The Association Between Thalassemia Major and Periodontal Health

Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 The Association Between Thalassemia Major and Periodontal Health Aliye Akcalı DDS, PhD*, Selda Kahraman Çeneli MD†, Pınar Gümüş DDS, PhD*, Nurcan Buduneli DDS, PhD, Professor*, David F. Lappin BSc, PhD, Research Fellow‡, Özgün Özçaka DDS, PhD, Associate Professor* * Department of Periodontology, School of Dentistry, Ege University, İzmir, Turkey. † ‡ Department of Haematology, School of Medicine, Aydın Government Hospital, Aydın, Turkey. Infection and Immunity Group, Dental Hospital and School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK. Aim: The aim of this cross-sectional study was to compare the local and systemic levels of sRANKL, OPG, APRIL, BAFF, IL-6 and IL-8 in biofluids of patients with thalassemia major (TM) with or without gingivitis. Materials & Methods: Seventy-seven patients were included in this study (TM; n=29, systemically healthy; n=48). Gingival crevicular fluid (GCF), saliva, serum levels of IL-6, IL-8, soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), osteoprotegerin (OPG), B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) were determined by ELISA. Data were analysed by appropriate non-parametric or parametric statistical tests. Results: Median GCF, serum and saliva: BAFF (p<0.001), IL-6 and IL-8 (p<0.005) were higher in TM gingivitis than in ‘systemically healthy’ gingivitis (p<0.001). GCF, serum, saliva APRIL, sRANKL, IL-6, IL-8 levels were higher in TM than in systemically and periodontally healthy comparison group (p<0.05). Positive correlations were found between BOP, PI scores and GCF APRIL, serum sRANKL, serum OPG, sRANKL concentrations in TM groups (p<0.05). Several significant positive correlations were found between BOP, PI scores and biofluid parameters also in systemically healthy groups. Conclusion: TM may have a role in the underlying systemic hematologic condition and potentially affect gingival inflammation via dysregulation of lymphocytes and increased activation of osteoclasts. MESH KEYWORDS: Cytokine(s); Gingivitis; Interleukin(s); Thalassemia major. Thalassemia is the most common genetic disease worldwide and The World Health Organization reports that approximately 60.000 infants with thalassemia major (TM) are born each year.1 TM is characterised by mutations of the β globin gene with a various degrees of defective β chain production, an imbalance in globin chain synthesis.2 Hemolysis and ineffective erythropoiesis together cause an anemia in TM that needs to present medical attention and patients required blood transfusion for survival.3 Chronic iron overload leading to multiple organ damage including liver, heart and bone is inevitable in TM. Recently, it was reported that periodontal tissues are also affected by iron accumulation.4 Erythroid cells are highly dependent on iron for survival and iron absorption and its recycling are regulated by hypoxia, inflammation, and erythropoiesis, possibly through distinct mechanisms.5 Altered bone metabolism such as osteopenia and osteoporosis is the major cause of morbidity in TM, however, underlying pathogenic mechanisms of bone mineral loss have not been fully understood.6 Receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), and 1 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 osteoprotegerin (OPG) are members of tumour necrosis factor (TNF) receptor superfamily and they play a central role in bone remodelling together with various cytokines.7 Alterations in the RANK/RANKL/OPG system are important in the impaired bone turnover in TM patients with complicated mechanisms involving chronic anaemia, iron toxicity, and endocrine complications.6 It is known that both RANKL and OPG can be detected in gingival tissue as well as in biofluids including gingival crevicular fluid (GCF), saliva and serum.8,9 Higher RANKL:OPG ratio has been reported in patients with gingival inflammation than those with healthy periodontium.8,9 Patients with TM may have various immunological defects in neutrophil and macrophage phagocytic and killing functions and production of some cytokines.10 A proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) are members of the