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This is to certify that the

dissertation entitled

MURINE RESPONSES TO CANDIDA ALBICANS: IN

VIVO FOOTPAD AND _I__lt_l_ VITRO PHAGOCYTIC STUDIES

 

presented by

JAMES MICHAEL VESELENAK

has been accepted towards fulfillment

 

of the requirements for
PhD degree in M01 093’
(Botany)

W

Date November 9, 1981

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

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MURINE RESPONSES TO CANDIDA ALBICANS: ///

1/

I VIVO FOOTPAD AND IN_VITRO PHAGOCYTIC STUDIES

By

James Michael Veselenak

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

l981

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j v . r l A
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ABSTRACT
MURINE RESPONSES TO CANDIDA ALBICANS: I VIVO FOOTPAD AND I VITRO

 

PHAGOCYTOSIS STUDIES

by

James Michael Veselenak

In_yjyg_footpad responses to injected Candida albicans were
characterized and compared in non-sensitized, sensitized and cylophos-
phamide (CY) pretreated mice. Non-sensitized mice reacted to an
injection of live yeast cells with a maximal 4 h footpad swelling
response. Greater than 99% of the injected yeast cells were killed by I
72 h. Mice were sensitized by prior subcutaneous injections of heat-
killed yeast cells and showed specific footpad responses as well as
antibodies to the antigen. Sensitized mice were not immune to
systemic infection when compared to non-sensitized mice. Greater
footpad responses were seen in sensitized mice after injection of live
yeasts although the rate of killing was no different than in
non-sensitized mice. Cyclophosphamide pretreatment resulted in a
severe granulocytopenia. These animals were more susceptable to systemic
candidosis. Footpad swelling responses were similar to those of
non-pretreated animals, but the ability to control the localized
infection was impaired.

Histopathological examination of footpad sections after infection
revealed an early infiltration which was exclusively granulocytic in

non-sensitized mice while sensitized mice showed a predominantly

James Michael Veselenak

granulocytic infiltrate although later a mononuclear component was
evident. Responses seen in CY pretreated mice were similar in cellular
makeup but less intense than in non-drug treated mice. Fungal
proliferation within necrotic areas was seen.

Polymorphonuclear neutrophils were used in the jg_yjtrg_phagocyto-
sis and candidacidal assays. Approximately 50% of the phagocytosed
yeasts were inhibited in germ tube formation while CY pretreatment
resulted in 29% and l6% germ tube inhibition for the 100 mg/kg and
200 mg/kg groups, respectively. Yeast cells and PMN incubated for 2 h
showed approximately 43% of the yeasts killed in the non-CY groups
while up to 90% were still viable in the 200 mg/kg groups. No effect
was seen with PMN from non-drug treated mice when incubated in various
concentrations of CY.

Cumulatively these data suggest that non-specific host defense
mechanisms are primarily responsible for the control of infection with
Q, albicans. Any alteration of this system resulting from the use of
cytotoxic or immunosuppressive agents will predispose the host to
infection by this organism. In addition, immunological hypersensitivity

to this yeast does not necessarily imply immunity to infection.

ACKNOWLEDGEMENTS

The author wishes to thank Dr. E.S. Beneke for serving as my
graduate chairman. His careful and critical review of this manuscript
and help and guidance throughout my graduate career is much appreciated.
Special thanks is extended to Dr. A.L. Rogers for his support and
ideas during this study. The author also wishes to thank Dr. R.J. Moon
and Dr. H.N. Tvedten for serving as members of my graduate committee
and for their careful review and comments in the preparation of this
manuscript.

I extend special thanks to Dr. Paul A. Volz of Eastern Michigan
University for his constant encouragement throughout my graduate
career. I am indebted for his interest in my professional development.

This study was supported by the Alumni Development Fund,
Department of Botany and Plant Pathology at Michigan State University.

I especially wish to thank my friends for their reinforcement when
the going got rough. Among them are: Sandy Herman, Mary Lu Hellie,
Dick Sawyer, Gary Mills, Kerry O'DOnnell, Rick Friedman, Eugene Britt,
Jeff Vincent, Brian Curry, Tom Burton, Dan Taylor, Steve Simon,

Jim Zlydaaszyk, and Julie. Thank you, all. Grateful thanks are
extended to Jude Johnson and Patti Perkins for typing this thesis.

DEDICATION

To my parents, Michael and Rita Veselenak, for understanding
why I wanted to go to school for so long.

TABLE OF CONTENTS

 

Egg;
LIST OF TABLES ....................... vi
LIST OF FIGURES ...................... viii
INTRODUCTION ........................ l
LITERATURE REVIEW ..................... 3
Candida albicans ................... 3
Mouse Footpad Model .................. l2
Cyclophosphamide ................... l8
MATERIALS AND METHODS ................... 26
Animals ........................ 26
Microorganisms .................... 26
Sensitization ..................... 27
L050 Procedure .................... 27

Preparation of Bovine Anti-Candida albicans Antiserum . 27

 

Footpad Sensitivity Assay ............... 28
Serological Tests ................... 29
Cyclophosphamide Pretreatment ............. 30
Leukocyte Determination ................ 30
Footpad Assay with Live Candida albicans ....... 30
Histopathology .................... 3l

Isolation of Mouse Polymorphonuclear neutrophils . . . 3l
Phagocytosis by Peritoneal Cells ........... 32
PMN Candidacidal Assay ................ 33

iv

TABLE OF CONTENTS Continued

flA_GE_
Statistics ...................... 34
RESULTS .......................... 35
Immune Responses in Sensitized Mice ......... 35
Survival of Mice After intravenous injection with
viable g, albicans ................. 39

White Blood Cell Kinetics in Non-sensitized,
sensitized and Cyclophosphamide Pretreated Mice . . . 39

Footpad Reactivity in Non-sensitized and sensitized
Mice Injected with Live Yeast Cells ......... 42

Footpad Reactivity in Cyclophosphamide Pretreated
Mice ......................... 45

In vitro Phagocytosis and Killing of C. albicans

Yeast Cells by PMN From Non-sensitized, Sensitized
and Cyclophosphamide Pretreated Mice ......... 5l

 

Footpad Histopathology in Non-sensitized, Sensitized
and Cyclophosphamide Pretreated Mice Injected with

Live g, albicans ................... 57
DISCUSSION ........................ 73
SUMMARY .......................... 93
BIBLIOGRAPHY ....................... 95

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

10

ll

LIST OF TABLES
PAGE

Footpad reactivity in non-sensitized and
sensitized mice after injection with 2 x l05
heat-killed C, albicans ............. 37
Specificity of footpad response in non-sensitized
and sensitized mice after injection with 2 x 105
heat-killed yeast cells ............. 38
L050 values in non-sensitized, sensitized and
cyclophosphamide (CY) pretreated mice, after
intravenous injection .............. 40
White blood cell kinetics in non-sensitized,
sensitized and cyclophosphamide (CY) pretreated

mice ......................
Footpad‘reactivity in non-sensitized and sensitized
mice after injection with 2 x l05 viable

C, albicans ................... 43
Viable yeast cells present in foot of non-sensitized
and sensitized mice after injection with 2 x l05

viable C, albicans 44

Footpad reactivity in cyclophosphamide (CY)
pretreated non-sensitized and sensitized mice
after injection of 2 x l05 heat-killed C, albicans 46

Footpad reactivity in cyclophosphamide (CY)
pretreated non-sensitized and sensitized mice after
injection of 2 x l05 live C, albicans ...... 48
Viable yeast cells present foot homogenate of
cyclophosphamide (CY) pretreated non-sensitized

and sensitized mice after injection with 2 x 105

live C, albicans ................
Germ tube production by g, albicans yeast cells
phagocytosed by PMN of non-sensitized, sensitized
and cyclophosphamide pretreated mice ......
Germ tube production by g, albicans yeast cells
phagocytosed by PMN preincubated with cyclophos-

phamide ..................... 54

vi

LIST OF TABLES Continued

PAGE
TABLE l2 Killing of C, albicans yeast cells in the presence
of PMN from non-sensitized, sensitized and
cyclophosphamide pretreated mice ......... 55

TABLE l3 Killing of g, albicans yeast cells by PMN
preincubated with cyclophosphamide ........ 56

vii

LIST OF FIGURES
PAGE

FIGURE l Germination of phagocytosed C, albicans after Th) 52
FIGURE 2 Histopathological section of a mouse footpad . . . 58

FIGURE 3 Section of a mouse footpad immediately after
injection with 2 x 105 live C, albicans ..... 60

FIGURE 4 Section of a non-sensitized mouse footpad 4 h
after injection with 2 x l05 live C, albicans . . 61

FIGURE 5 Section of a non-sensitized mouse footpad 24 h
after injection with 2 x 105 live C, albicans . . 62

FIGURE 6 Section of a non-sensitized mouse footpad 48 h
after injection with 2 x l0s live g, albicans . . 64

FIGURE 7 Section of a non-sensitized mouse footpad 72 h s
after injection with 2 x 105 live C, albicans . . 65

FIGURE 8 Footpad sections of sensitized m ce 4 h and
24 h after injection with 2 x l0 live
C, albicans ................... 66

FIGURE 9 Footpad sections of sensitiged mice 48 h and 72 h
after injection with 2 x 10 live 9, albicans . . 67

FIGURE l0 Footpad sections of non-sensitized, cyclophos-
phamidg pretreated mice 4 h after injection of
2 x l0 live C, albicans ............. 69

FIGURE ll Footpad sections of non-sensitized, cyclophos-
phamide pretreated mice 24 h - 72 h after injection
of 2 x 105 live C, albicans ........... 70

FIGURE l2 Footpad sections of sensitized, cyclophosphamide

pretreated mice 24 h - 72 h after injection of
2 x 10 live C, albicans . . . 72

viii

INTRODUCTION

Resistance to infection by the myriad of microorganisms in the
environment is based upon non-specific factors and specifically
acquired immunity which frequently operates in concert with these
non-specific factors, thereby greatly increasing their effectiveness
(Roitt, 1974).

Infection caused by the opportunistic yeasts, especially Candida
albicans, are becoming more common due to advances in antimicrobial
and chemotherapeutic agents. This yeast is a commensal in the intestinal
tract of a significant portion of the population and therefore any
alteration of the host may predispose the host to infection by this
organism. Thus, a better understanding of the basic host-microbe
interaction is necessary.

Penetration of a fbreign microorganism into the tissues of the
body results in an inflammatory response. Phagocytic cells migrate
from the bloodstream to the site of the injury. This is a non-specific
process, in that no previous exposure to the microbe is necessary for
proper function. These cells phagocytose the invading organism and
either the infection is limited and resolved or the microbe escapes
killing and the infection proceeds, possibly to other body sites. The
polymorphonuclear leukocyte (PMN) is a short-lived, highly phagocytic
cell that comprises a major portion of this non-specific defense system.
These cells are usually the first phagocytic cells to migrate to the
area of infection or injury.

The immune response is a remarkably versatile adaptive mechanism

in which animals form specifically reactive proteins and cells in

response to a variety of compounds, from macromolecules to whole
organisms. It constitutes a principle means of defense not only
against infection by pathogenic microorganisms and viruses, but
probably also against host cells which transform into cancer cells.
Prior exposure, either natural or active, with a particular agent
is usually necessary to induce specific immune responses directed to
the agent.

An in_yitrg_mouse footpad assay model for quantitatively studying
intralesional numbers of viable yeast cells was developed. Using
this model along with histopathological observations, the intralesional.

events after infection can be followed. In vitro studies of the

 

phagocytic and killing properties of the PMN showed the important role
of this cell in host defense to Candida.

IThe primary objective of this study is to characterize the host
response to a localized g, albicans infection. Sensitization as well
as debilitation of the host prior to challenge were used to better
understand the host-parasite relationship regarding this unique

microorganism.

LITERATURE REVIEW

Candida albicans

 

The first recorded account of disease caused by Candida albicans

 

was that of Hippocrates (Rippon, 1974). The aphthae (white patches)
in debilitated patients were described in his "Epidemics". The term
"thrush" probably arose as an ancient Scandinavian or Anglo-Saxon
word (Rippon, 1974; Odds, l979). The first concise descriptions of
oral and gastrointestinal candidosis were by Rosen von Rosenstein in
l77l and Underwood in l784 (Odds, l979). Candida infections of
virtually every tissue in the human body have been reported. Although
the most common manifestations of candidosis are superficial lesions
of mucocutaneous surfaces, the incidence and severity of systemic
disease is increasing due to advances in modern medicine and the use
of antibiotics (Seelig, l966), immunosuppressive and cytotoxic therapies
(Green, 1968), and intravenous infusions (Klainer and Beisel, l969).

The taxonomic position of the genus Candida was established by
the Eighth Botanical Congress at Paris in l954 (Rippon, 1974). Members
of this genus belong to the Form-class Deuteromycetes, Form-subclass
Blastomycetidae, and Form-order Cryptococcales (Alexopoulos and Mims,
1979). Despite the validity of this genus, the terms "Monilia" and
"moniliasis" still appear regularly in present-day textbooks and
learned journals.

The ecology of C, albicans is the alimentary tract of mammals and

birds (Rippon, T974) and all such species are susceptible to invasion

by this fungus (Smitten, 1967). Rarely, has 9, albicans been isolated
from the soil, but in these instances it probably represents fecal
contamination.

