RESUMEN

OBJECTIVES: Various sealant materials have been suggested to decrease decalcification during orthodontic treatment. However, only a few in vitro studies on the cytotoxicity of resinous pit and fissure sealants have been published, and to the best of our knowledge no similar studies are available for the enamel sealants used in orthodontics. Therefore, we aimed to characterize the possible adverse effects of enamel sealants, especially on the gingival epithelium. METHODS: Organotypic cultures of the human gingival mucosa were used to assess the possible impact of six enamel sealants. Differentiation and apoptosis were determined by immunofluorescent staining. The pro-inflammatory cytokines IL-1ß and IL-6 were quantified by ELISA. Cytotoxicity was measured using MTS assays in monolayer cultures of human gingival fibroblasts. Leaching of monomers from enamel sealants was quantified using HPLC. RESULTS: The differentiation of the organotypic gingival mucosa remained unaffected. All under-cured and several standard-cured sealants (Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal®) significantly induced apoptosis in the organotypic model. Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal® caused a significant induction of pro-inflammatory cytokines. Reducing curing time had an influence on cytotoxicity in monolayer cultures of primary human oral cells. All resin-based sealants leached monomers. SIGNIFICANCE: Enamel sealants might exert adverse effects on the gingival epithelium. Due to the vicinity of the enamel sealant to the gingival epithelium, and the large surface area of applied sealants, these materials should be carefully applied and sufficiently cured.

Asunto(s)

Encía/efectos de los fármacos , Modelos Biológicos , Selladores de Fosas y Fisuras/efectos adversos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Encía/citología , Encía/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo

RESUMEN

OBJECTIVES: Root resorptions due to a reduced repair function of cementoblasts are common side effects during orthodontic tooth movement (OTM). The mechanisms, which lead to an impaired cementoblast function, are not fully understood. Therefore, we aimed to investigate changes in the gene expression of cementoblasts during mechanical stimulus under inflammatory conditions. MATERIALS AND METHODS: Human primary cementoblasts (HPCB) were exposed to compression for 6 h or stimulation with IL-1ß for 96 h and subsequent 6 h compression. Genome-wide expression analysis was performed using microarray analysis. Prominent gene expression alterations (COX2, AXUD1, FOSB, CCL2, IFI6, and PTGES) were verified by quantitative RT-PCR (qRT-PCR) in two HPCB populations. A caspase 3/7 activity assay was used to determine caspase-3 and caspase-7 activity in stressed cells. RESULTS: Gene expression cluster analysis revealed apoptosis as an important process induced under both conditions. Apoptosis (pro- and anti-apoptotic) related gene expression was most relevant after pro-inflammatory stimulation and compression. qRT-PCR analysis confirmed significant up-regulation of COX2, AXUD1, and FOSB in both HPCB populations after compression, while selected genes significantly increased after pro-inflammatory stimulation and compression. Compression of cementoblasts increased caspase. The combination of pro-inflammatory stimulation and compression led to a slightly smaller increase of caspase activity. CONCLUSIONS: Gene ontology analysis showed that compressed HPCB up-regulate genes that are associated with apoptosis. Combining compression with a pro-inflammatory stimulus (IL-1ß) augmented the positive regulation of apoptosis-related pathways. The induction of apoptosis related gene expression (pro- and anti-apoptotic genes) in cementoblasts suggests an involvement of apoptosis in cementoblast regulation during OTM. CLINICAL RELEVANCE: As apoptosis is induced in HPCB after compression and inflammation, it is conceivable that HPCB cell death might contribute to root resorptions due to a loss of repair activity of cementoblasts. Further studies should be conducted to clarify the implication of the identified genes on root resorptions in order to develop therapeutic strategies to prevent a shortening of roots.

