Toll-like receptors 2 and 5 in human gingival epithelial cells co-operate with T-cell cytokine interleukin-17

Background/aim:  Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll-like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells.
Methods:  Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme-linked immunosorbent assays were performed to detect the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL-17.
Results:  Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P  < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis , and flagellin, a TLR5 ligand that is also found in Treponema denticola ) produced both IL-1β and TNF-α. To mimic T-cell help, IL-17 was added. This further greatly enhanced TLR ligand-induced IL-1β ( P  < 0.001) and TNF-α ( P  < 0.01) production.
Conclusions:  These findings show how pathogen-associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR-dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T-cell help in intercellular cooperation.     

Serum interleukin-8 level is a more sensitive marker than serum interleukin-6 level in monitoring the disease activity of recurrent aphthous ulcerations

BACKGROUND: Recurrent aphthous ulcerations (RAU) are common oral inflammatory lesions. Interleukin (IL)-8 is a pro-inflammatory cytokine of host response to injury and inflammation. Our recent study has found that measurement of serum IL-6 level can detect only 24% RAU patients with an abnormal serum level. In this study, we examined both the serum IL-6 and IL-8 levels in a group of RAU patients. The abilities of IL-6 and IL-8 to detect patients with an abnormal serum level were compared in order to find out whether IL-8 was a more sensitive serum marker than IL-6 in monitoring the disease activity of RAU. METHODS: In this study, we used a solid-phase, two-site sequential chemiluminescent immunometric assay to determine the baseline serum levels of IL-6 and IL-8 in 146 patients with RAU, 9 patients with traumatic ulcers (TU), and 54 normal control (NC) subjects. Eighty-two RAU patients, with the serum IL-6 or IL-8 levels higher than the upper limit of normal serum concentration, were treated with levamisole for 0.5-3.5 months, and their serum IL-6 and IL-8 levels were measured after treatment. RESULTS: We found that 25% (37/146) RAU patients, as well as 33% (20/61) major-type, 19% (13/69) minor-type, and 25% (4/16) herpetiform-type RAU patients, had a serum level of IL-6 greater than the upper normal limit of 4.7 pg/ml. In contrast, 60% (87/146) RAU patients, as well as 59% (36/61) major-type, 59% (41/69) minor-type, and 63% (10/16) herpetiform-type RAU patients, had a serum level of IL-8 greater than the upper normal limit of 8.7 pg/ml. In 82 RAU patients with the serum IL-6 or IL-8 levels higher than the upper limit of normal serum concentration, treatment with levamisole for a period of 0.5-3.5 months could significantly reduce the serum IL-6 level from 12.0 +/- 1.6 to 3.0 +/- 0.5 pg/ml (P < 0.001), and could significantly lower the serum IL-8 level from 70.9 +/- 11.2 to 13.8 +/- 3.1 pg/ml (P < 0.001). CONCLUSIONS: Because measurement of serum IL-8 level can detect 60% RAU patients with an abnormal serum level, while measurement of serum IL-6 level can detect only 25% RAU patients with an abnormal serum level, we conclude that serum IL-8 level is a more sensitive marker than serum IL-6 level in monitoring the disease activity of RAU. Levamisole can modulate both the serum IL-6 and IL-8 levels in RAU patients. IL-8, like IL-6, is also a useful serum marker in evaluating therapeutic effects of levamisole on RAU patients.     

