Academic Year 2013 - 2014

Measurement of local IgE in allergic rhinitis and chronic rhinosinusitis with and without

nasal polyposis

Sander De Bruyne

Eveline Van Mulders

Promotor: Prof. Dr. Philippe Gevaert

Mentor: Dr. Margot Berings

Dissertation presented in the 2nd Master year in the programme of

Master of Medicine in Medicine

FACULTY OF MEDICINE AND

HEALTH SCIENCES  

  II  

“The author and the promotor give the permission to use this thesis for consultation

and to copy parts of it for personal use. Every other use is subject to the copyright

laws, more specifically the source must be extensively specified when using results

from this thesis.”

14/04/2014

Sander De Bruyne Prof. Dr. Philippe Gevaert Eveline Van Mulders

  III  

FOREWORD

This thesis was written within the context of our Master degree in Medicine at Ghent

University. It is a composition of statistical analysis on datasets acquired at the

Department of Otorhinolaryngology of the University Hospital of Ghent and data we

collected in our own experiment.

We compare nasal measurement with different techniques and we discuss their role.

Literature research started in September 2012 and was continued until February 2014.

Statistical analysis was performed from June 2013 till March 2014.

In addition, experiments occurred from November 2013 until March 2014. Later on,

we performed the necessary statistical analyses in order to interpret the collected data.

We wish to thank those without whose help this work could not have been

accomplished: (1) the patients who participated in the experiments; (2) Prof. Dr.

Philippe Gevaert and Dr. Margot Berings for their guidance, assistance and

encouragements; last but not least our friends and family who supported us

throughout the course of this assignment.

Sander De Bruyne

Eveline Van Mulders

April 2014

  IV  

LIST OF ABBREVIATIONS

AR = Allergic rhinitis ARIA= Allergic Rhinitis and its Impact on Asthma BDL= Below detection level CRS= Chronic rhinosinusitis CRSsNP= Chronic rhinosinusitis without nasal polyposis CRSwNP= Chronic rhinosinusitis with nasal polyposis CSR= Class switch recombination CUAI= Chronic upper airway inflammation DF= Dilution factor EPOS= European Position paper on Rhinosinusitis and Nasal polyps ESS= Endoscopic sinus surgery FLC= Free light chains Gx3 IgE= Grass pollen specific IgE HDM= House dust mite IQR= Interquartile range NAPT= Nasal provocation test NCA= Neutrophil chemotactic activity NP= Nasal polyposis NSA= Nasal secretory activity RR= Receptor revision RSV= Respiratory syncytial virus SAE= Staphylococcus aureus enterotoxin SPA= Staphylococcal protein A SPT= Skin prick test RAST= Radio-Allergo-Sorbent-Test TSST= Toxic shock syndrome toxin VAS= Visual analogue scale WNS= Weight of nasal secretions collected

  V  

LIST OF FIGURES

Figure 1: SPT procedures. ............................................................................................ 18  

Figure 2: Material and venipuncture. ........................................................................... 19  

Figure 3: Material for sampling of nasal secretions. .................................................... 19  

Figure 4: Sampling of nasal secretions. ....................................................................... 20  

Figure 5: Processing of nasal secretions. ..................................................................... 21  

Figure 6: Correlations between tissue, serum and Merocel nasal secretions. .............. 23

Figure 7: Comparison of the change in total IgE concentrations in placebo and

omalizumab treated groups in nasal secretions collected with Merocels...…………..24

Figure 8: Comparison of change in total IgE (A) and SAE-IgE concentrations (B) in

placebo and mepolizumab treated groups in nasal secretions collected with

Merocels. .............................................................................................................. 25  

Figure 9: Total IgE in nasal secretions after treatment with methylprednisolone (solid

triangles), doxycycline (solid squares) or placebo (solid circles) in nasal

secretions collected with Merocels. ..................................................................... 26  

Figure 10: Comparison of inflammatory measurements in nasal secretions collected

with filter discs. .................................................................................................... 29  

Figure 11: Comparison of total IgE and HDM sIgE concentrations in serum (E and F)

and nasal secretions collected with Merocels (A and B) and filter discs (C and D)

in control patients and AR patients. ..................................................................... 31  

Figure 12: Correlations between HDM sIgE concentrations in serum and in nasal

secretions collected with Merocels and filter discs.. ............................................ 32  

  VI  

LIST OF TABLES

Table 1: Descriptives of total IgE measurement by means of Merocel, tissue and

serum .................................................................................................................... 24  

Table 2: Descriptives of total IgE measurements at baseline and after treatment with

placebo or omalizumab in nasal secretions collected with Merocels. ................. 24  

Table 3: Descriptives of total IgE and SAE-IgE measurements at baseline and after

placebo or mepolizumab treatment in nasal secretions collected with Merocels 25  

Table 4: Descriptives of total IgE change from baseline in placebo, doxycycline and

methylprednisolone treated groups in nasal secretions collected with Merocels 26  

Table 5: Inflammatory measurements in nasal secretions collected with filter discs in

control patients, grass pollen sensitive AR patients, CRSwNP patients and

CRSwNP with grass pollen allergy patients. ....................................................... 28  

Table 6: Baseline demographic and clinical characteristics of control patients and

HDM sensitive AR patients. ................................................................................ 29  

Table 7: Total IgE and HDM sIgE in nasal secretions (Merocel and filter disc) and

serum in control patients and HDM sensitive AR patients. ................................. 30  

Table 8: Total IgE and HDM sIgE measurements in HDM sensitive AR patients in

serum and nasal secretions (filter disc and Merocel). .......................................... 33  

Table 9: Summary strengths and weaknesses tissue, Merocels and filter discs……...38  

LIST OF APPENDICES

Appendix 1: Overview of the ‘superantigen hypothesis’ of CRS. ................................ X  

Appendix 2: Management scheme for adults with CRS with NP for ENT specialists

based on the EPOS guidelines. ............................................................................. X  

Appendix 3: Position of a Merocel inside the nasal cavity between the inferior

turbinate and the septum. .................................................................................... XI  

Appendix 4: Classification of allergic rhinitis according to ARIA ............................ XI  

  VII  

TABLE OF CONTENTS

FOREWORD………………………………………………………………….…... III

LIST OF ABBREVATIONS………………………………………………...……. IV

LIST OF FIGURES…………………………………………………………………V

LIST OF TABLES………………………………………………………………….VI

LIST OF APPENDICES…………………………………………………………...VI

1 ABSTRACT ........................................................................................................... 1

2 INTRODUCTION ................................................................................................. 5

2.1 Chronic upper airway inflammation ........................................................................ 5 2.1.1 Allergic rhinitis ..................................................................................................... 6

2.1.1.1 Classification ............................................................................................................... 6 2.1.1.2 Epidemiology .............................................................................................................. 6 2.1.1.3 Comorbidity ................................................................................................................ 6

2.1.1.3.1 Asthma ................................................................................................................ 6 2.1.1.3.2 Allergy ................................................................................................................. 6 2.1.1.3.3 Chronic rhinosinusitis ......................................................................................... 7

2.1.1.4 Diagnosis ..................................................................................................................... 7 2.1.1.5 Pathophysiology .......................................................................................................... 7

2.1.1.5.1 IgE-dependent mechanisms ................................................................................ 7 2.1.1.5.2 Role of IgE and the inflammatory infiltrate ........................................................ 8

2.1.1.6 Treatment .................................................................................................................... 8 2.1.2 Chronic rhinosinusitis with nasal polyposis .......................................................... 9

2.1.2.1 Epidemiology .............................................................................................................. 9 2.1.2.2 Diagnosis ..................................................................................................................... 9

2.1.2.2.1 Symptoms ............................................................................................................ 9 2.1.2.2.2 Examination ........................................................................................................ 9

2.1.2.2.2.1 Anterior rhinoscopy and nasal endoscopy ................................................... 9 2.1.2.2.2.2 Imaging ........................................................................................................ 9

2.1.2.3 Pathophysiology ........................................................................................................ 10 2.1.2.3.1 Nasal polyps ...................................................................................................... 10 2.1.2.3.2 Local IgE ........................................................................................................... 10 2.1.2.3.3 Inflammatory triggers ........................................................................................ 10 2.1.2.3.4 Local receptor revision and class switch recombination ................................... 11

2.1.2.4 Comorbidity .............................................................................................................. 11 2.1.2.4.1 Allergy ............................................................................................................... 11 2.1.2.4.2 Asthma .............................................................................................................. 12

  VIII  

2.1.2.5 Medical treatment options ......................................................................................... 12 2.1.2.5.1 Corticosteroids .................................................................................................. 12 2.1.2.5.2 Antibiotics ......................................................................................................... 12 2.1.2.5.3 Anti-IgE ............................................................................................................. 12 2.1.2.5.4 Anti-IL-5 ........................................................................................................... 13

2.2 Measurement of local IgE ........................................................................................ 13 2.2.1 Merocels .............................................................................................................. 14 2.2.2 Filter discs ........................................................................................................... 14 2.2.3 Tissue .................................................................................................................. 15

2.3 Aims of the study ...................................................................................................... 15

3 MATERIALS AND METHODS ........................................................................ 16

3.1 Analyzed datasets ..................................................................................................... 16 3.2 Experiment ............................................................................................................... 17

3.2.1 Patient recruitment .............................................................................................. 17 3.2.2 Skin prick testing ................................................................................................ 17 3.2.3 Blood testing ....................................................................................................... 18 3.2.4 Procedure for collection of nasal secretions ........................................................ 19

3.2.4.1 Sampling of nasal secretions ..................................................................................... 19 3.2.4.2 Processing of nasal secretions ................................................................................... 20

3.3 Literature .................................................................................................................. 21 3.4 Statistical analysis .................................................................................................... 22

4 RESULTS ............................................................................................................. 23

4.1 Correlations between Merocel, tissue and serum total IgE measurements ........ 23 4.2 Dynamic local IgE measurements by means of Merocels .................................... 24

4.2.1 Comparison omalizumab versus placebo ............................................................ 24 4.2.2 Comparison mepolizumab versus placebo .......................................................... 25 4.2.3 Comparison doxycycline, methylprednisolone and placebo ............................... 26

4.3 Local measurements by means of filter discs ........................................................ 27 4.4 Comparison of Merocels and filter discs for the measurement of local IgE

(experiment) ........................................................................................................................ 29 4.4.1 Demographic and clinical features ...................................................................... 29 4.4.2 Total IgE and HDM sIgE are increased locally in nasal secretions in AR patients

with AR ............................................................................................................................ 30

5 DISCUSSION ....................................................................................................... 34

5.1 Merocels for local IgE measurement ...................................................................... 34 5.2 Filter discs for local IgE measurement .................................................................. 35

  IX  

5.3 Comparison Merocels versus filter discs ............................................................... 36

6 REFERENCES .................................................................................................... 39

APPENDICES…………………………………………………………………...…..X

1    

1 ABSTRACT

Introduction: Local production of IgE in the respiratory mucosa has been

demonstrated in allergic rhinitis (allergen specific IgE) and in chronic rhinosinusitis

with nasal polyposis (polyclonal IgE), suggesting a role for local IgE in the

pathogenesis of these inflammatory diseases of the upper airway. It has been suggested

that the majority of allergen-specific IgE in the peripheral blood is not derived from

IgE-secreting cells in the blood, but is produced locally in target organs of allergy such

as the nasal mucosa. Considering the importance of local IgE production, it seems to

be essential to develop non-invasive, reproducible and easy to perform approaches for

measurement of local mucosal IgE production.

