From the Microbiological Research Institute (N.Y., M.S., M.K.), Tokushima
Research Institute (M.O.), Otsuka Pharmaceutical Co, Ltd, and Third Department
of Internal Medicine (Y.S.), Faculty of Medicine, University of Tokyo.
Correspondence to Norimi Yamamoto, PhD, Microbiological Research Institute, Otsuka Pharmaceutical Co, Ltd, 463–10 Kagasuno, Kawauchi-cho, Tokushima 771–01, Japan.
Methods and Results—To investigate the possibilities of IFN
therapy in viral myocarditis, we analyzed the effects of
recombinant murine interferon (mIFN)-
Conclusions—Our data demonstrate that IFN therapy, especially
intranasal administration of IFN-
Among the antiviral therapies currently available in clinical practice,
IFNs have become one of the most important and are under investigation
to explore other potential uses. It has been demonstrated that
treatment of EMC virus–infected mice with anti-IFN globulin resulted
in the death of all animals with marked virus replication in the
visceral organs.3 This indicates that IFNs are
one of the essential components of the host immune system against viral
infection. Regarding in vivo anti-EMC virus activity, human IFN-
Recent advances in medical science have revealed that the lungs are
capable of absorbing various substances of high or low molecular weight
that are inhaled or administered for local or systemic
distribution.9 10 In fact, the bioavailability of
IFN-
This study was conducted to compare the protective effect of mIFN-
Virus and Inoculation
For histopathological examination of the infected mice, the virus was
diluted to 300 pfu/mL and administered to 6-week-old DBA/2 slc mice in
a volume of 0.1 mL IP.
Administration of IFNs
In the experiment to determine the effect of timing of mIFN
administration, treatment started 2 days before virus inoculation (days
-2 to 4), 1 day after virus inoculation (days 1 to 7), or 3 days after
inoculation (days 3 to 9).
Survival time was monitored until 14 days after virus inoculation.
Determination of EMC Virus Titer in Viscera
The heart, brain, and pancreas were removed from individual animals 4
days after inoculation, rinsed with physiological
saline, and preserved at -80°C until plaque assay. Tissues of the
infected viscera were homogenized with 9 vol of 10%
Eagle's MEM using a Polytron and centrifuged at 4000 rpm for
10 minutes at 4°C. The supernatant was serially diluted in 10 vol of
Eagle's MEM, and 0.1-mL aliquots of the dilutions were overlaid on the
monolayered L929 cells. The confluent L929 cell monolayers were
incubated with 0.1 mL of serial 10-fold dilutions of virus-containing
supernatant in 10% Eagle's MEM. The viruses were adsorbed onto the
L929 cell monolayers for 60 minutes at 37°C in 5%
CO2 with tilting at 10-minute intervals. After
adsorption, the cells were washed with phosphate-buffered saline
without Ca2+ and Mg2+.
Then, 2 mL of the 10% Eagle's MEM containing 1% methylcellulose was
overlaid and incubated for 4 days at 37°C in 5%
CO2. After the overlay medium was discarded, the
cells were exposed to 0.05% crystal violet in 10% buffered formalin,
pH 6.3, overnight at ambient temperature for fixing and staining. After
the dye solution was discarded and the cell plates were rinsed with
chilled physiological saline, the plates were
dried, plaques formed on the monolayer were counted, and the virus
titer was calculated (detection limit, <102
pfu/g of tissue). The virus yield in the IFN-treated group was compared
with that in the infected control group.
Histopathology of the Heart
On the basis of microscopic examination of the ventricular
tissue appearance, myocardial damage was evaluated as follows: the
absence of damage was scored as 0; lesions in <25% of the
myocardium, +1; lesions in 25% to 50% of the tissues, +2;
lesions in 50% to 75% of the tissues, +3; and lesions in >75% of
the tissues, +4. Pathological scores were assessed independently by
three experts in pathology.
