Title: Association of salivary inflammatory biomarkers with primary Sjögren’s syndrome. Running title: Salivary IL-6, 5 and 4 in Sjögren's syndrome. Authors: Luz A. Moreno-Quispe1, Julia Serrano2, Leire Virto3, Mariano Sanz2, Lucía Ramírez2, Mónica Fernández-Castro4, Gonzalo Hernández2, Rosa María López-Pintor2 1Researcher, Department of Dental Clinical Specialties, School of Dentistry, Complutense University, Madrid, Spain. Department of Investigation, Innovación y Desarrollo Perú SAC, Ancash, Perú. 2Department of Dental Clinical Specialties, School of Dentistry, Complutense University, Madrid, Spain. 3Researcher, Etiology and Therapy of Periodontal Diseases (ETEP) Research Group, Complutense University and Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain. 4Rheumatology Service, Hospital Puerta de Hierro, Madrid, Spain. Corresponding Author: Rosa María López-Pintor Muñoz Departamento de Especialidades Clínicas Odontológicas Facultad de Odontología Universidad Complutense de Madrid Plaza Ramón y Cajal s/n, 28040-Madrid, Spain E-mail: rmlopezp@ucm.es Acknowgledments This article has been developed thanks to the financing of Fondecyt, initiative of the National Council of Science, Technology and Technological Innovation (CONCYTEC). Contract N° 227-2015-FONDECYT. We thank Peter Christensen and Elizabeth Christensen for reviewing the English language version of the manuscript. mailto:rmlopezp@ucm.es Conflict of interests The authors declare no conflict of interest Abstract Background: Primary Sjogren’s syndrome (pSS) is an autoimmune disease that leads to salivary and lacrimal gland dysfunction. The adaptive immune response associated with T helper-2 lymphocytes appears to be altered in these patients. Therefore, the objective of this study was to determine the salivary levels of IL-6, 5 and 4 in patients with pSS when compared to a healthy control (HC) group. The secondary objectives were to study whether ILs levels in pSS patients were associated with salivary flow, patient-reported outcomes (PROMs) for xerostomia and oral health quality of life (OHIP-14), pSS classification criteria and presence of extraglandular manifestations. Methods: A case-control study was conducted in 36 patients with pSS and 35 HCs. Cytokine levels were measured using high-sensitivity multiplex map human immunoassays. Unstimulated and stimulated whole saliva were collected and patients filled out the questionnaires. The U-Mann-Whitney test, chi-squared and Spearman correlation test were used. Results: IL-6 was significantly higher in pSS patients than in HCs (p=0.0001). IL-6 was significantly higher in pSS patients with a positive salivary gland biopsy (p=0.04), whole stimulated saliva hyposalivation (p=0.02) and presence of musculoskeletal disorders (p=0.03). There was a non-significant positive correlation between IL-6 levels and PROMs for xerostomia (r=0.31; p=0.06) and OHIP-14 (r=0.07; p=0.68) in pSS patients. Levels of IL4 and IL5 were not detected in both pSS and HCs patients. Conclusions: Salivary IL-6 levels were significantly associated with pSS patients and therefore, it is hypothesized that this biomarker may be useful in the diagnosis and follow-up of this disease. Keywords: Primary Sjögren's syndrome, Salivary interleukins, Salivary biomarkers, Visual Analog Scale for xerostomia, Oral Health Impact Profile-14. Introduction Primary Sjogren’s syndrome (pSS) is a chronic inflammatory and autoimmune disease with complex and heterogeneous course that leads to salivary and lacrimal gland dysfunction manifested by xerostomia and xerophthalmia(1,2). Although the specific aetiology of this disease has not been fully elucidated, it has been attributed to alterations in the immune response, mainly the adaptative immunity since there is an increase in the inflammatory infiltrate in the salivary glands of SS patients mainly composed by CD4+ T lymphocytes(3). This disease affects mainly women between the fourth and fifth decade of life and its prevalence rate is 60.82 cases per 100,000 people(4), what implies that other genetic, endocrine, and environmental risk factors may be involved. Humoral adaptive responses are characterized with a T helper (Th2) lymphocyte response with a predominant cytokine profile of IL-4, IL-5, IL-6 and IL-10. In fact, several reports(3,5,6,7), have attributed high cytokine levels of IL-6, IL4 and IL-5 in the initiation and progression