THE JOURNAL OF INFECTIOUS DISEASES • VOL. 125, NO.2·
© 1972 by the University of Chicago. All rights reserved.
FEBRUARY 1972
Anti-Candida Factors in Serum and Their Inhibitors.
II. Identification of a Candida-Clumping Factor and the
Influence of the Immune Response on the Morphology of
Candida and on Anti-Candida Activity of Serum in Rabbits
J. Kelly Smith and Donald B. Louria
The ability of normal rabbit serum to reduce populations of Candida albicans in
vitro is due in part to the clumping of viable blastospores by a macroeuglobulin
of fast beta mobility. Gradual loss of this clumping activity occurs during active
immunization of rabbits with Candida, and this loss is clearly related to the appearance of humoral antibody to Candida rather than to a reduction in clumping factor.
The antibody may interfere with clumping by competing successfully for binding
sites on the blastospores and by being present in quantities sufficient to produce
antibody excess. The 7S antibody to Candida also appears to promote mycelial
transformation, possibly by mitigation of the activity of mitochondrial disulfide
reductase or by induction of enzymes.
The effect of serum on the viability and growth
of Candida albicans has recently been the subject
of rather intense interest. Roth and Goldstein [1]
have shown that normal serum from humans inhibits the growth of C. albicans if small numbers
of yeast cells are added to the serum in vitro;
Caroline and co-workers [2] have attributed this
effect to the action of transferrin, a protein with
broad antimicrobial activity [3, 4]. In addition,
we have shown that normal serum from humans
will reduce populations of C. albicans in vitro in
a manner that appears to be related to the activity
of a heat-stable alpha or beta globulin [5]. Initially, this anticandidal activity was thought to be
a candidacidal effect, but recent studies suggest
that the lowered candidal census results from
clumping of the yeast and mycelial forms rather
than from killing [6]. Reduction of candida popu-
lations also occurs in the sera of rabbits, rats,
and guinea pigs (unpublished data).
The ability of serum from patients with mucocutaneous candidiasis to reduce populations of
C. albicans in vitro is frequently diminished or
absent [7], raising the question as to whether the
lack of anticandida activity is important in the
pathogenesis of infections involving Candida. Interestingly, Chilgren, Hong, and Quie [6] have
obtained evidence that the vitiation of anticandidal
activity is related to the presence of IgG antibody
to Candida.
In the present study, attempts have been made
to identify the factors responsible for the in-vitro
reduction of populations of C. albicans by normal
serum from rabbits. The effect of active immunization with Candida on the anticandidal properties
of rabbit serum has also been investigated.
Received for publication December 29, 1970, and in
revised form June 13, 1971.
This research was supported by NIAID grant no.
I-F3-AI-18-735-01 and American Cancer Society grant
no. PF 364 (Dr. Smith) and by NIAID grant no.
AI-08467 (Dr. Louria).
Please address requests for reprints to Dr. J. K. Smith,
North Shore Hospital, Manhasset, New York 11030.
Materials and Methods
Immunization of rabbits. Two strains of C.
albicans, designated A 2 and E 3 , were used for
immunization. Mouse-passed yeasts were inoculated onto Sabouraud's medium and incubated
for 18 hr at 25 C. The cells were collected in
115
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From the Division of Medicine (Infectious Diseases),
North Shore Hospital, Manhasset, New York;
Department of Medicine, Cornell University
Medical College, New York, New York;
and the Department of Public Health and
Preventive Medicine, College of Medicine and
Dentistry of New Jersey, Newark, New Jersey
116
scopically. Broth that had been inoculated with
candida served as a zero-hour control in each
test.
All samples except those containing unfractionated serum were fortified with an equal volume of a nutrient medium containing equal
amounts of a saline extract of heat-killed Candida
and 5% glucose in 0.9% NaCI. The extract was
prepared by heating washed yeast-phase Candida
at 65 C for 30 min, suspending them in saline,
and freezing the mixture at -20 C for 48 hr.
