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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2031-2036
IMMUNOBIOLOGY
The opioid antagonist naloxone induces a shift from Type 2 to Type
1 cytokine pattern in BALB/cJ mice
Paola Sacerdote,
Barbara Manfredi,
Leda Gaspani, and
Alberto E. Panerai
From the Department of Pharmacology, University of Milano, Milan,
Italy.
 |
Abstract |
Opioid peptides affect different immune functions. We present
evidence that these effects could be mediated by the modulation of
TH1/TH2 cytokine production. BALB/cJ mice were
immunized with 50 or 100 µg of the protein antigen keyhole-limpet
hemocyanin (KLH), and treated acutely or chronically with the opioid
antagonist naloxone. One and 2 weeks after immunization, the production
of cytokines by splenocytes was evaluated by in vitro restimulation with KLH. The acute and chronic treatment with the opioid receptor antagonist naloxone decreased the production of interleukin (IL)-4 by
splenocytes of BALB/cJ mice. In contrast, IL-2 and interferon- levels increased after naloxone treatment. Finally, the opioid antagonist diminished the serum immunoglobulin G anti-KLH antibody titers. These results suggest that naloxone increases TH1
and decreases TH2 cytokine production. The effect of
naloxone could be ascribed to the removal of the regulatory effects
exerted by endogenous opioid peptides, which could therefore activate
TH2 and suppress TH1 cytokines.
(Blood. 2000;95:2031-2036)
© 2000 by The American Society of Hematology.
| |
Introduction |
Evidence for the involvement of the endogenous opioid
peptides  -endorphin (BE) and met-enkephalin in the regulation of
immune function is continuing to accumulate. Although in vitro
experiments gave rise to contradictory results, showing both
immunosuppressive and immunoenhancing effects of opioid
peptides,1 most data indicate that, in vivo, particularly
BE possesses immunosuppressive properties.1-4
We have shown that BE exerts a physiological inhibitory effect on
cellular-mediated immune responses5 and that the
administration in the rodent of the opiate receptor antagonist naloxone
or of immunoglobulins, which neutralize the activity of BE, increases mitogen-induced splenocyte proliferation within
minutes.5,6
Moreover, previous work from our group suggested a possible role of the
endogenous opioids in the modulation of TH1/TH2
balance. In fact, the opioid receptor antagonist naloxone has been
shown to affect TH1-mediated phenomena, such as skin graft
rejection in mice7,8 and autoimmune encephalomyelitis in
rats.9,10
The target of the effects of BE in the immune system could be the
balance of the 2 types of mature T helper cells, TH1 and TH2. The TH1 and TH2 cells produce
different patterns of cytokines: TH1 cells produce
interleukin (IL)-2, interferon (IFN)-, and lymphotoxins, whereas
TH2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13.
TH1 cells are involved mostly in cell-mediated reactions, while the TH2 cytokines are commonly found in association
with strong antibody and allergic responses.11,12
In the attempt to better define a possible physiological role for
opioid peptides in the modulation of the balance between TH1 and TH2 cell types, in the present paper we
analyze the effect of the in vivo treatment with the opioid receptor
antagonist naloxone on splenocyte production of the TH1
cytokines IFN- and IL-2 and of the TH2 cytokine IL-4 in
BALB/cJ mice immunized with the protein antigen keyhole-limpet
hemocyanin (KLH). Serum IgM and IgG anti-KLH antibody titers were also evaluated.
Materials and methods
Animals
BALB/cJ male mice, 18 to 20 g body weight (Charles River, Calco,
Italy), were used in the study. Animals were kept on a 12-hour light-dark cycle with water and food ad libitum and were housed 5 mice
to a cage. Mice were allowed to acclimate to the laboratory conditions
for a minimum of 10 days before experimental manipulations. Each
experimental group consisted of 12 to 16 animals.
Procedures involving animals and their care were in conformity with the
institutional guidelines, which are in compliance with national and
international laws and policies.13,14,15
Immunization and naloxone treatment
Mice were injected intraperitoneally with 50 or 100 µg of the protein antigen KLH (Sigma, St Louis, MO) in a volume of
0.2 mL of saline and were killed for cytokine evaluation 7 or 14 days after immunization.
