Cilj istraživanja je usporediti koncentracije interleukina-6 i TNF-α u krvi i slini bolesnika s inaktivnom multiplom sklerozom i Hashimotovim tireoiditisom i onih bolesnika s inaktivnom MS i zdravom štitnjačom. Uzeti su uzorci sline i krvi 130 bolesnika u inaktivnoj fazi MS na Klinici za neurologiju, a laboratorijske analize uzoraka su izvršene u laboratoriju Klinike za onkologiju i nukearnu medicinu Kliničkog bolničkog centra Sestre Milosrdnice. Bolesnici su podijeljeni u dvije skupine: oni koji imaju zdravu štitnjaču i bolesnici s HT-om. Ne postoji statistički značajna razlika između razine IL-6 i TNF-α u krvi i slini u zadanim grupama.
Međutim, postoji statistički značajna korelacija između razine IL-6 iz sline i IL-6 iz krvi (r = 0,363; p < 0,01), statistički značajna korelacija između razine IL-6 iz sline i TNF-α iz krvi (r = 0,187; p < 0,01) i postoji statistički značajna korelacija između razine IL-6 iz krvi i TNF-α iz krvi (r = 0,432; p < 0,01). Također, postoji statistički značajna razlika u razini TSH u skupini eutireoidnih bolesnika s povišenom razinom protutijela na štitnjaču (TPO i TgA) u odnosu na bolesnike s razinom protutijela u referentnom intervalu (t = 3,324; df = 128, p < 0,01). Indeks tjelesne mase, proširena ljestvica statusa onesposobljenosti i pušenje ne koreliraju s razinom IL-6 i TNF-α u krvi i slini. Iako nismo dokazali razliku u razini interleukina 6 i TNF-α u krvi i slini u dvije skupine, dokazali smo značajnost sline kao dijagnostičkog uzorka jer u više parametara korelira s razinom u krvi. Također smo pokazali važnost određivanja TPO i TgA protutijela štitnjače jer je razina TSH u eutireoidnih bolesnika statistički značajno viša kod bolesnika s povišenim protutijelima.
|Abstract (english)|| |
Aim: The aim of the study was to compare the concentration of interleukin - 6 and TNF - α in the blood and saliva of patients with inactive multiple sclerosis and Hashimoto's thyroiditis and those patients with inactive MS and healthy thyroid.
Materials and methods: This study was approved by the Ethics Committee of Sestre Milosrdnice University Hospital Centre and by Ethics Committee of the School of Dental Medicine, University of Zagreb, Zagreb, Croatia. All participants signed informed consent according to Helsinki II. All patients fill out the questionnaire with general data, age of diagnosis, duration of remission, therapy, supplements, diet, malignancy, smoking and alcohol abuse. Patients with other autoimmune diseases and dental works are excluded. Also, inadequate specimens were excluded. Saliva and blood samples were taken from 130 patients in the inactive phase of multiple sclerosis at the Department of Neurology, and laboratory measurements were performed in the laboratory of the Department for Oncology and Nuclear Medicine of the University Hospital Center Sestre Milosrdnice. Patients are divided into two groups: those who have a healthy thyroid and patients with Hashimoto’s thyroiditis. In all patients, routine blood tests, TSH, fT4, fT3, TPO, TGA, CRP were made. Saliva and serum were collected for IL-6 and TNF-α. Whole saliva samples were collected with SalivaBio's 2 mL cryovials and the Saliva Collection Aid (exclusively from Salimetrics, State College, PA), a collection device specifically designed to improve volume collection and increase participant compliance and validated for use with salivary. Unstimulated , whole saliva, is gold standard in saliva collection, was collected by passive drool technique in order to maintain consistency in the type of sample collected. Immediately after collection, samples are frozen at -20° C. On the day of assay, the samples were thawed, vortexed and centrifuged for 15 minutes at 396 xg on a Rotina 35R centrifuge (Hettich, Kirchlengern, Germany). IL-6 and TNF-α in serum were measured on an Immulite 1000 automatic immunochemistry analyzer (Siemens Healthcare Diagnostics, Erlanger, Germany), with chemiluminescent immunoassay method as well as TNF-α in saliva. IL-6 in saliva was measured by commercial kit (Salimetrics, PA, SAD) with ELISA (Enzyme - Linked ImmunoSorbent Assay). TNF-α and IL-6 levels were expressed in pg/mL. Peripheral blood samples were collected by venipuncture in two Vaccuete® red cap serum tubes with clot activator (Greiner Bio-One, Kremsmünster, Austria), for CRP, TSH, fT4, fT3, A-TPO, A-TG, IL-6, TNF-α, and one tube with K2EDTA, lavender cap for complete blood count (CBC). Blood for serum testing was centrifuged for 10 minutes at 3500 g in a minute. CRP, TSH, FT4, A-TPO, A-TG were analysed immediately, and for IL-6 and TNF-α, two aliquots (2x550um) of serum samples were immediately frozen at -20°C until analysis. CRP was determined by immunoturbidimetry on an clinical chemical analyzer Architect c8000 (Abbott, Illinois, SAD), using calibrators and controls. The recomended reference value for CRP is < 5 mg/L. TSH, FT4 and FT3 were determined at immunoassay analyzer Abbott i2000
(Abbott, Illinois, SAD), chemiluminescence method, and A-TPO i A-TG with analyzer Cobas e601 (Roche Diagnostics, Basel, Switzerland), electrochemiluminescence method. CBC was performed at DxH 520 (Beckman-Coulter, Brea, SAD) analyzer. The recomended reference values fo TSH is 0.35 - 4.94 mIU/L, FT4: 9.01 - 19.05 pmol/L, A-TPO: < 34 kIU/L and A-TG: < 115 kIU/L. IL-6 and TNF-α in serum and salival TNF-α, after were defrosted by leaving at room temperature until liquid state, were analyzed on Immulite1000 (Siemens Healthcare Diagnostics, Erlangen, Germany) by the chemiluminescent enzyme immunometric method using original Siemens reagents and adjustors according to manufacturer's instructions. Teeth were washed one hour before colecting specimen and fasting for 12 hours, without any oral disease, injuries and inlammation of oral cavity. Statistical analysis was performed using the IBM SPSS Statistics, version 22. Results: There is no statistically significant difference between the levels of IL-6 and TNF-α in blood and saliva in the given groups. However, there is a statistically significant correlation between salivary IL-6 level and blood IL-6 (r = 0,363; p < 0,01), a statistically significant correlation between salivary IL-6 levels and blood TNF-α (r = 0,187; p < 0,01) and there is statistically significant correlation between blood IL-6 levels and blood TNF-α (r = 0,432; p < 0,01). Also, there is a statistically significant difference in TSH levels in the group of euthyroid patients with elevated antibody levels compared to patients with antibody levels in the reference interval (t = 3.324; df = 128, p < 0,01). BMI, EDSS, and smoking do not correlate with blood and saliva levels of IL-6 and TNF-α. Conclusion: Although we did not prove a difference in the level of interleukin-6 and TNF-α in blood and saliva in the two groups, we proved the significance of saliva as a diagnostic sample
because in several parametars it correlates with the level in the blood. We also demonstrated the importance of determing TPO and TgA thyroid antibodies because TSH levels in euthyroid patients were statistically significantly higher in patients with elevated antibodies.