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ABSTRACT: Synovial fibroblasts of temporomandibular joint (TMJ) are poorly characterized, although they have important roles in progression of temporomandibular disorders (TMD). In this study, we investigated responses of synovial fibroblasts to interleukin (IL)-1beta.
We examined gene expression profiles of synovial fibroblasts in response to IL-1beta, using Affymetrix GeneChip. Regulated upon activation normal T-cell expressed and secreted (RANTES) gene expression was confirmed by polymerase chain reaction (PCR) and real-time PCR. RANTES protein levels were measured by enzyme-linked immunosorbent assay (ELISA).
The RANTES was preferentially up-regulated in synovial fibroblasts by IL-1beta. The increase in RANTES gene expression in response to IL-1beta was confirmed by PCR and real-time PCR. Protein level of RANTES in synovial fibroblasts was also increased by IL-1beta.
The RANTES, a cc-type chemokine, has chemotactic effects on lymphocytes and monocytes. Increased gene expression and protein production of RANTES in synovial fibroblasts, in response to IL-1beta, may play an important role in recruitment of inflammatory cells into synovium and progression of synovitis in TMD.
Journal of Oral Pathology and Medicine 12/2004; 33(10):629-33. · 1.63 Impact Factor
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ABSTRACT: Although chronic caffeine exposure during pregnancy has been shown to affect fetal growth, adverse effects of caffeine on embryogenesis are not only well understood, but also controversial. We have used gene chip technology in an attempt to identify to what extent, if any, caffeine could possibly alter gene expressions in the cytotrophoblast-like cell line BeWo. Few down-regulated genes were found; most of the genes were up-regulated, suggesting that chronic caffeine exposure during the gestational period could exert certain influences on embryogenesis. The highest up-regulated gene expression of BeWo cells by caffeine was angiotensin II type 2 (AT(2)) receptor gene. We focused the genes of the renin-angiotensin system (RAS), angiotensin II type 1 (AT(1)) and AT(2)receptors and angiotensin I converting enzyme, for study on caffeine's responsive gene expression in BeWo cells and in the placentae of pregnant rats that were fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analysed the gene expressions using RT-PCR and LightCycler system. A significantly increased AT(2)receptor gene expression and a slight decreased AT(1)receptor gene expression demonstrated the caffeine's effect to the placental RAS.
Placenta 08/2003; 24(6):638-47. · 3.69 Impact Factor
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ABSTRACT: Porphyromonas gingivalis is a Gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis. We previously cloned a gene encoding dipeptydilaminopeptidase IV (DAPIV) from P. gingivalis. In the present study, for immunological diagnosis and development of passive immunization, we produced a mouse monoclonal antibody (MAb) capable of inhibiting the DAPIV activity of P. gingivalis using highly purified recombinant DAPIV as an immunogen. The constructed MAb, designated as MAb-Pg-DAP-1, significantly inhibited DAPIV activity in P. gingivalis, as well as slightly inhibited that in other gram-negative bacteria such as Porphyromonas endodontalis and Prevotella loesheii, whereas no inhibition was seen in the gram-positive bacteria Streptococcus mutans and Actinomyces viscosus. Furthermore, the MAb did not inhibit DAPIV enzyme activity in human serum. This novel MAb may be useful for the development of immunological diagnosis capability and in passive immunization.
Hybridoma and Hybridomics 07/2003; 22(3):147-51.
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ABSTRACT: Many studies have attempted to elucidate the mechanism of the biostimulatory effects of low-level laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5-2-fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.
Lasers in Medical Science 02/2003; 18(2):78-82. · 2.00 Impact Factor
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ABSTRACT: Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.
Journal of Dental Research 11/2002; 81(10):679-82. · 3.49 Impact Factor
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ABSTRACT: It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets. In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis.
Genomic DNA extracted from cultured P.g. 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR). We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g. The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe.
Fewer than 100 P.g. bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay. This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF). Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g. from which the template DNA was extracted semiquantitatively. It is noteworthy that the PCR assay could also be applied to detect P.g. from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay.
The PCR assay established in this study can be a powerful tool to detect P.g. in periodontal pockets and monitor the colonization and/or recolonization of P.g. at the very early phase.
Journal of Periodontology 10/2001; 72(9):1228-35. · 2.60 Impact Factor
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ABSTRACT: Porphyromonas gingivalis is a gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis. Hemagglutinin may mediate the adsorption and invasion of bacteria into host cells. Furthermore, the hemagglutinin plays a role in the agglutinate and lyse erythrocytes intake of heme which is an absolute requirement for this bacterial growth. We previously cloned the gene encoding the 130-kDa hemagglutinin protein domain (130-kDa HMGD) and identified the functional motifs of agglutination of erythrocytes. Bacterial cell attachment to erythrocytes is an important initial step in the expression of hemolytic activity. In this study, we highly purified recombinant r130-kDa HMGD and prepared the specific antiserum. Further, the effect of the antibody on the hemolytic activity of P. gingivalis cells was examined. The polyclonal antibody recognized 43,49-kDa major bands in P. gingivalis cells and r130-kDa HMGD, and significantly inhibited the hemagglutinating and hemolytic activities of P. gingivalis cells. The findings suggest that the antibody may be useful in the development of the passive immunization against periodontal diseases caused by P. gingivalis infection.
Journal of Oral Science 10/2001; 43(3):159-63.
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ABSTRACT: Individuals with Down syndrome (DS) have a high prevalence of periodontal disease, which develops early and progressed rapidly and extensively, in comparison with healthy controls. The severe periodontal disease in individuals with DS has been considered to result from abnormal factors in their host responses. The mechanisms involved in the periodontal inflammatory processes in individuals with DS are not fully understood. Plasminogen activators (PA) are serine proteases that are well known for their part in the initiation of the fibrinolytic cascade leading to the generation of plasmin in periodontal homeostasis, including fibrinolysis and connective tissue remodeling. The PA-plasmin system affects the progression of periodontal disease. In the present study, we examined the effects of the levels of PA activity stimulated with lipopolysaccharide (LPS) in the gingival fibroblasts from donors with DS (DGF). The levels of PA activity without LPS were low in the DGFs, the same as that in the gingival fibroblasts from donors of healthy controls (NDGF). In contrast, the levels of PA activity with LPS in DGFs were significantly higher than that in the NDGFs. These results suggested that PA plays an important role in inducing extensive and rapid inflammation in the periodontal disease in individuals with DS.
Journal of Oral Science 10/2001; 43(3):207-12.
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ABSTRACT: To measure the activities of plasminogen activator (PA), plasmin and kallikrein, multiple synovial fluid samples were taken from 32 patients with internal derangement (ID) and osteoarthrosis (OA), and nine asymptomatic volunteers. The enzyme activity in synovial fluid from the temporomandibular joint (TMJ) was quantitated by a fluorogenic substrate assay using an enzyme substrate. In fluid samples from the patient group, PA was detected in 24 (31.5%), plasmin in 20 (26.3%) and kallikrein in 53 (96.4%), while none of these enzymes were found in the synovial fluid samples from the control group. There were positive correlations found among PA, plasmin and kallikrein. These results clearly demonstrated increased levels of PA, plasmin and kallikrein activities in the synovial fluid of patients with ID and OA, and suggest that these enzymes may be involved in the pathogenesis of synovitis, as well as the resorption of cartilage and bone in TMJ.
International Journal of Oral and Maxillofacial Surgery 09/2001; 30(4):323-8. · 1.51 Impact Factor
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ABSTRACT: A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate.
Archives of Oral Biology 09/2001; 46(8):759-66. · 1.60 Impact Factor
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ABSTRACT: The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) in various cell types has been proposed as an important feature of many cellular processes requiring extracellular proteolysis, cell adhesion, motility, and invasion. uPAR attaches to the cell surface with a glycosylphophatidylinositol (GPI) anchor, and serves to localize and accelerate the proteolysis cascade. In this study, we examined both uPA and uPAR levels in human gingival fibroblasts treated with an inflammatory cytokine, interleukin-1beta (IL-1beta). PA activity in the cell lysate was increased by treatment with IL-1beta. Further, PA activity released by phosphatidylinositol-specific phospholipase C, which detaches the GPI anchor, was also increased by IL-1beta. The activity was inhibited by amiloride, a specific inhibitor of uPA. In addition, IL-1beta increased the protein and mRNA levels of both uPA and uPAR in gingival fibroblasts. These findings suggest that the enhancement of uPA and uPAR levels by IL-1beta may play an important role in the progression of periodontal diseases through pericellular proteolysis, and subsequent cellular behavior.