The opioid receptor antagonist naloxone HCl (S.A.L.A.R.S., Como, Italy)
was injected subcutaneously at a dose of 5 mg/kg. The dose
was chosen on the basis of our previous studies, which showed that this
dose induces a relevant modulation of some immune parameters.5,6,16 One group of mice received only 1 acute injection of naloxone at the moment of immunization, while a second group of animals was chronically treated with subcutaneous
naloxone once daily starting from the immunization day. The animals
received the last naloxone injection 30 minutes before
sacrifice. Control animals were immunized with KLH and were treated
acutely or chronically with saline. Moreover, in order to rule out the
possibility that the stress caused by handling and injections had an
effect on cytokines, a group of animals were immunized with KLH and
were not further handled or injected until the day of sacrifice.
Splenocyte cultures
One or 2 weeks after immunization, animals were killed by
decapitation, and spleens were aseptically removed. Cells were teased from the spleens with the use of 20-gauge sterile needles through an
incision made in the spleen capsule,5 centrifuged, and
washed twice in Hanks balanced salt solution. Cells were
suspended in RPMI supplemented with 10% fetal calf serum
(FCS), 1% glutamine, 2% antibiotics, 10 mmol/L Hepes,
and 50 mmol/L 2-ME (all from Sigma) and were plated at
7 × 106 cells in 24-well plates containing a final
concentration of 80 µg/mL KLH in a total volume of 1 mL. Plates were
incubated at 37°C in 5% CO2 and 95% air. Supernatants were collected after 48 and 72 hours in culture and stored frozen at
 80°C for cytokine analysis. The concentration of 80 µg/mL used in vitro was chosen on the basis of previous pilot experiments. This concentration, in fact, induced an easily measurable, but not
maximal, stimulation of cytokine production, in order to be able to
detect a possible stimulation as well as any inhibition induced by naloxone treatment.
Measurement of interleukin-2, interleukin-4, and interferon-
The levels of IL-2 in 48- and 72-hour supernatants were determined
by enzyme-linked immunosorbent assay (ELISA) protocol as standardized
by Pharmingen (San Diego, CA). Briefly, the anti-IL-2 capture
monoclonal antibody (mAb) 1(µg/mL) was absorbed on a polystyrene 96-well plate, and the IL-2 present in the sample was bound to the
antibody-coated wells. The biotinylated anti-IL-2 detecting mAb (0.5 µg/mL) was added to bind the IL-2 captured by the first antibody.
After washing, avidin-peroxidase (Sigma) was added to the wells to
detect the biotinylated detecting antibody, and finally 2,2'-azino-bis (3ethylbenthiazoline-6-sulfonic acid) (ABTS,
Sigma) substrate was added, and a colored product was formed in
proportion to the amount of IL-2 present in the sample, which was
measured at optical density 405 nm.
IL-4 production was measured in 48- and 72-hour supernatants with the
use of the ELISA protocol outlined above with mAb anti-IL-4 at the
same concentrations used for IL-2. IFN- was also evaluated with the
same ELISA protocol except for the use of anti-IFN- capture and
detecting antibody at 2 µg/mL and 1 µg/mL, respectively (all mAbs
were from Pharmingen).
Anti-keyhole-limpet hemocyanin antibody enzyme-linked immunosorbent
assay
Blood was collected at the time of sacrifice, and sera were stored
at 20°C. Plates were coated overnight with 10 µg/mL KLH in
a carbonate coating buffer, pH 9.6. Mice sera were diluted 1:25, 1:50,
and 1:100 in phosphate-buffered saline (PBS)/Tween containing 1 mol/L
NaCl and incubated for 3 hours at 37°C.
Alkaline-phosphate-conjugated goat anti-mouse IgM (chain specific,
Sigma), diluted 1:6000 in PBS/Tween, or anti-mouse IgG (chain specific,
Sigma), diluted 1: 6000 in PBS/Tween, was then added, and plates were
incubated overnight at 4°C. After washing, p-nitrophenyl-phosphate
substrate at 1 mg/mL in carbonate buffer was added, and the colored
product formed was measured at OD 405 nm. Serum from
nonimmunized mice served as control.
