Sakornrat Khongkhunthian

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

BACKGROUND AND OBJECTIVE Oral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts. MATERIAL AND METHODS Cultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. RESULTS Ten and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. CONCLUSION These findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.

Raised chondroitin sulphate WF6 epitope levels in gingival crevicular fluid in chronic periodontitis.

AIM To determine the levels of chondroitin sulphate (CS) WF6 epitope, recognized by WF6 monoclonal antibody, in gingival crevicular fluid (GCF) from different stages of periodontal disease and healthy periodontium, and to correlate those levels with clinical parameters. MATERIAL AND METHODS GCF samples, collected from 389 sites, were analysed for the WF6 epitope levels by the competitive enzyme-linked immunosorbent assay. RESULTS The median WF6 epitope level was significantly higher in chronic periodontitis sites (n=185) than in healthy and gingivitis sites (n=204) (p<0.001), whereas the median levels did not significantly differ between healthy (n=65) and gingivitis sites (n=139). The median level in severe periodontitis sites (n=60) was significantly higher than that in moderate periodontitis sites (n=63) (p=0.019). Similarly, the median level in moderate periodontitis sites was significantly higher than that in slight periodontitis sites (n=62) (p=0.001). The WF6 epitope levels significantly correlated with probing depth (r=0.777, p=0.001) and loss of clinical attachment level (r=0.814, p=0.001). CONCLUSION Elevated CS WF6 epitope levels in GCF are associated with severity of periodontitis. The WF6 antibody may therefore be clinically applied to monitor disease severity and progression.

Positive correlations between hCAP18/LL‐37 and chondroitin sulphate levels in chronic periodontitis

AIM To measure the levels of hCAP18/LL-37 in gingival crevicular fluid from patients with periodontal diseases compared with healthy controls and to determine the correlation between hCAP18/LL-37 and chondroitin sulphate (CS) levels in patients with periodontitis. MATERIAL AND METHODS Gingival crevicular fluid samples from 51 patients and 25 healthy volunteers were analysed for the hCAP18/LL-37 levels by immunoblotting and were determined for the CS levels by the competitive enzyme-linked immunosorbent assay. RESULTS Tris buffer pH 9.85 was selected to recover hCAP18/LL-37 from Periopaper strips, in which the percentages of recovery were around 70%. The median levels of hCAP18/LL-37 in the aggressive and the chronic periodontitis (CP) groups were significantly greater than those in the gingivitis and the healthy groups (p < 0.05). Significant correlations between the unprocessed 18-kDa fragment and CS levels (r = 0.650; p < 0.001) and between the mature 4.6-kDa fragment and CS levels (r = 0.502; p < 0.001) were observed only in the CP group. CONCLUSION The significant correlations between the hCAP18/LL-37 and the CS levels were found in CP, but not in aggressive periodontitis. The presence versus absence of such correlations may be clinically applicable to help clinicians distinguish between two distinct types of periodontitis.

Comparisons between two biochemical markers in evaluating periodontal disease severity: a cross-sectional study

BackgroundThe purpose of this study was to compare two biochemical markers, which have been previously used to determine the degrees of alveolar bone destruction, in evaluating periodontal disease severity.MethodsThe WF6 epitope of chondroitin sulfate (CS) and the alkaline phosphatase (ALP) levels were determined in gingival crevicular fluid (GCF) samples collected from patients with various degrees of disease severity, including ten patients with gingivitis (50 gingivitis sites) and 33 patients with chronic periodontitis (including gingivitis, slight, moderate, and severe periodontitis sites; n = 50 each), as well as from ten healthy volunteers (50 healthy sites) by Periopaper strips. The levels of CS and ALP were measured by an ELISA and a fluorometric assay, respectively.ResultsThe results demonstrated low levels of CS and ALP in non-destructive and slightly destructive periodontitis sites, whereas significantly high levels of these two biomolecules were shown in moderately and severely destructive sites (p < 0.05). Although a significant difference in CS levels was found between moderate and severe periodontitis sites, no difference in ALP levels was found. Stronger correlations were found between CS levels and periodontal parameters, including probing depth, loss of clinical attachment levels, gingival index and plaque index, than between ALP levels and these parameters.ConclusionsIt is suggested that the CS level is a better diagnostic marker than the ALP level for evaluating distinct severity of chronic periodontitis.

Favorable interleukin-8 induction in human gingival epithelial cells by the antimicrobial peptide LL-37.