International Union of Biochemistry and Molecular Biology Life 07/2001; 51(6):381-5. · 3.51 Impact Factor
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ABSTRACT: One glucosyltransferase (GTF) -I deficient mutant of Streptococcus sobrinus strain B13N was isolated through chemical mutagenesis with ethyl methanesulfonate, and characterized. This mutant, designated as B13N-Id, readily allowed us to purify a homogeneous oligo-isomaltosaccharide synthase (GTF-S) from its culture fluid. The purified GTF-S was only recognized with rabbit polyclonal antibody against recombinant GTF-S from an Ecsherichia coli MD124 clone expressing the B13N gtfS gene, and showed the almost same enzymatic properties as the recombinant enzyme. A double reciprocal plot of the B13N GTF-S for sucrose was biphasic, and the affinity for this substrate was high compared to that of GTF-S enzymes from other strains.
Bioscience Biotechnology and Biochemistry 07/2001; 65(6):1290-5. · 1.28 Impact Factor
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ABSTRACT: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis. This bacterium possesses hemagglutinating and hemolytic activities to attach and lyse erythrocytes. Hemolysis by this oral pathogen functions to provide heme-containing molecules for growth in the periodontal pocket. We previously constructed a monoclonal antibody using P. gingivalis vesicles as the immunogen, designated as MAb-Pg-vc, which inhibited vesicle-associated hemagglutinating activity. Furthermore, we cloned the gene encoding 130-kDa hemagglutinin (130-kDa HAG) and identified its functional motif for attachment to erythrocytes. Generally, bacterial cell attachment to erythrocytes is an important initial step for expressing hemolysis activity. In the present study, we examined the effect of MAb-Pg-vc on the hemolytic activity of P. gingivalis cells. The MAb-Pg-vc significantly inhibited the hemolytic activity and, further, this inhibitory activity was reduced by the synthetic peptide of the 130-kDa HAG functional motif.
European Journal Of Oral Sciences 05/2001; 109(2):109-13. · 1.88 Impact Factor
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ABSTRACT: Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system. To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid. Next, the secretion domain of the B. subtilis alpha-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR. Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed. The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion. The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was isolated.
Bioscience Biotechnology and Biochemistry 03/2001; 65(2):389-95. · 1.28 Impact Factor
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ABSTRACT: Biostimulatory effect of cell proliferation and bone formation by laser irradiation has been reported, however, very little is known about the molecular basis of mechanisms. We previously constructed the cDNA library of mouse osteoblastic cells (MC3T3-E1) which enhanced gene expression by laser irradiation using a subtracted gene cloning procedure. In the present study, we focused on a gene clone, designated as MCL-140, which exhibited the high homology of DNA sequence with mouse minichromosome maintenance (MCM) 3 gene. MCM3 is involved in the initiation of DNA replication as licensing factor in eukaryotic cells. Nucleotide sequence of MCL-140 insert was determined and assessed in the nucleic acid databases. The transcription level of MCL-140 was examined by Northern blot analysis. The DNA sequences of clone MCL-140 insert exhibited 96.2% homology with MCM 3 gene coding P1 protein. Higher MCM3 mRNA levels were observed in laser-irradiated cells compared to the levels in non-irradiated cells: furthermore, radiolabelled thymidine incorporation was increased by laser irradiation. These findings suggest that low-level laser irradiation may enhance DNA replication and play a role in stimulating proliferation of osteoblast through the enhancement of the MCM3 gene expression.
Lasers in Medical Science 02/2001; 16(3):213-7. · 2.00 Impact Factor
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ABSTRACT: Human gingival fibroblast (hGF) cells reside in gingival tissues which are challenged frequently by oral bacteria. Lipopolysaccharide (LPS) from periodontal pathogens can penetrate gingival tissues and stimulate the production of interleukin-1beta (IL-1beta), which has been implicated in inflammation and bone resorption. The anti-inflammatory effects of low-energy laser irradiation have been reported, but the mechanisms of this biostimulatory effect have not been fully elucidated. Primary cultured hGF cells were challenged with LPS isolated from Campylobacter rectus, a known periodontal disease-associated pathogen, and irradiated by a Ga-Al-As diode low-energy laser (830 nm, 3.95-7.90 J/cm2). The hGF cells cultured medium showed a marked elevation of IL-1beta production by LPS, which was significantly inhibited by laser irradiation in a dose-dependent manner. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, this inhibitory effect was involved in the reduction of IL-1beta mRNA levels but not that of the IL-1beta converting enzyme.
Lasers in Medical Science 02/2001; 16(3):218-23. · 2.00 Impact Factor
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ABSTRACT: This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.
