Plasminogen activators and plasminogen activator inhibitors in gingival crevicular fluid of cyclosporin A-treated patients

BACKGROUND: The plasminogen activator (PA) system plays many roles in the inflammatory process and tissue remodelling and repair and is considered to play a significant role in periodontal tissue destruction and healing. The aim of this study was to evaluate the role of the PA system in cyclosporin A (CsA)-induced gingival overgrowth in renal transplant patients. METHODS: Eighteen renal transplant patients exhibiting moderate to severe CsA-induced gingival overgrowth, 10 other renal transplant patients receiving CsA therapy but showing no sign of CsA-induced gingival overgrowth (CsA-H), 16 chronic gingivitis patients (CG) and 16 systemically and periodontally healthy control subjects (H) were included in the study. Gingival crevicular fluid (GCF) samples were obtained from four randomly selected sites in each subject with the exception of the CsA-induced gingival overgrowth group, where four GCF samples were harvested from sites with severe overgrowth (CsA GO+) and from four sites without any gingival overgrowth (CsA GO-). The GCF levels of albumin, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) were analysed by enzyme-linked immunosorbent assay. The results were tested for statistical differences. RESULTS: In CsA GO+ sites t-PA levels were significantly elevated in comparison with gingivitis and healthy sites, while PAI-2 levels in these sites showed statistically significant differences only with CsA-H and gingivitis sites (p<0.05). The levels of t-PA and PAI-2 were significantly higher in CsA GO- sites compared with those of CsA-H, gingivitis and healthy sites (p<0.05). The levels of u-PA and PAI-1 failed to show significant differences between the study groups. CONCLUSIONS: The findings of the present study indicate alterations in GCF t-PA and PAI-2 levels in CsA-induced gingival overgrowth and might suggest involvement of the plasminogen activating system in the pathogenesis of this side-effect of CsA therapy. However, to what extent these molecules contribute to the pathogenesis of CsA-induced gingival overgrowth remains to be determined.     

Evaluation of transforming growth factor-beta 1 level in crevicular fluid of cyclosporin A-treated patients

BACKGROUND: The aim of the present study was to investigate the level of transforming growth factor-beta 1 (TGF-beta 1) in gingival crevicular fluid (GCF) samples of cyclosporin A (CsA)-treated patients and to compare the results with control groups. METHODS: Fourteen renal transplant patients exhibiting severe CsA-induced gingival overgrowth, 10 patients with chronic gingivitis, and 10 subjects with clinically healthy periodontium were included in the study. In CsA-treated patients, GCF samples were harvested from sites exhibiting gingival overgrowth (CsA GO+) and sites not exhibiting gingival overgrowth (CsA GO-). The TGF-beta 1 levels in a total of 96 GCF samples from the 34 participants were analyzed by enzyme-linked immunosorbent assay. The results were expressed in terms of total amount (pg/2 sites) and concentration (ng/ml). RESULTS: TGF-beta 1 total amounts in CsA GO+ and CsA GO- sites were similar and significantly higher than that of healthy sites (P < 0.02 and P < 0.01, respectively). The total amount of TGF-beta 1 was also higher in gingivitis sites compared to the healthy sites, but the difference was not statistically significant (P > 0.05). CsA GO+ and CsA GO- sites exhibited higher total amount and concentration of TGF-beta 1 than that of gingivitis sites, but the differences were insignificant (P > 0.05). CONCLUSIONS: The results of the present study support the theory that CsA increases the synthesis of TGF-beta 1 in GCF. However, since the difference between CsA GO+ and CsA GO- sites was not statistically significant, it seems unlikely that GCF TGF-beta 1 level is the sole factor responsible for the CsA-induced gingival overgrowth. Complex interactions between various mediators of inflammation and tissue modeling are possibly involved in the pathogenic mechanisms of this side effect.     