Statistical analysis
Cytokine data were analyzed by means of 2-way analysis of variance
(ANOVA), with treatments and hours of culture, doses of KLH, and time
of immunization as factors, followed by a Tukey t test for
multiple comparisons. Acute and chronic saline groups were treated as a
single group. Serum antibody titers were analyzed with 2-way ANOVA,
with treatments and serum dilution as factors.
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Results |
Keyhole-limpet hemocyanin-stimulated cytokine production
The stress linked to simple manipulation and handling of animals did
not modify cytokine secretion, since levels of all cytokines did not
differ in acutely or chronically saline-injected mice, in comparison
with mice that were not further injected after KLH immunization (data
not shown). Moreover, since no difference was observed in acute or
chronically saline-treated animals, in the statistical analysis they
were considered as a single group. Naloxone treatment induces
significant changes. Figure 1 reports the
effect of the acute and chronic treatment with naloxone on IL-2
production by splenocytes.
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| Fig 1.
Effect of acute and chronic naloxone treatment on
KLH-stimulated IL-2 production in vitro by splenocytes.
Animals were immunized with either 50 µg KLH (panels A-D) or 100 µg
KLH (panels E-H) and killed after 1 week (panels A,B,E,F) or 2 weeks
(panels C,D,G,H). Spleen cells were cultured with 0 or 80 µg/ml KLH
for 48 hours (panels A,C,E,G) and 72 hours (panels B,D,F,H). Acute
saline control is represented by white bars; chronic saline control is
represented by right-slanted striped bars; acute naloxone treatment is
represented by left-slanted striped bars; chronic naloxone treatment is
represented by hatched bars. Results are expressed as mean ± SE. * = P < .01 versus saline controls.
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In the acute experiments, animals were treated with 1 injection of
naloxone or saline at the moment of in vivo immunization with 50 or 100 µg KLH, and 7 or 14 days later, splenocytes were cultured in vitro
with or without KLH for 48 and 72 hours. In the chronic experiments,
animals were treated daily with naloxone or saline from the day of
immunization to the day of sacrifice. With respect to
the levels of IL-2 in all of the in vitro KLH-stimulated cultures, the
2-way ANOVA on data revealed a main effect of both acute and chronic
treatment (F2,300 = 37.2, P < .001), with no significant difference between 48- and 72-hour cultures. Also, no
significant difference was observed between 50 and 100 µg KLH (F1,300 = 0.6, P = .4) or
between 1 and 2 weeks of immunization (F1,300 = 0.83,
P = .36). The effect of naloxone
treatments on spontaneous IL-2 production was still significant,
although less evident (F2,300 = 4.54,
P = .011). Moreover, in the saline group, the IL-2 levels
were lower in 72-hour culture than in 48-hour culture (Tukey,
q = 2.83, P < .05). In terms of the results in detail,
when animals were immunized in vivo with 50 µg KLH, 1 week after
immunization no effect of naloxone treatment was observed in the
48-hour cultures (Figure 1A); however, in the 72-hour supernatant, a
significant increase in KLH-stimulated production was present after
acute treatment with naloxone (Figure 1B). After 14 days of
immunization with 50 µg KLH, a significant increase of IL-2 in
KLH-stimulated cells was observed in 48-hour (Figure 1C) and 72-hour
cultures (Figure 1D) after chronic naloxone treatment. The production
of this cytokine showed an increase after naloxone treatment in the
animals immunized with 100 µg KLH: 1 week after immunization, the
enhancement of IL-2 production was evident in the 48-hour cultures
after acute naloxone treatment in the unstimulated cultures and after
both treatments in the stimulated ones (Figure 1E), while an increase
was present after acute naloxone treatment in the 72-hour stimulated
supernatant (Figure 1F). At 14 days after immunization, the increase
was present after chronic naloxone treatment in KLH-stimulated cells in
the 48-hour cultures, while in the 72-hour cultures the effect of
chronic naloxone was also present in the unstimulated cell cultures
(Figure 1G, 1H).