In the human, carriage rates for C, albicans vary with the many
reports published. In the epidemiological studies reported, variables
such as patient selection, methods of obtaining specimens, identifi-
cation procedures, and geographical location contribute to the wide
range of results obtained. Recovery of C, albicans from the oral
cavity, although ranging from 2 to 37%, show that in a majority of
studies the carrier rate is less than 20% for normal individuals
(Schmitt, 1971) while the recovery rate for g, albicans in hospital
patients is significantly higher. Few studies show an isolation
rate of less than 30% in hospitalized patients with the mean being
around 42% (Odds, 1979). Recovery of g, albicans from the intestinal
tract also shows differences between normal subjects and hospitalized
patients, although not as marked as the recovery rates from the oral
cavity. Weighted mean recoveries of C, albicans were 14.6% and 22%,
respectively, for normal subjects and patients (Mackenzie, 1962;
Hughes and Kim, 1973; Odds, 1979). Recovery rates for g, albicans
from the vagina show 7.8, 14.9, and 25.7% from normal subjects,
patients without vaginitis and patients with vaginitis respectively
(Odds, l979). Practically every other site on the human body has
been sampled for presence of yeasts, specifically C, albicans. Results

of these studies show a very low (<5%) recovery rate indicating that

these organisms are probably not resident flora of these sites but
merely transients (Rippon, 1974; Odds, 1979). The important point
in the above studies is that many people harbor an ecosystem of
commensal yeasts which have a potential to become pathogenic.

The manifestations of infections with C, albicans are as protean
as those seen with syphilis. Rippon (1974) defines three major
types of infections caused by C, albicans: '1) mucocutaneous disease
2) cutaneous involvement, and 3) systemic disease. Infection of the
mucocutaneous membranes includes vaginitis, balinitis and alimentary
tract disease usually due to improper diet (Gentles and LaTouche,
1969), poor hygiene (Rippon, 1974), antibiotic therapy (Seelig, 1966a,
1966b), or endocrine disorders (Sonck and Somersalo, 1963). These
infections may clear spontaneously once the underlying factor is
corrected or can be treated with tapical medications such as 1%
crystal violet, 0.1% hanmycin or 1% nystatin (Conant gt_al,, 1971).
Two types of patients are associated with cutaneous infection by
C, albicans, those with metabolic disorders such as obesity or diabetes
and those whose skin is predisposed by environmental conditions such
as moisture, occlusion, and maceration. Patients with occupations
such as dishwashers, fruit canners, and barmaids generally belong
in this latter group (Rippon, 1974). Treatment for these afflictions
is the same as that for mucocutaneous disease and elimination of the
predisposing environmental factor is usually sufficient. The third

major type of infection is systemic disease. This is a relatively

rare condition in an otherwise healthy individual and is usually the
result of some other debilitating illness. The prognosis in this
condition is poor and the death rate is high. Treatment consists

of intravenous (i.v.) infusions of amphotericin B (Medoff and Dismakes,
1970), miconazole (Bannatyne and Cheung, 1978), 5-f1uorocytosine

(Fass and Perkins, 1971), and recently ketoconazole (Cuce §t_al,,
1980).

Immunity to C, albicans has been the subject of a great many
research reports as well as one recent review (Rogers and Balish,
1980). Since C, albicans is a microorganism of relatively low
virulence (Mourad and Friedman, 1961) it becomes obvious that an
intact microbial defense system, both innate as well as specific, is
sufficient to control this organism. It is only when this defense
system is compromised that infection and disease caused by C, albicans
may occur. As has been mentioned, 9, albicans is a member of the
resident flora of a large portion of the population so that the
potential for infection is present especially when systems that
otherwise control the growth and spread of C, albicans are compromised.

When a microorganism gains entrance into the body, non-specific
host defense mechanisms are the first line of defense (Smith, 1968).
The blood is rapidly cleared of invading organisms by the liver,
lungs, spleen, lymph nodes, and to a lesser extent, the bone marrow
(Rogers, 1960). Baine gt_al, (1974) showed clearance of more than

90% of intravenously injected C, albicans within ten minutes in rabbits.

The vast majority of yeasts were trapped in the liver and lungs, while
Sawyer gt_al, (1976) reported similar results in rats. They also
reported that, although the yeasts were rapidly removed from the
bloodstream, they were not killed. In a later paper-(Sawyer gt a1,,
1981) they showed that non-specific activation of the reticuloendo-

thelial system by prior injection of Corynebacterium parvum, resulted

 

in killing of the yeasts using the in_!itrg_perfused liver model.
Scanning electron microscopy showed an influx of leukocytes into the
liver after 9, paryum.vaccination and light microscopy of the livers
showed the infiltrate to be primarily mononuclear (Sawyer gt_al,, 1981).
When invading microorganisms gain access to tissues, phagocytic
cells must move to the site of challenge from the bloodstream. The
polymorphonuclear neutrophil (PMN) is the first such phagocytic cell
to emigrate followed by blood monocytes which differentiate into
phagocytically active macrophages (Spector and Willoughby, 1963;
Elsbach, 1980). Emigration of leukocytes, specifically PMN, from
blood vessels to the site of infection has been the subject of many
reports. It is not clear if the localization of PMN at the tissue
lesion is due to chemotaxis. Harris, in 1953, showed unequivocally
that colonies of living Salmonella typhi, Staphylococcus albus and
Mycobacterium tuberculosis were chemotactic for granulocytes, whereas
killed microorganisms were found to be non-chemotactic as were minced
tissues. He concluded that emigration of white cells was a consequence

of the changes in the vessel wall associated with increased vascular

permeability. Therein lies the problem, whereas chemotaxis can be
unequivocally demonstrated only in_vitro, leukocyte emigration from
blood vessels can be shown only in_vivo (Spector and Willouhby, 1963).

Chemotaxis of PMN jn_vitro was shown by Boyden in 1962. The

 

availability of the technique he described has resulted in the
characterization of several chemotactic factors as well as delineation
of abnormalities of leukocyte chemotaxis in certain disease states
(Snyderman §t_al,, 1975). Some of the reported chemotactic factors
fer PMN include zymosan (Laxalt and Kozel, 1979), complement factor
,CSa (Ward and Newman, 1969) and aggregated human gamma globulin
(Snyderman §t_al,, 1975). Microorganisms that generate chemotactic

activity include Escherichia coli and Staphylococcus epidermidis

 

 

(Cutler, 1974), Cryptococcus neoformans (Laxalt and Kozel, 1979) and
C, albicans (Denning and Davies, 1973; Cutler, 1977) as well as those
mentioned above by Harris (1953).

Phagocytosis is facilitated by opsonins i.e. serum components
such as immunoglobulins IgG and IgM (specific antibodies) and comple-
ment factor C3, since PMN and mononuclear phagocytes have receptors
on their cell membrane for the Fc portion of IgG and for the C3b part
of this complement factor (Van Furth gt_al,, 1978). The binding of
antibodies to antigens is known to activate the serum complement system,
one of whose functions is to facilitate phagocytosis (Roitt, 1974).
Morelli and Rosenburg (1971) showed that complement-deficient mice

did not survive as long after a lethal challenge with C, albicans as

mice with an intact complement system. In addition, Ferrante and Thong
(1979) showed that following the removal of heat-labile opsonins by
heating pooled human serum at 56 C for 30 minutes, approximately 50%
loss in phagocytic activity by PMN occurred, while Ehlenberger and
Nussenzweig (1977) found that C3 and IgG have synergistic roles in
phagocytosis.

The action of PMN and macrophages on C, albicans has been

extensively studied jn_vitro. Louria and Brayton (1964) showed that

 

mouse PMN jn_yjtrg_ingest virtually all C, albicans blastospores to
which they are exposed within 30 minutes. This same effect was also
seen with macrophages (Ozato and Uesaka, 1974). In both these studies,
though, some of the yeasts grew out of the phagocytes after engulfment
(and thus escape intracellular destruction.

Among pathogenic SPECIES of Candida, C: albicans is found to
possess the greatest ability to resist intracellular killing by out-

growth from PMN and macrophages, and Q, guilliermondii is the least

 

able (Louria and Brayton, 1964). The abilities of various Candida spp.
to escape host phagocyte defenses therefore correlate with their
relative virulence for laboratory animals, and with their ability to
produce a filamentous growth form (Odds, 1979).

Studies which have attempted quantitative determinations of
the candidacidal activities of phagocytes have shown considerable
variability, with the average killing rate being about 50% of injested

yeasts (Rogers and Balish, 1980).

10

The biochemical mechanisms of killing by phagocytes have been
investigated, and several substances have been found which are
candidacidal. Lehrer and Cline (1969) found that the myeloperoxidase-’

peroxide-halide ion system is candidacidal in_vitro and that this

 

system is the major component for intracellular killing of C, albicans.
Lehrer (1972) later reported a second candidacidal mechanism in human
PMN independent of the myeloperoxidase system. Elin at al, (1974)
provided further evidence that PMN are important for defense against
candidosis. Using Chediak-Higashi mice which have neutrophils with
impaired capacity to kill C, albicans, they found an increased
susceptibility to infection by g, albicans when compared to normal mice.
Many reports to date have dealt with acquired immunity and
subsequent resistance to challenge with C, albicans. Injections of
large amounts of high-titered anti-Candida antiserum have been shown
to be somewhat protective in mice prior to challenge with usually
lethal amounts of C, albicans (Mourad and Friedman, 1968) and effective
in the clearing of g, albicans infeCtions in humans (Hiatt and Martin,
1946). Numerous investigators have immunized experimental animals
with doses of live or killed C, albicans yeast cells or with other
microbial antigens, and then noted whether or not such immunizations
protected the animals against subsequent challenge with lethal doses
of yeasts. The effects of such immunizations usually appeared as a
delay in mortality, rather than a prevention of mortality (Odds, 1979).
Collins (1974) has suggested that the best way to study the immune

11

response in a vaccinated animal is to compare the rate of growth or
inactivation of a sublethal challenge population of microbes in test
and control groups throughout the entire infectious period.

The histopathological responses noted in experimental animal
infections has been reviewed by Rogers and Balish (1980). In most
cases of human candidosis, the inflammatory exudate seen in cutaneous
disease is mixed, i.e., it is composed of intense infiltrates of PMN,
histiocytes, and some lymphocytes (Rippon, 1974). In cases of human
disseminated disease, microabcesses containing both intact and
degenerating neutrophils surrounded by a zone of histiocytes are typical
(Rippon, 1974). In experimental animal infections the inflammatory
reaction is similar to that in humans. Pearsall and Lagunoff (1974)
have described a thigh muscle model for candidosis in mice. The
histopathological responses seen in this model showed an initial PMN
response followed by a modest infiltration by macrophages, eosinophiles
and lymphocytes. Neutrophils remained the predominant cell type and
a predominance of mononuclear cells was not observed until very late
in the infection, when few yeast cells remained visually (Pearsall,
and Lagunoff, 1974). Rogers and Balish (1977, 1978) reported that in
their kidney model for infection, the initial cellular response was
neutrophilic. When the numbers of viable Candida cells are beginning
to wane, the cellular infiltrate is still almost exclusively
polymorphonuclear.

Engulfment and destruction by tissue macrophages or circulating

PMN and monocytes appears to be the ultimate fate of most microorganisms

12

that penetrate as far as the tissues of the host with intact cellular
defenses. Phagocytosis is a non-specific process, but its effects
may be controlled and enhanced by host immune responses. As best
written by Rogers and Balish (1980), "it is important that we obtain
more basic information concerning 9, albicans-host interactions and
elucidate those innate or acquired host defense mechanisms which

control the diseases caused by this unique opportunistic yeast".

Mouse footpad model

 

The mouse footpad model for the detection of delayed type
hypersensitivity (DTH) was first described by Gray and Jennings in
1955. As originally devised, the procedure consists of inserting a
short-beveled 26-guage needle into a metatarsel pad of the tensed
plantar surface of a hindfoot and injecting enough antigen solution
to swell an area of the foot 3 mm in diameter. In more recent
applications, 27 or 30 guage needles are used and a measured volume
(e.g. 0.04 ml) of antigen solution is injected (Kong at al,, 1966).
This technique enjoys wide adoption because it is technically easier
(Crowle, 1975). Additionally, the footpad test may be more sensitive
than the classic skin flank test because the antigen is held jg_§jtu_
longer (Crowle, 1959). This was confirmed by Oghiso and Matsuoka
(1979) who followed distribution of colloidal carbon in mice injected

by various routes. A typical footpad 0TH reaction will become visible

due to induration at about 6 h after challenge, peak at 18 to 24 h,

13

still persist strongly at 48 h, and then recede, to disappear after
another 1 to 2 days (Collins and Mackaness, 1968). Most frequently,
this delayed reaction will be preceded by a soft edematous humoral
antibody swelling, first appearing at about 2 h, peaking at 3 to 4 h,
and usually disappearing by 6 h (Collins and Mackaness, 1968).

Footpad reactions can be usually read subjectively by comparing
the test foot with the opposite foot, which either is untreated or
has been injected with antigen solvent only (Gray and Jennings, 1955).
Alternatively, the response can be measured objectively with calipers
(Youdim eta_l_., 1973), a skin thicknessdial guage (Rifkind gal”
1976) or, for the finest distinction, by the amount of a fluid that
the swollen foot will displace in a plethysmograph (Pearson gt_al,,
1971). These objective measurements can be made by comparing swelling
in the experimental foot with that of the control foot or by subtract-
ing the control value (measurement taken before injection) from the
experimental readings (Rifkind gt_al,, 1976). The nature and quantity
of the antigen are important. Tests with undiluted old tuberculin
(OT) are unsatisfactory because the glycerin in OT causes severe
nonspecific swelling (Gray and Jennings, 1955) while Flynt gt_al, (1975),
showed detectable responses to l:10,000 dilutions of merthiolate which
is used as a preservative in coccidioidin and other antigens. Because
some antigens are toxic (Anacker gt_al,, 1969), the lowest test doses
are used that will reliably detect DTH. Effective quantities must be

determined empirically for the antigen used. For most antigens employed,

14

such test doses are only a few micrograms (Anacker et a1., 1969;
Collins and Mackaness, 1968) or a few organisms (Patel and Lefford,
1978).