TNF-alpha and IL-4 levels in generalized aggressive periodontitis subjects

Objectives:  The aim of this study was to evaluate tumor necrosis factor (TNF)-α and interleukin (IL)-4 levels in healthy sites and sites exhibiting signs of moderate and advanced generalized aggressive periodontitis (GAgP) in the same subject.
Methods:  The following sites were selected for crevicular fluid sampling in the same AgP subject ( n  = 14): Healthy sites (HS): no marginal bleeding or bleeding on probing (BOP) and probing depth (PD) ≤ 3 mm; Moderate sites (MS): BOP and PD between 4 and 6 mm; Advanced sites (AS): BOP and PD ≥ 7 mm. One site from periodontally healthy subjects ( n  = 13) was sampled for use as a control. TNF-α and IL-4 levels were measured using ELISA.
Results:  The total amount of TNF-α was lower for control sites, while there were no differences among healthy and diseased sites from GAgP subjects ( P  < 0.05). The concentration of TNF-α was higher in HS, in relation to the other sites ( P  < 0.05). There were no significant differences among the groups regarding total amounts of IL-4 ( P  > 0.05), while IL-4 concentration was significantly higher in control sites, when compared with sites from GAgP subjects ( P  < 0.05).
Conclusion:  In conclusion, high levels of TNF-α and low levels of IL-4 were observed in both healthy and diseased sites within the same generalized AgP individuals.     

Phosphatidylinositol-3-kinase inhibitor LY 294002 blocks Streptococcus mutans-induced interleukin (IL)-6 and IL-8 gene expression in odontoblast-like cells

Aim  To investigate the involvement of the phosphatidylinositol-3-kinase (PI3K) in interleukin-6 (IL-6) and interleukin-8 (IL-8) gene expression in odontoblast-like cells when exposed to heat-killed Streptococcus mutans .
Methodology  Cultured human odontoblast-like cells (Dulbecco's modified Eagle's medium) were pre-treated with a specific inhibitor for PI3K (LY 294002) and subsequently stimulated with heat-killed S. mutans for 6 and 24 h. After stimulation, RNA was extracted from the cells and cDNA synthesis was performed. Gene expression of IL-6 and IL-8 was analysed by real-time polymerase chain reaction and normalized to the gene expression of beta-actin. Cell survival was determined for stimulation experiments.
Results  The gene expression of IL-6 and IL-8 was significantly increased in response to heat-killed S. mutans ( P  = 0.002 and P  < 0.001, respectively). After 6 h, the mRNA expression of IL-6 and IL-8 was significantly higher than after 24 h of stimulation ( P  = 0.019 and P  < 0.001, respectively). Pre-treatment with the inhibitor LY 294002 blocked the induced gene expression of IL-6 and IL-8 ( P  = 0.002 and P  < 0.001, respectively). No differences in viable cell counts were found after stimulation with heat-killed S. mutans and/or pre-treatment with LY 294002 compared with the unstimulated control.
Conclusion  Gene expression of IL-6 and IL-8 was induced by heat-killed S. mutans via signalling pathways mediated by PI3K. These findings indicate that odontoblasts respond to cariogenic bacteria with increased gene expression of pro-inflammatory mediators and hence participate in immune processes.     

Levamisole can modulate the serum tumor necrosis factor-α level in patients with recurrent aphthous ulcerations

BACKGROUND: Recurrent aphthous ulcerations (RAU) are common oral inflammatory lesions. Tumor necrosis factor (TNF)-alpha is an important inflammatory mediator and a critical cytokine for adequate host defense. Our previous studies have shown that 14-43% and 59-63% of patients in the ulcerative stage of major, minor or herpetiform RAU have significantly higher than normal serum levels of interleukin (IL)-6 and IL-8, respectively. In this study, we examined whether RAU patients in the ulcerative stage had a significantly higher than normal serum level of TNF-alpha and assessed whether treatment with levamisole can modulate serum TNF-alpha levels in RAU patients. METHODS: This study used a solid phase, two-site sequential chemiluminescent immunometric assay to determine the baseline serum levels of TNF-alpha in 146 patients with RAU, nine patients with traumatic ulcers (TU), and 54 normal control subjects. Fifty-five RAU patients with serum TNF-alpha levels higher than 5.0 pg/ml were treated with levamisole for 0.5-4 months and their serum TNF-alpha levels were measured after treatment. RESULTS: We found that 29% (42 of 146) RAU patients as well as 39% (24 of 61) major type, 20% (14 of 69) minor type, and 25% (four of 16) herpetiform type RAU patients had a serum level of TNF-alpha greater than the upper normal limit of 7.4 pg/ml. The mean serum level of TNF-alpha in patients with RAU (9.1 +/- 1.0 pg/ml, P < 0.001), major type RAU (11.6 +/- 1.9 pg/ml, P < 0.001), minor type RAU (6.9 +/- 0.9 pg/ml, P < 0.005), or herpetiform type RAU (9.6 +/- 2.7 pg/ml, P < 0.001) was higher than that (3.8 +/- 0.2 pg/ml) in normal control subjects. The mean serum TNF-alpha level was significantly higher in patients with major type RAU than in patients with minor type RAU (P < 0.05) and was significantly higher in major type RAU patients in the exacerbation stage than in the post-exacerbation stage (P < 0.05). In 55 RAU patients with serum TNF-alpha levels higher than 5.0 pg/ml, treatment with levamisole for a period of 0.5-4 months could significantly reduce the serum TNF-alpha level from 16.4 +/- 1.9 to 5.8 +/- 0.6 pg/ml (P < 0.001). CONCLUSIONS: We conclude that a significantly higher than normal serum level of TNF-alpha can be detected in 20-39% of patients in the ulcerative stage of major, minor or herpetiform RAU. The serum TNF-alpha level may be associated with the severity and the stage of RAU. Levamisole can modulate serum TNF-alpha levels in RAU patients.     