Independently of treatment, IFN- levels were higher in the 72-hour
than in the 48-hour cultures (spontaneous secretion:
F1,313 = 175.3, P < .001; KLH stimulated:
F1,317 = 10.123, P < .001). Naloxone
treatments induce a significant increase of IFN- levels in the in
vitro KLH-stimulated cultures (F2,317 = 10.1,
P < .001). Moreover, an interaction between the dose of KLH
used for immunization and naloxone effect was evident, as the
treatments were effective only in the animals immunized with 100 µg
KLH (F2,317 = 11.26, P = .001). In fact, in the
animals immunized with 50 µg KLH, the treatment with naloxone did not
affect the production of IFN- (Figure
2A-2D). On the contrary, in the 100 µg
KLH-immunized animals, a significant increase of the cytokine was
present. When IFN- was measured 7 days after immunization, a
significant enhancement was evident in both the 48- and the 72-hour
supernatant after acute treatment with naloxone for unstimulated as
well as in vitro KLH-stimulated cultures (Figure 2E, 2F). At 2 weeks
after immunization with 100 µg KLH, in 48-hour stimulated cultures a
significant increase of IFN- was observed after chronic naloxone
treatment in unstimulated cells, and after both acute and chronic
naloxone treatment in KLH-stimulated cells (Figure 2G). In the 72-hour culture, an increase after chronic naloxone treatment in KLH-stimulated cells was evident (Figure 2H).
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| Fig 2.
Effect of acute and chronic naloxone treatment on
KLH-stimulated IFN- production by splenocytes.
Animals were immunized with either KLH (panels A,B,C,D) or 100 µg KLH
(panels E,F,G,H) and killed after 1 week (panels A,B,E,F) or 2 weeks
(panels C,D,G,H). Spleen cells were cultured with 0 or 80 µg/ml KLH
for 48 h (panels A,C,E,G) and 72 h (panels B,D,F,H). Acute saline
control is represented by white bars; chronic saline control is
represented by right-slanted striped bars; acute naloxone treatment is
represented by left-slanted striped bars; chronic naloxone treatment is
represented by hatched bars. Results are expressed as mean ± SE. * = P < .01 versus corresponding saline
controls.
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Independently of treatment, the production of IL-4 was barely
detectable in the 48-hour cultures (data not shown). Figure 3 reports the levels of IL-4 in the 72-hour
cultures. The levels of IL-4 in the unstimulated cultures were also
very low. The effect of naloxone treatment on KLH-stimulated IL-4
production is indeed opposite to the one observed on IL-2 and IFN-.
A significant main effect of naloxone is in fact present
(F2,151 = 38.29, P < .001), since both acute
and chronic naloxone decrease IL-4 production. The effect is present
with both doses of in vivo KLH and is present 1 week as well as 2 weeks
after immunization.
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| Fig 3.
Effect of acute and chronic naloxone treatment on
KLH-stimulated IL-4 production in vitro by splenocytes.
Animals were immunized with either 50 µg KLH (panels A,B) or 100 µg
KLH (panels C,D) and killed after 1 week (panels A,C) or 2 weeks
(panels B,D). Spleen cells were cultured with 0 or 80 µg/ml KLH for
72 h. Acute saline control is represented by white bars; chronic saline
control is represented by right-slanted striped bars; acute naloxone
treatment is represented by left-slanted striped bars; chronic naloxone
treatment is represented by hatched bars. Results are expressed as mean ± SE. * = P < .01 versus saline controls.
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Keyhole-limpet hemocyanin antibody response
KLH IgM serum titers are reported in Figure
4. The IgM titers were higher
(F1,71 = 8.21, P = .006) in the groups of
animals immunized with 100 µg (Figure 4B) in comparison with those
immunized with 50 µg (Figure 4A). Moreover, higher titers were
present 1 week after immunization (Figure 4A, B) than 2 weeks after
immunization (Figure C, D) (F1,71 = 21.7,
P < .001). Figure 4 also shows that neither acute nor
chronic naloxone treatment modified IgM serum titers.
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| Fig 4.
