RESUMEN
OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.
Asunto(s)
Infecciones por Bacteroidaceae , Caspasas Iniciadoras , Encía , Periodontitis , Porphyromonas gingivalis , Infecciones por Bacteroidaceae/enzimología , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Caspasas Iniciadoras/biosíntesis , Células Cultivadas , Epitelio/enzimología , Epitelio/microbiología , Epitelio/patología , Encía/enzimología , Encía/microbiología , Encía/patología , Humanos , Interleucina-18/biosíntesis , Interleucina-8/biosíntesis , Periodontitis/enzimología , Periodontitis/microbiología , Periodontitis/patología , Porphyromonas gingivalis/metabolismo , ARN Mensajero/metabolismoRESUMEN
Thanatochemistry also known as chemistry of death and is used to determine post mortem interval (PMI). It is arguably one of the critical steps in forensic investigation. Recent addition of analyzing biochemical changes along with the traditional methods have gained importance, as they help us to record very early changes in the tissue specimens. In this view, our study aimed to correlate both histological changes and enzymatic changes in gingival tissue samples at intervals of immediate, 1 h, 5 h, 24 h and 48 h after death. Histologic changes noted were loss of epithelial architecture, chromatin clumping, nuclear vacuolation, karryopyknosis, eosinophilia and wide intercellular junctions. Two enzymes which differentiate between the autolytic phase (acid phosphatase) and putrefactive phase (ammonia) of decomposition were evaluated using UV spectrometer. Results in our study demonstrated there were variations as in gradual increase in ammonia levels (1.13±0.24-26.6±2.09) and gradual decrease in acid phosphatase levels (5.61±0.67-1.25±0.53) at different time intervals till 48 h. The cellular changes in gingival tissue could also be related to time. The result of our study helps us to identify potential of enzymatic changes which when correlated with histological reports helps us to predict the time of death accurately. Replicating this experiment in various known taphonomic conditions and other enzymes could highlight the usefulness of gingival tissue samples in determining time of death.
Asunto(s)
Encía/enzimología , Encía/patología , Cambios Post Mortem , Fosfatasa Ácida/metabolismo , Adulto , Amoníaco/metabolismo , Apoptosis , Núcleo Celular/patología , Cromatina/patología , Eosinofilia/patología , Células Epiteliales/patología , Femenino , Patologia Forense/métodos , Humanos , Uniones Intercelulares/patología , Masculino , Necrosis , Espectrofotometría Ultravioleta , Vacuolas , Adulto JovenRESUMEN
Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25-Hydroxyvitamin D3 (25(OH)2D3 ) on diabetic periodontitis. 25(OH)2D3 photo-affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D3 , which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony-stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss-related diseases.
Asunto(s)
Pérdida de Hueso Alveolar/enzimología , Calcifediol/uso terapéutico , Diabetes Mellitus Experimental/complicaciones , Periodontitis/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Pérdida de Hueso Alveolar/tratamiento farmacológico , Pérdida de Hueso Alveolar/microbiología , Pérdida de Hueso Alveolar/fisiopatología , Animales , Calcifediol/química , Células Cultivadas , Citocinas/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/patología , Encía/citología , Encía/enzimología , Encía/metabolismo , Encía/patología , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Periodontitis/tratamiento farmacológico , Periodontitis/metabolismo , Periodontitis/microbiología , Porphyromonas gingivalis , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , ARN Interferente Pequeño , Tirosina/metabolismoRESUMEN
Smokers have a risk of developing periodontal disease. Matrix metalloproteinases (MMP) play a significant role in periodontal tissue destruction. In this study possible relationship between smoking and gingival tissue expression of gelatinases in chronic periodontitis patients relative to periodontally healthy subjects was investigated. Forty chronic periodontitis patients (20 smokers and 20 non-smokers) and forty periodontally healthy subjects (20 smokers and 20 non-smokers) were enrolled. The clinical periodontal measurements recorded, and gingival tissues harvested after that. After histologic evaluation, matrix metalloproteinases -2 and -9 expressions were analyzed immunohistochemically. In nonsmokers, higher expression of metalloproteinases -2 and -9 detected in chronic periodontitis group compared to the periodontally healthy group. In the smoker chronic periodontitis group, the expression of metalloproteinases-2 was lower than nonsmoker chronic periodontitis group. Statistically significant differences detected between smoker and nonsmoker periodontally healthy groups in metalloproteinases-2 expression. For metalloproteinases-9 expression, smoker chronic periodontitis group has lower values than nonsmoker chronic periodontitis group. In periodontally healthy group smokers showed higher metalloproteinases -9 expressions than non- smokers. Present findings support the role of gelatinases in chronic periodontitis pathogenesis. Based on the current results we conclude that smoking alters the expression of gelatinases in gingival tissues.