Matrix metalloproteinases (MMP-8 and -9) and neutrophil elastase in gingival crevicular fluid of cyclosporin-treated patients

BACKGROUND: Gingival overgrowth (GO) is one of the most important side effects of cyclosporin A (CsA) medication, but its pathogenesis is not completely understood. The aim of this study was to identify and compare collagenase-2 (MMP-8), gelatinase-B (MMP-9), and neutrophil (PMN)-elastase levels in gingival crevicular fluid (GCF) from 15 renal transplant patients receiving CsA therapy and exhibiting CsA GO, 14 patients with gingivitis, and 10 periodontally healthy subjects. METHODS: Clinical data were obtained on plaque index, papilla bleeding index, and hyperplastic index from each site studied. GCF samples and clinical data were collected from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. CsA GO+ and CsA GO- sites were divided into 2 subgroups as clinically not inflamed (PBI = 0) and inflamed (PBI > or =1). GCF MMP-8, MMP-9, and PMN-elastase levels were analyzed by immunofluorometric assay. RESULTS: GCF MMP-8 and -9 levels and clinical degrees of gingival inflammation in CsA GO+ sites were similar to those in diseased sites. However, GCF elastase levels were significantly lower in CsA GO+ sites compared to those in diseased sites. GCF MMP-8, -9 and PMN-elastase levels were not different between CsA GO- sites and healthy sites. Additionally, GCF MMP-8 and -9 levels in inflamed CsA GO+ sites were higher but not statistically significantly than those in diseased sites. In contrast, GCF PMN-elastase levels in inflamed CsA GO+ sites were significantly lower than the levels in diseased sites. CONCLUSIONS: These results show that CsA therapy does not have a significant effect on GCF MMP-8 and MMP-9 levels, but the gingival inflammation seems to be the main reason for their elevations. However, low GCF PMN-elastase levels can be an important factor in the pathogenesis of CsA-induced gingival overgrowth. CsA therapy does not eliminate the potential use of GCF MMP-8 and -9 as future diagnostic markers of gingival inflammation.     

Levels of leukotriene B4 and platelet activating factor in gingival crevicular fluid in renal transplant patients receiving cyclosporine A

BACKGROUND: Cyclosporine A (CsA) is a potent immunosuppressant effectively used to prevent organ transplant rejection and also to treat several systemic diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of the present study was to identify the role of leukotriene B4 (LTB4) and platelet activating factor (PAF) in the pathogenesis of CsA GO. METHODS: LTB4 and PAF levels were detected in gingival crevicular fluid (GCF) samples from renal transplant patients receiving CsA therapy and exhibiting CsA GO, from patients with gingivitis and from periodontally healthy subjects. Plaque index, papilla bleeding index, and hyperplastic index were recorded at each study site. GCF samples and clinical data were obtained from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. LTB4 was extracted from the samples by solid-phase method using C18 cartridge and purified by high-performance liquid chromatographic (HPLC) method and analyzed by radioimmunoassay (RIA). PAF was extracted from GCF samples passing through amberlit resin columns, purified by HPLC, and analyzed by RIA. RESULTS: Total amounts of LTB4 and PAF in GCF were higher in CsA GO+ sites compared to the healthy sites from healthy controls. However, the amount of LTB4 and PAF elevation in CsA GO+ sites was not significantly higher than those in diseased sites. Clinical degrees of gingival inflammation were also similar between CsA GO+ and diseased sites. LTB4 and PAF total amounts in GCF were higher in CsA GO+ sites compared to CsA GO- sites in the same subjects, but this difference just failed to reach significance. Similar findings were obtained with concentration data. CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on GCF LTB4 and PAF levels and that gingival inflammation seems to be the main reason for their elevation. In CsA-treated patients, alterations in LTB4 and PAF levels might play a role in CsA GO through some asyet unknown mechanism. To our knowledge, this is the first report describing the levels of lipid mediators in GCF of CsA-treated patients. We assume that further studies will contribute to the understanding of the pathogenesis of CsA-induced gingival overgrowth.     

