HUMMING, NITRIC OXIDE AND PARANASAL SINUS VENTILATION

Full text

From the Department of Physiology and Pharmacology
Karolinska Institutet, Stockholm (Sweden)
HUMMING, NITRIC OXIDE AND
PARANASAL SINUS VENTILATION
Mauro Maniscalco M. D.
Stockholm 2006

All previously published papers were reproduced with permission from the publisher.
Published and printed by Karolinska University Press
Box 200, SE-171 77 Stockholm, Sweden
© Mauro Maniscalco, 2006
ISBN 91-7140-753-7

To my family

1
ABSTRACT
The paranasal sinuses are air-filled bony cavities surrounding the nose. They
communicate with the nose via the sinus ostia through which fluid and gases pass in
both directions. A proper ventilation is crucial for sinus integrity and blockage of the
ostia is a major risk factor for development of sinusitis.
In this thesis we have explored an entirely new approach to monitor sinus ventilation
- the nasal humming test. We show in human studies in vivo and in a sinus/nasal
model that the oscillating airflow generated during humming produce a dramatic
increase in sinus ventilation. Interestingly, this increased gas exchange can be readily
monitored on-line by simultaneously measuring the levels of the gas nitric oxide
(NO) in nasally exhaled air. The sinuses constitute a major natural reservoir of NO
and when gas-exchange increases during humming NO escapes rapidly into the nasal
cavity thereby creating a highly reproducible peak in exhaled NO.
When exploring the different factors that determine the humming peak in NO we
found that sinus ostium size was the most important but the humming frequency also
influenced the sinus NO release. In patients with severe nasal polyposis and
completely blocked sinus ostia the humming peak in NO was abolished. Moreover, in
patients with allergic rhinitis, absence of a NO peak was associated with endoscopic
signs suggestive of ostial obstruction. In the last study we went on to study if an
oscillating airflow could be used not only to wash a gas out from the sinuses but also
to enhance passage of substances into the sinuses. Indeed, we found evidence of an
increased intra-sinus drug deposition by adding a sounding airflow to an aerosol.
In conclusion, the ventilation of the paranasal sinuses increases greatly when a person
is humming; a finding that could have both diagnostic and therapeutic implications.
Measurements of nasal NO during humming may represent a test of sinus ostial
function. In addition, aerosol in combination with a sounding airflow could possibly
be useful to increase the delivery of drugs into the paranasal sinuses.

2

3
LIST OF PUBLICATIONS
This thesis is based on the following articles, which will be referred to in the text by
their Roman numerals:
I. M. Maniscalco, E. Weitzberg, J. Sundberg, M. Sofia, J.O. Lundberg.
Assessment of nasal and sinus nitric oxide output using single-breath
humming exhalations.
Eur Respir J 2003; 22: 323-9
II. J.O. Lundberg, M. Maniscalco, M. Sofia, L. Lundblad, E. Weitzberg.
Humming, nitric oxide, and paranasal sinus obstruction.
JAMA 2003; 289:302-3
III. M. Maniscalco, M. Sofia, E. Weitzberg, G. de Laurentiis, A. Stanziola, V.
Rossillo, J.O. Lundberg.
Humming-induced release of nasal nitric oxide for assessment of sinus
obstruction in allergic rhinitis: pilot study.
Eur J Clin Invest 2004; 34: 555-560
IV. M. Maniscalco, M. Sofia, E. Weitzberg, L. Carratù, J.O. Lundberg.
Nasal nitric oxide measurements before and after repeated humming
maneuvers.
Eur J Clin Invest 2003; 33: 1090-94
V. M. Maniscalco, M. Sofia, E. Weitzberg, J.O. Lundberg.
A sounding airflow enhances aerosol delivery into the paranasal sinuses.
Accepted for publication in Eur J Clin Invest

4

5
CONTENTS
INTRODUCTION .........................................................................................................................9
Anatomy and Physiology of the Paranasal Sinuses..............................................................9
Nitric Oxide in the Upper Airways.....................................................................................10
NO Chemistry and Synthesis ..................................................................................10
Sources of Airway NO ............................................................................................11
Physiological Role of NO in the airways................................................................11
NO and Upper Airway Diseases .............................................................................12
NO Measurement Methods .....................................................................................14
AIMS............................................................................................................................................17
MATERIAL AND METHODS ..................................................................................................19
Study Subjects .....................................................................................................................19
Sinus/Nasal Model ..............................................................................................................19
Measurement of Exhaled NO..............................................................................................20
Nasal Endoscopy.................................................................................................................21
Clinical Questionnaire.........................................................................................................21
Aerosol System ...................................................................................................................21
Statistics...............................................................................................................................21
RESULTS AND COMMENTS ..................................................................................................23
Characterization of Nasal NO during Humming (Paper I) ................................................23
Humming, NO and Nasal Disorders (Paper II and III) ....................................................24
Nasal polyps (II) ......................................................................................................24
Allergic rhinitis (III) ................................................................................................25
Reproducibility of NO Measurements in Relation to Humming (Paper IV).....................26
Sounding Airflows and Aerosol Delivery into the Sinuses (Paper V)..............................27
GENERAL DISCUSSION..........................................................................................................29
Source of the Nitric Oxide Measured during Humming ....................................................29
How Does Humming Increase Sinus Ventilation?.............................................................30
Factors Which Influence NO Levels during Humming .....................................................31
Humming Improves Accuracy of Nasal NO Measurements .............................................31
A Humming Test to Diagnose and Monitor Sinus Disease? .............................................32
Humming to Treat Rhino-Sinusitis?...................................................................................33
Humming to Enhance Drug Penetration into the Sinuses? ................................................33
CONCLUSIONS..........................................................................................................................35
ACKNOWLEDGEMENTS ........................................................................................................37
REFERENCES.............................................................................................................................39

6

7
LIST OF ABBREVIATIONS
AP allergic nasal polyposis
AR allergic rhinitis
CRS chronic rhino-sinusitis
cGMP cyclic guanosine monophosphate
cNOS constitutive nitric oxide synthase
eNOS endothelial nitric oxide synthase (NOS-1)
F
E
NO
50
fraction of exhaled nitric oxide
HS healthy subjects
iNOS inducible nitric oxide synthase (NOS-2)
L-NAME N
G
-nitro-L-arginine methylester
NAR Nasal airway resistance
nNOS neuronal nitric oxide synthase (NOS-3)
NO nitric oxide
NO
2
nitrogen dioxide
NO
2
-
nitrite
NO
3
-
nitrate
PCD primary ciliary dyskinesia
SD Standard deviation
SEM standard error of the mean

Introduction
9
INTRODUCTION
The paranasal sinuses are hollow, air-filled cavities located in the skull bone of many
mammals including humans. Their physiological function remains completely
unknown but millions of people are painfully reminded of their existence each year
during episodes of sinusitis.
The sinuses communicate with the nasal cavity through bony channels and this
connection is crucial for sinus integrity
1
. A proper ventilation of the sinuses ensures
entry of fresh oxygen and removal of waste products and invading bacteria. During a
common cold or allergic rhinitis, swelling of the nasal mucosa may obstruct the sinus
ostiae, an event considered to be central in the pathogenesis of sinusitis. Despite the
connection with the nasal mucosa, which is heavily colonized by bacteria, healthy
sinuses are considered to be sterile. One explanation for this could be the constantly
ongoing local generation of nitric oxide (NO), a gas with antibacterial and ciliary
stimulating properties. Parts of the NO gas generated inside the sinuses will leak out
into the nose where it can be measured with non-invasive techniques.
Humming is identified in this thesis as a simple manoeuvre to greatly enhance the
ventilation of the paranasal sinuses. By using nasal NO measurements we here further
explore the physiological determinants of humming-induced sinus ventilation as well as
potential diagnostic and therapeutic possibilities.
ANATOMY AND PHYSIOLOGY OF THE PARANASAL SINUSES
The biological function of the paranasal sinuses is still an enigma, although several
theories have been put forward. These include humidification and warming of inspired
air, lightening of the skull, improvement of vocal resonance, absorption of shock to the
face or skull, and secretion of mucus to assist with air filtration
2-5
.
The four paranasal sinuses (maxillary, sphenoid, frontal and ethmoid) develop as
outpouchings of the nasal mucosa. At birth they are fluid filled and pneumatization
occurs gradually during childhood so that by the age of 12 years all sinuses are fully
developed. They remain connected to the nasal
cavity via narrow ostia with a lumen diameter of
1 to 3 mm (Figure 1). The sinuses are lined with
a mucosal membrane, which is thinner and less
richly supplied with blood vessels and glands
than the mucosa of the nasal cavity. The ostia of
the frontal, maxillary and anterior ethmoid
sinuses open into the osteomeatal complex,
which lies in the middle meatus lateral to the
middle turbinate. The posterior ethmoid and
sphenoid sinuses open into the superior meatus
and sphenoethmoid recess. The osteomeatal
complex is important because the frontal,
ethmoid and maxillary sinuses all drain through
Fig. 1. The paranasal sinuses

Mauro Maniscalco
10
this area. An effective ciliary transport system sweeps mucus towards the ostia which
helps to to keep the sinuses free of invading pathogens
6
. In addition, a variety of
antibacterial compounds are released from the sinus mucosa including lactoferrin,
lysozyme and secretory antibodies
7
. For a satisfactory function of these protective
mechanisms, the mucosa is dependent on adequate ventilation and blood flow. A
normal ostial patency is the main prerequisite for adequate ventilation of the sinuses
5
,
and indeed an impaired ostial function is a central event in the pathophysiology of
sinusitis
8
.
NITRIC OXIDE IN THE UPPER AIRWAYS
Until less than a couple of decades ago the gas NO was considered to be only a noxious
component of air pollution present in car exhaust and cigarette smoke
9, 10
. Although the
discovery of an endogenously produced endothelium-derived relaxant of arterial
vessels dates back to 1980
11
, only in 1987 this molecule was identified as NO by two
independent groups
12, 13
. Since then this research field has been continuously growing,
and NO is now regarded as a key regulatory factor implicated in the control of several
different physiological and pathophysiological processes including blood flow, platelet
aggregation, neurotransmission, immunity
14
and inflammation
15
.
In 1991 Gustafsson and co-workers
16
discovered that NO is present in exhaled breath of
humans and two years later Alving and colleagues
17
could show that exhaled NO is
increased in patients with asthma. These findings triggered a great interest in studying
various aspects of exhaled NO and as of today more than 1000 papers have been
published in this area.
Before I continue with a detailed description of the specific projects included in this
thesis I would like to give a short background of NO in the airways including also a
general overview of NO’s chemistry, physiology and pathophysiology.
NO Chemistry and Synthesis
NO is a colourless gas characterized by a complex chemistry
18
. It is a small lipophilic
molecule, which diffuses readily through over biological membranes. In normal
conditions only a small fraction remains in aqueous solution, while the vast majority
enters the gas phase
19
. Because of its chemical structure, characterized by the presence
of an unpaired electron (making it a free radical), NO reacts rapidly with a number of
biological compounds which helps to explain its diverse biological effects
14
.
NO is synthesized from the semi-essential amino-acid L-arginine and molecular
oxygen, in a reaction catalyzed by one of several forms of nitric oxide synthases
(NOS). To date, three isoforms of NOS have been identified. Two of these are
constitutively expressed, while the third is expressed only in activated cells. One form
was initially identified in neurons of both brain and enteric nervous system, thus being
termed neuronal NOS (nNOS)
20
, whereas the other was originally found in endothelial
cells lining the vasculature and therefore termed eNOS
21
. These two forms have been
fully characterized and also found to be distributed more widely than originally
thought, thus being renamed NOS-1 and NOS-3, respectively. The third isoform,
originally named iNOS and now also known as NOS-2, is not expressed in resting cells