Upon thawing, the cells were removed by centrifugation at 5,860 g for 1 hr. The supernatant
fluid was standardized to an optical density of
1.80 (280 urn) and added to an equal volume
of 5% glucose in 0.9% NaCI; the mixture was
frozen at -20 C until use. The extract was effective in supporting the growth of Candida in cultures that were otherwise devoid of nutrients
essential for the growth of the organism.
Pevikon electrophoresis. Six-ml samples of
serum were fractionated by block electrophoresis
on Pevikon with a barbital buffer of 0.1 ionic
strength, pH 8.2. Separations were done at 4 C
for 30-36 hr at 350--400 v. One-em fractions
were removed and the protein was eluted with
5-15 ml of 0.9% NaCI. Protein determinations
were made on the eluates by the method of
Lowry [10].
Column chromatography. Two- to 5-ml samples of serum or protein were applied to a 5 X
50-em column of Sephadex G-200 buffered with a
mixture of Tris and NaCI (0.05 M Tris, 0.10
M NaCl, pH 7.5), and 5-ml eluates were collected at a flow rate of 50-100 ml/hr. Gradient
elution on carboxymethyl cellulose was done at
pH 5.0, starting with a 0.04 M sodium acetate
buffer. Concentrations of protein in the eluates
were estimated by readings of optical density at
280 nm.
Salt fractionation. Fractions of euglobulin
and pseudoglobulin were prepared by the method
of Mayer [11].
Immunoelectrophoresis. Immunoelectrophoresis was performed by the method of Scheidegger
[12], using goat antiserum to rabbit serum
(Hyland) or goat antiserum to rabbit gamma
globulin (Hyland) as the antibody source. Mobilities of precipitating antibodies to Candida
were determined by electrophoretically separating
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0.9% NaCl, washed three times in this saline,
and killed by heating at 56 C for 1 hr. Eight
albino rabbits were immunized by the iv route
with 5 X 10 7 cells three times per week for two
to five weeks. The rabbits were bled before immunization and at weekly, bi-weekly, or monthly
intervals thereafter for a period extending from
four to 31 weeks.
Assays for agglutinins and precipitins. Sera
were assayed for agglutinating antibody to a Tyson strain of C. albicans by the method of Buck
and Hasenclever [8]. The highest dilution that
produced visible aggregated particles was considered to be the titer of agglutination. Assays for
precipitins were done with 0.5 % agarose and a
sonicated extract of the E 3 or A 2 strains of
Candida (S antigen). The extract was prepared
according to the method of Taschdjian et aI. [9],
and was used in concentrations of 0.5 to 1.0 g of
protein/100 mI. Precipitins were read after diffusion for 72 hr at room temperature.
Absorptions. Cultures of C. albicans or Aspergillus niger were suspended in saline and killed
by heating to 65 C for 30 min. The organisms
were washed three times with normal saline and
added to the samples of serum in a ratio of two
parts serum to one part packed cells (v/v). The
mixtures of serum and fungi were rotated for 2
hr at 37 C and incubated at 4 C for 12-18 hr;
the cells were then removed by centrifugation at
2,000 rpm for 30 min.
Skin testing. Both before immunization and
at intervals during the first eight weeks after the
initiation of immunization, rabbits were skintested intradermally with 0.1 ml of S antigen,
oidiomycin (Hollister-Stier), or mannan. Reactions were recorded as mm of induration at 48
and 72 hr after skin testing; induration of greater
than 10 mm was considered significant.
Reduction of candida populations by serum.
1.0-3.9 X 10 6 blastospores from a 12-18-hr culture of A 2 or E 3 strains of C. albicans were added
to 1.0-ml samples of serum or other material
under study, and the mixtures were rotated at 22
rpm at 37 C. Samples were removed after 6 hr
and mixed by vigorous pipetting; pour plates were
made using Sabouraud's medium. Growth of
Candida was measured by colony counts after 4872 hr of incubation of the plates at room temperature. Preparations of 6-hr rotary samples
were mounted on coverslips and examined micro-
Smith and Louria
Anti-Candida Factors II
immune sera and allowing them to diffuse against
S antigen for 72-96 hr at room temperature.