Effect of cyclosporin A on apoptosis and expression of p53 and bcl-2 proteins in the gingiva of renal transplant patients

BACKGROUND: Gingival overgrowth (GO) is a common side effect of cyclosporin A (CsA) therapy, but the exact mechanism for this is unknown. Apoptosis plays an important role in the maintenance of tissue homeostasis and mediators of this process may be involved in the pathogenesis of drug-induced GO. This study compared p53 expression, bcl-2 expression, and apoptosis in gingival samples from CsA-treated renal transplant recipients to findings in controls with gingivitis. METHODS: Twenty-two kidney recipients with CsA-induced GO and 15 systemically healthy subjects with gingivitis were included in the study. The 15 systemically and periodontally healthy volunteer control group were immunohistochemically analyzed for grades of p53 and bcl-2 expression, and were processed using terminal TdT-mediated dUTP-biotin nick-end labeling (TUNEL) technique to identify and grade levels of apoptosis. RESULTS: There were no differences between the CsA group and the control group with respect to grades of p53 and bcl-2 expression (P >0.05 for both). However, the CsA group showed a lower apoptosis grade than the control group (P <0.05). None of the clinical parameters was significantly correlated with any of the immunohistochemical findings for p53 or bcl-2 (P >0.05 for all). Similarly, grade of apoptosis was not correlated with any of the clinical parameters (P >0.05). There was a significant positive correlation between serum CsA level and level of bcl-2 expression, but serum CsA was not significantly correlated with level of apoptosis or level of p53 expression. CONCLUSION: The results indicate that the pathogenesis of CsA-induced GO might involve inhibition of apoptosis, and overexpression of bcl-2 in the setting of high serum CsA.     

Gingival crevicular fluid and serum matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 levels in renal transplant patients undergoing different immunosuppressive therapy

Aim: We investigated gingival crevicular fluid (GCF) and serum matrix metalloproteinase‐8 (MMP‐8) and tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) levels from renal transplant patients receiving cyclosporine‐A (CsA) and having gingival overgrowth (GO), from patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy. Material and Methods: GCF samples were collected from sites with GO (GO+) and without GO (GO?) in CsA patients having GO; and GO? sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum MMP‐8 and TIMP‐1 levels were determined by a time‐resolved immunofluorometric assay (IFMA) and enzyme‐linked immunosorbent assay. Results: GO+ sites in CsA patients having GO had elevated GCF MMP‐8 levels compared with those of CsA patients having no GO, tacrolimus and healthy subjects (p<0.005), but these levels were similar to those of gingivitis. The GCF MMP‐8 level was higher in GO+ compared with GO? sites in CsA patients having GO (p<0.05). GCF TIMP‐1 levels were similar between groups. Tacrolimus patients had lower GCF MMP‐8 levels than gingivitis (p<0.005), but levels similar to the healthy group. Conclusion: These results show that CsA and tacrolimus therapy has no significant effect on GCF MMP‐8 levels, and gingival inflammation seems to be the main reason for their elevations.     

Gingival crevicular fluid osteocalcin, N-terminal telopeptides, and calprotectin levels in cyclosporin A-induced gingival overgrowth