Cytokine and chemokine levels in radicular and residual cyst fluids

Background:  Cytokines were thought to play an important role for the expansion of odontogenic cysts. The purpose of this study was to evaluate the cytokine and chemokine levels of radicular and residual cyst fluids.
Methods:  Cyst fluids were aspirated from 21 patients (11 radicular and 10 residual cysts) and the levels of interleukin-1 alpha (IL-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and regulated upon activation normal T cell expressed and secreted (RANTES) were determined by ELISA using commercially available kits.
Results:  Both radicular and residual cyst fluids contained IL-1α, TNF-α, MCP-1, and RANTES, concentrations of which were significantly higher in the radicular cyst fluids than those in the residual cysts ( P  < 0.001 for IL-1α, TNF-α, and RANTES; P  < 0.01 for MCP-1). Compared to the other mediators, the concentration of IL-1α was found to be highest in both of the cyst fluids. In addition, positive correlations were found between IL-1α, TNF-α, MCP-1, and RANTES in radicular and residual cyst fluids.
Conclusion:  If the radicular cyst is inadvertently left behind following tooth extraction, some degree of inflammation may carry on. Residual cysts, although to a lesser extend than radicular cysts, have the potential to expand.     

An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin-6 in an ovariectomized mouse model

Background/aims:  In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis.
Methods:  Fifty-three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide ( P. gingivalis -LPS, ATCC 33277) or Escherichia coli lipopolysaccharide ( E. coli -LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and the receptor activator of nuclear factor-κB ligand (RANKL) in serum were subsequently analyzed using an enzyme-linked immunosorbent assay (ELISA).
Results:  Under stimulation with P. gingivalis -LPS or E. coli -LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis -LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P  < 0.05). The serum level of IL-6 in the experimental group significantly increased 1–6 h after administration of E. coli -LPS and 1–3 h after administration of P. gingivalis -LPS ( P  < 0.05).
Conclusions:  A single booster injection of P. gingivalis -LPS induced short-term changes in OPG, RANKL, and IL-6 serum levels in this ovariectomized mouse model.     

The relationship between periodontal condition and serum levels of resistin and adiponectin in elderly Japanese