Asunto(s)

Cemento Dental/metabolismo , Perfilación de la Expresión Génica , Interleucina-1beta/administración & dosificación , Humanos

RESUMEN

AIM: The aim of this study was to investigate the response of primary human cementoblasts to conditions as they occur on the pressure side during orthodontic tooth movement. METHODS: In our previous study, the cementoblasts were characterized using markers for osteoblastogenic differentiation and the cementoblast-specific marker CEMP-1. Initially, primary human cementoblasts were compressed for 1 h, 4 h, and 6 h (30 g/cm(2)). In the second experiment, the cementoblasts were stimulated with interleukin (IL)-1ß for 24 h and for 96 h with 1 ng/ml and 10 ng/ml and subsequently compressed for 1 h and 6 h. Changes in mRNA expression for receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), and cyclooxygenase-2 (COX-2) were measured by quantitative real-time polymerase chain reaction (RT-PCR). RANK and RANKL were also examined by immunocytochemical staining at the protein level. RESULTS: Compression (30 g/cm(2)) led to a significant increase in RANKL expression after 6 h. OPG expression in compressed cementoblasts was significantly reduced after 1 h. RANK remained unchanged during the course of the experiment. Stimulation with IL-1ß induced RANKL and OPG expression. However, IL-1ß-dependent induction of RANKL was more prominent than the induction of OPG, leading to a (significant) increase in the RANKL/OPG ratios. The expression of RANK remained unchanged after 24 h of stimulation with IL-1ß and decreased significantly after 96 h. Compression of the prestimulated cells resulted in a further increase in RANKL expression significant after 6 h. OPG and RANK expression remained unchanged compared to the unstimulated sample. COX-2 increased significantly after both compression and stimulation with IL-1ß. Combined stimulation and compression resulted in a significant further increase after 6 h compared to IL-1ß stimulation alone. CONCLUSION: Primary human cementoblasts in vitro express increased levels of RANKL, in particular during the combination of inflammation and compression. The increase in RANKL expression is not compensated by an increase in OPG expression. The induction of RANKL expression was associated with a significant increase in COX-2 expression. Since RANKL attracts osteoclasts, its increase might be associated with the progression of root resorption. The in vitro alterations in cementoblasts we observed may be indicators of cellular mechanisms that lead to the increased root resorption during orthodontic treatment.

Asunto(s)

Cemento Dental/fisiología , Interleucina-1beta/metabolismo , Mecanotransducción Celular/fisiología , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Células Cultivadas , Fuerza Compresiva/fisiología , Humanos , Presión , Estrés Mecánico , Regulación hacia Arriba

RESUMEN

During orthodontic tooth movement, the application of adequate orthodontic forces allows teeth to be moved through the alveolar bone. These forces are transmitted through the periodontal ligaments (PDL) to the supporting alveolar bone and lead to deposition or resorption of bone, depending on whether the tissues are exposed to a tensile or compressive mechanical strain. Fibroblasts within the PDL (PDLF) are considered to be mechanoresponsive. The transduction mechanisms from mechanical loading of the PDLF to the initiation of bone remodeling are not clearly understood. Recently, members of the ephrin/Eph family have been shown to be involved in the regulation of bone homeostasis. For the first time, we demonstrate that PDLF exposed to tensile strain induce the expression of ephrin-B2 via a FAK-, Ras-, ERK1/2-, and SP1-dependent pathway. Osteoblasts of the alveolar bone stimulated with ephrin-B2 increased their osteoblastogenic gene expression and showed functional signs of osteoblastic differentiation. In a physiological setting, ephrin-B2-EphB4 signaling between PDLF and osteoblasts of the alveolar bone might contribute to osteogenesis at tension sites during orthodontic tooth movement.