Asunto(s)
Periodontitis Crónica/enzimología , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Fumar/efectos adversos , Adolescente , Adulto , Análisis de Varianza , Biopsia , Estudios de Casos y Controles , Estudios Transversales , Femenino , Fibroblastos/enzimología , Encía/enzimología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Adulto JovenRESUMEN
We first discovered human airway trypsin-like protease (HAT) in human mucoid sputum. Precursor HAT (47 kDa), a cell surface type â ¡ transmembrane serine protease, is proteolyzed to mature HAT (27 kDa). Hitherto, HAT has not been detected in other biological fluids except for human sputum. We aimed to clarify whether human saliva contains mature HAT. Trypsin-like protease was isolated from saliva of healthy volunteers by a method adopted for isolation of HAT from sputum using Boc-Phe-Ser-Arg-MCA as the substrate. Biochemical properties of purified protease were similar to those of recombinant HAT (rHAT). HAT concentration in saliva was measured by ELISA, and immunoreactive HAT:total protein ratio (ng/mg) in saliva samples from healthy subjects was similar to that in mucoid sputum. RT-PCR showed that HAT mRNA was expressed in human gingival epithelial cells but not in gingival fibroblasts. Both indirect immunofluorescence and western blotting using monoclonal antibody for α-smooth muscle actin (α-SMA;a myofibroblast marker) showed that HAT enhanced α-SMA fiber expression in gingival fibroblasts. These results indicate that both mucoid sputum and saliva from healthy subjects have similar concentrations of mature HAT, and HAT is related to certain physiological functions and pathological states of myofibroblasts in the oral cavity. J. Med. Invest. 65:258-267, August, 2018.
Asunto(s)
Saliva/enzimología , Serina Endopeptidasas/metabolismo , Actinas/metabolismo , Adulto , Células Cultivadas , Células Epiteliales/enzimología , Femenino , Fibroblastos/enzimología , Encía/citología , Encía/enzimología , Voluntarios Sanos , Humanos , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/genética , Esputo/enzimología , Adulto JovenRESUMEN
BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.
Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Citrulinación , Encía/enzimología , Encía/microbiología , Periodontitis/enzimología , Periodontitis/microbiología , Porphyromonas gingivalis/fisiología , Desiminasas de la Arginina Proteica/metabolismo , Adulto , Artritis Reumatoide/microbiología , Artritis Reumatoide/patología , Exotoxinas/metabolismo , Encía/patología , Humanos , Inflamación/patología , Linfocitos/patología , Persona de Mediana Edad , Periodontitis/genética , Periodontitis/patología , Desiminasas de la Arginina Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In this study, we investigated the promoter DNA methylation (DNAm) status of the MMP-9 and TIMP-1 genes in patients with chronic periodontitis to evaluate disease progression. Using pyrosequencing technology, DNAm levels of MMP-9 and TIMP-1 CpG islands were measured in 88 chronic periodontitis patients and 15 healthy controls. We found a positive correlation between methylation levels of MMP-9 CpG islands and the severity of chronic periodontitis. Methylated CpG islands were also closely associated with the duration of chronic periodontitis. Moreover, female patients exhibited lower methylation levels of MMP-9 but higher methylation levels of TIMP-1 compared with male patients, and the methylation levels of TIMP-1 gradually decreased with age. The findings of gender disparity in the DNAm of MMP-9 and TIMP-1 genes provide novel insights into chronic periodontitis.
