size filters. The filters were removed, and the amount of cell-bound 125I-C3 was determined. Binding data are represented as the number of C3 molecules bound per yeast cell.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An initial experiment assessed the abilities of two anti-GXM
mAbs of differing epitope specificites and different molecular groups
to produce capsular quellung-type reactions visible by DIC microscopy.
Serotype A C. neoformans cells (CN6) were incubated with mAb
3C2 or mAb 302. The results (Fig. 1)
showed two capsular binding patterns. An annular or "rim"-type
pattern, in which the capsule interior appears transparent with a
highly refractive outer edge, was produced by mAb 3C2. A hallmark of
the rim pattern is an increase in the optical gradient at the capsular
edge followed directly by a decrease in the optical gradient. The
results in Fig. 1 suggest that mAb 3C2 produces a highly refractile
"shell" at the capsular surface. A second pattern, designated
puffy, is generated by binding of mAb 302. The hallmark of the puffy
pattern is an increase in the optical gradient at the capsular surface
and the absence of the immediate decrease that is characteristic of the
rim pattern. Thus, mAb 302 produces an increase in the refractive
properties of the capsule but does not produce the highly refractile
outer shell found when mAb 3C2 is incubated with the yeast cells.
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. If it is assumed that the
yeast cells are randomly oriented, one can deduce that all yeast cells
have a similar spherical shape. Second, collection of optical sections
along a z-axis through the cells using FITC-labeled mAb 3C2
or mAb 302 showed the images expected from a spherical object (not
shown). As a consequence, we conclude that the rim and puffy patterns
are due to distinct effects of the Abs on the refractive index of the
capsule rather that effects of the Ab on the shape of the cells.
To determine whether the rim and puffy patterns were due simply to the
effects of different Ab concentrations, the capsular reactions were
determined at different mAb doses. The results (Fig. 2) indicate that whereas the intensity of
the capsular reaction is dose dependent, the particular pattern
generated by a mAb (puffy vs rim) is concentration independent.
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). In an effort to more firmly establish the
role of capsular epitope expression and mAb epitope specificity in
producing a given capsular binding pattern, we examined the effect of
capsular serotype on the binding pattern produced by a single mAb.
Cells of three strains of serotype A and three strains of serotype D
were incubated with mAb 3C2. The results (Fig. 3) showed a rim pattern for all strains
of serotype A and a puffy pattern for all cells of serotype D. Study of
additional mAbs that included one more mAb of molecular group II (mAb
471), two mAbs of molecular group III (mAbs 339 and 1255), and mAb 302
(molecular group IV) showed that the capsular binding pattern is
determined by the serotype specificity of the mAb and the serotype of
the target capsular polysaccharide (Table I). Abs from group II
produced a rim pattern with cells of serotype A and a puffy pattern
with serotype D cells; Abs of group III produced a rim pattern with
cells of both serotypes; and Abs of group IV produced a puffy or
puffy-like (strain 34875) pattern with cells of both serotypes.
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Encapsulated cryptococci are potent activators of the alternative
complement pathway (34, 35). We previously reported that
anticapsular mAbs having different epitope specificities and belonging
to different molecular groups have distinct effects on the kinetics for
activation and binding of C3 to serotype A cryptococci via the
alternative pathway (21). Because mAb 3C2 and mAb 302
produced different capsular binding patterns with cells of serotype A
(rim and puffy, respectively; Fig. 1) and had different effects on C3
deposition via the alternative pathway (suppression and no effect,
respectively (21)), an initial experiment assessed the
effect of a single Ab (mAb 3C2) on the kinetics for C3 deposition via
the alternative pathway onto cells of serotype A (rim pattern) or
serotype D (puffy pattern). Cells of serotype A (strain CN6) or
serotype D (strain 34875) were incubated with 40% NHS containing
125I-C3 and mAb 3C2 (50 µg/ml). Samples were
withdrawn at various times, and the amount of bound C3 was determined.
The experiment was done in GVB-Mg-EGTA to limit C3 deposition to the
action of the alternative pathway. The results (Fig. 4) showed that mAb 3C2 markedly
suppressed the amount of C3 deposited on serotype A cells via the
alternative pathway but had little effect on C3 deposition on serotype
D cells. In an effort to further assess the congruence between 1)
production of the rim pattern and suppression of C3 deposition via the
alternative pathway and 2) production of the puffy pattern and an
absence of suppression of C3 deposition, we examined the effects of
additional mAbs from molecular groups II, III, and IV on the kinetics
for C3 deposition. The binding experiments were done as in Fig. 4 using
two additional strains each of serotypes A (388 and 62066) and D (127
and 9375). The results, summarized in Table II, showed an absolute correlation
between the capsular binding pattern and the ability of an Ab to
suppress (rim pattern) or fail to suppress (puffy pattern) C3
deposition via the alternative pathway.
