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RESEARCH REPORT |
1 Department of Operative Dentistry and Periodontology, Dental School, University of Regensburg, 93042 Regensburg, Germany; and
2 School of Dental Sciences, University of Newcastle, England;
* corresponding author, stefan.ruhl{at}klinik.uni-regensburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(IL-1), IL-6, IL-8, epidermal growth factor (EGF), nerve growth factor (NGF), and albumin were detected, but in glandular saliva only EGF and NGF were identified. Albumin and IL-6 differed significantly between responders and controls, although the overall profile of salivary proteins remained unchanged. Thus, inflammatory cytokines and albumin are confined to whole saliva and are associated with GO, whereas its content of EGF and NGF appears unaffected by cyclosporin.
KEY WORDS: cyclosporin • gingiva • overgrowth • saliva • proteins • cytokines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Drug-induced gingival overgrowth (GO) is associated with chronic usage of cyclosporin, phenytoin, and calcium channel-blockers (Hassell and Hefti, 1991; Hallmon and Rossmann, 1999). Although the common mechanism of action of these drugs relative to GO remains uncertain, there is evidence that gingival cytokines and growth factors are involved in the pathogenesis of this disease (Williamson et al., 1994; Nares et al., 1996; Das et al., 2002). Despite the fact that gingival tissue is continuously bathed in salivary secretions that are known to exhibit tissue-protective and antimicrobial functions (Mandel, 1987), a possible involvement of saliva and its cytokines or growth factors in drug-induced GO has been investigated in only a few studies (Nieuw Amerongen et al., 1993; Markopoulos et al., 2001; Das et al., 2002). In these studies, it was not determined if increased cytokine concentrations are a consequence of local gingival disease activity or are an effect of the medication on the salivary glands. Thus, in the present investigation, cyclosporin-induced GO was used as a model for the study of proteins, cytokines, and growth factors in whole saliva and in secretions of the major salivary glands. Albumin was used as a reference for the contribution of local gingival disease activity.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Gel Electrophoresis and Blotting
The concentrations of total protein in salivary samples were determined according to the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Bovine serum albumin was used as the standard. Samples were denatured in sample buffer containing 5% 2-mercaptoethanol and subjected to SDS-PAGE (0.75 µg per lane or 15 µg per lane for blotting) on 4–20% gradient gels (Invitrogen Corp., Carlsbad, CA, USA). Salivary proteins were visualized with the use of an ammoniacal silver stain kit (Invitrogen Corp., Carlsbad, CA, USA). Glycosylated salivary components were identified on nitrocellulose transfers according to a modification of the hydrazide method and were further characterized by lectin blotting as previously described (Ruhl et al., 1996; Ruhl et al., 2000).
Analysis of Similarity between Protein Banding Patterns
Gels and blots were scanned with the use of a desktop scanner (Sharp Color Image Scanner Model JX-330). The resulting TIFF files were analyzed with the use of Image Master 1D software, version 2.0 (Amersham Pharmacia Biotech GmbH, Freiburg, Germany). For the analysis of similarity between individual banding patterns, lanes were scored for the presence or absence of bands. We compared banding patterns using the Image Master 1D database software, version 2.0 (Amersham Pharmacia Biotech GmbH). Similarity coefficients were calculated according to the method of Pearson. Dendrograms were calculated by means of the unweighted pair group method, with the arithmetic average (UPGMA) (Sneath and Sokal, 1973). Samples taken from two healthy individuals not belonging to the study population were run on each gel as references for reproducibility.
Albumin Determinations
Albumin concentrations were determined with the use of a nephelometric assay unit (BN PROSPEC; Dade Behring Marburg GmbH, Marburg, Germany; Host Driver software, version 1.2). According to the manufacturer’s instructions, albumin was detected with a rabbit anti-human albumin antiserum (N Antiserum to Human Albumin) with human serum albumin protein reference preparation CRM 470.
Cytokine and Growth Factor Determinations
Concentrations of interleukin-1 (IL-1), IL-6, IL-8, epidermal growth factor (EGF), nerve growth factor (NGF), transforming growth factor- (TGF), tumor necrosis factor- (TNF), platelet-derived growth factor (PDGF-AB), basic fibroblast growth factor (bFGF), and keratinocyte growth factor (KGF) were measured by means of immunoassay kits (TGF, Oncogene Research Products, San Diego, CA, USA; NGF, Promega Corp, Madison, WI, USA; all others, R&D Systems, Minneapolis, MN, USA). Plates were measured by means of a THERMOmax multiplate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm (FGF and TGF, 490 nm), and data were calculated from triplicate measurements with the use of SoftMaxPro software (Molecular Devices). Standard curves were derived according to a four-parameter logistic curve-fit.
Data Treatment and Statistics
Median concentrations for experimental groups (responders, non-responders, and controls) were calculated together with the 25 and 75 percent quantiles, and further statistical evaluation was performed by the non-parametric Mann-Whitney U Test (SPSS PC+ software, version 5.01, SPSS Inc., Chicago, IL, USA) at the 0.05 level of significance. Correlation analyses were performed with TableCurve 2D, version 4, software (SPSS Inc., Chicago, IL, USA).
Patients
Salivary samples were collected from all patients enrolled in two consecutive parts of the study, which received ethical approval from the combined Health Authority/University Ethics Committee of the University of Newcastle upon Tyne, England. Subjects participated after providing informed consent to this ethics-Board-approved protocol. The transplant subjects were more than 6 mos post-transplantation and were medicated with cyclosporin. Only male subjects who possessed a minimum of 6 of the 8 most anterior teeth in both upper and lower arches participated in the study. Overgrowth was scored as described by Seymour et al.(1985). Transplant subjects were assigned to experimental groups with marked GO, termed "responders" (overgrowth scores greater than 30%), and those with little or no gingival changes, termed "non-responders" (overgrowth scores less than 10%). The control group was comprised of males who were not medicated with cyclosporin or other drugs known to induce GO, did not show any signs of gingival inflammation, and whose probing pocket depths did not exceed 3 mm. For analyses of protein composition of whole saliva by SDS-PAGE and image analysis (first part of the study), the experimental groups consisted of nine responders, nine non-responders, and 10 controls. Based on these findings, we performed a consecutive study to further analyze cytokines, growth factors, and albumin in whole, parotid, and SMSL saliva. For this second part of the study, another 10 responders, 11 non-responders, and 10 controls were recruited.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The dendrogram in Fig. 1B, graphically depicting the similarities between individuals, indicates that all samples were already at a high level of similarity, with an overall correlation coefficient of 0.9. On this high level of correlation, no clustering of responders, non-responders, and controls was found. A similar lack of clustering (with overall correlation coefficients ranging from 0 to 0.76) was seen when nitrocellulose transfers were stained for glycoproteins by the hydrazide method or by lectin blotting (data not shown).
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, 4.5 pg/mL for IL-6, 1323 pg/mL for IL-8, 975 pg/mL for EGF, and 9644 pg/mL for NGF (Fig. 2). The median concentration for TGF was 14 pg/mL, with 3 out of 10 values under the assay’s detection limit of 6 pg/mL. For FGF, 8 out of 10 values were below the assay’s detection limit of 0.25 pg/mL. Only 2 samples showed PDGF-AB concentrations above the assay’s detection limit of 8.4 pg/mL. TNF and KGF in all samples were below the detection limits (4.4 and 15 pg/mL, respectively) of the assays.
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). Besides EGF and NGF, only IL-8 was detected in parotid (35 pg/mL) and SMSL (90 pg/mL) saliva of controls. All other cytokines ranged below or close to the detection limits (IL-1, 1.5 pg/mL) of the assays. The median concentration of total salivary protein in controls was higher in whole saliva (2.0 mg/mL) than in either parotid (1.0 mg/mL) or SMSL (0.7 mg/mL) secretions (Fig. 2A). There was no significant relation, however, between the concentrations of the cytokines selected and the concentrations of whole salivary protein as tested by correlation analyses (data not shown).
In responders, a tendency to increased median concentrations in whole saliva was found only for IL-1 (1604 pg/mL), IL-6 (46 pg/mL), and IL-8 (3297 pg/mL), as compared with controls (Fig. 2). This difference was found to be statistically significant only for IL-6. No statistically significant differences were found between non-responders and controls. In responders, median concentrations in SMSL secretions were 13 pg/mL for IL-1, 5 pg/mL for IL-6, and 101 pg/mL for IL-8. These concentrations were not significantly different from those in non-responders. In parotid secretions, the median concentrations for responders were 2.5 pg/mL for IL-1, ~ 0.5 pg/mL for IL-6, and 44 pg/mL for IL-8, again with no significant difference in comparison with non-responders.
Albumin in Whole Saliva and Glandular Secretions
In whole saliva of controls, the median concentration of albumin was 38.5 µg/mL (Fig. 2G), whereas in responders it was 88 µg/mL and in non-responders 52 µg/mL. The differences between responders and controls as well as between responders and non-responders were shown to be statistically significant. Between non-responders and controls, no significant difference could be detected. In secretions of the major salivary glands, almost all samples ranged at or below the detection limit of 18.9 µg/mL.
Correlations
No correlation was shown between the concentrations of EGF and NGF in glandular secretions or in whole saliva. There was no correlation between any of the inflammatory cytokines (IL-1, IL-6, and IL-8) in whole saliva with any of the other cytokines. None of the cytokines or albumin correlated with the total protein concentration (correlations not shown). Only IL-6 correlated with albumin (r2 = 0.89) in whole saliva of control patients (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-6, and IL-8 are predominantly present in whole saliva, whereas growth factors such as EGF and NGF are found in secretions of the major salivary glands. Growth factors are not influenced by either cyclosporin medication or GO, whereas inflammatory cytokines such as IL-6 in whole saliva appear to be disease-associated. Furthermore, cyclosporin-induced GO or medication with cyclosporin appears to have no influence on the overall composition of salivary proteins and glycoproteins. It can be concluded that cyclosporin medication, regardless of its clinical effect on the gingiva, alters neither the general profile of proteins in saliva nor the excretion of the investigated cytokines and growth factors by the major salivary glands. Because cyclosporin affects numerous cellular functions (Ho et al., 1996), this drug might also affect the function of salivary glands, resulting in an altered composition of salivary proteins. This may, in turn, be related to the pathogenesis of GO. Image analysis of protein patterns in individual samples resolved after SDS-PAGE showed, however, that the overall qualitative composition of proteins and glycoproteins in whole saliva was unchanged among patients and controls. Thus, it would appear that cyclosporin either does not affect the protein composition of saliva or does so at a level that is below that of detection with the current methodology.
Since cyclosporin is known to modulate cytokine expression by various cells (Ho et al., 1996), it may particularly influence cytokines excreted by the major salivary glands. Analysis of parotid and SMSL saliva, however, showed that the cytokines that were elevated in the whole saliva of patients medicated with cyclosporin were not derived from the major salivary glands. This is in clear contrast to what has been suggested for primary Sjögren’s syndrome, where it is mainly the glandular saliva that is responsible for the observed increase in salivary inflammatory cytokines (Fox et al., 1994; Tishler et al., 1999). However, in this disease, the primary site of inflammation is located in the salivary glands and not in the gingival tissue as in cyclosporin-induced GO. In the present study, only EGF and NGF were shown to be derived from glandular sources, with, in the case of EGF, the parotid gland producing slightly higher amounts. This confirms a previous report for EGF (Thesleff et al., 1988). Two- to five-fold-higher concentrations of EGF in whole saliva were measured than in a previous report (Markopoulos et al., 2001), and no statistically significant differences in EGF concentrations among responders, non-responders, and controls were found. Therefore, the results of the present study indicate that cyclosporin may not exert an effect on cytokine or growth factor excretion by the major salivary glands.
Because GO is typically associated with gingival inflammation (Seymour et al., 2000), inflammatory cytokines in whole saliva might be derived from either gingival crevicular fluid (GCF) or from leakage of serum through gingival tissue. This is supported by the finding that increased concentrations of inflammatory cytokines in whole saliva are paralleled by the appearance of albumin, a marker for leakage of plasma proteins into saliva (Oppenheim, 1970; Henskens et al., 1996). Furthermore, albumin concentrations, analogous to IL-6, were significantly elevated in responders as compared with controls, which suggests that both components may be derived from a common source. However, their production does not seem to be regulated by the same mechanism, because IL-6 and albumin concentrations do not correlate in responders. The increased IL-6 concentration in patients with GO is in accordance with the functions of IL-6 in periodontal inflammation, such as induction of immunoglobulin-secreting plasma cells and activation of osteoclasts (Okada and Murakami, 1998). In addition to GCF, the minor salivary glands could be a potential source of inflammatory cytokines (Fox et al., 1999). In view of the observed occurrence of GO in the buccal anterior region of the dentition, a future study on cytokine production of labial minor salivary glands during drug-induced GO would seem appropriate.
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ACKNOWLEDGMENTS |
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Received April 8, 2003; Last revision February 6, 2004; Accepted February 9, 2004
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REFERENCES |
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