of SS, resulting in high concentrations of B cells located at the inflammatory infiltrate within the salivary glands. Other studies have detected high levels of cytokines present in biopsy samples of minor salivary glands, as well as serum and saliva of pSS patients, although their diagnosis validity has not been demonstrated (3,5,8,9,10,11,12). In most of these studies the levels of measured cytokines were significantly higher than matched controls, but it was not clear whether the elevated levels were related to the progression of the disease, the decreased salivary flow and/or the reduced quality of life. Furthermore, these studies have utilized different biochemical methods to assess these proteins and only two studies(9,10) have used highly sensitive methodologies, which are especially useful when the volume of saliva is small, as it occurs in the case of patients with SS. The search of biomarkers in saliva as potential diagnostic indicators is increasingly sought for the diagnosis of many chronic diseases due to their simplicity and non- invasiveness, particularly in debilitating diseases such as SS(9,13). It was, therefore, the primary objective of this study to determine whether the cytokine levels of ILs 4, 5 and 6 in saliva would have any diagnostic value when comparing samples from pSS patients versus matched healthy controls (HC). Secondarily, the study aimed to assess whether the obtained cytokine levels were associated to hyposalivation, salivary flow rates (stimulated and unstimulated), pSS classification criteria and extra-glandular manifestations and patient reported outcomes (PROMs) in pSS patients. Materials and methods Patients In this cross-sectional case-control study 36 pSS were selected once diagnosed according to the criteria of the 2002 European-American Consensus(14). As matched controls 35 healthy subjects (HC) were recruited from volunteers, with similar age and sex, attending the postgraduate dental clinics at the Faculty of Odontology in the University Complutense of Madrid. All participants were recruited between December 2015 and March 2018 and signed an informed consent form. The study fulfilled the Helsinki Declaration and its subsequent revisions and had been previously approved by the Ethical Committee at the San Carlos University Hospital in Madrid (# 17/246 E). Subjects were excluded from participating when fulfilling any of these criteria: under 18 years of age; presence or history of hepatitis C infection, human immunodeficiency virus infection, sarcoidosis, cancer, lymphoma, head and neck radiation, graft versus host disease and any other uncontrolled systemic diseases; untreated periodontitis (with presence of nine or more sites with probing deep ≥ 5mm and with full-mouth bleeding score> 25%); subjects being treated with immunosuppressants or anticholinergic, immunological or xerostomizing drugs; and finally pSS patients with SWS salivary levels <0.02 ml/min. Selected pSS patients had a full examination by their rheumatologist reporting the following variables: time from the diagnosis to the visit to this study, criteria for the 2002 European-American Consensus, serological data (rheumatoid factor, antinuclear autoantibodies, immunoglobulins alteration), and presence of systemic manifestations of pSS. A complete orofacial examination was carried out in all patients by a specialist in Oral Medicine (RMLP), including a full periodontal examination and a focused examination of the oral mucosa and salivary glands. Saliva collection Unstimulated and stimulated saliva (UWS and SWS) was collected between 9:00 to 11:00 am. having asked the participants not to brushing their teeth, or eat, drink or smoke in the previous 90 minutes before the appointment. In a relaxed environment, subjects rinsed with water before the test and first, the UWS was collected during 15 minutes, and then using paraffin (Paraffin Pellets, Ivoclar Vivadent, Schaan, Lichtenstein) SWS was collected during 10 minutes. In these collections participants were asked to push the head forward in order to accumulate the saliva and then to drop it into a sterile container without making any effort with the mouth. The obtained UWS and SWS salivary flows were quantified in ml/min. Hyposalivation was defined as a flow rate <0.1ml/min for UWS and <0.7ml/min for SWS. Saliva collection and quantification were performed by a specialist in Oral Medicine (JS). The SWS flow was stored at -80° C until and processed at a later stage by full trained investigators (LV and LAM). Biochemical determinations The stored SWS was thawed and centrifuged at 3500 rpm before analysis. The concentrations of IL 4, 5 and 6 were measured using high-sensitivity multiplex map human immunoassays (Millipore® corporation, Cat. # HSTCMAG-28SK, Billerica, MA, USA) using the Luminex-200 System and the XY platform (Luminex® Corporation, Oosterhout, the Netherlands). Prior to processing the samples, the technique was calibrated using sheath fluid and microspheres obtained from Luminex® Corporation for classification and reporter readings. The detection limits for IL-4, IL-5 and IL-6 were 1.24, 0.46 and 0.11 pg/ml, respectively. The obtained results were analysed with the xPonent® software and were expressed as picograms per millilitre (pg/ml). Patient reported outcomes (PROMs) To assess the symptoms of xerostomia, we used the VAS validated for this purpose that contains 8 questions about oral dryness(15). VAS was filled by the participants prior to saliva collection, indicating the level of oral dryness they perceived, by marking a vertical line on a straight line measuring 10 cm (being the mark in 0 when no dryness was perceived and in 10 when the greatest imaginable oral dryness was perceived). To measure the impact of xerostomia in their oral health quality of life, participants filled the validated OHIP-14 questionnaire in Spanish(16). The questionnaire was completed by patients prior to saliva collection and consisted of 14 elements using a Likert scale of five points ranging from "never" (coded 0) to "very often" (coded 4), evaluating oral function and quality of life. Statistical analysis IL-6 concentrations were considered the primary outcome variable. The sample size was calculated using data from a previous study that compared salivary levels of IL-6, IL- 17A and nitric oxide between pSS patients and a control group(17). Considering a difference of 3 pg/mL in IL-6 levels between pSS and HC, with a standard deviation of 2.9 (pg/mL) obtained from the previously mentioned investigation, 24 subjects in each group would be required to detect statistically significant differences using an = 5% and a statistical power of 95%. Data was analysed with the Kolmogorov-Smirnov goodness-of-fit test to determine their normality. The U-Mann-Whitney test was used to detect differences between the pSS and HC group in the continuous variables and the chi-squared test for the qualitative variables. To associate two quantitative variables in the pSS group, the Spearman correlation test was used. All statistical tests were performed using the SPSS 25.0 software (SPSS Inc., Chicago, IL, USA) and the level of significance was established if p≤0.05. Results A total of 36 patients with pSS and 35 HCs were included. They were all women with mean ages of 56.58 ± 12.35 in the pSS group and 54.40 ± 9.16 in the HC group. The characteristics of the pSS patients are depicted in Table 1. Salivary flow levels were lower in the pSS group than in the HC group for both UWS (UWS 0.14 ± 0.16 vs. 0.36 ± 0.21; p=0.0001) and SWS (0.83 ± 0.73 vs. 1.50 ± 0.78; p=0.0001) (Table 2). The VAS and OHIP-14 scores were significantly greater in pSS patients than HCs, VAS 45.25 ± 13.83 vs. 14.31 ± 4.57 (p=0.0001) and OHIP-14 score 19.97 ± 13.23 vs. 0.80 ± 2.81 (p=0.0001) (Table 2). Table 3 depicts the results from the biochemical analysis. The concentrations of IL-6 were significantly higher in pSS patients compared to HCs, 9.73 ± 23.45 vs. 0.93 ± 2.70 (p=0.0001). Levels of IL-4 and IL-5 were almost undetectable in both groups (Table 3). We observed that pSS patients that suffered SWS hyposalivation had significantly higher salivary levels of IL-6 (19.11 ± 34.07 vs. 3.02 ± 6.01; p=0.02). Patients with UWS hyposalivation had also higher salivary IL-6 levels, but not statistically significant (14.67± 32.45 vs. 5.30±9.45; p=0.59). Table 4 depicts the possible associations between salivary levels of IL-6 and pSS classification criteria, extraglandular and serological manifestations. We observed that pSS patients with positive histological lesions in minor salivary glands and musculoskeletal disorders had significantly higher levels of IL-6 (p=0.04 and p=0.03, respectively). Furthermore, IL-6 levels were also correlated with PROMs, although these associations were