Background and Objective:  Diabetes and periodontitis are associated with each other. Adipokines, specifically adiponectin and resistin, are secreted from adipocytes and are thought to cause insulin resistance in rodents. Additionally, adiponectin and resistin may play a role in inflammation and immune responses. The aim of this study was to clarify the relationship between serum levels of adipokines and periodontal conditions in elderly Japanese people with and without periodontitis.
Material and Methods:  A total of 158 Japanese men and women (76 years old) with or without periodontitis were selected for the study. Serum adiponectin, resistin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were compared between subjects with and without periodontitis.
Results:  Serum resistin levels and total leukocyte counts in subjects with periodontitis were higher than in control subjects. No significant differences were observed in adiponectin, IL-6 and TNF-α levels between subjects with and without periodontitis. Logistic regression analysis showed that periodontitis with at least one tooth that displayed a probing pocket depth of ≥6 mm was significantly associated with higher serum resistin levels (odds ratio, 2.0; 95% confidence interval, 1.0–4.0). When excluding periodontitis subjects with ≤10% of bleeding on probing and excluding control subjects with >10% bleeding on probing, differences between groups and odds ratio increased. Serum adiponectin tended to decrease in patients with periodontitis, albeit not significantly.
Conclusion:  Increased serum resistin levels were significantly associated with periodontal condition, especially when considering bleeding on probing, in elderly Japanese people. There was also a trend, though non-significant, toward decreased levels of adiponectin in subjects with periodontitis.     

The gingival crevicular fluid Interleukin-1β and tumour necrosis factor-α levels in patients with rapidly progressive periodontitis

Cytokines are believed to play an important role in the pathogenesis of periodontal diseases. In the present study, gingival crevicular fluid (GCF) levels of two important cytokines, interleukin 1-/3 (IL-1β) and tumour necrosis factor-α (TNF-α) and, in addition, serum IL-1β levels, were determined in patients with severe and rapid periodontal breakdown by use of ELISA. While IL-1β was detected in all of the GCF samples studied, TNF-α could only be detected in about half the samples. The mean GCF IL-1β level was 38.45 ± 13.99 pg/mL, and the mean TNF-α level was 3.20 ± 1.39 pg/mL, respectively. The GCF IL-1β levels also presented a strong positive correlation with the mean pocket depths. Although weak, both of the cytokines also presented correlations with the presence of bleeding on probing. Additionally GCF samples contained increased IL-1β levels when compared with the serum samples suggesting local production mechanisms. The findings of the present study suggest that these cytokines may be involved in the pathogenesis of periodontal diseases (IL-1β being more significant), and also may help in defining the active phase of periodontal breakdown.     

Naringenin inhibits human osteoclastogenesis and osteoclastic bone resorption

Background and Objective:  Naringenin, a naturally occurring flavonoid, possesses a wide range of pharmacological properties. The purpose of this study was to investigate the effect of naringenin on human osteoclastogenesis and osteoclastic bone resorption.
Material and Methods:  Naringenin was tested in a human osteoclastogenesis model using primary osteoclast precursor cells activated by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for 6 days. Osteoclastogenesis was assessed by determining the number of tartrate-resistant acid phosphatase (TRAP)-stained multinuclear cells, while the secretion of factors involved in osteoclastogenesis was assessed using enzyme-linked immunosorbent assays. The effect of naringenin on bone resorption was investigated using an OsteoAssay human bone plate coupled with an immunoassay to evaluate the release of helical peptide 620–633 from the α1 chain of type I collagen.
Results:  Naringenin was non-toxic at the highest concentration used (50 μg/ml). Naringenin (10, 25 and 50 μg/ml) significantly inhibited osteoclastogenesis (by 29 ± 5, 57 ± 8 and 96 ± 1%, respectively). Naringenin also markedly inhibited the secretion of interleukin (IL)-1α (by 59%), IL-23 (by 87%) and monocyte chemoattractant protein-1 (by 58%). Lastly, naringenin (10, 25 and 50 μg/ml) significantly decreased the release of helical peptide 620–633, an indicator of bone resorption activity (by 44 ± 0.5, 73 ± 0.5 and 86 ± 1%, respectively).
Conclusions:  Naringenin can inhibit human osteoclastogenesis and osteoclastic bone resorption. It thus holds promise as a therapeutic or preventive agent for bone-related diseases such as periodontitis.     

