Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Antibacterial and cytotoxic evaluation of copper and zinc oxide nanoparticles as a potential disinfectant material of connections in implant provisional abutments: An in-vitro study.

OBJECTIVE: This study evaluates the antibacterial activity against mono and multispecies bacterial models and the cytotoxic effects of zinc oxide and copper nanoparticles(ZnO-NPs/Cu-NPs) in cell cultures of human gingival fibroblasts(HGFs). DESIGN: The antibacterial activities of ZnO-NPs and Cu-NPs against 4 bacteria species were tested according to their minimum inhibitory concentrations(MICs) and against mature multispecies anaerobic model by spectral confocal laser scanning microscopy. The viabilities and cytotoxic effects of ZnO-NPs and Cu-NPs to HGFs cell cultures were tested by MTT, LDH assays, production of ROS, and the activation of caspase-3. The results were analyzed using one-way ANOVA followed by Tukey tests, considering p < 0.05 as statistically significant. RESULTS: For all strains, MICs of ZnO-NPs and Cu-NPs were in the range of 78.3 µg/mL-3906 µg/mL and 125 µg/mL-625 ug/mL, respectively. In a multispecies model, a significant decrease in the total biomass volume(µ3) was observed in response to exposure to 125 µg/mL of each NPs for which there was bactericidal activity. Significant differences were found between the volumes of viable and nonviable biomass exposed to nanostructures with Cu-NPs compared to ZnO-NPs. Both NPs induced mitochondrial dose-dependent cytotoxicity, ZnO-NPs increases LDH release and intracellular ROS generation. Cu-NPs at a concentration of 50 µg/mL induced production of cleaved caspase-3, activating the apoptotic pathway early and at low doses. CONCLUSIONS: After 24 h, ZnO-NPs are biocompatible between 78-100 µg/mL and Cu-NPs below 50 µg/mL. Antibacterial activity in a monospecies model is strain dependent, and in a multispecies model was a lower doses after 10 min of exposure.

Evaluation in vitro of cytotoxicity of dentin desensitizers on human gingival fibroblasts / Evaluación in vitro de la citotoxicidad dedesensibilizantes dentinarios en fibroblastos gingivales humanos

The purpose of this study is to compare the cytotoxic effect of threematerials, which have been used for treating dental hypersensitivity. Materialand method: In vitro study. Clinpro (3M Co, St. Paul, MN. USA), Seal & Protect(Dentsply, DeTrey GmbH. Germany) and UltraEZ (Ultradent Products,Inc., S. South Jordan UT. USA) were used at concentrations of 0.1, 0.05, 0.01 and 0.001g/ml on human gingival fibroblasts. Furthermore, Clinpro and Seal & Protect were applied to this cell culture as polymerized disks. Toxicity was assessed at 24 and 48 hours by the use of the cell viability assay (MTT). Statistical analysis for cell viability was performed using two-way ANOVA and Tukey’s post hoc test. Statistical significance was set at 5 percent. Results: Seal & Protect and Clinpro were found to be highly toxic at 24 and 48 hours, reaching 70 percent toxicity at concentrations over 0.01g/ml. Seal & Protect and Clinpro polymerized disks were toxic at 24 and 48 hours. UltraEZ showed an increased between 46 percent and 67 percent in cell viability at 24 hours and between 8 percent and 45 percent at 48 hours. Statistical analysis showed differences between these three desensitizers when comparing concentration and control group (p<0.05). Discussion: UltraEZ did not have a cytotoxic effect and may be considered a compatible and safe material, whereas polymerized and non-polymerized Clinpro and Seal & Protect should be used with caution...