Asunto(s)
Periodontitis Crónica/genética , Metaloproteinasa 9 de la Matriz/genética , Regiones Promotoras Genéticas , Inhibidor Tisular de Metaloproteinasa-1/genética , Adulto , Factores de Edad , Estudios de Casos y Controles , Periodontitis Crónica/metabolismo , Islas de CpG , Metilación de ADN , Represión Enzimática , Femenino , Encía/enzimología , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Caracteres Sexuales , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Abstract Smokers have a risk of developing periodontal disease. Matrix metalloproteinases (MMP) play a significant role in periodontal tissue destruction. In this study possible relationship between smoking and gingival tissue expression of gelatinases in chronic periodontitis patients relative to periodontally healthy subjects was investigated. Forty chronic periodontitis patients (20 smokers and 20 non-smokers) and forty periodontally healthy subjects (20 smokers and 20 non-smokers) were enrolled. The clinical periodontal measurements recorded, and gingival tissues harvested after that. After histologic evaluation, matrix metalloproteinases -2 and -9 expressions were analyzed immunohistochemically. In nonsmokers, higher expression of metalloproteinases -2 and -9 detected in chronic periodontitis group compared to the periodontally healthy group. In the smoker chronic periodontitis group, the expression of metalloproteinases-2 was lower than nonsmoker chronic periodontitis group. Statistically significant differences detected between smoker and nonsmoker periodontally healthy groups in metalloproteinases-2 expression. For metalloproteinases-9 expression, smoker chronic periodontitis group has lower values than nonsmoker chronic periodontitis group. In periodontally healthy group smokers showed higher metalloproteinases -9 expressions than non- smokers. Present findings support the role of gelatinases in chronic periodontitis pathogenesis. Based on the current results we conclude that smoking alters the expression of gelatinases in gingival tissues.
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Adulto Joven , Fumar/efectos adversos , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Periodontitis Crónica/enzimología , Biopsia , Inmunohistoquímica , Estudios de Casos y Controles , Estudios Transversales , Análisis de Varianza , Estadísticas no Paramétricas , Fibroblastos/enzimología , Encía/enzimología , Persona de Mediana EdadRESUMEN
Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.
Asunto(s)
Antígenos de Superficie/fisiología , Encía/enzimología , Metaloproteinasa 12 de la Matriz/fisiología , Monocitos/enzimología , Periodontitis/enzimología , Receptores de Superficie Celular/fisiología , Adulto , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , División Celular , Células Cultivadas , Técnicas de Cocultivo , Inhibidores de la Ciclooxigenasa/farmacología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Encía/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inflamación , Queratinocitos/metabolismo , Metaloproteinasa 12 de la Matriz/biosíntesis , Metaloproteinasa 12 de la Matriz/genética , Monocitos/patología , Receptores de Orexina , Periodontitis/patología , Pirazoles/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genéticaRESUMEN
In recent years, nanomaterials have been used in the medical-dental field as new alternative antimicrobial agents. Bismuth subsalicylate (BSS) has been used as an antimicrobial agent, but the effect of BSS in the form of nanoparticles (BSS-nano) as a potential antimicrobial agent has not been tested, in specific against bacteria responsible for periodontal disease. The aim of this study was to evaluate the antibacterial effect of BSS-nano against oral anaerobic bacteria and to assess the safety of BSS-nano by evaluating their cytotoxicity in human gingival fibroblast (HGF-1) cells. BSS-nano were synthesized by laser ablation and were previously physico-chemically characterized using in vitro assays. The antibacterial activity was measured using the tetrazolium-based XTT assay, and cytotoxicity was determined using lactate dehydrogenase (LDH) and MTS assays in HGF-1 cells. Transmission electron microscopy of HGF-1 exposed to BSS-nano was also performed. BSS-nano was shown to have a primary size of 4-22 nm and a polygonal shape. Among the tested bacterial strains, those with a greater sensitivity to BSS-nano (highest concentration of 21.7 µg ml-1) were A. actinomycetemcomitans, C. gingivalis, and P. gingivalis. BSS-nano at a concentration of 60 µg ml-1 showed low cytotoxicity (6%) in HFG-1 cells and was mainly localized intracellularly in acidic vesicles. Our results indicate that the concentration of BSS-nano used as an effective antibacterial agent does not induce cytotoxicity in mammalian cells; thus, BSS-nano can be applied as an antibacterial agent in dental materials or antiseptic solutions.
