Download Enhancement of in Vivoand in Vitro Murine

[CANCER RESEARCH 48, 41-45, January 1, 1988]
Enhancement of in Vivo and in Vitro Murine Immune Responses by the
Cyclophosphamide Metabolite Acrolein1
Thomas T. Kawabata2and lumber L. White, Jr.
Departments of Pharmacology and Toxicology and Biostatistics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-06I3
ABSTRACT
immunoenhancement have been extensively investigated, the
pharmacological basis for the greater sensitivity of Ts precur
sors to CY has not been determined. The effects of CY on the
immune system, however, are known to be mediated by its
metabolites rather than the parent compound (24-26). Under
the influence of the cytochrome P-450 monooxygenase system,
CY is converted to 4-hydroxy-CY which rearranges to aldophosphamide. Aldophosphamide undergoes .''elimination to
form acrolein (2-propenal) and the alkylating compound, PAM
(24-26). The binding of PAM to DNA is purported to mediate
the antitumor and immunosuppressive effects of CY by inhib
iting cell proliferation (27-29). Cells of the immune system
that are proliferating at a high rate were observed to be the
primary targets of CY when administered at immunosuppres
sive doses (30, 31). It has been suggested that Ts precursors are
proliferating at a greater rate than other cell types of the
immune system and therefore are more sensitive to the antiproliferative effects of CY (32). This hypothesis suggests that PAM
is responsible for the immunoenhancement produced by CY.
On the other hand, since acrolein is known to readily bind to
sulfhydryl groups of important cellular macromolecules (25,
29,33), this reactive aldehyde metabolite should also be consid
ered to mediate the CY inhibition of Ts generation.
The objective of this study was to determine whether the
immune response may also be affected by acrolein and/or PAM.
This was accomplished by examining the humoral and DTH
responses of mice administered acrolein or CY before sensiti
zation with sRBC. In addition, the in vitro T-dependent AFC
responses of splenocyte cultures exposed to 4-HC, acrolein, or
PAM prior to the addition of sRBC were evaluated. 4-HC is
known to spontaneously break down in aqueous solutions to 4hydroxy-CY. Since splenocyte cultures are unable to metabolically activate CY, 4-HC can be used to circumvent the need for
a metabolic activation system (e.g., liver S-9 preparation, hepatocytes). Several investigators have used 4-HC in vitro and
have replicated the immunoenhancing actions of CY that were
observed in vivo (16-20).
Cyclophosphamide (CY)-mediated immunoenhancement has been at
tributed to the inhibition of suppressor T-cell generation. In order to
exert its effects on the immune system, metabolic activation of C'Y is
required. The metabolite of CY responsible for its immunoenhancing
properties are not known. Two reactive metabolites of CY which may
inhibit suppressor T-cell generation are phosphoramide mustard and
acrolein, compounds known to primarily bind to DNA and sulfhydryl
groups, respectively. The objective of this study was to determine whether
acrolein and/or phosphoramide mustard are capable of enhancing the
immune response in a manner similar to CY. Administration of 100 /mimi/
kg of acrolein, i.v., to female C57BL/6 x Oil F, mice 1 day before
antigen exposure (sheep red blood cells) resulted in a 50% increase in
the delayed-type hypersensitivity response (foot pad swelling). The Day
4 primary humoral immune response to sheep red blood cells was also
increased by 88 and 60% after the administration of 30 and 100 /xmol/kg
acrolein 1 day before sensitization with sheep red blood cells. Exposure
of splenocyte cultures to 3 x 10~7 and 10"* M 4-hydroperoxy-CY
or
acrolein produced significant increases in the in vitro T-dependent anti
body-forming cell response. In contrast, the antibody-forming cell re
sponse of cultures exposed to 3 x IO""- IO'"1M phosphoramide mustard
did not increase above control levels. These results suggest that acrolein
is responsible for the CY-induced enhancement of the immune response.
Moreover, the enhancement may be produced by the binding of acrolein
to sulfhydryl groups of molecules of cells required for the generation of
suppressor T-cells.