Introduction
11
but is activated in response to various inflammatory stimuli such as bacterial products
and cytokines.
18,22
NOS-1 and NOS-3 are calcium-calmodulin dependent and are
activated in response to a calcium signal. Enzyme activation occurs rapidly and
transiently providing a rapid pulse-like NO release. NOS-2 is calcium-calmodulin
independent and NO synthesis is controlled at the transcriptional level. Once the
enzyme is expressed it will produce large amounts of NO for prolonged periods. NOS-
2 expression is dependent on transcription factors such as nuclear factor-κB
23
and
activated by pro-inflammatory cytokines including tumor necrosis factor-α and
interleukin-1β
24
.
Soon after the L-arginine/NO pathway was described, several NOS inhibitors were
developed and these have been useful tools in exploring the various physiological
effects of NO. The majority of these compounds are L-arginine analogues with no
selectivity for the different NOS isoforms
25, 26
. Anti-inflammatory
glucocorticosteroids have been shown to inhibit NOS-2 expression
27
while leaving
the constitutive enzymes unaffected.
Sources of Airway NO
After the discovery of exhaled NO in 1991, it was surprisingly shown that the upper
airway is the major source of NO in adult healthy subjects
17
. Measurements in
tracheostomized individuals clearly demonstrated that more than 90 % of all exhaled
28
NO originates from the nasal region
29
. The exact origin of the NO found in nasal air, as
well as the relative contribution from different sources within the nasal airways are still
debated. The paranasal sinuses are a particularly important source of NO, as shown by
Lundberg et al.
30
, who punctured maxillary sinuses and detected a continuous NO
synthesis yielding very high concentrations (3000-25000 ppb). Interestingly, the NOS
found in the sinuses is predominantly calcium independent
31
, a characteristic usually
attributed to the NOS-2, but studies showed that it is constitutively expressed and not
inhibited by steroids, the latter being typical features of constitutive NOS. Immuno-
histochemical and mRNA in situ hybridization experiments confirmed a high
expression of NOS-2 in healthy sinus epithelium while this enzyme was much less
expressed in the nasal mucosa. All together these findings led to the suggestion that
much of the NO measured in the nasal cavity is originating from the paranasal sinuses.
On the other hand, other studies suggested only a partial contribution from the sinuses.
Thus, in one healthy volunteer, the occlusion of both osteomeatal complex and spheno-
ethmoidal recess, decreased nasal NO concentrations by only 12%, thereby suggesting
only a limited role of paranasal sinuses as sources of nasal NO
32
.
All three NOS isoforms have been identified in the upper airways. Several cells in the
upper airways can express NOS-1 and 3 including parasympathetic neurons innervating
nasal vessels, endothelial cells, and ciliated epithelial cells
33
. NOS-2 has been detected
in epithelial cells, macrophages, neutrophils, endothelial and vascular smooth muscle
cells.
33-35
Physiological Role of NO in the airways
It has been suggested that NO contributes to non-specific host defences against
bacterial, viral and fungal infections. Indeed, some bacteria are sensitive to the action

Mauro Maniscalco
12
of NO at concentrations of only 100-1000 ppb
36
. Therefore, NO may help to maintain a
sterile microenvironment within the paranasal sinuses, where the concentration of this
gas can exceed 30 000 ppb
30
. NO could also contribute to the airway host- defence by
regulating ciliary motility
37
. Indeed, it has been shown that application of NO donors to
the human nasal mucosa stimulates ciliary beating
38
, and that low nasal NO levels are
associated with an impaired mucociliary function
39
. Furthermore, the NO substrate L-
arginine is able to increase ciliary beat frequency in vitro
10, 40
.
A role of NO has been hypothesized in the regulation of nasal airway resistance to
airflow (NAR), and in the mechanisms of humidification and warming of inhaled air
flowing through the nose. As a powerful vasodilator, NO could control the filling of
nasal capacitance vessels, thus contributing to the total resistance to nasal airflow.
Vascular tone also regulates nasal temperature, and variations of NO levels are indeed
associated with thermic changes of nasal air in humans. Holden et al.
41
observed that
inhibition
of NO release is associated with cooling of nasal air. These findings suggest a
potential role for NO in modulation of the vascular changes necessary for temperature
conditioning
of nasal air and, as a consequence, NO could also be involved in
thermoregulation. However, in normal conditions NO does not seem to play an
important role in regulation of vascular tone. In fact, Silkoff et al.
42
reported that local
application of NOS inhibitors does not modify nasal patency measured by acoustic
rhinomanometry. Accordingly, no effect on NAR was demonstrated after topical
administration of L-NAME to healthy subjects, at doses capable of significantly
reducing nasal NO levels
43
.
NO has also been thought to exert other important physiological effects, such as the
improvement of ventilation–perfusion ratio in the lungs after auto-inhalation
44,
34, 45
. In
this sense, NO is believed to be an “aerocrine messenger” between the upper and lower
airways.
NO and Upper Airway Diseases
Allergic Rhinitis
The nasal mucosa of rhinitic patients is characterized by a high expression of the
inducible isoform of NOS-2
34
similarly to what is found in the lower airways of
asthmatics
46-49
. In these subjects, NOS has been detected in the mucosal epithelium as
well as in glandular cells and in the endothelium of sinusoids
50
. Kang et al.
51
suggested a role for NOS-2-derived NO in the increased mucus secretion occurring in
rhinitis. It is also possible that NO plays a role in the control of NAR, and in the
mechanisms of plasmatic microvascular leakage induced by pro-inflammatory
substances in both healthy individuals and rhinitic patients subjected to allergen
challenges
43, 52, 53
.
An intricate picture arises from the studies assessing nasal NO levels in allergic
rhinitics, where the results are quite contradictory. Some authors have shown variable
nasal NO levels after exposure to allergens
54-57
. In these patients, an increased
mucosal expression of NOS-2
34
was found as well as an increase in NO metabolites
(nitrate and nitrite) in nasal lavage fluid
58
. Martin et al.
57
detected high nasal and oral
NO in allergic rhinitic patients. Kharitonov et al.
56
reported that during the pollen
season nasal NO, but not oral NO concentrations, were significantly increased in
subjects with seasonal allergic rhinitis. These authors also showed that one hour after

Introduction
13
nasal challenge with allergens, nasal NO decreased and this correlated with the
severity of rhinitic symptoms. In contrast, Palm et al.
59
revealed no change in nasal
NO levels in patients with allergic rhinitis, whereas in that study orally exhaled NO
was increased. Also other groups failed to find an evident increase in nasal NO in
seasonal rhinitis out of the pollen season
60
and during the pollen season
55
.
Nasal polyposis
A high NOS-2 expression and activity has been demonstrated in nasal polyps
50
. Parikh
et al. found that NOS activity was also increased in nasal polyps from aspirin-sensitive
asthmatics
61
. They formulated the hypothesis that the overproduction of cytokines
occurring in aspirin-sensitive patients stimulates NOS-2 expression, thus promoting an
elevated synthesis of NO. The latter might inhibit the apoptosis of eosinophils, leading
to an increased survival of these cells, which in turn would release a great amount of
cytokines responsible for the perpetuation and propagation of nasal inflammation.
Studies have shown that nasal NO levels are lower in patients with polyps when
compared to controls or individuals with uncomplicated allergic rhinitis
62
. These
results, which are apparently paradoxical with regard to the already mentioned high
levels of NOS-2 expression, were attributed to the blockage of the osteomeatal complex
and to the consequent inability, for newly generated NO, to reach the nasal cavity. The
authors of this study suggested that nasal NO synthesis depends on iNOS expressed by
the inflamed nasal mucosa, as well as on constitutive sinus production by normal
tissues.
Sinusitis
In both acute and chronic sinusitis, a reduction of nasal NO levels and NOS-2
expression has been reported
63, 64
. In children with acute sinusitis, Baraldi et al.
demonstrated that nasal NO concentrations increased when
clinical conditions
improved as a consequence of antibiotic therapy
64
. It is still uncertain whether the low
NO levels detected in acute and chronic sinusitis result from a reduced maxillary NO
production, or are rather mainly due to an obstruction of sinus ostia caused by local
oedema, nasal congestion, and mucus accumulation within the sinus cavities. Indeed,
the nasal mucosa swelling which develops during
rhinitis might also lead to a partial
blockage of sinus ostia, thus resulting
in a reduced passage of NO from sinuses to the
nasal
cavity, where it is
measured. The latter interpretation is corroborated by the results
of studies performed on patients with nasal polyposis, showing that nasally measured
NO concentrations were inversely correlated with the number of occluded paranasal
sinuses. However, the hypothesis of a reduced maxillary NO synthesis is supported by
the finding of strongly reduced NOS-2 expression in the sinus mucosa during sinusitis.
Primary Ciliary Dyskinesia (PCD)
PCD is a genetic disorder characterized by abnormal structure and/or function of
ciliated cells in the airways and elsewhere
65
. Clinical presentations include neonatal
respiratory distress, recurrent lower respiratory tract infections, chronic rhino-sinusitis
and male infertility. It is associated with visceral mirror image arrangement (situs
inversus) in 50% of cases. PCD should be diagnostically distinguished from
bronchiectasis, atypical asthma, cystic fibrosis and unusually severe upper airway
disease
66
. Diagnosis is difficult and often requires complex investigations, aimed to

