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OBJECTIVE: The aim of this study was to evaluate the effects of both testosterone depletion and supraphysiological testosterone supplementation in the early phase of an animal cutaneous wound healing model in comparison with the physiological hormonal condition. MATERIAL AND METHODS: Forty rats were distributed into the following four groups: Control, Orchiectomy (OCX), Durateston (Dura) and OCX+Dura. On day 1, the testicles were removed (OCX group) and the rats (Dura group) received a supraphysiological dose (250 mg/kg) of exogenous testosterone weekly. After 15 days a full-thickness excisional skin wound was created in all animals, which was healed by the second intention for 7 days. On day 22, the rats were euthanatized and the wounds were harvested for histopathological evaluation, immunohistochemistry analyses and multiplex immunoassay. One-way ANOVA and post-hoc Tukey tests were performed. RESULTS: It was found that the supraphysiological testosterone level increased extracellular matrix deposition, promoted higher blood vessel formation and reduced wound contraction (p < 0.05). Additionally, it also stimulated PCNA-positive fibroblasts and KGF-positive cells (p < 0.05), while orchiectomy reduced the expression of IL-6 and TNF-α and increased VEGF and PDGF (p < 0.05) . CONCLUSION: In conclusion, the results provide evidence that supraphysiological testosterone supplementation plays a positive role in the early phase of cutaneous wound healing, thus improving granulation tissue maturation through the enhancement of angiogenesis.
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Testosterona , Cicatrización de Heridas , Animales , Suplementos Dietéticos , Modelos Animales de Enfermedad , Tejido de Granulación , Neovascularización Patológica , Ratas , Piel , Testosterona/farmacologíaRESUMEN
The aim was to assess the influence of local application of curcumin-loaded nanoparticles on an experimental model of periodontal repair. Periodontitis was induced by ligatures on both lower first molars of rats. After 15 days, ligatures were removed ("treatment") and animals were randomly allocated to three experimental groups (n = 8/group): (i) 0.05 mg/ml curcumin-loaded nanoparticles, (ii) empty nanoparticles (vehicle control), and (iii) sterile saline (negative control). Experimental treatments were administered locally on days 0, 3, 5, 7, 9, and 11 after ligature removal. Animals were euthanized at 7 and 14 days. Bone repair was assessed by microcomputer tomography (µCT). Histological sections were stained with hematoxylin/eosin (H/E), Picrosirius Red, and Masson's trichrome. Expression of Runx-2 was studied by immunohistochemistry. Gene expression of Itgam, Arg1, and Inos was assessed by RT-qPCR. At 7 days, there was increased gene expression of Itgam and Arg1 and of the relative expression of Arg1/Inos in curcumin-treated animals, but no difference in any other outcomes. At 14 days, curcumin-loaded nanoparticles significantly increased bone repair and collagen content, as well as the number of osteocytes, percentage of extracellular matrix, and expression of Runx2. The results demonstrate that local administration of curcumin-loaded nanoparticles enhanced tissue repair in an experimental model of periodontal repair. Nanoparticle-encapsulated curcumin enhances early post-treatment repair of periodontal tissues.
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Pérdida de Hueso Alveolar , Curcumina , Nanopartículas , Periodontitis , Ratas , Animales , Curcumina/farmacología , Periodontitis/tratamiento farmacológico , Periodontitis/patologíaRESUMEN
Lipid peroxidation (LPO) has been associated with periodontal disease, and the evaluation of malondialdehyde (MDA) in the gingival crevicular fluid (GCF), an inflammatory exudate from the surrounding tissue of the periodontium, may be useful to clarify the role of LPO in the pathogenesis of periodontal disease. We describe the validation of a method to measure MDA in the GCF using high-performance liquid chromatography. MDA calibration curves were prepared with phosphate-buffered solution spiked with increasing known concentrations of MDA. Healthy and diseased GCF samples were collected from the same patient to avoid interindividual variability. MDA response was linear in the range measured, and excellent agreement was observed between added and detected concentrations of MDA. Samples' intra- and interday coefficients of variation were below 6.3% and 12.4%, respectively. The limit of quantitation (signal/noise=5) was 0.03 µM. When the validated method was applied to the GCF, excellent agreement was observed in the MDA quantitation from healthy and diseased sites, and diseased sites presented more MDA than healthy sites (P<0.05). In this study, a validated method for MDA quantitation in GCF was established with satisfactory sensitivity, precision, and accuracy.