Asunto(s)

Efrina-B2/biosíntesis , Fibroblastos/metabolismo , Osteogénesis/fisiología , Ligamento Periodontal/metabolismo , Estrés Fisiológico/fisiología , Diente/metabolismo , Regulación hacia Arriba/fisiología , Adolescente , Adulto , Diferenciación Celular/fisiología , Células Cultivadas , Niño , Fibroblastos/citología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Masculino , Mecanotransducción Celular/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Movimiento/fisiología , Osteoblastos/citología , Osteoblastos/metabolismo , Ligamento Periodontal/citología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor EphB4/metabolismo , Diente/citología

RESUMEN

On the mechanistic level, response of periodontal fibroblasts permanently exposed to mechanical strain forces in vivo still lacks in clarity. Therefore, we first investigated putative strain modulation of proteins by combined 1D gel electrophoresis-based protein profiling and electrospray tandem mass spectrometry (ESI-MS). Thereafter, the exponential-modified protein abundance index (emPAI) identified strain modulation of cytoskeleton-associated molecules, including decrease in talin and microtubule-associated protein 4 (MAP4), and significant increase in myosin IC (Myo IC), the latter ones regulated by Ca(2+). These findings were corroborated by western blotting (WB) and indirect immunofluorescence (IIF). Regarding the dual function of Myo IC as actin-based cytoplasmic motor protein and nuclear transcription factor NM1, WB and IIF revealed inverse correlation for Myo IC and NM1. During strain application, cytoplasmic increase of Myo IC was counteracted by nuclear NM1 deprivation, the latter coinciding with a decline in RNA quantity. Independent on strain, cytoplasmic Myo IC and nuclear NM1 abundance could be abrogated by the Ca(2+) channel blocker nifedipine, suggesting Ca(2+) dependency of cytoplasmic and/or nuclear Myo IC/NM1 expression. Mechanistically, we conclude that, application of strain appears as causative for the decline in RNA by impacting NM1, thereby indicating the possible role of NM1 in RNA synthesis.

Asunto(s)

Calcio/metabolismo , Fibroblastos/metabolismo , Miosina Tipo I/fisiología , ARN/biosíntesis , Estrés Mecánico , Factores de Transcripción/fisiología , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Nifedipino/farmacología , Proteoma/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Talina/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated. RESULTS: In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-kinases p42/44, and p38 stress kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits beta3 and beta1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit alpha3, MMP-12, MAP-kinases and associated kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis. CONCLUSIONS: Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-kinases p42/44, and p38 stress kinase in conjunction with the amount of MMP-13, and integrin subunits beta1 and beta3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.

Asunto(s)

Metaloproteinasa 13 de la Matriz/metabolismo , Mecanotransducción Celular , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ligamento Periodontal/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adolescente , Niño , Regulación hacia Abajo , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Ligamento Periodontal/citología , Fosforilación , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética

RESUMEN

Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

Asunto(s)

Células Epiteliales/citología , Etanol/farmacología , Fibroblastos/citología , Encía/citología , Queratinocitos/citología , Ingeniería de Tejidos/métodos , Cadherinas/genética , Cadherinas/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo/métodos , Colágeno Tipo IV/metabolismo , Células Epiteliales/metabolismo , Epitelio , Fibroblastos/metabolismo , Expresión Génica/genética , Regulación de la Expresión Génica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Queratina-14/genética , Queratina-14/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Laminina/metabolismo , Vimentina/genética , Vimentina/metabolismo

RESUMEN

There is growing evidence that apoptosis involves the nuclear transcription factor NF-kappaB in conjunction with related genes. However, in the context of mechanical orthodontic forces, force-sensing target genes assigned to pathways of NF-kappaB and apoptosis have not been fully characterised. To contribute to the identification of putative target genes, we used cDNA arrays specific for NF-kappaB and apoptotic pathways and analysed elevated gene expression in primary human periodontal ligament fibroblasts (PDL-F) after a 6 h application of mechanical force. Among several identified genes (including several caspases), interleukin-1 beta (IL-1 beta) and NF-kappaB displayed significantly higher expression on the NF-kappaB array, whereas higher expression was obtained for BCL2-antagonist of cell death (BAD), member 6 of the TNF-receptor superfamily (FAS) and CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD) on the apoptosis array. Based on a defined cut-off level of a more than 1.5-fold higher expression, this significance in elevated gene expression was corroborated by reverse transcription/polymerase chain reaction (RT-PCR). Here, semi-quantitative (sq) PCR revealed a more pronounced elevation of mRNA gene expression in PDL-F after 6 h of stretch, when compared with 12 h. Moreover, the elevation after 6 h as observed by sq-PCR was convergent with quantitative PCR (q-PCR). q-PCR yielded levels of 5.8-fold higher relative gene expression for IL-1 beta and 1.7-fold for NF-kappaB, whereas that computed for BAD indicated a 5.2-fold, for CRADD a 2.1-fold and for FAS a 2.0-fold higher expression. The data obtained from the expression analysis thus indicate a stretch-induced transcriptional elevation of genes assigned to the NF-kappaB and apoptotic pathways. This elevation may render them target candidates for being addressed by mechanical orthodontic forces.