Mauro Maniscalco
14
evaluate the beating frequency of cilia, their light microscopy morphologic pattern as
well as ciliary ultrastructure and orientation, assessed by electron microscopy
66
. In
1994 Lundberg et al. reported markedly reduced nasal NO levels in 4 children with
PCD
28
, a finding that has now been confirmed by other groups
67, 68
. Consistently, an
almost absence of nasal NO has been noticed, thus suggesting that NO measurement
may be used as a screening test for PCD, with a high degree of diagnostic accuracy.
NO Measurement Methods
General considerations of measurement technique
The most common way of measuring exhaled and nasal NO is by
chemiluminescence
69
, where detection depends on the photochemical reaction between
gaseous NO and ozone generated in the analyzer. In brief, NO contained in the sampled
air reacts with an excess of ozone to produce NO
2
with an electron in an excited state
(NO
2
*). This NO
2
* changes back to ground state while emitting electromagnetic
radiation in the 600 to 3000 nm wavelength range. The chemiluminescence is detected
by a photomultiplier tube that proportionally converts the intensity of luminescence
into an electric signal for display.
Other techniques that have been used to establish that NO
is present in the exhaled
breath of humans include mass spectroscopy
and gas chromatography-mass
spectroscopy.
16, 29
Methods for nasal NO measurement
The development of standardized and reproducible methods for measurement of nasal
NO has come on the heels of methods developed for measurements of exhaled NO.
While the American Thoracic Society (ATS) and European Respiratory Society have
agreed on a highly standardized procedure for measurements of lower respiratory tract
exhaled NO, it has not been possible to define one single standardized measurement
procedure for nasal NO measurements
69, 70
. Nasal NO measurement techniques have
been addressed by a European taskforce, and more recently by an official statement of
the ATS, reflecting the growing interest in these measurements
71
.
The simplest and most used method for measuring nasal NO has been to aspirate nasal
air directly from the nasal cavity using the intrinsic flow of the NO analyser
59, 72-74
.
Typically, the sampling probe is connected to a nasal olive, which is gently introduced
into one nostril. Then the subject is asked to hold his/her breath and air circulates from
one naris to the other around the posterior nasal septum.
Another method to measure nasal NO involves a nasal single-breath exhalation at a
fixed flow rate using a face-mask
75, 76
. In this case an oral NO exhalation is also
performed using the same exhalation flow and the value is subtracted from that
obtained during the nasal exhalation
77
.
The sampling technique sometimes needs to be adjusted depending on the situation and
the patients to be studied. For example, breath-holding or single-breath measurements
are not possible in non-cooperating individuals e.g. infants and sedated patients.
With all methods, a constant trans-nasal flow rate produces a washout phase followed
by the establishment of a steady NO plateau seen in the profile of NO versus time,

Introduction
15
analogous to that seen in the lower respiratory tract. Nasal NO concentration is
inversely related to the trans-nasal airflow rate. However, different flow rates may
have different aerodynamic profiles resulting in changes in the physics of airflow (e.g.
laminar versus turbulent flow)
78
. The aerodynamics of this flow may affect the nasal
NO output. For all the above reasons, any standardized method used should rigorously
control trans-nasal airflow rate. The product of trans-nasal flow rate and measured NO
concentration allows calculation of NO output. Present evidence suggests that NO is
relatively constant over a range of trans-nasal flow rates between 0.25-3 liters/minute
78
.
There is reasonable agreement, using different measurement techniques, that nasal NO
output is in the range of 200-450 nL/min in healthy primates. At higher flow rates, NO
may increase progressively
77
.
It is becoming clear that, due to the complexity of anatomical structures, measurements
of nasal NO output or concentration cannot provide evidence as to the source of the gas
(e.g. nasal cavity and/or paranasal sinuses) or the biochemical processes, which
generate the NO. The sinuses communicate with the nasal cavity through ostia, and
there is a continuous gas exchange between these cavities. Therefore, a high NO flux
from sinuses could easily blunt slight alteration of NO originating from nose. This may
explain the large intra and inter-individual variation in nasal NO has been reported both
in healthy people and in subjects with rhinitis.
At present there is no simple and non invasive tool to explore the great NO reserve
contained in the sinuses and the exchange occurring between sinuses and nose via
sinus ostia. In 2002 Lundberg and Weitzberg
79
reported that nasal humming (which is
the production of a tone without opening the lips or forming words) resulted in an
enormous increase in exhaled nasal NO levels, likely due to a rapid washout of sinus
NO caused by the oscillating air flow. This thesis is a direct continuation of that
finding and here we tried to explore if measurements of NO during humming could
be used to overcome some of the problems associated with the currently used
methods.

Mauro Maniscalco
16

Aims
17
AIMS
The overall aim of this thesis was to investigate the diagnostic and therapeutical
potential of nasal humming. The specific aims were:
• To explore the various factors influencing the nasal NO increase induced by
humming.
• To study nasal NO during humming in patients with nasal and/or sinus diseases.
• To assess if humming could be used to improve the reproducibility of nasal NO
measurements.
• To test if a sounding airflow could be used to enhance the penetration of a drug into
the sinuses.

Mauro Maniscalco
18

Material and Methods
19
MATERIAL AND METHODS
For a detailed account of materials and methods the reader is referred to the
individual papers.
STUDY SUBJECTS
All studies were approved by the local ethics committees and all subjects gave their
informed consent.
In paper I, II, IV and V, a total of 38 healthy non-smoking volunteers (aged 20–47
years) without any history of allergy, nasal disease, asthma or any other chronic lung
conditions were recruited from hospital personnel.
In paper II we studied 10 subjects with nasal polyposis and in paper IV 5 subjects
with allergic rhinitis associated with nasal polyposis were studied. All patients had
bilateral polyps and completely opaque sinuses according to a recent computed
tomography scan and they were on the waiting list for sinus surgery.
In paper III and IV, 15 + 59 consecutive untreated subjects with mild to moderate
allergic rhinitis were studied. All had positive skin prick testing for common allergens.
They were referred from their general practitioner to the skin-testing facility because of
a previous clinical history of nasal obstruction, rhinorrea, itching and sneezing, lasting
≤ 5 years and occurring at least for two consecutive months/years. They required
medical treatment with nasal decongestants and/or antihistamines for less than one
week per month and they had nasal symptoms not troublesome enough to interfere with
normal daily activity or night time sleep.
SINUS/NASAL MODEL
In paper I and V a two-compartment model resembling the nasal cavity and one
sinus was used (Figure 2).
Description of the model.
A syringe (representing the sinus) was filled with various NO gas concentrations
between 2 – 10 ppm and connected horizontally to a plastic cylinder (representing the
nasal cavity) via a luer fitting. The diameter of the syringe tip (representing the
ostium) was varied between 0.8 – 4.0 mm. The volume of the syringe was varied
between 5 – 20 mL. The distal end of the cylinder (nasal cavity) was left open or
connected to a Hans Rudolph resistor of 50 cmH
2
O/L s, thereby generating cylinder
pressures of either 1 or 10 cmH
2
O. Flow and pressure were measured by a linear
pneumotachymeter. Resulting NO output was measured at the distal end of the
cylinder by a rapid-response chemiluminescence system (Aerocrine AB, Stockholm,
Sweden). The signal output from these devices were connected to a computer-based
system (Aerocrine NO system; Aerocrine AB) and yielded an instant on-screen
display of flow, pressure, NO concentration and NO output.
Artificial generation of humming in the model.
Pressurised NO-free air was set to generate three different flow rates (0.20, 0.25 and
0.30 L/s). The air was led through the plastic cylinder (nasal cavity) either via a

Mauro Maniscalco
20
rubber duck call, which yielded a pulsating airflow, or via a rubber duck call without
the sound generating membrane (quiet control). Three duck calls with different
fundamental frequencies (120, 200 and 450 Hz) were used. NO was measured during
a 10-s period and all experiments were repeated five times. In an additional
experiment, a turbulent flow was generated by leading pressurised NO-free air
through a plastic mesh connected to the cylinder and NO was measured as described
above. This experiment was carried out without a sound-generating device.
Human humming in the model.
In the same model, the pulsating airflow was also generated by a subject performing
oral exhalation through the cylinder, with or without phonation, at two fixed flow
rates (0.20 or 0.25 L/s) and three different frequencies (130, 150 or 450 Hz). NO
output was calculated from the entire exhalation with subtraction of oral NO output.
All experiments were repeated five times. To estimate the rate of NO exchange
between the two cavities, the remaining NO concentration in the syringe at the end of
each experiment was also measured.
Measurement of artificial and human humming sound frequency.
The audio signal of humming was picked up by a TCM 110 Tiepin electret condenser
microphone placed on the plastic cylinder in the model (Figure 2) and recorded
directly onto a PC by the Soundswell Signal Workstation. The fundamental frequency
was extracted by its Corr module, which computes the autocorrelation of the audio
signal in two adjacent time windows. The mean fundamental frequency and SD were
then determined by means of its histogram module. The resonance frequency of the
model system was calculated according to Durrant and Lovrinic.
Figure 2. Schematic presentation of a model resembling the sinus (syringe), the ostium
(syringe tip) and the nasal cavity (plastic cylinder). NO: nitric oxide.
MEASUREMENT OF EXHALED NO
The exhaled NO measurements were performed using chemiluminescence technique
according to the ATS guidelines (papers I - V). NO levels were measured in oral and
nasal exhalations either with phonation (humming) or during quiet expiration. A tight
fitting mask covering the nose was used for nasal measurements, and a mouthpiece was
used for oral exhalations. The subjects performed a single-breath exhalation against an

Material and Methods
21
expiratory resistance at a fixed flow rate (0.2 L/s) for ten seconds. To calculate nasal
NO levels the value of oral NO was subtracted from the nasally exhaled NO.
The exhaled air was led into the Aerocrine NO System or Niox® (Aerocrine AB,
Solna, Sweden) or 280 NOA (Sievers Instruments, Boulder, Sensor Medics, Milan,
Italy) for measurement of NO. Ambient NO levels were below 10 ppb during the
measurements.
NASAL ENDOSCOPY
In paper III nasal endoscopy was performed with a rigid endoscope 2.7 mm 0°. For
each side of the nose endoscopic images were evaluated by the following scoring
system. 0 = absence of significant nasal obstruction, 1 = turbinate hypertrophy or
polyps with a free visible passage to the osteomeatal region, 2 = severe obstruction
resulting from nasal polyposis or marked middle turbinate hypertrophy and no visible
passage to the osteomeatal region. Using this system the maximum total score was 4.
CLINICAL QUESTIONNAIRE
In paper III a clinical questionnaire about the patients present symptoms was used.
This included registration of the following symptoms: nasal obstruction, rhinorrea,
sneezing, itching, headache/facial pain and was scored as follows: 0 = absence, 1 =
very mild, 2 = mild, 3 = moderate, 4 = troublesome. Maximal score with this system
was 20. Subjects with a total score equal or less than 2 were classified as asymptomatic
at the time of the study.
AEROSOL SYSTEM
In paper V an aerosol system was used to administer aerosol to the healthy subjects.
In brief a jet nebulizer (Nebula) was connected via a T valve to a rubber duck call,
which yielded a sound with pulsating airflow or to a rubber duck-call without the
sound generating membrane. The rubber duck call was joined to a nasal cannula,
while a flow generator was connected to the opposite extremity of the T valve.
STATISTICS
The NO output (nL/min) was calculated for all sampling modalities in all papers.
Nasal NO was calculated using a subtraction method, where NO in orally exhaled air
is subtracted from that in nasally exhaled air. Results were presented as mean ± SEM
in paper I and as mean ± SD in paper II, III and IV. In paper III the 99%
confidence intervals (CI.) were calculated for silently exhaled nasal NO values and
net humming NO values. For analysis of unpaired data in papers I-IV the Mann-
Whitney U test was used. For paired data the Friedman’s test was used in paper I, the
Wilcoxon’s test in paper I, III and IV and ANOVA for repeated measurements in
paper V. Correlations were analysed by Spearman correlation test.
In Paper IV the coefficient of variation for the different breathing modes was
calculated by dividing the within subject standard deviation by the mean. The limit of
agreement was calculated according to Bland & Altman
80
. Analysis of variability
before and after humming was performed using Friedman test.
A p value less than 0.05 was considered significant.