Methods: Different datasets with CRS patients (with and without nasal polyps) and

AR patients were analyzed to obtain more knowledge concerning local IgE and its

measurement techniques. Furthermore, a small experiment was performed: in AR

patients and non-allergic control patients, nasal secretions were collected by means of

filter discs and Merocels. The purpose of this experiment was to compare and optimize

both methods for collection of nasal secretions and measurement of local IgE.

Results: Measurement of IgE in serum, tissue homogenates and nasal secretions In a dataset of 112 patients with CRS (with and without NP) levels of total IgE in

serum, in tissue and in nasal secretions (collected with Merocels) were significantly

correlated (p<.0001).

Dynamic measurements of local IgE in nasal secretions collected by means of Merocels 24 patients with CRSwNP and comorbid asthma were treated with omalizumab or

placebo. With omalizumab treatment (n=16), a significant increase in total IgE in nasal

secretions was observed (p=.041). No significant change was found in the placebo

group (n=8). 30 patients with severe NP had a treatment with mepolizumab or placebo.

Mepolizumab treatment (n=20) did not result in a significant change in total IgE and

SAE-IgE in nasal secretions. In the placebo group (n=10) a significant increase in total

IgE (p=.028) and a non-significant increase in SAE-IgE were observed. 47 patients

with CRSwNP had a treatment with methylprednisolone, doxycycline or placebo. In

the placebo group (n=18), a significant change from baseline in total IgE was observed

at week 4 (p=.009). In the methylprednisolone group (n=14) an initial decrease in total

  2  

IgE was followed by an increase in total IgE, however, these changes were not

significant. In the doxycycline group (n=14) total IgE levels did not change during

treatment.

Local measurements in nasal secretions collected by means of filter discs Nasal secretions were collected by means of filter discs and different biomarkers of

inflammation were measured. The highest total IgE levels were seen in the AR patients

(n=12), followed by the allergic CRSwNP patients (n=12). Both in the AR and the

allergic CRSwNP group, total IgE levels were significantly higher (resp., p=.026 and

p=.017) compared to the control patients (n=12). In the AR patients and the allergic

CRSwNP patients, total IgE was BDL in resp. 3 and 2 subjects. In the CRSwNP

patients without allergy (n=12) and in the control patients, total IgE was BDL in resp.

7 and 8 subjects. Parallel with total IgE, gx3 IgE was the highest in the AR patients

followed by the allergic CRSwNP patients. Levels of Gx3 IgE were BDL in all of the

CRSwNP patients without allergy and the control patients. Gx3 IgE was significantly

higher in the AR patients in comparison with the control (p=.001) and the CRSwNP

patients without allergy (p=.001).

Comparison of Merocels and filter discs for the measurement of local IgE In nasal secretions collected with filter discs and with Merocels, total IgE and house

dust mite specific (HDM) IgE were significantly higher in AR patients (n=15)

compared to control patients (n=15). In the nasal secretions collected with Merocels

total IgE and HDM IgE were BDL in resp. 3 and 4 AR patients, with the filter discs in

resp. 2 and 6 AR patients. HDM sIgE levels in serum, in the nasal secretions collected

by means of Merocels and in nasal secretions collected by means of filter discs were

significantly correlated.

Conclusion: IgE levels in nasal secretions collected by means of Merocels seem to be

a good reflection of local IgE levels in nasal tissue. As collection of nasal secretions by

Merocels is a standardized and non-invasive method, it can be used for repeated

measurements of local IgE and other markers of mucosal inflammation, in order to

study nasal diseases and monitor their treatment. The use of filter discs seems to be a

feasible alternative for collection of nasal secretions and measurement of local IgE.

3    

Inleiding: In allergische rhinitis (allergeen-specifiek IgE) en in chronische

rhinosinusitis met neuspoliepen (polyklonaal IgE) is lokale IgE productie in de

respiratoire mucosa aangetoond. Dit veronderstelt een rol voor lokaal IgE in de

pathogenese van deze inflammatoire aandoeningen van de bovenste luchtwegen. Er is

gesuggereerd dat de meerderheid van allergeen-specifiek IgE in perifeer bloed niet

afgeleid is van IgE-secreterende cellen in het bloed, maar afkomstig is van lokale

productie in doelorganen van allergie zoals het neusslijmvlies. Gezien het belang van

lokale IgE productie lijkt het essentieel om niet-invasieve, reproduceerbare en

eenvoudige benaderingen te ontwikkelen waarmee lokale IgE productie kan gemeten

worden.

Methodologie: Verschillende datasets van CRS patiënten (met en zonder

neuspoliepen) en AR patiënten werden geanalyseerd om meer inzicht te verwerven in

lokaal IgE en zijn meettechnieken. Verder werd ook een klein experiment uitgevoerd:

bij AR patiënten en niet-allergische controlepatiënten werden neussecreties verzameld

door middel van filter discs en Merocels. Het doel was om beide methodes met elkaar

te vergelijken en te optimaliseren voor de collectie van neussecreties en lokale IgE

meting.

Resultaten: Meting van IgE in serum, weefselhomogenaten en nasale secreties In een dataset van 112 patiënten met CRS (met en zonder NP) was er een significante

correlatie (p<.0001) tussen de concentraties van totaal IgE in serum, in weefsel en

nasale secreties (verzameld met Merocels).

Dynamische metingen van lokaal IgE in neussecreties verzameld met Merocels 24 patiënten met CRSwNP en comorbide astma ondergingen een omalizumab of een

placebo behandeling. Bij de behandeling met omalizumab (n=16) werd een

aanzienlijke toename van totaal IgE in nasale secreties waargenomen (p=.041). Geen

significante wijzigingen werden gevonden in de placebo groep (n=8). 30 patiënten met

ernstige NP ondergingen een behandeling met mepolizumab of placebo. Mepolizumab

behandeling (n=20) resulteerde niet in een significante verandering in totaal IgE en

SAE-IgE in nasale secreties. In de placebo groep (n=10) werd er een significante

stijging van totaal IgE (p=.028) en een niet significante toename in SAE-IgE

waargenomen. 47 patiënten met CRSwNP ondergingen een behandeling met

methylprednisolone, doxcycline of placebo. In de placebo groep (n=18) werd een

  4  

significante verandering in totaal IgE ten opzichte van de baseline waargenomen in

week 4 (p=.009). In de methylprednisolone groep (n=14) werd aanvankelijk een

afname van het totaal IgE gezien, gevolgd door een toename, maar dit was niet

significant. In de doxycyclinegroep (n=14) veranderden de totale IgE niveaus niet

tijdens de behandeling .

Lokale metingen in neussecreties verzameld door middel van filter discs Neussecreties werden verzameld door middel van filter discs en verschillende

ontstekingsparameters werden gemeten. De hoogste totale IgE niveaus werden

waargenomen bij AR patiënten (n=12), gevolgd door CRSwNP allergische patiënten

(n=12). Zowel in de AR groep als in de allergische CRSwNP groep waren de totale

IgE concentraties significant hoger (resp. p=.026 en p=.017) vergeleken met

controlepatiënten (n=12). In de AR groep en de allergische CRSwNP groep was totaal

IgE onder de detectielimiet (BDL) in resp. 3 en 2 patiënten. In CRSwNP patiënten

zonder allergie (n=12) en de controlepatiënten was totaal IgE BDL in resp. 7 en 8

patiënten . Parallel aan totaal IgE was Gx3 IgE het hoogst bij de AR patiënten, gevolgd

door de allergische CRSwNP patiënten. Gx3 IgE concentraties waren BDL bij alle

CRSwNP patiënten zonder allergie en bij alle controlepatiënten. Gx3 IgE was

significant hoger bij de AR patiënten in vergelijking met de controlegroep (p=.001) en

de CRSwNP patiënten zonder allergie (p=.001).

Vergelijking tussen Merocels en filter discs voor het meten van lokaal IgE In neussecreties verzameld door middel van filter discs en Merocels waren totaal IgE

en huisstofmijt specifiek IgE (HDM sIgE) significant hoger bij AR patiënten (n=15) in

vergelijking met controlepatiënten (n=15). In neussecreties verzameld met Merocels

waren totaal IgE en HDM sIgE BDL in resp. 3 en 4 AR patiënten, met de filter discs in

resp. 2 en 6 AR patiënten. Significante correlaties werden gevonden tussen HDM sIgE

concentraties in serum, in neussecreties verzameld met Merocels en in neussecreties

verzameld met filter discs.

Conclusie: IgE niveaus in neussecreties verzameld met Merocels lijken een goede

weerspiegeling te zijn van deze in nasaal weefsel. De collectie van nasale secreties met

Merocels is een gestandaardiseerde en niet invasieve methode die gebruikt kan worden

voor herhaaldelijke metingen van lokaal IgE en andere mucosale inflammatie

parameters om zo nasale ziektes te bestuderen en hun behandeling op te volgen. Het

gebruik van filter discs lijkt een haalbaar alternatief.

5    

2 INTRODUCTION

2.1 Chronic upper airway inflammation

Chronic upper airway inflammation can be divided into two major clinical entities:

rhinitis and rhinosinusitis. Rhinitis and sinusitis usually coexist [1]. The Allergic

Rhinitis and its Impact on Asthma (ARIA) guidelines define rhinitis as an

‘inflammation of the lining of the nose which is characterized by nasal symptoms

including anterior or posterior rhinorrhoea, sneezing, nasal blockage and/or itching of

the nose being present during two or more consecutive days for more than 1 h on most

days’ [2]. Allergic rhinitis (AR) is an IgE-mediated inflammation of the nose after

allergen exposure such as house dust mite (HDM) and pollen from grass and trees

[2,3]. Many nonallergic conditions can cause nasal symptoms which mimic allergic

rhinitis: infections, physical agents, hormonal imbalance, the use of certain drugs and

anatomical anomalies [4]. According to the European Position Paper on Rhinosinusitis

and Nasal Polyps 2012 (EPOS 2012) [1], rhinosinusitis in adults is defined as an

‘inflammation of the nose and the paranasal sinuses, characterized by two or more

symptoms, one of which should be either nasal blockage/obstruction/congestion or

nasal discharge (anterior/posterior nasal drip), ± facial pain/pressure , ± reduction or

loss of smell and either endoscopic signs of: nasal polyps, and/or mucopurulent

discharge primarily from middle meatus and/or oedema/mucosal obstruction primarily

in middle meatus and/or CT changes: mucosal changes within the ostiomeatal complex

and/or sinuses.’ Acute rhinosinusitis (ARS) in adults is defined as a sudden onset of

two or more symptoms as mentioned above for less than 12 weeks. If the problem is

recurrent, there must be symptom free intervals to meet the definition. Chronic

rhinosinusitis (CRS) in adults is defined as a presence of two or more symptoms as

listed above for 12 or more weeks [1]. CRS can be divided into a group with and

without endoscopic evidence of nasal polyps [1,5,6]. Chronic rhinosinusitis with nasal

polyps (CRSwNP) is characterized by bilateral, endoscopically visualized nasal polyps

in the middle meatus. In chronic rhinosinusitis without nasal polyps (CRSsNP) no

visible polyps in the middle meatus can be visualized [1]. Nasal polyps are edematous

masses protruding from the nasal and paranasal mucosa which can lead to an

obstruction of the nasal cavities [7,8].

  6  

2.1.1 Allergic rhinitis

2.1.1.1 Classification AR can be divided into ‘intermittent’ and ‘persistent’. Intermittent means that the

symptoms are present less than 4 days a week or for less than 4 consecutive weeks.

Persistent means that the symptoms are present more than 4 days a week and for

more than 4 consecutive weeks. The severity of AR can be classified as mild or

moderate/severe [2,9]. The classification in severity involves the symptomatology,

but also includes the impairment on quality of life (appendix 4).

2.1.1.2 Epidemiology AR is a common condition affecting 10-20% of the adult population worldwide [10].

In some countries up to 50% of the adolescents register with symptoms of AR [2]. The

prevalence has increased during the past 50 years and is still increasing, especially in

those countries where there was a low or mediate prevalence. Recent increase in the

prevalence cannot be due to a change in gene pool [2]. Rhinitis and allergic diseases

are taken more seriously and the European Union has developed programs to

understand, manage and prevent allergic diseases in a better way [2,11].

2.1.1.3 Comorbidity

2.1.1.3.1 Asthma AR and asthma, both allergen allergic diseases, often coexist [12]. AR and asthma

have a corresponding etiology and show familial and intra-individual connection [2].

Rhinitis is a significant risk factor for adult-onset asthma in both atopic and nonatopic

subjects [2]. Over 80% of asthmatics have rhinitis and 10-40% of patients with rhinitis

have asthma [2]. So the majority of patients with asthma have rhinitis symptoms

suggesting the concept of one airway one disease [13,14]. How more severe the

rhinitis, the more severe the asthma will be [2]. However, not all the patients with

rhinitis have asthma and there are differences between the diseases [13,14]. The

prevalence of asthma in subjects without rhinitis is usually less than 2% [15-17].

2.1.1.3.2 Allergy Other allergic diseases are common for patients with AR. The complex indoor

environment may contribute to an increasing prevalence of atopic diseases. It is proven

that multiple indoor allergens have an additional effect on atopic comorbidities [18].

7    

2.1.1.3.3 Chronic rhinosinusitis Inflammation of the nasal mucosal in AR predisposes the atopic individual to develop

CRS. Both diseases show an increasing prevalence and are frequently associated [2].

The association with asthma is stronger in those patients reporting both CRS and AR.

There is an increased prevalence of AR in patients with CRS, but the role of allergy in

CRS remains unclear [19-21]. IgE-mediated mechanisms, responsible for nasal

inflammation, are a main factor in the development of acute and/or chronic sinus

disease [22,23]. In acute and chronic rhinosinusitis a high prevalence of sensitization to

inhalant allergens is found and these patients seem to experience more sinus related

symptoms [2].

2.1.1.4 Diagnosis The diagnosis of AR is usually easy, but is still underdiagnosed. Many patients do

not perceive their symptoms as an impairment in daily life. Normally the diagnosis

of AR is based on a questionnaire, examination and skin tests to common

aeroallergens [2]. In most studies, the diagnosis of allergy was based on the

presence of a positive skin prick test and/or serum specific IgE determinations. This

indicates atopy but may not be sufficient to diagnose AR, particularly the persistent

form [2]. In a study from Tschopp et al. [24] the skin prick test had the best positive

predictive value for the epidemiologic diagnosis of AR compared to Phadiatop or

total serum IgE. AR can also be defined by a positive RAST (Radio-Allergo-

Sorbent-Test) [1].

2.1.1.5 Pathophysiology

2.1.1.5.1 IgE-dependent mechanisms Allergy is caused by a continuous overproduction of IgE to environmental antigens.

This involves different antigens such as indoor or outdoor allergens, foods and other

antigens [25]. Rhinitis is associated with increased levels of total serum IgE [26,27].

Allergen-specific IgE binds to receptors on the membranes of mast cells and basophils.

Mast cell accumulation in the airway mucosa is an important pathophysiologic event in

AR. Inhaled allergens are captured in the mucosa of nose and/or lungs. The

accumulation of receptor-bound IgE molecules on exposure to specific allergen results

in the production of inflammatory mediators (histamine, leukotriene and others)

responsible for the allergic response [28]. The link between this IgE-mediated reaction

and rhinitis has been confirmed by the effect of an anti-IgE monoclonal antibody [29-

  8  

31].

2.1.1.5.2 Role of IgE and the inflammatory infiltrate AR is characterized by an inflammatory infiltrate and the release of mediators is

responsible for the symptoms. Eosinophils, mast cells, T-cells, macrophages,

fibroblasts and other cells form the inflammatory infiltrate of the nasal mucosa in

patients with AR [32-34]. IgE has several pathophysiologic roles such as antigen

presentation, increased mast cell survival, defense against viruses, bacteria, fungi and

parasites and mucosal homeostasis. IgE interacts with B-cells, T-cells, mast cells,

eosinophils and basophils. Cross linking of IgE (bound to the surface of mast cells), the

subsequent degranulation and the release of Th2 cytokines lead to recruitment of

inflammatory cells [2]. Th2-cells drive the synthesis of IgE and the recruitment,

maturation, survival and effector function of accessory cells such as eosinophils,

basophils and mast cells [35].

2.1.1.6 Treatment Standard treatment for IgE-mediated allergic diseases such as AR are

antihistamines. Histamine, one of the major effector cells of allergic reaction, is

released by mast cells and basophils [2].

9    

2.1.2 Chronic rhinosinusitis with nasal polyposis

2.1.2.1 Epidemiology There is a lack of valid epidemiological studies for CRSwNP. In a survey based on

endoscopy, Sködve et al. [36] reported a NP prevalence of 2,7% in Sweden. In Finland,

Hedman et al. [37] found in a postal questionnaire survey that 4.3% of the adult

population answered positively to the question as to whether polyps had been found in

their nose. Klossek et al. [38] reported a NP prevalence of 2,1% in France by using a

disease-specific questionnaire. NP can be found in all races and becomes more

prevalent with higher age [38-41]. The average age of onset is approximately 42 years.

NP is uncommon under the age of 20 and in general more frequently found in men

than in women [1].

2.1.2.2 Diagnosis

2.1.2.2.1 Symptoms Diagnostic tests are available to verify the clinical symptoms and signs of

rhinosinusitis. Nonetheless, for the majority of patients, the diagnosis is made in

primary care based on symptoms alone. Symptoms remain the mainstay of diagnosis in

primary care. Symptoms are mainly the same in acute and chronic rhinosinusitis with

and without nasal polyposis, but symptom pattern and intensity may vary. Acute forms

of infections have usually more distinct and often more severe symptoms. [1]

2.1.2.2.2 Examination

2.1.2.2.2.1 Anterior rhinoscopy and nasal endoscopy Anterior rhinoscopy is the first examination in a patient, but alone it has a limited value

[1]. The diagnosis of CRS is based on nasal endoscopy. Compared to anterior

rhinoscopy, nasal endoscopy provides significantly better illumination and

visualization. This allows to obtain detailed information of the nasal cavities [1].

2.1.2.2.2.2 Imaging The plain sinus x-ray features a low cost and availability, but its usefulness is limited

for diagnosing rhinosinusitis due to underestimation of bony and soft tissue pathology

compared to computed tomography (CT) and magnetic resonance imaging (MRI)

[1,42]. CT scanning is first choice for the paranasal sinuses due to optimal display of

air bone and soft tissue, but it should not be regarded as the primary step in the

diagnosis [1].

  10  

2.1.2.3 Pathophysiology

2.1.2.3.1 Nasal polyps Polyps are infiltrated with inflammatory cells, mainly eosinophils. The local Th2

polarization observed in these polyps is characterized by high levels of eosinophilic

markers, IL-5 and local polyclonal IgE [7]. IL-5 is the key driver of eosinophilic

differentiation and survival. It is assumed that, through release of toxic products,

eosinophils lead to tissue damage and growth of polyps. IL-5 inhibition is a potential

novel therapeutic approach in patients with severe eosinophilic nasal polyposis [43].

2.1.2.3.2 Local IgE In NP homogenates, the level of total or specific IgE is independent of the atopic status

of the patient [1,44-46]. Local IgE in NP is the result of two types of IgE production:

systemic allergic IgE formation and a local polyclonal IgE formation. The local

polyclonal IgE production is located in the stroma and the epithelium of nasal polyps

[7]. Local polyclonal IgE formation occurs mainly in the subgroup of polyps

containing Staphyloccous Aureus enterotoxin-specific IgE [46,47]. High total IgE

concentrations can also be associated with an increase of the soluble low affinity-IgE

receptor sCD23. Membrane-bound CD23 normally binds to IgE, which leads to a

downregulation of IgE production. sCD23 can capture IgE and prevents binding to his

receptor [46].

2.1.2.3.3 Inflammatory triggers Parasites, fungi, viruses and bacteria can stimulate polyclonal lymphocyte responses

and thereby obliterate the specific immune system, resulting in a deficiency of

specificity of antibodies. Two polyclonal stimuli can trigger B-lymphocyte

proliferation and differentiation in the absence of antigen: bacterial products and T-

cells activated by a third-party antigen. Activated T-cells stimulate B-cells in a non-

cognate fashion via CD40 ligand and cytokine production [44]. IgE correlates with the

presence of Staphylococcus aureus enterotoxins (SAE) [1,7,44,45]. Gevaert et al.

demonstrated in about half of the examined polyp specimens polyclonal IgE formation

specific to SAE and a high prevalence of asthma [45]. Stimulation of polyclonal IgE

synthesis could be a way for S.aureus to colonize the nasal mucosa and devoid the

allergic response [47]. Up to 73% of S.aureus strain isolates, produces one or more

enterotoxins [45]. These enterotoxins may act as superantigens [44,45,47,48]

(appendix 1). There are two prominent IgE positive areas in NP: the follicular

11    

structures (CD20 B cells and CD3 T cells) and the diffuse lymphoid accumulations

(CD38 plasma cells and CD3 T cells) [44,47]. Biotinylated SAE superantigens bind to

these areas and directly affect the activation of B cells and plasma cells. This activation

leads to isotype switching and synthesis of IgE [45]. These superantigens can also

interact with the T cell receptor (TCR), resulting in a polyclonal T-cell activation with

a Th2 cytokine polarization. [1,45]. Key markers of Th2 inflammation are increased in

NP [47]. These cytokines favour IgE production indirectly by triggering B cell class

switching towards IgE production [1,45]. Each enterotoxin can only bind to a certain

subset of TCR Vβ-chains [45]. The T-cell receptor Vβ repertoire corresponds to the

specific IgE in the polyp tissue [45]. SAE can exert direct effects on eosinophil

proliferation and survival which possibly results in a severe eosinophilic inflammation

[44,45,48]. Beside the enterotoxins, Staphylococcal protein A (SPA) as well as the

toxic shock syndrome toxin (TSST-1) from Staphylococcus aureus, possess B-cell

superantigen moieties which induces polyclonal IgE synthesis [44].

2.1.2.3.4 Local receptor revision and class switch recombination Evidence has been found for local receptor revision (RR), class switch recombination

(CSR) and B-cell differentiation into IgE-secreting plasma cells in NP. NP is

associated with an increase of IL-4. IL-4 stimulates the synthesis of ε-germline gene

transcript (GLT), which has a significant role in the class switch recombination (CSR)

to IgE. Gevaert et al. [47] detected Iε-Cγ switch circle transcript in NP. B-cells and

plasma cells are enhanced in NP, as well as the ratio of plasma cells to B cells [47].

2.1.2.4 Comorbidity

2.1.2.4.1 Allergy To date, there is no clear evidence for a direct relationship between allergy and

rhinosinusitis [49,50]. Increased local IgE synthesis can reinforce allergic reactivity by

triggering mast cell degranulation. However, when the IgE formation is extreme it can

suppress specific reactivity by flooding the high affinity IgE receptors (Fcε receptors)

on mast cells in the tissue through polyclonal IgE. This polyclonal IgE would

neutralize any effect of specific IgEs to environmental allergens [47,48]. This could

explain why the IgE level is independent of the atopic status of the patient [44]. Some

studies show an increase of atopy markers in CRS patients [1]. Between 10 to 54% of

subjects with CRSwNP have allergy. On the other hand, the prevalence of NP in AR

patients has been reported between 0,5 to 4,5%, which is comparable with the normal

  12  

population [1].

2.1.2.4.2 Asthma CRSwNP is a multifactorial disease that is frequently associated with asthma [46,51].

The group that coexists with asthma is particularly characterized with tissue

eosinophilia and high local IgE levels. In mucosal tissues, mRNA for the ε-chain of

IgE was associated with a significant proportion of B cells [44,47]. This strengthens

the hypothesis of local IgE synthesis [44]. Asthma is described by 26% of patients

with CRSwNP, compared to 6% of controls [1,38,39]. There is a direct correlation

between NP, bronchial hyperreactivity and asthma [51]. Asthmatic patients with

CRSwNP have more nasal symptoms and are associated with high levels of local IL-5

and specific IgE against SAE [1,45,52].

2.1.2.5 Medical treatment options The figure in appendix 2 represents the management of CRSwNP for ENT-specialists

as proposed by EPOS 2012 [8].

2.1.2.5.1 Corticosteroids Treatment with methylprednisolone results in a reduction of systemic or local free light

chains concentrations and can be related to its potent systemic anti-inflammatory

action [43]. Methylprednisolone has a short but dramatic effect on polyp size and

symptoms [1].

2.1.2.5.2 Antibiotics Antibiotics are a therapeutic option because Staphylococcus aureus plays a role in the

pathophysiology of CRSwNP. Doxycycline is an anti-staphylococcal antibiotic that

can be used [1]. A RCT showed a significant effect of oral methylprednisolone and

doxycycline on size of nasal polyps, nasal symptoms and mucosal and systemic

markers of inflammation [53].

2.1.2.5.3 Anti-IgE The local production of IgE is functional and has a role in the regulation of chronic

inflammation. Antagonizing IgE antibodies is a relevant strategy. Omalizumab is a

recombinant DNA-derived humanized IgG1k monoclonal antibody. It reduces serum

and tissue IgE-levels and is a treatment option for patients with allergic and non-

allergic NP [1,48]. Omalizumab binds selectively free human circulating IgE and

13    

inhibits the binding of IgE to the high-affinity IgE receptor, decreasing free IgE levels.

Omazilumab also leads to a decrease in IgE receptors on mast cells, basophils, and

dendritic cells [48].

2.1.2.5.4 Anti-IL-5 Mepolizumab is a humanized anti-IL-5 monoclonal antibody and forms a potential

novel therapeutic approach for patients with severe eosinophilic NP. Mepolizumab

achieved a statistically significant reduction in NP size for at least 1 month after dosing

in 12 of patients in a study of Gevaert et al. [43].

2.2 Measurement of local IgE

Local IgE can be measured in nasal tissue homogenates and secretions [44,48].

Different sorts of cells (expelled epithelial cells and immunocompetent cells), plasma

exudation and mucus can be found in nasal secretions. Interesting is the fact that nasal

fluid contains inflammatory mediators reflecting the activity within the nasal mucosa.

Therefore it is possible to detect specific inflammatory cells, inflammatory mediators

and cytokines with biochemical analyzes and to follow the evolution of nasal diseases

[54]. Earlier, blown secretions were the only techniques to investigate nasal secretion,

but the obtained quantities of secretions were extremely variable [55]. Several other

techniques like the brush method, nasal scraping [56], biopsies [57] and the imprint

technique are explored and described . The standardization for these techniques has

improved the quality of analysis, but they are painful for the patient and traumatizing

for the nasal mucosa [54]. Microsuction [58], aspiration [59] and nasal lavage were

designed to collect nasal secretion with the least discomfort and the best reproducibility

[54]. Aspiration and nasal lavage have been described as the most reliable methods for

nasal secretion sampling [60]. These techniques require the cooperation from the

patient, which may be a problem with children or comatose patients. Another

disadvantage is the fact that the collected quantity of material can fluctuate

significantly [54]. Alternative methods for sampling of nasal secretions are the use of

swabs and sponges [54,61]. The use of pre-humidified gauze [62], filter discs [63] and

sinus packs (Merocels) has been described. Measurement of local IgE in the collected

material (tissue and/or nasal secretions) can be done by means of the UniCAP system

[44,48].

  14  

2.2.1 Merocels Merocels have already been used for several different purposes. They are mostly used

in nasal surgery, epistaxis management, endoscopic sinus surgery (ESS), transseptal

suturing and septoplasty to control bleeding [54]. Merocels are also considered as a

non-invasive, atraumatic and reproducible technique for the collection of nasal

secretions in order to do biochemical analyses [54]. Examples of use of Merocels for

monitoring local inflammation, for monitoring medical and surgical treatment are the

following: In a study of Watelet et al. [64] Merocels were used to follow different

phases of wound repair by detecting changes in the secretion of growth factors. In

another study Merocel samples were collected to determine IgA titers to respiratory

syncytial virus (RSV) fusion and attachment glycoproteins [65]. Gevaert et al. [48]

measured ECP, IL-5, sCD23, sIL-5Rα, total IgE and tryptase in nasal secretions

collected with Merocels from one sample. Klimet et al. [61] used Merocels for

monitoring the therapeutic efficacy of immunotherapy in AR by detecting specific cell

activation markers like ECP or tryptase. In a twelve-year follow-up study after

endoscopic sinus surgery (ESS) in patients with CRSwNP, nasal secretions were

collected by placing Merocels in the nasal cavities to measure different proteins (IL-5,

IL-5Rα, TGF-β1, MPO, ECP, total IgE and specific IgE antibodies) [66]. In a study

from Cassona et al. [67] Merocels appeared to be well tolerated by patients and showed

the lowest percentage of cytological alteration of ciliated cells compared to Clauden,

two-fingered glove pack with gauzes inside and Lyofoam.

2.2.2 Filter discs A filter disc is a small round piece of paper, punched out from a commercially

available synthetic filter card, placed into the nose against the nasal septum. Filter discs

are the collection system the most comparable to Merocels. It is a non-invasive,

relatively simple, quantitative technique that can be removed atraumatically [68]. Filter

discs are smaller than Merocels and give less stimulation of the nasal mucosa. In a

study from Naclerio et al. [68] filter discs were used to observe the effects of

histamine, methacholine, ipratropium bromide and atropine on the nasal mucosa.

Baumann et al. [69] used the filter disc method to detect cytokine levels in AR patients.

In another study with AR patients, filter discs were used to investigate the role of IL-31

[70]. Filter discs have also been used to differentiate congestive hyperresponsiveness

in AR by applying nasal bradykinin unilateral [71]. The paper filter method was

15    

already used in the context of HDM allergy to measure neutrophil chemotactic activity

(NCA) and histamine content in nasal secretions [72]. Howarth et al. used an allergen

solution on the surface of a small filter disc to induce local allergen provocation in

order to stimulate inflammatory cells such as eosinophils, mastcells and neutrophils

[73]. Lien calus et al. [66], collected nasal secretions before and 15 minutes after

NAPT, by placement of filter discs in the nose, to measure total IgE, total IgG, specific

IgG4 and tryptase concentrations.

2.2.3 Tissue Tissue extraction is the most reliable local measurement technique and thereby the

golden standard. It is an invasive procedure, but can inform directly about a range of

mucosal tissue cellular events [73]. Gevaert et al. [44] obtained fresh tissue strips from

nasal polyps during routine septal and sinus surgery to determine concentrations of

total IgE and IgE antibodies to inhalant allergens and S. aureus enterotoxins. In a study

from Lien Calus et al. [66] nasal tissue, namely NP tissue from CRSwNP patients, was

collected during endoscopic sinus surgery (ESS) to measure different proteins (IL-5,

IL-5Rα, TGF-β1, MPO, ECP, total IgE and specific IgE antibodies).

2.3 Aims of the study Several datasets were analyzed to obtain more knowledge concerning local IgE in AR

patients and CRSwNP patients. Further, we wanted to demonstrate the validity of local

IgE measurements on nasal secretions collected with Merocels and illustrate the role of

filter discs as a possible alternative. The experiment was performed in order to

optimize the process of collection of nasal secretions by using Merocels and filter discs

and to make a comparison between them.

  16  

3 MATERIALS AND METHODS

3.1 Analyzed datasets Data of 112 study subjects with CRS (with and without NP) collected at the

Department of Otorhinolaryngology of the University Hospital of Ghent were used to

compare IgE measurement by means of Merocel, tissue and serum [74]. Data of a

study with omalizumab [48] were used to analyze differences in total IgE

concentration when subjects underwent a placebo or an omalizumab treatment. A

randomized, double-blind, placebo-controlled study of allergic and nonallergic patients

with NP and comorbid asthma (n= 24) was conducted. Subjects received 4 to 8

(subcutaneous) doses of omalizumab (n= 16) or placebo (n= 8). The primary end point

was a reduction in total nasal endoscopic polyp scores after 16 weeks. The study was

conducted at the Department of Otorhinolaryngology of the University Hospitals of

Ghent (n= 20 patients) and Leuven (n= 4 patients), Belgium. A dataset of thirty

patients with severe NP (grade 3 or 4 or recurrent after surgery), refractory to

corticosteroid therapy, was used to measure the differences in total IgE after a placebo

or mepolizumab treatment. Patients were randomized in a double-blind fashion to

receive either 2 single intravenous injections (28 days apart) of 750 mg of

mepolizumab (n= 20) or placebo (n= 10). Change from baseline in NP score was

assessed monthly until 1 month after the last dose (week 8). Computed tomographic

scans were also performed at week 8. The objective of this study was to investigate the

therapeutic potential of inhibiting IL-5 with a humanized mAb as treatment for severe

NP. This study was also conducted at the Department of Otorhinolaryngology of the

University Hospital in Ghent. At last, data were used to analyze differences in total IgE

concentration over the time during 6 visits (at baseline, week 1, week 2, week 4, week

8, week 12), in placebo (n= 18), in doxycycline (n= 14) and in methylprednisolone (n=

14) treated groups. In a double-blind, placebo-controlled, multicenter trial, 47

participants with bilateral NP were randomly assigned to receive either

methylprednisolone in decreasing doses (32–8 mg once daily), doxycycline (200 mg

on the first day, followed by 100 mg once daily) or placebo for 20 days. Participants

were followed for 12 weeks. Patients were assessed for nasal peak inspiratory flow and

symptoms and by nasal endoscopy. Markers of inflammation such as eosinophilic

cationic protein (ECP), IL-5, myeloperoxidase, matrix metalloproteinase 9, and IgE

17    

were measured in nasal secretions. Concentrations of eosinophils, ECP, and soluble IL-

5 receptor α were measured in peripheral blood samples. The original endpoint of this

study was to evaluate the effects of oral glucocorticoids and doxycycline on symptoms

and objective clinical and biological parameters in patients with CRSwNP. To

illustrate the role of filter discs for local measurements, different biomarkers (before

nasal allergen provocation) were analyzed (total IgE, gx3 IgE, IgG, IgG4 and tryptase)

based on a dataset [66] with 12 grass pollen allergic CRSwNP patients. The patients

underwent a nasal allergen provocation test (NAPT) with grass pollen and were

compared to 12 grass pollen AR patients and 12 control patients. Further, VAS scores

of different symptoms, serum and nasal secretions were collected before and after

NAPT. A skin prick test was performed on all the patients for different common

aeroallergens to diagnose grass pollen allergy. The primary endpoint of this study was

the NAPT. The study was conducted at the Department of Otorhinolaryngology of the

University Hospitals of Ghent.

3.2 Experiment

3.2.1 Patient recruitment 15 patients with AR to house dust mite (HDM) and 15 control patients were recruited

from the Department of Otorhinolaryngology at the Ghent University Hospital. All

patients submitted a skin prick test (SPT) for 8 different common aeroallergens.

Patients with HDM sensitive AR were selected based on symptoms and a positive SPT

for HDM (Derm. Pteronyssinus ± Derm. Farina). The control subjects had a negative

SPT and were free of acute nose or sinus disease at the moment of sampling, no

chronic nasal or sinus complaints were reported in their history. The local ethical

committee on human experimentation of the institution (Ghent University Hospital,

Belgium) had given its approval for this project, and each participant gave an informed

consent before enrolment.

3.2.2 Skin prick testing A skin prick test (SPT) was performed in control and patient groups to demonstrate an

allergic response to a specific allergen (figure 1). Controls were performed with

Histamindihydrochloride 0,1% (positive control) and NaCl 0.9% (negative control).

Eight allergens were selected: Grasses, Trees, Artemesia vulgaris, Dermatophagoides

pteronyssinus, Dermatophagoides farina, Cat, Dog and Alternaria alternate. The SPT

  18  

was carried out on the inner forearm 2-3 cm from the wrist and the antecubital fossae.

The location of each allergen was coded with a marker pen to identify the allergens to

be tested with a distance between two SPTs (≥ 2 cm) to avoid false-positive reactions

due to direct contamination of a nearby test or secondary to an axon reflex [75]. A drop

of the allergen solution was placed on the skin in identical order for each tested subject

and immediately pricked through the drop using the tip of a lancet. Positive and

negative controls were first measured. After 15 minutes the largest diameter of the

wheal of each particular test was measured. The result was considered as positive when

the wheal had a diameter of ≥ 3 mm.

Figure 1: SPT procedures. (A) Allergen solutions. (B) Lancets. (C) Prick testing with lancet through a drop of allergen extract. (D) Waiting for 15 minutes. (E) Measuring the largest diameter of the wheal of each particular test.

3.2.3 Blood testing All the control and AR patients had a blood test to measure the concentration of total

IgE and HDM sIgE. Blood was collected by performing a standard venipuncture

(Serum gel separator tubes Terumo Venosafe, Ref VF-109SDK, figure 2) and allowed

to clot for 15-30 minutes. After that the blood was centrifuged for 15 minutes at 1500g

at 4°C. Serum was stored in aliquots at -20°C until further analysis with the UniCAP

system (Pharmacia & Upjohn, Uppsala, Sweden).

19    

Figure 2: Material and venipuncture. (A) From the left to the right: garrot, serum tube, needle, holder, cotton pad and adhesive plaster. (B) Standard venipuncture.

3.2.4 Procedure for collection of nasal secretions

3.2.4.1 Sampling of nasal secretions Filter paper discs (figure 3 C) were punched from filter cards (Shandon, Pittsburgh,

PA) using a 10-mm punch. The filter cards are made from a blend of 100% cotton and

cellulose fibers and have excellent absorbent properties. Before the experiments, the

Merocels and filter discs were placed separately in pairs in Falcon tubes (Blue Max Jr.

15-ml polypropylene conical tube, 17×120 mm style FALCON, Becton Dickinson

Lab- ware, Franklin Lakes, N.J., figure 3 A) and weighed (figure 4 A), coded and

registered. Filter discs were first placed in both nasal cavities under direct visualization

using a surgical headlight, a speculum and bayonet forceps (figure 3 A) in control and

patient groups for 5 minutes (figure 4 B) and afterwards placed back in the respective

Falcon tube. Secondly, Merocels went trough the same procedure, but for 10 minutes

(figure 4 D). The filter discs and Merocels were inserted on the floor of the cavity

between the septum and inferior turbinate (appendix 3, figure 4 C).

Figure 3: Material for sampling of nasal secretions. (A) From the left to the right: syringe, Falcon tube with two filter discs, nose speculum and bayonet forceps. (B) Falcon tube with two swollen Merocels after insertion in the nasal cavities. (C) Filter disc.

  20  

Figure 4: Sampling of nasal secretions. (A) Filter discs in Falcon tube weighed before insertion. (B) Placing filter discs in the nasal cavities for 5 minutes. (C) Position of the filter disc in the nasal cavity. (D) Merocels went trough the same procedure, but were placed in the nasal cavities for 10 minutes.

3.2.4.2 Processing of nasal secretions The quantity of secretions collected was determined by comparing the weight of

Merocels and filter discs with the Falcon tube before and after insertion using the same

balance (A&D Instruments Ltd., type HR-120, Japan) (figure 4 A and figure 5 A). The

difference corresponds to the weight of nasal secretion collected (WNS). The Merocels

and filter discs were washed with 0.9% NaCl solution (Merck Eurolab, Belgium, figure

5 B) and incubated for two hours at 4°C (figure 5 C). The volume of 0.9% added NaCl

solution was measured by a formula based on a dilution factor (DF) and the WNS:

volume (ml) = (DF x WNS) – WNS. In previous experiments [54] with fixed amount

of 0,9% NaCl solution for dilution (e.g. 3 ml), the amount of added NaCl was

sometimes very high in comparison with the amount of nasal secretion that was

collected (weight difference). Therefore, it is likely that some of the measurements

were BDL due to excessive dilution. We chose to use fixed DFs: 10 for the Merocels

and 20 for the filter discs. With this method it is unlikely that measurements are BDL

due to excessive dilution. The filter discs and Merocels were placed into the shaft of a

syringe (Plastipak 5 ml, Becton-Dickinson S.A., Cno. De Valdeoliva S/N, Madrid) and

all the fluid was pushed out of it by moving the piston of the syringe (figure 5 D). After

that, the falcon tubes, with syringe and Merocels or syringe and filter discs, were

21    

placed in the centrifuge at 1500g/15 minutes at 4°C (figure 5 E). The supernatants

were divided in aliquots (500 µL) and stored at or below -20°C (figure 5 F). Total IgE

and HDM sIgE concentrations in nasal secretions were measured by the UniCAP

system.

Figure 5: Processing of nasal secretions. (A) Weighing filter discs in Falcon tubes after insertion in the nasal cavities. (B) Washing with 0.9% NaCl solution. (C) Incubating for two hours at 4°C. (D) Filter discs placed into the shaft of a syringe and pushing all the fluid out of it. (E) Centrifuging at 1500g/15 minutes at 4°C. (F) Dividing the supernatants in aliquots (500 µL) and storing at or below -20°C. The same procedure was applied for the Merocels.

3.3 Literature

Particularly PubMed was used to study literature. Other used search engines were Web

of Science and Google Scholar. Combinations of the following keywords were used:

allergic rhinitis, chronic rhinosinusitis, nasal polyps, IgE, nasal secretion, treatment,

nasal irrigations, corticosteroids, antibiotics, anti-IgE, anti-IL-5, surgery, skin prick

test, nasal swabs, Merocel, filter disc, filter paper, tissue, diagnosis, pathophysiology,

Staphylococcus aureus, superantigens, inflammation, immunology, cytokines, IL-5. A

selection of articles was made by evaluating their abstract.

  22  

3.4 Statistical analysis All statistics were performed using the SPSS 21.0 software. Data are presented as

median and interquartile range (IQR). Statistical significance was assessed using two-

tailed tests and was defined as p<0.05. Graphics were made by MedCalc 12.7.0.0

software. Non-parametric test were used because the populations were not normally

distributed as assessed by Shapiro-Wilk tests. Correlations between continuous data

were determined by the Spearman correlation coefficient (Rs). A Wilcoxon Signed-

Rank test was executed to determine changes from baseline in inflammatory

parameters in different treatment groups. A Friedman test was carried out to determine

differences in the concentration of inflammatory parameters over the time in different

treatment groups. Pairwise comparisons were performed with a Bonferroni correction

for multiple comparisons. A Kruskal-Wallis test was done to determine differences in

inflammatory parameters between more than two disease groups. Comparisons

between two disease groups were performed by the Mann-Whitney U test for

continuous data.

23    

4 RESULTS

4.1 Correlations between Merocel, tissue and serum total IgE measurements

An assessment of the relationship between total IgE measured in nasal secretions

collected with Merocels, tissue and serum for 112 patients with CRS (with and without

NP) was made (table 1). Preliminary analysis showed the relationship to be monotonic,

as assessed by visual inspection of scatterplots. A positive correlation between total

IgE in tissue and serum (figure 6 A), Merocel nasal secretions and serum (figure 6 B),

Merocel nasal secretions and tissue (figure 6 C) has been found (resp. rs= .623,

p<.0001; rs=.645, p<.0001 and rs=.595, p<.0001). Total IgE in Merocel nasal

secretions had the lowest value (Mdn= 4.65 kU/L). Tissue and serum total IgE

measurements were comparable (resp. Mdn= 50.16 kU/L and 50.18 kU/L).

A B

C

Figure 6: Correlations between tissue, serum and Merocel nasal secretions. Scatterplots on a logarithmic scale showing a correlation in the measurement of total IgE between (A) tissue and serum (B) Merocel nasal secretions and serum and (C) Merocel nasal secretions and tissue. Rs: Spearman's rank correlation coefficient; p: significance level.

  24  

Table 1: Descriptives of total IgE measurement by means of Merocel, tissue and serum

Total IgE (kU/L) Merocel Tissue Serum

Subjects (n) 100 81 108

Median (IQR) 4.65 (2.23 – 13.69) 50.16 (10.34 – 240.76) 50.18 (17.40 – 141.75)

Legends: IQR= Interquartile range.

4.2 Dynamic local IgE measurements by means of Merocels

4.2.1 Comparison omalizumab versus placebo Differences in total IgE concentrations (at week 18), in nasal secretions collected with

Merocels (table 2), were analyzed in 24 subjects with CRSwNP and comorbid asthma

who had a placebo (n=8) or an omalizumab (n=16) treatment (figure 7). Total IgE

decreased not significantly after placebo treatment (p=.686). In contrary, there was a

significant increase after omalizumab treatment (p=.041).

Figure 7: Comparison of the change in total IgE concentrations in placebo and omalizumab treated groups in nasal secretions collected with Merocels. In the box-and-whisker plot, the central box represents the lower to upper quartiles (25th to 75th percentiles). The middle line represents the median. The vertical line extends from the minimum to the maximum values. All measurements are marked with a black open circle.

Table 2: Descriptives of total IgE measurements at baseline and after treatment with placebo or omalizumab in nasal secretions collected with Merocels.

Placebo (n= 8) Omalizumab (n= 16)

Total IgE (kU/L) Median (IQR)

baseline 88.98 (26.44 – 146.99) 20.17 (9.31 – 56.63)

Week 18 74.85 (13.72 – 205.18) 72.375 (15.24 – 165.27)

Legends: IQR= Interquartile range.

25    

4.2.2 Comparison mepolizumab versus placebo Analyzing differences in total IgE and SAE-IgE concentrations (at week 12), in nasal

secretions collected with Merocels (table 3) in 30 patients with severe NP who had a

placebo (n= 10) or a mepolizumab (n= 20) treatment, revealed a significant increase in

total IgE after placebo treatment (p=.028). After mepolizumab treatment no significant

differences were noted (p=.199; figure 8 A). For SAE-IgE, there were no significant

differences after placebo and mepolizumab treatment (resp. p=.173 and p=.327; figure

8 B).

Figure 8: Comparison of change in total IgE (A) and SAE-IgE concentrations (B) in placebo and mepolizumab treated groups in nasal secretions collected with Merocels. SAE-IgE: Staphylococcus aureus enterotoxin-specific IgE. All measurements are marked with a black open circle.

Table 3: Descriptives of total IgE and SAE-IgE measurements at baseline and after placebo or mepolizumab treatment in nasal secretions collected with Merocels.

Placebo (n= 10) Mepolizumab (n= 20)

Median (IQR)

Total IgE (kU/l)

baseline 21.94 (10.85 - 177.73) 20.76 (6.64 - 95.93)

week 12 63.28 (14.38 - 273.77) 10.58 (4.34 - 29.00)

SAE-IgE (kU/l)

baseline 4.79 (2.32 - 15.20) 6.94 (4.77 - 17.57)

week 12 12.63 (9.04 - 20.23) 7.45 (3.72 - 15.89)

Legends: SAE-IgE: Staphylococcus aureus enterotoxin-specific IgE; IQR: Interquartile range.

A B

  26  

4.2.3 Comparison doxycycline, methylprednisolone and placebo Study of total IgE concentrations (change from baseline) during 6 visits (figure 9), in

nasal secretions collected with Merocels in 47 CRSwNP patients (table 4), showed

significant differences in the placebo group (n=18; p=.003). Post-hoc analysis revealed

significant differences at week 4 (p=.009). In the methylprednisolone group (n= 14) an

initial decrease in total IgE was followed by an increase in total IgE, however, these

changes were not significant (p=.130). In the doxycycline group (n=14) total IgE levels

did not change during treatment (p=.412).

Figure 9: Total IgE in nasal secretions after treatment with methylprednisolone (solid triangles), doxycycline (solid squares) or placebo (solid circles) in nasal secretions collected with Merocels. Table 4: Descriptives of total IgE change from baseline in placebo, doxycycline and methylprednisolone treated groups in nasal secretions collected with Merocels.

Placebo

(n=18)

Doxycycline

(n=14)

Methylprednisolone

(n=14)

Total IgE (kU/L) change from baseline Median (IQR)

week 1 0.62 (-2.98 - 23.88) -0.96 (-11.68 - 0.00) -0.25 (-27.32 - 12.62)

week 2 5.91 (-0.60 - 49.44) 0.00 (-8.01 - 27.37) -11.85 (-36.29 - 0.96)

week 4 29.81 (4.93 - 67.91) 0.00 (-8.01 - 1.28) -3.92 (-57.96 - 4.01)

week 8 6.98 (0.00 - 155.30) 0.00 (-4.14 - 0.00) 1.76 (-18.76 - 21.83)

week 12 29.74 (0.00 - 124.57) 0.00 (0.00 -11.52) 10.06 (0.00 - 34.63)

Legends: IQR= Interquartile range.

-30

-20

-10

0

10

20

30

40

50

0 1 2 4 8 12

Tota

l IgE

(ku/

L) c

hang

e fr

om

base

line

Time (weeks)

Total IgE (nasal secretions)

Placebo

Doxycycline

Methylprednisolone

27    

4.3 Local measurements by means of filter discs Filter discs are an alternative option for the collection of nasal secretions. Analyzes on

a dataset with 12 control patients, 12 grass pollen allergic CRSwNP patients and 12

grass pollen AR patients (table 5) revealed that the group of patients with AR had the

highest value of total IgE (Mdn= 43.13 kU/l), followed by the patients with CRSwNP

with allergy (Mdn= 23.00 kU/l), those with CRSwNP without allergy (Mdn= BDL)

and the control group (Mdn= BDL) in nasal secretions collected with filter discs

(figure 10 A). Total IgE concentrations were significantly different between the disease

groups (p=.005). Post-hoc analysis revealed significant differences between the control

and AR group (p=.026) and the control and CRSwNP with allergy group (p=.017).

There were no significant differences between the AR group and CRSwNP with

allergy group (p=1.00). In the control group, the measurement of total IgE was BDL in

8 subjects (n=11). In the AR group and CRSwNP with allergy group, total IgE was

BDL in resp. 3 (n=12) and 2 (n=12) subjects. In the CRSwNP without allergy group, 7

subjects (n=12) were BDL. Parallel with total IgE, gx3 IgE was the highest in the AR

group (Mdn= 14.11 kU/l), followed by the patients with CRSwNP with allergy (Mdn=

3.53 kU/l), those with CRSwNP without allergy (Mdn= BDL) and the control group

(Mdn= BDL) (figure 10 B). Gx3 IgE (kU/l) was significantly different between the

disease groups (p<0.05). Post-hoc analysis revealed that gx3 IgE was significant higher

in the AR group in comparison with the control group (p=.001) and the CRSwNP

without allergy group (p=.001). Total IgG and IgG4 concentrations were the highest in

the control group (resp. Mdn= 256.17 mg/l and 16.75 mg/l), followed by the CRSwNP

group (resp. Mdn= 212.01 mg/l and 13.11 mg/l ), AR group (resp. Mdn= 182.62 mg/l

and 12.87 mg/l) and the CRSwNP with allergy to grass pollen group (resp. Mdn=

173.50 mg/l and 11.34 mg/l), but the differences were not significant (resp. p=.837 and

p=.994; figure 10 C and D). Finally, in the control group and CRSwNP with and

without allergy group, tryptase was not measurable in the majority of patients (Mdn=

BDL). In the AR group, tryptase was measurable in 7 out of 12 patients (Mdn= 33,67

µg/l), but the differences were not significant (p=.057; figure 10 E).

  28  

Table 5: Inflammatory measurements in nasal secretions collected with filter discs in control patients, grass pollen sensitive AR patients, CRSwNP patients and CRSwNP with grass pollen allergy patients.

Legends: Gx3 IgE: IgE to mix of grass pollen; IQR: Interquartile range; BDL: below detection level.

A B

C D

Controls AR CRSwNP CRSwNP with allergy

Median (IQR)

Total IgE

(kU/l) 8 BDL; 1.65 (1.65-2.82) 3 BDL; 43.13 (4.01-

72.26) 7 BDL; 1.65 (1.65-33.19) 2 BDL; 23.00 (7.11-111.67)

gx3 IgE (kU/l) All BDL 3 BDL; 14.11 (2.95 -

24.02) All BDL 5 BDL; 3.53 (1.66-7.26)

Total IgG

(mg/l) 256.17 (193.12-425.45) 182.62 (87.62-290.74) 1 BDL; 212.01 (60.22-

581.42) 2 BDL; 173.50 (36.96-

702.39)

IgG4 (mg/l) 16.75 (4.26-31.40) 12.87 (5.25-48.03) 13.11 (3.29-102.83) 11.34 (3.62-73.56)

Tryptase (µg/l) 10/12 BDL 5 BDL, 33.68 (8.28-

78.32) 8 BDL; 8.28 (8.28-13.04) 9 BDL; 8.28 (8.28-14.62)

29    

E

Figure 10: Comparison of inflammatory measurements in nasal secretions collected with filter discs. Total IgE concentrations (A), gx3 IgE concentrations (B), total IgG concentrations (C), IgG4 concentrations (D) and tryptase concentrations (E) in control patients, grass pollen sensitive AR patients, chronic rhinosinusitis with NP without allergy patients (CRSwNP) and chronic rhinosinusitis with NP with allergy patients (CRSwNPwA). All measurements are marked with a black open circle and significant differences are marked with a line.

4.4 Comparison of Merocels and filter discs for the measurement of local IgE (experiment)

4.4.1 Demographic and clinical features 15 control patients and 15 HDM sensitive AR patients were recruited in order to

analyze total IgE and HDM sIgE concentrations in serum and nasal secretions

collected with both Merocels and filter discs. In the AR group, 13 patients were poly-

sensitized and had a positive SPT for one or more of the other tested allergens. Two

control patients had a SPT with a HDM wheal of 2 mm. Three patients had comorbid

asthma. Three AR patients used oral antihistamines during the days prior to sampling

of the nasal secretions (in these patients, SPT was performed on another occasion)

(table 6).

Table 6: Baseline demographic and clinical characteristics of control patients and HDM sensitive AR patients.

Control (n= 15) AR (n= 15)

Age (yr), median (IQR) 25.00 (23.25-33.00) 24.00 (23.00-25.00)

Men/Women, n/n 4/11 6/9

Asthma, n 0 3

HDM (SPT+), n 0 15

Other allergies (SPT+), n 0 13

Antihistaminica use (last 3 days), n 0 3

Legends: yr: years; IQR: Interquartile range; n: numbers; HDM: House dust mite; SPT: Skin prick test.

  30  

4.4.2 Total IgE and HDM sIgE are increased locally in nasal secretions in AR patients

Total IgE and HDM sIgE concentrations, in control patients and HDM sensitive AR

patients, in nasal secretions (Merocels and filter discs) and serum are shown in table 7

and illustrated in figure 11. For each AR patient, the individual weight of the collected

nasal secretions, the added NaCl volume, serum and nasal secretion measurements

(raw and final data) are pointed out in table 8. Three control patients had a slightly

increased concentration of HDM sIgE in serum. Total IgE and HDM sIgE in nasal

secretions collected with Merocels were BDL in resp. 3 and 4 AR patients, with filter

discs resp. 2 and 6 AR patients were BDL. Total IgE and HDM sIgE were significantly

increased in HDM sensitive AR patients in serum and nasal secretions collected with

both Merocels and filter discs (table 7). Significant positive correlations between HDM

sIgE measured in Merocel nasal secretions and filter disc nasal secretions (rs=.753,

p=.001, figure 12 A), Merocel nasal secretions and serum (rs=.569, p=.027, figure 12

B) and filter disc nasal secretions and serum (rs=.749, p=.001, figure 12 C) were found

in the AR patients.

Table 7: Total IgE and HDM sIgE in nasal secretions (Merocels and filter discs) and serum in control patients and HDM sensitive AR patients.

Control (n= 15) Control vs AR

AR (n= 15)

Nasal Secretions

Merocel

Total IgE (kU/L) 12 BDL <0.001 3 BDL; 10.50 (2.38-51.98)

HDM sIgE (kU/L) All BDL <0.001 4 BDL; 4.60 (1.25 - 12.15)

Filter disc

Total IgE (kU/L) 11 BDL; 2.00 (2.00 - 5.60) 0.002 2 BDL; 7.80 (4.45 - 30.90)

HDM sIgE (kU/L) All BDL 0.001 6 BDL; 4.00 (2.00 - 13.75)

Serum

Total IgE (kU/L) 11.75 (9.96 -66.00) 0.001 164.00 (57.50 - 233.75)

HDM sIgE (kU/L) 12 BDL <0.001 27.30 (8.62 - 35.18)

Legends: Differences between groups were analyzed for each parameter by a Mann-Whitney U Test. All numbers are represented as median (IQR). BDL= Below detection level.

31    

A B

C D

E F

Figure 11: Comparison of total IgE and HDM sIgE concentrations in serum (E and F) and nasal secretions collected with Merocels (A and B) and filter discs (C and D) in control patients and AR patients. Total IgE and HDM sIgE are significant increased in HDM sensitive AR patients with all the different measurement techniques. All measurements are marked with a black open circle.

  32  

A B

C

Figure 12: Correlations between HDM sIgE concentrations in serum and in nasal secretions collected with Merocels and filter discs. Scatterplots displaying the correlation between HDM sIgE levels in Merocel and filter disc nasal secretions (A), in Merocel nasal secretions and serum (B) and in filter disc nasal secretions and serum in HDM sensitive AR patients (C). Rs: Spearman's rank correlation coefficient; p: significance level.

33

Legends: WNS: weight of nasal secretion collected, DF: dilution factor, /: below detection level, volume= (DF x WNS) – WNS.

Table 8: Total IgE and HDM sIgE measurements in HDM sensitive AR patients in serum and nasal secretions (filter disc and Merocel).

Serum Filter disc (DF 20) Merocel (DF 10)

RAW DATA FINAL DATA RAW DATA FINAL DATA

AR patients (n=15)

Total IgE

(kU/l)

HDM sIgE

(kU/l)

WNS (g)

Volume (ml)

Total IgE

(kU/l)

HDM sIgE

(kU/l)

Total IgE

(kU/l)

HDM sIgE

(kU/l)

WNS (g)

Volume (ml)

Total IgE

(kU/l)

HDM sIgE

(kU/l)

Total IgE

(kU/l)

HDM sIgE

(kU/l)

Patient 1 472.00 45.80 0.078 1.48 4.53 0.80 90.60 16.00 0.54 4.88 6.54 1.29 65.40 12.90

Patient 2 595.00 82.80 0.13 2.41 1.44 0.50 28.80 10.00 0.20 1.80 2.21 0.57 22.10 5.70

Patient 3 65.50 7.67 0.10 1.92 0.20 / 4.00 / 0.75 6.72 / / / /

Patient 4 307.00 33.60 0.081 1.54 5.25 1.26 105.00 25.20 1.14 10.28 2.97 0.99 29.70 9.90

Patient 5 134.00 36.10 0.11 2.07 1.58 0.75 31.60 15.00 0.42 3.77 5.94 2.43 59.40 24.30

Patient 6 26.65 10.70 0.11 2.01 0.25 0.20 5.00 4.00 0.28 2.48 0.35 0.23 3.50 2.30

Patient 7 166.50 33.00 0.076 1.44 0.22 / 4.40 / 2.05 16.42 / / / /

Patient 8 194.00 20.20 0.063 1.19 0.39 0.20 7.80 4.00 1.90 15.1 1.43 0.48 14.30 4.80

Patient 9 62.00 8.28 0.20 3.75 0.39 / 7.80 / 0.44 3.96 7.25 1.39 72.50 13.90

Patient 10 56.00 2.87 0.041 0.79 0.23 / 4.60 / 0.17 1.52 / / / /

Patient 11 164.00 29.10 0.037 0.70 1.03 0.40 20.60 8.00 1.63 13.02 0.50 0.21 5.00 2.10

Patient 12 197.00 35.70 0.056 1.07 1.14 0.43 22.80 8.60 1.13 9.04 1.05 0.46 10.50 4.60

Patient 13 28.40 3.42 0.16 3.08 / / / / 0.23 4.29 0.23 / 2.30 /

Patient 14 28.30 9.63 0.16 3.05 / / / / 0.59 5.32 0.26 0.20 2.60 2.00

Patient 15 246.00 27.30 0.028 0.53 4.49 1.35 89.80 27.00 0.14 1.29 6.40 1.75 64.00 17.50

Median 164.00 27.30 0.081 1.54 0.39 0.20 7.80 4.00 0.54 4.88 1.05 0.46 10.50 4.60

  34  

5 DISCUSSION

Local production of IgE in the respiratory mucosa has been demonstrated in AR

(allergen specific IgE) and in CRSwNP (polyclonal IgE), suggesting a role for local

IgE in the pathogenesis of these inflammatory diseases of the upper airway. It has

been suggested that the majority of allergen-specific IgE in the peripheral blood is not

derived from IgE-secreting cells in the blood, but is produced locally in target organs

of allergy such as the nasal mucosa. Considering the importance of local IgE

production, it seems to be essential to develop non-invasive, reproducible and easy to

perform approaches for measurement of local mucosal IgE production.