INTRODUCTION
The antineoplastic drug, Cyclophosphamide, is commonly
used as an immunosuppressive agent in the treatment of au
toimmune diseases and to prevent graft rejection. However,
numerous investigators have demonstrated that when admin
istered in low doses before or soon after antigen exposure, CY3
will also enhance cellular and humoral immune responses ( 17). The immunoenhancing effects of CY have been attributed
to a selective inhibition of Ts generation (6-20), a cell popula
tion involved in the down regulation of a variety of immune
responses, including responses generated against neoplastic
cells (21, 22). Subsequent studies demonstrated that this selec
tive effect on Ts generation was due to a greater sensitivity of
precursor Ts to CY than effector Ts, helper T-cells, precursor
and effector cytolytic T-cells, and B-cells (8, 9, 15-20). In
addition, antigen-presenting cells involved in the induction of
Ts effector cells (Ts3) were found to be sensitive to low doses
ofCY(23).
Although the immunological mechanisms of the CY-induced
MATERIALS AND METHODS
Chemicals. Acrolein (99%) was obtained from Aldrich Chemical Co.
(Milwaukee, WI). Cyclophosphamide was purchased from Sigma
Chemical Co. (St. Louis. MO). 4-HC was generously provided by Dr.
Michael Colvin (Pharmacology Department, Johns Hopkins Oncology
Center, Johns Hopkins University) and PAM was provided by the Drug
Synthesis and Chemistry Branch, Division of Cancer Treatment, Na
tional Cancer Institute.
Animals. Female CS7BL/6 x C3H L, mice, hepatitis and Sendai
virus free, 8-12 wk of age, were used in this study (Frederick Cancer
Research Center, Frederick, MD). Mice were received at 5-7 wk of
age, randomized, and quarantined for at least 1 wk prior to use. Plastic
cages were used to house the mice with woodchip bedding. Food (Purina
Laboratory Chow) and tap water were provided ad libitum. Mice were
maintained on a 12-h light-dark cycle at 21-24°Cand 40-60% relative
Received 6/23/87; revised 9/23/87; accepted 9/28/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by USPHS Grants ES05317 and ES03434 and National
Institute of Environmental Health Sciences Contract ES55094.
2To whom requests for reprints should be addressed, at Box 613, Medical
College of Virginia, Richmond, VA 23298-0613. Recipient of a National Research
Service Award Postdoctoral Fellowship ESOS3I7.
3The abbreviations used are: CY, Cyclophosphamide; AFC, antibody forming
cells; DTH, delayed-type hypersensitivity; 4-HC, 4-hydroperoxycyclophosphamide; PAM, phosphoramide mustard; sRBC, sheep red blood cells; Ts, suppressor
T-cells; PFC, plaque-forming cells.
humidity.
Delayed-type Hypersensitivity Response. Acrolein was diluted in
saline (vehicle) and mice were administered 10, 30, and 100 «¿mol/kg
41
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MECHANISM OF CYCLOPHOSPHAMIDE
acrolein, i.v. All drugs were administered in a volume of 0.1 ml/10 g
body weight. Doses of 200 /tmol/kg were found to be lethal, whereas
the doses used in this study did not produce any behavioral signs of
toxicity. Spleen weight, cellularity, and body weight were not signifi
cantly affected. Another group of mice was administered 20 mg/kg CY
(72 /imol/kg), i.p. Although the DTH response may be more greatly
enhanced with higher doses of CY (100-200 mg/kg) (2, 5, 13), these
doses also result in a suppressed humoral immune response (2). A dose
of 20 mg/kg had been reported to enhance DTH (5) and AFC (7)
responses to sRBC. Mice of all treatment groups were sensitized with
sRBC 24 h after drug exposure. The method of sensitization and
challenge was similar to that described by LaGrange et al. (3). Mice
were sensitized with 10* sRBC/mouse i.v. and challenged with 10*
sRBC in the left hind footpad 6 days after sensitization. Swelling
responses were measured with a micrometer 24 h after challenge. The
footpad thickness of the right foot was subtracted from the left to obtain
the value of footpad swelling. Untreated mice that were challenged but
not sensitized had a footpad swelling of 0.2 mm.