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where cells of serotype A strain CN6 or serotype D
strain 34875 were incubated with 40% NHS in the presence or absence of
mAb 3C2 (50 µg/ml). The results showed early deposition (2–4 min) of
C3 when serotype A cells were incubated with mAb 3C2. However, overall
levels of C3 binding were suppressed in the presence of the mAb, a
result that is consistent with our previous studies (21).
In contrast, mAb 3C2 produced no early deposition of C3 on serotype D
strain 34875, a result that is consistent with a failure to initiate
the classical pathway.
Can the results shown in Fig. 4 can be generalized to state that
production of a rim pattern correlates with the ability of anti-GXM
mAbs to initiate the classical pathway, whereas production of a puffy
pattern correlates with a failure to initiate the classical pathway? To
address this question, we examined the effects of mAbs from molecular
groups II, III, and IV on C3 deposition onto additional strains of
serotypes A and D in the presence of 40% NHS and
GVB2+. The results, summarized in Table II,
showed that the effect of the other group II mAb (471) was identical
with that of mAb 3C2. One of the group III Abs (mAb 1255) did not
induce early classical pathway-dependent binding of C3 to either A or D
cells, whereas the other (mAb 339) activated the classical pathway only
on serotype D cells. mAb 302 was not able to facilitate early C3
binding to cells of either serotype. These results indicate that there
is a general correlation between production of the rim pattern and
early deposition of C3 via the classical pathway, but there are
exceptions in the behavior of group III mAbs.
Molecular basis and consequences of the rim pattern
One explanation for a correlation between the rim binding pattern
and suppression of complement deposition is the possibility that the
epitopes bound by rim pattern Abs are presented in a manner that allows
the mAb to cross-link polysaccharide at the capsular surface. Upon
cross-linking, the mAb could form an "Ab shell" that both accounts
for the refractile rim and prevents amplification of the complement
cascade within the capsular matrix (21, 36). If
cross-linking at the capsular surface accounts for the rim pattern, Fab
fragments should fail to produce the rim pattern and fail to suppress
C3 deposition via the alternative pathway. Indeed, we have previously
shown that Fab fragments of suppressive mAbs have no effect on the
accumulation of C3 on encapsulated cryptococci (36).
Accordingly, we examined the capsular reaction produced by serotype A
cells with F(ab')2 and Fab fragments of mAb 3C2.
The results (Fig. 5) showed that
F(ab')2 fragments produce the same rim binding
pattern as the parent IgG. In contrast, the Fab fragments produce a
puffy pattern.
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), mAb 3C2 showed binding only at the
outer edge of the capsule. In contrast, FITC-labeled mAb 302 bound to
sites distributed throughout the capsule.
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) showed that the Fab
fragments of mAb 3C2 bind to epitopes that are distributed throughout
the capsule. These results support the argument that binding of mAb 3C2
to the capsule exterior prevents subsequent binding of the Ab to the
capsule interior.
The apparent ability of mAb 3C2 to block its own binding to the capsule
interior through the formation of an Ab impermeable shell suggests that
mAb 3C2 would also block binding of mAb 302 to the capsule interior. To
test this hypothesis, mAbs 3C2 and 302 were labeled with TR and FITC,
respectively. These mAbs were added sequentially (50 µg/ml each) to
serotype A C. neoformans cells (CN6). When TR-mAb 3C2 was
added first, the TR label was found at the capsular perimeter (Fig. 7). However, immunofluorescence
microscopy showed little or no binding of FITC-mAb 302 to the capsule.
These results indicate that mAb 3C2 inhibits the binding of mAb 302 to
the capsule interior. Conversely, when FITC-mAb 302 was added first,
followed by TR-mAb 3C2, both FITC-mAb 302 and TR-mAb 3C2 bound to the
capsule (Fig. 7). An identical pattern of results was obtained if the
fluorescence label was reversed, i.e., TR-mAb 302 and FITC-mAb 3C2 (not
shown).
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). Use of the secondary Ab produced
cells with one of two capsular patterns. Most cells showed the typical
rim pattern. A second pattern was also found that was a hybrid between
the rim and the puffy patterns (not shown). This second pattern showed
a sharp increase in the optical gradient at the capsular edge typical
of the rim pattern. The gradient was more abrupt than is found with the
puffy pattern, but there was no immediate decrease in the optical
gradient characteristic of the rim pattern. Use of the secondary Ab at
a lower concentration (12 µg/ml) produced a puffy pattern. mAb 302
alone produced the expected puffy pattern, whereas the anti-IgG
alone showed no apparent binding to the capsule (not shown). An
evaluation of the effect of the anti-IgG secondary Ab on C3
deposition via the alternative pathway showed the expected result that
no suppression occurred when serotype A cells were treated alone with
mAb 302 or the anti-IgG F(ab')2. However,
addition of the second Ab to mAb 302-treated cells produced a
dose-dependent suppression of C3 binding (Fig. 9). These results indicate that both the
capsular reaction and the effect of the Ab on C3 deposition can be
modified by a cross-linking second Ab.