Asunto(s)

Fibroblastos/metabolismo , FN-kappa B/genética , Ligamento Periodontal/metabolismo , Estrés Mecánico , Apoptosis , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ligamento Periodontal/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Resistencia a la Tracción

RESUMEN

We employed topographical patterning to analyze early keratinocyte differentiation on top of microfabricated pillar arrays. Fibronectin immobilized on pillar "heads" yielded a nucleus-associated granular keratin 1 (K1) pattern in immortalized human gingival keratinocytes (IHGK) at pillar interspaces of 14 mum. Decreasing distances of 11and 8 mum revealed cytoplasmic extension of the early differentiation marker K1 on poly(dimethylsiloxane) (PDMS) pillars. The most extensive cytoplasmic K1 protein distribution noted at the smallest pillar scale coincided with higher ratios of K1 mRNA gene transcription. These experiments suggest that early keratinocyte differentiation was governed by the topographical characteristics of the pillar pattern. Moreover, they form the basis to study cell functions such as differentiation in a defined topologically structured environment.

Asunto(s)

Queratinocitos/citología , Nanotecnología/métodos , Materiales Biocompatibles , Adhesión Celular , Diferenciación Celular , Supervivencia Celular , Dimetilpolisiloxanos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queratina-1/metabolismo , Queratinocitos/metabolismo , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Siliconas , Propiedades de Superficie

RESUMEN

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.

Asunto(s)

Células Epiteliales/metabolismo , Encía/citología , Queratinocitos/metabolismo , Fenotipo , Animales , Transformación Celular Viral , Células Cultivadas , Técnicas de Cocultivo , Femenino , Encía/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos/citología , Citometría de Barrido por Láser , Ratones , Ratones Desnudos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética

RESUMEN

OBJECTIVES: It has been demonstrated that extracellular matrix molecules are involved in cyclosporine-induced gingival overgrowth (GO). However, for many of these molecules, it remains unclear whether their abundance is modulated on the protein and gene expression level. MATERIAL AND METHODS: To contribute to this clarification, we have analysed the protein and mRNA expression of type-I collagen (COL1) and decorin (DC) in native specimens obtained from five patients with GO, and matched normal tissue using indirect immunofluorescence (IIM), in situ hybridization (ISH) and quantitative polymerase chain reaction (PCR). RESULTS: IIF revealed a largely co-localized although remarkably increased abundance for COL1 and DC in GO. This increase coincided with an up-regulated gene expression observed for both molecules, as detected by ISH and quantitative PCR. CONCLUSIONS: Analysis of our data clearly demonstrates elevated levels for COL1 and DC and shows for the first time in native human tissue that involvement of these genes in GO is not confined to the protein level but also includes the transcriptional level.

Asunto(s)

Colágeno Tipo I/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Sobrecrecimiento Gingival/metabolismo , Proteoglicanos/biosíntesis , Adulto , Estudios de Casos y Controles , Colágeno Tipo I/genética , Ciclosporina/efectos adversos , Decorina , Proteínas de la Matriz Extracelular/genética , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Sobrecrecimiento Gingival/inducido químicamente , Sobrecrecimiento Gingival/genética , Humanos , Inmunosupresores/efectos adversos , Hibridación in Situ , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Proteoglicanos/genética , ARN Mensajero/análisis , Transcripción Genética , Regulación hacia Arriba , Vimentina/biosíntesis

RESUMEN

In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO.