not statistically significant (Table 5). Almost significant positive correlation was obtained between IL-6 and VAS for xerostomia (r=0.31; p=0.06). Similarly, there was a non-significant positive correlation between the total values of OHIP-14 and the levels of IL-6 (r=0.07; p=0.68). Discussion The main objective of this cross-sectional case-control study was to study the association between the cytokine (ILs 4, 5 and 6) levels in saliva and the diagnosis of pSS. Furthermore, to assess whether these biomarker levels in saliva were correlated with the severity of the disease measured by the salivary flow, or its progression measured by the extra-glandular manifestations and the patient’s own self-assessment. We found significant higher levels of IL-6 in pSS patients compared with matched HC controls, however the levels of the other two measured cytokines (IL4 and IL5) were almost undetected in both groups. When focusing on the pSS patients, significant association were found between IL-6 levels and SWS hyposalivation, positive histology in minor salivary glands and musculo-skeletal disorders. In addition, an almost significant positive correlation was observed between IL-6 levels and VAS for xerostomia in the pSS group. These results corroborate previous studies also reporting significant higher levels of IL- 6 in saliva of pSS patients when compared with HCs, although using other biochemical detection technologies, such as ELISA(8,12) or Multiplex(7) in samples of SWS and parotid salivary flow. We used in this investigation the highly sensitive Luminex® system, also used by Nguyen et al. and Kang et al. who reported similar significantly higher IL-6 levels in pSS patients when compared with matched controls. This technique offers advantages in sensitivity and specificity for the detection of proteins in small aliquots of saliva (≥0.02 ml/min). In summary, the obtained results corroborate with previous reports that IL-6 levels are significantly higher in pSS compared to a HC group, regardless of the analytical technique used. The lack of positive results for IL-4 and IL-5 are however, different from other studies(3, 7,10,18) that also reported higher levels of these cytokines in pSS patients, although always in lower levels compared with IL-6. Ohyama et al. in 2015 using a Multiplex system reported significantly higher levels of IL-4 and IL-5 in pSS patients compared with controls. Moriyama et al. using ELISA tests also reported significantly higher levels of IL-4, but not of IL-5. Kang et al. using Luminex® also reported significantly higher levels of IL-4 in pSS patients, but did not analyze IL-5. We found no reason to be able to justify the lack of detection of IL4 and IL5, since the same samples were used to detect all three ILs. Previous studies have also reported that IL-6 levels in saliva correlated significantly with the degree of lymphocytic infiltration in labial salivary glands (3,12). In the present study minor salivary gland biopsies were not performed, but the pSS patients previous records from the rheumatology units reported history of histological positivity and these positive patients had significantly higher levels of IL-6 in saliva. Similarly, equivalent results were found in pSS patients with SWS hyposalivation (SWS flow less than 0.7ml/min). These results indicate that increased IL-6 levels may be associated with the level of gland degeneration in these patients, corroborating the results reported in other studies (3,12). pSS patients can suffer lymphoproliferative, hematological, renal disorders and alterations of the central and peripheral nervous systems, lungs, and skin. These diseases often cause fatigue, pain and musculoskeletal disorders in pSS patients(18). In this study we found significantly higher levels of salivary IL-6 in pSS patients with musculoskeletal disorders, but no other association was found between IL-6 levels and other extra-glandular manifestations. This study also correlated IL-6 levels with UWS and SWS flow rates and VAS for xerostomia. These data suggest that the higher is oral dryness the higher are the salivary IL-6 levels, measured by determinations of the salivary flow and by the self-assessment of the patients. These results have not been reported in previous studies and further research should be made in well-designed prospective studies to further assess the diagnostic validity of IL-6 