Asunto(s)
Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Antibacterianos/farmacología , Bismuto/farmacología , Nanopartículas/química , Compuestos Organometálicos/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Salicilatos/farmacología , Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Anaerobiosis/efectos de los fármacos , Anaerobiosis/fisiología , Antibacterianos/química , Bismuto/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Composición de Medicamentos/métodos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Encía/citología , Encía/efectos de los fármacos , Encía/enzimología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Pruebas de Sensibilidad Microbiana , Nanopartículas/ultraestructura , Compuestos Organometálicos/química , Porphyromonas gingivalis/crecimiento & desarrollo , Salicilatos/químicaRESUMEN
BACKGROUND AND OBJECTIVE: No previous study has directly compared the levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) between smokers and individuals with diabetes mellitus (DM) with periodontitis. Therefore, the aim of this study was to evaluate the gene expression of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 in tissues with chronic periodontitis (ChP) of smokers and individuals with type 2 DM. MATERIAL AND METHODS: Gingival biopsies were harvested from: non-smokers and non-diabetic individuals with ChP (n = 18) (ChP group); non-diabetic smokers (≥ 10 cigarettes per day for at least the past 5 years) with ChP (n = 18) (SChP group); non-smoking individuals with type 2 diabetes (glycated hemoglobin levels ≥ 7.5%) and ChP (n = 18) (DMChP group). The tissue levels of mRNA of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 were evaluated by quantitative real-time polymerase chain reaction. RESULTS: The MMP-8 expression was the lowest in the ChP group (p < 0.05). The DMChP group presented increased mRNA levels of MMP-2 and MMP-9, when compared to the SChP group (p < 0.05). MMP-1 expression and the MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-8/TIMP-1, MMP-9/TIMP-1, MMP-1/TIMP-2 and MMP-2/TIMP-2 ratios were higher in the DMChP group than in the ChP and SChP groups (p < 0.05). The DMChP group presented lower mRNA levels of TIMP-1 than the ChP group (p < 0.05). The MMP-8/TIMP-2 ratio was the highest in the SChP group (p < 0.05). CONCLUSION: Uncontrolled type 2 DM upregulates the ratio of MMP/TIMPs in sites with ChP more than smoking, which may contribute to a greater extracellular matrix degradation and periodontal breakdown in DM-related periodontitis.
Asunto(s)
Periodontitis Crónica/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Metaloproteinasas de la Matriz/metabolismo , Fumar/efectos adversos , Adulto , Periodontitis Crónica/enzimología , Periodontitis Crónica/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Encía/enzimología , Encía/metabolismo , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
OBJECTIVE: Endogenous hydrogen sulfide (H2S) has recently been shown to play an important role in inflammation, but the role of endogenous H2S in the human gingival tissue is unknown. The aim of this study was to investigate whether gingiva had enzymes for H2S synthesis, and whether the effect of these enzymes for H2S production changed with periodontal inflammation. DESIGN: Gingival tissues were collected from patients undergoing periodontal operation including gingivitis, moderate chronic periodontitis, severe chronic periodontitis and normal controls. RT-PCR and western blotting were performed to measure mRNA and protein levels of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) for H2S production. Immunohistochemistry was carried out to detect the location of the enzymes. H2S levels and synthesis in gingival tissue were evaluated with modified methylene blue method. RESULTS: The mRNA and protein of CBS and CSE were both expressed in human gingiva and raised significantly in moderate and severe periodontitis compared of that in healthy control. CBS, but not CSE, increased in gingivitis (p<0.05). However, there was no significant difference of H2S level and synthesis among these groups (p>0.05). CONCLUSIONS: Both CBS and CSE were expressed in human gingival tissue. The mRNA and protein levels of CBS and CSE were up-regulated in periodontitis.