Asunto(s)

Ciclosporina/efectos adversos , Sobrecrecimiento Gingival/enzimología , Inmunosupresores/efectos adversos , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloendopeptidasas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/enzimología , Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Sobrecrecimiento Gingival/inducido químicamente , Sobrecrecimiento Gingival/patología , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 10 de la Matriz , Metaloendopeptidasas/genética , Persona de Mediana Edad , Inhibidor Tisular de Metaloproteinasa-1/genética

RESUMEN

A case of necrotizing ulcerative periodontitis (NUP), the most severe inflammatory periodontal disorder caused by plaque bacteria, is shown. Clinically, the gingiva showed distinct signs of ulceration, and radiography revealed horizontal bone loss. Indirect immunofluorescence, carried out on frozen sections of tissue specimens obtained from the NUP lesion, exhibited clear expression of atypical keratin K19, particularly in basal cells, when compared to noninflamed gingiva. Moreover, NUP tissue showed extensive intraepithelial abundance for the basement membrane component laminin-1/10 and the extracellular matrix molecule tenascin. Strong expression of integrin subunit alphav and matrix metalloproteinase-13 in conjunction with interleukin 1-beta further discriminated NUP gingival epithelium from normal tissue. The results suggest that NUP is associated with changes in the expression and topography of the analyzed molecules in the gingival epithelium, which in turn may reflect the fast progression of the disease.

Asunto(s)

Encía/inmunología , Gingivitis Ulcerosa Necrotizante/inmunología , Adulto , Animales , Bovinos , Técnica del Anticuerpo Fluorescente , Gingivitis Ulcerosa Necrotizante/terapia , Humanos , Queratinas/análisis , Laminina/análisis , Masculino

RESUMEN

By using raft cultures of the polyclonal HaCaT cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference, we investigated the effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis. In comparison to conventional monolayer cultures this model system allows analysis of apoptosis under more tissue-like conditions by mimicking the stratified organization of a normal surface epithelium. Apoptosis was triggered either by FasL or TRAIL. Execution of the death program was checked at early and late stages by monitoring procaspase-3 cleavage and DNA fragmentation, respectively. Rafts of E5-expressing keratinocytes were completely protected from apoptosis and showed a background of apoptotic cells as low as the untreated cultures. In contrast, the HaCaT/pMSG cultures revealed a dramatic increase in apoptotic cells upon ligand treatment throughout the epithelial compartment. We conclude that the presence of the HPV-16 E5 protein in our tissue-like model prevents FasL- or TRAIL-mediated apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Queratinocitos/citología , Glicoproteínas de Membrana/farmacología , Proteínas Oncogénicas Virales/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Caspasas/fisiología , Células Cultivadas , Fragmentación del ADN , Proteína Ligando Fas , Humanos , Ligando Inductor de Apoptosis Relacionado con TNF

RESUMEN

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.

Asunto(s)

Células Epiteliales/efectos de los fármacos , Etanol/farmacología , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Proteínas Represoras , Animales , División Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Trasplante de Células , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/trasplante , Fibroblastos/citología , Fibroblastos/metabolismo , Encía/citología , Humanos , Immunoblotting , Queratina-14 , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinas/biosíntesis , Masculino , Ratones , Ratones Desnudos , Microscopía Confocal , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/fisiología , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus , Trasplante Heterólogo , Vimentina/biosíntesis

RESUMEN

Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.

Asunto(s)

Molécula 1 de Adhesión Intercelular/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Periodontitis/terapia , Secuencia de Bases , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Expresión Génica , Gingivitis/genética , Gingivitis/fisiopatología , Gingivitis/terapia , Humanos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/fisiología , Interleucina-1/fisiología , Mucosa Bucal/fisiopatología , Oligodesoxirribonucleótidos Antisentido/genética , Periodontitis/genética , Periodontitis/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo