Influence of different opacities and layering technique of nanotechnology composite resins regarding the wavelength and fluorescence intensity: in vitro study / Influencia de diferentes opacidades y la técnica de estratificación de resinas compuestas de nanotecnología en la longitud de onda e intensidad de fluorescencia: estudio in vitro

Objective: To evaluate the influence of opacity and the layering technique on the fluorescence of different composite resins. Materials and Methods: Two opacities (enamel and dentin) and the layering technique (enamel + dentin) of the composite resins: Filtek® Z350 and Palfique LX5 were evaluated in vitro. Composite resin discs were fabricated using a preformed matrix of 10 mm diameter and 0.5 mm thick for the single opacity groups and 10 mm thick for the layering technique groups, using 2 layers of 0.5 mm thickness of each opacity (n = 5). Specimens were analyzed using the Raman spectroscopy method. Data were analyzed using the Kruskall-wallis and Mann-Whitney U tests. Results: When evaluating the intensity of fluorescence, no statistically significant difference was found when comparing the layering technique and enamel opacity (p2> 0.05) and an increase in the dentin opacity value for both brands of composite resin. Regarding wavelength, no statistically significant difference was found when comparing the layering technique with enamel opacity and dentin opacity for both Filtek® Z350 and Palfique LX5® composite resins (p2 > 0.05). Conclusions: The fluorescence intensity of the layering technique is similar to enamel opacity for both composite resins. Likewise, the wavelength of the layering technique is similar to the enamel opacity and dentin opacity for both brands.

Objetive: Evaluar la influencia de la opacidad y de la técnica de estratificación en la fluorescencia de diferentes resinas compuestas. Materiales y Métodos: Se evaluó in vitro 2 opacidades (Esmalte y Dentina) y la técnica de estratificación (Esmalte + Dentina) de las resinas compuestas: Filtek® Z350 y Palfique LX5. Se fabricaron discos de resina compuesta, utilizando una matriz preformada de 10 mm de diámetro y 0,5 mm de grosor para los grupos de opacidad única y 10 mm de grosor para los grupos de técnica estratificada, utilizando 2 capas de 0,5 mm de cada opacidad (n = 5). Los especímenes se analizaron mediante el método de Espectroscopía Raman. Los datos se analizaron utilizando la prueba de Kruskall-wallis y Prueba U de Mann Whitney. Resultado: Al evaluar la intensidad de fluorescencia no se encontró diferencia estadísticamente significativa entre los pares: Técnica estratificada versus Opacidad Esmalte para ambas marcas de resina compuesta Filtek® Z350 y para Palfique LX5® (p2 > 0,05). Para longitud de onda no se encontró diferencia estadísticamente significativa entre los pares: Técnica estratificada versus Opacidad Esmalte y Técnica estratificada VS Opacidad Dentina para ambas resinas compuesta Filtek® Z350 y Palfique LX5® (p2> 0,05). Conclusión: La intensidad de fluorescencia de la técnica estratificada es similar a la opacidad Esmalte para ambas resinas compuestas. De igual manera la longitud de onda de la técnica estratificada es similar a la opacidad Esmalte y opacidad Dentina para ambas marcas.

Modificação de uma resina composta Bulk Fill Flow com extrato acetônico da casca da romã / Modification of a Bulk Fill Flow composite resin with acetonic extract from pomegranate peel

