Ribavirin and cysteinyl leukotriene-1 receptor blockade as treatment for severe bronchiolitis.
ABSTRACT In this work we have evaluated the clinical responses of pneumovirus-infected mice to combination therapy with the antiviral agent, ribavirin, and the CysLT1 cysteinyl leukotriene receptor antagonist, montelukast. We observed substantial virus replication in our mouse model of pneumovirus infection and significant accumulation of cysteinyl leukotrienes in lung tissue, the latter detected at levels that correlate directly with granulocyte recruitment to the airways. While administration of the nucleoside analog, ribavirin, reduced virus replication approximately 2,000-fold, the clinical outcomes as measured by morbidity and mortality, in response to ribavirin monotherapy were indistinguishable from those of the no-treatment controls. Similarly, montelukast therapy alone did not reduce granulocyte recruitment nor did it improve the clinical outcome. However, combined therapy with ribavirin and montelukast resulted in a significant reduction in morbidity and a substantial reduction in mortality (50% survival at t = 14 days and onward, compared to 10-20% survival in response to montelukast alone or to ribavirin alone, respectively, p < 0.01). These findings define further the independent contributions made by virus replication and by the ensuing inflammatory response to the detrimental sequelae of pneumovirus infection in vivo.
- [show abstract] [hide abstract]
ABSTRACT: Human eosinophils secrete two distinct ribonucleases that have antiviral activity against pathogens of the family Paramyxoviridae. To examine the role of eosinophils and their ribonucleases in host defense against paramyxovirus pathogens in vivo, we have developed a mouse model involving a viral pathogen that naturally targets a rodent host. In this work we describe infection of Balb/c mice with pneumonia virus of mice (PVM, strain J3666), a paramyxovirus pathogen found frequently among rodent populations. We show here that pulmonary eosinophilia is an immediate response to infection with PVM, with bronchoalveolar lavage fluid containing 12-14% eosinophils obtained as early as day 3 postinoculation. Infection is accompanied by the production of macrophage inflammatory protein-1-alpha (MIP-1alpha), a chemokine that has been associated with the pulmonary eosinophilia observed in response to respiratory syncytial virus infection in humans and with enhanced clearance of influenza virus in mice. Interestingly, we observed no changes in expression of the chemoattractants eotaxin and RANTES in response to PVM infection, and interleukin-5 remained undetectable throughout. These responses-clinical pathology, viral recovery, pulmonary eosinophilia, and production of MIP-1alpha-will provide a means for exploring the role of eosinophils, eosinophil secretory ribonucleases, and eosinophil chemoattractants in host defense against PVM and related paramyxovirus pathogens in vivo.Cellular Immunology 04/2000; 200(2):98-104. · 1.74 Impact Factor
Article: Leukotrienes in respiratory disease.[show abstract] [hide abstract]
ABSTRACT: Arachidonic acid metabolism via 5-lipoxygenase gives rise to a group of biologically active lipids known as leukotrienes: leukotriene B(4), which is a potent activator of leukocyte chemotaxis, and cysteinyl leukotrienes (leukotriene C(4), D(4)and E(4)) which account for the spasmogenic activity previously described as slow-reacting substance of anaphylaxis. The biological actions of leukotrienes and the observations that leukotrienes are synthesised in the lung following antigen provocation and are elevated in asthma, stimulated considerable activity in the pharmaceutical industry to find drugs that modulate the synthesis or actions of leukotrienes. Three cysteinyl leukotriene antagonists (zafirlukast [Accolate], montelukast [Singulair] and pranlukast) and one 5-lipoxygenase inhibitor (zileuton) have received regulatory approval for the treatment of asthma. The clinical data obtained from using these drugs are generally consistent and complimentary. As a class the leukotriene modulators produce a rapid improvement in lung function after the first oral dose. Lung function improvements are maintained on chronic administration and are associated with reductions in a variety of asthma symptom scores. All of the available data are consistent with the hypothesis that all the leukotriene modulators exert their clinical benefit primarily through interference with cysteinyl leukotrienes. There are no compelling clinical data for an additional contribution by leukotriene B(4)in human asthma. In other respiratory conditions such as COPD, which are characterised by pronounced neutrophil infiltration, it may be that the chemotactic properties of leukotriene B(4)are more important and therefore evaluation of 5-lipoxygenase inhibitors in this condition is warranted. The introduction of the leukotriene modulators into clinical practice is the culmination of over 60 years of research since the initial discovery of the slow-reacting substances. The leukotriene modulators, and in particular the cysteinyl leukotriene antagonists, provide respiratory physicians with an oral therapeutic option and have set an efficacy standard which new oral anti-inflammatory approaches will have to beat.Paediatric respiratory reviews 10/2001; 2(3):238-44. · 2.79 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Samples of nasopharyngeal secretions from a group of 73 infants with bronchiolitis or upper respiratory illness alone during infection with respiratory syncytial virus were analyzed for leukotriene C4 (LTC4) content using a reverse-phase high-pressure liquid chromatography assay with confirmation by radioimmunoassay. Titers of respiratory syncytial virus (RSV)-specific IgE in nasopharyngeal secretion (NPS) specimens were determined using an enzyme-linked immunosorbent assay. The highest concentrations of LTC4 were found in the first 3 to 8 days after the onset of illness, and LTC4 was detectable in progressively lower concentrations in samples obtained up to 28 days after the onset of illness. LTC4 was detected in samples of NPS obtained in the acute phase of illness from 67% of infants with bronchiolitis due to RSV and in 33% of samples of NPS obtained during the same interval from infants with upper respiratory illness alone (p less than 0.025). Concentrations of LTC4 in children with bronchiolitis were 5-fold higher (1271 pg/ml) than the mean concentration of LTC4 in children with upper respiratory illness (224 pg/ml, p less than 0.02). LTC4 was detected in 83% of the children developing an RSV-IgE response and in 24% of subjects not developing an RSV-IgE response (p less than 0.001). Quantities of LTC4 measured in NPS were directly correlated with the magnitude of the RSV-IgE response in secretions (r = 0.33, p less than 0.02). These studies lend support to previous investigations suggesting that severe bronchiolitis due to RSV results from IgE-mediated hypersensitivity reactions to viral antigens, with release of chemical mediators of airway obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)Pediatric Research 11/1988; 24(4):504-7. · 2.67 Impact Factor
Antiviral Research xxx (2005) xxx–xxx
Ribavirin and cysteinyl leukotriene-1 receptor blockade as
treatment for severe bronchiolitis
Cynthia A. Bonvillea, Helene F. Rosenbergb, Joseph B. Domachowskea,∗
aDepartment of Pediatrics, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA
bLaboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
Received 13 July 2005; accepted 19 October 2005
the CysLT1 cysteinyl leukotriene receptor antagonist, montelukast. We observed substantial virus replication in our mouse model of pneumovirus
infection and significant accumulation of cysteinyl leukotrienes in lung tissue, the latter detected at levels that correlate directly with granulocyte
recruitment to the airways. While administration of the nucleoside analog, ribavirin, reduced virus replication ∼2000-fold, the clinical outcomes
as measured by morbidity and mortality, in response to ribavirin monotherapy were indistinguishable from those of the no-treatment controls.
Similarly, montelukast therapy alone did not reduce granulocyte recruitment nor did it improve the clinical outcome. However, combined therapy
with ribavirin and montelukast resulted in a significant reduction in morbidity and a substantial reduction in mortality (50% survival at t=14
days and onward, compared to 10–20% survival in response to montelukast alone or to ribavirin alone, respectively, p<0.01). These findings
define further the independent contributions made by virus replication and by the ensuing inflammatory response to the detrimental sequelae of
pneumovirus infection in vivo.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Pneumovirus; Chemokine; Montelukast; Ribavirin; Mice
Cysteinyl-leukotrienes (leukotrienes C4, D4 and E4) are
leukocyte-derived lipid proinflammatory mediators that make
prominent contributions to the pathophysiology of bronchial
asthma (Bigby, 2000; McMillan, 2001). Among these contribu-
tions, cysteinyl leukotrienes promote bronchospasm, wheezing
and enhanced eosinophil recruitment in response to allergen
provocation. Leukotriene antagonists, which function as selec-
tive, competitive inhibitors of these leukotrienes at serpentine
G protein coupled CysLT1 receptors found in bronchial smooth
muscle, lung macrophages, and peripheral blood cells, provide
significant clinical benefit (James and Sampson, 2001) and are
already approved for clinical use in the management of this dis-
The clinical similarities between acute episodes of bronchial
asthma and symptoms associated with primary respiratory syn-
∗Corresponding author. Tel.: +1 315 464 7505; fax: +1 315 464 7564.
E-mail address: firstname.lastname@example.org (J.B. Domachowske).
cytial virus infection (RSV; family Paramyxoviridae, subfam-
ily pneumovirus) suggested the possibility of pathophysiologic
and biochemical similarities. Several groups have reported on
cysteinyl-leukotriene synthesis and release and its association
with RSV infection and its related symptomatology (Volovitz
et al., 1988; VanSchaik et al., 1999; Behera et al., 1998), and
a recent randomized clinical trial suggested that leukotriene
antagonist therapy provided after emergence of primary RSV
symptoms could result in significant reduction in reactive air-
As part of an overall study of the pathogenesis of pneu-
movirus infection in vivo, we have developed a model of respi-
(Domachowske et al., 2000a, 2000b; Bonville et al., 2003). The
natural rodent pathogen, pneumonia virus of mice (PVM; also
family Paramyxoviridae, subfamily pneumovirus) is among the
closest known phylogenetic relatives of RSV, but, unlike RSV
when utilized in mouse models, PVM can replicate effectively
in mouse lung tissue and infection results in significant morbid-
0166-3542/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
AVR-2034; No. of Pages 7
C.A. Bonville et al. / Antiviral Research xxx (2005) xxx–xxx
ity and mortality. Similar to severe RSV infection in humans,
PVM infection in mice is accompanied by a profound, acute
inflammatory response, which includes a prominent pulmonary
eosinophilia. We have identified the CC-chemokine MIP-1?
(CCL3) as the crucial mediator of the antiviral inflammatory
response to PVM (Domachowske et al., 2000b), analogous to
what has been inferred for RSV infection in human infants
(Harrison et al., 1999; Garofalo et al., 2001).
Using the PVM-infection model, we have recently reported
on the clinical utility of combined antiviral and specific anti-
MIP-1? immunomodulatory therapy, findings that underscore
the independent contributions of both virus replication and the
inflammatory response to the pathogenesis of this respiratory
virus infection in vivo (Bonville et al., 2003, 2004). Here we
present our findings on the responses of PVM-infected mice to
antiviral therapy when it is administered in conjunction with
cysteinyl-leukotriene receptor blockade. Our results suggest a
role for cysteinyl-leukotrienes in promoting the detrimental
sequelae of this infection, and as a target for development of
novel rational therapies.
2. Materials and methods
2.1. Mouse and virus stocks
C57Black/6 mice were obtained from Taconic Laborato-
ries, Germantown, NY. Mouse-passaged stocks of PVM (strain
J3666, ∼106pfu/ml, original stock virus obtained from Dr. A.J.
Easton, University of Warwick, Coventry, U.K.) were obtained
from clarified mouse lung homogenates as described previ-
ously (Domachowske et al., 2000a, 2000b) and stored in liquid
nitrogen. Virus stocks were defrosted and diluted in phosphate
buffered saline (PBS) immediately prior to intranasal inocula-
2.2. Establishing PVM infections in mice and treatment
Six- to 8-week-old mice were used in all experiments.
Mice subjected to brief isoflurane anesthesia were inoculated
intranasally with 60 plaque-forming units (pfu) of mouse-
passaged PVM strain J3666 in a 50ul volume of PBS at day
0. This viral inoculum was chosen because it produces moder-
ate to severe clinically symptoms in 100% of infected mice,
and at least 80% mortality if therapeutic intervention is not
initiated. Animals were weighed and observed daily. Clinical
scoring of infected mice was as initially devised by Cook et al.
(1998) with modifications as previously described (Bonville et
al., 2003). This clinical scoring system is based on observed
symptoms using a scale from 1 to 6: 1, healthy; 2, ruffled fur at
gic with labored breathing; 5, pre-morbid, with emaciation and
cyanosis; 6, death. Mice were sacrificed on days 0 through 7
post-inoculation for bronchoalveolar lavage fluid analysis and
total lung chemokine and cysteinyl leukotriene concentrations.
Lung PVM titers were determined from total lung homogenates
ical scores and mortality included four groups of 10 mice each.
To collect virologic, biochemical and histologic data, identi-
cal experiments were performed using 60 additional mice. Six
mice were sacrificed on day 0, then six mice in each treatment
arm were sacrificed on days 3, 5 and 7 for viral lung titers,
chemokine (MIP-1? and MCP-1) concentrations, and cysteinyl
leukotriene concentrations. Using appropriate diluent controls,
Group 1 received daily intraperitoneal montelukast (10mg/kg;
of intraperitoneal ribavirin (37.5mg/kg/dose; ICN Pharmaceu-
ticals), group 2 received montelukast, group 3 received twice
daily ribavirin and group 4 received diluent injections only. For
and continued until day 14. All procedures were reviewed and
approved by the Committee on the Humane Use of Animals,
SUNY Upstate Medical University.
2.3. Bronchoalveolar lavage, differential cell counts and
was harvested from six mice by trans-tracheal instillation and
removal of pre-chilled phosphate-buffered saline with 0.25%
ery of 1.2–1.5ml per mouse). Total and differential leukocyte
counts were obtained by light microscopic quantitative analy-
sis of methanol-fixed cytospin preparations stained with Diff
Quik (Fisher Scientific, Pittsburgh, PA). For histologic evalua-
tion, lungs were inflated with 10% formalin, dissected en bloc,
set in paraffin, and sectioned onto glass slides.
2.4. Lung homogenates, chemokine, cysteinyl leukotriene
and plaque assays
Mice were sacrificed as described above (six mice per condi-
tion per time point) and lungs were removed and transferred
into 1ml pre-chilled Iscove’s Modified Dulbecco’s Medium.
Lung tissue suspensions were subjected to blade homogeniza-
tion (Tissumizer, Tekmar, Cincinnati OH) and cellular debris
and stored at −80◦C or liquid nitrogen prior to analysis. Assays
for mouse MIP-1?, mouse MCP-1, and cysteinyl leukotrienes
were performed as per the manufacturer’s instructions (R&D
Systems) and results were corrected for total protein as deter-
mined by the Bradford colorimetric assay using bovine serum
albumin standards. Viral recovery was determined by standard
plaque assay on the BS-C-1 epithelial cell line (American Type
Culture Collection, Manassas VA).
2.5. Statistical analysis
Data points represent the average±S.E.M. of samples from
six mice in two or more separate trials. Fisher’s exact test was
employed for categorical (clinical) data. Pearson’s correlations
were performed for paired sets of continuous data. One-tailed
t-tests were used to compare continuous data. Kaplan Meier
C.A. Bonville et al. / Antiviral Research xxx (2005) xxx–xxx
analyses were performed using Statistica Software (StatSoft,
Tulsa, OK), all other statistics were per the algorithms of the
Microsoft Excel data analysis program.
3.1. PVM induced cellular, chemokine and leukotriene
Table 1 documents the cellular inflammatory responses seen
tory response to acute infection with PVM is virtually 100%
granulocytic, with eosinophils representing 10–15% of the total
leukocyte count at the earliest time points. No differences were
tion of cellular influx, or in the patterns of increased leukotriene
centration (pg/ml) and total granulocyte count (cells/ml) was
observed (r2=0.70; Fig. 1A) together with a similar corre-
lation between cysteinyl leukotriene concentration and BAL
eosinophils (r2=0.74; Fig. 1B).
3.2. Ribavirin treatment inhibits PVM replication in mouse
Table 2 documents virus titers on days 0–7 determined from
total lung homogenates obtained from mice inoculated on day 0
with 60pfu PVM. In the absence of ribavirin, pulmonary virus
titers increased to 1.3±0.7×108pfu/g lung tissue by day 7.
Twice daily ribavirin administration decreased lung virus titers
alone (*p<0.01 compared to PBS or montelukast only con-
Total and differential leukocyte counts determined in bronchoalveolar lavage fluid, and leukotriene concentrations in lung homogenates from mice inoculated with
60pfu of PVM on day 0
Day Total cells (×103/ml)Neutrophils (×103/ml)Eosinophils (×103/ml)Lymphocytes (×103/ml) Leukotriene (pg/ml)
94 ± 4
90 ± 6
117 ± 6a
200 ± 19a
243 ± 18a
218 ± 21a
186 ± 9a,b
173 ± 17a,b
65 ± 29
82 ± 14
98 ± 12
242 ± 36a
245 ± 27a
184 ± 30a
207 ± 18a
211 ± 36a,b
88 ± 9
93 ± 7
124 ± 14
188 ± 16a
200 ± 41a
238 ± 33a
172 ± 35a
189 ± 48a,b
Ribavirin and montelukast
90 ± 8
92 ± 4
111 ± 12
208 ± 14a
252 ± 40a
230 ± 22a
179 ± 16a
185 ± 9a
Data are expressed as the mean±S.E. from n=6 mice.
ap<0.01 compared to day 0.
bConcentrations from n=2 mice only.
C.A. Bonville et al. / Antiviral Research xxx (2005) xxx–xxx
Fig. 1. Bivariate scattergram of cysteinyl leukotriene concentrations (pg/ml) vs.
bronchoalveolar lavage (BAL) fluid (A) granulocyte counts (cells/ml) and (B)
eosinophil counts. Regression lines with Pearson coefficients are indicated.
trols) and to 8.2±2.4×104(*p<0.01 compared to PBS or
telukast. We conclude that montelukast alone has no effect on
3.3. Ribavirin treatment reduces virus-induced pulmonary
Concentrations of the proinflammatory chemokines MIP-1?
documented in Table 3. When compared to PBS-treated mice,
mice treated with ribavirin had lower mean pulmonary MIP-
1? and MCP-1 concentrations on day 7 post-inoculation (both
two-fold reductions,*p<0.01). This is also consistent with pre-
Fig. 2. Mean clinical scores of mice (n=6 per point) infected with pneumo-
nia virus of mice (PVM, strain J3666) on day 0 and treated with twice-daily
intraperitoneal ribavirin (37.5mg/kg/dose×2 doses/day, filled circles), once
daily montelukast (10mg/kg, open squares), both ribavirin and montelukast
(filled squares) or diluent control (PBS, open circles) beginning on day 3. Error
bars indicate±standard error of the mean (S.E.). Clinical scores in the ribavirin
plus montelukast treatment arm were lower compared to each of the other three
treatment arms on day 8 and thereafter (p<0.05), except for the PBS arm on
day 12 (p=0.13 compared to montelukast plus ribavirin).
vious findings (Bonville et al., 2003), and can be contrasted
to the ∼2000-fold drop in lung virus titer. Mice that received
both ribavirin and montelukast had the lowest overall mean pul-
monary MIP-1? and MCP-1 concentrations (24±4.5pg/mlmg
and 160±70pg/mlmg lung protein on day 7 post-infection,
of the intervention combinations tested had a measurable effect
on total lung cysteinyl leukotriene concentrations at the time
points tested (Table 1).
3.4. Co-administration of montelukast and ribavirin results
in reduced severity of clinical symptoms and improved long
Shown in Fig. 2 are the mean clinical scores from two sep-
arate experiments that included 10 mice in each treatment arm
(drugs and doses as per Table 3). While clinical scores were
similar in mice receiving no therapy, montelukast alone, or rib-
avirin alone, mice treated with both ribavirin and montelukast
had overall a much less severe clinical course, statistically sig-
nificant by day 8 and thereafter (*p<0.05 as shown). Percent
Virus titers (pfu/g lung tissue) from mice (n=6 per point) inoculated on day 0 with 60pfu of PVM strain J3666
DayPBS Montelukast onlyRibavirin only Fold reductionMontelukast and ribavirinFold reduction
Virus titer (×104pfu/g lung tissue)
Montelukast (10mg/kg) was administered once daily; ribavirin (37.5mg/kg/dose), twice daily. All treatments were initiated on day 3 post-inoculation. Data are
expressed as the mean±S.E.
ap<0.01 compared to PBS-treated control, fold reduction calculated vs. PBS-treated control. ND is not detected.
C.A. Bonville et al. / Antiviral Research xxx (2005) xxx–xxx
Detection of proinflammatory chemokines MIP-1? and MCP-1 in lung tissue homogenates from mice (n=6 per point) inoculated on day 0 with 60pfu of PVM
DayPBSMontelukast only Ribavirin onlyFold reductionMontelukast and ribavirin Fold reduction
MIP-1? (pg/mlmg lung protein)
MCP-1 (pg/mlmg lung protein)
Montelukast (10mg/kg) was administered once daily; ribavirin (37.5mg/kg/dose), twice daily. All treatments were initiated on day 3, and continued until day 14
post-inoculation. NA, is not assayed. Data expressed±S.E.M. from six mice at each time point from two separate experiments.
ap<0.01 compared to PBS-treated controls.
bp<0.01 compared to the ribavirin only group.
mean weight loss in the control arm was 11±3% original body
telukast alone group, while only 2±1% in the ribavirin plus
montelukast group (*p<0.01). Results from a separate survival
study (n=10 per treatment arm) shown in Fig. 3 demonstrated
control and ribavirin only groups, with 50% long-term survival
*p<0.01 when compared to each of the other groups. Observed
pulmonary histology on day 5 is shown in Fig. 4. Compared
to uninfected lungs (Fig. 4A), untreated, PVM-infected lungs
(Fig. 4B) show intense granulocytic inflammation. The granu-
observed when both ribavirin and montelukast are administered
as therapeutics (Fig. 4C).
We have shown that during acute, severe PVM infection,
pulmonary cysteinyl leukotriene concentrations increased two-
fold in direct correlation with absolute bronchoalveolar lavage
fluid granulocyte counts. These results are analogous to those
obtained by VanSchaik et al. (1999), who reported a substan-
tial increase in cysteinyl leukotriene concentrations in respi-
ratory secretions of infants and children infected with RSV.
As cysteinyl-leukotrienes are potent pro-inflammatory medi-
Fig. 3. Survival analysis of mice inoculated with 60pfu PVM on day 0, and treated with ribavirin (37.5mg/kg/dose×2 doses/day, filled circles), montelukast
(10mg/kg, open squares), both ribavirin and montelukast (filled squares) or diluent control alone (same volumes, open circles) beginning on day 3; n=10 mice per
group. Significantly improved survival of combined ribavirin and montelukast-treated mice (*p<0.01) was observed when compared independently to each of the
other three groups.