Primary IgM Antibody-forming Cell Response. Mice were treated
with vehicle, acrolein, or CY as described for the DTH assay. One day
after drug exposure, mice were sensitized i.v. with 5 x 10* sRBC. An
additional group of mice was treated with acrolein (100 ¿imol/kg,i.v.)
or vehicle 1 day after sensitization. Four days after sensitization, mice
were sacrificed and the spleens were removed. The number of IgMproducing cells was determined by a modification of the Jerne plaque
assay (34). Briefly, spleen cells and sRBC were added to an agar
solution. Guinea pig complement was added and the mixture was
vortexed and poured on a Petri plate. A glass coverslip was placed on
the drop of agar to obtain an even spread of cells. Following a 3-h
incubation period at 37°C,cleared areas of hemolysis (plaques) occur
IMMUNOENHANCEMENT
RESULTS
Effects of Acrolein and CY on the Delayed-! ype Hypersensitivity Response. The footpad swelling response of mice admin
istered acrolein at doses of 10 and 30 /¿mol/kg1 day before
sensitization to sRBC did not significantly differ from the
response of vehicle-pretreated mice (Fig. 1). However, a dose
of 100 Ã-/mol/kgproduced a 50% increase in footpad swelling.
Although a slightly greater swelling response was observed in
CY-pretreated mice, the response was not statistically different
from the mice treated with 100 ¿unol/kgacrolein.
Effects of Acrolein and CY on the in Vivo I -dependent AFC
Response. Administration of 30 and 100 /¿mol/kgof acrolein 1
day before sensitization to sRBC increased the PFC response
by 88 and 60%, respectively (Fig. 2). A dose of 10 ¿¿mol/kg
did
not produce a significant increase. Cyclophosphamide pretreat
ment was found to also significantly enhance the number of
PFC generated, a response similar to that obtained with 100
/¿mol/kgacrolein. In contrast, the AFC response of splenocytes
of mice administered 100 /mio I/kg acrolein 1 day after sensiti
zation with sRBC did not differ from the control response.
Effects of 4-HC, Acrolein, and PAM on the in Vitro I dependent AFC Response. The AFC response of splenocyte
cultures exposed to 3 x 10~8 or IO"7 M 4-HC was not altered,
whereas the addition of 3 x 10~7 or 10~* M 4-HC resulted in
an AFC response 62 and 65% above control levels, respectively
(Fig. 3A). At higher concentrations, 10~5 and 3 x 10~5 M, the
number of AFC/106 splenocytes decreased 50 and 99%, respec
tively. A similarly shaped concentration-response
curve was
obtained with the exposure of splenocyte cultures to acrolein
ring around each AFC were enumerated under magnification. The AFC
response was expressed as the number of PFC per IO6splenocytes.
In Vitro 1-dependent Antibody-forming Cell Response. Spleen cell
suspensions were prepared to 1.1 x IO7cells/ml in RPMI 1640 con
taining 2 HIML-glutamine (Gibco Laboratories, Grand Island, NY)
antibiotic/antimycotic (Gibco Laboratories, Grand Island, NY; 100
units/ml penicillin and streptomycin and 0.25 jig/ml fungizone), 2%
fetal calf serum (HyClone Laboratories, Inc., Logan, UT), and 50 /IM
2-mercaptoethanol. The cell suspensions were added to 24-well cluster
plates (0.9 ml/well) and 100 n\ of the test compound dissolved in
Earle's balanced salt solution containing 1 mM 4-(2-hydroxyethyl)-l-
T?•6541
8
::.v'm%Wi-Wlï
„LTt*T•:.;.
piperazine ethane sulfonic acid were added (pH 7.2). Drug solutions
were kept at 4°Cand prepared 10-20 min before adding them to the
cell suspensions. The cultures were incubated in 5% CO: at 37°Cfor
60 min while rocking. The cells were centrifuged and washed twice in
the 24-well plates with the same media used to initially prepare the cell
suspensions. The cultures were sensitized with 10' sRBC/well and
o
o
incubated for 5 days (peak day) IgM response in a special gas mixture
of 10% CO2, 7% O2, and balanced nitrogen. The number of AFC
generated in these cultures was determined by the Jerne plaque assay
as described above. Cell viability of the splenocyte cultures was deter
mined by the pronase method (34, 35). Viability measurements by this
method were found to be more sensitive than those obtained with
conventional trypan blue uptake methodology.