salivary levels as a biomarker for pSS progression. A similar trend was reported when assessing the IL-6 salivary levels and the impact of this disease in the patient’s self-reported quality of life, since higher IL-6 levels were observed in patients with poorer quality of life. Even though other previous studies have not studied this association using the OHIP-14 questionnaire, similar results were reported using the Short Form 36 Health Survey (SF-36), which also assess the impact of the disease in the patient’s quality of life (19). The results reported in this clinical study need to be considered with caution in light of the evident limitations of this cross-sectional study, since the diagnostic validity of the determination of these biomarkers in saliva can only be ascertained in prospective cohort studies. Similarly, the fact that two of the studied cytokines, were almost undetected, preclude strong conclusions on the possible role of cytokines as diagnostic biomarkers for this disease. The results for IL-6, however, were very robust, and corroborate previous reports using this cytokine as biomarker for SS. 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Health-related quality of life in patients with primary Sjogren's syndrome: relationship with serum levels of proinflammatory cytokines. Scand J Rheumatol 2009; 38(5): 386-389. doi: 10.1080/03009740902973821 https://doi.org/10.1067/moe.2001.111551 http://dx.doi.org/10.1016/S0140-6736(05)66990-5 http://dx.doi.org/10.1016/S0140-6736(05)66990-5 Table 1. Clinical characteristics of patients with pSS (n=36) Characteristics of pSS patients n (%) or mean±SD Duration of the disease (years) 8.57±7.70 Oral symptoms 36 (100%) Ocular symptoms 36 (100%) Oral signs 17 (47.2%) Ocular signs 32 (88.9%) Histopathology 20 (55.6%) Autoantibodies Anti-Ro/La(+) 30 (83.3%) Extraglandular manifestations Whatever systemic manifestation 26 (72.2%) Parotid enlargement 9 (25%) Musculoskeletal involvement 16 (44.4%) Skin involvement 9 (25%) Lung involvement 3 (8.3%) Central nervous system involvement 1 (2.8%) Peripheric nervous system involvement 1 (2.8%) Haematological alteration 10 (27.8%) Gastrointestinal involvement 3 (8.3%) pSS serological manifestations Positive rheumatoid factor 16 (44.4%) ANA positive 34 (94.4%) Modified inmunoglobulins 20 (55.6%) n: number; %: percentage; SD:Standard Deviation Table 2. Results of UWS, SWS, VAS for xerostomia and OHIP-14 score in pSS and HCs. Variables pSS Score (mean±SD) HC Score (mean±SD) P-value Flow rates UWS 0.14±0.16 0.36±0.21 0.0001 SWS 0.83±0.73 1.50±0.78 0.0001 Score VAS for xerostomia (cm) VAS speech difficulties 4.33±2.97 0.20±0.53 0.0001 VAS swallowing difficulties 5.28±2.57 0.14±0.43 0.0001 VAS lack of saliva in mouth 4.28±2.65 9.03±1.77 0.0001 VAS dry mouth sensation 6.22±2.64 0.54±0.85 0.0001 VAS dry throat sensation 6.11±2.64 0.63±1.29 0.0001 VAS dry lip sensation 6.64±2.33 0.74±1.63 0,0001 VAS dry tongue sensation 6.22±2.45 0.57±1.22 0.0001 VAS level of thirst 6.17±2.82 2.46±2.31 0.0001 VAS Total 45.25±13.83 14.31±4.57 0.0001 Score OHIP-14 Functional limitation 3.33±1.96 0.26±0.85 0.0001 Question 1: Do you have discomfort to pronounce some words due to oral dryness? 1.72±1.30 0.17±0.71 0.0001 Question 2: Do you have felt that your taste sensation has worsened due to oral dryness? 1.39±1.40 0.09±0.28 0.0001 Physical pain 4.58±2.09 0.17±0.51 0.0001 Question 3:Do you have any painful discomfort in your mouth? 1.81±1.31 0.14±0.43 0.0001 Question 4:Do youou have felt discomfort when eating some foods due to oral dryness? 2.64±1.25 0.03± 0.17 0.0001 Psychological Discomfort 3.69±2.96 0.14±0.43 0.0001 Question 5:Have you been worried about oral dryness? 2.11±1.58 0.03±0.17 0.0001 Question 6: Do you have felt nervous due to the presence of oral dryness? 1.75±1.59 0.11±0.32 0.0001 Physical disability 2.64±2.30 0.06±0.34 0.0001 Question 7: Has your diet been unsatisfactory due to oral dryness? 1.22±1.22 0.03±0.17 0.0001 Question 8: have you had to interrupt your meals due to oral dryness problems? 1.19±1.37 0.03±0.17 0.0001 Psychological disability 2.36±2.46 0.06±0.34 0.0001 Question 9: Did you find it difficult to relax due to oral dryness? 1.36±1.46 0.03±0.17 0.0001 Question 10: Did you have felt disturbed in front of other people due to oral dryness? 0.97±1.28 0.03±0.17 0.0001 Social disability 2.06±2.34 0.06±0.34 0.0001 Question 11:Have you felt irritable in front of 0.92±1.34 0.03±0.17 0.0001 other people due to oral dryness problems? Question 12: Did you have difficulty doing your usual jobs due to problems with your oral dryness? 1.06±1.37 0.03±0.17 0.0001 Handicap 2.08±2.40 0.06±0.34 0.0001 Question 13: Have you felt that your life is generally less satisfactory due to oral dryness problems? 1.25±1.36 0.03±0.17 0.0001 Question 14: Have you felt totally unable to function due to problems with oral dryness? 0.72±1.19 0.03±0.17 0.0001 Overall OHIP 14 19.97±13.23 0.80±2.81 0.0001 Mann-Whitney U test; HC: Healthy controls; UWS: Unstimulated Whole Saliva; SWS: Stimulated Whole Saliva; VAS: Visual Analogue Scale; OHIP-14: Oral Health Impact Profile-14. Table 3. Levels of IL 4, 5 and 6 in saliva of patients with pSS compared with healthy controls (HC) pSS (n=36) HC (n=35) p-value Patients 35 36 1 Gender F/M 36/0 35/0 1 Age 56.58±12.35 54.40±9.16 0.3 IL-4 (pg/mL) 0.0 0.0 1 IL-5 (pg/mL) 0.03±0.18 0.0 0.32 IL-6 (pg/mL) 9.73±23.45 0.93±2.70 0.0001 Mann-Whitney U test; pSS: Primary Sjögrens Syndrome; HC: Healthy controls; n: number of patients; IL: Interleukins. Table 4. Levels of IL 6 and pSS classification criteria, extraglandular and serological manifestations. Criteria for pSS-2002 positive negative P-value Oral symptoms 100% (36) -- -- Ocular symptoms 100% (36) -- -- Oral signs 14.67±32.45 5.30±9.45 0.59 Ocular signs 10.49±24.79 3.65±3.33 0.90 Histology 10.83±30.41 8.35±10.40 0.04 Autoantibodies Anti-SSA and Anti-SSB(+) 7.39±14.69 21.41±48.99 0.98 Extraglandular manifestations Systemic manifestations 6.57±14.78 17.93±37.78 0.34 Parotid inflammation 2.28±2.85 12.21±26.68 0.40 Musculoskeletal disorders 6.76±18.30 12.09±27.11 0.03 Skin involvement 7.16±10.37 10.58±26.53 0.49 Lung involvement 2.17±2.99 10.41±24.39 0.58 Central nervous system involvement 25.14 9.28±23.63 0.22 Peripheric nervous system involvement 0.94 9.98±23.74 0.89 Hematological alterations 4.44±7.55 11.76±27.09 0.43 Gastrointestinal involvement 0.45±0.46 10.57±24.34 0.16 Altered immunoglobulins 9.71±26.78 9.73±19.17 0.96 Serological manifestations Positive rheumatoid factor 8.31±17.93 10.85±27.49 0.91 ANA positive 10.15±24.07 2.50±2.45 0.97 Modified inmunoglobulins 9.71±26.88 9.73±19.17 0.96 Mann-Whitney U test; pSS: Primary Sjögrens Syndrome. Table 5. Correlation of IL 6 and UWS, SWS results, VAS for xerostomia and OHIP-14 score. Variables r P-value Salivary flow UWS -0.16 0.50 SWS -0.06 0.73 VAS for xerostomia VAS speech difficulties 0.23 0.19 VAS swallowing difficulties 0.13 0.45 VAS lack of saliva in mouth 0.34 0.04 VAS dry mouth sensation 0.26 0.13 VAS dry throat sensation 0.13 0.46 VAS dry lip sensation 0.03 0.88 VAS dry tongue sensation 0.27 0.11 VAS level of thirst 0.18 0.30 VAS total 0.31 0.06 OHIP-14 Functional limitation 0.16 0.36 Question 1: Do you have discomfort to pronounce some words due to oral dryness? 0.24 0.16 Question 2: Have you felt that your taste sensation has worsened due to oral dryness? 0.06 0.74 Physical pain 0.11 0.53 Question 3:Do you have any painful discomfort in your mouth? 0.06 0.72 Question 4: Have you felt discomfort when eating some foods due to oral dryness? 0.02 0.90 Psychological Discomfort -0.08 0.64 Question 5:Have you been worried about oral dryness? -0.09 0.62 Question 6: Have you felt nervous due to the presence of oral dryness? -0.03 0.85 Physical disability -0.01 0.94 Question 7: Has your diet has been unsatisfactory due to oral dryness? -0.06 0.74 Question 8: Have you had to interrupt your meals due to oral dryness problems? 0.06 0.72 Psychological disability 0.06 0.72 Question 9: Did you find it difficult to relax due to oral dryness? 0.09 0.60 Question 10: Have you felt disturbed in front of other people due to oral dryness? -0.08 0.62 Social disability 0.12 0.5 Question 11: Have you felt irritable in front of other people due to oral dryness problems? 0.07 0.69 Question 12: Do you have difficulty doing your usual jobs due to problems with your oral dryness? 0.28 0.10 Handicap 0.16 0.34 Question 13: Have you felt that your life is generally less satisfactory due to oral dryness problems? 0.10 0.57 Question 14: Have you felt totally unable to function due to problems with oral dryness? 0.25 0.14 Overall OHIP 14 0.07 0.68 r: Spearman Rho test; IL: Interleukins; UWS: Unstimulated Whole Saliva; SWS: Stimulated Whole Saliva; VAS: Visual Analogue Scale; OHIP-14: Oral Health Impact Profile-14.