RESUMEN
OBJECTIVE AND MATERIAL AND METHODS: This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 - Oragene™ commercial kit, protocol 2 - QIAamp DNA mini kit, protocol 3 - DNA extraction using ammonium acetate, protocol 4 - Instagene™ Matrix and protocol 5 - Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. RESULTS: Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. CONCLUSIONS: In summary, a complicated picture emerges when taking into account the extracted DNA's quantity, purity and quality; depending on a given researchers needs, one protocol's particular strengths and costs might be the deciding factor for its employment.
Asunto(s)
ADN/aislamiento & purificación , Saliva/química , Electroforesis , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Control de Calidad , Juego de Reactivos para Diagnóstico , Valores de Referencia , Reproducibilidad de los Resultados , Manejo de Especímenes/métodos , Espectrofotometría , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5'-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC) after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo - USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg) and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5'-hydroxypiroxicam was only detected in 2 out of 10 volunteers.
Asunto(s)
Antiinflamatorios no Esteroideos/sangre , Cromatografía Líquida de Alta Presión/métodos , Extracción Líquido-Líquido/métodos , Piroxicam/análogos & derivados , Piroxicam/sangre , Antiinflamatorios no Esteroideos/farmacocinética , Humanos , Naproxeno/sangre , Naproxeno/farmacocinética , Piroxicam/farmacocinética , Valores de Referencia , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Saliva sampling used to quantify piroxicam and 5'-hydroxypiroxicam is a noninvasive and painless method when compared to sequential blood sampling. For that, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometric method for simultaneous determination of piroxicam and 5'-hydroxypiroxicam in saliva and human plasma was developed and validated. Piroxicam and its major metabolite were separated using a LiChroCART 125-4 RP Select-B Sorbent C18 column using a mixture of methanol and 2% phosphoric acid (pH 2.7) (70:30, v/v) for the mobile phase with a flow injection of 1mL/min. The run time was 4min. Volunteers had saliva and blood sampled before, 1, 2, 3, 4, 5, 6, 8, 11, 24, 48 and 72h after taking a 20mg oral dose of piroxicam. The pharmacokinetic parameters of piroxicam in plasma samples were as follows: AUC0-72 (64819hng/mL), predicted clearance (0.2L/h), distribution volume (14.8L), elimination half-life (50.7h) and saliva/plasma concentration ratio (0.003). The estimation of all pharmacokinetic parameters for 5'-hydroxypiroxicam would require collections beyond 72h; however, it was possible to quantify the mean maximum concentration (133ng/mL), time to peak concentration (53.6h), mean AUC0-72 (6213hng/mL), predicted clearance (110.3L/h) and saliva/plasma concentration ratio (0.04). The developed methods proved effective and sensitive for determining the lower quantification limit of piroxicam in plasma (6.1ng/mL) and saliva (0.15ng/mL) and of 5'-hydroxypiroxicam in plasma (1.2ng/mL) and saliva (0.15ng/mL).
Asunto(s)
Piroxicam/análogos & derivados , Piroxicam/administración & dosificación , Piroxicam/análisis , Saliva/química , Espectrometría de Masas en Tándem/métodos , Administración Oral , Cromatografía Liquida/métodos , Humanos , Piroxicam/sangreRESUMEN
BACKGROUND: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). METHODS: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. CONCLUSION: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.
Asunto(s)
Técnicas de Silenciamiento del Gen , Encía/metabolismo , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Ligamento Periodontal/metabolismo , Receptor Toll-Like 2/genética , Adolescente , Adulto , Análisis de Varianza , Células Cultivadas , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Femenino , Fibroblastos/metabolismo , Encía/citología , Humanos , Inflamación/genética , Interleucina-6/genética , Interleucina-8/genética , Masculino , Ligamento Periodontal/citología , Interferencia de ARN , Transducción de Señal , Estadísticas no Paramétricas , Receptor Toll-Like 4/genética , Adulto JovenRESUMEN
Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment.
Asunto(s)
Humanos , Masculino , Femenino , Saliva/química , ADN/aislamiento & purificación , Control de Calidad , Juego de Reactivos para Diagnóstico , Valores de Referencia , Manejo de Especímenes/métodos , Espectrofotometría , Factores de Tiempo , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , ElectroforesisRESUMEN
OBJECTIVE: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens. MATERIAL AND METHODS: Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively. RESULTS: The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60%x10%; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95% CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR. CONCLUSIONS: Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes.
Asunto(s)
Bacilos Gramnegativos Anaerobios Facultativos/patogenicidad , Enfermedades Periodontales/complicaciones , Porphyromonas gingivalis/patogenicidad , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/microbiología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Índice de Placa Dental , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Periodontales/microbiología , Índice Periodontal , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de RiesgoRESUMEN
The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-ß), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 µg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-ß protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 µg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-ß when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 µg/mL and 10 µg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-ß and IL-8 but not IL-10.
Asunto(s)
Fibroblastos/metabolismo , Encía/citología , Interleucina-10/biosíntesis , Interleucina-8/biosíntesis , Ligamento Periodontal/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Encía/inmunología , Humanos , Lipopolisacáridos/análisis , Porphyromonas gingivalis/inmunología , Factores de TiempoRESUMEN
Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5'-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC) after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo - USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg) and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5'-hydroxypiroxicam was only detected in 2 out of 10 volunteers.
Asunto(s)
Humanos , Piroxicam/análogos & derivados , Piroxicam/sangre , Antiinflamatorios no Esteroideos/sangre , Cromatografía Líquida de Alta Presión/métodos , Extracción Líquido-Líquido/métodos , Valores de Referencia , Factores de Tiempo , Piroxicam/farmacocinética , Antiinflamatorios no Esteroideos/farmacocinética , Naproxeno/sangre , Naproxeno/farmacocinética , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens. MATERIAL AND METHODS: Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively. RESULTS: The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60 percentx10 percent; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95 percent CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR. CONCLUSIONS: Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes.
Asunto(s)
Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Bacilos Gramnegativos Anaerobios Facultativos/patogenicidad , Enfermedades Periodontales/complicaciones , Porphyromonas gingivalis/patogenicidad , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/microbiología , Factores de Edad , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Índice de Placa Dental , Índice Periodontal , Enfermedades Periodontales/microbiología , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de RiesgoRESUMEN
The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 µg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 µg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 µg/mL and 10 µg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.