/home/opencode/cpanel/stomaeduj_hacked/uploads/2018/03/Article_5_3_4.pdf ORAL MEDICINE SALIVARY MATRIX METALLOPROTEINASES ACTIVITY LEVELS IN Original Article AUTOIMMUNE DISEASES Iulia-Ioana Stănescu1a , Alina Dima2b, Eugenia Bălănescu3b, Daniela Miricescu1c, Alexandra Totan1c, Radu Rădulescu1d, Bogdan Calenic1e* , Paula Perlea4f, Daniela Bălan5g, Maria Greabu1h 1 Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, RO-050474 Bucharest, Romania 2 Department of Internal Medicine, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, RO-020125 Bucharest, Romania 3 Laboratory of Clinical Immunology, Colentina Clinical Hospital, RO-020125 Bucharest, Romania 4 Department of Endodontics, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, RO-010221 Bucharest, Romania 5 Department of Physiology, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, RO-010221 Bucharest, Romania a DDS, Assistant Professor b MD, PhD c PhD, Assistant Professor d DDS, PhD e DDS, PhD, Assistant Professor f DDS, PhD, Professor g MD, PhD, Assistant Professor h PhD, Professor, Head ABSTRACT DOI: 10.25241/stomaeduj.2018.5(3).art.4 OPEN ACCESS This is an Open Access article Introduction: It is widely recognized that saliva represents a solid alternative as a under the CC BY-NC 4.0 license. diagnostic fluid in a wide range of oral and general diseases. Autoimmune diseases Peer-Reviewed Article constitute an important health threat to both men and women worldwide with Citation: Stănescu I-I, Dima A, Bălănescu E, Miricescu D, matrix metalloproteinases (MMPs) playing significant roles in pathogenesis and Totan A, Rădulescu R, Calenic B, Perlea P, Bălan D, Greabu development of these diseases. The specific aim of the present study is to asses, M. Salivary matrix metalloproteinases activity levels in autoimmune diseases. Stoma Edu J. 2018;5(3):168-172. for the first time, salivary levels of a selected panel of MMPs in several autoimmune diseases. Academic Editor: David Wray, MD (Honours), BDS, MB ChB, FDS, RCPS (Glasgow), FDS RCS (Edinburgh) F Med Methodology: The study included 30 patients divided into groups such as: Sci, Professor Emeritus, Professor, University of Glasgow, Glasgow, UK systemic sclerosis, vasculitis and healthy subjects. Salivary levels of MMP-1, -2, -7, -9 and -10 were analyzed using magnetic bead-based multiplex assays and Luminex Received: September 03, 2018 Revised: September 10, 2018 technology. Acccepted: September 24, 2018 Results: MMP-2 salivary levels were statistically elevated in systemic sclerosis, Published: September 25, 2018 while MMP-10 were also increased in vasculitis patients. Salivary levels of MMP-9 *Corresponding author: Assist. Professor Bogdan were found significantly increased in all analyzed groups. Calenic, DDS, PhD, Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Conclusion: Taken together, our results promote saliva as a reliable diagnostic Pharmacy, RO-050474 Bucharest, Romania fluid for quantifying MMPs in autoimmune diseases. Tel.:+40755044047, Fax: +40.213.110.984; e-mail: bcalenic@yahoo.co.uk Keywords: saliva, autoimmune diseases, matrixmetalloproteinase, vasculitis, systemic Copyright: © 2018 the Editorial Council for the sclerosis. Stomatology Edu Journal. 1. Introduction Scleroderma is considered a rare disease that affects Over the past decades, saliva has become a hot topic the connective tissue. Depending on the extent of in medical research related to discovering alternative the manifestations, scleroderma can be classified diagnostic methods. Whole saliva is a complex fusion either as localized- when only the skin and in of the fluid produced by major and minor salivary some cases the tissues underneath are involved, or glands with gingival crevicular fluid, oral desquamated systemic sclerosis, a more complex form in which cells, bacteria, as well as with food remains [1,2]. cutaneous manifestations are associated with visceral The advantages provided by saliva as a diagnostic impairment. Theories regarding the etiology of this fluid include non-invasive and effortless collection affliction have incriminated genetic factors, trauma, procedures, the need for small samples for analysis, viral or bacterial infections, neurological damage or along with easy storage and an established correlation vascular abnormalities. However, an increased level between salivary and blood biomarkers’ levels [3]. of antinuclear antibodies, as well as the frequent In addition to these benefits, its rich composition association with autoimmune diseases such as consisting of electrolytes, proteins and hormones systemic vasculitis or systemic sclerosis, plead for an deriving not only from oral sources, but also from autoimmune disease characterized by an inflammatory systemic production, promote saliva as a remarkable phase followed by fibrosis, causing atypical collagen to diagnosis and monitoring fluid for both local and replace normal dermis [9]. Vasculitis is a rare disease general diseases [4-6]. However, few studies have characterized by an inflammatory process located focused on the potential saliva has as a diagnostic fluid within the walls of blood vessels, affecting mainly small for autoimmune diseases [7,8]. calibre vessels. The great diversity of the vessels affected 168 Stoma Edu J. 2018;5(3): 168-172 http://www.stomaeduj.com SALIVARY MATRIX METALLOPROTEINASES ACTIVITY LEVELS IN AUTOIMMUNE DISEASES explains the clinical heterogeneity of this disease that Original Article Table 1. Clinical data of patients included in the study (autoimmune involves a great variety of organs [10]. Meanwhile, disease group and control group). matrix metalloproteinases (MMPs) represent a group Inclusion Disease BMI, of proteases produced by several types of cells, Cases age, duration, kg/m2 including pro-inflammatory cells with multiple roles Years Years such as leukocyte infiltration in conditions with an Total 63 53.0 8.0 25.5 inflammatory component [11]. To the best of our subjects (37.5 – 60.5) (4.0 – 14.0) (21.8 – 29.9) knowledge up to the present, there are no scientific 52.0 2.0 25.9 SS 10 (40.0 – 60.0) (0.5 – 9.0) (22.4 – 30.9) studies focusing on the salivary MMP levels in patients with autoimmune diseases. 47.0 8.0 25.8 VA 10 (42.0 – 70.0) (2.0 – 14.0) (24.8 – 35.1) Taking all this into consideration, the main objective of 45.0 21.9 the present research is to assess the potential use of saliva Control 10 (30.0 – 50.5) - (19.5 – 22.6) as a diagnosis and monitoring fluid for autoimmune SS – systemic sclerosis; VA - vasculitis; BMI – body mass index; kg – kilogram; diseases by evaluating salivary levels of a group of MMPs m – meter; in patients with systemic sclerosis and vasculitis and compare them with those of healthy subjects. Table 2. MMP levels in Systemic Sclerosis. SS n-10 cases. MMP1 MMP2 * MMP7 MMP9 * MMP10 * 2. Material and methods 1424.43 24.3125 1093.68 6571.25 1796.62 2.1. Patient selection SS (1009.22- (19.32- (1003.02- (5894.31- (1221.33- 1994.30) 29.33) 1903.10) 7043.44) 1943.01) Our study included a total of 30 patients: 10 patients with systemic sclerosis and 10 vasculitis patients. 3565.167 10.3 1521.45 1148.5 943.82 10 healthy subjects, with no autoimmune disease, Control (1237.5- (3.45- (854.3- (843.4- (754.28- represented the control group. Clinical data including 4422.5) 14.32) 2321.59) 2749.48) 1232.37) age, gender, current treatments, as well as a series of Data are expressed as median (inferior; superior limits); * Mann-Whitney test; biochemical and immunological parameters have p – value < 0.05 is considered statistically significant been collected for all the participants to the study. All Table 3. MMP levels in Vasculitis. VA n-10 cases. subjects included in our research agreed to participate voluntarily and signed an informed consent. The MMP1 MMP2 MMP7 MMP9 * MMP10 * present research was approved by the ethics board of Colentina Clinical Hospital, Bucharest, Romania. All 1899.7 25.4 1071 5551.7 2372.2 VA (1002.54- (10.22- (920.32- (4988.38- (2082.10- patients were diagnosed by the same experienced 2540.9) 38.43) 1643.39) 5903.80) 3002.48) medical professional. 3565.167 10.3 1521.45 1148.5 943.82 Control (1237.5- (3.45- (854.3- (843.4- (754.28- 2.2. Samples collection 4422.5) 14.32) 2321.59) 2749.48) 1232.37) For all participants in the study, saliva samples Data are expressed as median (inferior; superior limits); * Mann-Whitney test; were collected at the moment of inclusion in the p – value < 0.05 is considered statistically significant present study. Their written consent was taken prior to collecting the samples. The collection method 20,000 pg/mL; MMP-2: 68 – 50,000 pg/mL; MMP-7: 548 followed a well-established protocol and took place as – 400,000 pg/mL; MMP-9: 14 – 10,000 pg/mL; MMP- follows: 2-5 mL of unstimulated whole saliva was given 10: 27 – 20,000 pg/mL. The assay involved a two-hour by each participant in the morning around 9 AM. The multistep procedure involving preparation of reagents, participants were asked to refrain from eating, drinking samples and controls, preparation of the 96 well-plate or smoking prior to saliva collection. All subjects rinsed and analysis using a Luminex® 200™ and the xPONENT® with 5 mL of distilled water before collection. The software for data acquisition and analysis. patients were also asked to sit down and be relaxed