Effect of acute and chronic naloxone treatment on serum
IgM anti-KLH antibody response.
Either animals were immunized with 50 µg KLH and killed after 1 week
(A) or 2 weeks (C), or they were immunized with 100 µg KLH and killed
after 1 week (B) or 2 weeks (D). Results are expressed as mean ± SE
of 10 animals per group. * = P < .01 versus
saline control.
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As reported in Figure 5, in the
saline-treated animals immunized with 50 µg KLH
(Figure 5A), the titers of anti-KLH IgG were lower than in the
animals treated with 100 µg (Figure 5B)
(F1,83 = 9.08, P = .003); moreover, higher
titers were present 2 weeks after immunization (Figure 5C, D) in
comparison with 1 week after (Figure 5A, B)
(F1,83 = 30.17, P < .001).
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| Fig 5.
Effect of acute and chronic naloxone treatment on serum
IgG anti-KLH antibody response.
Animals were immunized with either 50 µg KLH and killed after 1 week
(panel A) or 2 weeks (panel C), or with 100 µg KLH and killed after 1 week (panel B) or 2 weeks (panel D). Results are expressed as mean ± SE of 10 animals per group.
* = P < .01 versus saline control.
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A significant overall effect of naloxone treatment on IgG was present
(F2,251 = 10.29, P < .001 (Figure 5).The post
hoc comparison indicated that the antibody titers were in fact reduced
in the animals chronically treated with naloxone 1 week after
immunization with 50 µg KLH (P < .05) (Figure 5A, B),
while in animals immunized with 100 µg KLH the IgG titers appeared to
be reduced after both acute and chronic naloxone treatment
(P < .05) (Figure 5D). No significant interactions (week × KLH dose × treatment) were present (F2,251 = .765, P = .466).
| |
Discussion |
The administration of the opioid antagonist naloxone appears to
affect the pattern of cytokine production by splenocytes of BALB/cJ
mice immunized with the protein antigen KLH. Acute as well as chronic
naloxone treatments decrease IL-4 production in both unstimulated and
in vitro KLH-stimulated splenocyte cultures. On the contrary, the
production of the TH1 cytokines IL-2 and IFN- is
increased by naloxone. Although slight differences are present
depending on the dose of KLH used to immunize animals, the duration of
immunization, and the duration of in vitro culture, no qualitative
difference is observed: TH1 cytokines show a trend to
increase and TH2 cytokines to decrease after naloxone.
Independently of treatment, although higher levels of IFN- and IL-4
are present after 72 hours of in vitro stimulation, the opposite is
true for IL-2, since a decline of the levels of the cytokine is
observed. IL-2 acts as an autocrine growth factor for T cells: IL-2,
secreted by TH cells following stimulation by antigen,
upregulates the expression of its own receptors, and the subsequent
binding of IL-2 to this high-affinity receptor results in proliferation
of the antigen-activated T cells. However, the half-life of cell-bound
IL-2 is 25 to 30 minutes, with the IL-2/IL-2R complex being removed
from the cell surface by internalization.17 The fact that
lower amounts of free IL-2 are found at cells cultured for longer times
could therefore be explained by massive utilization of the cytokine by
the activated cells.
When TH1 cytokines are evaluated 7 days after
immunization with the highest dose of KLH, acute treatment with
naloxone is sufficient to increase TH1 cytokines, while
chronic treatment is more efficacious in increasing TH1
cytokines 2 weeks after immunization.
We do not at the moment have a satisfactory explanation for this time course.
IL-4 production is greatly affected by naloxone treatment. A
significant decrease of this cytokine is in fact present at both doses of antigen, at both 1 and 2 weeks after immunization, and after
treatment with both acute and chronic naloxone. In BALB/cJ mice, one
type of T helper population dominates over the other. BALB/cJ mice are
susceptible to infection by intracellular pathogens, have a weak
cell-mediated immune response, and consistently present a
TH2 dominance.18,19 We cannot say, at the
moment, whether naloxone could primarily act on the dominant
TH2 population, affecting IL-4 secretion. Since IL-4 is
inhibitory for the differentiation and effector function of the
TH1 subset,11,12 the IL-4 decrease induced
by naloxone could thereafter permit the production of TH1 cytokines. Alternatively, the effect
on TH1 cytokines can precede and thereafter affect lL-4
production.Further studies are in progress in order to find a
potential temporal dependence.