Results
terial was resuspended in 0.9% NaCI in concentrations of protein ranging from 7.8 mg/IOO
ml to 2.0 g/IOO ml, and was tested in standard
rotary experiments with Candida. A solution containing 1.0 g of macrogiobulin/IOO ml was assayed for agglutinins and precipitins to Candida.
Concentrations of 15 mg or more of 100 ml
macro-euglobulin reduced populations of Candida 20-fold or more and induced clumping typical of that in whole normal serum (figure 2).
The preparation had no agglutinating or precipitating antibody activity against formalin-killed
Candida.
Immune rabbit serum. Relation of population
reduction and clumping activity to immune response. Eight rabbits were immunized with
Candida and their sera examined for agglutinating
antibody to whole Candida and precipitating antibody to S antigen, as well as for their ability to
reduce populations of Candida in 6-hr rotary experiments; all rotary experiments were done with
Figure 2.
Clumping of Candida albicans by normal rabbit serum.
Figure 1.
Clumping of Candida albicans by a
macro-euglobulin preparation with mobility of fast
beta globulin (31 mg of proteinll 00 ml).
Downloaded from http://jid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 11, 2016
Normal rabbit serum. Population reduction
and clumping activity. The ability of normal
rabbit serum to reduce populations of Candida
in 6-hr rotary experiments was determined.
Twenty-two samples of serum from nonimmunized animals reduced colony counts from fiveto l23-fold; the average was a 30-fold reduction
over zero-time controls. This reduction was associated with clumping of yeasts and mycelia.
Typically, the clumps consisted of tight central aggregates of yeasts with some central intertwining
and peripheral outgrowth of mycelial elements
(figure 1). Clumping did not occur in two rotary
experiments using heat-killed Candida.
Isolation of clumping activity. Pooled normal
rabbit serum was fractionated by salting-out, electrophoresis on Pevikon, and chromatography on
Sephadex G-200; the various fractions were assayed in standard rotary experiments. Chromatographic fractions were concentrated by lyophilization to the original volume of the applied sample.
Clumping and population-reduction activity comparable to that of normal rabbit serum was found
in euglobulin fractions, in Pevikon eluates of
high mobility (beta globulins), and in macroglobulin fractions. Based on these findings, partially purified clumping factor was obtained by
electrophoresis of a preparation of euglobulin
from pooled normal rabbit serum, followed by
chromatographic fractionation of active betamobility fractions; eluates of macroglobulin were
then pooled and lyophilized. The lyophilized ma-
117
118
Skin Test
25
1j
"'s
20 :::'
15 1i5
I
180
10
:: 160 ~
E
~
~
o
Xl
§ 120 L
~
I~
100
:;;
60
!
5 1' ~
140'
1
~
80 l
320 ~
i=
Antl-S
14
40
ex:
....:
I
1.8
pr~IPM!DgjlntIQojlxt .
u
.f
1280
1640
E
E
/
.....-
....
0 ---.--~
o
2 4 5
I I. I I
,,P-
8
I 2 20
II 0
31
Weeks
7
.- 3 i. v, doses of 5x10 candida
Figure 3. Kinetic relation between activity of serum from a single rabbit in reducing populations of
Candida and immune response. 0
Oidiomycin,
S antigen, and 0
mannan.
•=
=
=
tion activity were reached again 11-12 weeks
after the period of immunization had ended. As
with the other immunized animals, the capacity of
the serum of this rabbit to clump Candida diminished as the humoral antibody response intensified (figure 4).