TNF superfamily and they regulate survival and activation of lymphocytes.11 Increased levels of cytokines such as IL-6, IL-8, APRIL and BAFF have been reported in biofluids during the shift from gingival health to periodontal disease.12,13 Oral health problems in patients with TM are mostly related with varying degrees of facial deformities, malocclusions or dental arch dimensions.14,15,16 To date, there is limited data available on the periodontal health status of patients with TM.17,18,19 It was hypothesized that TM may cause increased levels of inflammatory cytokines in biofluids particularly when there is accompanying gingival inflammation, which in turn may modify the clinical signs of both chronic diseases. Therefore, the aim of the present study was to investigate the local and systemic levels of sRANKL, OPG, APRIL, BAFF, IL-6 and IL-8 comparatively in patients with TM with or without gingivitis. MATERIALS AND METHODS Study Population and Clinical Examination Seventy-seven patients (38 females and 39 males; age range between 18 and 58 years) were recruited for this study from the outpatient clinic of Department of Haematology, Aydın Government Hospital, Turkey, between September 2012 and April 2013. Patients were divided into four study groups; 14 patients with TM and healthy periodontium (TMh), 15 patients with TM and gingivitis (TMg), 20 systemically and periodontally healthy comparison group (Hh), 28 systemically healthy individuals with gingivitis (Hg). The study was conducted in full accordance with ethical principles, including the World Medical Association’s Declaration of Helsinki, as revised in 2008. The study was approved by the Ethics Committee of the Ege University with the protocol number 13-11/72. The study conforms to STROBE guidelines for observational studies.20 The study protocol was explained and written informed consent was received from each individual before enrolment in the study. Complete medical and dental histories were obtained from each individual. Demographic, anthropometric data and history of all past treatments were collected. Independent factors likely to be associated with low bone mass such as history of gonadal or pubertal dysfunction, history of iron chelating therapy, history of treatment with calcium and vitamin D, pre-transfusion haemoglobin level, serum levels of calcium, phosphorus, alkaline phosphatase, and thyroid function indices (T3, T4 and TSH) were determined. Inclusion criteria for TM patients were age ≥18 years, absence of hepatitis B, C or HIV infection, and treatment with chelation therapy using 2 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 deferasirox and regular erythrocyte transfusion, vitamin B and C. The comparison group consisted of sex and age-matched systemically healthy individuals. Pregnancy, presence of any other known systemic disease and usage of antibiotics or antiinflammatory drugs within the last 6 months were the exclusion criteria. Smoking status was determined by self-reporting but current or former smokers were not excluded. Eligible patients were returned to the clinic for clinical periodontal measurements including probing depth (PD), plaque index (PI)21, and bleeding on probing (BOP; +/-). BOP was deemed positive if it occurred within 15 seconds after probing. Clinical periodontal measurements were performed at 6 sites/tooth (mesiobuccal, mid-buccal, disto-buccal, mesio-lingual mid-lingual and disto-lingual locations), except the third molars, using a Williams periodontal probe∗ and by the examiners calibrated initially and at 6-month intervals during the study (AA, PG, ÖÖ). Diagnosis of gingivitis was assigned according to the clinical and radiographic criteria proposed in the 1999 International World Workshop for a Classification of Periodontal Disease and Conditions22 BOP scores exceeded 50% of all sites, with PD <3 mm at 90% of the measured sites and no more than one site had a PD >4 mm or clinical attachment level (CAL) ≥1 mm, and no clinical and/or radiographic sign of periodontitis was evident. Collection of Biofluid Samples To minimise the effect of diurnal variation on biochemical parameter levels all biofluid samples were collected in the morning between 8:00 am and 9:00 am and immediately