during saliva sampling as well as not to communicate 2.4. Statistical analysis with each other. Immediately after the collection, The statistical analysis was performed using SPSS the samples were kept on ice and transported in an software. The characteristics were expressed as median isotherm box and subsequently, they were centrifuged (quartile 1; 3). Statistically significant correlations at 5000 rpm for 10 minutes. Samples were aliquoted were found using the Mann-Whitney test (a two- and stored at -80ºC until further determinations. sided p-value less than 0.05 was noted as statistically significant). A Spearman test was used to evaluate 2.3. Matrix metalloproteinases detection possible correlations between salivary MMPs levels, All MMPs were detected using Luminex technology and as well as for other significant bivariate (p-value < 0.05 Magnetic Bead-Based Multiplex assays. This method was considered statistically significant). enables the simultaneous detection of multiple salivary human biomarkers. Thus salivary levels of MMP-1, -2, -7, -9, -10 were assessed using MILLIPLEX MAP Human 3. Results MMP Magnetic Bead Panel 2 - Immunology Multiplex 3.1. Clinical data Assay. All samples and controls were processed Several clinical parameters such as body mass index, age following the manufacturer's specifications. The assay at the moment of the diagnostic and at the inclusion as required 25 μL of saliva diluted at 1:20; each MMP well as disease duration were recorded for all subjects in- had its own detection interval as follows: MMP-1: 27 – cluded in the study (see Table 1). Stomatology Edu Journal 169 SALIVARY MATRIX METALLOPROTEINASES ACTIVITY LEVELS IN AUTOIMMUNE DISEASES 3.2. MMP-1, -2, -7, -9, 10 levels in Systemic Sclerosis Original Article In the present study patients with SS showed a Table 4. Increased salivary MMPs levels in autoimmune diseases (increments are based on own results and are statistically significant at a p statistical increment in salivary levels of MMP-2, -9 and < 0.05). -10 when compared with respective controls. MMP-7 SS VA was also increased, but with no statistical significance. MMP-1 were statistically lower in the SS group vs the MMP-1 - - control group (Table 2). MMP-2 ↑ 2 fold - MMP-7 - - 3.3. MMP-1, -2, -7, -9, 10 levels in Vasculitis Patients suffering from vasculitis had statistically MMP-9 ↑ 6 fold ↑ 4 fold increased levels of MMP-9 and -10. At the same time, MMP-10 ↑ 2 fold ↑ 2 fold the data were not statistically relevant for MMP-1, -2 and MMP-7 (Table 3). autoimmune disease. Moreover, salivary MMP-1 was decreased SS and VA when compared to their controls, but the results were not statistically significant. These 4. Discussion findings corroborated with existing scientific literature In the past, blood serum has routinely been used in may suggest that MMP-1 does not play an active the diagnostic process. However, most of the blood role in the pathogenesis of autoimmune diseases. constituents are found in the saliva, as they pass Considering their organization, MMP-2 and MMP-9 are through transcellular and paracellular routes. Nowadays considered gelatinases. MMP-2 plays important roles in saliva could be introduced as a diagnostic tool in many both physiological processes such as angiogenesis and medical fields including microbiology, immunology, in pathological situations like varicose veins disease, oncology, endocrinology, etc. Saliva is easy to collect, inflammation, and cardiovascular diseases. Meanwhile, ship and store; it could be obtained in sufficient MMP-9 in involved in cell apoptosis and osteoarthritis. quantities at a low cost via non-invasive methods [12,13]. MMP-9 was shown to present higher levels in patients Saliva-testing kits have been presented on the market, with systemic sclerosis [18]. Interestingly, in our study although some of them are still waiting for approval. salivary MMP-9 levels were found statistically higher in Intense efforts have been undertaken to detect markers all patient groups included in the study when compared that reflect tumor pathologies such as oral cancers. The to the respective controls. Knowing that MMP-9 is composition of saliva also reflects pathological changes generally involved in programmed cell death we can in salivary glands. Salivary tumors can release in the infer that this marker is involved in tissue remodeling salivary flow important levels of stathmin or maspin, processes occurring in these diseases. MMP-7 belongs tumor necrosis factor, Dim1p, v-Ha-ras oncogene, type I to the matrilysins subgroup and has been linked to collagen pro alpha or pirin [14]. Systemic cancers such as inflammation, lung disease, cardiovascular afflictions breast, gastric or larynx neoplasm can also be detected and to physiological cell apoptosis. In the present study, in saliva. p53 is a tumor suppressor protein capable of MMP-7 salivary levels were not statistically elevated in blocking the tumor formation. Mutations in p53 gene any studied disease. MMP- 10 or stromelysin 2 has been are found in most of the tumors and are associated linked to liver disease, viral infections, lung disease, with tumor development and poor prognosis. p53 tissue remodeling and cell apoptosis and is considered antibodies are found in the serum and saliva of patients to play an important role in peripheral arterial disease. with oral squamous cell carcinoma [15]. Another marker Our findings show that MMP-10 salivary levels were for oral squamous cell carcinoma identification, with significantly elevated when compared to controls in significantly increased levels in saliva, is cortisol. The both systemic sclerosis and vasculitis patients. marker is even frequently used for tumor staging. Some The present study has several limitations that will be growth factors concentrations in saliva also correlate addressed in further studies: a larger number of patients with the development of neoplasms. Elevated levels will be needed in the future to validate the present of Fibroblast growth factor 2 (FGF2) correspond to findings. Future correlations with MMP serum levels in salivary gland tumor growth. Sjögren syndrome, an all patients groups are needed in order to address the autoimmune disease which among other effects alters possible utility of salivary MMPs in clinical settings. the composition of saliva and tears is also studied in association with reactive species of oxygen (ROS) changes [16]. Several recent studies have shown that 5. Conclusion the biomarker composition is different in saliva from In conclusion, the present study shows for the first time, patients with Sjögren syndrome versus controls. Other that important matrix metalloproteinase is increased studies analyzed saliva in connection with diseases such and can be detected in saliva from patients with as Alzheimer, anorexia, bulimia of mental stress [17]. autoimmune diseases. Thus, salivary determination of MMP-1 is a collagenase that normally can be found MMP-1, -2, -7, -9 and -10 promotes saliva as a viable in low quantities in the cells, but increases its levels in alternative when determining the activity of these autoimmune diseases as well as in inflammation. This enzymes in systemic sclerosis or vasculitis. protease that degrades collagen plays an important role in wound healing, immune response, inflammation, fibrotic disorders and cardiovascular diseases. Moreover, Conflicts of Interest MMP-1 could be involved in cancer. Our data did not The authors declare no conflict of interest. show an increased activity of MMP-1 in any included 170 Stoma Edu J. 2018;5(3): 168-172 http://www.stomaeduj.com SALIVARY MATRIX METALLOPROTEINASES ACTIVITY LEVELS IN AUTOIMMUNE DISEASES 8. Bonamico M, Nenna R, Montuori M, et al. 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She graduated from the Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy in dentistry and her field of interest in her research as a PhD candidate is salivary diagnosis in autoimmune diseases. Stomatology Edu Journal 171 SALIVARY MATRIX METALLOPROTEINASES ACTIVITY LEVELS IN AUTOIMMUNE DISEASES Questions Original Article 1. Saliva is: qa. A reliable diagnostic fluid containing biomarkers that statistically correlate with those found in blood; qb. Not a reliable diagnostic fluid containing biomarkers that statistically correlate with those found in blood; qc. A fluid produced by major and minor salivary glands with gingival crevicular fluid, oral desquamated cells, but not oral bacteria; qd. An invasive method of collection. 2. Vasculitis: qa. Is a common autoimmune disease; qb. Usually affects large blood vessels; qc. Can affect only a few organs; qd. Is located in the walls of blood vessels. 3. MMP-2 is: qa. Increased 2 fold in saliva from patients with vasculitis; qb. Increased 2 fold in saliva from patients with systemic sclerosis; qc. Not statistically increased in saliva samples taken from patients with autoimune diseases; qd. Not a gelatinase. 4. MMP-9 is: qa. Increased 6 fold in saliva from patients with systemic sclerosis; qb. Increased 2 fold in in saliva from patients with systemic sclerosis; qc. Statistically decreased in saliva samples taken from patients with autoimune diseases; qd. Not involved in apoptosis. http://www.quintpub.com/grunder/ 172 Stoma Edu J. 2018;5(3):168-172 http://www.stomaeduj.com