Asunto(s)
Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Encía/enzimología , Sulfuro de Hidrógeno/metabolismo , Adolescente , Adulto , Anciano , Periodontitis Crónica/enzimología , Periodontitis Crónica/metabolismo , Cistationina betasintasa/genética , Cistationina gamma-Liasa/genética , Femenino , Encía/patología , Gingivitis/enzimología , Gingivitis/metabolismo , Humanos , Sulfuro de Hidrógeno/análisis , Inmunohistoquímica , Inflamación , Persona de Mediana Edad , Embarazo , ARN Mensajero/análisis , Regulación hacia Arriba , Adulto JovenRESUMEN
OBJECTIVE: Type I plasminogen deficiency (Plgdef) is an uncommon chronic inflammation of mucous membranes. Gingival enlargements usually proceed with progressive periodontal destruction and tooth-loss. Plasmin(ogen)-independent enzymatic mechanisms for fibrin clearance have already been discussed in the literature. Our primary objective was to verify, immunohistochemically, the occurrence of different enzymatic factors involved in tissue breakdown of inflamed compared to healthy gingiva. Secondly, we tried to find out, if these patients have a similar microbiological profile to the patients with known gingivitis and periodontitis. MATERIALS AND METHODS: Immunohistochemical analysis of enzymes elastase, plasminogen (plg), cathepsin G, matrix-metalloproteinase (MMP)-3 and MMP-7 and of glycoprotein fibrinogen were performed with gingival tissues from 3 healthy controls, 8 patients with Plgdef and 3 patients with gingivitis and periodontitis. Furthermore, plaque from 5 patients with plasminogen deficiency were also obtained to determine the microbiological profile. RESULTS: Significantly high numbers of elastase positive leukocytes were detected in all samples. Staining for MMP-3 and MMP-7 was seen in samples with gingivitis and periodontitis with a stronger staining in samples with periodontitis by Plgdef. Fibrinogen was detectable in all samples. Staining for plg was stronger in samples with periodontitis than in other samples. Staining for cathepsin G was weak in gingivitis and periodontitis. Subgingival microbial flora showed elevated colony forming units of Prevotella intermedia/nigrescens, Fusobacterium spp., Eikenella corrodens, Porphyromonas gingivalis and viridans streptococci. CONCLUSION: Strong staining of elastase, MMP-3 and MMP-7 and weak staining of plg in Plgdef samples supports the plasmin(ogen) - independent fibrin clearance. Similar subgingival microbiological flora was observed in periodontitis with Plgdef as in other periodontal diseases. Further investigations should determine the exact pathomechanism and focus on effective treatment methods of this entity.