Analytical Biochemistry 02/2001; 288(2):168-175. · 3.00 Impact Factor
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ABSTRACT: Although the severity of periodontal disease is known to be affected by the age of the host, the pathological role of aging in periodontal disease, especially that attributable to trauma from occlusion, has not been well characterized. Prostaglandin (PG)E2 and interleukin (IL)-1beta are key mediators involved in periodontal diseases, potent stimulators of bone resorption, and are produced by human periodontal ligament (PDL) cells in response to mechanical stress. To investigate age-related changes in the biosynthetic capacity of PGE2 and IL-1beta in PDL cells, we examined the effects of in vivo aging with mechanical tension on PGE2 and IL-1beta expression by rat PDL cells. PDL cells obtained from the incisors of 6-week (young) and 60-week (old) rats were cultured on flexible-bottomed culture plates. The cells were deformed by causing a 9% or 18% increase in surface area at 6 cycles per minute for 1 to 5 days. We found an approximately twofold increase in PGE2 and IL-1beta production by old PDL cells subjected to mechanical tension compared with that by young cells, although the constitutive levels were similar in both. The expression of cyclooxygenase (COX)-2 and IL-1beta mRNA (messenger ribonucleic acid) was enhanced by mechanical tension as determined by use of reverse transcription-polymerase chain reaction (RT-PCR), whereas COX-1 and IL-1beta-converting enzyme mRNA remained unchanged. It is possible that the large amount of PGE2 and IL-1beta produced by PDL cells from an aged host in response to mechanical force may be positively related to the acceleration of alveolar bone resorption.
The Journals of Gerontology Series A Biological Sciences and Medical Sciences 11/2000; 55(10):B489-95. · 4.60 Impact Factor
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ABSTRACT: We have cloned the gene for a 40-kDa outer membrane protein (40-kDa OMP) from Porphyromonas gingivalis 381. The recombinant (r)40-kDa OMP has become the subject of considerable interest because of its potential role in the development of a vaccine useful for passive immunization. To develop such a vaccine, it is essential to fully understand the functions of anti-r40-kDa OMP antibody in the host defense against P. gingivalis. To that end, we developed a panel of monoclonal antibodies by immunizing mice with purified r40-kDa OMP. The objective of this study was to determine the bactericidal activity on P. gingivalis by the IgG1 monoclonal antibody Pg-ompA2.
Bacterial growth measurement, a complement-mediated anti-P. gingivalis assay based on [3H]thymidine uptake, and a 14C-release assay were performed to test the bactericidal activity of Pg-ompA2 to P. gingivalis.
In the presence of complement, Pg-ompA2 was lethal to P. gingivalis 381 as well as to the more virulent P. gingivalis strains, including ATCC 53977 and W83. Using component-deficient complement, we determined that Pg-ompA2 killed P. gingivalis by activating both the classical and alternative complement pathways.
Pg-ompA2 has an in vitro complement-mediated bactericidal activity to P. gingivalis. Pg-ompA2 may contribute to the development of a local immunotherapy that can be applied in the gingival crevice of a patient with P. gingivalis-related periodontitis, or be a vaccine candidate.
Journal of Periodontology 04/2000; 71(3):368-75. · 2.60 Impact Factor
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ABSTRACT: It has been reported that lipopolysaccharide (LPS) from periodontal pathogens can penetrate gingival tissues and stimulate the production of prostaglandin E2 (PGE2), which is known as a potent stimulator of inflammation and bone resorption. Although biostimulatory effects of low-level laser irradiation such as anti-inflammatory results have been reported, the physiological mechanism is not yet clarified. The purpose of the present study was to determine the effect of laser irradiation on PGE2 production and cyclooxygenase (COX)-1 and COX-2 gene expression in LPS-challenged human gingival fibroblast (hGF) cells in vitro. hGF cells were prepared from healthy gingival tissues and challenged with LPS, and Ga-Al-As diode laser was irradiated to the hGF cells. The amount of PGE2 released in the culture medium was measured by radioimmunoassay, and mRNA levels were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Irradiation with Ga-Al-As diode low-level laser significantly inhibited PGE2 production in a dose-dependent manner, which led to a reduction of COX-2 mRNA levels. In conclusion, low-level laser irradiation inhibited PGE2 by LPS in hGF cells through a reduction of COX-2 mRNA level. The findings suggest that low-level laser irradiation may be of therapeutic benefit against the aggravation of gingivitis and periodontitis by bacterial infection.
European Journal Of Oral Sciences 03/2000; 108(1):29-34. · 1.88 Impact Factor