Background: The aim of this cross‐sectional study is to investigate gingival crevicular fluid (GCF) osteocalcin, cross‐linked N‐terminal telopeptide (NTx), and calprotectin levels in cyclosporin A (CsA)–induced gingival overgrowth (GO). Methods: Forty medicated patients with CsA including 20 with GO (CsA GO+), 10 without GO (CsA GO?), 10 with GO and chronic periodontitis (CsA CP) and 60 patients with CP alone, 20 patients with gingivitis, and 20 healthy patients were enrolled. Probing depth, clinical attachment level, plaque index, and papillary bleeding index were recorded. GCF calprotectin, osteocalcin, and NTx levels were analyzed by enzyme‐linked immunosorbent assay. Parametric tests were used for statistical analysis. Results: The CsA GO+ and CP groups had significantly lower GCF osteocalcin levels and osteocalcin/NTx ratio than the healthy group, whereas GCF osteocalcin levels and osteocalcin/NTx ratio in the gingivitis group were higher than the CsA GO+, CsA GO?, CsA CP, and CP groups (P <0.05). The CP group had elevated GCF calprotectin levels compared to the other study groups (P <0.05). The CsA GO+ and CsA GO? groups also had higher GCF calprotectin levels compared to the CsA CP, gingivitis, and healthy groups (P <0.05). Conclusions: Increased GCF calprotectin and decreased GCF osteocalcin levels in the CsA GO+ and CsA GO? groups might suggest that CsA plays a role on the levels of these markers. The similarity of GCF osteocalcin, NTx, and calprotectin levels in the CsA GO+ and CsA GO? groups might suggest that these molecules are not involved in the pathogenesis of GO.     

Gingival crevicular fluid transforming growth factor-beta1 in cyclosporine and tacrolimus treated renal transplant patients without gingival overgrowth

BACKGROUND: Gingival crevicular fluid (GCF) levels of transforming growth factor-beta(1) (TGF-beta(1)) have been previously investigated in relation to the pathogenesis of cyclosporine-A (CsA)-induced gingival overgrowth (GO) but no clinical data are available regarding the GCF levels of TGF-beta(1) in patients treated with tacrolimus (Tac). However, as gingival inflammation is pronounced at sites of GO and this consequently may lead to an elevation in TGF-beta(1) levels the present study aimed to evaluate gingival crevicular fluid (GCF) TGF-beta(1) levels in renal transplant patients using CsA or Tac without GO. METHODS: GCF TGF-beta(1) levels were investigated in 30 renal transplant patients without GO medicated with either CsA (n=15) or Tac (n=15). Sixteen gingivitis patients and 15 periodontally healthy subjects were selected as controls. Periodontal status was evaluated by measuring probing depth, plaque index and papilla bleeding index. The TGF-beta(1) levels were analysed by enzyme-linked immunosorbent assay. RESULTS: Both CsA and Tac groups had significantly elevated GCF TGF-beta(1) total amount compared to gingivitis and healthy groups (p<0.008). GCF TGF-beta(1) total amount of CsA and Tac groups was similar (p>0.008). Gingivitis and healthy groups had also similar GCF TGF-beta(1) total amount (p>0.008). CONCLUSIONS: Within the limits of the present data it is unlikely that TGF-beta(1) is an exclusive mediator of CsA- or Tac-induced GO. However, pathogenesis of GO is multifactorial and contribution of TGF-beta(1) to the interrelations between cytokines and growth factors with fibrogenic potential cannot be disregarded.     

GCF and serum myeloperoxidase and matrix metalloproteinase-13 levels in renal transplant patients

Aim

The rationale of this study was to address whether local or systemic changes reflect proteolytic (matrix metalloproteinase-13) or oxidative (myeloperoxidase) stress in renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), in patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy.

Material and methods

Gingival crevicular fluid (GCF) samples were collected from sites with (GO+) and without GO (GO−) in CsA patients having GO; GO− sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum myeloperoxidase (MPO) and matrix metalloproteinase-13 (MMP-13) levels were determined by ELISA.

Results

GO+ sites in CsA patients having GO had elevated GCF MPO levels than those of CsA patients having no GO, tacrolimus and healthy subjects (p < 0.005), but comparable to those of gingivitis. GCF MPO levels were higher in GO+ compared to GO− sites in CsA patients having GO (p < 0.05). Patient groups had similar, but higher GCF MMP-13 levels than healthy group.

Conclusions

These results show that CsA and tacrolimus therapy have not a significant effect on GCF MPO and MMP-13 levels, and gingival inflammation seems to be the main reason for their elevations.     