Mauro Maniscalco
22

Results and comments
23
RESULTS AND COMMENTS
CHARACTERIZATION OF NASAL NO DURING HUMMING (PAPER I)
We have characterised nasal NO during humming and explored various factors that
could determine gas exchange between the sinuses and the nasal cavity. Moreover, we
have investigated if NO measurements during humming could give additional
information about NO production at different sites in the upper airways. This was
achieved by studying healthy volunteers, as well as creating a model of the sinus and
the nose (Figure 2), in which the influence on NO release from the sinus could be
investigated in relation to ostium size, humming frequency, sinus volume, sinus NO
concentration, air flow and pressure.
In all subjects humming caused an increase in exhaled NO output compared to silent
exhalation (Figure 3). In contrast, oral phonation had no effect on exhaled NO.
Furthermore, in all the experiments the shape of the NO curve was characterized by a
large initial peak followed by a progressive decline towards a plateau (Figure 3). Our
interpretation of these findings is that humming induces a rapid release of air from the
sinuses, containing high concentrations of NO, which is then followed by a plateau
representing the combined continuous release of NO from the nasal and sinus
epithelium. Topical nasal application of the NOS inhibitor L-NAME reduced silently
exhaled nasal NO by > 50%, but had no effect on the humming-induced increase in
nasal NO output.
When consecutive humming
manoeuvres were performed at
5 sec. intervals nasal NO output
decreased during each
manoeuvre mainly because of
the gradual disappearance of the
initial peak. Within four nasal
humming manoeuvres the peak
was completely absent. A silent
period of 3 minutes was
necessary to obtain a complete
recovery of the nasal NO output
during humming. Nasal NO
output during silent exhalation
was reduced by 30 % when
performed immediately after a
humming procedure. These findings together with those from the L-NAME
experiments point towards a substantial contribution of sinus NO to the levels found in
nasally exhaled air. Furthermore, it suggests that humming procedures before nasal NO
measurements may enhance this method when used in patients with nasal diseases. This
is further explored in paper IV.
Fig. 3. Representative tracing of nitric oxide output (NO)
during a single-breath nasal exhalation performed at
constant flow with (dotted line) or without (solid line)
humming.

Mauro Maniscalco
24
In the model, all the factors tested,
which are known to influence
normal sinus ventilation had effect
on NO output during humming.
However, ostium size was the most
critical, shown by a dramatic
increase in NO output after
enlarging the ostium size (Figure 4).
In fact, with an ostium diameter
above 2 mm more than 80% of the
sinus gas volume was exchanged by
one single humming manoeuvre.
Also modification of humming
frequency caused significant changes
in NO output.
Fig. 4. Influence of ostium size on sinus
gas exchange in a sinus/nasal model
during silent exhalation (○) or humming (●).
When studying the effect of
different humming frequencies on
NO output in the model, we found
the greatest effects where achieved
when the humming frequency was
close to the calculated resonance
frequency of the particular sinus
(Figure 5). Turbulent flow did not
change NO output during humming.
The results from the model indicate
that the sinus ostium is the major
determinant for NO release from
the sinuses during humming.
Fig. 5. Effect of humming frequency on nitric oxide
output in a sinus/nasal model using sinuses with
different resonance frequency.
HUMMING NO AND NASAL DISORDERS (PAPER II AND III)
Nasal polyps (II)
Based on the findings in paper I we hypothesized that patients with obstructed sinus
ostia would exhibit less of an increase in exhaled nasal NO levels upon humming.
Therefore we studied patients affected by bilateral nasal polyps, chronic sinusitis and
completely opaque sinuses and compared the results to those obtained in healthy
controls. NO levels were measured in single-breath nasal exhalations first silently and
then during humming.

Results and comments
25
During silent nasal
exhalation, NO output was
similar in control patients
and patients with sinusitis
(189 ± 27 nL/min vs. 162 ±
22 nL/min, respectively).
Mean output of nasal NO
increased 7-fold during
humming (to 1285 ± 189
nL/min) in control patients
but remained completely
unchanged in the patients
with sinusitis (169 ± 21
nL/min, Figure 6). The most
likely explanation is a lack of
air passage between the
sinuses and the nasal cavity,
which was evident from the
computed tomography
showing obstructed sinuses
bilaterally in all patients.
Interestingly, one of the
patients had endoscopic sinus
surgery during the course of
this study, and in this patient
NO levels increased during
humming to almost normal
levels 2 weeks after the
operation (figure 7).
It is noteworthy that humming
was necessary to reveal differences in nasal NO between patients and controls since
silent nasal exhalation levels of NO were similar in these groups.
Allergic rhinitis (III)
Sinusitis and nasal polyposis are increasingly recognized as a common event occurring
during upper allergic respiratory diseases such as allergic rhinitis. Indeed, as many as
45-60 % of patients with allergic rhinitis have radiological evidence of sinus
abnormalities and allergic rhinitis is considered a major risk factor for sinusitis
81
.
In the paper III we studied if nasal NO measurement during humming could be used
to identify sinus obstruction in patients with allergic rhinitis. Nasal NO levels were
measured during humming in rhinitis patients and the levels were compared to nasal
symptoms and endoscopic findings.
Consecutive patients with mild to moderate persistent allergic rhinitis were studied and
their present nasal symptoms were recorded. Then NO levels were measured during
Fig. 6. Effect of humming on nasal nitric oxide output in
healthy controls and in patients with bilateral nasal
pol
y
posis.
Fig. 7. Effect of humming on exhaled nasal NO
concentrations in a patient with nasal polyposis before (b)
and two weeks after sur
g
er
y
(
a
)
.

Mauro Maniscalco
26
silent single-breath nasal exhalations and humming. Based on the NO results the
patients were divided in two groups; those with a great increase in nasal NO during
humming (humming responders, n = 46) and those without a significant increase
(humming non-responders, n = 13).
A significant humming response was then defined as a humming NO increase above 3
standard deviations from the mean NO levels measured during quiet nasal exhalation in
the study population. Subjects who showed a humming NO level of at least 3 standard
deviations above the mean NO levels during quiet exhalations were therefore
categorized as humming responders while those who did not increase above this level
were categorized as humming non-responders.
In 11 of the non-responders and in 22 responders, the passage to the osteomeatal
complex area was assessed and scored by nasal endoscopy. This was done by an oto-
rhino-laryngologist unaware of the NO results. Among the non-responders 9 out of 11
patients (80%) had endoscopic signs suggestive of bilateral sinus obstruction, compared
to 1 out of 22 (< 5%) of the humming responders. Baseline nasal symptom score and
NO levels during quiet exhalation were not significantly different between the groups.
In conclusion the absence of a nasal NO increase during humming is associated with
endoscopic findings suggestive of sinus ostial obstruction in subjects with allergic
rhinitis. Measurement of nasal NO during humming may be a simple method to detect
sinus abnormalities in these patients.
REPRODUCIBILITY OF NO MEASUREMENTS IN RELATION TO
HUMMING (PAPER IV)
Large intra and inter-individual variations in exhaled nasal NO have been reported both
in healthy people and in subjects with rhinitis. We hypothesized that nasal NO
measurements would have a better reproducibility if they were performed immediately
after repeated humming procedures, since pre-treatment with humming would reduce
the contribution from the paranasal sinuses in the following silent exhalations.
NO output was measured before and after repeated humming manoeuvres in orally and
nasally exhaled air in healthy subjects (HS), patients with allergic rhinitis (AR) and
patients with allergic nasal polyposis (AP). NO measurement were performed in each
subject during silent nasal exhalations, either preceded by a period of silence/free
speaking or immediately after five consecutive humming manoeuvres (post-humming).
Mean nasal NO output after a period of silence/free speaking was 231 nL/min (95% CI:
178 – 284) in HS, 434 nL/min (347 - 522) in AR (p<0.001) and 262 nL/min (163 - 361)
in AP. After repeated humming manoeuvres nasal NO output was 16% (5 to 50%)
lower in HS and 14 % (1 to 49%) lower in AR, while it remained unchanged in the
subjects with AP. Intra-subject coefficient of variation during silent nasal exhalation
was 12%, 13% and 5% in HS, AR and AP, respectively. After humming intra-
individual coefficient of variation significantly decreased in both HS and AR, but was
unchanged in AP (Figure 8).

Results and comments
27
These data show that nasal NO levels measured immediately after repeated humming
manoeuvres are consistently lower and more reproducible than nasal NO levels
measured after a period of silence or free speaking. The humming procedure may be
useful to better estimate NO output from the nasal cavity mucosa in health and disease.
SOUNDING AIRFLOWS AND AEROSOL DELIVERY INTO THE SINUSES
(PAPER V)
The use of aerosol therapy in the treatment of paranasal disorders has been suggested
by several authors. Administration with a nebulizer is not without difficulties and
several factors such as particle size, aerosol pressure and other factors may limit the
ability of proper delivery of solutions. Since an oscillating airflow improves the gas
exchange in the paranasal sinuses we wanted to test if it could also enhance the
deposition of an aerosol into these cavities.
In healthy subjects the NOS inhibitor L-NAME was administrated into the nostrils by a
jet nebulizer connected to a duck pipe, which could be manipulated to generate either
an oscillating or silent airflow. The degree of penetration of L-NAME into the sinuses
was estimated by calculating the reduction in nasal NO during humming exhalations
performed after nebulization.
Sinus drug deposition was also studied in a model of the nose and sinus, in which
changes in sinus volume, ostium size and sound frequency was evaluated. The
calculated resonance frequency of the model was 200 Hz.
Fig. 8. Mean value (horizontal bar) and 95% confidence interval (box) of intra-subject
coefficient of variation of nasal nitric oxide output measurements taken during standard quiet
nasal exhalation before and after five consecutive humming manoeuvres. P< 0·05. NS = not
significant.
*

Mauro Maniscalco
28
the weight of the syringe (sinus) as compared to the
rflow (4.4 ± 0.3 mg and 1.0 ± 0.1 mg, respectively, p <
mproves sinus deposition during
eight of the syringe when the aerosol was delivered by
e, a significant change in aerosol deposition was
by modifying the sound frequency. The maximum deposition was found at a
frequency of 200 Hz as compared to 120 Hz and 450 Hz (4.4 ± 0.3 mg, 3.5 ± 0.2 mg
nd 3.0 ± 0.3 mg, respectively; p < 0.05) . No difference in aerosol deposition was
noted when syringe volume was modified, regardless of mode of delivery.
In humans the nasal delivery of L-NAME with a silent airflow had no effect on the NO
levels during humming (from 201.4 ± 75.3 ppb to 181.0 ± 82.3 ppb; p= 0.67). In
contrast, the delivery of L-NAME with an oscillating airflow caused a significant
reduction in nasal NO during
humming (from 225.6 ± 61.5
ppb to 147.5 ± 40.1 ppb;
p<0.05). The effect on
humming-induced nasal NO
release lasted at least 40
minutes (Figure 9). The two
different modes of L-NAME
delivery reduced silently
exhaled NO from the nose to a
similar degree. Control
experiments with saline
showed no effect on nasal NO
levels. These findings suggest
that nebulization with an
oscillating airflow may
enhance the delivery of a drug
into the sinuses.
In the model of the nose and
caused a significant increase in
delivery without an oscillating ai
0.05). This further supports that oscillation i
nebulization.
Ostial diameter influenced the w
an oscillating airflow. Furthermor
observed
hu
sinus the aerosol delivery with an oscillating airflow
a
Fig. 9. Exhaled nasal nitric oxide measured during
mming after topical aerosolized L-NAME
delivered
with (filled squares) or
without (unfilled squared) an
oscillating airflow.