Effect of purified antibody to Candida on activity of clumping factor. Support for what appeared to be a paradoxical interference with the
clumping of Candida by antibody to Candida was
obtained in a demonstration of the capacity of
semipurified antibody to Candida to interfere
with the activity of the clumping factor. Globulin
fractions containing agglutinin activity were prepared by Pevikon electrophoresis; the fractions
were pooled and further purified by chromatography on carboxymethyl cellulose and Sephadex
G-200 as previously described. Active fractions
were lyophilized, and dissolved in 0.9% NaCI to a
concentration of protein of 1.0 g/100 ml. Purity
of the preparation was determined by immunoelectrophoresis using caprine antiserum to whole
serum and gamma globulin of the rabbit. Precipitins to Candida and mobility of precipitating
antibody were determined in the manner previously described. The solution of antibody was assayed in standard rotary experiments against
viable Candida both in the presence and in the
absence of 15 mg/ 100 ml of semipurified clumping factor. Controls included the same concentra-
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sera both undiluted .and at dilutions of 1: 2 and
1:5, in 0.9% NaCI or Hanks' solution. Skin
reactivity of seven of these animals to S
antigen and oidiomycin was determined; skin
testing with mann an was performed on one rabbit. The results of these studies are summarized
in table 1.
A rise in agglutinating antibody to Candida was
evident in the sera of all eight animals by the
second week of immunization; titers peaked by
the fourth or fifth week. Low titers of precipitating antibody were detected only during the
peak agglutinin response. Skin reactivity to S
antigen became manifest in seven to 14 days;
none of the animals developed skin reactivity to
oidiomycin or mannan.
In each animal, immunization resulted in a
gradual decrease in population-reduction activity of whole serum, and this decrease paralleled
the appearance of agglutinating and precipitating
antibody. Significant loss of activity occurred only
when titers of agglutinins reached 1: 80-1: 160 or
more. In each instance, loss in population-reduction activity was also associated both with a corresponding decrease in the number, size, and
tightness of clumps, and with the appearance of
elongated mycelia and pseudomycelia, both
loosely interwoven and free. In all the animals,
both the population-reduction and clumping activities could be partially or completely restored
by diluting the immune sera before rotating them
with Candida; in general, the higher the titer of
antibody, the greater was the dilution necessary
to restore activity. Absorption with heat-killed
Candida restored population-reduction and
clumping activity to six hyperimmune sera,
whereas similar absorptions with Aspergillus niger
did not.
The kinetics of this relationship in a rabbit
immunized with Candida for 41;2 weeks and followed for 31 weeks (rabbit no. 8) is illustrated
in figure 3. It is apparent that populations of
Candida in rotary mixtures of serum and Candida increased as the titers of antibody increased, and fell as the titers of antibody fell,
with appreciable changes in population-reduction
activity only when titers of agglutinin reached
levels of 1: 80 or greater. The skins of the
animals reacted to S antigen at seven weeks, but
not to oidiomycin or to mannan. Preimmunization levels of antibody and population-reduc-
Smith and Louria
Anti-Candida Factors II
Table 1.
119
Relation between effect of serum on growth of Candida and immune response.