A The histological features of DTH is a predominantly mononuclear
cell infiltrate (Gell and Hinde, 1954) whereas a reaction in which
polymorphonuclear cells predominate is widely interpreted as an Arthus
reaction (Crowle, 1975). There are important exceptions. In DTH
reactions to polysaccharides in mice, monocyte infiltration is heavy,
but the polymorphonuclear cell maintains numerical superiority from
early in its reaction throught its peak (Kong gt_al,, 1966). This
is not due to an Arthus reaction, for it is passively transferred with
lymphoid cells but not antiserum. Rifkind §t_al. (1976) reported that
in mice sensitized with Coccidioides immitis mycelial antigen, there
was primarily a neutrophilic leukocyte response at 24 and 48 h along
with a significant mononuclear component in the infiltrate which was
not seen in non-sensitized mice. The cellular infiltrate in mice
actively sensitized with antigen was indistinguishable from that seen
in mice passively sensitized with immune spleen cells. Essentially,
no mononuclear cells were observed in footpads 0f mice given only
immune serum (Rifkind gt_al,, 1976).

The DTH reaction observed in mice is antigen specific. Mice
immunized with C, immitis antigen reacted when footpad challenged with
homologous antigen but failed to react with C, albicans antigen

(Rifkind at 11., 1976).1 Although there may be cross-reactivity between

15

similar antigens (e.g., Salmonella enteritidis and S, gallinarum)

 

most DTH reactions are appropriately specific (Crowle, 1975). For
example, mouse DTH can demonstrate the differences between bacterial
species of the same genus (Collins and Mackaness, 1968), discriminate
mumps from influenza viruses (Feinstone gt al,, 1969) and help to
identify haptenic molecules (Bekierhunst and Yarkoni, 1973).

There have been other models devised for detection of DTH in the
mouse, some before the footpad technique and some after. The first
satisfactory evidence that mice could develop DTH was by Hart at al,
in 1952. They showed that mice chronically infected with virulent
tubercle bacilli could be fatally shocked with purified protein
derivative (PPD).

Later, Kircheimer and Malkiel (1953), demonstrated that mice
could be sensitized to tuberculin without virulent infection. This
reactivity was elicited by skin testing the mice, inherently difficult
due to the thinness of the mouse skin. Although this skin test
technique is readily mastered, it is not as widely used as footpad
tests despite the advantages of offering more test sites on an animal,
allowing easy quantitative objective measurement, and being simpler
and quicker to perform (Crowle, 1959).

A more recent model for DTH detection, the mouse-thigh lesion has
been developed by Pearsall and Lagunoff (1974) and Pearsall £3 31,
(1978). With this technique, a large number of viable C. albicans is

injected intramuscularly into the thigh. The hypersensitive response

16

is measured as the subsequent swelling of the thigh. The thigh lesions
can also be followed histologically. A criticism of this model can be
made because a very large number of viable g, albicans yeast cells

(5 x 108) must be used. Leunk and Moon (1979) reported toxin-like
properties of C, albicans when administered i.v. to mice in high doses

(4.5 x 105).

These effects were not seen with the same dosage of
heat-killed cells.

Attempts to follow the numbers of intralesional organisms have,
until recently, been mostly qualitative observations, although a common
procedure used in assessing systemic spread of organisms after i.v.
challenge is to homogenize selected organs and determine viable cell

counts. Patel and Lefford (1978) noted the presence or absence of

acid-fast bacilli in tissues of Mycobacterium leprae sensitized mice

 

after footpad challenge while Giger gt a1. (1978) reported relative
numbers of fungal elements in cutaneous lesions of mice sensitized

with live 9, albicans. Louria gt_al, (1963) reported, that in i.v.
injected animals, organisms became harder to detect microscopically
as the inflammatory reaction intensified.

In 1974, Lepper studied qualitatively as well as quantitatively
the cellular components of the inflammatory response in the skin during
the course of a primary experimental dermatophyte infection but only
noted the presence or absence of fungal structures in relation to the
types of leukocyte infiltrate. Green and Balish (1979) took this

experiment further by quantitatively following fungal proliferation

17

and elimination during lesion development by homogenizing and culturing

skin infected with Trichophyton mentagrophytes. They showed that

 

lesion severity, in terms of gross tissue damage, occurred at the same
time as peak fungal load and that reinfection with the same organism
resulted in accelerated lesion development, probably as a result of

an anamnestic hypersensitivity reaction, but with no corresponding

peak fungal load. The infecting organism was eliminated at the same
rate in both primary and secondary infections. They suggested that
skin homogenization may be a more sensitive method of determining fungal
persistence because: 1) organisms may be difficult to see microscop-
ically, and 2) the inability of differentiating viable from non-viable
organisms (Green and Balish, 1979).

The first report using mouse footpad injections to produce a
self-limiting localized infection was that of Shepard in 1960. The
technique has been of inestimable value in the development of new
chemotherapeutic agents and in assessing the rate at which Mycobacterium
lggrag_are killed during treatment of patients (Shepard, 1971 and
Shepard gt__l,, 1968). The jn_yjyg_footpad microbicidal technique
has also been used by Patel and Lefford (1978) as evidence for cell-
mediated immunity (CMI) to M, lgp§a§_antigens in sensitized mice.

After sensitization, mice were footpad challenged with viable Listeria
monocytogenes. Twenty-four hours later, the mice were sacrificed,
the injected foot was homogenized and appropriate dilutions were plated.

Reduction in viable cells per foot was evidence for CMI since one of

18

the hallmarks of CMI is nonspecific resistance to listeria (Mackaness
gt_al,, 1974). Collins gt 31,, (1978) also used the mouse footpad
culture method to study immunity to mycobacterial infection. They

injected footpads of mice with a readily cultivable Mycobacterium

 

strain, M, marinam. The onset of cellemediated immunity was well
correlated with a concurrent decrease in viable cells cultured from
the homogenized foot. The above experiments show that the jn_yjyg_
mouse footpad assay model lends itself to further study with different
microorganisms which can lead to a better understanding of host-

parasite interactions.

Cyclophosphamide

 

Efforts to modify the chemical structure of the nitrogen mustards
to achieve greater selectivity for neoplastic tissues led to the
development of cyclophosphamide (Arnold gt_al,, 1958). The first use
of this family of compounds was in gas warfare during World War I.
Modern interest in these compounds as anticancer agents is a byproduct
of studies on war gases during World War II. Gilman and his colleagues,
after noting the toxicity of simple alkylating agents to lymphoid
tissues and rapidly dividing cells, suggested that alkylating agents
might have a therapeutic effect against tumors of lymphoid origin
(Gilman and Philips, 1946). The suggestion led to studies on neoplasms
in experimental animals and subsequently to clinical tests, which

demonstrated that one of the mustards had activity against Hodgkin's

19

disease. This agent was the first synthetic compound clearly shown to
possess antitumor activity in humans. These results led to the
synthesis of hundreds of other compounds possessing alkylating activity.
It was not until 1965 that Schmidt and colleagues critically evaluated
94 alkylating agents against 25 different rat and mouse neoplasms
(Schmidt gt_al,, 1965). The outstanding representatives in nearly
every test system were the isomeric phenylalanine mustards and cyclo-
phosphamide (2-[bis(2-chloroethyl)amino] tetrahydro-Zflrl,3,2-oxazaphos-
phorine 2-oxide). Among the trade names are the following: Cytoxan
(United States), Exdoxana (England) and Procytox (Canada).

Cyclophosphamide is prepared by treating N, H-bis (2-chloroethyl)
phosphoramidic dichloride with an equimolar amount of 3-amino-l-propanol
in the presence of two molar equivilents of triethylamine (Arnold gt_al,,
1958). It is soluble in water, ethanol, benzene, acetone, and ether
(Arnold gt_al,, 1958). As determined by an assay with leukemic mice,
cyclophosphamide is stable in solution for at least 4 weeks (Vietti
gt_al,, 1973).

The initial success of cyclophosphamide as an antitumor agent in
experimental tumor systems (Arnold gt_al,, 1958) and the demonstration
that it was ineffective against cells jn_!jtrg_(Foley gt_al,, 1960)
led to numerous attempts to explain the enzymatic mechanism by which
activation occurs. The most favored mechanism for activation of
cyclophosphamide was based on the elevated phosphoramidase activity

in certain tumor tissues which would cleave the compound and release

20

a biologically active (cytostatic) product (Hill, 1975). Foley gt a1,
(1960, 1961) tested blood serum and homogenates of various normal and
neoplastic tissues of rats previously injected with cyclophosphamide
and noted a product that was toxic to mammalian cells in culture, but
found only in the liver and the blood. Incubation of cyclophosphamide
with homogenates showed only the liver produced an inhibitory
substance, indicating that this organ was the primary site of activa-
tion for the agent. Perfusion of an isolated rat liver with whole

rat blood containing cyclophosphamide gave a high concentration of
toxic material. Liver slices produced active metabolites only when
incubated in the presence of oxygen while slices of other tissues were
essentially ineffective (Brock and Hohorst, 1967).

Chromatography of serum from rats injected with labeled
cyclophosphamide revealed two metabolites, which could also be found
after incubation of the drug with slices of rat liver (Hill, 1975).
Further analysis showed that the major metabolite contained phosphorus.
While the structure of the activated form of cyclophosphamide is still
unsettled, studies using isophosphamide have shown that this compound
is metabolized by mouse liver microsomes in a manner similar to that
for cyclophosphamide (Hill gt_al,, 1973). The activated product of
isophosphamide oxidation binds to deoxyribonucleic acid and inhibits
thymidine incorporation into deoxyribonucleic acid by leukemic L1210
cells (Allen and Creaven, 1972).

After injection into mice cyclophosphamide is rapidly metabolized

with the rate based on the size of the dose applied versus the

21

cytostatic activity demonstrable in serum (Hill, 1975). The half-life
of cyclophosphamide in mouse serum is about 20 min and only 5% of a

100 mg/kg dose is excreted unchanged in a 24 h period (Hill, 1975).
Cyclophosphamide is found in the brain of mice as soon as five min

after an i.p. injection (Mellet, 1966). The whole-body autoradiographic
technique revealed that cyclophosphamide accumulated within 30 min

of an i.p. injection in the liver, gallbladder, small intestine and
kidneys of mice with a later accumulation in the thymus, spleen and
testes (Hill, 1975).

The presence of a foreign compound in the tissues of an animal
undoubtedly causes many changes in metabolism. During the past 20
years, the influence of cyclophosphamide on tissues and organ systems
has been extensively studied. Tumors of mice treated with cyclophos-
phamide have a lower rate of in_yjtgg_glycolysis 4 h after treatment
(Wright at _l-: 1960) but after 72 h glycolysis is no longer operative.
The conclusion was that tumor inhibition by cyclophosphamide is a
process independent of glycolytic activity. Preliminary experiments
in the field of lipid metabolism have given no clues to the mechanism
of action of cyclophosphamide. The effect of this agent on lipid
content of tumors is dependent on the type of tumor studied (Hill,
1975). Cyclophosphamide has demonstrable effects on the cellular
processes involved in the production of ATP. Some of these have been

implicated as fundamental to the action of the agent. The nicotinamide

adenine dinucleotide (NAD) levels of tissues of animals treated with

22

cyclophosphamide have been the subject of several investigations.
In mice, high does of cyclophosphamide decrease the NAD glycohydrolase
activity of the spleen (Tsukagoshi §__al,, 1968). Liss and Palme
(1965) reported a decrease in NAD levels in Ehrlich ascites cells,
but this fall in NAD content occurred only after a change in the
deoxyribonucleic acid/ribonucleic acid ratio and is thus probably not
the primary mechanism for toxicity.

There seems to be common agreement that cyclophosphamide inhibits
the incorporation of precursors into DNA; this has been considered
as a possible primary effect of the drug. Liss anchalme (1963) found

3-thymidine into the deoxyribonucleic

that incorporation of H
acid (DNA) of Ehrlich ascites cells and spleen of mice is reduced

50% after treatment with a moderate dose of cyclophosphamide, while
Short §£_§l: (1972) showed decreased DNA synthesis by mouse embryos

at all times after 6 h post-injection. The effects of cyclophosphamide
on chromosomes are probably related to its action on cellular DNA.
Chromosomal disturbances have been observed in ascites tumor cells

and mouse bone marrow (Arrighi gt_al,, 1962). Leukocyte cultures

from patients treated with cyclophosphamide show chromosomal abnormal-
ities (Arrighi gt_al,, 1962). In_yjtrg_treatment of human leukocytes
does not result in chromosomal damage (Hampel gt_al,, 1966) while
another publication states that chromosomal and polyploidy changes

are evident in such cells after exposure to cyclophosphamide

(Nasjleti and Spencer, 1967). The results of this last study imply

23

that cells in culture are capable of converting cyclophosphamide to
an active form, a fact which has not been conclusively demonstrated
(Hill, 1975). The list of effects continues to grow, but as yet the
primary action responsible for cytotoxicity of cyclophosphamide is
not known with certainty.

In all animals tested, cyclophosphamide produces severe and
potentially lethal depression of hematopoietic stem cells. Also,
affected are cells of the lymph nodes (Turk and Poulter, 1972), spleen
(Turk and Poulter, 1972), thymus (Castaldi et a1., 1972) and bone
marrow (DeWys gt_a1,, 1970). The total marrow cell count and the
peripheral blood leukocyte count of mice fall progressively for three
days after cyclophosphamide administration but both return to normal
after a few more days (DeWys gt_al,, 1970). The number of erythrocytes
in mice is not greatly changed by cylophosphamide treatment (Valeriote
gt_al,, 1968) but rapidly proliferating erythroid cells are more
sensitive (Blackett and Adams, 1972). Peripheral granulocytopenia
is seen in rats and mice following injection of cyclophosphamide
(Shadduck and Nunna, 1971) but granulocytosis is seen during hematologic
recovery (Host, 1966). Neutropenia is also seen in cyclophosphamide
treated dogs and monkeys (Schmidt gt_al,, 1965). The hematological
effects of cyclophosphamide on humans are similar to those seen with
experimental animals (Pegg, 1963).

Cyclophosphamide has been used extensively as an immunosuppressant

of both the humoral and cellular immune systems in experimental

24

animals. Early experiments showed decreased antibody production to
human erythrocytes in cyclophosphamide pretreated mice (Frisch and
Davies, 1966). Small doses are most immunosuppressive when given on
the same day while large doses are effective even after three days
subsequent to antigen injection (Frisch and Davies, 1966). In a now
classical experiment they showed that a large, single dose of
cyclophosphamide given to mice can result in complete suppression of
the immune response to a previously administered erythrocyte antigen
but not to a different one given 1 to 2 days later. Aisenberg and
Wilkes (1967) using the Jerne plaque assay showed that cyclophosphamide~
alone kills the hemolysin-producing cells (B lympocytes), while
non-proliferating cells are killed at a much lower rate. Total
recovery of immune competence may require more than 30 days (Marbrook
and Baguley, 1971). Mice treated with cyclophosphamide will respond
normally to sheep erythrocytes if immunocompetent cells from other
mice are transferred at the same time (Santos, 1966).

The effects of cyclophosphamide on the cellular immune system
are similar to those of the humoral immune system. The drug prolongs
the survival of both skin allografts and homografts on mice,
especially if it is given to the animals between one day before and
two days after transplantation (Fox, 1964; Floersheim, l969). Graft
vs. host disease induced in mice by injection of spleen cells from
another strain is not as lethal in animals treated with cyclophospha-

mide (Levy, 1973). Sharma and Lee (1977) showed functional T-cell

25

deficiency accompanied by a marked degeneration in the thymus dependent
areas of the spleen as well as the thymus itself in chickens injected
with cyclophosphamide.

Most recent reports refer to the exclusivity of cyclophosphamide
as a B-cell suppressant but since cooperation between B and T cells
is required in the immunological response, the destruction of either
could lead to tolerance.

The effect of cyclophosphamide pretreatment on infections is a
general enhancement of the infectious process. Numerous reports in
the literature show, in general, a decreased resistance to microbial
challenge. This effect is seen with microorganisms such as
Staphylococcus aureus (Sharbaugh and Grogan, 1969) influenza virus
(Hurd and Heath, 1975) Mycoplasma pulmonis (Singer gt_al,, 1972)

Nocardia asteroides (Beamon and Maslan, 1977) and Candida albicans

 

(Moser and Domer, 1980).

MATERIALS AND METHODS

Animals

Female HA(ICR) mice weighing 28 to 32 g were purchased from
Harlan Laboratories, Indianapolis, Ind. Animals were maintained under
standard laboratory conditions with Wayne Lab-Blox (Allied Mills, Inc.,

Chicago, Ill.) and water available ag_libitum.

Microorganisms

 

The strain of Candida albicans used in this study was a fecal
isolate obtained from the Olin Health Center, Michigan State University.
Identity was confirmed by typical assimilation patterns using the
method of Land et_al, (1975). Adjunct tests, such as germ tube
formation and chlamydospore production, were also performed. Stock
cultures were maintained on Sabouraud dextrose agar (SDA) slants at
25 C. When live cells were used, the inoculum was prepared by trans-
fer of a loopful of the stock to 100 m1 of trypticase soy broth
(Difco) supplemented with 4% d-glucose and incubated for 16-20 h at
37 C on a rotary shaker. Yeast-phase cells were harvested and washed
three times in sterile saline (0.85% NaCl). Haemocytometer counts
were used to standardize the inoculum and pour plates of 10-fold
dilutions in SDA confirmed any adjustments made.

Killed cells were prepared by heating the yeast suspension in a
60 C water bath for 4-5 h. Non-viability was ensured by plating an
aliquot onto SDA and incubation at 35 C for three days. The

26

27

suspensions of heat-killed cells were frozen at -20 C and thawed
immediately before use.

Another species, C, kru§g1_was obtained from the stock collection of
Dr. E.S. Beneke of Michigan State University. This species was used for
comparative studies with g, albicans. Preparation of inoculum was the

same as for C, albicans.

Sensitization

Four weekly doses of l x 106

heat-killed g, albicans in 0.1 ml
sterile saline were injected subcutaneously at alternating ventral areas
of mice. Sensitivity was assayed 1 week after the final injection using

random animals picked from the group.

L050 procedure

 

The computation of the LD50 was according to the procedure of Reed
and Muench (1938). Mice were injected (six per group) via a lateral tail

3, 104 5 6

vein with a 0.1 ml suspension of 10 , 10 , or 10 , washed C, albicans
yeast-phase cells per ml using a 1 m1 plastic syringe with a 27 guage
needle. The animals were monitored daily and the experiment was termin-
ated after 30 days. The accumulated total of deaths per groups during

this period was used as the basis for computing the median lethal dosei

Prepartion of bovine anti-Candida albicans antiserum

 

A yearling heifer (MSU #228) was vaccinated by the following pro-

cedure. Two weekly subcutaneous injections of 2.5 x 109 heat-killed

28

C, albicans suspended in sterile saline were administered at different
shaved sites. Two weeks after the second injection, the cow was skin
tested intradermally with 1 ml of a 1:5 saline dilution of the same
antigen. Increases in skin thickness and areas of inflammation were
measured with calipers at O h (control) and at 24, 48, and 72 h after
antigen injection. Results showed a definite increase in skin thick-
ness surrounded by an area of inflammation at all times subsequent

to injection. A control site injected with sterile saline showed no
measurable reaction. Serum collected at the time of skin testing
showed a tube agglutination titer of 2560. Fetal bovine serum

(Grand Island Biological 00., Grand Island, N.Y.) was used as the
negative bovine control in both the immunodiffusion and agglutination

procedures.

Footpad sensitivity assay
The sensitivity of mice to g, albicans was assayed by means of
the footpad swelling test (Rifkind gt_al,, 1976). In footpad tests,

2x105

heat-killed C, albicans suspended in 0.04 ml sterile saline
was injected intradermally into the right hind footpad. The left
hind footpad, injected with an equal amount of sterile saline, served
as the control. The thickness of both feet were measured with a -
micrometric caliper (Scientific Products, Rochester, Mich.).

Measurements were made prior to and immediately after (0 h), followed

by 4, 24, 48, and 72 h after injection. The net increase in footpad

29

response was determined by subtracting the increase in the control

foot from that of the experimental foot.

Serological tests

 

Sera from mice before and after sensitization were tested for
the presence of precipitating and agglutinating anti-candidial
antibodies. The method of Ouchterlony (1949) was used in the immuno~
diffusion assay for precipitating antibodies. The diffusion medium
consisted of 1.0% agarose, 7.5% glycine and 0.85% NaCl. The medium
was autoclaved, cooled to 50 C and 2.5 ml pipetted onto clean, level
microscope slides (3 x 1 inch). After the agar had solidified, wells
were cut using a gel diffusion punch (Diagnostic Products, Inc.,
Cincinnati, Ohio). The agar plugs were removed from the wells using
a pasteur pipette and mild suction. Antigen (Oidiomycin; Hollister-
Stier, Inc., Spokane, Wash.) was added to the outer wells. Serum to
be tested was placed in the center well. Positive control serum from
the hyperimmune cow was included with each run. The slides were
incubated at 37 C in a moist chamber for 24 h and examined for
precipitin lines.

Agglutinin titers with homoldgous and heterologous antigens were
determined by the tube agglutination test (Sweet and Kaufmann, 1970)
in parallel with the immunodiffusion procedure. Antigens were adjusted

to 5 x 107

heat-killed cells per ml with a haemocytometer. Two-fold
dilutions of the test serum in 0.5 ml volumes were mixed with an equal

volume of the standardized cell suspension. The tubes were incubated

30

in a 37 C waterbath for 48 h after which they were read for visible

agglutination.

Cyclophosphamide_pretreatment

Mice were injected intraperitoneally (i.p.) with appropriate
doses of cyclophosphamide (Cytoxan; Mead Johnson Laboratories,
Evansville, Ind.) dissolved in saline. In all cases, the mice were
used in experiments at least 3 days and no longer than 5 days after
injection.
Leukocyte determination

Bldod samples were obtained from a lateral tail vein. White cell
counts were performed using Unopettes (#5853; Becton Dickinson and
Co., Rutherford, N.J.). Cells were counted in an improved Neubauer
chamber (haemocytometer). Differential leukocyte counts were performed
on air dried smears stained by the Wright method. Cell types were
expressed as the percentage of 100 total cells counted multiplied by

the total white cell count.

Footpad assay with live Candida albicans

 

 

Mice were challenged with a live dose of C, albicans injected
into the right hind footpad. At appropriate time intervals, groups
of mice were killed by cervical dislocation. The thickness of the
foot was measured (see above) and foot removed with sterile scissors.

The foot was surfaced sterilized by dipping in 70% ethyl alcohol and

31

placed in a 100 ml graduate cylinder. The volume was adjusted to
100 ml with sterile saline and the foot was homogenized in a Virtis
"45" Hi-Speed Homogenizer (The Virtis Co., Gardiner, N.Y.) for 2
minutes at medium speed. Quantitative SDA pour plates were made, in
duplicate, using 10-fold serial dilutions of the homogenate in saline.
The plates were incubated at 37 C for 48 h and the number of colony
forming units (CFU) were counted manually on a Quebec Colony Counter
(American Optical, Buffalo, N.Y.). The surviving yeasts were expressed
as the Log1O present in the homogenate.

Control experiments were performed to ascertain dissemination of
the yeast inoculum to other organs. Samples of liver, lung, spleen
and kidneys were homogenized with a glass-teflon homogenizer in 10 ml

sterile saline, and counted as above.

Histopathology,

The foot that had been inoculated with antigen was removed. The
plantar surface (footpad) was carefully excised and fixed in buffered
10% formalin. The tissues were embedded in paraffin, sectioned at
6 microns, and stained with both hematoxylin-eosin (H & E) and periodic

acid-Schiff (PAS) stains.

Isolation of mouse polymorphonuclear neutrophils
The procedure for preparation of mouse polymorphonuclear neutro-
phils (PMN) used in these experiments was similar to that of Cutler

(1974) with minor modifications.

32

Mice were injected i.p. with 3 ml of 0.5% glycogen (Sigma Chemical
Co., St. Louis, Missouri) in 0.85% NaCl. After 3 to 4 h, 3 ml of tissue
culture medium 199 (M199; Grand Island Biological Co., Grand Island,
N.Y.) plus a 5 USP units/ml sodium heparin (United States Biochemical
Corp., Cleveland, Ohio) was injected i.p. After 5 to 10 minutes, the
mice were killed and the abdominal skin was cut away, being careful not
to cut the peritoneal membrane. The peritoneal fluid, which consisted
of tissue culture medium with peritoneal exudate cells, was aspirated
into Siliconized test tubes using a 3 ml syringe with an 18 guage
needle. Siliconized tubes were used throughout these experiments to
prevent adherance of leukocytes to the test tube walls. The exudate
cells were pelleted by centrifugation at about 200 x g for 10 min.,
washed once in fresh M199 and resuspended in M199 + 5 units/ml heparin
+ 10% fetal calf serum. Viability was determined by trypan blue dye
exclusion and the percent PMN in the peritoneal cell suspension was
determined by differential counts of Wright stained smears. Cells
were counted using a haemocytometer. The suspension was kept on ice

until ready for use in either the phagocytosis or candidacidal assay.

Phagocytosis by peritoneal cells
Neutrophils were allowed to adhere to glass by the following
procedure. One ml aliquots of the cell suspensions containing

6 PMN were placed onto sterile 22 x 22 mm glass

approximately 1 x 10
cover slips. The cover slips were incubated for l h at 37 C after

which nonadherent cells were removed by gentle, repeated rinses of

33

fresh M199. One ml portions of M199 containing 1-2 x 105 viable yeast
cells were placed onto the cover slips and incubated at 37 C for l h.
After incubation, the cover slips were fixed in 100% methanol for

10 min, stained by the Wright method, and attached to microscope slides
with Permount (Fischer Scientific Co., Fair Lawn, N.J.). The cover
slips were examined under oil immersion for ingested yeast cells. For
estimating germ tube production, 100 phagocytosed yeast cells were
counted and germ tube producing cells were expressed as a percentage.
For a control, 100 extracellular yeast cells were examined for germ
tube production.

An additional experiment was designed to determine if incubation
of PMN from normal mice in various concentrations of cyclophosphamide
would alter their germ tube inhibition ability. In this experiment,
the PMN were harvested as before but were incubated for 1 hr in Ml99*
plus 0.01, 0.1 and 1 mg/ml cyclophosphamide while allowed to adhere
to glass cover slips. The yeast cells were added after this time and
the cover slips were incubated for an additional hour.. The cover

slips were then fixed, stained and counted as above.

PMN candidacidal assay

 

Neutrophils were assessed for killings of yeast cells in the
following manner. To a 0.9 ml suspension containing 1 x 106 mouse
peritoneal PMN was added 1 x 105/0.1 ml viable C, albicans cells. The

leukocyte and yeast cell suspension was incubated at 37 C on a rotary

34

shaker for 2 h. A control tube contained medium with all components
except leukocytes.