Discussion
29
GENERAL DISCUSSION
Measurements of exhaled NO are now becoming a part of clinical practice in the
diagnosis and therapy monitoring of allergic asthma
82-86
. This simple non-invasive test
reveals the degree of lower airway inflammation and can help the physician when
titrating anti-inflammatory treatment, monitoring compliance or in differential
diagnosis
87-89
. There has been hope that also nasal NO measurements could be similarly
useful e.g. in monitoring allergic rhinitis but to date the results have been somewhat
disappointing mainly due to methodological issues.
In this thesis we have explored an entirely new approach to nasal NO measurements -
the nasal humming test. These studies hopefully open up new opportunities to use nasal
NO measurements in diagnosis and therapy monitoring of upper airway inflammatory
disease. In addition, therapeutic opportunities using humming can also be foreseen.
Below, our specific findings are discussed in greater detail. I have divided this
discussion into three major areas: first a section on the mechanisms behind the increase
in nasal NO during humming, thereafter a section on the possible diagnostic application
of a humming test and finally a more speculative section on the possible therapeutical
application of humming.
SOURCE OF THE NITRIC OXIDE MEASURED DURING HUMMING
The great peak in nasal NO is seen only during humming whereas a silent exhalation at
the same flow rate does not produce any increase in nasal NO. So clearly in some way
the sound waves are facilitating NO release from the airways. The source of this NO
must be within the nasal airways as the NO peak only occurs during nasal exhalations
with humming and not during oral exhalations. Where then is the NO coming from, and
what is the mechanism of the release? In this thesis several proofs have been given to
support that the nasal peak in NO during humming is a result of a rapid washout of NO
from the sinuses. First, the profiles of the nasal exhalation curves (peak and progressive
decline) in the model (paper I) and in the human in vivo studies (paper I to IV) were
almost identical and showed characteristic patterns. In addition the NO release during
humming was dependent on the same prime parameter (ostial size). A rapid increase
in NO occurs when a subject starts humming, and after the
initial maximum, NO
starts to decrease. Naturally, in the model NO reaches zero when the syringe is
emptied while in the in vivo studies this does not happen because of the continuously
ongoing production of NO. Second, after removal of the accumulated NO from the
paranasal sinuses during humming, the
concentration is gradually rebuilt again. The
longer time allowed between two humming manoeuvres, the higher the
NO peak in
nasal NO. Indeed, both the peak and the total nasal NO output were markedly
decreased following repeated consecutive humming manoeuvres and a complete
recovery was observed after a 3-min period of silence. This pattern fits well with the
notion that humming rapidly empties the sinuses and that a period of silence will
allow for NO to accumulate again. Third, topical nasal administration of a NOS
inhibitor (L-NAME) did not affect nasal NO during humming, while it significantly
decreases silent nasal NO. This suggests that the great NO amount released during
humming is likely to come from the sinuses which were not targeted by the aerosol
administration.
In recent studies other authors have made very similar observations on humming and
nasal NO using slightly different methods
90, 91
.
Again, a rapidly increasing NO

Mauro Maniscalco
30
concentration was found when the
subjects started humming. After reaching a
maximum, the emission
started to decrease with the shape of an exponential decay
and
finally reached a constant level. The time constant of this
decay (NO washout)
was 3 sec. The peak height of
the NO emission during humming increases when the
time between
two humming processes increases. When no humming-induced NO
emission
takes place, the NO concentration in the sinuses re-build again.
HOW DOES HUMMING INCREASE SINUS VENTILATION?
In our study significant changes were found in NO output by modifying the frequency
of humming. In particular when studying the effect of different humming frequencies in
the nasal/sinus model, it was found that the NO output was greatest when the frequency
of humming was close to the calculated resonance frequency of the particular sinus
(Figure 5). The
influence of frequency was also analyzed in human
subjects by
modulating the sound frequencies of humming and
measuring NO output.
The results
confirm a dependence on the frequency that
varied considerably between subjects.
It is likely that the sinus cavity behaves like a kind of Helmholtz
resonator
92
, which is
a container of gas with an open hole (neck or port). Such a container has a specific
resonance frequency. At that frequency the air within the neck (airplug) will be most
easily moved. An outside variation in air pressure causes the airplug in the neck to
oscillate in and out. This oscillation is explained by the following: When air is pushed
into a cavity through the neck (ostium) it will be compressed, its pressure will increase
and it will tend to expand back to its original volume, thereby driving the air back out
of the cavity. The momentum of this driving force will create an overshoot and produce
a slight vacuum in the cavity which will then suck air back in. The air will oscillate in
and out of the cavity for a few cycles at a natural frequency. It is comparable to a mass
on a spring, which is pressed down and then released (Figure 10). During humming the
oscillating sound will move the air in and out through the ostium, with the highest
efficacy near the resonance frequency of the particular sinus. The vibration of the
airplug will pump NO out of the cavity
resulting in the observed peak in nasal NO
seen during humming.
Several factors will determine the resonance
frequency of the system including the area
of the opening port, the cavity volume and
the length of the opening port. Very
recently, Granqvist et al.
93
developed a
physical model where airflow from a
pressure tank was modulated
at different
frequencies. This airstream was passed
through a tube
with a radial hole constituting
the neck of a Helmholtz
resonator which
contained NO. The NO content of
the air
streaming out of the tube was measured.
This
NO content varied when the location of
the resonator, its
air volume or the modulation
frequency of the airflow were
changed.
Fig. 10. During humming the sinus
cavity behaves as a kind of Helmholtz
resonator. The humming oscillations
induce movement back and forth of the
airplug in the neck of the container like
a mass on a spring. This movement
increases the gas exchange from the
container.

Discussion
31
The relevance of these three factors was also confirmed in a computer model of the
system. The computer model was surprisingly efficient in replicating data obtained
from the physical model of the nasal tract and maxillary sinuses.
It is clear from the present raw modelling of the nasal resonator system that the
increased NO ventilation that occurs during humming can be explained by the acoustic
flow in and out of the maxillary sinuses.
We cannot exclude that humming could increase NO output either from other sources
in the main nasal airways or with other mechanisms such as turbulence of flow or
vibrations of skull. However, oral
NO output does not increase during humming
indicating that pulsating sound waves do not increase
NO diffusion from NO-producing
respiratory epithelial surfaces
in general. Furthermore the application of a turbulent
flow in the model did not have any effect on NO.
FACTORS WHICH INFLUENCE NO LEVELS DURING HUMMING
In paper I we attempted to explore the factors that determine the rate of air exchange
between the sinuses and the nose. For this we created a sinus/nasal model in which we
could vary the studied parameters, e.g. ostium size, sinus volume, sinus NO
concentration and humming frequency. We found that all these factors influenced the
nasal NO release during the humming, but the ostium size was the most important. In
fact when doubling the ostium size the increase in NO washout was almost 10 fold.
This confirms earlier studies which have shown that ostium size is the most important
factor determining sinus ventilation. Aust and Drettner
94
were the first to measure sinus
ostial patency in vivo in man. They used an invasive technique to measure pressure
differences in the sinuses after injecting air and then calculated the resistance to outflow
via the natural sinus ostium
95
. More recently, Paulsson et al.
96
used a non-invasive
133-xenon washout technique to evaluate the ventilation of the sinuses in healthy
subjects and in patients with sinus disease. The washout halftimes were used as a
measure of the ventilation of the sinuses. They found that the ostial size was the most
important factor determining the washout of 133-xenon from the sinuses
97
. In polyposis
patients the washout times were significantly longer than in healthy subjects, and
became significantly shorter after sinus surgery
96
.
It should be noted that the model we used in our study does not mimic the continuous
NO production occurring normally in the human sinuses and the complex dynamics of
production and absorption from nasal airway mucosa. Moreover, the sinus ostium
diameter could not be directly measured in the healthy subjects. Nevertheless, the
experiments looking at remaining NO levels in the syringe after single-breath
exhalations indicate that humming is an enormously effective means of increasing sinus
ventilation. This is also supported by the in vivo experiments (paper I), where the rapid
decline in NO during humming indicated sinus gas exchanging.
HUMMING IMPROVES ACCURACY OF NASAL NO MEASUREMENTS
Attempts to standardize nasal NO measurements have been recently made
70
. However,
the possibility to detect increases in nasal NO output is limited by the high background
NO levels in the upper airways originating from several sources in the nose and
sinuses. As we have discussed above, a high NO flux from the sinuses could easily