Expression of hypoxia-inducible factor-1α is significantly associated with the progression and prognosis of oral squamous cell carcinomas in Taiwan

Background:  Overexpression of hypoxia-inducible factor-1α (HIF-1α) has been found to be significantly associated with the tumor invasion, lymph node metastasis, clinical stage, and prognosis of a variety of human cancers.
Methods:  This study examined the expression of HIF-1α in 57 specimens of oral squamous cell carcinoma (OSCC), 41 specimens of oral epithelial dysplasia (OED, 12 mild, 17 moderate, and 12 severe OED cases), and 14 specimens of normal oral mucosa (NOM) by immunohistochemistry.
Results:  We found that the mean nuclear HIF-1α labeling indices (LIs) increased significantly from NOM (9 ± 6%) through mild OED (25 ± 18%), moderate OED (41 ± 27%), and severe OED (42 ± 22%) to OSCC samples (55 ± 23%, P  < 0.001). A significant correlation was found between the higher mean nuclear HIF-1α LI and OSCCs with larger tumor size ( P  < 0.001), regional lymph node metastasis ( P  < 0.001), or more advanced clinical stages ( P  < 0.001). Only larger tumor size ( P  = 0.002) and nuclear HIF-1α LI ≥ 60% ( P  = 0.048) were identified as independent unfavorable prognosis factor by multivariate analyses with Cox regression model. Kaplan–Meier curve showed that OSCC patients with a nuclear HIF-1α LI ≥ 60% had a significantly poorer cumulative survival than those with a nuclear HIF-1α LI < 60% (log-rank test, P  = 0.022).
Conclusions:  We conclude that the expression of HIF-1α is an early event in oral carcinogenesis. The nuclear HIF-1α LI in OSCC samples can predict the progression of OSCCs and the survival of OSCC patients.     

Serum interleukin-6 level is a useful marker in evaluating therapeutic effects of levamisole and Chinese medicinal herbs on patients with oral lichen planus

Background: Oral lichen planus (OLP) is a T cell‐mediated inflammatory disease. Interleukin‐6 (IL‐6) is a pro‐inflammatory cytokine that has effects on cellular and humoral immunities. Previous studies have shown that keratinocytes and tissue‐infiltrating mononuclear cells from OLP lesions can secrete IL‐6. In some OLP patients, the high serum IL‐6 levels are reduced after treatment, suggesting that IL‐6 may be a useful marker in evaluating therapeutic effects and in monitoring the disease status of OLP. Methods: In this study, we used a solid phase, two‐site sequential chemiluminescent immunometric assay to determine the baseline serum levels of IL‐6 in a group of 180 patients with erosive OLP (EOLP), nonerosive OLP (NEOLP), erythema multiforme (EM), traumatic ulcers (TU), oral submucous fibrosis (OSF), pemphigus vulgaris (PV), or Sjögren's syndrome (SS), and in 77 normal control subjects. Some OLP patients were treated with levamisole plus Chinese medicinal herbs or levamisole only for 0.5–5.5 months and their serum IL‐6 levels were measured after treatment. Results: We found that approximately 99% of the normal control subjects and the patients with EM, TU, or OSF had a normal serum IL‐6 level less than 5.0 pg/ml. However, 15% (22/149) OLP patients, 15% (20/136) EOLP patients, 20% (5/25) major type EOLP patients, 14% (15/111) minor type EOLP patients, 15% (2/13) NEOLP patients, 14% (1/7) EM patients, 43% (3/7) PV patients, and 100% (6/6) SS patients had a serum IL‐6 level greater than 5.0 pg/ml. The mean serum IL‐6 level in patients with OLP (3.4 ± 3.1 pg/ml, P < 0.001), EOLP (3.4 ± 3.2 pg/ml, P < 0.001), major type EOLP (4.9 ± 3.5 pg/ml, P < 0.001), minor type EOLP (3.0 ± 3.0 pg/ml, P < 0.01), or NEOLP (4.2 ± 1.5 pg/ml, P < 0.001) was significantly higher than that in normal control subjects (2.0 ± 1.5 pg/ml). A significant difference in the mean serum IL‐6 level was also found between major type and minor type EOLP patients (P < 0.01). The mean reduction of serum IL‐6 level in OLP patients treated with levamisole plus Chinese medicinal herbs was significantly higher (7.4 ± 4.7 pg/ml) than that in OLP patients treated with levamisole only (3.8 ± 2.3 pg/ml, P < 0.05), suggesting that the combination therapy was superior to levamisole only. Conclusion: We conclude that levamisole and levamisole plus Chinese medicinal herbs can modulate the serum IL‐6 level in OLP patients. IL‐6 may be a useful marker in evaluating therapeutic effects and in monitoring the disease status of OLP.     