5.1 Merocels for local IgE measurement Tissue IgE is only partially related to IgE concentrations in serum [1,45]. We

identified a significant positive correlation between total IgE measured by means of

Merocels, serum and tissue in 112 patients with CRS (with and without NP).

Nevertheless, total IgE concentrations measured with Merocels were much lower than

those measured in serum or tissue. We already know that there is an initial increase in

total IgE concentration due to omalizumab. Omalizumab binds selectively free IgE

[48] and forms more slowly eliminating IgE complexes [76]. Besides free IgE,

complexed IgE is also included in the measurements, as a result total IgE

concentration increases shortly after commencing omalizumab therapy. This

phenomenon is also demonstrated in nasal secretions collected with Merocels. We

found a significant increase in total IgE after omalizumab treatment in patients with

CRSwNP and comorbid asthma at week 18. Patients with CRSwNP and comorbid

asthma are particularly characterized with high local IgE levels [48]. We also found

high baseline values of total IgE in this group. Recent research demonstrated that

omalizumab was equally efficacious in both allergic and non-allergic patients (SPT

negative) with NP and asthma [48]. This strengthens the hypothesis that blood tests

and skin prick tests are misleading us and that local IgE is more important. IL-5

seems to play a key role in the chemotaxis, activation, and survival of eosinophils

[77,78]. Bachert et al. [46] found an association between levels of both total and

specific IgE and eosinophilic infiltration in NP. These findings were unrelated to skin

prick test results. There is an increasing evidence that Staphylococcus aureus–derived

enterotoxins stimulate eosinophilic inflammation through production of TH2

35    

cytokines and local IgE formation [79]. In vitro studies indicate that SAE induced

cytokine release tends to be pro-inflammatory and Th2 skewed, promoting IL-4 and

IL-5 [80,81]. In our results, no significant differences were found in total IgE and

SAE-IgE concentrations after mepolizumab (anti-IL5) treatment for patients with

severe NP.

5.2 Filter discs for local IgE measurement We found total IgE, measured in nasal secretions with filter discs, in 9 out of 12 grass

pollen sensitive AR patients and in 10 out of 12 grass pollen allergic CRSwNP

patients. Gx3 IgE was measurable in 9 out of 12 grass pollen sensitive AR patients

and 7 out of 12 grass pollen allergic CRSwNP patients. IgE in CRSwNP is polyclonal

and allergen-specific IgE forms only a small fraction of the total IgE concentration. In

contrast, a big fraction of total IgE in nasal secretions in AR patients is allergen-

specific, resulting in sensitization of resident mast cells for immediate

hypersensitivity on exposure to allergen [66]. Total IgE and Gx3 IgE were not

significantly different in the AR group compared to the CRSwNP with allergy group.

Although IgE antibodies characterize the response to allergens, it also includes other

parameters like total IgG, IgG4 and tryptase [82]. A model [82] suggests that a route

to IgE production is a switch from IgM to IgG to IgG4 inside germinal centers with

subsequent switch from IgG4 to IgE production in the periphery. Zhang et al. found

evidence that the switch to IgE could occur sequentially through IgG4 [83]. IgG can

inhibit histamine release and IgG4 antibodies can block IgE receptor-facilitated

allergen binding to B cells and diminishes activation of mast cells and basophils

[82,84]. Total IgG and IgG4 concentrations were measurable with the filter discs, but

there were no significant differences between the different disease groups. Mucosal

IgE in NP tissue is functional by activating mast cells [85]. Tryptase is a marker of

mast cell activation, but no significant differences were found.

  36  

5.3 Comparison Merocels versus filter discs The datasets mentioned above were collected for other primary endpoints than local

IgE measurements. In those studies, local IgE concentrations were mostly determined

to research trends and to function as a possible activity predictor. In our experiment,

data were collected to investigate the presence of total IgE and HDM sIgE in nasal

secretions collected with Merocels and filter discs in control patients and AR patients

and to analyze differences between those groups. Further, we wanted to make a

comparison between the Merocel and the filter disc technique.

AR patients had highly increased total IgE and HDM sIgE concentrations in serum

and in nasal secretions collected with both Merocels and filter discs. HDM sIgE levels

in serum and in nasal secretions collected by means of Merocels and filter discs were

significantly correlated. Together, these observations support the validity of Merocels

and filter discs for local IgE measurements. In contrast with our expectations, filter

discs showed relatively good results. Total IgE concentrations in nasal secretions

collected with Merocels and filter discs were BDL in resp. 3 and 2 out of 15 AR

patients. HDM sIgE was BDL in 6 out of 15 AR patients, in nasal secretions collected

with filter discs, whereas in nasal secretions collected with Merocels HDM sIgE was

BDL in 4 AR patients. At this point, we can conclude that we can be optimistic about

the use of filter discs in clinical trials.

The Merocel technique is highly reproducible, as shown in both in vitro models and

humans. Unlike with filter discs, sufficient amounts of secretions can be collected

with Merocels to measure several mediators from one sample [54]. However, there

are also limitations to be kept in mind. Merocels are larger and can cause local

irritation and stimulation of the nasal mucosa. Stimulation of the mucosa may vary

from time to time, which makes Merocels probably less reproducible in comparison

with filter discs. For these reasons, Merocels are not recommended for repeated

measurements on the same day and in nasal provocation tests. Insertion of the

Merocel into the nose may be difficult and uncomfortable in patients with an anterior

septal deviation, a narrow vestibulum nasi or severe NP [54]. Filter discs are easier to

manipulate and its placement in the nasal cavity is simple. In a study from Kristiansen

et al., filter discs were used to detect serum IgA, IgM and IgG antibody activity. The

secretory IgA concentrations of multiple samples from one person were stable, which

proved reproducibility [63]. Filter discs also have disadvantages. They are not

37    

commercialized and thereby not medically approved. Another disadvantage is the fact

that filter discs require a higher dilution factor. As a result, total IgE and HDM sIgE

concentrations, which are BDL in nasal secretions collected with filter discs, are

sometimes just above the detection limit in nasal secretions collected with Merocels

(table 8, patient 13 and 14). With both Merocels and filter discs, a clear differentiation

and comparative analysis between nasal sides is possible. On the other hand, no clear

distinction can be made between nasal secretion coming from the septum, the

turbinates, the sinus cavities or local glands with both Merocels and filter discs [54].

The amount of nasal secretions depends on several factors. As an example, in upper

respiratory tract infections watery secretions are an early symptom [86]. As

mentioned before, Merocels can give a variable stimulation of the nasal mucosa and

thereby induce a natural dilution of the nasal secretion. Heikkinen et al. [87]

postulated that the interpretation of substances present in nasal specimens is difficult

because of the unknown dilution in these samples.

In summary, we can conclude that IgE levels in nasal secretions collected by means of

Merocels seem to be a good reflection of local IgE levels in nasal tissue. As collection

of nasal secretions by Merocels is a standardized and non-invasive method, it can be

used for repeated measurements of local IgE and other markers of mucosal

inflammation, in order to study nasal diseases and monitor their treatment. In the

experiment, Merocels performed slightly better than filter discs. The use of filter discs

seems to be a feasible alternative for collection of nasal secretions and measurement

of local IgE. Both Merocels and filter discs have advantages and disadvantages and

both methods will probably have their specific applications in future experiments and

clinical trials. Strengths and weaknesses of tissue, Merocels and filter discs are

summarized in table 9.

  38  

Table 9: Summary strengths and weaknesses tissue, Merocels and filter discs.

Strengths Weaknesses

Tissue Merocels Filter discs Tissue Merocels Filter discs

Golden standard

Highly reproducible

Probably more reproducible compared to

Merocels

Very invasive

Local irritation and stimulation

Not standardized and

commercialized

Superior local IgE

measurements

Sufficient amounts of secretions

Less discomforting

and local irritation

Can not be used several times on the

same day

Not recommended to use them several

times on the same day

Higher dilution factor needed

Clear differentiation between nasal

sides

Clear differentiation between nasal

sides

Clear differentiation between nasal

sides

No clear distinction

between different origins

No clear distinction

between different origins

Informs directly about a

range of mucosal tissue cellular events

Standardized and non-invasive Non-invasive Unknown

dilution Unknown dilution

Slightly better local IgE

measurements compared to filter discs

Easy to manipulate and

simple placement Expensive

Allow qualitative measurements

Allow qualitative measurements

Sometimes difficult and

uncomfortable insertion

Can be used for

follow-up in clinical trials

Can be used for follow-up in

clinical trials and in nasal

provocation tests

Good reflection of local IgE

levels in nasal tissue

Good reflection of local IgE

levels in nasal tissue

Lower dilution factor needed

39    

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APPENDICES Appendix 1: Overview of the ‘superantigen hypothesis’ of CRS. Retrieved from [1].

Appendix 2: Management scheme for adults with CRS with NP for ENT specialists based on the EPOS guidelines. Retrieved from [8].

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European Position Paper on Rhinosinusitis and Nasal Polyps 2012

8.5.3.2. Treatment For treatment evidence and recommendations for CRSsNP see

Table 8.3 and 8.4.

Treatment should be based on severity of symptoms

Decide on severity of symptomatology using VAS and

endoscope. See Figure 8.5.

Acute exacerbations of CRS should be treated like acute

rhinosinusitis.

8.5.4. Evidence based management for adults with CRS with NP for ENT specialists8.5.4.1. DiagnosisSymptoms present longer than 12 weeks

Two or more symptoms one of which should be either nasal

blockage/obstruction/congestion or nasal discharge (anterior/

posterior nasal drip):

± facial pain/pressure,

± reduction or loss of smell;

Signs

ENT examination, endoscopy;

review primary care physician’s diagnosis and treatment;

questionnaire for allergy and if positive, allergy testing if it

has not already been done.

Figure 8.6. Management scheme for adults with CRS with NP for ENT specialists.

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Appendix 3: Position of a Merocel inside the nasal cavity between the inferior turbinate and the septum. Retrieved from [54].

Appendix 4: Classification of allergic rhinitis according to ARIA Retrieved from [10]. Classification of allergic rhinitis according to ARIA.

1. Intermittent means that the symptoms are present

<4 days a week Or for <4 consecutive weeks

2. Persistent means that the symptoms are present More than 4 days a week

And for more than 4 consecutive weeks 3. Mild means that none of the following items are present:

Sleep disturbance Impairment of daily activities, leisure and/or sport

Impairment of school or work

Symptoms present but not troublesome 4. Moderate/severe means that one or more of the following items are present:

Sleep disturbance Impairment of daily activities, leisure and/or sport

Impairment of school or work Troublesome symptoms