Functional Regulatory Mechanisms Underlying Bone Marrow Mesenchymal Stem Cell Senescence During Cell Passages.

Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.

Reparative bone-like tissue formation in the tooth of a systemic sclerosis patient.

AIM: To report a case of reparative bone-like tissue formation in the tooth of a patient with systemic sclerosis. SUMMARY: A 58-year-old Japanese female patient with systemic sclerosis was referred because of tooth fracture. Cone beam computerized tomography (CBCT) revealed multiple root resorption and the unclear transition from alveolar bone to root profile. A sample from a fractured tooth was analysed histologically. Haematoxylin and eosin-stained sections revealed the irregular replacement of pulp and dentine by bone-like tissue. Calcinosis was noted in various parts of the body and a histological analysis identified it as dystrophic calcification on sclerosed fibrous connective tissue. Bite force and the occlusal area were markedly weaker than the means for female of the same age. KEY LEARNING POINTS: CBCT may be more useful than dental radiography for diagnosing multiple root resorption in systemic sclerosis patients. When systemic sclerosis patients have calcinosis, their root status must be examined carefully. When root resorption is present in systemic sclerosis patients, reparative bone-like tissue formation in teeth needs to be taken into account prior to the initiation of dental treatment.

The induced RNA-binding protein, HuR, targets 3'-UTR region of IL-6 mRNA and enhances its stabilization in periodontitis.

RNA-binding proteins (RBPs) regulate mRNA stability by binding to the 3'-untranslated region (UTR) region of mRNA. Human antigen-R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)-6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL-6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in-vitro and in-vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)-derived from Pg (PgLPS) and tumour necrosis factor (TNF)-α in OBA-9, an immortalized human gingival epithelial cell. The luciferase activity of 3'-UTR of IL-6 mRNA was increased by TNF-α, Pg and PgLPS in OBA-9. Luciferase activity was also increased in HuR-over-expressing OBA-9 following a bacterial stimulation. Down-regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL-6. In contrast, the over-expression of HuR increased IL-6 mRNA expression and production in OBA-9. The HuR inhibitor, quercetin, suppressed Pg-induced HuR mRNA expression and IL-6 production in OBA-9. An oral inoculation with quercetin also inhibited bone resorption in ligature-induced periodontitis model mice as a result of down-regulation of IL-6. These results show that HuR modulates inflammatory responses by regulating IL-6.

MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption.

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP-monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis ( n = 6-7/group) where Pasteurella pneumotropica ( Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase-positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor-α, interleukin-1ß, and RANKL in the gingival tissue compared with the control site without ligature ( P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti- Pp IgG antibody nor in vitro anti- Pp T-cell response and resultant production of RANKL was affected by anti-DC-STAMP-mAb. This study illustrated the roles of DC-STAMP in promoting local OC cell fusion without affecting adaptive immune responses to oral bacteria. Therefore, it is plausible that a novel therapeutic regimen targeting DC-STAMP could suppress periodontal bone loss.

Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice.

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.

The differential expression of mgl mRNA by Porphyromonas gingivalis affects the production of methyl mercaptan.

OBJECTIVE: A large number of individuals have halitosis. The total amount of volatile sulfur compounds, which are the main cause of halitosis, has been correlated with periodontitis following bacterial infection. In this study, Porphyromonas gingivalis (Pg), a major periodontopathogenic bacterium, was isolated from patients with halitosis by the amplification of 16S rRNA, and the ability of isolated Pg to produce methyl mercaptan (CH3 SH) was determined to clarify the relationship between halitosis and Pg infection. MATERIALS AND METHODS: CH3 SH concentrations were measured in patients using Oral Chroma. The production of CH3 SH by Pg standard and clinical strains was also measured in vitro. Real-time PCR was performed to compare the expression of mgl mRNA (which encoded l-methionine-a-deamino-g-mercaptomethane-lyase) among the Pg strains. The production of CH3 SH and the expression of mgl mRNA were also determined to assess the effects of oriental medicine. RESULTS: The production of CH3 SH and the expression of mgl mRNA strongly correlated with each other in the presence of l-methionine. The expression of mgl mRNA by Pg W83 was strongly inhibited by magnoliaceae. CONCLUSION: The production of CH3 SH was correlated with the expression of mgl. Furthermore, the oriental medicine, magnoliaceae, may represent a potential treatment for halitosis.