Determination of ED50 In Vitro by Plaque Assay
Next, 2 mL of the mIFN suspension was added to three wells at each
concentration. Two milliliters of 10% Eagle's MEM was added to the
control wells. After the cells were exposed to mIFN for 24 hours, the
medium was discarded and the cells were rinsed with 10% Eagle's MEM.
EMC virus was placed in a volume of 0.2 mL each and allowed to adsorb
for 60 minutes at 37°C with tilting at 10-minute intervals. The cells
were rinsed with 10% Eagle's MEM, followed by incubation with 2 mL of
10% Eagle's MEM containing 1% methylcellulose at 37°C for 4 days
in 5% CO2/95% air. After the overlay medium was
discarded, the cells were fixed and stained with 0.05% crystal violet
in 10% buffered formalin solution, pH 6.3, overnight at an ambient
temperature. After rinsing with chilled
physiological saline, the numbers of plaques on the
monolayer cells were counted, and the 50% plaque reduction rate was
calculated as the ED50 value.
Statistical Analysis
In the intramuscular administration experiment, the survival rate of
the infected control mice through the 14-day observation period was
6.7% (Fig 1B
Effect of mIFN Treatment on Virus Yields in Visceral
Organs
In the intramuscular administration experiment, there was no
significant effect of virus yield reduction in the mIFN-
Other Visceral Organs
These results indicate that protection against EMC virus infection was
achieved through an antiviral effect of mIFN-
Effect of mIFN Treatment on Histology of the Heart
Necrosis of Cardiac Myocytes
Cellular Infiltration
Calcification in Ventricular Myocardium
These results indicate that protection against viral myocarditis
was achieved through the effect of mIFN-
Effect of Administration Route of IFN-
Effect of Timing of IFN-
In this study, we found that both mIFN-
The immunomodulating activities of mIFN-
Regarding murine viral myocarditis caused by coxsackievirus B3, IFN-
Thus, the striking effects of mIFN-
Regarding the effect of IFN therapy on human myocarditis, until now
there have been only a few reports. Siegener et
al19 reported that enteroviral RNA was detected
by in situ hybridization in 20 of 77 patients with dilated
cardiomyopathy who underwent
endomyocardial biopsy. Four enterovirus-positive
patients with hemodynamic deterioration were given
IFN-
It is well known that in acute viral myocarditis, cell-mediated
autoimmune mechanisms induced by viral infection as well as the direct
cytopathic effect of viruses are important in the pathogenesis of the
myocardial damage. Evidence has accumulated that the persistent
myocardial cell damage involved in viral myocarditis may cause
continuous destruction of contractile proteins and facilitate fibrosis,
which may lead to dilated cardiomyopathy.
Therefore, immunomodulating therapy against the cell-mediated
autoimmunity induced by virus infection along with IFN therapy would be
the best way to treat patients with acute myocarditis and chronic
ongoing myocarditis.
Received June 19, 1997;
revision received October 21, 1997;
accepted October 23, 1997.
2.
Kanda T, Nagaoka H, Kaneko K, Wilson JE, McManus BM,
Imai S, Suzuki T, Murata K, Kobayashi I. Synergistic effects of
tacrolimus and human interferon-
3.
Gresser I, Tovey MG, Bandu M, Maury C, Brouty-Boye D.
Role of interferon in the pathogenesis of virus diseases in mice as
demonstrated by the use of anti-interferon serum, I: rapid evolution of
encephalomyocarditis virus infection. J Exp Med. 1976;144:1305–1315.
4.
Weck PK, Rinderknecht E, Estell DA, Stebbing N.
Antiviral activity of bacteria-derived human alpha interferons against
encephalomyocarditis virus infection of mice. Infect Immun. 1982;35:660–665.
5.
Connel EV, Cerruti RL, Sim IS. Interactions between
recombinant interferons alpha and gamma in the treatment of
experimental virus infections in mice. In: Stewart WE, Schellekens H,
eds. The Biology of the Interferon System 1985. Amsterdam:
Elsevier Science Publishers BV; 1986;419–422.
6.
Matsumori A, Crumpacker CS, Abelmann WH. Prevention of
viral myocarditis with recombinant human leukocyte interferon
7.
Shalaby MR, Hamilton EB, Benninger AH, Marafino
BJ. In vivo antiviral activity of recombinant murine gamma
interferon. J Interferon Res. 1985;5:339–345.[Medline]
[Order article via Infotrieve]
8.
Iida J, Saiki I, Ishihara C, Azuma I. Protective
activity of recombinant cytokines against Sendai virus and
herpes simplex virus (HSV) infection in mice. Vaccine. 1989;7:229–233.[Medline]
[Order article via Infotrieve]
9.
Schanker LS. Drug absorption from the lung.
Biochem Pharmacol. 1978;27:381–385.[Medline]
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10.
Schanker LS, Mitchell EW, Brown RA Jr. Species
comparison of drug absorption from the lung after aerosol inhalation or
intratracheal injection. Drug Metab Dispos. 1986;14:79–88.[Abstract]
11.
Patton JS, Trinchero P, Platz RM. Bioavailability of
pulmonary delivered peptides and proteins:
12.
Rubin BY, Gupta SL. Differential efficacies of human
type I and II interferons as antiviral and antiproliferative agents.
Proc Natl Acad Sci U S A. 1980;77:5928–5932.
13.
Johnson HM, Bazer FW, Szente BE, Jarpe MA. How
interferons fight disease. Sci Am. 1994;270:40–47.
14.
Huang S, Hendriks W, Althage A, Hemmi S, Bluethmann H,
Kamijo R, Vil
15.
Kamijo R, Shapiro D, Le J, Huang S, Aguet M,
Vil
16.
Seko Y, Takahashi N, Yagita H, Okumura K, Yazaki Y.
Expression of cytokine mRNAs in murine hearts with acute
myocarditis caused by coxsackievirus B3. J Pathol. 1997;183:105–108.[Medline]
[Order article via Infotrieve]
17.
Seko Y, Matsuda H, Kato K, Hashimoto Y, Yagita H,
Okumura K, Yazaki Y. Expression of intercellular adhesion molecule-1 in
murine hearts with acute myocarditis caused by coxsackievirus B3.
J Clin Invest. 1993;91:1327–1336.
18.
Godeny EK, Gauntt CJ. Murine natural killer cells limit
coxsackievirus B3 replication. J Immunol. 1987;139:913–918.[Abstract]
19.
Siegener MS, Heim A, Figulla HR.
Subclassification of dilated cardiomyopathy and
interferon treatment. Eur Heart J. 1995;16(suppl
O):147–149.
20.
Miri
© 1998 American Heart Association, Inc.
Basic Science Reports
Effects of Intranasal Administration of Recombinant Murine Interferon-
on Murine Acute Myocarditis Caused by Encephalomyocarditis Virus
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Viral myocarditis has
been strongly implicated in the pathogenesis of dilated
cardiomyopathy as well as acute myocarditis. Among
the antiviral therapies, interferons (IFNs) have been widely studied
and become very important in clinical practice. and natural mIFN-
/ß by
the intranasal and intramuscular routes on the development of acute
murine myocarditis caused by encephalomyocarditis virus. Both mIFN-
and mIFN-/ß treatment by either route significantly increased the
survival rate; none of the mIFN-–treated mice died. The effect of
mIFN- was significantly greater than that of mIFN-/ß.
Furthermore, intranasal administration of mIFN- significantly
suppressed virus replication and inflammation in the heart. , dramatically improved the
prognosis of acute murine viral myocarditis by suppressing virus
replication and raises the possibility of antiviral therapy with
IFN- in patients with acute myocarditis.
Key Words: myocarditis • cardiomyopathy • viruses • immunology
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The EMC virus, which
is a member of Cardiovirus of the family of Picornaviridae
(which includes Coxsackievirus), causes acute myocarditis occasionally
with fatal outcome in humans and various animal species. DBA/2 mice
infected with EMC virus show marked dilatation and
hypertrophy of the heart in the chronic stage of
myocarditis, which is characterized by lesions similar to those seen in
dilated cardiomyopathy in
humans.1 2 A/D
administered intraperitoneally has been mainly
evaluated.2 4 5 6 In addition, there is a report
in which recombinant human leukocyte IFN- or mIFN- given
intraperitoneally prevented the death of mice that
were inoculated with a lethal dose of EMC virus.7
However, studies of IFN by the intranasal route are scarce. To our
knowledge, there is the only study in which mIFN- was protective
against Sendai virus replication in mice when given intranasally but
not intravenously.8 , which has a high molecular weight, was as high as 56% of the
dose given to anesthetized rats by the intratracheal
route.11
with that of natural mIFN-/ß on EMC virus–induced myocarditis by
the intranasal and intramuscular routes on the basis of survival rate
and duration, virus titers in the heart, and pathology of the heart
determined in DBA/2 mice.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
IFNs
Recombinant mIFN- (lot 004003; titer,
6.52x105 IU/mL; specific activity,
6.27x105 IU/mg of protein) and natural
mIFN-/ß (lot 005; titer, 6.95x107 IU/mL;
specific activity, 3.56x107 IU/mg of protein)
were generously given by Hayashibara Biochemical Laboratories, Inc
(Okayama, Japan) and stored at -80°C until use.
The M variant of EMC virus ATCC VR-1314 was obtained from the
American Type Culture Collection and was used after three passages in
DBA/2 slc mice (SLC Japan). Hearts of the infected mice were excised
and emulsified in 10% heat-inactivated FBS in 10%
Eagle's MEM, pH 7.6, through the use of a model K Polytron (Kinematica
AG) and was clarified by centrifugation at 4000 rpm for
10 minutes at 4°C. The supernatant was divided into aliquots and
frozen at -80°C. The mean titer of the virus preparation was
1.5x105 pfu/mL when determined on L929
monolayered cells. The diluent, which contained 500 pfu/mL, was
administered to 3-week-old mice in a volume of 0.1 mL IP.
Mice were anesthetized with ketamine HCl
(Ketaral-50; Sankyo Zoki Co); then, mIFN- or mIFN-/ß solution
was administered into the right nasal cavity once daily in a volume of
20 µL with the use of a micropipette (Pipetman p-20; Gilson) or
injected intramuscularly into the thigh in a volume of 0.1 mL.
Treatment with mIFNs lasted for 7 days, starting 2 days before virus
inoculation (days -2 to 4) to determine the life-prolonging effect and
for histopathological study.
To determine the effect of mIFNs in reducing virus yield in the
viscera, IFN treatment lasted for 6 days, starting from 2 days before
virus inoculation (days -2 to 3).
Treated, nontreated, and mock-infected mice were killed, and
their hearts were removed 10 days after virus inoculation. The hearts
were rinsed immediately with physiological saline
and sectioned transversely into three parts. The portion with the
largest ventricular circumference was fixed in 10%
buffered formalin solution. The tissues were embedded in paraffin and
stained with hematoxylin and eosin.
For the study of the antiviral efficacy of mIFN-/ß and
mIFN-, EMC virus was prepared by three passages in DBA/2 mice and
two passages in FL cells. The supernatant obtained was stored at
-80°C. The virus was thawed and diluted with 10% Eagle's MEM to a
concentration of 250 pfu/mL immediately before use. The host cells used
for this assay were monolayered L929 cells, which were prepared by
incubation of 1.25x105 cells on 12-well plates
for 2 days at 37°C in 5% CO2/95% air.
mIFN-/ß and mIFN- were prepared by twofold dilution from 50 to
0.4 IU/mL with 10% Eagle's MEM.
Survival curves was determined according to the Kaplan-Meier
method, and then probability values were calculated according to the
generalized Wilcoxon test to evaluate the life-prolonging
effects of IFNs. One-way ANOVA followed by two-way Dunnett's test was
used to evaluate the differences of virus yields in the viscera between
the groups. The Mann-Whitney U test was used to evaluate the
histopathological gradings in the heart. Bonferroni's correction was
used for the multiple comparisons. The level of significance was set at
P<.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Effect of mIFN Treatment on the Survival of Mice
In the intranasal administration experiment, all infected control
mice died 5 to 7 days after inoculation. In sharp contrast, none of the
mice that received mIFN- at 10 000 IU/d from days -2 to 4 died by
the end of the 14th day after inoculation. The life-prolonging effect
of mIFN- was significant at P=.0002. The survival rate
for the mice treated with mIFN-/ß at 100 000 IU/d for the same
treatment periods was only 10%, but the life-prolonging effect of
mIFN-/ß was also significant at P=.0352 (Fig 1A
). The effect of mIFN- at 10 000
IU/d was significantly (P=.0015) greater than that of
mIFN-/ß at 100 000 IU/d.
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Figure 1. Protective effect of mIFNs on EMC virus infection
in 3-week-old DBA/2 male mice. A, Intranasal administration: 
,
0.01% of murine albumin in physiological
saline (mAlb, n=15); 
, 100 000 IU/mouse of mIFN-/ß (n=10);
, 10 000 IU/mouse of mIFN- (n=8). B, Intramuscular injection:
, 0.01% of mAlb (n=15); , 100 000 IU/mouse of mIFN-/ß
(n=15); , 1000 IU/mouse of mIFN- (n=15).
). Again, none of the mice that received mIFN- at 1000
IU/d from days -2 to 4 had died by the end of the 14-day
postinoculation observation period, demonstrating a significant
(P=.0002) life-prolonging effect. The survival rate for the
animals treated with mIFN-/ß administered at 10 000 IU/d for the
same treatment periods was 53.3%, and the life-prolonging effect of
mIFN-/ß was significant at P=.0004 (Fig 1B). The effect
of mIFN- at 1000 IU/d was significantly (P=.0219) greater
than that of mIFN-/ß at 10 000 IU/d.
Heart
The antiviral efficacies of mIFN- and mIFN-/ß
administered intranasally or intramuscularly in the target organ, the
heart of infected mice, were analyzed through the use of the
plaque assay. The virus yield in the hearts of mice treated with
mIFN- 10 000 IU/d intranasally was 3.7±1.0 log10 pfu/g of tissue,
which is significantly (P<.01) lower than that of infected
control mice (6.3±0.2 log10 pfu/g of tissue) (Fig 2A). Treatment with mIFN-/ß at
100 000 IU/d intranasally also significantly (P<.05)
reduced the virus yield to 4.0±2.1 log10 pfu/g of tissue, but the
effect was less than that of mIFN- (Fig 2A).
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Figure 2. Virus yield reduction in the heart of 3-week-old
DBA/2 mice inoculated with EMC virus and treated with mIFN- or
mIFN-/ß 4 days after inoculation. A, Intranasal administration
(n=5). B, Intramuscular injection (n=5). or
mIFN-/ß treatment (Fig 2B).
We also analyzed the antiviral efficacies of mIFN- and
mIFN-/ß administered intranasally or intramuscularly in the brain
and pancreas through the use of the plaque assay. mIFN- at 10 000
IU/d intranasally also reduced the virus yield to 4.4±1.5 and 5.1±1.5
log10 pfu/g of tissue, respectively, which again was significantly
(P<.01 and <.05, respectively) lower than that of infected
control mice (7.0±0.3 and 7.2±0.3 log10 pfu/g of tissue,
respectively). mIFN-/ß at 100 000 IU/d intranasally also
significantly reduced the virus yield to 3.5±1.3 and 4.5±2.3 log10
pfu/g of tissue, respectively. Both again were
significantly(P<.01 and <.05, respectively) lower than
that of infected control mice. In the intramuscular administration
experiment, there again was no significant effect of virus yield
reduction in the mIFN- or mIFN-/ß treatment. or mIFN-/ß
administered intranasally in the major viscera.
There were extensive necrotic foci of myocytes with calcification
and cellular infiltration in the hearts of infected control mice (Fig 3B). The cellular infiltrates mainly
consisted of mononuclear cells. Polymorphonuclear leukocytes were
also observed near the large necrotic lesions, whereas only minimal
cardiac lesions were observed in the mice treated with mIFN- at
10 000 IU/d intranasally (Fig 3A).
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Figure 3. Histological findings in the heart
of 6-week-old DBA/2 male mice inoculated with EMC virus and treated
with mIFN 10 days after inoculation. A, Heart of mice treated with
mIFN- at 10 000 IU/mouse intranasally from days -2 to 4 after
virus inoculation (hematoxylin and eosin stain, x9.8). B, Heart of
infected control mice (hematoxylin and eosin stain, x9.8).
The administration of mIFN- at 10 000 IU/d intranasally
significantly (P=.0462) suppressed necrosis of myocytes in
the ventricular myocardium, as demonstrated by
the pathology scores of +0.1±0.4 in the mIFN-–treated group and
+1.0±0 in the control group (Table). The scores for
mIFN- and mIFN-/ß administered intramuscularly were comparable
to the scores for infected controls (Table).
View this table:
[in a new window]
Table 1. Histopathology Scores for Necrosis in Myocardium
of 6-Week-Old Male DBA/2 Mice Treated With mIFN- or mIFN-/ß at
10 Days After Inoculation
Similar to the examination of necrotic myocytes, the
administration of mIFN- to the animals at 10 000 IU/d intranasally
significantly suppressed (P=.00462) cellular infiltration
(inflammation) into the ventricular myocardium,
as demonstrated by the pathology scores of +0.1±0.4 in the
mIFN-–treated group and +1.0±0 in the infected control group
(Table). The scores for mIFN- and mIFN-/ß administered
intramuscularly were comparable to the scores of infected control
animals (Table).
The administration of mIFN- and mIFN-/ß to the animals
intranasally produced no significant differences in the pathology score
for calcification in the myocardium compared with the
infected control group (Table). The scores for mIFN by the
intramuscular route also were comparable to those of the infected
control animals (Table). administered
intranasally. on Survival of
Mice
In an experiment in which we used four doses by the intranasal
route and three doses by the intramuscular route, the intranasal route
was slightly more effective than the intramuscular route in prolonging
the life of infected animals in all doses, even at the lowest dose of
500 IU/d for 7 days starting from 2 days before virus inoculation. The
effect was much more apparent at the higher doses of 1000, 5000, and
10 000 IU/d intranasally at significance levels of P=.0028,
.0004, and .0004, respectively (Fig 4A).
Because all animals treated at the higher two doses survived throughout
the observation period, a dose-effect relationship was not established.
The administration of mIFN- by the intramuscular route also was
clearly effective at all tested doses of 100, 500, and 1000 IU/d for 7
days at significance levels of P=.0063, .0018, and .0018,
respectively (Fig 4B). The survival rates obtained by the intramuscular
route appeared to be dependent on the dose, but dose-dependency was not
demonstrated statistically.
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Figure 4. Comparison of protective effect of mIFN- by the
intranasal and intramuscular routes in EMC virus–infected 3-week-old
DBA/2 male mice. A, Intranasal administration: , 0.01% of murine
albumin in physiological saline (mAlb,
n=14); 
, 500 IU/mouse (n=14); , 1000 IU/mouse (n=11); , 5000
IU/mouse (n=8); 
, 10 000 IU/mouse (n=8). B, Intramuscular
injection: , 0.01% of mAlb (n=7); , 100 IU/mouse (n=7); , 500
IU/mouse (n=7); , 1000 IU/mouse (n=7). Administration on Survival of
Mice
mIFN- was administered to the infected mice at doses of
100 000 and 1000 IU/d by the intranasal or intramuscular routes for 7
days starting 2 days before, 1 day after, or 3 days after virus
inoculation (Fig 5A and 5B,
respectively). The life-prolonging effect was apparent by both routes
at a significance level of P=.0003 when the treatment was
started at 2 days before or 1 day after virus inoculation. The
protective effect was also seen when the treatment was started at 3
days after virus inoculation, but the difference between the treated
and the infected control group was not statistically significant.
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Figure 5. Comparison of protective effect of mIFN-
(10 000 IU/mouse intranasally or 1000 IU/mouse intramuscularly) by
treatment schedules in EMC virus–infected 3- week-old DBA/2 male mice.
A, Intranasal administration: , 0.01% of murine albumin in
physiological saline (mAlb, n=10); , 7-day
treatment starting from 2 days before inoculation (n=11); , 7-day
treatment starting from 1 day after inoculation (n=15); , 7-day
treatment starting from 3 days after inoculation (n=15). B,
Intramuscular injection: , 0.01% of mAlb (n=10); , 7-day
treatment starting from 2 days before inoculation (n=10); , 7-day
treatment starting fom 1 day after inoculation (n=10); , 7-day
treatment starting from 3 days after inoculation (n=10).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Although acute myocarditis usually is idiopathic, it has been
generally accepted that most cases of the disease are caused by
picornaviruses such as EMC virus. Because patients with acute
myocarditis present with acute heart failure, and some of them
develop dilated cardiomyopathy later in the course
of the disease, much effort has been made to develop new therapeutic
interventions. Various recombinant cytokines, including
mIFN-, have been recognized to significantly augment host resistance
against virus infection in mice when administered by the intranasal
route compared with the intravenous
route.8 The lung, which is known to be highly
permeable to high-molecular-weight molecules,9 10
seems to be an efficient administration route for bioactive peptides. A
study of intratracheal administration of peptides and proteins in rats
demonstrated that human IFN- had an absolute bioavailability of
>56%.11 Therefore, we tried to treat murine
acute myocarditis induced by EMC virus with mIFNs by the intranasal
route. and mIFN-/ß by either
intranasal or intramuscular route were effective in prolonging survival
time of the mice with viral myocarditis, when the treatment was started
from 1 day after virus inoculation. However, the intranasal inhalation
was found to be more effective than the pain-inflicting intramuscular
injection in suppressing viral growth in the target organs, including
heart, brain, and pancreas. In addition, intranasal administration of
mIFN- was more effective in suppressing the disease than that of
mIFN-/ß. In a separate in-house experiment, the 50% plaque
reduction assay of the antiviral activity using L929 cells and EMC
virus in this study provided ED50 values for
mIFN- and mIFN-/ß of 1.0 and 1.3 IU/mL (95% confidence limits,
0.75020 and 1.38097 and 0.80069 and 1.83707), respectively (Fig 6). The two types of mIFN exhibited
similar activity against the myocarditis-inducing virus. Similar
results have been reported regarding the antiviral efficacies of human
type I (IFN-/ß) and type II (IFN-) IFNs using human fibroblast
FS-4 cultures.12 Regarding IFN--therapy in
other animal models, Iida et al8 demonstrated
that intranasal but not intravenous administration of
IFN- protected mice against Sendai virus infection. This suggests
that factors other than bioavailability may be involved in the
differences in effectiveness among the administration routes. The
authors suggested that the activation of alveolar macrophages
and neutrophils by the intranasal route was one of the mechanisms
involved. However, the precise mechanism by which that intranasal route
was more effective than the intravenous or intramuscular
route is still unknown and remains to be clarified.
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Figure 6. Antiviral activities of mIFNs against EMC virus on
L929 monolayered cells. , Mean percentage for mIFN-/ß (n=3);
, mean percentage for mIFN- (n=3)., including
macrophage activation in lung alveoli, are unique and not
provided with mIFN- or -ß by the pulmonary route. Recent
advances in cell biology revealed that IFN- is the most potent
macrophage activator; it produces
macrophages to kill virus-infected cells and proteins to
destroy pathogens.13 14 The antiviral activity of
macrophages appears to be mediated by nitric oxide and possibly
other reactive nitrogen intermediates synthesized by nitric oxide
synthase, which is induced by macrophage-activating agents such
as IFN-. After ingestion by macrophages, microbes such as
viruses are digested and degraded into peptide fragments in the
cytoplasm and then presented on the cell surface by major
histocompatibility complex molecules. T cells specifically recognize
processed antigens through their T-cell receptors and then proliferate
and release chemicals that help other immune cells to combat or
eradicate infections. Because IFN- is a much more potent inducer of
major histocompatibility complex class II antigen expression on
macrophages than IFN-/ß,15 the
decreased virus population in the target organs revealed in the
present study seems to be well understood.
is mainly synthesized by the infiltrating natural killer (NK) cells in
the early stage of acute myocarditis.16 17 It was
shown that depletion of NK cells by treatment with anti-asialo GM1
antibody plus complement aggravates coxsackievirus B3–induced murine
myocarditis by enhancing virus replication in the
heart.18 This strongly suggests that NK cells
play a critical role in limiting virus replication by killing
virus-infected cells as well as synthesizing IFN-. These data
support the protective effect of IFN- in viral myocarditis because
IFN- is known to activate NK cells as well as directly
suppress virus replication. In the present study, we also
analyzed the relative distribution of phenotypic markers among
the infiltrating cells, especially NK cells, cytotoxic T lymphocytes
(CTLs), and T-helper cells in the heart with viral myocarditis with or
without IFN- treatment. We found that there was a tendency toward
decrease in the percentage of NK cells and CTLs among the infiltrating
cells as well as a marked decrease in the total number of the
infiltrating cells in the IFN-–treated group compared with the
control group (data not shown). This supports the virus-suppressing
effect of IFN- because NK cells and CTLs are thought to kill
virus-infected myocardial cells. achieved by the intranasal route
in suppressing virus replication in the heart and other organs,
improving myocardial inflammation, and prolonging life of the infected
mice have demonstrated that this cytokine is very promising as
a biotherapeutic agent against viral myocarditis. However, in contrast
to the experimental viral myocarditis, viruses frequently cannot be
detected in patients with acute myocarditis because such patients
usually come to the hospital after they developed clinical
manifestations that are mainly the result of inflammatory responses, by
which time the viruses have disappeared. In the present study, the
effectiveness of IFN therapy was significantly reduced when given after
virus inoculation. This may limit the clinical application of the IFN
therapy to some extent. subcutaneously for 6 months. Then, all 4 patients had improved
hemodynamic and clinical parameters, and in
2 of them enteroviral RNA was not detectable in a subsequent biopsy.
Miri
et al20 also reported that treatment
with IFN- subcutaneously was effective in improving cardiac function
in 11 of 14 patients with dilated cardiomyopathy.
Considering that these results were obtained through subcutaneous
administration of IFN-, intranasal administration of IFN- will
have a remarkable value in the treatment of patients with acute viral
myocarditis and dilated cardiomyopathy.
Selected Abbreviations and Acronyms
ED50
=
50% effective dose
EMC
=
encephalomyocarditis
IFN
=
interferon
MEM
=
minimal essential medium
mIFN
=
murine interferon
pfu
=
plaque-forming unit(s)
Acknowledgments
The authors thank Masahisa Kyogoku, MD, for his support of this
investigation. We also thank Masatoshi Sakashita, Tomoko Doi, Atsumi
Kawai, and Naoko Ise for their excellent technical assistance.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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