Following incubation, the suspension was diluted in 9 ml saline
and homogenized in a sterile glass-teflon homogenizer. Quantitative
SDA pour plates were made using lO-fold serial dilutions of the control
and experimental suspensions in saline. The plates were incubated
for 48 h at 37 C and counted manually. The percentage of viable
yeast cells was expressed according to the following formula:

N = number of CFU in experimental tube 100
number of CFU in contr01_t6bes
Statistics

Results were analyzed using the Students t test. All data was

reported with its standard deviation.

RESULTS

Immune responses in sensitized mice

 

Groups of mice were actively sensitized with C, albicans or
C, kru§g1_heat-killed yeast cells. Random animals from each sensitized
group were selected and assayed 1 week after the final subcutaneous
injection of antigen. Serum was collected from some of the animals
for serological studies while other mice were tested for footpad
reactivity.

Sera collected from mice before and after sensitization were

tested for precipitating and agglutinating antibodies. Precipitin
reactions with oidiomycin were strongly positive using sera from
sensitized mice while non-sensitized mouse sera showed no reaction.
The specific bovine antiserum, included as a positive control,
consistently show precipitins. Sera from mice sensitized with 9,.krgsej_
yeast cells showed no precipitins with the g, albicans antigen.
Agglutinating antibodies were not detected in any of the mouse sera
using the tube agglutination technique with heat-killed C, albicans
yeast cells. A strong agglutination titer (2560) was seen with the
bovine antiserum. In addition, a weak agglutination titer (20) was
seen using heat-killed p, kgpsgi_yeast cells and the bovine antiserum
indicating little antigenic cross-reactivity between C, albicans and
s. M.

Reactivity of mice to the sensitizing antigen was also assayed

utilizing the footpad swelling technique. Preliminary experiments

35

36

showed that the minimum amount of yeast antigen needed to elicit a

5 yeasts per 0.04 ml (data not shown).

response represented ca. 2 x 10
This concentration of antigen was used in all subsequent experiments.
The data in Table 1 show the net footpad increase at all times except

0 h is significantly greater in mice sensitized with antigen

(p < 0.001) than in those with no prior sensitization. At 4 h,

footpad reactivity in non-sensitized mice was approximately one-half
that seen in the sensitized group. Between 4 and 24 h, the footpad
reaction was still increasing in the sensitized group while the
reactions in the non-sensitized mice waned. By 48 h, the footpad
measurements in the non-sensitized mice had returned to normal. The

48 h measurement in the sensitized group showed the footpad still

very swollen but by 72 h, the footpad had essentially returned to

normal size. To show that this reaction was specific for the
sensitizing antigen, groups of mice were sensitized with another species
of Candida, C, krysej,

Table 2 shows the specificity of footpad reactions in mice
challenged with homologous antigen and no reactivity in those animals
challenged with heterologous antigen (p < 0.001).

Overall, these data indicate that mice sensitized with heat-killed
yeast cells: 1) produce detectable antibody, and 2) show specific
footpad reactivity when challenged with homologous antigens. In
addition, this specific footpad responsiveness remained for at least
18 weeks after the final sensitization dose although serum antibody

became undetectable by that time (data not shown).

37

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39

Survival of mice after intravenous injection with viable C, albicans

 

The L0 values for the non-sensitized group is approximately

50
the same as that of the sensitized group (Table 3). Pretreatment
with 200 mg/kg cyclophosphamide significantly decreased the animals'
ability to survive the challenge (> 100 fold). Mice pretreated with
100 mg/kg of cyclophosphamide showed a greater ability to survive
when compared to the 200 mg/kg group although the median lethal dose

value was much lower than observed in those groups not receiving

cyclophosphamide.

White blood cell kinetics in non-sensitized, sensitized and

 

cyclosphosphamide pretreated mice

 

The total white blood cell count (WBC) for non-sensitized mice
was approximately 8289 cells/mm3 of which 17% (1467) were PMN, 76%
(6563) were lymphocytes, and 3% (259) were monocytes (Table 4).
The WBC and differential for sensitized mice were essentially the
same as that for non-sensitized mice. Seventy-two hours after
injection with 100 mg/kg cyclophosphamide the WBC decreased to 5491

3 with the greatest decrease seen in the calculated number of

cells/mm
PMN. The total numbers of lymphocytes and monocytes, although
depressed when compared to non-drug treated animals was not statisti-
cally significant (p > 0.05). An even greater decrease in WBC was
observed in mice pretreated with 200 mg/kg of cyclophosphamide. The

use was 4255 cells/mm3 with 6% (253) PMN, 92% (3816) lymphocytes and

40

Table 3. L05 values in non-sensitized, sensitized and cyclophosphamide
(CY pretreated mice, after intravenous injection.

 

 

 

Experimental L050a

Non-sensitized 3.4 x 105
Sensitizedb . 3.3 x 105
CY (100 mg/kg)C 8 x 103
CY (200 mg/kg) <1 x 103

 

amice (6 per group) were injected with varied dilutions of live washed
C, albicans cells in saline and observed for 30 days.

b 6

mice were sensitized with 4 weekly injections of 10 heat- killed

C. albicans yeast cells.

cmice received a single intraperitoneal injection of cyclophosphamide

3-5 days prior to injection.

41

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42

2% (186) monocytes. Again, the greatest reduction in cell type was seen
in the PMN (p < 0.001) although all cell types were significantly reduced
(p < 0.02). Eosinophils were occasionally seen but always comprised

less than 1% of the total white cell count.

Footpad reactivity in non-sensitized and sensitized mice injected with

 

livepyeast cells

 

Mouse hind footpads were inoculated with washed viable C, albicans
yeast cells. At 0, 4, 24, 48, and 72 h, groups of mice were sacrificed.
The footpad thickness was measured, and the foot removed. Viable
yeast cells remaining in the foot were determined by pour plates of the
foot homogenate.

Table 5 shows the results of footpad.measurements in non-sensitized
and sensitized mice injected with live yeast cells. The non-sensitized
mice exhibited a maximum response at 4 h. The footpad decreased to
approximately the same thickness at both 24 and 48 h with further
decrease by 72 h. At 96 h, the foot had returned to its pre-injection
size (data not shown). The footpad thickness response in the sensitized
mice was significantly greater (p < 0.01) than that of the non-sensitized
group at all time points with the exceptions of the 0 h and the 72 h
readings. This group displayed an equally intense response at 4 h and
24 h followed by a moderate reduction at 48 h. At 72 h, the footpad

swelling was on the decline and returned to normal by 96 h.

43

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44

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45

Viable cell counts in non-sensitized mouse footpads are shown
in Table 6. The non-sensitized group showed a steady decrease in the
number of live yeast cells present in the injected foot. By 72 h,
less than 1% of the original inoculum remained. The sensitized group
showed a similar decrease in viable cell counts. As with the
non-sensitized group at 72 h, greater than 99% of the original inoculum
was no longerviable.

Control experiments were also performed to determine if any
dissemination of the injected inoculum occurred. Rarely were yeasts
cultured from homogenates of liver, lung, spleen or kidneys. In the
event that yeasts were cultured, the animal was not included in

computation of the above data.

Footpad reactivity in cyclpphosphamide pretreated mice

Both non-sensitized and sensitized, cyclophosphamide pretreated
mice exhibited varying footpad responses when injected with heat-killed
C, albicans as compared to non-drug treated controls (Table 7).
Non-sensitized mice which received 200 mg/kg cyclophosphamide prior
to challenge showed a similar response as non-treated controls. A
maximum response was seen at 4 h with a reduction thereafter and a
return to normal size by 48 h. The sensitized group that received
200 mg/kg cyclophosphamide showed a very different response. Whereas
the nontreated sensitized group showed a maximum response at 24-48 h
the cyclophosphamide pretreated mice showed a mild reaction at 4 h

with an increased response at 24, 48 and 72 h. The foot did not return

46

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47

to its preinjection size until 120 h post injection.

With cyclophosphamide pretreated mice, injection of live C, albicans
produced a generally more intense footpad reaction over the course of
the experiment than that seen in non-drug treated groups (Table 8).
The 4 h response in non-sensitized mice which received 100 mg/kg of
cyclophosphamide was slightly less than that seen in non-sensitized
mice which had not received cyclophosphamide while the response seen
in mice which had received 200 mg/kg of cyclophosphamide was approxi-
mately the same as the control group. By 24 h, the footpad reaction
in the non-pretreated group showed a 50% decrease while those animals
receiving cyclophosphamide exhibited a continued intense response.

The 48 h reaction showed a decrease in both cyclophosphamide pretreated
groups from the 24 h reading followed again by a slight decrease at

72 h. The non-pretreated mice showed similar 24 and 48 h responses
followed by a decrease at 72 h. The inflammatory reaction was still
greater at 72 h for the cyclophosphamide treated groups than seen in
the non-treated mice.

The sensitized, cyclophosphamide pretreated groups showed less
rapid increased 4 h and 24 h responses when compared to the sensitized
mice which had not received cyclophosphamide. By 48 h, the reaction
in this non-drug treated group was on the decline, while that seen in
the cyclophosphamide pretreated groups showed a continued increase,
more marked in the group that received 200 mg/kg of the drug. This

increased response remained at 72 h for the 200 mg/kg group compared

48

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49

to a decreased response in the 100 mg/kg group. The sensitized mice
which had not received the drug showed a gradual return to normal.

Overall, these results indicate an enhanced footpad response in
both non-sensitized and sensitized mice, when pretreated with
cyclophosphamide and injected with live C, albicans. This response
was, in general, more pronounced in those mice which had received
200 mg/kg of cyclophosphamide in contrast to the 100 mg/kg groups and
of longer duration. The footpad response was much greater in mice
sensitized with heat-killed yeast cells both non-drug treated as well
as drug treated than in mice not sensitized to p, albicans.

Viable cell count results in footpads of mice pretreated with
cyclophosphamide are shown in Table 9. By 4 h, the number of viable
cells had not decreased significantly in any of the cyclophosphamide
pretreated animals. At 24 h, mice which had received 100 mg/kg of
cyclophosphamide displayed a moderate decrease in live cells while
those mice which had received 200 mg/kg of the drug demonstrated an
increase in viable cell number. At 48 and 72 h, this trend continued
with the 100 mg/kg groups exhibiting a steady decrease, but not as
dramatic as in those animals not receiving cyclophosphamide. The
200 mg/kg groups displayed an increase in viable yeast cells through
72 h (Table 9). Cyclophosphamide pretreatment, seems to substantially
reduce the ability to control the localized infection. Sensitized and
non¥sensitized mice, both with and without cyclophosphamide pretreatment,
had no difference in the number of yeast cells present during the 72 h

period.

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50

 

 

 

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51

In vitro phagocytosis and killing of g, albicans yeast cells by PMN

 

 

from non-sensitized, sensitized, and pyclophpsphamide pretreated mice.
One hour after addition of g, albicans yeast cells to mouse
peritoneal PMN, many phagocytosed yeast cells were seen inside the PMN.
The percentage of germ tube production by phagocytosed (intracellular),
as well as extracellular yeast cells (Figure l) in the different groups
is shown in Table 10. The values observed between non-sensitized and
sensitized mice were not statistically different (p > 0.05) while the
percent germinating in either of the cyclophosphamide pretreated groups
was statistically significant (p < 0.001) when compared to either of
the groups which had not received the drug. There was no significant
observable differences in phagocytic activity in any of the groups.
The PMN from the cyclophosphamide (200 mg/kg) group were statistically
(p < 0.001) less able to inhibit germ tube production than PMN from
mice which had received cyclophosphamide (100 mg/kg). Virtually all
of the extracellular yeast cells were seen to produce germ tubes.
The results of the experiment to determine if incubation of
normal PMN in various concentrations of cyclophosphamide altered germ
tube inhibition capability are shown in Table 11. There i
was no difference in germ tube inhibition between non-pretreated PMN
and those pretreated with cyclophosphamide. Also, cyclosphosphamide
had no effect on the ability of extracellular yeast cells to produce

germ tubes (Table 11).

52

Figure l. Germination of phagocytosed C, albicans after 1 h
A. Mouse phagocyte showing 2 phagocytosed yeasts (arrows).
Note the lack of a germ tube. (Wright) x 1400.
B. Intracellular yeast (arrow) with germ tube initial.
Note germ tubes produced by extracellular yeasts.
(Wright) x 1400.
C. Intracellular yeast showing prominent germ tube

(Wright) x 1400.

 

53

Table 10. Germ tube production by C, albicans yeast cells phagocytosed
by PMN of non-sensitized, sensitized and cyclophosphamide
pretreated mice.

 

% Germinationa

 

 

 

Experimental Intracellular Extracellular
Non-sensitized 50 :_2.8 99
Sensitizedb so i 1.7 99
Cyclophosphamide (1oo mg/kg)C 71 : 1.4d 99
Cyclophosphamide (200 mg/kg) 84 :_2.9d’e 99

 

amean percentage of 100 counted yeast cells producing germ tubes
+ standard deviation.

bmice were sensitized with 4 weekly subcutaneous injections of 106

heat-killed C, albicans yeast cells.

cmice received a single intraperitoneal injection of cyclophosphamide
3-5 days prior to challenge.

dp < 0.001 vs. non-sensitized group.

ep < 0.001 vs. cyclophosphamide (100 mg/kg).

54

Table 11. Germ tube production by C, albicans yeast cells phagocytosed
by PMN preincubated with cyclophosphamide.