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In addition to the role of epitope specificity in determining the
potential of a mAb to suppress C3 deposition via the alternative
pathway, epitope specificity also determines the ability of
anti-GXM IgM mAbs to mediate protection in a murine model of
cryptococcosis (12, 20). Previous studies demonstrated
that IgM mAbs 12A1 and 2D10 are protective, whereas mAbs 21D2 and 13F1
are not. Evaluation of the capsular reaction by DIC showed that the
protective IgM mAbs produced the rim pattern with cells of serotype A
strain CN6; the nonprotective Abs produced the puffy pattern (Fig. 10). An evaluation of the capsular
reaction with serotype D strains, including strain 24067 which was
originally used to assess protective efficacy of the Abs, produced
similar results; a rim pattern was observed with cells incubated with
protective Abs and only the puffy pattern was observed with
nonprotective Abs (not shown).
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). Protective mAbs 12A1 and 2D10,
which produce the rim pattern on serotype A cells, suppressed the rate
and amount of C3 bound to encapsulated cryptococci when Mg-EGTA was
included in the reaction mixture. In contrast, the nonprotective mAbs
21D2 and 13F1, which produce the puffy pattern, showed very limited
suppression of C3 deposition in the presence of Mg-EGTA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A C. neoformans capsular reaction has been demonstrated by use of polyclonal (4, 5) and monoclonal (3) Abs. As visualized by DIC microscopy, we now report that mAbs of differing epitope specificities produce one of at least two capsular binding patterns (rim or puffy). Evidence that the capsular pattern is determined by epitope specificity is derived from two complimentary lines of study. First, Abs with different epitope specificities produce distinct patterns with cells of serotype A. The rim pattern was produced by group II mAbs, which recognize an epitope shared by all four serotypes, and by group III mAbs, which recognize an epitope found on serotypes A, B, and D. In contrast, group IV mAbs, which recognize an epitope found only on serotypes A and D, produce a puffy pattern on these same cells. Second, a single mAb can produce different capsular binding patterns on cells of different serotypes. For example, group II mAbs produce a rim pattern on serotype A cells and a puffy pattern on serotype D cells.
The capsular quellung reaction was one of the first means reported to study Ag-Ab interactions. Since its initial discovery, whether the reaction actually involves a change in volume has been the subject of some controversy (38, 39). In the case of the cryptococcal capsule, the capsular reaction does not involve capsular swelling (2, 4). Less well understood is the molecular mechanism for the capsular reaction. Indeed, to our knowledge, the physical and molecular basis for the capsular reaction has not been determined.
DIC microscopy provides insight as to the physical basis for the capsular reaction. DIC systems are designed to detect an optical gradient, preserve its sign, and convert the gradient into intensity variations. Opposite gradients will have opposite intensities by transmission microscopy. The optical gradient is proportional to the product of the geometric gradient and the difference in refractive index. Because encapsulated cryptococci that were treated with different mAbs were spherical and of a similar size, the geometric gradient is similar in all specimens. As a consequence, the observed variation in light intensity primarily reflects changes in the refractive index in the specimen (33). These changes appear as variations in light intensity (dark to light or light to dark) against a gray background. The rim pattern is characterized at the capsule edge by an initial increase (dark) followed by an immediate decrease (light) back to the background (gray), giving the capsule interior a transparent appearance, where no change occurs until the other side of the capsule edge where dark is again followed by light at the capsule/media interface. This appearance identifies a striking increase and a rapid decrease in the refractive index at the capsular edge. A likely explanation for this refractile rim is the accumulation of a large amount of protein, i.e., Ab, at the edge of the capsule. Alternatively, the refractile rim could be caused by an Ab-dependent change in hydration at the capsular surface. In contrast, the puffy pattern is characterized by an initial increase (darkening) at the capsule edge followed by a gradient of change throughout the capsule. These results suggest that the two classes of Abs (rim vs puffy) have fundamentally different effects on the capsular structure.
A molecular explanation for the rim pattern is cross-linking of the capsular surface by Ab. If Ab cross-linking were involved, a requirement for bivalency in production of the rim pattern would be expected. This is what was found when the binding patterns of Fab and F(ab')2 fragments of rim pattern-producing mAb 3C2 were examined. F(ab')2 fragments produced a rim pattern identical with that of the parent IgG. Fab fragments produced a puffy pattern.