Growth of Candidat
Antibody in serum
Rabbit
Day of
immuni- Aggluzation
tinins
Precipitins
0
I: 10
1:15
1:40
1: 160
1:80
0
0
0
0
1:2
1:1
0
3
6
16
28
35
0
1:5
1:15
1:60
1: 160
1:320
0
0
0
0
1:2
1:8
0
3
6
16
28
35
0
1:5
1:5
1:320
1:640
0
0
0
0
1:8
4
0
14
28
1:20
1:30
1: 160
5
0
14
28
6
0
14
28
7
0
14
28
35
56
70
84
98
112
140
217
0
14
28
35
56
70
84
98
112
140
217
2
3
8
* Induration
S
antigen
Oidiomycin
6-hr rotary mixtures
O-hr inoculum
Whole
serum
172
13
60
10
280
13
0
0
0
172
1:5 dilutions
Schedule
of immunizationt
Days 1-3,
6-8,22-24
7
7
280
11
9
132
16
93
69
380
15
1: 2 dilutions
Days 1-3,
6-8,22-24
7
35
258
17
8
246
0
172
15
Days 1-3,
6-8,22-24
5
80
320
129
5
20
108
20
17.5
0
0
0
1:2
0
10
8
0
0
0
163
6
10
91
5
9
27
9
1
13
Days 1-3,
16, 17, 20
1:5
1: 10
1:320
0
1: 1
1:2
5
15
21.5
0
0
0
163
26
9
245
7
3
149
8
2
96
Days 1-3,
16, 17, 20
1: 10
1:25
1:80
0
0
1:2
8
12.5
16
0
0
0
163
7
11
87
4
7
56
1
6
16
Days 1-3,
16, 17, 20
1:15
1:160
1:640
1:640
1:320
0
0
369
10
0
3
12
180
162
128
19
28
3
4
5
12
11
4
6
17
16
1
2
6
3
3
1
1
3
8
11
8
1
3
1
3
Days 1-3,
8-10, 1517, 22-24,
29-31
1: 160
1:80
1:80
1:40
1:40
0
0
0
1:1
0
0
0
0
0
0
0
1:30
1:320
1:640
1:640
1:320
1: 160
1: 160
1:40
1:20
1:30
1:30
0
0
0
1:2
0
0
0
0
0
0
0
0
0
397
0
2
11
73
101
9
2
4
4
5
2
1
14
11
37
5
29
0
4
7
7
Days 1-3,
8-10, 1517, 22-24,
29-31
10
7
54
162
168
104
52
16
28
10
6
28
(mm) 72 hr after skin test.
t Colony counts/ml X 104 •
:j: On each day rabbits received 5 X 107 heat-killed Candida intravenously.
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0
3
6
16
28
35
Reactivity of skin *
Smith and Louria
120
Relation between agglutinating-antibody response and clumping of Candida.
tion of clumping factor alone, and 7S gamma
globulins prepared from pooled sera of nonimmune rabbits by a procedure identical to that used
in purification of antibody.
The results are listed in table 2. Analysis of
the purified solution of antibody revealed predominantly IgG of gamma! mobility with minor
IgA contaminants. The titer of agglutinins was
1:80, and precipitins were detectable in a 1: 2
dilution; the precipitating antibody had the mobility of gamma! globulin. The control preparation
of 7S globulin was found on immunoelectrophoresis to have a similar content of protein, but
no activity of agglutinating or precipitating antibody. In rotary experiments, the antibody promoted a level of candida growth that was significantly above the zero- and 6-hr control levels;
this growth was associated with a total absence
of clumping and with marked mycelial and
pseudomycelial transformation (figure 5). Addition of antibody to the clumping factor significantly interfered with its population-reduction
and clumping capacities. The 7S globulin from
nonimmune rabbits had no appreciable effect on
populations of Candida, clumping or mycelial
transformation and did not alter the clumping
capacities of semipurified clumping factor.
Table 2. Effect of semipurified 7S antibody to Candida on activity of clumping factor.
Titers of antibody
to Candida
Growth Mycelial
of
transforCandida" mationt
Sample
Agglutinins
Precipitins
7Simmune
globulin
1:80
1:2
625
7S nonimmune
globulin
0
o
220
Clumping
factor t
0
o
9
Clumping
factorr +
immune
globulin
1:80
1:2
Clumping
factort +
nonimmune
globulin
0
o
6
Nutrient
control
0
o
226
* Colony
550
4+
3+
counts/rnl X 104 in 6-hr rotary cultures;
O-hr inoculum
192 X 104 colonies/ml,
t Numbers indicate percentage of Candida in mycelial or pseudomycelial phase: 4+
>70%; 3+
40-70%; 2+
10-40%; 1+
<10%.
:j: Concentration of macro-euglobulin
15 rng protein/
100 ml.
=
=
=
=
=
=
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Figure 4.