 

 

 

 

 

% Germinationa ‘_

Experimental Intracellular Extracellular
Normal PMN 50 :_2.8 99
Cyclophosphamide
pretreated PMNb

0.01 mg/ml 46 :.5 99

0.1 mg/ml 49 3: 7.2' 99 ‘

1.0 mg/ml 47 :_3.8 99

 

6mean percentage of 100 counted yeast cells producing germ tubes
:_standard deviation.

bPMN were incubated for l h in the various concentrations of

cyclophosphamide before addition of yeast cells.

55

Table 12. Killing of g, albicans yeast cells in the presence of PMN
from non-sensitized, sensitized and cyclophosphamide
pretreated mice.

 

 

 

Experimental % Recovered % Killed
Non-sensitized 55 i 8" 44 1 8
Sensitizedb 58 i 5 42 .t 5
Cyclophosphamide (100 mg/kg)c 78 :_4d 22 :_4
Cyclophosphamide (200 mg/kg) 90 :_6d 10 + 6

 

apercentage :_standard deviation.

bmice were sensitized with 4 weekly subcutaneous injections of 106

heat-killed C, albicans yeast cells.

cmice received a single intraperitoneal injection of cyclophosphamide
3-5 days prior to challenge.

dp < 0.001 vs. non-sensitized group

56

Table 13. Killing of C, albicans yeast cells by PMN preincubated
with cyclophosphamide.

 

 

 

Experimental % Recovered % Killed
Normal PMN) 56 1 8a 44 i 8
Cyclophosphamide
pretreated PMNb
0.01 mg/ml 58 :_5 42 :_5
0.1 mg/ml 55 :_7 45 :_7
1-0m9/m1 56:6 44:6

 

apercentage i_standard deviation.

bPMN were incubated for 1 hr in the various concentrations of

cyclosphosphamide before addition of yeast cells.

57

The in vitro ability of mouse PMN to kill 9, albicans yeast cells
was assessed. Yeasts and PMN at a ratio of 1:10 were incubated for
2 h, then the number of viable yeast cells remaining in the medium was
determined. The results (Table 12) showed no difference in the
ability of PMN from non-sensitized and sensitized mice to kill
C, albicans. Pretreatment of the mice with cyclophosphamide impaired
this killing ability. PMN from mice receiving 100 mg/kg of cyclo-
phosphamide showed better killing ability than those from mice which
had received 200 mg/kg.

As with the phagocytosis assay, PMN from non-pretreated mice were
exposed to cyclophosphamide. Results from this experiment showed no

suppression of candidacidal activity (Table 13) in cyclophosphamide

PMN when compared to normal PMN.

Footpad histopathology in non-sensitized, sensitized, and cyclpphospha-
'mide pretreated mice injected with live C. albicans.

The histological section from a normal, uninjected mouse footpad
is shown in Fig. 2. The outer desquammating layer of the epidermis,
the stratum corneum, consists of keratinized tissue which is sloughed
off during normal wear. Immediately below the stratum corneum are: ‘
the stratum lucidum and the stratum granulosum, the latter being
more prominent and consisting of 2 or 3 layers of somewhat flattened
cells containing granules of keratohyalin and eleiden. The innermost
layer of the epidermis is the stratum spinosum, also called the stratum

Malpighii or the germinal layer. Below the epidermis is the corium or

58

Figure 2. Histopathological section of a mouse footpad.

A. Sectioned mouse footpad showing stratum corneum (s),
epidermis (e), dermis (d) and muscle (m). (H + E) X
140

B. Higher magnification showing stratum corneum (sc),
stratum granulosum (sg), dermis (d), sebaceous gland
(sb) and hair follicle (h). (H + E) X 280.

C. Resident cells of footpad dermis including mast cell
(m), polymorphonuclear leukocyte (p) and fibroblast (f).
(H + E) X 630.

 

59

dermis. It is composed of a superficial thin layer that interdigitates
with the epidermis. The dermis contains blood and lymphatic vessels,
nerves and nerve endings, sebaceous glands and hair follicles.' Some of
the resident cells of the dermis include: fibroblasts, occasional
polymorphornuclear leukocytes, macrophages, and mast cells. The striated
muscle bundles are easily seen, the separation probably being artifact—
ual due to dehydration, embedding and sectioning.

Figure 3A shows a section of a footpad immediately after injection.
The epidermis and dermis have some thickening due to fluid. Also
evident is separation of the muscle bundles. Figure 3B is a higher
magnification of the dermis shading edema or increase due to fluid.
Figure 3C is a PAS stain showing injected yeast cells evident between
the muscle bundles.

Four h after injection of live 9, albicans into a non-sensitized
mouse footpad, the footpad had less edema than immediately after
injection along with a moderate to severe cellular infiltrate (Figure
4A). This infiltrate was almost exclusively neutrophilic (Figure 48).
Examination of PAS stained sections revealed very few yeast cells, none
of which were producing pseudohyphae. Figure 5A shows a more apparent
cellular infiltration of the infected footpad 24 h after injection.

Many PMN were present in the dermis as well as throughout the muscle
layer. The infiltrate was still exclusively neutrophilic, but some
eosinophils were also present (Figure 5B). Examination of PAS stained
sections reveal very rare yeast cells which were producing pseudohyphae.

These were seen within areas of localized severe cellular infiltration

60

Figure 3. Section of a mouse footpad immediately after injection with

2 X 105 live C, albicans.

A.

Mouse footpad showing moderate dermal edema (arrows)
and separating muscle fibers. (H + E) X 140.

Higher magnification of dermal edema. (H + E) X 630.
Injected yeast cells between muscle bundles (PAS) X
630.

 

61

Figure 4. Section of a non-sensitized mouse footpad 4 h after

injection with 2 X 105 live C, albicans.

A.

Mouse footpad showing some edema along with a moderate
to severe cellular infiltrate. (H + E) X 140.
Cellular infiltrate showing an exclusively granulocytic

component. (H + E) X 630.

 

62

Figure 5. Section of a non—sensitized mouse footpad 24 h after
I injection with 2 X 105 live 9, albicans.
A. Footpad section showing a moderate to severe cellular
infiltrate, more concentrated between the dermis and '
the muscle. (H + E) X 140.
B. Cellular infiltrate showing mostly PMN (p) along with
some eosinophils (e). (H + E) X 1400.

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63

microabscesses (not shown).

Forty-eight h after injection, footpads showed very intense
inflammation (Figure 6A). The PMN was still the predominant cell type
but small numbers of macrophages were also evident. Small microabscesses
were apparent (Figure 6B) and yeast cells with pseudohyphae were usually
associated with these abscesses. The yeast cells were, however, rare
in these areas. Figure 7 shows a footpad 72 h after injection. Although
an infiltrate was still apparent, the foot was beginning to return to
its preinjection appearance. The small microabscesses seen at 48 h
were no longer evident being replaced by early signs of fibrosis.
Examination of PAS stained sections showed no yeast cells or pseudohyphae
present.

Footpads from sensitized mice showed a similar but more intense
response to injected antigen. The cellular response seen at 4 h was
very similar to that seen in non-sensitized mice (Figure 8A). The
greatest histopathological differences seen in sensitized mouse footpads
compared to non-sensitized footpads were at subsequent time points.

There was a more severe infiltrate seen at 24 h in sensitized mice
(Figure 88). The infiltrate consisted of predominantly PMN but also
significant numbers of macrophages and eosinophils could be seen. Small
abscesses were also seen. At 48 h the infiltrate was very severe
(Figure 9A). Again this infiltrate consisted of predominantly PMN,

with macrophages and eosinophils present. Moderately large areas of
very severe infiltrate with associated necrosis were observed, with

rare PAS positive yeast cells present. Some early fibrosis was also

64

Figure 6. Section of a non-sensitized mouse footpad 48 h after
injection with 2 X 105 live C, albicans.
A. Mouse footpad showing severe cellular infiltrate.
(H + E) X 140.
8. Small microabscess. (H + E) X 630.

 

65

Figure 7. Section of a non-sensitized mouse footpad 72 h after
injection with 2 X 105 live C, albicans.
A. Mouse footpad showing mild residual cellular
infiltration. (H + E) X 140.

B. Evidence of early fibrosis (arrows). (H + E) X 630.

p
w\

.

. .L
.3.

E III

 

66

Figure 8. Footpad sections of sensitized mice after injection with
2 X 105 live C, albicans.
A. 4 h after injection. Moderate cellular infiltrate,
some edema. (H + E) X 140.
B. 24 h after injection. Severe infiltrate, layer of
cellulitis is apparent adjacent to muscle. (H + E) X

140.

 

67

Figure 9. Footpad sections of sensitized mice after injection with
2 x 10531ive g, albicans.
A. 48 h after injection. Very severe infiltration with
associated necrosis can be seen. (H + E) X 140.
B. 72 h after injection. Very severe infiltrate.

(H + E) x 140.

.. .N _
J;

c 1. .4 . ,
OP}...- . :..~l..huhw..fi
. .4... fl . “.4
In. an...”

I ‘

 

68

evident. Sections at 72 h post-injection still revealed a very heavy
infiltrate (Figure 9B) with more macrophages present.

Footpads from mice, both non-sensitized as well as sensitized,
pretreated with 200 mg/kg of cyclophosphamide also exhibited a cellular
response when injected with a suspension of live 9, albicans yeast cells.
This response was not nearly as intense as that seen in animals not
pretreated with the drug. In addition, large areas of necrosis had
developed and much yeast proliferation was evident in these areas.
Figure 10A shows the moderate edema and mild cellular infiltrate seen
in a non-sensitized, cyclophosphamide pretreated mouse footpad 4 h
after injection with live C, albicans. Figure 108 shows an area of
the dermis within the footpad and the proliferating yeast cells and
pseudohyphae are apparent in this area (Figure 10C). The cellular
infiltrate was composed of predominantly PMN. Twenty-four h after
injection, the inflammation seen was more severe than at 4 h (Figure 11A)
although not as intense as that seen in non-cyclophosphamide pretreated
mice. In addition, more macrophages seen in the infiltrate than were
seen in mice that had not received cyclophosphamide. As with the 4 h
sections, some microabscesses with yeast cells and pseudohyphae were
present. The 48 h sections showed more foci of infection as well as
severe muscle damage (Figure 118). Many macrophages were present in
the infiltrate. At 72 h (Figure 110) the infiltrate consisted primarily
of macrophages along with degenerating PMN. Large abscesses were

evident and examination of PAS stained sections showed many yeast cells

69

Figure 10. Footpad sections of non-sensitized, cyclophosphamide

pretreated mice 4 h after injection with 2 X 105 live

C, albicans.

A.

Mouse footpad showing dermal edema and moderate
cellular infiltrate (H + E) X 140

Area showing fungal proliferation (PAS). X 140
Yeast cells and pseudohyphae in tissue space. (PAS)

X 630.

 

70

Figure 11. Footpad sections of non-sensitized, cyclophosphamide

pretreated mice after injection with 2 X 105 live

C, albicans. I

A. Mouse footpad 24 h after injection. Severe infiltrate
(H + E) X 140

B. Mouse footpad 48 h after injection. Severe infiltrate
and much muscle damage. (H + E) X 140

C. Mouse footpad 72 h after injection. Large abscess

with necrotic center can be seen. (H + E) X 140.

 

71

with pseudohyphae. Sensitized, cyclophosphamide pretreated mice
showed more cellular infiltration than was seen in non-sensitized,
drug treated animals (Figure 12). As with the non-sensitized,
cyclophosphamide pretreated mice, large areas of focal suppurative
inflammation had formed by 48 h. Severe muscle damage was evident

and large aggregates of yeast cells and pseudohyphae were seen in
these areas. Macrophages did, however, comprise a significant portion
of the cellular infiltrate at 24 h, more so than in non-sensitized,

cyclophosphamide pretreated mice.,

72

Figure 12. Footpad sections of sensitized, cyclophosphamide pretreated

mice after injection with 2 X 105 live C, albicans.

A.

House footpad 24 h after injection. Severe inflamma-
tion consisting of PMN and macrophages. (H + E) X 140.
Mouse footpad 48 h after injection. Severe inflamma-
tion, much muscle damage. (H + E) X 140

Mouse footpad 72 h after injection. Cellular infiltrate
mostly macrophage with degenerating PMNs. (H + E) X
140. Part of an abscess is seen at bottom of

picture.

trite

 

DISCUSSION

The observation that infections by g, albican in healthy hosts are
very rarely encountered seems to indicate that non-specific host defense
mechanisms greatly contribute to resistance to infection. It is only
when this system is compromised, either naturally, experimentally or
the unfortunate result of treatment for some other disorder that
infection by this organism will proceed. In addition, a majority of
healthy adults show positive antibody and/or cell-mediated hypersensitivity
responses to C, albicans which indicate that specific acquired immunity
may play a role in defense from infection.

Over the years, various animal models of candidosis, representing ‘
the diverse clinical pictures presented by these infections have been
developed (Rogers and Balish, 1980). They include models for cutaneous,
vaginal and disseminated disease. Experimental manipulation of these
models, usually by means of pretreatment with agents which suppress or
activate innate or acquired defense systems, have led to a greater
understanding of host-parasite interactions regarding this organism.

'Of particular interest is the jp_yiye_footpad microbicidal technique
first described by Shepard in 1960. This self-limiting localized
infection was developed to follow intralesional populations of an
obligate parasite, Mycobacterium leprae, and in assessing various
chemotherapeutic agents used in the treatment of leprosy. The mouse
footpad.swelling test for detection of hypersensitivity, especially of
the delayed type, has been utilized extensively (Crowle, 1975). Rifkind

£2.21- (1976) have adapted this model for use with Q, albicans.

