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���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������LbjbjW�W�7R5�5��k7��������	�	���������8<L��RA��6����)@+@+@+@+@+@+@�C��FF+@��+@����KA�<�<�<
����)@�<)@�<�<�<�����0�u�\G�%5��<@"A0RA�<�F�8��F�<�F��<��<+@+@�<RA���������������������������������������������������������������������F�	�:Comparison of Clinical and Biochemical in Allergic versus Nonallergic Rhinitis
Ula_ Erayd1n1 , Ceren G�nel2,  Leyla Didem Kozac13, Muhan Erku_4, H.Sema Ba_ak2
1Ula_ Erayd1n, M.D. Ardahan Kahta State Hospital.
2Ceren G�nel, M.D. Assistant Professor. Department of Otolaryngology-Head and Neck Surgery, Adnan Menderes Univercity Medical School Hospital.
Adress: Adnan Menderes Universitesi T1p Fak�ltesi Hastanesi KBB AD, Aytepe Mevkii
09100, Ayd1n, Turkey
Cell: +90 533 7175693
Fax: + 90 256 2182037
 e-mail: drgunel@hotmail.com
3Leyla Didem Kozac1, M.D. Proffessor. Department of Medical Biochemistry, Adnan Menderes Univercity Medical School Hospital.
4Muhan Erku_, M.D, Proffessor. Department of Medical Pathology, Adnan Menderes Univercity Medical School Hospital.
5H.Sema Ba_ak, M.D, Professor. Department of Otolaryngology-Head and Neck Surgery, Adnan Menderes Univercity Medical School Hospital.

Paper was approved by the Institutional Review Board.







ABSTRACT 
Background: Rhinitis can be induced by different mechanisms and involves several etiological agents. It is important to differentiate between alergic rhinitis and nonalergic rhinitis, as management differs for each.

Objective: We aimed to study the several biomarkers of alergic rhinitis and nonalergic rhinitis to investigate their potential as outcome measures in clinical intervention trials. 

Materials and Methods: All study groups were compared in terms of life quality standards, mucociliary clearance time, nasal eosinophil percentage, serum total IgE levels, air flow level, various biomarkers in nasal fluid. All groups were evaluated for asthma by a respiratory medicine specialist performing pulmonary function testing.  
Results: Serum total IgE level, nasal irrigation IgE level and nasal mucociliary clearance time averages were significantly different in patients with alergic rhinitis compared to both nonalergic and control groups (p=0.018, p=0.044, p=0.025). Nasal eosinophillia percentages, nasal lavage IL-5 and Vasoactive intestinal peptide levels were significantly difference in the alergic and nonalergic groups (p=0.000, p=0.001). 
Conclusion: Detection of total IgE in nasal fluid when presence of a history of allergy promotes diagnosis of allergic rhinitis. Presence of high VIP and IL-5 levels in nasal fluid and presence of nasal eosinophillia can be useful in evaluating AR/NAR subgroups. Therefore novel therapies may improve in the future. 


Key Words: allergic rhinitis, Ig E, IL-5, nonallergic rhinitis, VIP.





Introduction
Nasal mucosal inflammation can occur with various mechanisms and therefore it is difficult to classify the causes leading to complaints of rhinitis and to make a differential diagnosis [1]. The diagnosis of AR is based on clinical manifestations and supported by a positive result for skin prick test (SPT) or serum specific immunoglobulin E (sIgE) antibodies to aeroallergens. In contrast, rhinitis is diagnosed as NAR when an allergic cause has been ruled out by the presence of an inconsistent clinical history, a negative SPT, and the absence of serum sIgE antibodies [2]. 
Our aim in this study was to analyze eosinophils in nasal smear, mucociliary clearance time (MCT), serum IgE levels and substance P (SP), Calcitonin gene related peptide (CGRP), vasoactive intestinal peptide (VIP), endothelin, interleukin 5 (IL5) and Immunoglobulin E (IgE) values in nasal irrigation fluid of the patients with AR and NAR to investigate their potential as outcome measures in clinical intervention trials. Therefore novel therapies may improve in the future. We also looked for an answer to question of whether AR and NAR affects daily life, mental health and physical functions and whether there is a difference between the two groups.