Data Analysis. Each of the in vivo studies presented is representative
of at least two studies. The in vitro AFC responses of control cultures
were found to vary between experiments. However, the variability of
responses of replicate control cultures within an experiment was usually
never greater than ±10% of the mean. Thus, the percentage of control
values of replicate cultures of at least 2 experiments (4 replicates/dose
group/experiment) was calculated and presented as the percentage of
control AFC/10* spleen cells. The data were analyzed for homogeneity
n-*T•kT
O
10
30
DOSE OF ACROLEIN
100
CY
(umol/kg)
Fig. I. Effects of acrolein and CY (20 mg/kg) on the DTH response. The
results are of a representative experiment of two experiments. Each of the values
represents the mean ±SE footpad swelling obtained from 8 mice per treatment
group. *. significant difference from the control values (/' < 0.05).
1800-1MJ
*TT^*0
DAY
-1
1600-ü
1400-Z
+1Ty0
1200-ÕÕ
1000-D
600-ü
0
*u.1
to determine whether parametric or nonparametric analysis were ap
propriate. When an analysis of variance of parametric data showed
significant differences, control groups were compared to treatment
groups by Dunnett's t test. When nonparametric data showed signifi
''-
0 •T
10
30
100
CYDAY
100
DOSE OF ACROLEIN (umol/kg)
cant differences, treated and control groups were analyzed by the
Wilcoxon rank sum test. When a comparison was made between two
groups, Student's t test was utilized. Groups differing from the control
Fig. 2. Effects of acrolein and CY (20 mg/kg) on the primary AFC response
(Day 4 IgM). Results were obtained from a representative experiment of two
experiments. Each value represents the mean ±SE. AFC/10* spleen cells of 8
mice per treatment group. *, significant difference from the control values (P <
0.05).
response at the level of P < 0.05 were considered significant.
42
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MECHANISM OF CYCLOPHOSPHAMIDE
IMMUNOENHANCEMENT
(Fig. 3B). Significant increases of 20-46% above the control
AFC response were observed with exposure to 3 x 10~7, 10~6,
and 3 x 10~6 M acrolein (Fig. 3B). The number of AFC
generated in cultures exposed to 10~5 and 3 x IO"5 M acrolein,
120r•;•.
100
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•
':'.-•'-.'•
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80
60
however, was suppressed by 50 and 99%, respectively. In con
trast to the effects of 4-HC and acrolein, exposure of splenocyte
cultures to PAM did not result in an enhanced AFC response.
Concentrations of 3 x 10~8to 10~4M PAM did not significantly
40
20¡IIIlili?TT¡¡¡i¡IIIA?:
3X10
ffl
alter the AFC response, while a 99% suppression was observed
with the addition of 10~3 M PAM (Fig. 3C).
<
>
Effects of 4-HC, Acrolein, and PAM on Cell Viability of
Splenocyte Cultures. Viabilities of splenocyte cultures used to
examine the effects of 4-HC, acrolein, and PAM on the in vitro
AFC response were also determined. At concentrations of me
tabolites in which AFC responses were observed to decrease by
99%, significant decreases in cell viability were also observed
(Fig. 4, A-C). At lower concentrations of 4-HC, acrolein, or
io'
3x16
'•"•"-'10»
10
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3X10
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CONCENTRATION OF 4-HC (MOLAR)
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CONCENTRATION OF ACROLEIN (MOLAR)
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10
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10
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3X1¿5
10
10*,
CONCENTRATION OF PAM (MOLAR)
•z.