73

74

By combining features of the reported uses of the model, i.e.
quantitative intralesional population changes and hypersensitivity
responses along with histopathological examination of infected footpads,
a better understanding of intralesional events after infection could be
realized. In addition, experimental manipulation of the host prior to
challenge could contribute more information regarding the infectious
process. The experimental strategies utilized in this research consisted
of following the course of a self-limiting fungal infection in normal,
immunologically hypersensitive and metabolically compromised mice.
Additionally, jn_yjt§p_experiments were used to help clarify in 11!g_
observations.

Immune responses in animals sensitized with heat-killed g, albicans
yeast cells indicated that immunological hypersensitivity specific
for this organism was induced. Precipitating anti-g, albicans antibody
was present in serum and specific footpad swelling responses to injected
antigen were seen. Sera from non-sensitized mice failed to show
detectable anti-g, albicans antibody or positive footpad responses
indicating little, if any, past exposure to Q, albicans.

Precipitating antibodies have been reported in mice previously
sensitized with live or heat-killed p, albicans (Giger et__l,, 1978;
Evron, 1980). The total quantity of yeasts used in the sensitization
procedures correlated well with the percentage of positive sera. Giger
et_e1, (1978), using counterimmunoelectrophoresis, demonstrated

precipitins in 92.3% of sera from mice sensitized twice with 106 live

75

C, albicans yeast cells and 54% of mice inoculated twice with 5 x 105

5

yeasts. Only 10% of mice inoculated twice with 5 x 10 heat-killed

yeasts showed precipitating antibddies. In the present study, when

6 heat-killed yeast cells

mice were sensitized with a total of 4 x 10
all sera tested positive for precipitins. A larger number of organisms
inoculated each time would probably favor greater antibody production
(Davis et,el,, 1973). The inoculation of smaller numbers of viable
cells could contribute to greater antibody production due to the less
rapid degradation and hence, a longer duration of stimulus for antibody
production. Evron (1980) reported precipitating antibodies present in
sera from mice sensitized once i.p. with 4 x 106 live C, albicans

6 to 8 x 107

suspended in saline or with s.c. injection of 6 x 10
heat-killed yeasts suspended in Freund's complete adjuvant (FCA).

Since the sera from these groups of mice were pooled, the percentage

of those positive for precipitins was not reported. In both of the
above reported studies, sera from mice not sensitized showed no
detectable precipitins.

No anti-g, albicans precipitating antibodies were detected in mice
sensitized with Q, k§g§e1_antigen using oidiomycin, a g, albicans
soluble extract. The lack of anti-C, albicans antibody could be
explained by the investigation of Sweet and Kauffman (1970). They
showed that of the 13 antigens recognized in medically important species

of Candida, p, albicans and C, krusei have only 2 common antigenic det-

erminants.

76

Agglutinating antibodies were not detected in any of the mouse
sera tested. This result is in agreement with those reported by Giger
et_el, (1978) but contrary to the report of Evron (1980). Specific
anti-g, albicans agglutinating antibodies have been demonstrated in other
mammalian systems, specifically rabbit (Sweet and Kauffman, 1970),
human (Rippon, 1974) and cow (see Materials and Methods, Preparation of
bovine anti-Q, albicans antiserum). Mouse serum has been reported to
agglutinate bacteria (Friedman and Moon, 1980).

One possible explanation for the results of Evron (1980) is her
use of FCA, a potent antibody production stimulator (Davis et_el,, 1973).
The possibility exists that continued production of antibodies of the
IgM class, shown to be better agglutinins, contributed to these results.
The problem of disparate results in agglutinating activity of mouse
sera and yeast cells deserves further attention.

Strong footpad reactivity was seen in all mice tested which had
been sensitized with heat-killed yeasts and challenged with the same
antigen (Table 1). At all times after injection footpad responses were
significantly greater in the sensitized group when compared with the
non-sensitized group. Rifkind et.el, (1976) were the first to adapt
the mouse footpad swelling test for use with fungal antigens. Their
results, utilizing heat-killed g, albicans are very similar to the
results obtained in this study at 48 h post-injection. They did not,
however, indicate swelling values for other time points as was reported

in this study. Giger et_el, (1978) and Evron (1980) also reported

 

77

footpad swelling values in mice sensitized with heat-killed C, albicans.
In these two studies, though, the challenge antigen used was a soluble
cytoplasmic extract whereas Rifkind et_el, (1976) and this investigation,
used heat-killed cells. Evron (1980) reported significant footpad
responses at 4 h, peaking at 48 h and waning thereafter while Giger
e__al, (1978) showed little swelling at 4 h and none thereafter. As
with the results in precipitating antibody production, the total amount
of sensitizing antigen used in their procedure may not have been adequate
to induce footpad reactivity.

Results of the present study show that the footpad response seen
is specific for the sensitizing antigen (Table 2). Mice only reacted
to the antigen with which they were sensitized and showed the same time
sequence of reactivity. Collins and Mackaness (1968) previously reported
the ability of the mouse footpad model to distinguish between different
species of Salmonella. Rifkind et_el, (1976) also showed specificity
for the sensitizing antigen.

The small footpad swelling value seen in non-sensitized mice at
4 b can probably be attributed to the use of a particulate antigen for
challenge (Table l). The cause of the swelling was most likely non-
specific inflammation due to the use of this antigen type, however the
presence of undetectable amounts of circulating antibody to yeasts that
contributed to this reactivity cannot be discounted. Mice may normally
harbor yeasts in their gastrointestinal tract, and these yeasts may

have common antigens with the challenge yeaSt (Savage and Dubos, 1967).

78

No increase in footpad size attributable to trauma from injection was
noted.

Allergic inflammation seen at 4 h post-injection is usually referred
to as an Arthus reaction, or immediate type hypersensitivity (ITH),
while inflammation that takes 24 to 48 h to peak is termed delayed type
hypersensitivity (DTH). The results presented in this study for
sensitized mice show a strong 4 h response peaking at 24 h and waning
thereafter. It is not unreasonable to postulate that both types of
hypersensitivity reactions are seen, an initial antibody-mediated ITH
followed by an overlapping DTH. Alternately, the reaction could be
thought of as a prolonged ITH. Due to continued presence of the

particulate antigen 1__§jtp, antibody may be produced locally and
therefore contribute to an inflammation due to continued production of
opsonins (e.g. complement factors C3a and 05a).

Rifkind et__l, (1976, 1977) showed that footpad reactivity to
C, albicans could be transferred to a naive animal via sensitized spleen
cells or Lawrence-type transfer factor and not with immune serum.
Evron (1980) demonstrated the presence of macrophage migration inhibition
factor (an jn_yjt[9_indicator of cell-mediated hypersensitivity) in
mice sensitized with either live or heat-killed C, albicans cells. In
the experiments reported by these investigators, the footpad reactions
were maximal at 24 to 48 h and were therefore classified as delayed

hypersensitivity responses. Based on the results of these published

reports, it is reasonable to assume that the footpad reactions seen in

79

the present study are of the delayed type although they probably contained
a strong Arthus component as suggested by the presence of circulating
antibodies as well as a significant 4 h response.

Footpad reactivity in non-sensitized and sensitized mice after
injection of viable p, albicans was similar to that seen with heat-killed
yeast cell challenge except the response was much more enhanced (Tables
1 and 5). Again, a much greater response was seen in sensitized mice
than in the non-sensitized group indicating the response was at least
in part, immunologically based. Three possibilities can be postulated
fer the greater response seen in mice injected with live cells as compared
to heat-killed cells. 1) An increase in fungal cell load due to
multiplication of the cells after injection, 2) the production of a
toxin by actively metabolizing yeast cells in tissue, or 3) pyrogens
excreted as a result of cell metabolism or present on the cell surface.
Although these pyrogens cannot be considered toxins, they do have toxic
properties and thereby can increase inflammation.

The first possibility can be discounted as shown by the results
presented in Table 6. There was a significant decrease in the numbers
of viable cells recovered from tissue 24 h after injection. At least
90% of the cells injected were not viable at this time and by 72 h
greater than 99% of the injected cells were killed. There was no differ-
ence in the ability of the animals of either group, non-sensitized or
sensitized, to control the infection. There was no dissemination of
the yeast cells from the foot to other parts of the body at any time

as proven by negative culture results of homogenized organs.

 

80

Oghiso and Matsuoka (1979) showed, that after injection of colloidal
carbon into mouse hind footpads, there was very slight egress of the
particles to other areas of the body. Essentially, all of the suspension
remained in the footpad or was trapped in the popliteal lymph node.
Since carbon particles are much smaller in size than intact yeast cells,
it can be assumed that the injected yeasts remained at or near the site
of injection. Care was taken, however, to include the area containing
this lymph node when the foot was excised and homogenized.

The second possibility, the production of a toxin by g, albicans
is a controversy as evidenced by the many published reports. This was
reviewed by Odds (1979). He concluded that there is no substantial
evidence to suggest that exotoxins, analagous to the toxins secreted
by bacterial pathogens, are produced by Q, albicans or other yeasts.

The third possibility seems more tenable. Candida surface glyco-
proteins have pyrogenic properties. Cutler et_el, (1972) obtained
pyrogenic reactions in rabbits which were elicited with whole cells
and cell walls of Q, albicans but not with cytoplasmic fractions. This
pyrogenic activity was not seen when the preparations were heated
previously. Holder and Nathan (1973) illustrated toxin-like properties
i.e., aggregation of platelets in sonically disrupted viable C, albicans
cells but none in disrupted heat-killed cells. One may conclude that
the toxic effects seen in candidal infections are manifestations of the
host response to intact viable cells of Candida or to some components

of Candida released in the process of digestion by phagolysosomes.

 

81

Results presented here indicate that there was no added protection
to lethal Q, albicans challenge as a result of prior sensitization with
heat-killed yeast cells. Both non-sensitized as well as sensitized
mice showed approximately equal mortality after i.v. injection of
viable p, albicans (Table 3).

Acquired immunity to infection with p, albicans in experimental
animals has been the subject of many reports. Mourad and Friedman
(1961) first showed some protection to lethal challenge in mice
previously inoculated s.c. with either live, merthiolate-killed, or
sonically disrupted 2- albicans. Merthiolate-killed~cells conferred the
least protection while sonicated cells gave the best. No data was ‘
presented on the immune status of the mice i.e., the presence of antibody
or skin reactivity at the time of challenge. Hasenclever and MitChell
(1963), using a different route of sensitization, i.p., and a dose of
approximately 2 x 107 live yeast cells given at 14 and 7 days before
challenge, showed increased survival. Intraperitoneal sensitization
with heat—killed yeast cells suspended in incomplete Fruend's adjuvant
conferred no protection. Again, the mice were not assessed for
specific immune responses. Giger et_el, (1978) showed that some
protection could be seen in mice previously sensitized's.c. with either
live or heat-killed cells compared to those not previously exposed to
Candida. This protection was short-lived as mice sensitized 2 weeks
before i.v. challenge showed protection while those sensitized 4 weeks

befbre challenge were not protected.

 

82

Other investigators, using non-specific immune activators, have
attempted to show protection to lethal Q, albicans challenge. Marra and

Balish (1974), using mice sensitized with Listeria monocytogenes, reported

 

short-lived immunity to p, albicans challenge as measured by faster
elimination of viable yeast cells from various organs. Rogers and
Balish (1977) showed no protection to Q, albicans challenge in mice

vaccinated with Mycobacterium bovis (strain BCG) but increased protection

 

against L, monocytogenes infection.

 

The literature cited above show that a variety of experimental
strategies have been used in attempts to confer protection to lethal
challenge with C, albicans. These attempts have resulted in limited
success and if protection was induced it was of short duration. Collins
(1974) has criticized these "protection studies" used as a basis for
development of vaccines. Increases in the mean time to death have very
little relevance if the majority of both vaccinated and control groups
eventually succumb to the challenge infection. Thus, he has suggested
comparing the fate, either elimination or multiplication, of the chall-
enge organism in vaccinated and control groups.

There seems to be some confusion in the literature concerning the
use of certain terminology, specifically immunization, vaccination
and sensitization. Immunization refers to the process or procedure by
which resistance to microbial challenge is conferred as the result of a
previous inoculation. For instance, laboratory mice inoculated with

M, leprae resulted in resistance to infection by L, monocytogenes and

83

M, tuberculosis (Patel and Lefford, 1978) as well as Salmonella enteriditis

 

(Collins, 1974). Vaccination, on the other hand, refers to the injection
of a suspension of a specific microbe (or some part of it) as a means of
prophylaxis or cure of the disease caused by that organism. An example
is the immunity to pertussis (whooping cough) after inoculation with

either killed Bordetella pertussis bacteria or of a bacterial fraction.

 

Sensitization refers to the process by which an individual has been
rendered susceptible to immunological reactions by previous exposure of
the immunological system to the antigens concerned. These reactions
may not be associated with resistance to infection. In the present
study, it seems justified to use the term "sensitized". Animals pre-
viously inoculated with heat-killed C, albicans show hypersensitivity
after antigen exposure as evidenced by specific circulating antibody
and strong footpad reactions (Table 1) but no acquired immunity to lethal
challenge (Table 3).

Pretreatment of mice with cyclophosphamide (CY) greatly increased
their susceptibility to Q, albicans infection (Table 3). This finding
is in agreement with published reports using other infectious agents.
Robinson e__el, (1969) showed that the nonlethal infection of mice with
Sendai virus was converted to a fatal pneumonic illness when CY was
administered while Hurd and Heath (1975) reported similar results using
mice infected with influenza virus. Moser and Domer (1980) reported
that the net effect of CY treatment was to nullify any ability of mice

to develop resistance to reinfection. As in the present study, they

84

also reported a significant increase in mortality after i.v. injection
of p, albicans in CY pretreated mice.