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Técnicas de Química Analítica/métodos , Cromatografía Líquida de Alta Presión , Líquido del Surco Gingival/química , Malondialdehído/análisis , Humanos , Peroxidación de Lípido , Enfermedades Periodontales/metabolismo , Enfermedades Periodontales/patologíaRESUMEN
BACKGROUND: Obesity may represent a chronic low-grade inflammation, but there is a lack of long-term longitudinal studies. The aim of this longitudinal study was to evaluate the recurrence of periodontal disease in obese and normal weight patients submitted to scaling and root planing. METHODS: The study included 22 patients who had received periodontal treatment 2 years previously, 13 obese and nine non-obese. The patients were evaluated for anthropometric measurements of body mass index, waist circumference, waist-to-hip ratio, and fat percentage through bioimpedance. The following periodontal parameters were recorded: visible plaque index (VPI), gingival bleeding index (GBI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). The immunological evaluation analyzed the proinflammatory cytokines interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in the gingival crevicular fluid (GCF). RESULTS: Obese and normal weight patients did not differ in relation to the periodontal parameters of VPI, GBI, PD, CAL, or BOP 2 years after completion of the periodontal therapy. Sites with periodontitis in obese individuals showed higher levels of IL-6 and TNF-α in the gingival fluid (P <0.05). CONCLUSION: Obese and normal weight individuals had similar periodontal behaviors, with low recurrence of the periodontal disease; however, obesity was related to increased inflammatory activity in gingival fluid, which may become a risk indicator for future greater recurrence of the disease in the presence of inadequate plaque control.
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Periodontitis Crónica , Enfermedades Periodontales , Periodontitis Crónica/complicaciones , Periodontitis Crónica/terapia , Índice de Placa Dental , Raspado Dental , Estudios de Seguimiento , Líquido del Surco Gingival , Humanos , Estudios Longitudinales , Obesidad/complicaciones , Pérdida de la Inserción Periodontal , Enfermedades Periodontales/complicaciones , Enfermedades Periodontales/terapia , Índice PeriodontalRESUMEN
The immune system detects shifts from homeostasis and eliminates altered cells. However, neoplastic cells can modulate the host response to escape immunosurveillance thereby allowing tumor progression. Head and neck squamous cell carcinoma (HNSCC) is one of the most immunosuppressive cancers but its role in co-opting the immune system to actively promote tumor growth has not been investigated. In this study, we investigated the influence of soluble factors secreted by HNSCC and non-neoplastic epithelial cells on proliferation, apoptosis, activation, cytokine gene expression and phenotypic polarization of immune cells of healthy donors. Then, we determined if the immunomodulation caused by HNSCC-derived soluble products leads to immunosubversion by assessing proliferation, migration and survival of tumor cells exposed to soluble products secreted by modulated immune cells or co-cultured with immune cells. Soluble products from HNSCC inhibited proliferation and cytokine expression in PBMCs, activation of T cells, and polarization of CD4+ towards the Th17 phenotype. These changes co-opted the immune cells to favor cell proliferation, survival and migration of HNSCC. This immunosubversion was observed both indirectly with secreted products and with direct cell-to-cell contact. We conclude that HNSCC-derived secreted products create an immunosuppressive environment that facilitates evasion of tumor cells and subverts the immune cells into a pro-tumoral phenotype.
