RESUMEN
BACKGROUND: Gingivitis is driven by plaque accumulation and, if left untreated, can progress to irreversible periodontitis. For many, the mechanical action of toothbrushing does not achieve adequate plaque control. The aim of this study was to investigate whether twice-daily use of a toothpaste containing 0.2% high molecular weight (HMW) sodium hyaluronate with 67% sodium bicarbonate and 0.221% sodium fluoride (experimental toothpaste) could improve gingival health compared with a regular fluoride toothpaste (negative control). The study also assessed whether the experimental toothpaste could provide additive gingival health benefit over a toothpaste containing only 67% sodium bicarbonate and 0.221% sodium fluoride (positive control). METHODS: This was a single-center, examiner-blinded, randomized, clinical study in healthy adults with mild-to-moderate gingivitis. At baseline, after abstaining from toothbrushing for 12 h, prospective participants underwent oral soft tissue (OST) and oral hard tissue examination followed by assessments for gingival inflammation (Modified Gingival Index [MGI]), gingival bleeding (Bleeding Index [BI]), and supra-gingival plaque (Turesky Plaque Index [TPI]). Eligible participants were stratified by gender and baseline number of bleeding sites (low: <45; high: ≥45 bleeding sites). Following randomization, participants underwent prophylactic dental treatment. Participants received a full OST examination, MGI, BI and TPI assessments after 3 days, 1, 2 and 6 weeks of product use. RESULTS: In total, 110 participants were screened for study entry and all were randomized to receive one of three toothpastes (experimental: sodium hyaluronate, sodium bicarbonate, sodium fluoride; positive control: sodium bicarbonate, sodium fluoride; negative control: regular fluoride toothpaste). For all measures, significant improvements were observed in participants receiving either sodium bicarbonate-containing toothpaste (experimental or positive control) compared with the regular fluoride toothpaste (negative control) at week 6. No significant difference was observed in any assessment or visit comparing the experimental toothpaste with the positive control. CONCLUSIONS: Both the experimental and the positive control toothpastes demonstrated clinically relevant improvements in gingival health compared with a regular fluoride toothpaste (negative control). However, no additional gingival health improvement was observed for the experimental toothpaste compared with the positive control, therefore, no additional gingival health benefit can be attributed to the inclusion of sodium hyaluronate in this formulation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04737538 (04/02/2021).
Asunto(s)
Placa Dental , Gingivitis , Adulto , Humanos , Pastas de Dientes/uso terapéutico , Fluoruro de Sodio/uso terapéutico , Bicarbonato de Sodio/uso terapéutico , Ácido Hialurónico/uso terapéutico , Fluoruros/uso terapéutico , Estudios Prospectivos , Gingivitis/prevención & control , Gingivitis/tratamiento farmacológico , Placa Dental/prevención & control , Índice de Placa Dental , Sodio/uso terapéutico , Método Doble CiegoRESUMEN
OBJECTIVE: The objective of this study was to investigate the antigingivitis and antiplaque treatment effect of a toothpaste containing 67% w/w sodium bicarbonate, at the individual tooth site, tooth region and whole mouth (overall) level, by way of a pooled analysis of data from similarly designed clinical trials. METHODS: Six randomized controlled GSKCH clinical trials, 12-24 weeks in duration, were selected based on pre-specified criteria which included access to patient level data, pre-treatment dental prophylaxis, >20 bleeding sites and mild-moderate gingivitis at screening and use of 67% sodium bicarbonate toothpaste and non-sodium bicarbonate (regular) toothpaste (negative control) for ≥4 weeks. Efficacy outcomes comprised plaque index (TPI), modified gingival index (MGI) and gingival bleeding (bleeding index (BI), number of bleeding sites (BS)). Treatment comparisons were made using ANCOVA for whole mouth (overall) scores and by tooth site region (facial overall, lingual overall; margin/body overall, facial margin/body, lingual margin/body; papillae/interproximal overall, facial papillae/interproximal, lingual papillae/interproximal). Pooled data for BI, MGI, and TPI at individual tooth sites was plotted as a mouthmap to summarize treatment response (change from baseline) by tooth site, at the 24-week timepoint. RESULTS: For all measures, whole mouth and for all tooth regions at all post-treatment timepoints, significant (p < 0.001) differences in favour of the 67% sodium bicarbonate toothpaste compared with control were observed. At the 24-week timepoint, facial regions demonstrated greater improvements than lingual regions, with the greatest between treatment improvement seen for the facial-papillae regions for bleeding (BS/BI), facial-margin regions for MGI and facial-body regions for TPI. All individual tooth sites demonstrated numerically greater reductions from baseline for the 67% sodium bicarbonate toothpaste than the control toothpaste following 24 weeks use, with the greatest improvements (change from baseline) seen for posterior and papillae tooth sites for bleeding, margin tooth sites for MGI and body tooth sites for TPI. CONCLUSION: This pooled analysis of patient level-data, limited to GSKCH long-term gingivitis clinical studies, demonstrates that twice daily use of a 67% sodium bicarbonate toothpaste effectively removes plaque from all tooth sites, and results in clinically significant improvements in measures of gingival health, overall and for all the tooth regions investigated, compared with a non-sodium bicarbonate (regular) toothpaste following 24 weeks twice daily use.