LL37 induces VEGF expression in dental pulp cells through ERK signalling.

AIM: To examine the in vitro effects of LL37 on the expression of vascular endothelial growth factor (VEGF) in human pulp cells and to identify the intracellular signalling pathway involved. METHODOLOGY: Pulp cells at passage 6 were treated with 10 µg mL(-1) synthesized LL37, and an inhibition assay was performed with MAPK or NF-κB inhibitors to determine the possible signalling pathway. VEGF mRNA, VEGF protein and phosphorylated ERK1/2 levels were determined by real-time PCR, ELISA and Western blot, respectively. Data were analysed using t-tests. RESULTS: LL37 significantly increased both the mRNA and protein levels of VEGF in pulp cells (P < 0.01). However, pre-treatment with an ERK kinase inhibitor suppressed these increases. Furthermore, the inhibitor blocked LL37-induced ERK1/2 phosphorylation. CONCLUSIONS: LL37 activated the ERK pathway to boost VEGF secretion from human pulp cells. Because of this angiogenic effect and its reported induction of pulp cell migration and antimicrobial activity against cariogenic bacteria, LL37 may be applicable as a pulp capping material.

Interleukin-8 induces DNA synthesis, migration and down-regulation of cleaved caspase-3 in cultured human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). MATERIAL AND METHODS: DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. RESULTS: IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. CONCLUSION: IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.

Irsogladine maleate inhibits Porphyromonas gingivalis-mediated expression of toll-like receptor 2 and interleukin-8 in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study. MATERIAL AND METHODS: OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production. RESULTS: The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9. CONCLUSION: These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.

Brain-derived neurotrophic factor prevents the endothelial barrier dysfunction induced by interleukin-1ß and tumor necrosis factor-α.

BACKGROUND AND OBJECTIVE: Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Although a local inflammatory step is required to initiate the subsequent process of tissue regeneration, excessive inflammation may inhibit or delay tissue regeneration. Therefore, the regulation of inflammation is essential for periodontal tissue regeneration. In the present study, we examined the influence of BDNF on the human microvascular endothelial cell (HMVEC) barrier dysfunction induced by interleukin (IL)-1ß or tumor necrosis factor (TNF)-α to determine the effects of BDNF on the regulation of local inflammation in periodontal tissue regeneration. MATERIAL AND METHODS: Endothelial permeability was analyzed using a Dextran transport assay with transwell plates. The expression of vascular endothelial (VE)-cadherin was assessed by immunoblotting and immunofluorescence microscopy. RESULTS: BDNF (25 ng/mL) inhibited increase induced in endothelial permeability by IL-1ß and TNF-α. IL-1ß and TNF-α decreased VE-cadherin protein levels, while BDNF recovered the reduction in HMVECs. BDNF protected the increase induced in endothelial permeability by IL-1ß and TNF-α through TrkB. The single addition of BDNF into the culture increased the expression of VE-cadherin in HMVECs. CONCLUSION: BDNF played an important role in inhibiting endothelial barrier dysfunction, which suggests that it may assist in enhancing periodontal tissue regeneration.

Smad2 is involved in Aggregatibacter actinomycetemcomitans-induced apoptosis.

Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-ß receptors (TGF-ßRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-ß type I receptor (TGF-ßRI) in OBA9 cells. SB431542 (a TGF-ßRI inhibitor) and siRNA transfection for TGF-ßRI, which reduced both TGF-ßRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-ßRI signaling cascade by SB431542 and siRNA transfection for TGF-ßRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-ßRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-ßR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis.