CONCENTRATION OF 4-HC (MOLAR)
Fig. 4. Effects of 4-HC (A), acrolein (B), and PAM (C) on cell viability of
splenocyte cultures. Each value represents the mean ±SE cell viability (% of
control) of replicate cultures of at least two experiments. Shaded horizontal bar,
SE of cell viability of control cultures. The viability of control splenocyte cultures
ranged from 45 to 65%. *, significant difference from control (P < 0.05).
UÌ
111
(f)
3X105
10
180
'o160ö
14°n
^n_j
PAM, cell viability did not significantly differ from control
levels.
1
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T*>
DISCUSSION
40"
Since Maguire and Ettore (1) first reported in 1967 that CY
pretreatment could enhance the contact sensitization response,
numerous investigations were conducted to examine the effect
of CY on other immunological responses and to determine the
immunological basis for this effect of CY (2-19). Precursors of
Ts were determined to be affected and to be more sensitive to
CY than other cell types involved in the immune response (8,
9, 15-20). Moreover, Lowy et al. (23) reported that APC are
required for the generation of antigen-specific effector Ts (Ts3)
(azobenzenearsonate system) and that these cells are CY sen
sitive. The mechanism for the greater sensitivity of precursor
Ts and Ts3-APC in comparison to other cell types, however,
has not been elucidated.
In order to mediate its effects on the immune system, it is
now well established that CY must first be metabolically acti
vated (24-26). The reactive metabolites of CY, PAM and
acrolein, are known to mediate their toxicity by different mech
anisms. PAM binds primarily to DNA (27-29), whereas acro
lein binds to sulfhydryl groups of proteins and peptides (29,
33). Thus, insight into the mechanisms of CY selectivity on Ts
generation may be obtained by determining which metabolite
of CY is responsible for its immunoenhancing actions. In this
study, the effects of acrolein on the in vivo DTH and primary
20
ILoT-ii1+T.,71il--:-•.•:•/:•:ï::li-Bi*
3X10
Ul
U
EC
Ul
CL
10'
10-6
"•"
3X10
-5
3X10
CONCtl. ¡RATIONOF ACROLEIN (MOLAR)
120T10060'6040'20-f*TT
Tti:
._
.üsi;i:*'
•3X10
j •"
•'
•
'
3X1"0
_
T
'3Xl"o '
'3X1"<Ti
10"
' 10
10"
CONCENTRATION OF PAM (MOLAR)
Fig. 3. Effects of 4-HC (A), acrolein (B), and PAM (C) on the in vitro Tdependent AFC response. Each value represents the mean ±SE. AFC/106
recovered spleen cells (% of control) of replicate cultures of at least two experi
ments. Four replicate cultures per each treatment group were used in each
experiment. Shaded horizontal bar, SE. AFC/10* recovered spleen cell of replicate
cultures of the control response. The control responses ranged from 600-3300
AFC/10* cells. *, significant difference from control (P < 0.05).
43
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MECHANISM OF CYCLOPHOSPHAM1DE IMMUNOENHANCEMENT
IgM AFC response and the in vitro T-dependent AFC response
were examined. In addition, the effects of 4-HC, acrolein, and
PAM on the in vitro AFC response were examined.
Administration of acrolein 1 day before sensitization was
found to enhance the DTH and AFC responses to sRBC to an
extent similar to that obtained with CY pretreatment. When
acrolein was administered 1 day after drug administration
rather than before sensitization, the enhancing effect of acrolein
on the primary AFC response was not observed. This timing of
drug and antigen exposure is one of the major characteristics
ofCY-mediatedimmunoenhancement(2,10-12).
After antigen
exposure, Ts precursors differentiate to Ts effector cells (21,
22) and become less sensitive to the effects of CY (15-20). AFC
responses of splenocyte cultures were also enhanced with ex
posure to 4-HC or acrolein at similar concentrations. The
concentrations of 4-HC which produced an enhanced AFC
response were similar to those used by other investigators to
selectively inhibit the generation of Ts (16-20). In contrast to
the effect of 4-HC and acrolein, PAM was found to suppress
only the AFC response at the highest concentration, a concen
tration which significantly decreased cell viability. These results
suggest that acrolein may mediate the CY-induced immunoenhancement rather than PAM.