Introdução: A cárie dentária ainda se constitui um problema de saúde pública. Áreas adjacentes a restaurações são frequentemente acometidas por cárie. Por mais que as resinas compostas estejam sendo estudadas e melhoradas, ainda não apresentam atividade antimicrobiana. As cascas da romã (Punica Granatum) são um recurso potencial para compostos bioativos como fenólicos, proantocianidinas e flavonoides, além de apresentarem atividade antioxidante e efeito inibitório contra bactérias Gram-negativas e Gram-positivas. Objetivo: modificar a resina composta Opus Bulk Fill Flow (FGM®) com o extrato acetônico da casca da romã em diferentes concentrações e avaliar a rugosidade da superfície e mudança de cor. Metodologia: foi realizada a extração de 5g de casca da romã utilizando 100mL de solvente acetona 70%. Após rotaevaporação, filtragem e liofilização do extrato, este foi macerado, peneirado e pesado em concentrações diferentes a partir da concentração inibitória mínima capaz de inibir o crescimento de Streptococcus mutans ATCC 700610. A resina composta Opus Bulk Fill Flow foi modificada com esse extrato em diferentes concentrações de forma a gerar 5 grupos: Controle 0 µg (n=10), G930 µg (n=10), G1860 µg (n=10), G3730 µg (n=10) e G7460 µg (n=10). Rugosidade (Ra), diferença de cor (ΔE00) e índice de brancura (WID) foram submetidos aos testes de normalidade. Os dados não-paramétricos de Ra foram submetidos ao teste de Kruskal-Wallis com pós-teste de Dunn e os dados paramétricos do ΔE00 e WID foram submetidos ao teste ANOVA 1 Fator com pósteste de Tukey por meio do software GraphPad Prism 8 e Microsoft Excel 2019. Resultados: Verificou-se que a média do índice de brancura (WID) diminuiu conforme o aumento da concentração do extrato na resina modificada (p<0,05), assim como, as resinas modificadas se tornaram, visivelmente a olho nu, um pouco mais amareladas. Após 1 mês, as amostras dos grupos experimentais sofreram a mesma variação de cor (ΔE00) que o grupo controle, uma vez que os valores das médias foram semelhantes. Portanto, todos os grupos apresentaram estabilidade de cor. A rugosidade superficial (Ra) não mostrou diferença estatisticamente significativa (p>0,05). Todos os grupos apresentaram médias com valores similares a 0,06µm. Conclusão: A alteração de cor na resina, com a inserção do extrato, ainda na maior concentração, se manteve na classificação do matiz A. O que não afeta as propriedades organolépticas e pode ser considerada uma cor similar à cor dos dentes naturais. A adição do extrato na resina manteve a rugosidade superficial de todos os grupos dentro do valor ideal, prevenindo a adesão de biofilmes e microrganismos e proporcionando conforto ao toque da língua (AU).

Introduction: Dental caries still constitutes a public health problem. Areas adjacent to restorations are often affected by caries. Even though resin composites are being studied and improved, they still do not have antimicrobial activity. Pomegranate peels (Punica Granatum) are a potential resource for bioactive compounds such as phenolics, proanthocyanidins and flavonoids, in addition to presenting antioxidant activity and inhibitory effects against Gram-negative and Gram-positive bacteria. Objective: to modify the Opus Bulk Fill Flow (FGM™) resin composite with the acetone extract of pomegranate peel in different concentrations and evaluate the surface roughness, and color change. Methodology: 5g of pomegranate peel was extracted using 100mL of 70% acetone solvent. After rotary evaporation, filtering and lyophilization of the extract, it was macerated, sieved and weighed at different concentrations based on the minimum inhibitory concentration capable of inhibiting the growth of Streptococcus mutans ATCC 700610. The Opus Bulk Fill Flow resin composite was modified with this extract in different concentrations to generate 5 groups: Control 0 µg (n=10), G930 µg (n=10), G1860 µg (n=10), G3730 µg (n=10) e G7460 µg (n=10). Roughness (Ra) and color difference (ΔE00) were subjected to normality tests and non-parametric data were subjected to the Kruskal-Wallis test with Dunn's post-test using GraphPad Prism 8 and Microsoft Excel 2019 software. Results: It was found that the average whiteness index (WID) decreased as the concentration of the extract in the modified resin increased (p<0.05), as well as the modified resins became, visibly to the naked eye, a little more yellowish. After 1 month, samples from the experimental groups suffered the same color variation (ΔE00) as the control group, since the average values were similar. Therefore, all groups showed color stability. Surface roughness (Ra) did not show a statistically significant difference (p>0.05). All groups presented average values similar to 0.06µm. Conclusion: The color change in the resin, with the insertion of the extract, even at the highest concentration, remained in the classification of hue A. This does not affect the organoleptic properties and can be considered a color similar to the color of natural teeth. The addition of the extract to the resin composite maintained the surface roughness of all groups within the ideal value, preventing the adhesion of biofilms and microorganisms and providing comfort to the touch of the tongue (AU).