An increased susceptibility to infection implies some alteration
in resistance mechanisms of the host. The possibility that this altera-
tion is single in nature seems remote. It is more probable that a
multitude of factors are involved. Although numerous side effects of
CY therapy occur such as hemorrhagic cystitis with hematuria and diarrhea,
severe leukopenia is the most marked (Sharbaugh and Grogan, 1969). In
the present study, all mice pretreated with CY showed significant depression
in the numbers of peripheral white blood cells especially the PMN
(Table 4). This cell type was significantly reduced in numbers in both
drug-pretreated groups when compared to non-treated controls. No other
side effects associated with CY pretreatment were observed in this study.
Moser and Domer (1980), using 200 mg/kg of CY to pretreat mice also
showed significantly lowered total leukocyte counts with the reduction
in numbers of PMN being the most marked.

Mice pretreated with 200 mg/kg of CY showed an even greater suppres-
sion of PMN than those which received 100 mg/kg of the drug (Table 4).
Sharbaugh and Grogan (1969) reported a similar dose dependent leukocyte
suppression in rats. By increasing or decreasing the dosage of CY used
to pretreat the animals, the severity of peripheral leukopenia could
be controlled.

Although the exact mode of action of CY is not known, it seems

likely that the drug inhibits the incorporation of precursors into DNA

85

(Hill, 1975). Since a clinical use of CY is to arrest the growth of
tumor cells, inherently rapidly dividing cells, this explanation seems
plausible. It is an unfortunate side effect of CY therapy that normally
rapidly dividing cells such as bone marrow stem cells are also affected.
Joyce and Chervenick (1977) observed that the number of marrow progenitor
cells was maximally depressed in mice 3-5 days after CY treatment as

was the number of peripheral blood PMN. For this reason, mice in the
present study were used experimentally at least 3 and no longer than 5
days after CY pretreatment.

The results discussed so far for CY pretreated mice show several
advantages for its use in studying infectious diseases. 1) Cyclophos-
phamide pretreatment results in selective depletion of white cells of
the granulocyte type, in addition, this effect appears to be dose
dependent although at high doses, significant reduction of all leukocyte
types is seen (Table 4). 2) Increased susceptibility to infection with
relatively non-virulent microorganisms can be induced. 3) There appears
to be a lack of noticeable side effects after drug treatment. It is
also suggested that the PMN is of prime importance in resistance to
infection with Q, albicans since depletion of this cell type correlated
well with mortality.

The footpad swelling responses seen after injection of heat-killed
yeast cells in CY pretreated non-sensitized mice showed a similar
response as that seen in non-treated controls (Table 7). The response
seen in the CY pretreated sensitized group was of lesser intensity than

that of non-treated sensitized mice. The response seen appeared to be

86

immunologically based since it was greater than that of the non-sensitized
CY pretreated group. This indicates that sensitized mice pretreated with
CY, even at the largest dosage, still retain immunological reactivity
albeit not as pronounced as in non-drug treated sensitized mice. In
addition, the ability to mount an immediate inflammatory response, as
evidenced by the 4 h swelling increase, is not affected by drug treatment.
The effects of CY on immune reactions have been the subject of
several reports. Cyclophosphamide appears to specifically affect B-cell
function (Stockman et_el,, 1973) although T-cell suppression has been
shown (Sharma and Lee, 1977). Beaman and Maslan (1977) reported a marked
increase in susceptibility to Nocardia asteroides infections in mice 1
after CY treatment. An intact humoral response is required for protect-
ion, from infection as well as recovery from the infection (Rippon,
1974). Mice were more susceptible to Histoplasma capsulatum infections
after CY pretreatment (Cozad and Lindsey, 1974). Recovery from this
infection is associated with the cellular (T-cell) component of the
immune system (Rippon, 1974). Moser and Domer (1980), using CY pretreated
live 9, albicans sensitized mice, showed significantly reduced precipita-
ting antibody production as compared to animals not treated with CY.
They suggested that the ability to produce antibody at the time of
challenge is crucial to defense against the infection. Based on the
results of these publised reports, it seems that CY affects both components

of the immune system although B-cell suppression seems to be the most

pronounced.

 

87

The footpad swelling responses after injection of viable C, albicans
observed in CY pretreated mice, both non-sensitized and sensitized, were
greater in magnitude and of longer duration than seen in those mice not
pretreated (Table 8). Mice pretreated with CY showed a dose dependent
inability to control this footpad infection as evidenced by viable yeast
cell counts from foot homogenates (Table 9). These counts seemed to‘
cerrelate with the swelling observed. Thus the increased size may have
been due, in part, to multiplication of the yeasts within the lesion.

Moser and Domer (1980), using a cutaneous Q, albicans infection
model, reported a similar inability of CY pretreated mice to control
the numbers of injected organisms. They showed up to lOO-fold increases
in numbers of viable cells recovered from lesion homogenates 3 days
after inoculation. There were larger lesion volumes after injection
than in non-CY pretreated mice. Sensitized mice which received CY 3
days before cutaneous challenge had larger lesion volumes than similarly
pretreated non-sensitized mice. There was no difference in the ability
of non-sensitized or sensitized CY pretreated mice to clear the infect-
ion. Dissemination to other body organs increased as a result of CY
pretreatment. No dissemination of inoculated yeasts was observed in
the present study, utilizing the footpad model.

Examination of histopathological sections of mouse footpads revealed
an acute inflammatory (granulocytic) cellular infiltrate soon after
injection (4 h) of viable yeast cells. No discernible differences

between the infiltrates of non-sensitized and sensitized mice were seen

 

88

at this time but differences were apparent at subsequent times.
Sensitized mice showed a mononuclear component in the cellular infilt-
rate not seen in non-sensitized mice. The PMN did, however, retain
numerical superiority throughout the entire experimental period in
sensitized mice.

Histopathological responses in footpads of mice previously sensi-
tized with heat-killed Q, albicans and injected with the same antigen
have previously been described (Rifkind et_el,, 1976). This response
was characterized by a cellular infiltration consisting of neutrophils
and mononuclear cells with the latter component being more intense at
48 h than at 24 h. Non-sensitized mouse footpads showed an exclusively
neutrophilic response to the antigen. Surprisingly, they reported no
swelling 4 h after antigen injection in either group. This is
inconsistent with the results of the present study (Table 1). Both
studies used the same antigen type (whole heat-killed yeast cells)
as well as the same sensitization procedure. Injection of a particular
suspension should result in a non-specific, but measurable, inflammatory
response (Davis et_el,, 1973; Roitt, 1974). Additionally, the results
of this study showed a significantly greater 4 h response in sensitized
mice than seen in non-sensitized mice. This, coupled with the
demonstration of specific anti-g, albicans antibody in sensitized mouse
sera, suggests an immunological basis for this early swelling increase.
Rifkind et_el, (1976) did not evaluate their sensitized animals for

humoral responses, i.e., antibody.

 

89

Histopathological responses to infection by p, albicans have been
described for a number of animal models. Giger et,el. (1978), in a
study of the pathology induced by cutaneous infection with g, albicans,
reported the cellular infiltrate to be composed of mixed acute and
chronic inflammatory cells in and around areas of dermal abscess.
Animals which were previously sensitized and then challenged showed a I
more intense infiltrate, of the same composition as that seen in non-
sensitized mice. The histopathological reactions to live and dead
yeasts were strikingly different. Abscesses frequently developed with
the former, whereas no abscesses were seen with latter. Pearsall and
Lagunoff (1974) reported in their mouse-thigh lesion model that the
cellular infiltration consisted primarily of neutrophils one week after
infection. At 2 weeks the infiltrate had increased in intensity and at
that time, more macrophlages, eosinophils and lymphocytes were seen.

In human infections, both cutaneous and systemic, the PMN is the largest
component of the cellular infiltrate (Rippon, 1974; Rogers and Balish,
1980).

The results of footpad homogenization when combined with the
histopathological observations suggest a correlation. As the intensity
of the cellular infiltrate increases, the number of viable yeast cells
correspondingly decreases. Twenty-four hours after injection the
numbers of viable yeast cells remaining in the foot was approximately
10% of the original inoculum, or up to 90% of the injected cells had

been killed. Hematoxylin and eosin stained footpad sections showed

 

90

at 24 h, in both non-sensitized and sensitized mice, was composed of
predominantly PMN although macrophage were more evident in sections
from sensitized mice. At 72 h after inoculation about 1% of the
injected yeast cells were still viable in both groups. These data
suggest that the PMN is the cell responsible for the elimination of the
microorganism early in the infection.

Further evidence for the significance of the PMN is seen when
results of CY pretreatment are analyzed. The histopathological
responses observed, soon after infection, showed a much'less intense
cellular infiltrate. As early as 4 h after injection, areas of fungal
proliferation within necrotic spaces were seen (Figures 108 and 10C).
Similar necrotic areas were evident at subsequent times after 4 h.

The corresponding viable yeast cell counts at the experimental times
(Table 9) confirmed fungal multiplication, during the course of the
infection.

. Is the inability of CY pretreated mice to control this localized
infection due to: 1) the smaller number of PMN available that can be
recruited to combat this infection, 2) PMN being produced that are
deficient in their ability to kill 9, albicans, or 3) both of the above?

A series of jn_yjtgp_experiments were designed to try to answer
the above questions using suspension of mouse PMN and g, albicans.
After incubation of PMN for non-drug treated mice with Q, albicans,
approximately 50% of the injested yeast cells produced germ tubes

(Table 12). This indicated the other 50% were either dead or still

 

91

viable although inhibited in their germ tube production ability. The
jn_yjt§p_candidacidal assay was used in an attempt to determine if these
non-germinated cells were viable. The results showed that approximately
57% of the yeasts were still viable after incubation of murine PMN and
yeast suspension (Table 12). Thus, there seemed to be good correlation
between inhibition of germ tube production and yeast cell death.
Identical experiments using PMN from CY pretreated mice showed both
the inability to prevent germ tube formation (Table 10) as well as
decreased candidacidal activity (Table 12). By preincubating PMN from
non-CY treated mice in various concentration of CY, these deficiencies
were not evident (Tables 13 and 15). This suggests that the PMN being
produced in CY pretreated mice may have altered candidacidal capabilities.

The.actions of PMN on Candida have been extensively studied
jn_yjt§p, These studies have been reviewed by Odds (1979) and Rogers
and Balish (1980). The salient facts of these studies are as follows:
1) C, albicans yeast cells are rapidly phagocytosed jn_gjtgp_by PMN,
2) some injested cells escape killing by producing germ tubes and/or
pseudohyphae and break out of the phagocyte, and 3) this ability corre-
lates well with the relative virulence for laboratory animals.

Sharbaugh and Grogan (1969) observed no significant changes in
the injestive powers of phagocytes from animals pretreated with CY.
They did observe striking decreases in the ability of phagocytes to kill

injested Pseudomonas aerugjnosa. Moser and Domer (1980) also suggested

 

altered functional capabilities of PMN in their cutaneous model of

 

92

Q, albicans infection. In addition, CY induces a significant decrease
in the levels of fibrinolytic and proteolytic enzymes of leukocytes
(Prokopowicz et_el,, 1967). It seems, therefore, that although a major
side effect of CY therapy is severe leukopenia, the cells that are
produced have altered function. A possibility exists that interaction
between the phagocytes membrane and lysosomes of phagocytic cells is
hindered in some manner, thus preventing lysosomal enzyme release and
therefore altered killing capacity of the cell.

These investigations using the footpad model have shown that the
PMN is the primary phagocytic cell responsible for the defense against
infection by g, albicans. Induction of specific ITH and/or DTH seem
to have no effect on the elimination of the infectious agent.
Cyclophosphamide therapy seems to not only depress PMN production but
also PMN.function. In addition, the footpad model as presented here,
can be further developed as an experimental tool to better understand
basic host-parasite interactions and the forces which decide infection

or health of the host.

SUMMARY

This study characterized the host response to a self-limiting
C, albicans infection. Groups of mice, non-sensitized, sensitized and
CY pretreated, were injected via a hind footpad with viable yeast cells.
Footpad swelling responses, numbers of viable yeasts remaining in the
lesion and histopathological examination of thin sections were eval-
uated over the course of the infection.

Non-sensitized mice exhibited maximal 4'h footpad swelling
responses after injection with a waning thereafter. By 72 h post-
injection, the foot had essentially returned to its pre-injection size
and approximately 99% of the injected yeasts had been killed. A
cellular infiltrate composed of PMN with few eosinophils was seen.

Mice previously inoculated with heat-killed C, albicans showed
circulating anti-C, albicans antibody as well as a specific footpad
swelling response to injected antigen. This footpad response had
immediate as well as delayed hypersensitive characteristics. There was
no difference in immunity to lethal infection between sensitized and
non-sensitized mice. Elimination of viable yeast cells from the infected
foot was at the same rate as in non-sensitized mice. The cellular
infiltrate was composed of predominantly PMN but included a significant
mononuclear component.

Treatment of mice with CY resulted in a severe peripheral leukopenia,
especially granulocytopenia. Resistance to lethal challenge was reduced

significantly. Specific footpad responses in CY pretreated sensitized

93

 

94

mice were still seen indicating retention of immune reactivity. Ability
to clear the localized infection was severely impaired.

Ip_yjt§p_experiments using murine PMN showed candidacidal activity.
However, PMN from CY pretreated mice showed an altered ability to kill
injested Q, albicans. This defect could not be induced by prior
incubation of normal mouse PMN in CY.

The importance of an intact non-specific host defense system has
been shown by the results of this study. Immunological hypersensitivity
may enhance the host response but have no effect on immunity. Among
the non-specific host defense factors, a functional PMN seems to be
of prime importance in resistance to infection by opportunistic

organisms.

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