Anti-Candida Factors II
Discussion
The results of this study indicate that the ability
of normal rabbit serum to reduce populations of
C. albieans in vitro is due in part to the clumping
of viable blastospores by a macro-euglobulin with
the mobility of a fast beta globulin. This preparation enhances the natural tendency of Candida to
clump during its blastospore phases, but it has no
demonstrable antibody activity. It is unclear as to
whether the protein itself or its nonprotein constituents (i.e., lipids) are responsible for the
clumping. It is apparent, however, that the clumping activity of normal rabbit serum is not simply
the result of a single factor acting on Candida,
but rather the end result of several variables acting in concert. The inability of normal serum to
clump nonviable candida stresses the importance
of cell-dependent factors on this process, and
may explain the lack of agglutinating activity of
normal serum in standard assays using nonviable
Candida.
The loss of serum-clumping and populationreduction activity during active immunization
with heat-killed Candida is clearly related to the
appearance of humoral antibody to Candida.
Supporting the contention that the antibodies
themselves are responsible for this loss are the
following findings: (l) There is a striking similarity between the kinetics of the response of
antibody to Candida and the loss of clumping.
(2) There is a positive correlation between the
degree of antibody response and the loss of
clumping. (3) Clumping activity can be restored
by prior absorption of immune sera with C. albieans but not with A. niger; and (4) Fractions
of purified 78 gamma globulin containing high titers of agglutinins to Candida vitiate the activity
of semipurified clumping factor, whereas fractions
of similarly purified 78 globulin from the same
nonimmunized animals do not.
Although it is likely that antibody to Candida
interferes with the clumping and populationreduction activity of normal rabbit serum, the
mechanism by which it does so is unclear. One
possibility is that the antibody prevents clumping
of blastospores by preferentially binding to the
yeast forms in quantities sufficient to produce excess of antibody, although definitive evidence to
support such an hypothesis is lacking.
When incubated with yeast-phase Candida,
both immune sera and semipurified antibody to
Candida appear to promote mycelial and pseudomycelial transformation. Nickerson and Falcone
[13] and Nickerson [14] have postulated that extension of yeast forms occurs as a result of persistent disulfide-protein-mannan linkages in the
cell walls of such forms, and that the plastic deformation and fibrillar ordering necessary for cell
division are dependent upon the reduction of the
disulfide linkages by mitochondrial protein disulfide reductase. It is interesting to note that
these protein-mannan complexes in cell walls are
among the most antigenic constituents in C. albieans [15, 16], and that rabbit 78 agglutinating
and precipitating antibodies similar in mobility
to those produced in these experiments have been
shown by Matthew et al. [16] to be directed
against mannan antigens. Hence it is possible that
antibody promotes mycelial transformation by
mitigating the activity of disulfide reductase, and
that it does so by binding to the mannan-proteindisulfide substrate and altering the usually rigid
physicochemical and stereochemical requirements
of interaction between enzyme and substrate.
An alternative explanation for the effect of
antibody on mycelial transformation may be
found in the studies of Chattaway and Holmes
[17], who theorized that mycelial transformation
is directed by a variation in the activity of enzymes that produce cell-wall components, and
that both an increase in the production of chitin
and alterations in the type of proteins synthesized
were essential to mycelial production. It is possi-
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Figure 5. Marked mycelial and pseudomycelial
transformation and lack of clumping in mixture of
Candida and semipurified antibody to Candida.
121
122
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Downloaded from http://jid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 11, 2016
bIe, therefore, that antibody to Candida promotes
mycelial transformation by activating membrane
or intracellular systems of enzymes in a manner
yet to be determined. Our preliminary studies indicate that arginine specifically induces mycelial
transformation in certain strains of C. albicans,
possibly by induction of enzymes. It should be
noted that arginine is the only amino acid that is
found in mycelial phases of C. albicans but not in
blastospore phases [17]. The possibility that antibody to Candida enhances mycelial transformation because of an unusually high content of Nterminal arginine is under study.
We are currently using rabbits and mice as experimental models in investigating the role of
clumping factor in defense of the host against
invasion by Candida and the intriguing possibility
that antibody to candida paradoxically promotes
infection with candida.
Smith and Louria