Antimicrobial peptide LL37 promotes vascular endothelial growth factor-A expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: LL37, originally found in the innate immune system, is a robust antimicrobial peptide. LL37 exhibits multiple bio-functions in various cell types, such as migration, cytokine production, apoptosis, and angiogenesis besides its antimicrobial activity Periodontal ligament (PL) cells play a pivotal role in periodontal tissue regeneration. Based on these findings, we hypothesized that LL37 can regulate PL cell function to promote regeneration of periodontal tissue. To prove this hypothesis, we investigated the effect of LL37 on the potent angiogenic inducer vascular endothelial growth factor (VEGF) expression in cultures of human PL (HPL) cells because neovascularization is indispensable for the progress of tissue regeneration. Moreover, we investigated the signaling cascade associated with LL37-induced VEGF expression. MATERIAL AND METHOD: HPL cells were treated with synthesized LL37 in the presence or absence of PD98059, a MEK-ERK inhibitor, or PDTC, an NF-κB inhibitor. VEGF expression levels were assessed by real-time polymerase chain reaction analysis and an enzyme-linked immunoassay. Phosphorylation levels of ERK1/2 or NF-κB p65 were determined by Western blotting. RESULTS: LL37 upregulated VEGF-A expression at the mRNA and protein levels in HPL cells, while VEGF-B mRNA expression was not affected. Both ERK and NF-κB inhibitors clearly abrogated the increase in VEGF-A levels induced by LL37 in HPL cells. Importantly, LL37 increased phosphorylated levels of ERK1/2 and NF-κB p65 in HPL cells. CONCLUSION: LL37 induces VEGF-A production in HPL cells via ERK and NF-κB signaling cascades, which may result in angiogenesis, thereby contributing to periodontal regeneration.

Detection of periodontal pathogens in newborns and children with mixed dentition.

We report the age-related prevalence of red complex periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, along with four strains of orange complex pathogens. The bacteria present in samples isolated from tongue, cheek, and subgingival sulcus in edentulous newborns and children with mixed dentition were monitored by polymerase chain reaction (PCR). P. gingivalis was not detected in any site of any subject in the two groups tested. However, T. denticola was not only found in the 6-13 years age group, but also in edentulous newborns at a relatively high prevalence, indicating non-dentition-related colonization by T. denticola. Campylobacter rectus, Prevotella intermedia, T. forsythia, Eikenella corrodens, and Parvimonas micra were found in the oral cavity of most subjects belonging to the 6-13 years age group compared to newborns. This suggested a pronounced association between these colonizing bacteria and the presence of teeth. There was also a strong relation between T. denticola and T. forsythia for their prevalence in the subgingival sulcus of the 6-13 years age group (p < 0.0001), but not in the other sites tested, suggesting that the colonization of dentition-related T. forsythia may be associated with the increased prevalence of non-dentition-related T. denticola in the subgingival sulcus. Overall, these results suggest that dentition is a key determinant of bacterial colonization, especially orange complex bacteria and the red complex bacterium T. forsythia.

A role of oral bacteria in bisphosphonate-induced osteonecrosis of the jaw.

No consensus has yet been reached to associate oral bacteria conclusively with the etio-pathogenesis of bisphosphonate-induced osteonecrosis of the jaw (BONJ). Therefore, the present study examined the effects of oral bacteria on the development of BONJ-like lesions in a mouse model. In the pamidronate (Pam)-treated mice, but not control non-drug-treated mice, tooth extraction followed by oral infection with Fusobacterium nucleatum caused BONJ-like lesions and delayed epithelial healing, both of which were completely suppressed by a broad-spectrum antibiotic cocktail. Furthermore, in both in vitro and in vivo experiments, the combination of Pam and Fusobacterium nucleatum caused the death of gingival fibroblasts (GFs) and down-regulated their production of keratinocyte growth factor (KGF), which induces epithelial cell growth and migration. Therefore, in periodontal tissues pre-exposed to bisphosphonate, bacterial infection at tooth extraction sites caused diminished KGF expression in GFs, leading to a delay in the epithelial wound-healing process that was mitigated by antibiotics.

Expression and possible immune-regulatory function of ghrelin in oral epithelium.