Levamisole and Chinese medicinal herbs can modulate the serum interleukin-6 level in patients with recurrent aphthous ulcerations

Background: Recurrent aphthous ulcerations (RAU) are common oral inflammatory lesions. Interleukin‐6 (IL‐6) is a pro‐inflammatory cytokine that has effects on cellular and humoral immunities. Previous studies have shown that the high serum IL‐6 levels in some RAU patients can be reduced by drug treatment. This finding suggests that IL‐6 may be a useful marker in evaluating therapeutic effects of RAU. Methods: In this study, we used a solid phase, two‐site sequential chemiluminescent immunometric assay to determine the baseline serum levels of IL‐6 in a group of 228 patients with RAU, erythema multiforme (EM), traumatic ulcers (TU), oral submucous fibrosis (OSF), pemphigus vulgaris (PV), or Sjögren's syndrome (SS), and in 77 normal control subjects. Some RAU patients were treated with levamisole plus Chinese medicinal herbs or levamisole only for 0.5–5 months and their serum IL‐6 levels were measured after treatment. Results: We found that about 99% of the normal control subjects and the patients with EM, TU, or OSF had a serum IL‐6 level within the normal limit of 5.0 pg/ml. However, 24% (48/197) RAU patients, 14% (1/7) EM patients, 43% (3/7) PV patients, and 100% (6/6) SS patients had a serum level of IL‐6 greater than 5.0 pg/ml. The mean serum level of IL‐6 in patients with RAU (3.6 ± 3.5 pg/ml, P < 0.001), minor type RAU (2.7 ± 2.0 pg/ml, P < 0.05), major type RAU (5.2 ± 4.6 pg/ml, P < 0.001), or herpetiform type RAU (4.1 ± 3.8 pg/ml, P < 0.01) was higher than that in normal control subjects. The mean serum level of IL‐6 in major type (P < 0.001) or in herpetiform type RAU patients (P < 0.05) was higher than that in minor type RAU patients. The mean reduction of serum IL‐6 level (10.0 ± 7.1 pg/ml) in RAU patients after treatment with levamisole plus Chinese medicinal herbs was significantly higher than that (5.1 ± 3.7 pg/ml) in RAU patients after treatment with levamisole only (P < 0.005), suggesting that the combination therapy is superior to the single therapy of levamisole only. Conclusion: We conclude that levamisole and levamisole plus Chinese medicinal herbs can modulate the serum IL‐6 level in RAU patients. Although the therapeutic effect of RAU can be assessed by a decrease in the frequency, duration and number of the oral ulcerations, it can also be monitored by a reduction of serum IL‐6 level in RAU patients.     

Alteration in peripheral blood mononuclear cell function and serum cytokines in oral lichen planus

Different activation parameters of peripheral blood mononuclear cells (PBMC) from 31 patients with oral lichen planus (OLP) were examined and compared with 23 healthy donors. Impaired spontaneous (450±241 vs 1290±480 cpm) and mitogen-induced (39580±14470 vs 67000±11810 cpm) lymphocyte blastogenesis was observed in OLP patients. Furthermore, reduced cytokine production was found after phytohemagglutinin A (PI1A) stimulation for all cytokines studied-tumour necrosis factor α(TNFα, 432.2±73.4 vs 979.8±46.3 units/ml), interleukin 2 (IL-2, (56.2±14.9 vs 572.6±12.9 pg/ml), interferon γ (IFNγ, 48.5±11.9 vs 82.6± 12.4 pg/ml) and interleukin 6 (IL–6. 253.6±57.7 vs 1.419.0±279.6 units/ ml)-except for interleukin 1ß (IL–lß) and lymphotoxin (LT). In contrast, unstimulated culture supernatants showed increased TNFα (38.2±13.1 vs 8.0±0.2 units/ml), LT (10.2±2.2 units/ml vs <0.4) and IL–6 (18.5±5.6 units/ml vs <0.5) activity. Similarly, elevated concentrations of TNFα (19.6±6.3 units/ml) and IL–6(22.9±4.7 units/ml) were detected in the sera of OLP patients. Combination of PHA and phorbol myristate acetate (PM A) could restore OLP proliferative T cell response and cytokine production to the level of healthy donors, whereas exogenous recombinant human IL-2 (rhuIL-2) plus PMA did not seem to be an effective stimulant for OLP T cells. These results indicate an alteration in the immune condition of OLP patients and an impairment in T lymphocyte function.     