Mauro Maniscalco
32
blunt slight alterations in NO originating from nasal mucosa. Accordingly, large intra
and inter-individual variation in nasal NO have been reported both in healthy people
and in subjects with rhinitis
54-57
. Interestingly, by introducing repeated humming
manoeuvres immediately before measurements the NO levels measured during silent
exhalation were reduced as was the intra-individual variation (paper IV). This is likely
explained by the rapid removal of NO accumulated in the sinuses thereby transiently
reducing sinus contribution to NO levels measured in nasally exhaled air. This is
interesting for several reasons. First, it gives support to the notion that sinus NO is
indeed continuously contributing to the levels of nasal NO. Secondly, it indicates that if
nasal NO is measured after repeated humming manoeuvres it may represent a more
reliable measure of the nasal mucosal NO output with less contribution from the
paranasal sinuses. Although we managed to decrease the variability in nasal NO by
humming prior to the measurements, it remains to explore if this procedure will
increase the diagnostic accuracy of nasal NO measurements.
A HUMMING TEST TO DIAGNOSE AND MONITOR SINUS DISEASE?
Acute or chronic inflammation of the paranasal sinuses and the adjacent nasal surface,
clinically termed rhino-sinusitis
98
is very common. It has been reported that sinusitis
has an incidence rate of 14% in USA, with an 18% increase over the past 11 years
99
.
Moreover, sinusitis is increasingly recognized as a common event occurring during
upper and lower allergic respiratory diseases such as allergic rhinitis and asthma
53, 100
.
Indeed, as many as 45-60% of patients with allergic rhinitis have radiological evidence
of sinus abnormalities and allergic rhinitis is considered a risk factor for sinusitis
81
. In
clinical practice, classic symptoms of blocked nose, rhinorrea, sneezing and itching are
commonly encountered either with rhinitis or sinusitis. This often requires further
objective testing such as nasal endoscopy and radiological investigation, which are time
and cost-consuming, thereby contributing to the high economic burden of allergic
rhinitis
100, 101
. A key pathogenic event in sinusitis is the impaired sinus ventilation
caused by obstruction of the osteo-meatal complex
101
. This area of the nose is difficult
to assess during routine clinical examination
102
.
We speculate that measures of nasal NO may represent a new approach to test
osteomeatal patency. In paper II we found a complete absence of the normal nasal NO
increase during humming in patients with bilateral nasal polyposis and CT-proven
occluded sinus ostia. Furthermore in paper III the humming peak was lost or markedly
reduced in allergic rhinitis patients with endoscopic signs suggestive of sinus
obstruction. It is likely that the absence of a nasal NO peak during humming is caused
by a partial or complete obstruction of the osteomeatal complex with reduced gas
exchange through the sinus ostium. Also, from the experimental model of the
nose/sinus in the paper I, the ostium size was shown to be the major factor determining
the NO increase seen during humming.
Interestingly, in the prospective study (paper III) in allergic rhinitis subjects there were
no significant differences in clinical nasal score between humming non-responders and
humming responders whereas the endoscopic score was significantly higher in the first
group. This indicates that a humming test could be used to reveal sub-clinical sinus
obstruction. If a humming test accurately reflects the degree of osteal function, the next
step would be to explore if this information has any clinical relevance. That is, will

Discussion
33
early interventions (e.g. with anti-inflammatory treatment) in certain risk patients
prevent later development of chronic sinus disease?
HUMMING TO TREAT RHINO-SINUSITIS?
As evident from this thesis, humming is an enormously
effective means of increasing
sinus ventilation. Previous works
have shown that the time needed to exchange all gas
in the sinuses
varies between 5 min up to 1 h
94, 96
, with much longer
time needed in
patients with sinus disorders
96
. The current
results indicate that almost the entire sinus
volume is exchanged
in one single exhalation if the subject is humming. Even when
using a small ostial diameter, humming was very effective at
increasing NO exchange
in the sinus model. This suggests that
humming could help to increase sinus ventilation
in patients
with sinusitis and partly obstructed ostia.
Whether this would
be beneficial in treatment or prevention of sinusitis remains
to be
studied. Nevertheless, it is interesting that medical,
as well as surgical treatment of
chronic sinusitis generally
aims to increase sinus ventilation, which is often impaired
in
this disorder.
In support of this, a case report was described very recently
103
proposing that strong
low pitch (130 Hz) humming was an effective means of treating chronic rhino-sinusitis
(CRS). In this report a subject hummed frequently for 4 days in an attempt to treat the
sinusitis. The morning after the first humming session, the subject awoke with a clear
nose and found himself breathing easily through his nose for the first time in over 1
month. By humming 60–120 times four times per day CRS symptoms were essentially
eliminated in 4 days.
Although the concept of using humming to prevent or treat sinusitis humming appears
attractive, evidence of its effectiveness is still lacking. A potential problem is in a
situation with completely blocked ostia when sinus ventilation obviously will remain
zero even during humming.
HUMMING TO ENHANCE DRUG PENETRATION INTO THE SINUSES?
The ability to deliver medications directly into sinuses could theoretically reduce the
need for systemic medications thereby reducing the risk for unwanted side effects
104
.
Several factors may influence how effectively a nebulizer will deliver solutions to the
target organ including the particle size, aerosol pressure and other factors
105, 106
. In spite
of the widespread use of aerosols in respiratory diseases
107-118
few studies have been
performed in order to assess aerosol deposition into the sinuses cavities
119-122
.
In paper V we have shown that the peak in nasal NO caused by humming is markedly
reduced if an aerosolized NOS inhibitor is delivered into the nose by the use of a
sounding airflow. In the control experiments, where the same NOS inhibitor was
delivered without the sounding airflow, the humming-induced increase in nasal NO
was unaltered. This suggests that the sounding airflow enhanced the penetration of the
NOS inhibitor into the NO producing cells in the sinuses. Furthermore, the aerosol
delivery with sounding airflow increased the aerosol deposition in a model of the
paranasal sinus, where the size of ostium and frequency of the sounding airflow
influenced the aerosol deposition.

Mauro Maniscalco
34
To our knowledge this is the first study, which gives a demonstration of the effect of
sound pulsated aerosol to enhance aerosol diffusion into sinuses both in vivo in humans
and in a model of a sinus. Kumazawa et al.
109
found that intermittent vocalisation
increased deposition rate of aerosol particles into the larynx of healthy subjects. Proof
of this concept for nasal cavities has arisen from several studies mainly performed in
artificial models and in animals or from deposition studies using radionuclide or more
recently from a plastinated model of the maxillary sinus
123
.

Conclusions
35
CONCLUSIONS
The overall conclusions of the present thesis are the following:
• Ventilation of the paranasal sinuses is greatly enhanced by humming
• The increased sinus ventilation is detected as a peak in exhaled nasal NO
• Ostium size is a major determinant of the NO peak seen during humming
• The humming peak NO is absent in patients with chronic sinusitis and
osteomeatal obstruction
• Endoscopic signs of sinus abnormalities are associated with reduced NO peaks
during humming in patients with allergic rhinitis
• Nasal NO measured immediately after repeated humming may represent a more
reliable measure of the nasal mucosal NO because contribution from the sinus
NO source is transiently minimized.
• Aerosol in combination with a sounding airflow may increase the delivery of a
drug into the paranasal sinuses.

Mauro Maniscalco
36

Acknowledgements
37
ACKNOWLEDGEMENTS
I wish express my sincere gratitude to all those who have helped and supported me
throughout these studies, and especially to:
Professor Jon Lundberg my brilliant supervisor, for giving me the opportunity to
work in your outstanding laboratory. For your help, generosity and friendship, but
overall for your great mind, your superb scientific guidance in combination with
fantasy and enthusiasm.
Professor Eddie Weitzberg my excellent co-supervisor for all your ideas, for your
creative and rigorous scientific guidance, for your constant support and enthusiasm
during my PhD period and for your friendship.
Professor Matteo Sofia, my co-supervisor and friend for your endless support. For
your excellent and creative scientific guidance, for your inspiring scientific and non
scientific discussions, for unfailingly being always present.
Professors Kjell Alving and Lars Gustafsson for your kindness and hospitality.
Professor Tim Higenbottam for introducing me to the fascinating field of nitric oxide
research.
Professor Luigi Carratù for introducing me into respiratory field and supporting me to
do research.
My co-workers Professor Johan Sundberg, Guglielmo de Laurentiis, Anna Stanziola
and Vincenzo Rossillo for the stimulating discussions and enjoyable collaboration.
Tanja and Peter for your friendship and for having introduced me in the Swedish
tradition and food and for your help during my staying in Sweden.
Former and present colleagues in the research group: Håkan Björne, Mirko Govoni,
Helena Marteus, Carina Nihlén, Jörgen Palm, Claudia Reinders, Margareta
Stensdotter, Daniel Törnberg, Emmelie Jansson for your support and hospitality.
My colleagues in the Hospital Santa Maria della Pieta: Anna Zedda and Stanislao
Faraone for your support and understanding.
My colleagues in the Monaldi Hospital: Mauro Mormile, Antonio Molino,
Alessandro Sanduzzi, Alessandro Vatrella, Antonello Ponticiello, Paolo Giacomelli
and Maria Luisa Bocchino for the stimulating discussions.
My mother and my father for your support, for always believing in me and
encouraging me, for your love.
My brothers Fabio and Rino, my sister Arabella and my parents in law for your
support and love.

Mauro Maniscalco
38
Finally I would like to thank Pia, my wife, for your endless love and for being the
person most important in my life. Roberta and Manuela our wonderful children for
the great serenity and happiness you bring to my life.
The studies were supported by the Swedish Research Council (Medicine) the Swedish
Heart-Lung Foundation, the Swedish Asthma and Allergy Association's Research
Foundation, the Swedish Knowledge Foundation, Karolinska Institutet and Aerocrine
AB. Conflict of interest: My supervisors Jon Lundberg and Eddie Weitzberg are co-
founders of Aerocrine AB, a company that develops equipment for measurements of
exhaled NO.

References
39
REFERENCES
1. Aust R, Falck B, Svanholm H. Studies of the gas exchange and pressure in the
maxillary sinuses in normal and infected humans. Rhinology 1979; 17:245-51.
2. Van Cauwenberge P, Sys L, De Belder T, Watelet JB. Anatomy and
physiology of the nose and the paranasal sinuses. Immunol Allergy Clin North
Am 2004; 24:1-17.
3. Jones N. The nose and paranasal sinuses physiology and anatomy. Adv Drug
Deliv Rev 2001; 51:5-19.
4. Cole P. Physiology of the nose and paranasal sinuses. Clin Rev Allergy
Immunol 1998; 16:25-54.
5. Aust R, Stierna P, Drettner B. Basic experimental studies of ostial patency and
local metabolic environment of the maxillary sinus. Acta Otolaryngol Suppl
1994; 515:7-10.
6. Alho OP. Nasal airflow, mucociliary clearance, and sinus functioning during
viral colds: effects of allergic rhinitis and susceptibility to recurrent sinusitis.
Am J Rhinol 2004; 18:349-55.
7. Fukami M, Stierna P, Veress B, Carlsoo B. Lysozyme and lactoferrin in
human maxillary sinus mucosa during chronic sinusitis. An
immunohistochemical study. Eur Arch Otorhinolaryngol 1993; 250:133-9.
8. Fokkens W, Lund V, Bachert C, Clement P, Helllings P, Holmstrom M, et al.
EAACI position paper on rhinosinusitis and nasal polyps executive summary.
Allergy 2005; 60:583-601.
9. Borland C, Higenbottam T. Nitric oxide yields of contemporary UK, US and
French cigarettes. Int J Epidemiol 1987; 16:31-4.
10. Pryor WA, Dooley MM, Church DF. Mechanisms of cigarette smoke toxicity:
the inactivation of human alpha-1-proteinase inhibitor by nitric oxide/isoprene
mixtures in air. Chem Biol Interact 1985; 54:171-83.
11. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the
relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288:373-
6.
12. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-
derived relaxing factor produced and released from artery and vein is nitric
oxide. Proc Natl Acad Sci U S A 1987; 84:9265-9.
13. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the
biological activity of endothelium-derived relaxing factor. Nature 1987;
327:524-6.
14. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol Rev 1991; 43:109-42.
15. Coleman JW. Nitric oxide in immunity and inflammation. Int
Immunopharmacol 2001; 1:1397-406.
16. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S.
Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs
and humans. Biochem Biophys Res Commun 1991; 181:852-7.
17. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in
exhaled air of asthmatics. Eur Respir J 1993; 6:1368-70.
18. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell
1994; 78:915-8.