Asunto(s)
Conjuntivitis/patología , Encía/patología , Gingivitis/patología , Periodontitis/patología , Plasminógeno/deficiencia , Enfermedades Cutáneas Genéticas/patología , Adolescente , Estudios de Casos y Controles , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Encía/enzimología , Encía/microbiología , Gingivitis/enzimología , Gingivitis/microbiología , Humanos , Inmunohistoquímica , Masculino , Periodontitis/enzimología , Periodontitis/microbiología , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
The periodontium undergoes age-related cellular and clinical changes, but the involved genes are not yet known. Here, we investigated age-related genetic changes in gingiva at the transcriptomic level. Genes that were differentially expressed between young and old human gingiva were identified by RNA sequencing and verified by real-time PCR. A total of 1939 mRNA transcripts showed significantly differential expression between young and old gingival tissues. Matrix metalloprotease (MMP) regulation was the top pathway involved in gingival aging. MMP3, MMP9, MMP12, and MMP13 were upregulated in old gingival tissues, concomitantly with interleukin-1 beta (IL1B) expression. In vitro experiments using human gingival fibroblasts (hGFs) showed that MMP12 was upregulated in old hGFs compared to young hGFs. Moreover, the MMP3, MMP9 and IL1B levels were more highly stimulated by infection with the oral bacterium, Fusobacterium nucleatum, in old hGFs compared to young hGFs. Collectively, these findings suggest that, in gingiva, the upregulation of MMP12 may be a molecular hallmark of natural aging, while the upregulations of MMP3, MMM9, and IL1B may indicate externally (e.g., infection)-induced aging. These findings contribute to our understanding of the molecular targets involved in gingival aging.
Asunto(s)
Envejecimiento/metabolismo , Fibroblastos/enzimología , Gelatinasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Encía/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Infecciones por Fusobacterium/enzimología , Fusobacterium nucleatum , Perfilación de la Expresión Génica , Encía/microbiología , Gingivitis/enzimología , Gingivitis/microbiología , Humanos , Interleucina-1beta/biosíntesis , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS: A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS: We have shown that ß-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION: This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.
Asunto(s)
Periodontitis Crónica/enzimología , Inflamación/genética , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/genética , Periodontitis Periapical/genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/genética , Actinas/biosíntesis , Actinas/genética , Periodontitis Crónica/genética , Complejo II de Transporte de Electrones/análisis , Complejo II de Transporte de Electrones/genética , Encía/enzimología , Granuloma/enzimología , Granuloma/genética , Humanos , Inflamación/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/biosíntesis , Granuloma Periapical/enzimología , Granuloma Periapical/genética , Periodontitis Periapical/enzimología , Quiste Radicular/enzimología , Quiste Radicular/genética , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Transcripción GenéticaRESUMEN
Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on ß-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of ß-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for ß-catenin processing. The ß-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3ß were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that ß-catenin fragments were translocated to the nucleus. The accumulation of ß-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the ß-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of ß-catenin and disassociation of the ß-catenin destruction complex by gingipain-dependent proteolytic processing. ß-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Infecciones por Bacteroidaceae/metabolismo , Cisteína Endopeptidasas/metabolismo , Porphyromonas gingivalis/enzimología , Adhesinas Bacterianas/genética , Infecciones por Bacteroidaceae/enzimología , Infecciones por Bacteroidaceae/genética , Infecciones por Bacteroidaceae/microbiología , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/genética , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Cisteína-Endopeptidasas Gingipaínas , Encía/enzimología , Encía/metabolismo , Encía/microbiología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Porphyromonas gingivalis/genética , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , beta Catenina/metabolismoRESUMEN
Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFß in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFß. We previously showed that Src, JNK, and Smad3 mediate TGFß1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFß. In this study, we demonstrated that TGFß1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFß1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFß1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFß1-induced NOX4 expression in HGFs.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/fisiología , Fibroblastos/enzimología , Encía/citología , NADPH Oxidasas/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Acetilcisteína/farmacología , Técnicas de Cultivo de Célula , Células Cultivadas , Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Silenciador del Gen , Encía/enzimología , Sobrecrecimiento Gingival/enzimología , Sobrecrecimiento Gingival/patología , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Naftoquinonas/farmacología , Oxidación-Reducción , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Superóxidos/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: The aim of this study is to evaluate the expression of human telomerase reverse transcription (hTERT) enzyme in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared with healthy individuals. METHODS: A total of 79 individuals consented to participate in the study. The study sample comprised healthy individuals (n = 30), patients with CP (n = 30), and patients with AgP (n = 19). Gingival tissue was collected and evaluated for hTERT expression by Western blot and immunohistochemical methods. Reverse transcription polymerase chain reaction was performed using the gingival crevicular fluid (GCF) samples. RESULTS: The hTERT messenger RNA (mRNA) and protein expression was significantly higher in AgP compared with CP (P <0.001). In GCF, 53.33% of patients with CP and 68.42% of patients with AgP were showing hTERT mRNA expression, but it was not detected in the control group. The AgP tissue showed higher hTERT expression compared with CP (P <0.001). The hTERT mRNA expression did not show a correlation with gingival index (GI), plaque index (PI), probing depth (PD), and clinical attachment loss (AL) in patients with AgP, whereas hTERT protein expression was strongly correlated with GI, PI, PD, and AL in patients with AgP. The protein expression of hTERT shows significant but moderate correlation with GI and AL in patients with CP. CONCLUSION: High expression of hTERT might be associated with periodontal disease progression, suggesting that hTERT could be a potential prognostic marker.