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

Background:  The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation.
Methods:  Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1β), IL-6, IL-8 and tumour necrosis factor alpha (TNF-α) and immunoregulatory cytokines: transforming growth factor-beta (TGF-β) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry.
Results:  Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8⁺ T cells and produced higher levels of TNF-α, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-β and proinflammatory cytokines. TGF-β, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions.
Conclusion:  Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-β is more potent and different from IL-10.     

Activation of nuclear factor-kappa B correlates with tumor necrosis factor-alpha in oral lichen planus: a clinicopathologic study in atrophic-erosive and reticular form

Backgroud:  Nuclear factor-kappa B (NF-κB) is believed to be involved in the pathogenesis of various inflammatory diseases, including oral lichen planus (OLP). The objective of the present study was to investigate the possible relationship between NF-κB activation and expression of tumor necrosis factor-alpha (TNF-α) in OLP and their expression pattern in relation to several clinical features.
Methods:  Thirty OLP cases were divided into atrophic-erosive form (14 cases) and reticular form (16 cases) according to their clinical manifestations. The expression of NF-κB p65 and TNF-α of both two groups were investigated by immunohistochemical staining, and the percentage of positive cells was calculated in each case. Biopsies of 10 normal oral mucosa (NOM) also underwent the same procedure as controls.
Results:  Nuclear factor-kappa B p65 nuclear staining was found in nuclei of basal and suprabasal epithelial keratinocytes in OLP, however, no positive staining was found in NOM. Positive TNF-α staining was detected in cytoplasm of basal epithelial keratinocytes in OLP, and only scattered staining was detected in NOM. Expression of NF-κB p65 and TNF-α were significantly different with respect to clinical forms and lesion sites ( P  < 0.05), except for genders ( P  > 0.05) in 30 OLP cases. NF-κB nuclear staining positively correlated ( r  = 0.676, P  < 0.01) with TNF-α overexpression in OLP.
Conclusions:  Nuclear factor-kappa B activation and its correlation with overexpression of TNF-α may play an important role in pathogenesis of OLP. There might be a positive regulatory loop between NF-κB and TNF-α, which may contribute to inflammation in OLP; NF-κB may also protect epithelial keratinocytes from excessive apoptosis.     

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-lα, IL-1β and tumor necrosis factor (TNF)-α stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-lα, IL-lβ, or TNF-α-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-l or TNF-α-induced IL-6 production, and that the enhancement of IL-6 production by IL-l or TNF-α may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kB) activation, markedly inhibited IL-l (α or β) or TNF-α-induced IL-6 production; so this production may be partially mediated through NF-kB. IL-l (α or β) and TNF-α enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-lβ was augmented by the addition of interferon (IFN)-(gama), but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-l (α or β)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-l or TNF-α, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-(gama) or IL-4, and glucocorticoids.     

The effect of periodontal therapy on serum TNF-α and HbA1c levels in type 2 diabetic patients

Background:  To determine the effect of non-surgical periodontal therapy on serum TNF-α and HbA1c levels in poorly and well-controlled type 2 diabetic patients.
Methods:  In total, 45 patients were enrolled in the study; 30 patients with type 2 diabetes mellitus with periodontitis (15 with poorly controlled diabetes, HbA1c ≥ 7%, group 1A and 15 with well-controlled diabetes, HbA1c < 7%, group 1B) and 15 patients that were systemically healthy with periodontitis (group 2). The plaque index, gingival index, probing depth, clinical attachment loss, gingival bleeding index, HbA1c value, and circulating TNF-α concentration were measured at baseline and three months after the non-surgical periodontal therapy.
Results:  All periodontal parameters and serum TNF-α levels were significantly decreased three months after the non-surgical periodontal therapy compared to the baseline values in all groups. The HbA1c values were significantly decreased only in well-controlled diabetic patients. We found no significant differences in the periodontal parameters or TNF-α levels at baseline and after three months between the two groups.
Conclusions:  Although non-surgical periodontal therapy eliminates local/systemic infection and inflammation via decreases in TNF-α, it is insufficient for significantly reducing HbA1c levels without strict glycaemic control in poorly controlled diabetic patients in a short time period.     

Capsular polysaccharide from Actinobacillus actinomycetemcomitans inhibits IL-6 and IL-8 production in human gingival fibroblast

We previously reported that a capsular polysaccharide (CP) from Actinobacillus actinomycetemcomitans Y4 induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures. However, the effects of A. actinomycetemcomitans Y4 CP on human gingival fibroblasts (HGF) are still unclear. The present study was undertaken to test the hypothesis that A. actinomycetemcomitans Y4 CP alters the production of inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 by HGF. When HGF were cultured with various concentrations of Y4 CP for 24 h, IL-6 and IL-8 production decreased in a concentration-dependent manner. Y4 CP (100 microg/ml) suppressed the release of IL-6 from 9.09 +/- 0.08 ng/ml to 0.34 +/- 0.21 ng/ml (P < 0.01) and IL-8 production decreased from 3.76 +/- 0.03 ng/ml to 0.09 +/- 0.01 ng/ml (P < 0.01). Y4 CP suppressed 70-80% of the release of IL-6 and IL-8 from HGF stimulated with Y4 lipopolysaccharide (LPS), too. Interestingly, anti-A. actinomycetemcomitans Y4 CP completely inhibited the effect of A. actinomycetemcomitans Y4 CP on IL-6 and IL-8 production from HGF. These results indicate that Y4 CP inhibits the release of IL-6 and IL-8 from HGF, suggesting that A. actinomycetemcomitans Y4 modulates the inflammatory response in periodontitis. Remarkably, this inhibitory effect was reversed by specific anti-A. actinomycetemcomitans Y4 CP suggesting an important relationship between the organism and the humoral host response.     

Immune-expression of HSP27 and IL-10 in recurrent aphthous ulceration

Background:  Recently, abnormal cellular immune response has been considered responsible for the oral lesion in the recurrent aphthous ulceration (RAU). For reasons not yet defined, antigens of the oral microbiota would trigger abnormal Th1 immune response against epithelial cells. On the other hand, studies have demonstrated that heat shock proteins (HSP) can block the production of proinflammatory cytokine through inhibition of NF-κB and mitogen-activated protein kinase pathways or activate anti-inflammatory cytokines and therefore control the magnitude of the immune response. HSP27 has been considered a powerful inductor of IL-10, a major inhibitor of Th1 response.
Methods:  Using immunohistochemistry, we studied the expression and location of HSP27 and IL-10 in ulcerated lesions clinically diagnosed as RAU ( n  = 27) and to compare it with that of oral clinically normal mucosa (CT; n  = 6) and of other inflammatory chronic diseases such as oral fibrous inflammatory hyperplasia (FIH; n  = 18), Crohn's disease (CD; n  = 10) and ulcerative colitis (UC; n  = 9).
Results:  A lower proportion of HSP27-positive epithelial cells in RAU and CD were observed when compared with CT and FIH ( P  < 0.001**; P  = 0.013**). A lower proportion of IL-10-positive interstitial cells in RAU was observed when compared with FIH, UC, CT and CD ( P  < 0.001**; P  < 0.001**; P  < 0.001**; P  = 0.034*).
Conclusion:  Altogether the data suggest that a reduced cellular expression of HSP27 and IL-10 in RAU might be related with the aetiopathogenesis of the ulcerated oral lesions.