frozen at - 40°C. Participants were asked to avoid oral hygiene measures (flossing, brushing and mouth-rinses), eating, and drinking 2 h prior to saliva sampling. All individuals were asked first to rinse their mouth with distilled water for 2 min, wait for 10 min and then expectorate into sterile 50 ml tubes for 5 min. Five ml of venous blood were obtained in tubes with a silicone-coated interior† utilizing a standard venipuncture method. The collected blood samples were left at room temperature to allow clotting to occur and then centrifuged at 1500 x g for 15 min at +4°C to remove the fibrin clot and cellular elements. GCF samples were obtained from the buccal aspects of two interproximal sites with obvious plaque accumulation and visible signs of inflammation at single-rooted teeth and at least one multi-rooted tooth from each individual. Filter paper strips were used for GCF sampling.‡ First, supragingival plaque was removed carefully by sterile curettes and the surfaces were gently air-dried and isolated by cotton rolls. Then filter paper strips were placed in the orifices of gingival sulcus/pocket for 30 seconds. Care was taken to avoid mechanical trauma, and strips visually contaminated with blood were discarded. The absorbed GCF volume was estimated by a calibrated instrument.§ The two strips from each patient were placed into one polypropylene tube and frozen. Measurement of sRANKL, OPG, APRIL, BAFF, IL-6, IL-8 Levels in Biofluid Samples Commercial ELISA kits were purchased for the measurement of APRIL,** sRANKL,†† OPG,‡‡ IL-6§§ and IL-8.*** The BAFF††† assay was developed in-house from antibody pairs (Goat polyclonal as a capture antibody and biotinylated mouse monoclonal for detection) and recombinant BAFF‡‡‡ was used as the assay standard. GCF samples from each patient were eluted in 1ml of PBS containing 0.05% Tween 3 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 20 with a 60 min incubation on a rotary mixer at +4oC. ELISA. The ELISA assays were carried out in duplicate on 50µl samples of GCF and in triplicate on 25 - 50µl of serum or saliva according to the manufacturers’ recommendations. The optical densities were read at 450 nm with a background subtraction at 570 nm and the samples were compared with the standards. The minimum detection limits in the assays were: IL-6, 0.79 pg/ml; IL-8 1.59 pg/ml; APRIL, 15.8 pg/ml; BAFF, 15.8 pg/ml; OPG, 7.9pg/ml; and sRANKL, 7.9pg/ml .The GCF results were expressed as total amounts per sampling time (pg/30s) and also as concentrations (pg/µL). The findings in saliva and serum samples were expressed as concentrations (pg/mL). Statistical Analysis The distribution of the data was evaluated by D’Agostino-Pearson omnibus normality test. Comparisons between all groups for non-normally distributed variables (biochemical data) were performed by the Kruskal-Wallis test and Dunn’s test was used in order to correct for multiple comparisons. For normally distributed variables (age, PI, BOP and PD) one-way ANOVA test with Holm-Sidak’s multiple comparison test (family-wise significance and confidence level 0.05) was used. Correlations between clinical and biochemical data were analysed by Spearman’s correlation test. The statistical analyses were conducted using the statistical software,§§§ and statistical significance was considered at p<0.05. RESULTS Periodontal Clinical Findings Demographics and full mouth clinical periodontal recordings are presented in Table 1. Mean PD and CAL were below 3 mm in all the study groups. The One-way ANOVA indicated that clinical periodontal parameters were not significantly different when comparing the groups in the two gingivitis groups with or without TM (p>0.05). Biochemical Data in the Serum Samples Circulating levels of investigated cytokines are presented in Fig. 1. Serum BAFF (Fig. 1A), APRIL (Fig. 1B), and IL-6 (Fig. 1F) concentrations were significantly higher (p<0.05) in the TM groups than the comparison groups. Serum OPG concentrations were similar in the study groups (Fig. 1C). sRANKL was higher in the TMg group than in the TMh (Fig. 1D) and also higher in TMh than Hh and the sRANKL:OPG ratio was greater (Fig. 1E) (p<0.01) than the comparison groups. Serum IL-8 levels (Fig. 4G) were