Do oral antiseptics used in the dental routine have anti-viral efficacy? a systematic review of in vitro studies / Os antissépticos orais utilizados na rotina odontológica têm eficácia anti-viral? Uma revisão sistemática de estudos in vitro / ¿Los antisépticos orales utilizados en la rutina odontológica tienen eficacia antiviral? Una revisión sistemática de estudios in vitro

Introduction: Mouthwashes play an important role in the dental clinic, but their role on viruses requires investigation. Objective:to review in vitro studies to identify the effect of different mouthwashes on the main viruses associated with routine dental care. Methodology:The following databases were searched in September 2023: PubMed, Embase, Scopus and Web of Science databases; the Cochrane Library and the Virtual Health Library (VHL); and grey literature. In vitro studies that used mouthwashes to reduce the viral load were selected. The PICOS strategy was considered to define eligibility criteria: the Population (viruses involved in the etiology of oral infection), the Intervention (oral antiseptics), the appropriate comparator (positive and negative controls), the Outcomes of interest (reduction of viral load) and the Study design (in vitro studies). Results:Considering the eligibility criteria, 19 articles were included in this review. The efficacy of povidone-iodine (PVP-I), chlorhexidine, Listerine®, essential oils, and cetylpyridinium chloride (CPC) rinses were investigated. PVP-I (0.23%) had its effects mainly associated with coronaviruses SARS(Severe Acute Respiratory Syndrome),demonstrating a significant reduction in viral load after 15 seconds of exposure. Chlorhexidine (0.05%; 0.1% and 0.5%) was ineffective against adenovirus, poliovirus, and rhinovirus respiratory viruses. Listerine® demonstrated superior efficacy against HSV-1 and 2 viruses and influenza A, and cetylpyridine chloride also demonstrated virucidal activity against influenza A. Conclusions:The type, concentration, and time of exposure to antiseptics varied between studies. PVP-I and chlorhexidine digluconate were the most studied substances, butin general, PVP-I was more effective in reducing viral titers, especially concerning coronaviruses. Other antiseptics such as CPC, H2O2 and Listerine® have also shown significant reduction in viral load, but this is a limited number of studies (AU).

Introdução: Os enxaguantes bucais desempenham um papel importante na clínica odontológico, porém seu papel sobre os vírus requer investigações. Objetivo: revisar estudos in vitro para identificar o efeito de diferentes colutórios sobre os principais vírus associados ao atendimento odontológico de rotina. Metodologia: As seguintes bases foram pesquisadas até setembro de 2023: PubMed, Embase, Scopus e Web of Science; a Biblioteca Cochrane e a Biblioteca Virtual em Saúde (BVS); e literatura cinzenta. Foram selecionados estudos in vitro que utilizaram bochechos com o objetivo de reduzir a carga viral. A estratégia PICOS foi considerada para a definição dos critérios de elegibilidade: População (vírus envolvidos na etiologia da infecção oral), Intervenção (antissépticos orais), Comparador (controles positivos e negativos), os Desfechos de interesse (redução da carga viral) e o desenho do estudo (estudos in vitro). Resultados: Considerando os critérios de elegibilidade, 19 artigos foram incluídos para esta revisão. A eficácia da povidona-iodo (PVP-I), clorexidina, Listerine®, óleos essenciais e lavagens com cloreto de cetilpiridínio foram investigadas. O PVP-I(0.23%)teve seus efeitos principalmente associados ao coronavírusSARS (Síndrome Respiratória Aguda Severa),demonstrando uma redução significativa da carga viral após 15 segundos de exposição. A clorexidina mostrou-se ineficaz contra vírus respiratórios de adenovírus, poliovírus e rinovírus. Listerine® demonstrou eficácia superior contra vírus HSV-1 e 2 e vírus influenza A, e cloreto de cetilpiridinio também demonstrou atividade virucida contra influenza A.Conclusões:O tipo, concentração e tempo de exposição aos antissépticos variaram entre os estudos. O PVP-I e o digluconato de clorexidina foram as substâncias mais estudadas, mas no geral, o PVP-I foi mais eficaz na redução dos títulos virais, principalmente no que diz respeito aos coronavírus. Outros antissépticos como CPC, H2O2 e Listerine® também mostraram redução significativa da carga viral, mas trata-se de um número limitado de estudos (AU).

Introducción: Los enjuagues bucales son importantesen la clínica dental, sin embargo, su efecto sobre los virus requiere investigaciones. Objetivo: Revisar estudios in vitro para identificar el efecto de enjuagues bucales sobre los principales virus asociados con larutinaodontológica. Metodología: Las siguientes bases de datos fueron investigadas hasta septiembrede 2023: PubMed, Embase, Scopus y Web of Science; Biblioteca Cochrane y Biblioteca Virtual en Salud (BVS); yliteratura gris. Se seleccionaron estudios in vitro que utilizaron enjuagues bucales con el objetivo de reducir la carga viral. Se consideró la estrategia PICOS para definir los criterios de elegibilidad: Población (virus implicados en la etiología de la infección oral), Intervención (antisépticos bucales), Comparador (controles positivos y negativos), Resultados de interés (reducción de la carga viral) y diseño del estudio (in vitro). Resultados: Considerando los criterios de elegibilidad, se incluyeron 19 artículos.Se investigó la eficacia de povidona yodada (PVP-I), clorhexidina, Listerine®,aceites esenciales y enjuagues de cloruro de cetilpiridinio (CPC). PVP-I(0.23%)mostró sus efectos principalmente asociados al coronavirus SARS(Síndrome Respiratorio Agudo Severo), demostrando una reducción significativa de la carga viral después de 15 segundos. Se ha demostrado que la clorhexidina es ineficaz contra losvirus respiratorios adenovirus, poliovirus y rinovirus. Listerine® demostró una eficacia superior contra los virus HSV-1 y 2 y el virus de la influenza A, y el CPCtambién mostró actividad virucida contra la influenza A.Conclusiones: El tipo, la concentración y el tiempo de exposiciónvariaron entre los estudios. PVP-I y digluconato de clorhexidina fueron las sustancias más estudiadas, pero,PVP-I fue más efectiva en la reducción de los títulos virales, especialmente en lo que respecta a los coronavirus. Otros antisépticos como CPC, H2O2 y Listerine® también mostraron una reducción significativa de la carga viral, pero se trata de un número limitado de estudios (AU).

Whitening toothpastes effect on enamel surface morphology, chemical profile and color: An In Vitro study / Efecto de las pastas dentales blanqueadoras sobre la morfología, el perfil químico y el color de la superficie del esmalte: un estudio In Vitro

Abstract To assess the effect of different whitening toothpastes on enamel surface morphology, chemical profile and their whitening efficiency. Sixty teeth were divided into 5 groups (12 teeth/group); Gp I: brushed with no toothpaste. Gp II: brushed with Pearl-based toothpaste. Gp III: brushed with Charcoal- based toothpaste. Gp IV: brushed with Alumina-based toothpaste. Gp V: brushed with salt and lemon- based toothpaste. Each tooth was brushed with a bean sized toothpaste wetted with distilled water twice daily by using standard electronic toothbrush for four weeks. Enamel surface morphology, chemical profile and color of each tooth were investigated. Chemical profile results and color measurements were analyzed statistically. Gp I revealed normal enamel surface morphology and chemical profile. Gp II and Gp III demonstrated surface morphology close to Gp I with insignificant reduction in mineral content. Gp IV showed obvious shallowing of perikymata ridges, exposure of fish-scale appearance, with pits and depressions, alongside to the significant reduction in mineral content. Gp V presented the most enamel surface alteration with widening of prism sheath and depressions all over the surface, besides the highest significant reduction in mineral content. Gp V, followed by Gp IV, demonstrated the highest color and whiteness changes, followed by Gp III, then Gp II, while the lowest value was in Gp I. Salt & lemon-based toothpaste, followed by Alumina-based, exerted the most considerable changes in the morphology and chemical profile of the enamel surface, beside to the higher whitening effects on teeth than the others.

Resumen El objetivo del presente estudio fue evaluar el efecto de diferentes pastas dentales blanqueadoras sobre la morfología y el perfil químico de la superficie del esmalte y su eficacia blanqueadora. Sesenta dientes se dividieron en 5 grupos (12 dientes/grupo); Grupo I: cepillado sin pasta de dientes. Grupo II: cepillado con pasta de dientes a base de perlas. Grupo III: cepillado con pasta de dientes a base de carbón. Grupo IV: cepillado con pasta de dientes a base de Alúmina. Grupo V: cepillado con sal y pasta de dientes a base de limón. Cada diente se cepilló con una pasta de dientes del tamaño de un frijol humedecida con agua destilada dos veces al día utilizando un cepillo de dientes electrónico estándar durante cuatro semanas. Se investigaron la morfología de la superficie del esmalte, el perfil químico y el color de cada diente. Los resultados del perfil químico y las mediciones de color se analizaron estadísticamente. El Grupo I reveló una morfología de la superficie del esmalte y un perfil químico normales. Los Grupos II y III demostraron una morfología de superficie cercana a la del Grupo I con una reducción insignificante en el contenido mineral. El grupo IV mostró una evidente reducción de las crestas perikymatas, exposición de apariencia de escamas de pez, con hoyos y depresiones, junto con una reducción significativa en el contenido mineral. El grupo V presentó la mayor alteración de la superficie del esmalte con ensanchamiento de la vaina del prisma y depresiones en toda la superficie, además de una significativa reducción en el contenido mineral. El grupo V, seguido del grupo IV, demostró los mayores cambios de color y blancura, seguido del grupo III, luego el grupo II, mientras que el valor más bajo se presentó en el grupo I. Las pastas dentales a base de sal y limón, seguidas de las de alúmina, ejercieron los cambios más considerables en la morfología y el perfil químico de la superficie del esmalte, además de tener mayores efectos blanqueadores sobre los dientes que las demás.

Phytochemical Screening and in vitro Anticancer Activity of Some Medicinal Plants Grown in the Kingdom of Saudi Arabia / Cribado Fitoquímico y Actividad Anticancerígena in vitro de Algunas Plantas Medicinales Cultivadas en el Reino de Arabia Saudita

SUMMARY: This study evaluated the phytochemical screening, antioxidant capacity, and in vitro anticancer activities of four plants namely, Gypsophila capillaris, Anabasis lachnantha, Haloxylon salicornicum, and Horwoodia dicksoniae which belong to four different families: Caryophyllaceae, Amaranthaceae, Chenopodiaceae, Brassicaceae, respectively. The total phenolics, anthocyanins, saponins, total antioxidant capacity (TAC), and DPPH assays were determined by spectrophotometer. In vitro anticancer activity was assessed using two human cancer cell lines; hepatocellular carcinoma (HepG-2) and breast adenocarcinoma (MCF-7) to estimate the inhibition concentration 50 % (IC50). The results showed that H. dicksoniae has the highest concentrations of phenolics and saponins, while H. salicornicum has the highest DPPH. The highest concentration of TAC was found in G. capillaries. Among the tested extracts, G. capillaries and H. salicornicum have the potential activity against MCF-7 and HepG-2 cell lines in vitro. The content of polyphenols in G. capillaries was profiled by high-performance liquid chromatography (HPLC). The highest concentration among the phenolic compounds was chlorogenic (60.8 µg/ml) while the highest concentration among the flavonoid compounds was hesperidin (1444.92 µg/ml). In summary, G. capillaries and H. salicornicum extracts have potent anticancer activity against HepG-2 and MCF-7 cell lines.