Mauro Maniscalco
40
19. Chung SJ, Fung HL. Identification of the subcellular site for nitroglycerin
metabolism to nitric oxide in bovine coronary smooth muscle cells. J
Pharmacol Exp Ther 1990; 253:614-9.
20. Nakane M, Schmidt HH, Pollock JS, Forstermann U, Murad F. Cloned human
brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett
1993; 316:175-80.
21. Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, et al.
Structure and chromosomal localization of the human constitutive endothelial
nitric oxide synthase gene. J Biol Chem 1993; 268:17478-88.
22. Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC,
et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of
inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A
1993; 90:522-6.
23. de las Heras B, Navarro A, Diaz-Guerra MJ, Bermejo P, Castrillo A, Bosca L,
et al. Inhibition of NOS-2 expression in macrophages through the inactivation
of NF-kappaB by andalusol. Br J Pharmacol 1999; 128:605-12.
24. Kuemmerle JF. Synergistic regulation of NOS II expression by IL-1 beta and
TNF-alpha in cultured rat colonic smooth muscle cells. Am J Physiol 1998;
274:G178-85.
25. Hibbs JB, Jr., Vavrin Z, Taintor RR. L-arginine is required for expression of
the activated macrophage effector mechanism causing selective metabolic
inhibition in target cells. J Immunol 1987; 138:550-65.
26. Joly GA, Ayres M, Chelly F, Kilbourn RG. Effects of NG-methyl-L-arginine,
NG-nitro-L-arginine, and aminoguanidine on constitutive and inducible nitric
oxide synthase in rat aorta. Biochem Biophys Res Commun 1994; 199:147-
54.
27. Knowles RG, Salter M, Brooks SL, Moncada S. Anti-inflammatory
glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in
the lung, liver and aorta of the rat. Biochem Biophys Res Commun 1990;
172:1042-8.
28. Lundberg JO, Weitzberg E, Nordvall SL, Kuylenstierna R, Lundberg JM,
Alving K. Primarily nasal origin of exhaled nitric oxide and absence in
Kartagener's syndrome. Eur Respir J 1994; 7:1501-4.
29. Lundberg JO, Weitzberg E. Nasal nitric oxide in man. Thorax 1999; 54:947-
52.
30. Lundberg JO, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggaard
A, et al. High nitric oxide production in human paranasal sinuses. Nat Med
1995; 1:370-3.
31. Lundberg JO, Weitzberg E, Rinder J, Rudehill A, Jansson O, Wiklund NP, et
al. Calcium-independent and steroid-resistant nitric oxide synthase activity in
human paranasal sinus mucosa. Eur Respir J 1996; 9:1344-7.
32. Haight JS, Djupesland PG, Qjan W, Chatkin JM, Furlott H, Irish J, et al. Does
nasal nitric oxide come from the sinuses? J Otolaryngol 1999; 28:197-204.
33. Kawamoto H, Takumida M, Takeno S, Watanabe H, Fukushima N, Yajin K.
Localization of nitric oxide synthase in human nasal mucosa with nasal
allergy. Acta Otolaryngol Suppl 1998; 539:65-70.
34. Kawamoto H, Takeno S, Yajin K. Increased expression of inducible nitric
oxide synthase in nasal epithelial cells in patients with allergic rhinitis.
Laryngoscope 1999; 109:2015-20.
35. Giannessi F, Fattori B, Ursino F, Giambelluca MA, Soldani P, Scavuzzo MC,
et al. Ultrastructural and ultracytochemical study of the human nasal

References
41
respiratory epithelium in vasomotor rhinitis. Acta Otolaryngol 2003; 123:943-
9.
36. Kudeken N, Kawakami K, Saito A. Different susceptibilities of yeasts and
conidia of Penicillium marneffei to nitric oxide (NO)-mediated fungicidal
activity of murine macrophages. Clin Exp Immunol 1998; 112:287-93.
37. Chen JH, Takeno S, Osada R, Ueda T, Yajin K. Modulation of ciliary activity
by tumor necrosis factor-alpha in cultured sinus epithelial cells. Possible roles
of nitric oxide. Hiroshima J Med Sci 2000; 49:49-55.
38. Runer T, Cervin A, Lindberg S, Uddman R. Nitric oxide is a regulator of
mucociliary activity in the upper respiratory tract. Otolaryngol Head Neck
Surg 1998; 119:278-87.
39. Lindberg S, Cervin A, Runer T. Low levels of nasal nitric oxide (NO)
correlate to impaired mucociliary function in the upper airways. Acta
Otolaryngol 1997; 117:728-34.
40. Runer T, Lindberg S. Ciliostimulatory effects mediated by nitric oxide. Acta
Otolaryngol 1999; 119:821-5.
41. Holden WE, Wilkins JP, Harris M, Milczuk HA, Giraud GD. Temperature
conditioning of nasal air: effects of vasoactive agents and involvement of
nitric oxide. J Appl Physiol 1999; 87:1260-5.
42. Silkoff PE, Roth Y, McClean P, Cole P, Chapnik J, Zamel N. Nasal nitric
oxide does not control basal nasal patency or acute congestion following
allergen challenge in allergic rhinitis. Ann Otol Rhinol Laryngol 1999;
108:368-72.
43. Maniscalco M, Sofia M, Faraone S, Carratu L. The effect of platelet-
activating factor (PAF) on nasal airway resistance in healthy subjects is not
mediated by nitric oxide. Allergy 2000; 55:757-61.
44. Gerlach H, Rossaint R, Pappert D, Knorr M, Falke KJ. Autoinhalation of
nitric oxide after endogenous synthesis in nasopharynx. Lancet 1994;
343:518-9.
45. Lundberg JO. Airborne nitric oxide: inflammatory marker and aerocrine
messenger in man. Acta Physiol Scand Suppl 1996; 633:1-27.
46. Howarth PH, Redington AE, Springall DR, Martin U, Bloom SR, Polak JM,
et al. Epithelially derived endothelin and nitric oxide in asthma. Int Arch
Allergy Immunol 1995; 107:228-30.
47. Wilson N. Measurement of airway inflammation in asthma. Curr Opin Pulm
Med 2002; 8:25-32.
48. Barnes PJ, Liew FY. Nitric oxide and asthmatic inflammation. Immunol
Today 1995; 16:128-30.
49. Donnelly LE, Barnes PJ. Expression and regulation of inducible nitric oxide
synthase from human primary airway epithelial cells. Am J Respir Cell Mol
Biol 2002; 26:144-51.
50. Ramis I, Lorente J, Rosello-Catafau J, Quesada P, Gelpi E, Bulbena O.
Differential activity of nitric oxide synthase in human nasal mucosa and
polyps. Eur Respir J 1996; 9:202-6.
51. Kang BH, Chen SS, Jou LS, Weng PK, Wang HW. Immunolocalization of
inducible nitric oxide synthase and 3-nitrotyrosine in the nasal mucosa of
patients with rhinitis. Eur Arch Otorhinolaryngol 2000; 257:242-6.
52. Maniscalco M, Ferrara G, Carratu L, Sofia M. Nitric oxide attenuates platelet-
activating factor induced nasal airway plasma extravasation in healthy
subjects. Eur J Clin Invest 2002; 32:858-61.

Mauro Maniscalco
42
53. Krouse JH. Allergy and chronic rhinosinusitis. Otolaryngol Clin North Am
2005; 38:1257-66.
54. Arnal JF, Didier A, Rami J, M'Rini C, Charlet JP, Serrano E, et al. Nasal nitric
oxide is increased in allergic rhinitis. Clin Exp Allergy 1997; 27:358-62.
55. Henriksen AH, Sue-Chu M, Holmen TL, Langhammer A, Bjermer L. Exhaled
and nasal NO levels in allergic rhinitis: relation to sensitization, pollen season
and bronchial hyperresponsiveness. Eur Respir J 1999; 13:301-6.
56. Kharitonov SA, Rajakulasingam K, O'Connor B, Durham SR, Barnes PJ.
Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and
may be modulated by nasal glucocorticoids. J Allergy Clin Immunol 1997;
99:58-64.
57. Martin U, Bryden K, Devoy M, Howarth P. Increased levels of exhaled nitric
oxide during nasal and oral breathing in subjects with seasonal rhinitis. J
Allergy Clin Immunol 1996; 97:768-72.
58. Sato M, Fukuyama N, Sakai M, Nakazawa H. Increased nitric oxide in nasal
lavage fluid and nitrotyrosine formation in nasal mucosa--indices for severe
perennial nasal allergy. Clin Exp Allergy 1998; 28:597-605.
59. Palm JP, Alving K, Lundberg JO. Characterization of airway nitric oxide in
allergic rhinitis: the effect of intranasal administration of L-NAME. Allergy
2003; 58:885-92.
60. Maniscalco M, Sofia M, Carratu L, Higenbottam T. Effect of nitric oxide
inhibition on nasal airway resistance after nasal allergen challenge in allergic
rhinitis. Eur J Clin Invest 2001; 31:462-6.
61. Parikh A, Scadding GK, Gray P, Belvisi MG, Mitchell JA. High levels of
nitric oxide synthase activity are associated with nasal polyp tissue from
aspirin-sensitive asthmatics. Acta Otolaryngol 2002; 122:302-5.
62. Colantonio D, Brouillette L, Parikh A, Scadding GK. Paradoxical low nasal
nitric oxide in nasal polyposis. Clin Exp Allergy 2002; 32:698-701.
63. Deja M, Busch T, Bachmann S, Riskowski K, Campean V, Wiedmann B, et
al. Reduced nitric oxide in sinus epithelium of patients with radiologic
maxillary sinusitis and sepsis. Am J Respir Crit Care Med 2003; 168:281-6.
64. Baraldi E, Azzolin NM, Biban P, Zacchello F. Effect of antibiotic therapy on
nasal nitric oxide concentration in children with acute sinusitis. Am J Respir
Crit Care Med 1997; 155:1680-3.
65. Noone PG, Leigh MW, Sannuti A, Minnix SL, Carson JL, Hazucha M, et al.
Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir
Crit Care Med 2004; 169:459-67.
66. Bush A, O'Callaghan C. Primary ciliary dyskinesia. Arch Dis Child 2002;
87:363-5.
67. Bush A. Primary ciliary dyskinesia. Acta Otorhinolaryngol Belg 2000;
54:317-24.
68. Karadag B, James AJ, Gultekin E, Wilson NM, Bush A. Nasal and lower
airway level of nitric oxide in children with primary ciliary dyskinesia. Eur
Respir J 1999; 13:1402-5.
69. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide
measurements: recommendations. The European Respiratory Society Task
Force. Eur Respir J 1997; 10:1683-93.
70. ATS/ERS Recommendations for Standardized Procedures for the Online and
Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal
Nitric Oxide, 2005. Am J Respir Crit Care Med 2005; 171:912-30.