significantly higher in TMh and Hg groups compared to Hh group (p<0.001). Biochemical Data in the Saliva Samples The biochemical data obtained in saliva samples are presented in Fig. 2. Saliva BAFF (Fig. 2A), APRIL (Fig. 2B), sRANKL (Fig.2D), IL-6 (Fig. 2F) levels were significantly higher in the TM groups than the comparison groups (p<0.01). Saliva OPG concentrations were similar in the study groups (Fig. 2C). Saliva sRANKL concentrations were higher in the Hg group than the Hh group (Fig. 2D). The ratio of sRANKL:OPG (Fig. 2E) was greater in TMg than in Hg (p<0.05). Saliva concentrations of IL-8 were significantly higher in the TMh group and the Hg group compared to the Hh group (Fig. 2G). 4 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 Biochemical Data in the GCF Samples The biochemical data obtained in the GCF samples are presented in Fig. 3. Greater amounts of APRIL and OPG were observed in the TMh than in the TMg group (p<0.05) (Fig. 3C). The amounts of sRANKL (Fig. 3D) and IL-6 (Fig. 3F) were significantly greater in the TM groups than in the comparison groups (p<0.01) and the sRANKL amount was higher in the TMg than in the TMh group (p<0.05). Concentrations of APRIL, BAFF, OPG, sRANKL, IL-6 and IL-8 in GCF are presented in Fig. 4. BAFF concentrations (Fig. 4A) in GCF were greater in the healthy comparison groups than in the TM groups (p<0.05). BAFF concentrations were also significantly higher (p<0.05) in the GCF of the TMh group compared to the TMg group. GCF APRIL (Fig. 4B), sRANKL (Fig. 4D), and IL-6 (Fig. 4F) concentrations were significantly greater (p<0.01) in TM groups than the healthy groups (p<0.001) and in the TMh than the TMg group (p<0.001). OPG (Fig. 4C) and IL-8 (Fig. 4F) concentrations were similar in the study groups. GCF sRANKL concentration was higher in the TMg than in the TMh group (p<0.05). Correlations Between the Clinical Periodontal Parameters and the Biochemical Data In the TM group, significant positive correlations were found between BOP, PI scores and GCF concentrations of BAFF, sRANKL, GCF APRIL (total), serum OPG and sRANKL (Table 2). In the systemically healthy comparison group significant positive correlations were detected between BOP, PI scores and GCF BAFF, serum and GCF APRIL (concentration and level), OPG & IL-6 in all biofluids, sRANKL & IL-8 in saliva and serum. Additionally, BOP scores significantly correlated with serum BAFF and GCF IL-8 concentrations. DISCUSSION It is likely that there is an association between TM and periodontal health status. However, very few studies have been published on this issue and to our knowledge, this is the first study to investigate the salivary and GCF levels of RANKL, OPG, APRIL, BAFF, IL-6, and IL-8 in patients with TM and gingivitis. The present study revealed that patients with TM had elevated serum and saliva levels of APRIL, sRANKL, IL-6, and sRANKL:OPG ratio as well as higher GCF sRANKL, IL-6, IL-8 levels than the systemically healthy comparison groups. Osteopenia and osteoporosis are major causes of morbidity in patients with TM due to imbalanced osteoclastic bone resorption and the direct iron toxicity on osteoblasts.23 The balance between RANKL and OPG has a controlling role on bone remodelling and bone loss.24 Local stimuli from hormones, inflammatory mediators and bacterial products contribute to the pathogenesis of osteoporosis in patients with thalassemia and lead to increased RANKL levels, decreased OPG:RANKL ratio and altered microenvironment of the over stimulated red marrow.25,26 Increased serum OPG and sRANKL concentrations have been reported as biochemical markers of bone turnover27 although the diagnostic value of increased or decreased serum levels of these proteins remains controversial. Significantly lower serum OPG concentrations have been reported in patients with TM than the systemically health controls28,29 but the present data indicated similar OPG levels in the study groups in all biofluids. Various other studies reported conflicting data, such as increased OPG concentrations.27,30 The present findings of serum RANKL levels are in line with a 5 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 previous investigation, which reported increased serum RANKL levels and