Puffy and rim pattern Abs localize differently in the capsule, as shown
by direct immunofluorescence microscopy (Fig. 6). An important question
is whether the apparent peripheral binding site for Abs producing the
rim pattern reflects localization of the epitope only at the capsular
surface or is a consequence of cross-linking of the capsular surface.
Several lines of evidence indicate that peripheral binding is due to
the formation of a shell with reduced permeability to macromolecules at
the capsular surface. First, FITC-Fab fragments of the rim
pattern-producing mAb 3C2 bound throughout the capsule (Fig. 6).
Second, mAbs that produce the rim pattern suppress the rate and amount
of C3 deposition through the alternative pathway, suggesting reduced
access for complement components to the capsule interior
(21). Third, preincubation of C. neoformans
cells with mAb 3C2 completely blocks subsequent binding of mAb 302, an
Ab that normally binds throughout the capsule (Fig. 7). Fourth, the
presence of a cross-linked Ab shell that excludes binding of C3 and mAb
302 binding is supported by the requirement for Ab bivalency for both
rim pattern production (Fig. 5) and suppression of C3 deposition
(36). Finally, cross-linking of the puffy
pattern-producing, nonsuppressive mAb 302 with a secondary Ab results
in both production of the rim pattern (Fig. 8) and a dose-dependent
suppression of C3 deposition (Fig. 9).
IgM mAbs that are protective in a murine model of cryptococcosis
produce a rim binding pattern (Fig. 10) and suppress C3 deposition
(Fig. 11). Nonprotective IgMs produce the puffy pattern and do not
influence complement deposition. Previous studies reported that the
nonprotective IgM Abs form punctate patterns when a FITC-conjugated
secondary Ab is added (37). The relationship between the
puffy pattern observed by DIC and the punctate pattern observed by
indirect immunofluorescence is uncertain. The differences in appearance
may be a consequence of the imaging methods or use of a secondary Ab.
The rim pattern observed by DIC has striking similarity to the
appearance of the annular pattern observed by indirect
immunofluorescence for protective mAbs (37). These two
patterns are likely due to similar molecular mechanisms; however, the
role of cross-linking in production of the annular pattern described by
Nussbaum et al. has not been determined.
In contrast to the IgM Abs, IgG1 mAbs producing either capsular pattern (rim or puffy) are protective (40). This raises a question as to why capsular binding pattern is a determinant of protective efficacy for IgM, but not IgG mAbs. A likely explanation involves the interaction of Ab and C3 with phagocytes. Cryptococci opsonized with IgG and C3 would be bound by both Fc and C3 receptors on macrophages, neutrophils, and monocytes. However, yeast cells opsonized with IgM and C3 can be bound only through C3 receptors, given that an Fc receptor for IgM Abs on these cells has not been found. We hypothesize that cross-linking by rim pattern IgM Abs decreases mobility of the capsule-bound C3 and thereby increases the stability of the interaction between C3 and its corresponding receptor, enhancing phagocytosis, and as a result determines protective efficacy by IgM Abs.
In this study, we present data suggesting Ab-mediated cross-linking of the capsular surface as a unifying hypothesis to explain differences in mAb capsular localization, influence on the kinetics for activation and binding of C3, and protective efficacy. The large capsule of C. neoformans and the availability of an extensive library of anti-GXM mAbs provided a unique system in which to evaluate mAb-capsular interactions. The value of the large capsule of C. neoformans for study of fundamental aspects of the capsular reaction was noted 50 years ago by Neill et al. (4) and remains true today. The capsule reaction observed after Ab binding to C. neoformans is due to Ab-induced changes in the refractive index of the capsule. Ab-mediated capsular cross-linking may also be important to the efficacy of Abs reactive with other encapsulated pathogenic organisms.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas R. Kozel, Department of Microbiology/320, School of Medicine, University of Nevada, Reno, NV 89557.
3 Abbreviations used in this paper: GXM, glucuronoxylomannan; DIC, differential interference contrast; NHS, normal human serum; TR, Texas Red.
Received for publication November 30, 1999. Accepted for publication February 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated murine macrophage-like J774.16 cells. J. Leukocyte Biol. 57:657.[Abstract]
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D. O. Beenhouwer, R. J. May, P. Valadon, and M. D. Scharff High Affinity Mimotope of the Polysaccharide Capsule of Cryptococcus neoformans Identified from an Evolutionary Phage Peptide Library J. Immunol., December 15, 2002; 169(12): 6992 - 6999. [Abstract] [Full Text] [PDF]
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G. R. McLean, M. Torres, N. Elguezabal, A. Nakouzi, and A. Casadevall Isotype Can Affect the Fine Specificity of an Antibody for a Polysaccharide Antigen J. Immunol., August 1, 2002; 169(3): 1379 - 1386. [Abstract] [Full Text] |
100 µg/ml).