BACKGROUND LL-37, the only member of the antimicrobial peptide cathelicidin family in humans, exerts a variety of biological activities, especially immunomodulation through either direct chemotactic activity or up-regulation of several cytokines and chemokines in various cell types. In this study, we aimed to determine the immunoregulatory effect of LL-37 on Th1/Th2 cytokine expression and production in human gingival epithelial cells (HGECs). METHODS Cultured HGECs were treated with different concentrations of LL-37 for different numbers of times. The cytotoxicity of LL-37 was determined by an MTT assay. Total RNA was isolated for RT-PCR and real-time PCR analyses of cytokine expression. Cell-free culture supernatants were assayed for Th1/Th2 cytokine levels by a cytokine bead array. RESULTS Out of eleven Th1/Th2 cytokines tested, treatment of HGECs with non-toxic doses of LL-37 (2-6 μM) significantly raised only IL-8 levels in the cell-free culture supernatants, when compared to control untreated cells (P <0.05). Consistent with the elevated IL-8 levels, IL-8 mRNA expression was remarkably and significantly induced by LL-37 treatment (P < 0.05), when compared to the modest mRNA induction of other three cytokines, including IL-1β, IL-6, and TNF-α. The time-course study demonstrated a cumulative IL-8 mRNA induction by LL-37 treatment within a 24-hour interval. CONCLUSIONS These findings indicate that LL-37 favorably induces IL-8 expression and secretion in HGECs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Development of mucoadhesive buccal films from rice for pharmaceutical delivery systems

The aim of this work was to investigate the suitable rice varieties for developing pharmaceutical buccal films. Two rice varieties with extreme difference in amylose content were used. Rice powders were chemically modified to yield the carboxymethyl rice prior to film preparation. Scanning electron microscope (SEM) and X-ray diffractometer (XRD) were used to investigate the solid structure of rice powders. The results indicated that amylose content in the rice grains played the effects on the morphology and crystalline structure of the modified rice powders as well as the film properties. The modified rice powders of low amylose content showed halo pattern XRD whereas some crystalline peaks could be observed from the high amylose content modified rice powders. Adding of glycerin caused the films better properties of more transparency and getting rid of air bubbles. High amylose rice films showed more transparency and higher mucoadhesive property and was considered to be suitable for incorporating the drug. Adding of surfactant caused the increase in tensile strength and decrease in elongation of the rice films. The most suitable surfactant for diclofenac buccal rice film is Tween 20. This study demonstrates that rice grains are the promising natural source for pharmaceutical film forming agent. Suitable pharmaceutical buccal films could be developed from the rice with high amylose content.

Elevated Levels of a Disintegrin and Metalloproteinase 8 in Gingival Crevicular Fluid of Patients With Periodontal Diseases

BACKGROUND A disintegrin and metalloproteinase 8 (ADAM8) is involved in inflammation and is essential for osteoclastogenesis. Elevated ADAM8 levels are detected in human serum and other body fluids in several inflammatory conditions. Therefore, we hypothesized that ADAM8 levels are also raised in gingival crevicular fluid (GCF) of patients with periodontal diseases. METHODS Forty-five patients with periodontal diseases (n = 15 for each group: the group of patients with gingivitis, the group with aggressive periodontitis [AgP], and the group with chronic periodontitis [CP]) and 15 volunteers who exhibited healthy gingiva were recruited. Four periodontal parameters, gingival index, plaque index, probing depth, and clinical attachment level, were recorded before GCF collection. The presence of ADAM8 in GCF was shown by immunoblotting using anti-human ADAM8 polyclonal antibody against its prodomain, and the ADAM8 levels were measured by an enzyme-linked immunosorbent assay. RESULTS Four immunoreactive bands at 120, 70, 50, and <30 kDa were detected in the groups of patients with periodontitis, whose intensities were stronger than those in the group of patients with gingivitis, consistent with significantly greater ADAM8 levels in both groups of patients, with either CP or AgP, than those in the group of patients with gingivitis and in the group that was healthy (P <0.001). Moreover, the ADAM8 levels correlated significantly with the four periodontal parameters (P <0.001), indicating that ADAM8 levels are positively associated with the degree of periodontal tissue inflammation and destruction. CONCLUSIONS The ADAM8 levels are elevated in the GCF of patients with periodontal diseases, including gingivitis, CP, and AgP, in comparison to control participants who are healthy, and they correlate with four clinical parameters that reflect the degree of disease severity.

Effect of rice variety on the physicochemical properties of the modified rice powders and their derived mucoadhesive gels

In the present study; the glutinous Niaw Sanpatong (NSP) and Niaw Koko-6 (NKK), and the non-glutinous Jasmine (JM) and Saohai (SH) were chemically modified. The difference of these rice varieties on the physicochemical characteristics of the modified rice powders and the properties of the derived gels were evaluated. X-ray diffractometer was used for crystalline structure investigation of the rice powders and gels. A parallel plate rheometer was used to measure the rheological property of the gels. It was found that the non-glutinous varieties produced gels with higher mucoadhesive properties than the glutinous rice. Rheological behavior of JM and SH gels was pseudoplastic without yield value whereas that of NSP and NKK gels was plastic with the yield values of 1077.4 ± 185.9 and 536.1 ± 45.8 millipascals-second (mPas), respectively. These different properties are considered to be due to the amylose content in different rice variety. The results suggest that the non-glutinous rice varieties with high amylose content are the most suitable for preparing gels as local delivery systems via the mucosal membrane.

The antimicrobial peptide, human β-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases

&NA; Previous studies have demonstrated increased expression and raised levels of human &bgr;‐defensin (hBD)‐1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri‐implantitis, oral bone‐resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD‐1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD‐1 and cell viability was assayed by MTT. PBMCs were treated with macrophage‐colony stimulating factor and receptor activator of nuclear factor kappa‐B ligand (RANKL) in the presence or absence of non‐toxic doses of hBD‐1. In vitro osteoclastogenesis was analyzed by tartrate‐resistant acid phosphatase (TRAP) staining, osteoclast‐specific gene expression, and a resorption pit assay. Involvement of mitogen‐activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD‐1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP‐positive multinuclear cells and resorption areas on the dentin slices, and further up‐regulated expressions of osteoclast‐specific genes compared to those by RANKL treatment (p < 0.05). However, hBD‐1 treatment without RANKL failed to induce formation of osteoclast‐like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD‐1 co‐treatment (p < 0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD‐1‐enhanced osteoclastogenesis. Collectively, hBD‐1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs. HighlightsHBD‐1 potentiates the inductive effect of RANKL on human osteoclast formation and function in vitro.However, hBD‐1 has no inductive effect on human osteoclastogenesis.The potentiation effect of hBD‐1 on in vitro osteoclastogenesis is mediated by transient activation of the p44/42 MAPKs.

Inducible expression of A Disintegrin and Metalloproteinase 8 in chronic periodontitis and gingival epithelial cells.

BACKGROUND AND OBJECTIVES The expression of A Disintegrin and Metalloproteinase 8 (ADAM8) is associated with several inflammatory diseases. Elevated ADAM8 levels have been shown in gingival crevicular fluid of patients with chronic periodontitis. The objective of this study was to investigate ADAM8 expression in chronic periodontitis tissues compared with that in normal tissues. ADAM8 expression and its inductive mechanism were examined in human gingival epithelial cells (HGECs) and human gingival fibroblasts. MATERIAL AND METHODS Total RNA and protein were extracted from gingival biopsies of 33 patients with chronic periodontitis and those of 23 healthy volunteers. ADAM8 mRNA and protein expression was analyzed by real-time polymerase chain reaction, immunoblotting and immunohistochemistry. ADAM8 expression in control and stimulated cells in the presence or absence of specific inhibitors for mitogen-activated protein kinase pathways was assayed by real-time polymerase chain reaction, immunoblotting, flow cytometry and immunofluorescence. RESULTS ADAM8 mRNA and protein expression in chronic periodontitis tissues was significantly greater than that in normal tissues (p < 0.01). Significantly increased ADAM8 expression was detected in the gingival epithelium of chronic periodontitis tissues (p < 0.001). ADAM8 mRNA expression in HGECs, but not in human gingival fibroblasts, was significantly induced by stimulation with Fusobacterium nucleatum (p < 0.05), partially via the p44/42 mitogen-activated protein kinase pathway. ADAM8 expression in the cell lysates and on the surface of HGECs was induced by stimulation with F. nucleatum. CONCLUSION ADAM8 expression is increased in inflamed chronic periodontitis tissues and localized within gingival epithelium, consistent with an upregulation of ADAM8 expression in F. nucleatum-stimulated HGECs, suggesting a possible role of ADAM8 in innate immunity of periodontal tissue.