Introduccion: El proposito de este estudio es comparar el efecto citotoxico de tres materiales que se han utilizado para el tratamiento de la hipersensibilidad dental. Material y metodo: Estudio in-vitro. Los desensibilizantes dentinarios Clinpro (3M ESPE), Seal&Protect (Dentsply) yUltraEZ (Ultradent) fueron utilizados a concentraciones de 0,1; 0,05; 0,01 y 0,001 g/ml sobre cultivos celulares de fibroblastos gingivales humanos. Ademas, Clinpro y Seal&Protect se aplicaron a este cultivo celular como discos polimerizados. La toxicidad se evaluo a 24 y 48 horas mediante ensayo de viabilidad (MTT). El analisis estadistico para la viabilidad celular se realizo mediante ANOVA de dos vias seguido de analisis Tukey. La significancia estadistica se fijo al 5 por ciento. Resultados: Clinpro y Seal&Protect resultaron ser altamente toxicos a las 24 y 48 horas, alcanzando un 70 por ciento de toxicidad aconcentraciones superiores de 0,01 g/ml. Los discos polimerizadosde Clinpro y Seal&Protect fueron toxicos a 24 y 48 horas. UltraEZ produjo un aumento de la viabilidad celular entre un 46 por ciento y 67 por ciento a las 24 horas y entre un 8 por ciento y 45 por ciento a las 48 horas. El analisis estadistico mostro diferencias entreestos tres desensibilizantes al comparar la concentracion y su grupo control (p<0,05). Discusion: UltraEZ no tuvo efecto citotoxico y puede ser considerado como un material compatible y seguro para ser utilizado, mientras que Clinpro y Seal&Protect en su estado polimerizado y no polimerizado deberian ser utilizados con precaucion...

Bioregulation of kallikrein-related peptidases 6, 10 and 11 by the kinin B1 receptor in breast cancer cells.

The sera of patients with breast cancer have higher levels of des[Arg(9)]bradykinin, a kinin B1 receptor (B1R) agonist, than that from healthy individuals. Stimulation of breast cancer cells with the analog Lys-des[Arg(9)]bradykinin causes release of metalloproteinases-2 and -9 and increases cell proliferation. We examined the possibility that breast cancer cells, in addition to B1R, express the kinin-forming protease true tissue kallikrein (KLK1) and the endogenous proteins termed kininogens from which kinins are enzymatically released. Furthermore, we investigated whether stimulation of breast cancer cells with a B1R agonist would modify the cellular levels of KLK6, KLK10 and KLK11, three kallikrein-related peptidases with a still poorly-understood biological role in breast cancer. We found that breast cancer cells expressed KLK1 and kininogens, and that stimulation of estrogen-sensitive breast cancer cells with the B1R agonist produced down-regulation of KLK10 (a protease associated with growth suppression) but up-regulation of KLK11 and KLK6 (peptidases related to increased cell proliferation and invasiveness, respectively). Furthermore, we showed that the B1R agonist acts as a functional stimulus for the secretion of KLK1 and KLK6, an event relevant for kinin production and cell invasion, respectively.

Therapeutic potential of a low-cost device for wound healing: a study of three cases of healing after lower-extremity amputation in patients with diabetes.

Diabetic foot ulcers constitute a tremendous challenge for patients, caregivers, and health care systems. The high incidence and high financial costs associated with their treatment have transformed them in a health and economic worldwide problem. The increase in population life expectancy and lifestyle changes have facilitated the spreading of diabetes, rising diabetic foot ulcer incidence. Only 60%-80% of the patients achieve healing of ulcers, and the incidence of a second ulcer, in the same or different site of the foot that has had a previous ulcer, is approximately 50% in 2-5 years. In addition, ulcers with duration longer than 4 weeks are commonly associated with bad results in healing and an increased risk of amputation. Three patients with type 2 diabetes mellitus have been subjected to treatment with NL.1.2, a low-cost, biocompatible solid device that presented pro-angiogenic properties. The selected patients had undergone amputation, and their wounds, classified as Wagner II, did not show a significant progress in healing after a period of 2-5 months before treatment with NL.1.2. Complete closure of their wounds was achieved in 42-60 days.

Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.