indifferent OPG levels in patients with TM and systemically healthy comparison groups.23 The RANKL:OPG ratio has been suggested as a promising rapid and cheap screening marker for osteopenia or osteoporosis in patients suffering from thalassemia.29 The discovery of the RANK/RANKL/OPG system has brought significant progress in the understanding of the regulatory mechanisms of osteoclast differentiation and activation exerted by the immune system.7 RANKL:OPG pathway is involved in the pathogenesis of periodontitis.8,9,31 Accordingly, the present findings indicated higher GCF, serum and saliva sRANKL levels in patients with TM and gingivitis than systemically and periodontally healthy individuals. Whether gingival inflammation worsens the existing impaired osteoclast differentiation in patients with TM in this study is unclear, but worthy of consideration for future investigation. It is interesting to note the significant negative correlation between PI, BOP and the GCF sRANKL concentration of the TM patients when such a difference is not evident in the comparison groups. Considering the present findings, the increase in GCF sRANKL concentration of TM patients is not likely to be solely related with the level of clinical gingival inflammation and the microbial plaque levels. The patients in the TMh group may have subclinical gingival inflammation. The positive correlations between inflammatory markers and clinical periodontal parameters only in systemically healthy individuals may also support this speculation. Increased levels of inflammatory markers in patients with TM, who have low plaque scores and clinically healthy periodontium may explain these findings. IL-6 and IL-8 are important components of the pro-inflammatory response. Markedly increased plasma IL-6 and IL-8 levels were reported in patients with TM.32 The present data provides further support for the previous findings and increased production of IL-6 and IL-8 might be explained by abnormalities in iron metabolism due to overstimulation of macrophages. The present study revealed the highest serum IL-6 concentrations in patients with TM and gingivitis and IL-8 concentrations in periodontally healthy TM patients. Serum levels of these cytokines may be relevant in the pathophysiology of TM. Levels of inflammatory cytokines such as IL-6 and IL-8 are elevated in oral fluids in the presence of gingival inflammation compared to periodontal health.13 According to the present findings, gingival inflammation seems to have an enhancing effect on the levels of these cytokines, which in turn are capable of upregulating the chemotactic, secretory and phagocytic functions of macrophages33 and neutrophils.34 While IL-8 is principally involved in increased neutrophil and monocyte recruitment to sites of infection, both IL-633 and IL-8 are known to influence intracellular killing by these cells and particularly IL-8 stimulates secretion of B-cell activating BAFF and APRIL.35 BAFF and APRIL in return exert reverse signalling on the human monocytes (THP-1 cell-line) and macrophages to stimulate IL-8 and MMP-9 production36,37 and stimulate phagocytosis.37 Thus, at a localised periodontal tissue site, there is potential for these molecules to accentuate the activity induced by each other. In the short term this may be beneficial for eradication of infection, but longer duration may result in significant tissue damage and bone loss. A plausible hypothesis for when levels are perturbed and the balance upset, is that this leads to a defect in intracellular killing and other damaging consequences due to changes in the necessary cell functions for resolving disease and promoting tissue repair.38 6 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 Patients with TM may present with various immunological defects, such as impairment of neutrophil- macrophage- phagocytic and killing functions and altered production of various cytokines. However, the role of B/T-lymphocyte stimulatory cytokines such as APRIL, BAFF has not been investigated before in a similar patient group. The present findings revealed higher saliva and serum BAFF and APRIL levels in patients with TM compared to systemically healthy individuals with or without gingivitis. Although GCF APRIL levels were higher in the TM group, BAFF concentrations in GCF were lower than those in the healthy comparison groups. The reason for this finding may be a significant change in the periodontal inflammatory cell population and/or an indication of leukocyte dysfunction in TM, but this has yet to be elucidated. In order to delineate the present changes in BAFF/APRILL levels in this particular systemic condition, BAFF/APRIL antagonists, which are reported to be successful in clinical trials,39 may be investigated in future studies. CONCLUSION The present findings suggest a possible association between BAFF/APRIL system and TM. Alterations in APRIL and BAFF may be related with impaired regulation and activation of lymphocytes, which are associated with autoimmune response in patients with TM that can be possibly potentiated by gingival inflammation. Furthermore, evaluation of RANKL, OPG levels in oral biofluids may provide relevant information not only on periodontal disease state but also pathogenesis of bone and mineral metabolism in patients with TM. Further longitudinal prospective studies are warranted to better clarify the role of these cytokines in the pathogenesis of TM itself and its complications such as impaired bone metabolism. Elucidating the underlying pathogenic mechanisms may allow the design of optimal therapeutic and preventive measures for patients with this haematological disease. ACKNOWLEDGEMENTS The authors declare no conflicts of interest with respect to authorship and/or publication of this article. REFERENCES 1. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ 2008;86:480-487. 2. Cooley TB, Lee OP. Series of cases of splenomegaly in children with anemia and peculiar bone changes. Trans Amer Pediatr Soc 1925;37:29. 3. Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med 2005;15:1135-1146. 4. Calişkan U, Tonguç MO, Ciriş M, et al. The investigation of gingival iron accumulation in thalassemia major patients. J Pediatr Hematol Oncol 2011;33:98-102. 5. Ginzburg Y, Rivella S. β-thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism. Blood 2011;20:4321-4230. 6. Toumba M, Skordis N. Osteoporosis syndrome in thalassaemia major: an overview. J Osteoporos 2010;26:1-7. 7. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-176. 8. Buduneli N, Kinane DF. Host-derived diagnostic markers related to soft tissue destruction and bone degradation in periodontitis. J Clin Periodontol 2011;38:85-105. 7 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 9. Belibasakis GN. Bostanci N. The RANKL-OPG system in clinical periodontology. 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Periodontal condition and orofacial changes in patients with Thalassemia Major: A clinical and radiographic overview. J Clin Pediatr Dent 2012;36:301-308. 17. Al-Wahadni AM, Taani DQ, Al-Omari MO. Dental diseases in subjects with b-thalassemia major. Community Dent Oral Epidemiol 2002;30:418-22. 18. Tsalikis LE, Kaklamanos EG, Kavadia-Tsatala S, Chasapopoulou E, Pidonia-Manika I. Association of gingival crevicular fluid and serum intracytoplasmic enzyme levels in periodontally healthy homozygous (major) beta-thalassemia patients. J Clin Periodontol 2004;31:356-363. 19. Singh J, Singh N, Kumar A, Kedia NB, Agarwal A. Dental and periodontal health status of Beta thalassemia major and sickle cell anemic patients: a comparative study. J Int Oral Health 2013;5:53-58. 20. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. PLoS Med 2007;4:e296. 21. Löe H. The gingival index, the plaque index and the retention index systems. J Periodontol 1967;38:610-616. 22. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6. 23. Morabito N, Gaudio A, Lasco A, et al. Osteoprotegerin and RANKL in the pathogenesis of thalassemia-induced osteoporosis: new pieces of the puzzle. J Bone Miner Res 2004;19:722-727. 24. Lerner UH. New molecules in the tumor necrosis factor ligand and receptor superfamilies with importance for physiological and pathological bone resorption. Crit Rev Oral Biol and Med 2004;15:64-81. 25. Lerner UH. Inflammation-induced bone remodeling in periodontal disease and the influence of post-menopausal osteoporosis. J Dent Res 2006;85:596-607. 26. Liu YC, Lerner UH & Teng YT. Cytokine responses against periodontal infection: protective and destructive roles. Periodontol 2000 2010;52:163-206. 27. Angelopoulos NG, Goula A, Katounda E, et al. Circulating osteoprotegerin and receptor activator of NF-kappaB ligand system in patients with beta-thalassemia major. J Bone Miner Metab 2007;25:60-67. 28. Voskaridou E, Terpos E, Spina G, et al. Pamidronate is an effective treatment for osteoporosis in patients with beta-thalassaemia. Br J Haematol 2003;123:730-737. 8 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 29. Salah H, Atfy M, Fathy A, Atfy M, Mansor H, Saeed J. The clinical significance of OPG/sRANKL ratio in thalassemia patients suffering from osteopenia or osteoporosis in Egyptian patients. Immunol Invest 2010;39:820-832. 30. Pietrapertosa AC, Minenna G, Colella SM, Santeramo TM, Renni R, D'Amore M. Osteoprotegerin and RANKL in the pathogenesis of osteoporosis in patients with thalassaemia major. Panminerva Med 2009;51:17-23. 31. Cochran DL. Inflammation and bone loss in periodontal disease. J Periodontol 2008;79:15691576. 32. Oztürk O, Yaylim I, Aydin M, et al. Increased plasma levels of interleukin-6 and interleukin-8 in beta-thalassaemia major. Haematologia 2001;31:237-244. 33. Flesch IE, Kaufmann SH. Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6). Infect Immun 1990;58:269-271. 34. Nibbering PH, Pos O, Stevenhagen A, Van Furth R. Interleukin-8 enhances nonoxidative intracellular killing of Mycobacterium fortuitum by human granulocytes. Infect Immun 1993;61:3111-3116. 35. Scapini P, Bazzoni F, Cassatella MA. Regulation of B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS) expression in human neutrophils. Immunol Lett 2008;116:1-6. 36. Lee WH. BAFF and APRIL induce inflammatory activation of THP-1 cells through interaction with their conventional receptors and activation of MAPK and NF-κB. Inflamm Res 2011a;60:807815. 37. Lee SM, Kim EJ, Suk K, Lee WH. Synthetic peptides containing ITIM-like sequences of IREM-1 inhibit BAFF-mediated regulation of interleukin-8 expression and phagocytosis through SHP-1 and/or PI3K. Immunology 2011b;134:224-233. 38. Sorsa T, Tjäderhane L, Konttinen YT et al. Matrix metalloproteinases: contribution to pathogenesis, diagnosis and treatment of periodontal inflammation. Ann Med 2006;38:306-21. 39. Mackay F, Schneider P. Cracking the BAFF code. Nat Rev Immuno 2009;9:491-502. Correspondence Address: Dr. Aliye Akcalı, Department of Periodontology, School of Dentistry, Ege University, 35100 Bornova, İzmir / Turkey, Tel: + 90 232 388 11 05, Fax: + 90 232 388 03 25, E-mail: [email protected] Submitted November 12, 2014; accepted for publication April 15, 2015. Figure 1. Serum levels of biochemical data. Concentrations (pg/mL) of BAFF (A), APRIL (B), OPG (C), sRANKL (D), sRANKL:OPG (E), IL-6 (F) and IL-8 (G) in serum. The horizontal lines in the boxplots represent the median values and the whiskers represent the 5-95 percentiles. Values below and above the whiskers are drawn as individual dots. Significant differences between groups are shown as follows: * TMh significant difference with TMg; † TMh significant difference with Hh; ‡ TMg significant difference with Hg; § Hh significant difference with Hg. Figure 2. Salivary levels of biochemical data. Concentrations (pg/mL) of BAFF (A), APRIL (B), OPG (C), sRANKL (D), sRANKL:OPG (E), IL-6 (F) and IL-8 (G) in saliva. The horizontal lines in the boxplots represent the median values and the whiskers represent the 5-95 percentiles. Values below and above the whiskers are drawn as individual dots. Significant differences between groups are shown as follows: * TMh significant difference with TMg; † TMh significant difference with Hh; ‡ TMg significant difference with Hg; § Hh significant difference with Hg. 9 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 Figure 3. GCF total amounts of biochemical data. Amounts (pg/30S) of BAFF (A), APRIL (B), OPG (C), sRANKL (D), sRANKL:OPG (E), IL-6 (F) and IL-8 (G) in GCF. The horizontal lines in the boxplots represent the median values and the whiskers represent the 5-95 percentiles. Values below and above the whiskers are drawn as individual dots. Significant differences between groups are shown as follows: * TMh significant difference with TMg; † TMh significant difference with Hh; ‡ TMg significant difference with Hg; § Hh significant difference with Hg. Figure 4. GCF concentrations of biochemical data. Concentrations (pg/µL) of BAFF (A), APRIL (B), OPG (C), sRANKL (D), sRANKL:OPG (E), IL-6 (F) and IL-8 (G) in GCF. The horizontal lines in the boxplots represent the median values and the whiskers represent the 5-95 percentiles. Values below and above the whiskers are drawn as individual dots. Significant differences between groups are shown as follows: * TMh significant difference with TMg; † TMh significant difference with Hh; ‡ TMg significant difference with Hg; § Hh significant difference with Hg. 10 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 Table 1. Demographics and clinical periodontal measurements of the study based on periodontal status Systemically Healthy Thalassemia (n=29) (n=48) Periodontal status Healthy (n=14) Gingivitis (n=15) Healthy (n=20) 24.1 ± 5.1 27.9 ± 10.3 26.6 ± 4.2 Age (Years) 7/7 8/7 9/11 Male/Female 6/8 5/10 6/14 Smoker/ Non-smoker 0.64 ± 0.29 2.21 ± 0.45‡ 0.6 ± 0.5 PI (Score 0-3) 8.80 ± 10.73 73.45 ± 17.31‡ 10.21 ± 13.82 BOP (%) Gingivitis (n=28) 27.2 ± 6.1 15/13 11/17 1.29 ± 0.94† 68.52 ± 21.73* Values are shown as mean ± standard deviation. Significant differences between the gingivitis and periodontally healthy groups are indicated as follows: * p < 0.05, † p < 0.001, ‡ p<0.0001 11 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 Table 2. Correlation between the clinical periodontal parameters and the biochemical data Thalassemia Systemically Healthy Biochemical Data PI BOP PI BOP Serum BAFF r 0.072 0.033 0.340 0.263 p 0.714 0.868 0.093 0.028* Saliva BAFF r -0.049 0.012 0.394 0.288 p 0.800 0.951 0.049* 0.006† GCF BAFF (total amount) r -0.326 -0.252 0.613 0.522 p 0.120 0.235 <0.001† <0.001† GCF BAFF (concentration) r -0.477 -0.397 0.387 0.368 † p 0.055 0.019* 0.010 0.015* Serum APRIL r -0.026 -0.072 0.736 0.611 p 0.899 0.722 <0.001† <0.001† Saliva APRIL r 0.215 0.243 0.220 0.234 p 0.273 0.213 0.133 0.110 GCF APRIL (total amount) r -0.747 -0.835 0.355 0.395 p <0.001† <0.001† 0.020* 0.009† GCF APRIL (concentration) r -0.276 -0.257 0.392 0.392 † p 0.164 0.196 0.006† 0.006 Serum OPG r -0.334 -0.431 -0.460 -0.416 p 0.083 0.005† 0.022* 0.001† Saliva OPG r -0.008 -0.072 -0.493 -0.477 p 0.967 0.714 0.001† 0.001† GCF OPG (total amount) r 0.108 0.179 -0.470 -0.370 p 0.584 0.361 0.014* 0.001† GCF OPG (concentration) r -0.166 -0.090 -0.580 -0.527 p 0.397 0.649 <0.001† <0.001† Serum sRANKL r 0.372 0.438 0.646 0.625 p 0.047* 0.018* <0.001† <0.001† Saliva sRANKL r -0.002 0.060 0.428 0.437 p 0.993 0.762 0.002† 0.003† GCF sRANKL (total amount) r -0.087 -0.174 -0.211 -0.172 p 0.661 0.376 0.165 0.259 GCF sRANKL r -0.402 -0.390 -0.159 -0.053 (concentration) p 0.297 0.729 0.034* 0.040* Serum IL-6 r 0.190 0.151 0.619 0.456 p 0.364 0.471 <0.001† 0.001† Saliva IL-6 r 0.142 0.006 0.502 0.302 p 0.500 0.977 <0.001† 0.041* GCF IL-6 (total amount) r 0.057 -0.079 0.576 0.464 p 0.791 0.713 0.001† <0.001† GCF IL-6 (concentration) r -0.353 -0.263 0.300 0.260 p 0.066 0.177 0.092 0.050* Serum IL-8 r -0.125 -0.192 0.330 0.318 p 0.527 0.328 0.027* 0.033* Saliva IL-8 r 0.059 -0.026 0.332 0.251 p 0.765 0.896 0.093 0.024* GCF IL-8 (total amount) r 0.052 0.038 0.294 0.175 p 0.792 0.846 0.244 0.047* GCF IL-8 (concentration) r -0.113 -0.099 0.168 -0.054 p 0.568 0.617 0.276 0.727 12 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 * Correlation is significant at the 0.05 level (2-tailed). † Correlation is significant at the 0.01 level (2tailed). * Hu-Friedy, Chicago, IL, USA † BD Diagnostics, Franklin Lakes, NJ ‡ PerioPaper, ProFlow, Amityville, NY § Periotron 8000, Oraflow, Plainview, NY ** R & D Systems Abingdon UK †† Peprotech London UK ‡‡ R & D Systems Abingdon UK §§ R & D Systems Abingdon UK *** eBioscience Hatfield UK ††† R & D Systems Abingdon UK ‡‡‡ R & D Systems Abingdon UK §§§ GraphPad Prism version 6.00c for Mac OS X, GraphPad Software, La Jolla California USA 13 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 14 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 15 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 16 Journal of Periodontology; Copyright 2015 DOI: 10.1902/jop.2015.140639 17