Asunto(s)
Periodontitis Agresiva/enzimología , Periodontitis Crónica/enzimología , Telomerasa/análisis , Adulto , Biomarcadores/análisis , Tejido Conectivo/enzimología , Índice de Placa Dental , Epitelio/enzimología , Femenino , Encía/enzimología , Líquido del Surco Gingival/enzimología , Humanos , Masculino , Persona de Mediana Edad , Pérdida de la Inserción Periodontal/clasificación , Pérdida de la Inserción Periodontal/enzimología , Índice Periodontal , Bolsa Periodontal/clasificación , Bolsa Periodontal/enzimología , ARN Mensajero/análisis , Adulto JovenRESUMEN
AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.
Asunto(s)
Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Polietilenglicoles/farmacología , Ácidos Polimetacrílicos/farmacología , Streptococcus mitis/efectos de los fármacos , Técnicas de Cocultivo , Fibroblastos/citología , Fibroblastos/enzimología , Encía/citología , Encía/enzimología , Humanos , Inflamación/metabolismo , Integrina beta1/metabolismo , Proteína Quinasa C-alfa/metabolismo , Transducción de Señal , Streptococcus mitis/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The present study aims to examine the inhibitory effect of cyclosporin-A (CsA) on periodontal breakdown and to further explore the correlations of CsA-induced attenuation of periodontal bone loss with the expressions of gelatinases (i.e., matrix metalloproteinase [MMP]-2 and MMP-9) and extracellular matrix metalloproteinase inducer (EMMPRIN). METHODS: Forty Sprague-Dawley rats were randomly divided into four groups: 1) control; 2) CsA; 3) ligature (Lig); and 4) ligature plus CsA (Lig + CsA). The CsA group received 10 mg â Kg(-1) â d(-1) CsA for 8 days. The Lig group received silk ligature on selected molars. The Lig + CsA group received silk ligature and CsA treatment. The inhibitory effects of CsA on the ligature-induced periodontal breakdown was examined with microcomputed tomography (micro-CT) and histometric analyses to analyze the amount of attachment loss, crestal bone loss, connective tissue attachment, and the surface area with inflammatory cell infiltration. The effects of CsA on ligature-induced expressions of gelatinases and EMMPRIN in gingival tissues were examined with Western blotting and zymography, respectively. RESULTS: By micro-CT and histology, the Lig + CsA group had significantly more periodontal breakdown than the control and CsA groups but less periodontal breakdown than the Lig group. Consistent results were found for the expressions of gelatinases and EMMPRIN among the groups demonstrating that the Lig + CsA group had significantly less gingival protein expression of gelatinases and EMMPRIN than the Lig group. CONCLUSIONS: CsA inhibited the expressions of gelatinase MMPs and EMMPRIN and partially prevented the periodontal breakdown in ligature-induced experimental periodontitis. The CsA-induced attenuation of periodontal bone loss was strongly correlated positively with the expressions of MMP-2, MMP-9, and EMMPRIN in gingiva.