References
43
71. ATS Workshop Proceedings: Exhaled Nitric Oxide and Nitric Oxide
Oxidative Metabolism in Exhaled Breath Condensate: Executive Summary.
Am J Respir Crit Care Med 2006; 173:811-3.
72. Qian W, Djupesland PG, Chatkin JM, McClean P, Furlott H, Chapnik JS, et
al. Aspiration flow optimized for nasal nitric oxide measurement. Rhinology
1999; 37:61-5.
73. Daya H, Qian W, McClean P, Haight J, Zamel N, Papsin BC, et al. Nasal
nitric oxide in children: a novel measurement technique and normal values.
Laryngoscope 2002; 112:1831-5.
74. Struben VM, Wieringa MH, Mantingh CJ, Bommelje C, Don M, Feenstra L,
et al. Nasal NO: normal values in children age 6 through to 17 years. Eur
Respir J 2005; 26:453-7.
75. Kharitonov SA, Barnes PJ. Nasal contribution to exhaled nitric oxide during
exhalation against resistance or during breath holding. Thorax 1997; 52:540-4.
76. Tornberg DC, Marteus H, Schedin U, Alving K, Lundberg JO, Weitzberg E.
Nasal and oral contribution to inhaled and exhaled nitric oxide: a study in
tracheotomized patients. Eur Respir J 2002; 19:859-64.
77. Palm JP, Graf P, Lundberg JO, Alving K. Characterization of exhaled nitric
oxide: introducing a new reproducible method for nasal nitric oxide
measurements. Eur Respir J 2000; 16:236-41.
78. Djupesland PG, Chatkin JM, Qian W, Cole P, Zamel N, McClean P, et al.
Aerodynamic influences on nasal nitric oxide output measurements. Acta
Otolaryngol 1999; 119:479-85.
79. Weitzberg E, Lundberg JO. Humming greatly increases nasal nitric oxide. Am
J Respir Crit Care Med 2002; 166:144-5.
80. Bland JM, Altman DG. Statistical methods for assessing agreement between
two methods of clinical measurement. Lancet 1986; 1:307-10.
81. Kaliner M. Medical management of sinusitis. Am J Med Sci 1998; 316:21-8.
82. Ricciardolo FL, Sterk PJ, Gaston B, Folkerts G. Nitric oxide in health and
disease of the respiratory system. Physiol Rev 2004; 84:731-65.
83. Colice GL. Categorizing asthma severity and monitoring control of chronic
asthma. Curr Opin Pulm Med 2002; 8:4-8.
84. Bates CA, Silkoff PE. Exhaled nitric oxide in asthma: from bench to bedside.
J Allergy Clin Immunol 2003; 111:256-62.
85. Ashutosh K. Nitric oxide and asthma: a review. Curr Opin Pulm Med 2000;
6:21-5.
86. Kharitonov SA. Exhaled markers of inflammatory lung diseases: ready for
routine monitoring? Swiss Med Wkly 2004; 134:175-92.
87. de Jongste JC. Surrogate markers of airway inflammation: inflammometry in
paediatric respiratory medicine. Paediatr Respir Rev 2000; 1:354-60.
88. Zitt M. Clinical applications of exhaled nitric oxide for the diagnosis and
management of asthma: A consensus report. Clin Ther 2005; 27:1238-50.
89. Dinakar C. Exhaled nitric oxide in the clinical management of asthma. Curr
Allergy Asthma Rep 2004; 4:454-9.
90. Struben VM, Wieringa MH, Mantingh CJ, Bruinsma SM, de Jongste JC,
Feenstra L. Silent and humming nasal NO measurements in adults aged 18-70
years. Eur J Clin Invest 2005; 35:653-7.
91. Menzel L, Hess A, Bloch W, Michel O, Schuster KD, Gabler R, et al.
Temporal nitric oxide dynamics in the paranasal sinuses during humming. J
Appl Physiol 2005; 98:2064-71.

Mauro Maniscalco
44
92. Dang J, Honda K. Acoustic characteristics of the human paranasal sinuses
derived from transmission characteristic measurement and morphological
observation. J Acoust Soc Am 1996; 100:3374-83.
93. Granqvist S. SJ, Lundberg J.O., Weitzberg E. Acoustic modeling of NO gas
evacuation from the maxillary sinuses. JASA 2006; in press.
94. Aust R, Drettner B. Experimental studies of the gas exchange through the
ostium of the maxillary sinus. Ups J Med Sci 1974; 79:177-86.
95. Aust R, Drettner B, Falck B. Studies of the effect of peroral
fenylpropanolamin on the functional size of the human maxillary ostium. Acta
Otolaryngol 1979; 88:455-8.
96. Paulsson B, Dolata J, Larsson I, Ohlin P, Lindberg S. Paranasal sinus
ventilation in healthy subjects and in patients with sinus disease evaluated
with the 133-xenon washout technique. Ann Otol Rhinol Laryngol 2001;
110:667-74.
97. Paulsson B, Dolata J, Lindberg S, Ohlin P. Factors influencing 133-xenon
washout in a nose-sinus model. Clin Physiol 2001; 21:246-52.
98. Lanza DC, Kennedy DW. Adult rhinosinusitis defined. Otolaryngol Head
Neck Surg 1997; 117:S1-7.
99. Kaliner MA, Osguthorpe JD, Fireman P, Anon J, Georgitis J, Davis ML, et al.
Sinusitis: bench to bedside. Current findings, future directions. Otolaryngol
Head Neck Surg 1997; 116:S1-20.
100. Ray NF, Baraniuk JN, Thamer M, Rinehart CS, Gergen PJ, Kaliner M, et al.
Healthcare expenditures for sinusitis in 1996: contributions of asthma, rhinitis,
and other airway disorders. J Allergy Clin Immunol 1999; 103:408-14.
101. Wagenmann M, Naclerio RM. Anatomic and physiologic considerations in
sinusitis. J Allergy Clin Immunol 1992; 90:419-23.
102. Devaiah AK. Adult chronic rhinosinusitis: diagnosis and dilemmas.
Otolaryngol Clin North Am 2004; 37:243-52.
103. Eby GA. Strong humming for one hour daily to terminate chronic
rhinosinusitis in four days: A case report and hypothesis for action by
stimulation of endogenous nasal nitric oxide production. Med Hypotheses
2006; 66:851-4.
104. Desrosiers MY, Salas-Prato M. Treatment of chronic rhinosinusitis refractory
to other treatments with topical antibiotic therapy delivered by means of a
large-particle nebulizer: results of a controlled trial. Otolaryngol Head Neck
Surg 2001; 125:265-9.
105. Hollie MC, Malone RA, Skufca RM, Nelson HS. Extreme variability in
aerosol output of the DeVilbiss 646 jet nebulizer. Chest 1991; 100:1339-44.
106. Hess D, Fisher D, Williams P, Pooler S, Kacmarek RM. Medication nebulizer
performance. Effects of diluent volume, nebulizer flow, and nebulizer brand.
Chest 1996; 110:498-505.
107. Christoforidis AJ, Tomashefski JF, Mitchell RI. Use of an ultrasonic nebulizer
for the application of oropharyngeal, laryngeal and tracheobronchial
anesthesia. Chest 1971; 59:629-33.
108. Mitchell DM, Solomon MA, Tolfree SE, Short M, Spiro SG. Effect of particle
size of bronchodilator aerosols on lung distribution and pulmonary function in
patients with chronic asthma. Thorax 1987; 42:457-61.
109. Kumazawa H, Asako M, Yamashita T, Ha-Kawa SK. An increase in laryngeal
aerosol deposition by ultrasonic nebulizer therapy with intermittent
vocalization. Laryngoscope 1997; 107:671-4.

References
45
110. Fuloria M, Rubin BK. Evaluating the efficacy of mucoactive aerosol therapy.
Respir Care 2000; 45:868-73.
111. Fink JB, Rau JL. New horizons in respiratory care: the pharmacology of
inhaled aerosol drug therapy. Respir Care 2000; 45:824-5.
112. Roche N, Huchon GJ. Rationale for the choice of an aerosol delivery system.
J Aerosol Med 2000; 13:393-404.
113. Groneberg DA, Witt C, Wagner U, Chung KF, Fischer A. Fundamentals of
pulmonary drug delivery. Respir Med 2003; 97:382-7.
114. Everard ML. Inhalation therapy for infants. Adv Drug Deliv Rev 2003;
55:869-78.
115. Fiel SB. Aerosol delivery of antibiotics to the lower airways of patients with
cystic fibrosis. Chest 1995; 107:61S-4S.
116. Mathur RS, Shah AA, Shah JR. Aerosol therapy in respiratory diseases. J
Assoc Physicians India 1991; 39:705-8.
117. Summers QA. Inhaled drugs and the lung. Clin Exp Allergy 1991; 21:259-68.
118. Huchon G. Aerosol deposition in the alveolar space. Lung 1990; 168
Suppl:672-6.
119. Negley JE, Krause H, Pawar S, Reeves-Hoche MK. RinoFlow nasal wash and
sinus system as a mechanism to deliver medications to the paranasal sinuses:
results of a radiolabeled pilot study. Ear Nose Throat J 1999; 78:550-2, 3-4.
120. Senocak D, Senocak M, Bozan S. Sinonasal distribution of topically applied
particles: computerized tomographic detection and the effects of topical
decongestion. Otolaryngol Head Neck Surg 2005; 133:944-8.
121. van Wijngaarden HA, Fokkens JK. The principle of the 'aerosol sonique'.
ORL J Otorhinolaryngol Relat Spec 1973; 35:111-6.
122. Saijo R, Majima Y, Hyo N, Takano H. Particle deposition of therapeutic
aerosols in the nose and paranasal sinuses after transnasal sinus surgery: a cast
model study. Am J Rhinol 2004; 18:1-7.
123. Durand M, Rusch P, Granjon D, Chantrel G, Prades JM, Dubois F, et al.
Preliminary study of the deposition of aerosol in the maxillary sinuses using a
plastinated model. J Aerosol Med 2001; 14:83-93.