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BACKGROUND: Arachidonate-5-lipoxygenase (5-LO) activity and increased leukotriene B4 (LTB4) production have been implicated in various inflammatory conditions. Increased production of leukotrienes has been associated with periodontal diseases; however their relative contribution to the tissue destruction is unknown. We used an orally-active specific 5-LO inhibitor to assess its role in inflammation and bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontal disease. METHODS: Periodontal disease was induced in Balb/c mice by direct injections of LPS into the palatal gingival tissues adjacent to the upper first molars 3 times/week for four weeks. Animals were treated with the biochemical inhibitor (2 mg/Kg/day) or the same volume of the vehicle by oral gavage. µCT analysis was used to assess bone resorption. EIA determined leukotriene B4, and ELISAs quantified TNF, IL-12 and IL-10 in the gingival tissues. Histological sections were used for the morphometric analysis (number neutrophils and mononuclear cells). Osteoclasts were counted in TRAP-stained sections. RESULTS: Administration of 5-LO inhibitor effectively reduced the production of LTB4 (23.7% decrease) and significantly reduced TNF and IL-12 levels in the gingival tissues. Moreover, reduction of LTB4 levels in the gingival tissues was associated with a significant decrease in bone resorption and a marked reduction in the number of osteoclasts and inflammatory cells. CONCLUSION: 5-LO activity plays a relevant role in inflammation and bone resorption associated with the LPS model of experimental periodontal disease.
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BACKGROUND: The effect of the interaction between type 2 diabetes and dyslipidemia on inflammation and lipid peroxidation (LPO) has not been assessed. AIM: To investigate whether diabetes coupled with dyslipidemia alters oxidative metabolism leading to increased LPO products and inflammatory status. METHODS: 100 patients were divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetes with dyslipidemia (DM-PC/D), well-controlled diabetes with dyslipidemia (DM-WC/D), normoglycemic individuals with dyslipidemia (NG/D), and normoglycemic individuals without dyslipidemia (NG/ND). Plasma was evaluated for an LPO product (MDA), antioxidant levels and inflammatory cytokines. RESULTS: Diabetics presented significantly higher levels of LPO (p<0.05) and the DM-PC/D had higher levels of proinflammatory cytokines and MDA in the plasma in comparison with normoglycemics (p<0.05). Interestingly IL1-ß, IL-6, and TNF-α in DM-WC/D were not statistically different from those in DM-PC/D. Normoglycemic individuals with dyslipidemia presented significantly increased levels of IL-6 and TNF-α when compared to normoglycemic without dyslipidemia (p<0.05). MDA levels were also positively correlated with the presence of DM complications (r=0.42, p<0.01). CONCLUSIONS: These findings show that dyslipidemia is associated with an increased inflammatory status, even in well-controlled diabetics and in normoglycemics. Our results suggest that lipid metabolism and peroxidation are important for the development of inflammation, which is elevated in several complications associated with diabetes.
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Diabetes Mellitus Tipo 2/complicaciones , Dislipidemias/complicaciones , Inflamación/complicaciones , Peroxidación de Lípido , Adulto , Antioxidantes/metabolismo , Estudios Transversales , Citocinas/metabolismo , Humanos , Persona de Mediana Edad , Estrés Oxidativo , Factor de Necrosis Tumoral alfaRESUMEN
The myeloid differentiation factor 88 (MyD88) plays a pivotal role in Toll-like receptor (TLR)- and interleukin-1 receptor (IL-1R)-induced osteoclastogenesis. We examined the role of MyD88 on p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and nucleotide-binding oligomerization domain (Nod) induction by lipopolysaccharide (LPS) and IL-1 beta, and their effect on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production in bone marrow stromal cell (BMSC). RANKL, Nod1, Nod2, NF-κB, and p38 protein levels were determined by Western blot. Nod2 was stimulated with muramyl dipeptide (MDP) prior to TLR4 stimulation with LPS. MyD88 deficiency markedly inhibited RANKL expression after LPS stimulation and increased OPG messenger RNA (mRNA) production. Also, MyD88 was necessary for NF-κB and p38 MAPK activation. MDP alone did not induce RANKL and OPG expressions; however, when combined with LPS, their expressions were significantly increased (p < 0.05). Our results support that MyD88 signaling has a pivotal role in osteoclastogenesis thought NF-κB and p38 activation. Nod2 and especially Nod1 levels were influenced by MyD88.
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Células Madre Mesenquimatosas/metabolismo , Factor 88 de Diferenciación Mieloide/biosíntesis , Osteoprotegerina/biosíntesis , Ligando RANK/biosíntesis , Receptores de Interleucina-1/biosíntesis , Receptores Toll-Like/biosíntesis , Animales , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/biosíntesis , Proteína Adaptadora de Señalización NOD2/biosíntesis , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: Local invasion of bone is a frequent complication of oral squamous cell carcinoma (OSCC). Development of these osteolytic lesions is mediated by osteoclasts. Receptor activation of NF-κB ligand (RANKL) signaling, counteracted by osteoprotegerin (OPG), regulates osteoclastogenesis. Previous studies in rodent models have demonstrated that inhibition of RANKL decreases tumor growth and lesions within bone. However, the contributory role of OSCC cells to this disease process has yet to be defined. METHODS: RANKL expression was assessed in a panel of OSCC cell lines by qPCR, flow cytometry, and ELISA. Induction of osteoclastogenesis was assessed by co-culture with macrophages or with OSCC-derived conditioned medium. In an animal model of bone invasion, nude mice were injected intratibially with UMSCC-11B cells expressing a RANKL luciferase promoter to detect tumor-derived RANKL activity. Osteolytic lesions were analyzed by X-ray, micro-CT, and histological methods. RANKL expression was assessed in human OSCC tissues by immunohistochemistry. RESULTS: We demonstrated that OSCCs express varied levels of all RANKL isoforms, both membrane-bound and soluble RANKL. Both co-culture and treatment with OSCC-conditioned media induced osteoclastogenesis. In mice, we demonstrated human RANKL promoter activity during bone invasion. Over the course of the experiment, animals suffered osteolytic lesions as RANKL-driven luciferase expression increased with time. After 8weeks, human-derived RANKL was detected in areas of bone resorption by immunohistochemistry. Similar epithelial RANKL expression was detected in human OSCC tissues. CONCLUSION: These data demonstrate the ability of OSCCs to produce RANKL, directly altering the tumor microenvironment to increase osteoclastogenesis and mediate local bone invasion.
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Resorción Ósea , Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/metabolismo , Osteólisis , Ligando RANK/metabolismo , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/fisiopatología , Línea Celular Tumoral , Técnicas de Cocultivo , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Neoplasias de la Boca/fisiopatología , Osteoclastos/citología , Reacción en Cadena de la Polimerasa , Tomografía Computarizada por Rayos XRESUMEN
Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.
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Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/uso terapéutico , Terapia Molecular Dirigida/métodos , Enfermedades Periodontales/terapia , Transducción de Señal/efectos de los fármacos , Biopelículas , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Quinasas Janus/inmunología , Quinasas Janus/metabolismo , Proteínas Quinasas Activadas por Mitógenos/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Enfermedades Periodontales/etiología , Enfermedades Periodontales/inmunología , Factores de Transcripción STAT/inmunología , Factores de Transcripción STAT/metabolismoRESUMEN
This study evaluated the effects of bFGF and TGF-beta, individually and combined, on cell proliferation and collagen metabolism. Primary human periodontal ligament cells were stimulated with two concentrations (1 and 10 ng/ml) of each growth factor, both individually and combined. Proliferation was determined by a commercial biochemical assay. Real time RT-PCR determined gene expression of MMP-1 and -2, collagen types I and III, TIMP-1, -2 and -3. Autocrine effects on synthesis of bFGF and TGF-beta were evaluated by ELISA. Only TGF-beta, either isolated or associated with bFGF, significantly increased cell proliferation. TGF-beta had anabolic effects, increasing expression of type I and III collagen as well as of TIMPs, whereas bFGF had opposite effects. When bFGF and TGF-beta were associated, the anabolic effects prevailed. Synthesis of TGF-beta was induced only by the association of lower concentrations of the growth factors, whereas there was a dose-dependent production of bFGF. It is concluded that bFGF had a predominantly catabolic effect, and TGF-beta exerted an anabolic effect on hPDL cells.