Materials and Methods
Skin prick test was performed [3]with the initial diagnosis of rhinitis on the patients who admitted to Ear Nose and Throat (ENT) outpatient clinic with at least two of the following complaints: nasal obstruction, rhinorrhea, nasal itching and sneezing. Those with a response to at least one allergen in skin prick test were included in AR group, while those who did not display response to any allergen were included in NAR group. Besides, a control group was constructed from healthy individuals. Each group consisted of 13 individuals, total of 39 people were evaluated in the study. Subjects with any other clinically relevant disorders were excluded.. Informed consent was obtained from the participants. The study had ethical approval from the hospital institutional review board.
History and physical examination findings of all study groups were recorded in prepared registry forms. Nasal obstruction, rhinorrhea, sneezing, nasal itching, nocturnal mouth-breathing, snoring, loss of taste and smell, hearing disorder and asthma diagnosis were interrogated. Onset of the symptoms, factors aggravating the symptoms, accompanying diseases, family history of atopy, allergens at home and at work and previous medical treatments were recorded in the forms. 
Standard life quality forms (SF-36) [4] were filled for all individuals who participated in the study. The SF-36 is a multipurpose general health survey that measures eight domains of health: physical functioning, role limitations due to physical health, bodily pain, general health perceptions, vitality, social functioning, role limitations due to emotional problems, and mental health (0-100 point range). Besides, average of the first four headings were calculated as physical health component (PHC) and average of the last four headings were calculated as mental health component (MHC) and groups were compared to each other.
All groups were evaluated by a chest specialist and respiratory function test (RFT) was performed.
 Serum total IgE (IU/mL) was measured on an Immulite 2000 automated analyzer (DPC) using the chemiluminescent enzyme immunoassay method. Nasal smear was taken from medial side of the inferior concha using disposable brush. Groups were compared in terms of eosinophil percentages in the nasal smear. Nasal irrigation samples were placed in eppendorf cups and all samples were collected and kept at -80oC until the study was completed.  IL-5 measurement was done with  human IL-5 ELISA kit (BenderMed Systems, Cat. No:  BMS278), IgE levels with DRG company kit (Cat. No: EIA-1788), VIP with human VIP ELISA kit (Phoenix Pharmaceuticals, Cat No: EK-064-16), CGRP with human CGRP ELISA kit (SPI-BIO, bertin Pharma, Cat No: A05481),  Endothelin with human Endothelin ELISA kit (Cayman, Cat No: 583151), Substance P with human substance P ELISA kit(Cayman, Cat No: 583751); all measurements were performed in accord to instructions given in the producer company�s prospectus. Results were recorded at 450 nm using ELISA reader (ThermoLab Systems). 
Nasal mucociliary clearance time  was assessed with the saccharin test as described previously. (Greenstone M, Cole PJ. Ciliary function in health and disease. Br J Dis Chest 1985;79:9Y26) Nasal peak flow meter measurements was performed by make an expirium from the nose rapidly into the peak flow meter equipment.  Results was read and recorded for all patients.

Statistical Method
Accordance of the groups for normal distribution was analyzed with Kolmogorov-Simirnoff Test. Since flowmeter and saccharine levels do not display a normal distribution, they were showed as median value (25%-75%). For statistical comparison, Kruskal Wallis ANOVA test was used. Since other variables had normal distribution, they were displayed as mean � standard deviation and single way- variant analysis was performed. In comparison of categorical variables, Chi-square test was used and definitive statistics were showed as number and percentage. A value of pd"0,05 was considered as statistically significant. 
RESULTS
Demographic characteristics and family history of allergy  of the study cases are shown in Table 1. The distribution and duration of symptoms are summarized in Table 2. 
SF-36 form scores of the groups are summarized in Table 3. Highest scores in all of the 8 subcomponents in the SF-36 form belong to control group (p=0,001). But there were not significant difference between AR and NAR group. 
Peak flowmeter and saccharine test results are summarized in Table 4. Nasal MCT of patients with AR were significantly prolonged compared in NAR group and control group (p=0,033).
Respiratory function test results in all study cases were evaluated by a pulmonologist and are shown Table 5. In patient groups that were evaluated in terms of presence of asthma, 1 of the 13 (8.3%) patients who underwent respiratory function test was diagnosed as asthma in AR group, whereas in NAR group, 2 of the 13 patients (15,4%) who underwent respiratory function test was diagnosed as asthma. In control group, none of the 13 patients (0%) who underwent respiratory function test was diagnosed as asthma. Differences of asthmatic case numbers among the groups was not statistically significant (p>0,05). 
Groups were evaluated considering serum total IgE levels of 100 IU/ml and below as normal and above 100 IU/ml as high (Table 6). Serum total Ig E levels were significant higher in AR group compared to NAR and control groups (p=0,018). 
In nasal smears, presence of eosinophilia was shown Table 7. Allergic rhinitis group had higher number of eosinophilia compared to the control group (p=0,044).
Mean levels of IgE, IL 5, VIP, SP and Endothelin in nasal irrigation fluids are shown in Table 8. Ig E levels were significantly higher in AR group compared to NAR and control groups (p=0,004) while IL-5 and VIP levels were significantly higher in AR and NAR groups compared to control group (p=0,004, p=0,002, p=0,005, p=0,001). 
When the groups were compared in substance P, CGRP and Endothelin-1 levels in nasal irrigation fluid, the difference was not significant (p>0,05). 
Discussion
Allergic rhinitis and NAR affect the quality of life in all aspects for the patients [5,6]. Evaluation of life quality related to health can be performed using general or specific survey forms. In our study, we used SF-36, which is a general survey. In terms of quality of life, two subgroups of the rhinitis were markedly different from each other, however, we did not find any difference between two rhinitis groups (Table 3). Consistent with the literature, our findings established that quality of life was affected similarly in patients with AR and in patients with NAR [7,8].
Can the history and accompanying diseases give a clue for differential diagnosis between AR and NAR? 
One our findings that attracts attention is that family history of atopy in AR group was 38,5% while it was 61,5% in NAR group. Veskitkul et al found that 45.7% of the patients with NAR had a family history [9]. These results suggest that familial atopy does not indicate only AR, but they can be important for NAR�s as well. 
Another finding was the fact that majority of the patients in AR group defined intermitant complaints, while those with NAR defined perennial complaints. Most common complaint was sneezing in AR group and nasal obstruction in NAR group. Besides there was not any difference between the patients in terms of asthma. Our findings on frequency of complaints, characteristics of being intermitant and perennial, and relation with asthma in AR and NAR patients were similar to those in the study done by Kalpakl1olu et al [7].
Which laboratory tests could be helpful for differential diagnosis of two major subgroups of the rhinitis? 
Mucociliary clearance time; studies showed that eosinophils and their products can prolong MCT by damaging the mucosa epithelium [10,11]. In our study, MCT was significantly prolonged in AR group compared to NAR and the control groups. This inconsistency can occur because we did not make a subclassification in NAR group in terms of nasal eosinophilia.  
Nazal eosinophilia; It was shown in many studies that most dominant cells in allergic diseases are eosinophils [12-14]. Nasal eosinophilia is a nonsensitive but specific test in diagnosis of AR [15]. In SPT positive patients presence of nasal eosinophilia supports the diagnosis of AR but an eosinophilia-negative nasal smear does not rule out the diagnosis [16]. In our study we found nasal eosinophilia in 61% of the patients in AR group, 30,7% in NAR group and 15,3% in the control group. We also believe that nasal eosinophilia supports the diagnosis of AR.
Serum total IgE; total IgE level was significantly high in AR patient group compared to the other two groups. However, while high IgE levels support the diagnosis of atopic disease, normal IgE levels do not rule out the diagnosis of allergic disease. They can be used for diagnosis in cases with asthma and rhinitis, dermatologic diseases, allergic bronchopulmonary aspergillosis, immune deficiencies and drug reactions [17]. 
Local/nasal IgE; Although rhinitis symptoms mainly occur due to local changes in sinonasal region, diagnostic tests search for systemic response. Local IgE production is increasingly recognized as an important factor in sinonasal inflammation [18]. Systemic allergy testing methods may not detect increased levels of IgE produced locally [4]. Increased levels of locally produced IgE may be anticipated in the setting of allergic rhinitis [19]. Local allergic rhinitis describes nasal production of specific immunoglobulin E antibodies  in the absence of serum specific IgE antibodies and a negative SPT [2,19]. Rondom et al reported that patients with local allergic rhinitis shares AR symptoms [20]. We measured total IgE levels not specific Ig E in nasal irrigation fluid and found that nasal IgE averages were significantly higher in AR group compared to other groups. High IgE averages in nasal secretions of the patients with AR can arise from local IgE secretion. We think that total IgE values in nasal fluid    may helpful in diagnosis of AR at the countries which SPT and serum specific IgE antibodies are not paid by the insurance companies as well as in our country.
Local/nasal inflammatory factors; It is known that IL-5 has many effects on eosinophils. These include effects on differentiation and proliferation, chemotactic properties, accumulation of eosinophils in inflammation region by inducement of adhesion molecules and antiptotic effects [18]. Also IL-5 has a role in early and late phase of IgE related hypersensitivity [21]. In our study nasal irrigation IL-5 levels were significantly high in both AR and NAR groups, while we did not find any significant difference between in the two groups. We believe that detection of IL-5 in nasal secretion supports the presence of sinonasal inflammatory pathology, however it can not be used to differentiate AR and NAR.
Airway nerves are involved in the regulation of airway function under physiological conditions and in the pathophysiological events of allergic airway inflammation. Sensory nerve fibers contain various sensory neuropeptides, including the tachykinin, SP and neurokinin A, whereas parasympathetic nerve endings contain VIP. Substance P acts on vascular permeability via the NK1 receptor, thus leading to nasal obstruction and mucus secretion from the submucosal glands [22]. In addition, SP has cellular effects on mast cells, T and B cells, and macrophages. CGRP is a potent vasodilator with its receptors being concentrated on a few veins, small arteries and arterioles with none being found on the gland cells [23]. While in some studies it was reported that VIP and SP played an important neuroimmunologic role in pathogenesis of AR, with no role of CGRP [24], some studies reported that these three neuropeptides had no role [24,25].  In our study highest level of VIP were in NAR group, followed by AR group, and when both groups were compared with the control group, results were significantly higher. Detection of high VIP levels in AR and NAR groups without any provocation that induces nasal secretion is a valuable finding. On the other hand, there was not any difference among the groups in terms of substance P and CGRP averages in our study. According to us, VIP may play a role in the cystic degeneration of glandular cells and lead to the pathogenesis of the hypertrophic and hypersecretive changes both of AR and NAR. Developing specific antagonist of VIP will further the treatment of rhinitis.
There are limited number of studies in literature on search for endothelin-1 in nasal secretion. When the groups were evaluated in terms of endothelin levels in nasal irrigation fluid, although highest value was detected in AR group, there was not statistically significant difference among the groups. We think that presence of endothelin-1 in nasal irrigation is not valuable in diagnosis of rhinitis. 
Conclusion
As a result, detection of total IgE in nasal fluid when presence of a history of allergy promotes diagnosis of allergic rhinitis especially at the countries which SPT and serum specific IgE antibodies are not paid by the insurance companies as well as in our country. Presence of high VIP and IL-5 levels in nasal irrigation fluid and presence of nasal eosinophillia can suggest sinonasal inflammatory pathologies. Besides, they can illuminative in phenotyping NAR and can be leading in rhinitis treatment. We support the opinion that determination of SP, endothelin-1 and CGRP in nasal irrigation fluid has no place in sinonasal pathologies. Our study also showed that AR and NAR affected the quality of life negatively in every aspect of lives of the patients.
















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TABLES

Patient groupMaleFemaleAge(years)P valueFamily history of atopyAllergic rhinitis 4(%30,8)9(%69,2)31,23�11,54>0,05    5 (38,5%)Nonallergic rhinitis5 (%38,5)8 (%61,5)30,92�11,70>0,05    8 (61,5%)Control5 ((%38,5)8 (%61,5)37,38�8,88>0,05     0

Table 1. Patient demographics.














SymptomAllergic groupNonallergic
groupControl
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��$Ifgdl2�Ff>	$Ifgdl2�646,213100Nazal itching+861,5969,2000,000-538,5430,813100Sneezing+131001076,9000,000-00323,1131000ntermitant
969.2646.1500Persistent
431.8753.8500

Table 2. The distribution and duration of symptoms.











AR Score AvaregeNAR Score AvaregeControl Score AvaregeP valuePhysical Functioning78,0777,6989,61Physical Health63,4644,2388,46Bodily Pain74,3067,3881,69General Health Perception56,7645,6965,00Vitality58,4655,0075,38Mental Health66,1566,7677,23Social Functioning72,1188,4692,30Emotional Problems58,9861,5569,23Physical Health Component 47,2442,5352,33p=0,001Mental Health Component44,5047,7050,71p>0,05

Table 3: SF-36 form scores of the groups.











Allerjic Rhinitis Group
Median(%25-%75)Non-allerjik Rhinitis Group
Median(%25-%75)Control Group
Median(%25-%75) P valueSaccharine test8,5(7-10,75)4(3-8,25)5,5(4,75-9,5)0,033Peak flowmeter72,50(70-90)95(65-115)95(85-107,5)0,080
Table 4: Results of saccharin test and peak flowmeter analyse.





































Allerjic 
Rhinitis GroupNon-allerjik Rhinitis Group
Control Group
P valueFEV1 %107,90109,08109,930,918FEV1/FVC %97,1399,8199,200,383FEF 2599,42100,30106,700,646FEF 5093,0598,7895,060,807FEF 7568,1080,1163,840,202
Table 5: Results of respiratory function test .














 Allerjic 
Rhinitis Group       Non-allerjik Rhinitis Group
Control Group
P valueNumber%Number%Number%
Ig EHigher 1001076,9323,1538,5
0,018Lower
100323,11076,9861,5 
Table 6: Results of  Ig E levels.














Nasal SmearAllerjic 
Rhinitis Group     Non-allerjik Rhinitis Group
Control Group
P valuenumber%number%number%Eosinophiliapresence861,5430,8215,40,044absence538,5969,21184,6
Table 7: Results of  eosinophilia in the nasal smear.















AR Score AvaregeNAR Score AvaregeControl Score AvaregeP  valueIg E2,942,522,500,025IL 559,12132,075,800,000VIP0,34830,37780,10570,001Substance P136,28139,57137,330,566Endothelin-121,5716,8917,170,687CGRP24,6821,9327,330,585
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