Evaluation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 levels in gingival fibroblasts of cyclosporin A-treated patients

BACKGROUND: Cyclosporin A (CsA) is a potent immunosuppressant used to prevent organ transplant rejection and to treat various autoimmune diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of this study was to identify and compare matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in gingival fibroblast cultures of tissues derived from renal transplant patients receiving CsA and exhibiting gingival overgrowth and from periodontally healthy control subjects. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with CsA, and control tissues were obtained from systemically healthy donors. Gingival fibroblasts were grown using explant cultures. Three different study groups were identified: 1) CsA GO fibroblast culture; 2) CsA-treated healthy gingival fibroblast culture (H+CsA); and 3) healthy gingival fibroblast culture (H). The levels of MMP-1 and TIMP-1 in these groups of gingival fibroblasts were analyzed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: The levels of TIMP-1 were significantly lower in CsA GO than H (P < 0.05). There was no statistically significant difference in the levels of MMP-1 between H and CsA GO (P = 0.505). The ratio of MMP-1 to TIMP-1 was significantly higher in CsA GO than H (P < 0.05). CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on MMP-1 levels. However, low TIMP-1 levels can be an important factor in the pathogenesis of CsA GO, since the balance between MMP-1 and TIMP-1 levels was changed by CsA.     

Relationship between IL-1A polymorphisms and gingival overgrowth in renal transplant recipients receiving Cyclosporin A

AIM: Levels of interleukin-1alpha (IL-1alpha) are elevated in periodontal inflammation. IL-1A gene polymorphisms are associated with inflammatory diseases. This study aimed to investigate IL-1A gene polymorphism in Cyclosporin A (CsA)-treated renal transplant patients and investigate the association between this polymorphism and gingival crevicular fluid (GCF) levels of several cytokines. MATERIALS AND METHODS: Fifty-one renal transplant patients on CsA treatment (25 with and 26 without gingival overgrowth) and 29 healthy controls were recruited for the study. Demographic, pharmacological and periodontal parameters were recorded and gingival overgrowth was assessed. RESULTS: Multiple regression analysis showed that genotype was significantly associated with gingival overgrowth (p=0.02). Carriage of the IL-1A (-889) T allele was strongly protective [95% confidence interval (CI): 0.046-0.77], although not significantly associated with IL-1alpha protein levels in GCF. IL-1alpha, IL-1beta and IL-8, but not IL-6, were detected in GCF of CsA-treated patients, but none of them was significantly associated with gingival overgrowth. CONCLUSIONS: This study is the first to associate a gene polymorphism as a risk factor for CsA-induced gingival overgrowth in renal transplant patients, demonstrating that IL-1A polymorphism might alter individual susceptibility to CsA. However, there was no association between GCF cytokine levels and the presence of gingival overgrowth or patient IL-1A genotype.     

Immunohistochemical evaluation of Ki-67 expression and apoptosis in cyclosporin A-induced gingival overgrowth

BACKGROUND: This study was planned to evaluate cell division rate and apoptosis by immunohistochemical and in situ hybridization techniques in cyclosporin A (CsA)-induced gingival overgrowth tissue samples to determine whether these processes played a role in the pathogenesis of this condition. METHODS: Fourteen CsA-induced overgrowth tissues from renal transplant recipients, 10 control tissues from patients with plaque-induced gingivitis, and 14 control tissues from systemically and periodontally healthy subjects were evaluated. In patient groups, clinical periodontal recordings and tissue sampling were performed before initiation of any periodontal intervention. Numbers of Ki-67-positive cells/field and apoptotic cells/field in formalin-fixed/paraffin-embedded tissue sections were determined. Data were evaluated by one-way analysis of variance, post hoc Sidak test with modified Bonferroni correction, and Pearson correlation analysis. Three phenytoin- and five nifedipine-induced overgrowth tissues were also processed in the same way, and findings in these tissue specimens were evaluated as case series. RESULTS: The number of keratinocytes was significantly greater in the CsA-induced gingival overgrowth group than in the healthy control group (P <0.05). Cells labeled by in situ end labeling, namely the apoptotic cells, were significantly fewer in the CsA group than in the gingivitis and healthy control groups (P <0.01). Overall, statistically significant positive correlations were found between the numbers of Ki-67-positive cells and probing depth and hyperplastic, bleeding, and plaque indices (P <0.01). Phenytoin and nifedipine samples exhibited obviously higher expression of Ki-67-positive cells than the CsA, gingivitis, and healthy control groups. CONCLUSION: Our findings suggest that decreased apoptosis may have a more prominent role than increased cell division in the pathogenesis of CsA-induced gingival overgrowth.     

Toll like receptor 4 and membrane-bound CD14 expressions in gingivitis, periodontitis and CsA-induced gingival overgrowth

Objective

Immune cell recognition of lipopolysaccharides via CD14 and Toll-like receptor 4 (TLR4) complexes plays a crucial role in linking innate and adaptive immune responses. This study was aimed to investigate the expression of TLR4 and membrane-bound CD14 (mCD14) in the gingival tissues of patients with gingivitis, periodontitis and CsA-induced gingival overgrowth.

Design

Gingival tissues were obtained from 10 renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), 10 patients with chronic periodontitis, 10 generalized aggressive periodontitis, 10 gingivitis and 10 healthy subjects. Immunohistochemistry was performed in order to determine the localization of TLR4 and mCD14 in tissue specimens.

Results

TLR4 and mCD14 expressions were detected in all tissues including healthy gingival biopsies. TLR4 and mCD14 positive cells were predominantly confined to the epithelium–connective tissue interface area, and were highly expressed in the basal cell layer of patients with CsA GO and chronic periodontitis, compared to healthy group (P < 0.05).

Conclusion

The present study suggests that TLR4 and mCD14 protein expressions may be interrelated and appear to be associated with periodontal disease. CsA usage seemed not to affect TLR4 and mCD14 expressions in CsA induced GO tissues.     

Interleukin‐6 Family of Cytokines in Crevicular Fluid of Renal Transplant Recipients With and Without Cyclosporine A–Induced Gingival Overgrowth

Background: Interleukin (IL)‐6 family of cytokines, including IL‐6, oncostatin M (OSM), leukemia inhibitory factor (LIF), and IL‐11, have fibrogenic features. The current study determines gingival crevicular fluid (GCF) levels of fibrosis‐related IL‐6–type cytokines in cyclosporine A (CsA)–induced gingival overgrowth (GO). Methods: Eighty non‐smokers were included (40 CsA‐medicated renal transplant patients with GO [GO + ; n = 20] or without GO [GO?; n = 20], 20 individuals with gingivitis, and 20 healthy participants). Probing depth and plaque, papilla bleeding, and hyperplastic index scores were recorded. GCF samples were obtained from the mesio‐buccal aspects of two teeth. GCF IL‐6, IL‐1β, OSM, LIF, and IL‐11 levels were analyzed by enzyme‐linked immunosorbent assay. Results: The GO+ and GO? groups had higher IL‐6 total amounts than the healthy group (P <0.008). IL‐1β total amounts in the GO+ group were significantly higher than in both the healthy and GO? groups (P <0.008). OSM total amount was elevated in the GO+ and GO? groups compared with both the gingivitis and healthy groups (P <0.008). All groups had similar LIF and IL‐11 total amounts (P >0.008). Moderate positive correlations were detected among IL‐6, IL‐1β, OSM, and IL‐11 total amount in GCF and clinical parameters (P <0.05). Conclusions: IL‐6 and OSM increases in GCF as a result of CsA usage or an immunosuppressed state irrespective of the severity of inflammation and the presence of GO. The IL‐6 family of cytokines might not be directly involved in biologic mechanisms associated with CsA‐induced GO. Lack of an association between assessed IL‐6 cytokines and CsA‐induced GO might indicate distinct effects of these cytokines on fibrotic changes of different tissues.     

Immunohistochemical study of cyclosporin-induced gingival overgrowth in renal transplant recipients

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressant widely used to treat transplant patients and various systemic diseases with immunological components. Gingival overgrowth (GO) is a common side effect of CsA administration; however, the pathogenesis of drug-induced GO is poorly understood. The aim of this study was to evaluate the expression of Ki-67, activation molecules (CD71, CD98), leukocytes activation antigens (CD45, CD45RA, CD50, CD11a, CD162, CD227, CD231), neurothelin (CD147), and novel endothelial cell antigens (B-F45, SCF87, B-D46, B-C44, VJ1/6) in gingival tissue in renal transplant recipients treated with CsA. METHODS: Tissues from 15 renal transplant patients with significant GO and 10 systemically healthy control subjects with gingivitis were studied. Frozen-section biopsies were stained with monoclonal antibodies specific for the above-mentioned antigens using an indirect immunoperoxidase technique. RESULTS: Comparison of the CsA-treated and control groups revealed no significant differences with respect to expression of Ki-67; CD50; activation molecules; neurothelin; or novel endothelial cell antigens B-D46, B-C44, and VJ1/6. However, expression patterns of CD45, CD45RA, CD11a, CD162, CD227, CD231, B-F45, and SCF87 were significantly different in CsA and control groups. CONCLUSION: Leukocyte activation antigens play an important role in CsA-induced gingival overgrowth.     

Apoptosis in cyclosporin A-induced gingival overgrowth: a histological study

BACKGROUND: Cyclosporin A (CsA) is known to induce gingival overgrowth. Apoptosis plays a critical role in the regulation of inflammation and the host immune response. The aim of this study was to investigate apoptosis in CsA-induced gingival enlargement using electron microscopy examination of keratinocytes. METHODS: Gingiva specimens were collected from 12 CsA-treated renal transplant patients with gingival overgrowth and eight healthy controls with gingivitis. Clinical findings (probing depth, gingival index, and plaque index) were compared in the two groups. Histological and ultrastructural features of the specimens were also compared, and extent of keratinocyte apoptosis was scored on a three-tier scale: 0 = no apoptotic cells; 1 = one or two apoptotic cells; 2 = more than two cells. RESULTS: There were no significant differences between groups with respect to gingiva-related clinical findings or extent of keratinocyte apoptosis. CONCLUSIONS: The results indicate that the extent of keratinocyte apoptosis in the gingiva of kidney recipients with CsA-induced gingival overgrowth is similar to that observed in inflamed gingiva of healthy individuals. Further studies on apoptosis of different cell types in the presence of CsA should clarify this agent's role in the pathogenesis of drug-induced gingival enlargement.     

Gingival tissue proteoglycan and chondroitin-4-sulphate levels in cyclosporin A-induced gingival overgrowth and the effects of initial periodontal treatment