Originally found in stomach mucosa, ghrelin is a peptide appetite hormone that has been implicated as an immuno-modulatory factor. Ghrelin has also been found in salivary glands and saliva; however, its expression patterns and biological properties in the oral cavity remain unclear. Therefore, we investigated the expression patterns of ghrelin in saliva, gingival crevicular fluid (GCF), and gingival tissue, as well as its in vitro effects on IL-8 production by TNF-α or LPS-stimulated oral epithelial cells. In the clinical samples obtained from 12 healthy volunteers, the concentration of ghrelin in GCF remarkably exceeded that detected in saliva. The expression of ghrelin mRNAs and growth hormone secretagogue (GHS) receptors could be detected in human oral epithelial cells. Immunohistochemical analysis revealed the expression of ghrelin in gingival epithelium, as well as in fibroblasts in the lamina propria. Ghrelin increased intracellular calcium mobilization and cAMP levels in oral epithelial cells, suggesting that ghrelin acts on epithelial cells to induce cell signaling. Furthermore, synthetic ghrelin inhibited the production of IL-8 from TNF-α or LPS-stimulated oral epithelial cells. These results indicate that ghrelin produced in the oral cavity appears to play a regulatory role in innate immune responses to inflammatory infection.

Irsogladine maleate abolishes the increase in interleukin-8 levels caused by outer membrane protein 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans through the ERK pathway in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Irsogladine maleate (IM) suppresses the increase in interleukin (IL)-8 production induced by outer membrane protein (OMP) 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans in cultures of human gingival epithelial cells (HGEC). However, how IM suppresses the OMP29-induced increase in IL-8 expression remains unknown. In this study, we focused on intracellular signaling pathways to elucidate the mechanism behind the suppression. MATERIAL AND METHODS: HGEC, which had been pretreated with inhibitors of intracellular signaling molecules, were exposed to OMP29 (1 microg/mL) with or without IM (1 microM). IL-8 expression at the mRNA and protein levels was examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Extracellular signal-regulated kinase (ERK) activity was measured with a p44/42 mitogen-activated protein kinase assay kit. RESULTS: An ERK inhibitor, PD98059, as well as IM, obviated the OMP29-induced increase in IL-8 levels in HGEC. A Jun kinase inhibitor, SP600125, and a nuclear factor kappaB inhibitor, PDTC, did not influence the OMP29-induced increase in IL-8 mRNA expression. The OMP29 stimulated phosphorylation of ERK in HGEC. Irsogladine maleate inhibited the phosphorylation. CONCLUSION: The suppression of the phosphorylation of ERK by IM in HGEC culminates in inhibition of the OMP29-induced increase in IL-8.

Irsogladine maleate counters the interleukin-1 beta-induced suppression in gap-junctional intercellular communication but does not affect the interleukin-1 beta-induced zonula occludens protein-1 levels in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1 beta is involved in periodontal disease. Little is known, however, about the effect of interleukin-1 beta on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1 beta. In this study, we examined how interleukin-1 beta affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1 beta-induced changes in the intercellular junctional complexes in human gingival epithelial cells. MATERIAL AND METHODS: Human gingival epithelial cells were exposed to interleukin-1 beta, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. RESULTS: Interleukin-1 beta decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1 beta-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. CONCLUSION: The effect of interleukin-1 beta on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis.

[Ectopic production of human chorionic gonadotropin by poorly differentiated transitional cell carcinoma of the renal pelvis].

Herein we report a case of ectopic production of hCG by poorly differentiated transitional cell tumor of the renal pelvis. The patient was a 55-year-old male who had been diagnosed at another hospital as having giant hydronephrosis and renal stones and was referred to our hospital. The plain abdominal CT showed a low-density mass at the lower pole of the right kidney. His serum hCG level was as high as 120 mIU/ml. Transperitoneal nephrectomy was performed on July 7, 1987. Histopathological examinations showed the presence of squamous metaplasia within a high-grade transitional cell carcinoma, and immunohistochemical studies revealed the presence of chorionic gonadotropin in some giant cells. Two courses of combination chemotherapy with methotrexate, vinblastine, adriamycin and cisplatin (M-VAC regimen) were given to him from the third week after the operation. However, he died of debility with distant metastasis 6 months after the operation. As far as we know, this is the third reported case in Japan.