Asunto(s)
Gingivitis , Pastas de Dientes , Humanos , Pastas de Dientes/uso terapéutico , Cepillado Dental , Bicarbonatos , Bicarbonato de Sodio/uso terapéutico , Resultado del Tratamiento , Gingivitis/prevención & control , Índice de Placa DentalRESUMEN
AIM: To compare efficacy of an anhydrous 0.454% w/w stannous fluoride/sodium fluoride toothpaste (Test) versus a sodium monofluorophosphate toothpaste (Negative control) and a stannous chloride/sodium fluoride toothpaste (Positive control) for dentine hypersensitivity relief after 8 weeks' twice-daily use. MATERIALS AND METHODS: In this randomized, examiner-blind, stratified, parallel study, primary and secondary efficacy variables were mean changes in Schiff score (evaporative [air] sensitivity) and tactile threshold (Yeaple probe), respectively, from baseline to Week 8 between Test (n = 62) and Negative control (n = 62). Test and Positive control (n = 61) comparisons were exploratory objectives. RESULTS: All groups significantly improved from baseline on both dentine hypersensitivity measures (p < .0001). Difference between adjusted mean changes from baseline in Schiff sensitivity scores at Week 8 for Test versus Negative control groups was 0.19 (95% CI 0.002, 0.374), in favour of the Negative control (p = .0476; 12.57% difference). Difference in tactile threshold was -7.20 g (95% CI -16.376, 1.975), and this was not statistically significant (p = .3715; -21.83% difference). Test group showed no significant difference versus Positive control for either measure. Toothpastes were generally well tolerated. CONCLUSION: While twice-daily use of Test toothpaste significantly reduced dentine hypersensitivity from baseline, there was no significant advantage over negative or positive controls. STUDY REGISTRATION: Clinicaltrials.gov; NCT03310268.
Asunto(s)
Desensibilizantes Dentinarios , Sensibilidad de la Dentina , Desensibilizantes Dentinarios/uso terapéutico , Sensibilidad de la Dentina/tratamiento farmacológico , Sensibilidad de la Dentina/prevención & control , Método Doble Ciego , Fluoruros/uso terapéutico , Humanos , Fosfatos , Fluoruro de Sodio , Fluoruros de Estaño/uso terapéutico , Pastas de Dientes/uso terapéutico , Resultado del TratamientoRESUMEN
PURPOSE: To monitor extrinsic stain formation over 24 weeks with twice-daily use of an anhydrous 0.454% SnF2 dentifrice containing 5% sodium tripolyphosphate (STP) ('Test'), compared to a standard fluoride dentifrice (0.76% fluoride as sodium monofluorophosphate [SMFP\) ('Comparator') following a professional prophylaxis. METHODS: Primary efficacy variables were between-treatment differences in extrinsic dental stain of anterior teeth after 4, 8, 12, and 24 weeks' use using mean area and intensity scores of the MacPherson modification of the Lobene stain index (MLSI), and to monitor oral tolerability. RESULTS: Overall, 214 subjects were randomized to treatment. The Test dentifrice group demonstrated statistically significantly less stain compared with the Comparator at each timepoint for all outcome variables (MLSI - Area × Intensity, Area only, and Intensity only) with the exception of MLSI (Area × Intensity) at Week 12. Products were generally well tolerated with 26 treatment-related adverse events (TRAEs) reported (10 with Test, 16 with Comparator). Five subjects withdrew from the 24-week study due to TRAEs, three in the Test group, two in the Comparator group. Statistically significantly less anterior tooth staining was observed with up to 24 weeks twice-daily brushing with a 0.454% SnF2/5% STP anhydrous dentifrice compared to a marketed fluoride dentifrice with 0.76% SMFP. CLINICAL SIGNIFICANCE: Long term use of a SnF2/STP dentifrice demonstrated minimal stain build-up after 24 weeks, twice-daily use; products were generally well-tolerated.
Asunto(s)
Dentífricos , Fluoruros de Estaño , Decoloración de Dientes , Colorantes , Dentífricos/efectos adversos , Dentífricos/uso terapéutico , Método Doble Ciego , Humanos , Fosfatos , Fluoruro de Sodio , Fluoruros de Estaño/efectos adversos , Fluoruros de Estaño/uso terapéutico , Pastas de Dientes , Resultado del TratamientoRESUMEN
AIM: To determine peripheral blood neutrophil superoxide release and C-reactive protein (CRP) concentration in chronic periodontitis patients, before and after non-surgical periodontal treatment. MATERIALS AND METHODS: Neutrophils were isolated from patient and control volunteers (n = 20) and superoxide measured by lucigenin-enhanced chemiluminescence with and without stimulation with unopsonized Porphyromonas gingivalis, unopsonized Fusobacterium nucleatum and phorbol 12-myristate 13-acetate (PMA) before and 2-months following non-surgical therapy. Corresponding high-sensitivity plasma CRP concentrations were also determined. RESULTS: At pre-treatment baseline, patient neutrophils released more superoxide in the absence (p ≤ 0.032) and presence of periodontal bacteria (p ≤ 0.013) and after PMA stimulation (p = 0.041) compared to control cells. Post-therapy, patient neutrophil superoxide release was reduced to control cell levels. Median patient plasma CRP concentrations were non-significantly higher than control values and were reduced after therapy (1.80-1.36 mg/l). Patient pre-treatment baseline, unstimulated neutrophil superoxide release showed a significant, positive correlation with plasma CRP concentration (p = 0.01). CONCLUSIONS: Chronic periodontitis is characterized by peripheral neutrophils exhibiting superoxide hyperactivity and hyper-reactivity to periodontal pathogens that is not a constitutive feature of periodontitis patients. The positive, pre-therapy relationship between unstimulated neutrophil superoxide release and plasma CRP is consistent with a protective role for CRP in reducing oxidative stress and systemic inflammation in vivo.
Asunto(s)
Proteína C-Reactiva/análisis , Fusobacterium nucleatum , Humanos , Neutrófilos , Porphyromonas gingivalis , SuperóxidosRESUMEN
AIM: To investigate the chemotactic accuracy of peripheral blood neutrophils from patients with chronic periodontitis compared with matched healthy controls, before and after non-surgical periodontal therapy. MATERIAL & METHODS: Neutrophils were isolated from patients and controls (n = 18) by density centrifugation. Using the Insall chamber and video microscopy, neutrophils were analysed for directional chemotaxis towards N-formyl-methionyl-leucyl-phenylalanine [fMLP (10 nM), or CXCL8 (200 ng/ml)]. Circular statistics were utilized for the analysis of cell movement. RESULTS: Prior to treatment, neutrophils from patients with chronic periodontitis had significantly reduced speed, velocity and chemotactic accuracy compared to healthy controls for both chemoattractants. Following periodontal treatment, patient neutrophils continued to display reduced speed in response to both chemoattractants. However, velocity and accuracy were normalized for the weak chemoattractant CXCL8 while they remained significantly reduced for fMLP. CONCLUSIONS: Chronic periodontitis is associated with reduced neutrophil chemotaxis, and this is only partially restored by successful treatment. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to their disease by increasing tissue transit times, thus exacerbating neutrophil-mediated collateral host tissue damage.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Periodontitis Crónica/patología , Neutrófilos/fisiología , Desbridamiento Periodontal/métodos , Adulto , Estudios de Casos y Controles , Factores Quimiotácticos/farmacología , Periodontitis Crónica/terapia , Índice de Placa Dental , Raspado Dental/métodos , Femenino , Estudios de Seguimiento , Humanos , Interleucina-8/farmacología , Masculino , Persona de Mediana Edad , N-Formilmetionina Leucil-Fenilalanina/farmacología , Índice Periodontal , Aplanamiento de la Raíz/métodosRESUMEN
The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.
Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales , Humanos , Pruebas Inmunológicas , Epitelio , Investigación , Enfermedad CrónicaRESUMEN
Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of lifestyle factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.
Asunto(s)
Antioxidantes , Micronutrientes , Humanos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Estrés Oxidativo , Biomarcadores/metabolismo , Oxidación-ReducciónRESUMEN
Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils as well as of oral neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.
Asunto(s)
Trampas Extracelulares , Neutrófilos , Endonucleasas , PlasmaRESUMEN
AIM: To determine the effect of cigarette smoke extract, nicotine and cotinine on lucigenin-detectable neutrophil superoxide production. MATERIALS & METHODS: Neutrophils from periodontally healthy individuals were treated with aqueous smoke extract, nicotine and cotinine, prior to stimulation or at the same time as stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli Lipopolysaccharide (LPS). Superoxide generation was determined by lucigenin chemiluminescence. RESULTS: Smoke extract induced superoxide release from neutrophils (p <0.0001) in a dose-dependent manner. By contrast, superoxide generation by neutrophils in response to pathologically relevant stimuli was inhibited by pre-treatment with smoke extract (p <0.01). This inhibition did not require the continued presence of the extract. A similar reduction in stimulated superoxide production by smoke extract was detected when neutrophils were simultaneously exposed to the extract and stimuli. Nicotine and cotinine (0-10 µg/ml) had no effect on superoxide release from unstimulated or stimulated neutrophils. CONCLUSIONS: Stable water-soluble components of cigarette smoke directly induce superoxide generation by otherwise unstimulated neutrophils, but reduce superoxide responses of cells to pathologically relevant stimuli. These data suggest potential neutrophil-mediated mechanisms by which smoking may initiate and maintain oxidative stress at periodontally healthy sites and participate in disease progression, by reducing innate immune responses.
Asunto(s)
Mezclas Complejas/farmacología , Cotinina/farmacología , Neutrófilos/efectos de los fármacos , Nicotiana , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Oxidantes/metabolismo , Humo , Superóxidos/metabolismo , Acridinas , Relación Dosis-Respuesta a Droga , Escherichia coli , Fusobacterium nucleatum/fisiología , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoglobulina G/inmunología , Lipopolisacáridos/farmacología , Sustancias Luminiscentes , Neutrófilos/microbiología , Estrés Oxidativo/efectos de los fármacos , Staphylococcus aureus/inmunología , Staphylococcus aureus/fisiologíaRESUMEN
The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.
Asunto(s)
Gingivitis/metabolismo , Inflamación/metabolismo , Proteoma/química , Proteómica/métodos , Enfermedad Aguda , Adulto , Cromatografía Liquida , Análisis por Conglomerados , Femenino , Fusobacterium/química , Líquido del Surco Gingival/química , Gingivitis/microbiología , Humanos , Inflamación/microbiología , Marcaje Isotópico , Masculino , Metagenoma , Modelos Biológicos , Proteoma/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Estadísticas no Paramétricas , Espectrometría de Masas en TándemRESUMEN
OBJECTIVES: This proof-of-principle, single-center, randomized, examiner-blind, crossover study compared two experimental polyvinyl acetate (PVA)-based denture adhesives (Test Adhesives 1 and 2) with a marketed reference polymethyl vinyl ether/maleic anhydride (PMV/MA)-based adhesive and no adhesive using incisal bite force area over baseline over 12 hr (AOB0-12 ) in participants with an at least moderately well-fitting complete maxillary denture. Previous in vitro studies suggested the experimental denture adhesives provided superior performance. MATERIALS AND METHODS: Participants were randomized to a treatment sequence such that each received each treatment once. Prior to treatment application (baseline) and at 0.5, 1, 3, 6, 9, and 12 hr following the application, participants bit on a force transducer until their maxillary denture dislodged. Between-treatment differences in AOB0-12 were analyzed using analysis of covariance. For study validity, the reference adhesive was compared with no adhesive. Participants were asked to rate sensory experiences and ease of denture removal. RESULTS: Twenty-three participants were included in the modified intent-to-treat population. Although Test Adhesives 1 and 2 had a higher mean AOB0-12 than no adhesive, differences were not statistically significant. No statistically significant difference was also found between the reference adhesive and no adhesive; hence, study validity was not attained. Participants did not report any clear differences between the test or reference adhesives in terms of taste or feel; however, dentures were easier to remove with the test adhesives versus reference. No treatment-related adverse events were reported. CONCLUSION: Neither the experimental PVA-based denture adhesives nor the PMV/MA-based reference product demonstrated a statistically significant difference in incisal bite force AOB0-12 compared with no adhesive. The reasons for these unexpected results is unclear; they suggest that findings of in vitro tests for denture adhesive performance are not always translated to in vivo performance (Clinicaltrials.gov: NCT02937870).
Asunto(s)
Adhesivos/administración & dosificación , Fuerza de la Mordida , Retención de Dentadura/métodos , Dentadura Completa , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios Cruzados , Femenino , Humanos , Incisivo , Masculino , Maxilar , Persona de Mediana Edad , Polivinilos/administración & dosificación , Prueba de Estudio Conceptual , Adulto JovenRESUMEN
OBJECTIVE: To evaluate the efficacy of a dissolvable strip containing 15% w/w calcium sodium phosphosilicate (CSPS) (Novamin®) in providing rapid relief from dentine hypersensitivity (DH). METHODS: In this examiner-blind, proof-of-principle study, 120 healthy adults with DH were randomized 1:1 to the Test strip, professionally applied to facial surfaces of two selected teeth, or to No treatment. Sensitivity was assessed at baseline and 10min, 2h and 4h post-application in response to evaporative (air) and tactile stimuli (measured by Schiff sensitivity scale/a numeric rating scale and tactile threshold, respectively). Change from baseline was analyzed by ANCOVA. RESULTS: At 10min post-application, mean Schiff score change from baseline (primary endpoint) was statistically significant with the Test strip (-0.46; 95% confidence intervals [CI]: -0.563, -0.356; p<0.0001) but not with No treatment (-0.02; 95% CI: -0.119, 0.088; p=0.7664). The between-treatment group difference favored the Test strip (difference: -0.44; 95% CI: -0.591, -0.297; p<0.0001). Similar improvements with the Test strip were reported for all other evaporative (air) and tactile sensitivity endpoints (p<0.0001 vs no-treatment) at all timepoints (10min, 2h, 4h). Test strips were considered by most staff and participants slightly/moderately easy to apply (98%). Many participants rated the overall usage experience as "like moderately" (40%) or "like extremely" (20%). There were no treatment-related adverse events. CONCLUSION: This new CSPS-based technology may provide a novel treatment option for rapid relief from DH (Clinicaltrials.gov NCT02937623). CLINICAL SIGNIFICANCE: A dissolvable strip containing 15% w/w calcium sodium phosphosilicate (CSPS) demonstrated significantly greater dentine hypersensitivity reductions following a single application compared with no treatment. Strips were well-liked by participants and generally well tolerated. A strip containing CSPS, which dissolves within 10min, may provide rapid relief from dentine hypersensitivity.
RESUMEN
Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of life-style factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Micronutrientes/metabolismo , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Ácido Ascórbico/sangre , Ácido Ascórbico/metabolismo , Biomarcadores , Carotenoides/sangre , Carotenoides/metabolismo , Ensayo Cometa , Daño del ADN , Ácido Deshidroascórbico/sangre , Ácido Deshidroascórbico/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Desoxiguanosina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Líquido del Surco Gingival , Humanos , Micronutrientes/sangre , Oxidación-Reducción , Plasma , Especies Reactivas de Oxígeno , Saliva , SueroRESUMEN
The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.
Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Biomarcadores , Enfermedad Crónica , Biología Computacional/métodos , Expresión Génica , Humanos , Inmunohistoquímica , Inflamación/patología , Reacción en Cadena de la PolimerasaRESUMEN
Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.
Asunto(s)
Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Biomarcadores , Catepsina G/metabolismo , Citotoxicidad Inmunológica , Trampas Extracelulares/inmunología , Trampas Extracelulares/microbiología , Humanos , Elastasa de Leucocito/metabolismo , Viabilidad Microbiana/inmunología , Microscopía Fluorescente , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Neutrófilos/ultraestructura , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pro-inflammatory cytokine release (IL-8, IL-6, TNF-α, IL-1ß) by peripheral blood neutrophils, isolated from periodontitis patients (before/after therapy) and matched controls, was determined after 18 h culture in the presence/absence of Escherichia coli LPS, opsonised Staphylococcus aureus, heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis. All cultures demonstrated differences in the amounts of each cytokine detected (P < 0.0001), with a clear release pattern (IL-8 > IL-6 > TNF-α = IL-1ß). Median cytokine release from unstimulated patient neutrophils was consistently, but non-significantly, higher than from control cells. Stimulated cytokine release from untreated patient neutrophils was also consistently higher than from control cells. This hyper-reactivity was significant for all tested cytokines when data for all stimuli were combined (P < 0.016). In terms of individual stimuli, significant hyper-reactivity was detected with LPS (IL-8), F. nucleatum (IL-8, TNF-α), opsonised S. aureus (IL-8, TNF-α, IL-1ß) and P. gingivalis (IL-8, IL-1ß). Cytokine production by patient neutrophils did not reduce following successful non-surgical periodontal therapy and, except for responses to F. nucleatum, the cytokine hyper-reactivity detected pre-therapy was retained. These data demonstrate that chronic periodontitis is characterised by neutrophils that constitutively exhibit cytokine hyper-reactivity, the effects of which could modulate local and systemic inflammatory-immune responses and influence the risk and severity of periodontitis-associated systemic inflammatory diseases.
Asunto(s)
Infecciones Bacterianas/inmunología , Periodontitis Crónica/inmunología , Citocinas/metabolismo , Activación Neutrófila , Neutrófilos/inmunología , Adulto , Infecciones Bacterianas/complicaciones , Circulación Sanguínea , Células Cultivadas , Periodontitis Crónica/etiología , Citocinas/genética , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/microbiología , Regulación hacia ArribaRESUMEN
This study determined the influence of physiologically relevant concentrations of C-reactive protein (CRP) on reactive oxygen species (ROS) production by neutrophils. Neutrophils from healthy individuals were incubated with soluble pentameric CRP prior to TLR stimulation with Fusobacterium nucleatum, or FcγR stimulation with IgG-opsonised Staphylococcus aureus or heat-aggregated IgG. ROS generation by unstimulated cells and those after stimulation were determined using luminol, isoluminol and lucigenin chemiluminescence, detecting predominantly intracellular hypochlorous acid (HOCl), extracellular hydrogen peroxide (detected as HOCl) and extracellular superoxide respectively. Baseline (unstimulated) neutrophil ROS generation and release was reduced compared with vehicle control by 10 µg/ml CRP. There was no consistent effect of CRP on FcγR-stimulated HOCl production, but the extracellular superoxide response was reduced by 10 µg/ml CRP. By contrast, CRP reduced intracellular (10 µg/ml) and extracellular (3 and 10 µg/ml) HOCl generation, but increased superoxide release (1-10 µg/ml) in response to TLR stimulation. Physiologically relevant concentrations of CRP inhibited baseline ROS generation and reduced FcγR-stimulated extracellular superoxide and TLR-stimulated HOCl release, suggesting that CRP may offer some degree of host protection from neutrophil-associated, low-level oxidative stress. However, CRP enhanced TLR-mediated superoxide release from neutrophils, potentially increasing oxidative stress but aiding host protection from infection.