OBJECTIVES: Cyclosporin A (CsA) is a potent immunosuppressive drug used in organ transplant patients to prevent graft rejection. CsA-induced gingival overgrowth is one of the side effects of this drug and its pathogenesis is still unclear. The present study was planned to comparatively analyse total proteoglycan (PG) and chondroitin-4-sulphate (C4S) levels in CsA-induced overgrown gingival tissue samples obtained before and after initial periodontal treatment and to compare these findings with the situation in healthy gingiva. MATERIAL AND METHODS: Gingival tissue samples were obtained from nine patients with CsA-induced gingival overgrowth before and 4 weeks after initial periodontal treatment including oral hygiene instruction and scaling and also from 10 healthy control subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analysed using modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using non-parametric tests. RESULTS: All clinical measurements in the CsA-induced gingival overgrowth group demonstrated significant reductions 4 weeks after initial periodontal treatment (p<0.05). There was no significant difference between the levels of baseline total PG in CsA-induced gingival overgrowth and healthy control groups (p>0.05). The gingival tissue levels of PG in CsA-induced gingival overgrowth group decreased significantly 4 weeks after treatment (p=0.043). Gingival tissue C4S levels in the overgrowth group were significantly higher than the healthy control group at baseline (p=0.000). C4S levels of the overgrowth group were significantly reduced after treatment (p=0.033), but these levels were still significantly higher than the healthy control group (p=0.000). CONCLUSION: The observed prominent increase in gingival tissue C4S levels may be interpreted as a sign of an increase in C4S synthesis in CsA-induced gingival overgrowth. Furthermore, remission of clinical inflammation by means of initial periodontal treatment had a positive effect on tissue levels of these extracellular matrix molecules.     

Cyclosporin A regulates interleukin-1beta and interleukin-6 expression in gingiva: implications for gingival overgrowth

BACKGROUND: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA); however, the cellular mechanisms remain poorly understood. CsA's immunosuppressant properties involve the regulation of synthesis and cellular response to cytokines. A CsA-induced alteration in the cytokine profile within gingival tissue could provide a mechanism for gingival hyperplasia. The aim of this study was to investigate the effects of CsA on the production of 2 cytokines - interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) - by both gingival fibroblasts and peripheral blood mononuclear cells (PBMC). METHODS: Cells were stimulated for 24 hours in the presence of CsA over a concentration range of 100 to 2,000 ng/ml and the resultant cytokine production determined by ELISA. In addition, levels of both cytokines within normal, inflamed, and overgrown gingival tissue were determined. RESULTS: CsA inhibited IL-6 production by gingival fibroblasts in a dose-dependent manner. In contrast, at a concentration of 2,000 ng/ml, CsA stimulated IL-6 production by PBMC (P <0.05). Fibroblasts derived from overgrown gingiva produced significantly higher levels of IL-6 than their normal counterparts (P <0.05). CsA inhibited IL-1beta production by PBMC over the whole concentration range (P <0.05). IL-1beta was not found in measurable quantities in any of the fibroblast cultures. Levels of IL-6 extracted from overgrown gingival tissue were significantly higher than in inflamed or normal tissue. In contrast IL-1beta levels in overgrown tissue were not statistically significantly greater than those in inflamed tissue. CONCLUSIONS: These results show that CsA does regulate cytokine expression in gingival tissue. This effect may play an important role in the pathogenesis of CsA-induced gingival overgrowth.     

Cyclosporin A upregulates phospholipase C beta1 in fibroblasts from gingival overgrowth

BACKGROUND: In an attempt to evaluate the influence of cyclosporin A (CsA) on fibroblast metabolism, the phospholipase C beta1, (PLC beta1) nuclear expression was evaluated in fibroblasts from heart transplantation patients treated with CsA who exhibited gingival overgrowth (GO) and from controls. METHODS: PLC beta1 was assessed by immunoblotting and immunocytochemistry means. RESULTS: Findings did not show any difference in terms of PLC beta1 expression between the 2 groups when fibroblasts were incubated in media without CsA, while the addition of CsA highly stimulated the fibroblasts from CsA-treated patients compared to controls. The abnormal fibroblastic response in CsA-treated patients was detected both in cells from enlarged gingival sites and in cells from clinically healthy gingival sites. CONCLUSIONS: These results do not explain whether the exaggerated reactivity to in vitro CsA is the consequence of a genetically transmitted susceptibility to CsA that identifies those subjects at risk for developing GO, or whether it is a secondary effect of the long-term in vivo exposure to CsA. However, the present data underline the lack of any close relationship between enhanced fibroblast activity and clinical signs of GO and support the hypothesis that some other factors, together with CsA, are involved in the pathogenesis of CsA-induced GO.