Consistent with the decrease of IL-4 production, naloxone also affects
the primary antibody response, as shown by the low serum IgG levels
that are present in the mice after naloxone treatment, confirming the
link between IL-4 and antibody production.11,12 On the
whole, the data obtained suggest that treatment with naloxone tends to
skew the T-cell balance toward a TH1 pattern.
Given that naloxone is an almost pure antagonist at the µ-opioid
receptor and is devoid of any intrinsic activity at the µ receptor,
the effects of the drug are likely to be due to the removal of a
regulatory tone exerted by endogenous opioid peptides. We previously
showed that, in rat and human, naloxone increased T-lymphocyte
proliferation, increased NK activity, and worsened the
development of inflammatory responses.5,20 Similar effects were also shown by our laboratory with the use of a neutralizing antibody against the opioid peptide BE.6,20 It can
therefore be hypothesized that the modulation of the
TH1/TH2 cytokine pattern induced by naloxone
could be due to the removal of BE effects, although the involvement of
other opioid peptides (eg, met-enkephalin) could also be possible.
We and others have demonstrated that BE is produced and released by
the cells of the immune system1,3,4,21,22 and that it can
bind specific opioid receptors present on immunocytes.1,5 Evidence therefore exists for an autocrine/paracrine activity of the opioid.
The data reported in this paper contribute a new insight on the role
of opioid peptides in the modulation of the immune system. It
becomes in fact questionable to claim a unique immunosuppressive or
immunostimulatory role for opioid peptides and BE. It can rather be
suggested that the opioid might exert an inhibitory control of
TH1 cell populations, probably through the stimulation of
TH2 cell types. In line with this hypothesis, the
literature is often contradictory on the effects of opioid peptides on
the immune system.1,3 Depending on the immune function
evaluated (eg, cellular versus humoral) and on the preexisting
TH1/TH2 balance (eg, after previous exposure of
animals to different pathogens or by genetic predisposition),
inhibition or stimulation of classical laboratory immune parameters
has been reported.
The effect of chronic naloxone treatment on TH1 cytokines
seems to be in contrast with what we previously reported in a different experimental model.5 In the earlier experiments, in fact,
naloxone was given chronically to naive rats with a resting immune
system, and lymphoproliferation, tested in vitro upon mitogen
stimulation, was decreased. In the present experiments, the opioid
antagonist is administered to immunized animals that present an in vivo
stimulated immune response. Similarly, chronic naloxone treatment was
able to increase TH1 cytokines in skin graft experiments.
In this case, the drug was also given to an already activated immune
system. These observations indicate that the status of the immune
system of the host can be relevant in order to direct the opioid
control of the immune responses.
The disruption of a correct TH1/TH2 balance is
involved in the development of several immune diseases.23
As a consequence, the effects that opioid peptides and naloxone exert
on TH cell types can be relevant in immunopathology.
Consistently, we demonstrated that the administration of naloxone,
which, as shown in the present paper, increases the production of
TH1 cytokines, worsened the development of experimental
autoimmune encephalomyelitis9 where the
TH1-type effector cell response is dominant.10
Moreover, in a murine model of skin allograft rejection, which is
driven by the development of a TH1 response,7
we showed that naloxone significantly anticipated, while BE delayed,
the onset of the rejection.8 We can hypothesize that the
use of opioid peptides or their modulation and/or the use of opioid
antagonists might be interesting new tools to achieve immune deviation.
In conclusion, our data indicate a role for opioid peptides in the
modulation of TH1/TH2 balance in the complex
network of immunoregulatory signals and offer a clue to the
interpretation of conflicting results present in the literature.
| |
Footnotes |
Submitted April 22, 1999; accepted November 11, 1999.
Reprints: Paola Sacerdote, Dept. Pharmacology, via Vanvitelli
32, 20129 Milano, Italy; e-mail: paola.sacerdote{at}unimi.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction