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Combined fluticasone propionate and salmeterol reduces RSV
infection more effectively than either of them alone in
Rajeswari Singam, Prasanna K Jena, Sumita Behera, Gary R Hellermann,
Richard F Lockey, Dennis Ledford and Shyam S Mohapatra*
Address: Division of Allergy and Immunology, Joy McCann Culverhouse Airway Disease Research Center, University of South Florida College of
Medicine and James A. Haley VA Hospital, Tampa, FL, USA
Email: Rajeswari Singam - firstname.lastname@example.org; Prasanna K Jena - email@example.com; Sumita Behera - firstname.lastname@example.org;
Gary R Hellermann - email@example.com; Richard F Lockey - firstname.lastname@example.org; Dennis Ledford - email@example.com;
Shyam S Mohapatra* - firstname.lastname@example.org
* Corresponding author
Background: Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in
infants and is a risk factor for the development of asthma. Allergic asthmatics are more susceptible
to RSV infection and viral exacerbation.
Methods: Since the effectiveness of corticosteroids in treating RSV infection has been
controversial, we tested fluticasone propionate (FP) and salmeterol (Sal) alone versus FP plus Sal
(FPS) on RSV-induced airway inflammation. Mice were sensitized and challenged with ovalbumin
(OVA) and infected with RSV. Following infection they were treated with FP, Sal, or FPS intranasally
and airway hyperreactivity (AHR), inflammation and RSV titers were examined.
Results: The group treated with FPS showed significantly lower AHR compared to the group
treated with FP or Sal alone. The group treated with FP alone showed slightly decreased (non-
significant) AHR compared to controls. Treatment with FPS resulted in significant decreases in the
percentage of eosinophils and neutrophils in bronchoalveolar lavage fluid and in lung pathology
compared to FP or Sal. FP alone decreased eosinophils but not neutrophils or lymphocytes, while
Sal alone decreased eosinophils and neutrophils but not lymphocytes. FPS treatment of mice
infected with RSV in the absence of allergen sensitization resulted in a 50% decrease of RSV titer
in the lung and a reduction in neutrophils compared to FP or Sal.
Conclusion: Together, these results indicate that fluticasone in combination with salmeterol is a
more effective treatment for decreasing airway hyperreactivity and inflammation than either of
them alone in allergen-sensitized, RSV-infected mice.
Asthma is a chronic lung disease with two distinct features
– airway inflammation and airway hyperresponsiveness
[1,2]. An association between viral upper-respiratory
infections (URIs) and exacerbations of asthma has been
reported [3,4]. The most commonly identified viruses in
Published: 23 May 2006
Virology Journal 2006, 3:32doi:10.1186/1743-422X-3-32
Received: 15 February 2006
Accepted: 23 May 2006
This article is available from: http://www.virologyj.com/content/3/1/32
© 2006 Singam et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Virology Journal 2006, 3:32http://www.virologyj.com/content/3/1/32
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these studies include rhinovirus, coronavirus, influenza
virus and respiratory syncytial virus (RSV) . RSV is the
predominant cause of URIs in infants below 2 years of age
and infection may result in bronchiolitis, which is a risk
factor for asthma [6-12]. RSV may constitute the earliest
trigger for the development of a T-helper type 2 (Th2)-
dominant immune response, which is the hallmark of
immunopathology in allergic subjects including asthmat-
ics, and also in rodent models . URIs cause a decrease
in peak flow that lags behind upper airway symptoms by
1–2 days, with 46% of subjects in one study reporting a
two day lag in peak flow reduction .
A combination therapy involving a long-acting β2 agonist
and an inhaled corticosteroid (ICS) has emerged as an
effective asthma management strategy to control persist-
ent asthma . A combination of salmeterol (Sal) and
fluticasone propionate (FP) was found to be superior to
either of them alone [16,17]. The combination is also sig-
nificantly more effective than montelukast plus FP or
monotherapy with inhaled budesonide . The
increased effectiveness of FPS has been attributed to
increased activation and translocation to the nucleus of
glucocorticoid receptors [19,20]. However, the effect of
these drugs on viral exacerbation in allergic asthmatics has
not been studied.
The conclusion of the Cochrane Review of available con-
trolled trials of ICS in children with a history of mild epi-
sodic viral wheeze was that high dose ICS was partially
effective for the treatment of mild episodic viral wheeze of
childhood . Since URIs induce exacerbations, β2-ago-
nists may be of specific value in reducing such exacerba-
tions. In an in vitro study of Pseudomonas aeruginosa
infection, a combination of FP and Sal reduced infection
and preserved ciliated cells to a greater degree than either
alone suggesting synergy between the two agents .
Because 80–85% of asthma exacerbations in children are
associated with viral infections, early intervention with a
combination therapy should have beneficial effects on
viral asthma exacerbations.
Since virus-induced exacerbation is accompanied by air-
way inflammation, we reasoned that the combination of
a steroid and a β-2 agonist might provide protection from
severe RSV infection and the ensuing asthma exacerba-
tion. This hypothesis was tested in a mouse model of aller-
gen sensitization and RSV infection using OVA as the
allergen . Mice with chronic or acute sensitization to
OVA were RSV infected and then treated with FP or Sal or
the two together (FPS). Airway hyperreactivity (AHR) and
pulmonary inflammation were measured five days after
infection. The results show that the combination of FP
and Sal provides significant protection in terms of both
airway hyperreactivity and pulmonary inflammation
compared to either of them alone.
Female BALB/c mice, 4–6 weeks of age were obtained
from Charles River and housed under pathogen-free con-
ditions at the University of South Florida Vivarium. All
treatment protocols were approved by the USF Institu-
tional Animal Care and Use Committee (IACUC).
RSV preparation and infection of mice
The A2 strain of human RSV (American Type Culture Col-
lection, Manassas, VA) was propagated in HEp-2 cells
(American Type Culture Collection) grown in Eagle's min-
imal essential medium (Gibco) with 2% FBS. At maxi-
mum cytopathic effect, the cells were harvested in the
same medium. The suspension was clarified by centrifuga-
tion at 700 × g for 10 min at 4°C and the resulting super-
natant was layered onto a glycerol gradient and
centrifuged at 14,000 × g for 3 hrs at 4°C. The pellet con-
taining virus was resuspended in pre-cooled (4°C) buffer
(0.22 µ-filtered 50 mM HEPES (pH 7.5), 100 mM MgSO4,
and 150 mM NaCl) and stored in aliquots in liquid nitro-
gen. Viral titers were determined by standard plaque assay
combined with immunostaining for RSV. Mice were
infected under light anesthesia by intranasal inoculation
of RSV (5 × 106 PFU).
OVA sensitization, RSV infection and drug treatment
For experiments in which only a single RSV infection was
used (therapeutic regimen), mice were sensitized by intra-
peritoneal injection (i.p.) of OVA on day 1 and by intra-
nasal (i.n.) administration of OVA on days 7 and 9. On
day 9 the mice were infected i.n. with of RSV. From day 10
to day 13 they were treated daily i.n. with fluticasone (FP)
propionate, salmeterol (Sal), or the two in combination
(FPS) at 10 µg per mouse (FP and Sal were obtained from
GlaxoSmithKline). Airway hyperreactivity (AHR) was
measured on day 14 and on day 15 the animals were sac-
rificed. For the prophylactic experiments, mice were sensi-
tized by intraperitoneal injection (i.p.) of OVA on day 1
and by intranasal (i.n.) administration of OVA on days 9,
12 and 14. On day 19 the mice were infected i.n. with of
RSV. From day 21 to day 27 mice were treated daily i.n.
with FP, Sal, or the two in combination (FPS) at 10 µg per
mouse. On day 28, mice were reinfected with RSV and
then challenged i.n. with OVA. AHR was measured on day
29. On day 30 the mice were sacrificed and BAL fluid,
lungs and spleens were taken. All experiments were
repeated at least twice.
Determination of airway hyperreactivity (AHR)
AHR, expressed as enhanced pause (Penh), was measured
in unrestrained mice by whole body plethysmography
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Virology Journal 2006, 3:32http://www.virologyj.com/content/3/1/32
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