Investigators have suggested that precursors to Ts may be
proliferating at a greater rate (32) or are less capable of repairing
the DNA damage (15), thereby making this cell population
more sensitive to CY. This would imply that the DNA alkylating metabolite, PAM, is responsible for the inhibition of Ts
induction. This hypothesis is supported by the findings that a
variety of antineoplastic drugs are also capable of enhancing
the immune response (36). More in-depth studies, however,
have demonstrated that Adriamycin (37, 38) and bleomycin
(39) mediate their immunoenhancing effects through a mecha
nism that does not involve Ts. Moreover, results of studies on
Ts-precursor proliferation rates in relation to the development
of Concanavalin A-induced suppressor activity (antigen-non
specific) indicate that Ts precursors are not a highly prolifer
ating cell type (40). Treatment of cells with mitomycin C, to
inhibit DNA synthesis, did not prevent the induction of sup
pressor cell activity of Concanavalin A-exposed cultures (40).
These observations suggest that the antiproliferative actions of
CY may not be the mechanism for selectivity on precursor Ts
and further support the role of acrolein.
The greatest sensitivity of Ts precursors to CY has also been
suggested to be attributed to lower levels of aldehyde dehydrogenase activity (15). CY is metabolized to 4-hydroxy-CY, which
rearranges to aldophosphamide and decomposes to PAM and
acrolein (24-26). Aldophosphamide may be converted to the
inactive carboxyphosphamide by aldehyde dehydrogenase (24,
25). Differences in the susceptibilities of hemopoietic stem cells
to CY have been demonstrated to be mediated by differences in
aldehyde dehydrogenase activity (41). However, since acrolein
was found in this study to mimic the immunoenhancing effects
of CY and 4-HC, differences in aldehyde dehydrogenase activity
may not be the major factor in determining the relative sensi
tivities of Ts precursors and/or Ts-APC.
Acrolein is known to mediate its toxic effects on cells by
binding to sulfhydryl groups of important cellular molecules
(29, 33). Utilizing [14C]CYlabeled at the carbon associated with
nucleic acids. Thus, inhibition of Ts generation may be me
diated by the binding of acrolein to important sulfhydryl groups
of molecules in Ts precursors and Ts3-APC. The sensitivity of
these cell types to acrolein may be due to the presence of certain
sulfliyclryl containing molecules unique and/or critical to the
function of these cells (e.g., cell membrane receptors), a lower
concentration of intracellular glutathione, or to the inability to
regenerate sufficient amounts of glutathione.
In addition to providing evidence for the immunoenhancing
effects of acrolein, the in vitro AFC studies demonstrated that
higher concentrations (3 x 10~5 M) of 4-HC and acrolein
suppressed the AFC response without a significant decrease in
cell viability. A 33-fold greater concentration of PAM (10~3 M)
was required to inhibit the AFC response. High concentrations
of PAM were also reported to be required to decrease the
survival of K562 tumor cells (42) and to inhibit mitogeninduced proliferation of murine splenocytes (43). Differences
in potency of PAM and 4-HC in inhibiting tumor cell growth
were also reported by Powers and Sladek (44). These results
suggest that the immunosuppressive effects of CY may in part
be mediated by acrolein and that the antiproliferative effects of
PAM may not be the only contributing factor.
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acrolein, Mannello et al. (29) demonstrated that the radioactiv
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chloroethyl group was found to be primarily associated with
44
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MECHANISM OF CYCLOPHOSPHAMIDE
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45
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research.
Enhancement of in Vivo and in Vitro Murine Immune Responses
by the Cyclophosphamide Metabolite Acrolein
Thomas T. Kawabata and Kimber L. White, Jr.
Cancer Res 1988;48:41-45.
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