Este estudio evaluó la detección fitoquímica, la capacidad antioxidante y las actividades anticancerígenas in vitro de cuatro plantas, Gypsophila capillaris, Anabasis lachnantha, Haloxylon salicornicum y Horwoodia dicksoniae, que pertenecen a cuatro familias diferentes: Caryophyllaceae, Amaranthaceae, Chenopodiaceae y Brassicaceae, respectivamente. Los ensayos de fenólicos totales, antocianinas, saponinas, capacidad antioxidante total (TAC) y DPPH se determinaron mediante espectrofotómetro. La actividad anticancerígena in vitro se evaluó utilizando dos líneas celulares de cáncer humano; carcinoma hepatocelular (HepG-2) y adenocarcinoma de mama (MCF- 7) para estimar la concentración de inhibición del 50 % (IC50). Los resultados indicaron que H. dicksoniae tiene las concentraciones más altas de fenólicos y saponinas, mientras que H. salicornicum tiene el DPPH más alto. La mayor concentración de TAC se encontró en G. capillaries. Entre los extractos probados, G. capillaries y H. salicornicum tienen actividad potencial contra líneas celulares MCF-7 y HepG-2 in vitro. El contenido de polifenoles en G. capillaries se perfiló mediante cromatografía líquida de alta resolución (HPLC). La concentración más alta entre los compuestos fenólicos fue clorogénica (60,8 µg/ml), mientras que la concentración más alta entre los compuestos flavonoides fue la hesperidina (1444,92 µg/ml). En resumen, los extractos de Gypsophila capillaris y H. salicornicum tienen una potente actividad anticancerígena contra las líneas celulares HepG-2 y MCF-7.

Papel dos receptores farmacológicos AT1 e AT2 da Angiotensina II em doença periodontal induzida: aspectos moleculares / Role of pharmacological receptors AT1 and AT2 of Angiotensin II in caused periodontal disease: molecular aspects

O sistema renina angiotensina (SRA) é de grande importância para o equilíbrio hídrico e regulação da pressão arterial do organismo, além de estar associado ao estimulo de vias próinflamatórias. Seu principal peptídeo é a angiotensina II, que interage principalmente com os receptores do tipo 1 (AT1) e do tipo 2 (AT2). Foi encontrado interrelação entre as doenças cardiovasculares e a periodontite. Este estudo teve como objetivo avaliar os aspectos moleculares em camundongos submetidos a um modelo experimental de periodontite, observando a influência dos receptores de Ang II tipo 1 (AT1(-)) e Ang II tipo 2 (AT2(-)) na periodontite. Métodos: A periodontite experimental foi induzida colocando-se uma ligadura com fio de nylon 5.0 ao redor do segundo molar superior esquerdo de camundongos knockoutAT1(-), AT2(-) e selvagem (WT), subdivididos 2 grupos para cada linhagem: sem ligadura e ligadura, totalizando seis grupos: três controles e três experimentais. Após 15 dias da indução da doença os animais foram submetidos à eutanásia. Com o intuito de avaliar se as variações genéticas teriam influência sobre a periodontite foram realizadas as análises de citocinas, peptídeos e enzimas foram analisados a partir de tecidos gengivais por ELISA e RT-PCR. Resultados: Os animais WT e AT2(-) apresentaram resultados semelhantes em relação às citocinas IL-1ß, IL-6, TNF-α, com aumento dos níveis em relação aos saudáveis (p < 0,001). Houve diferenças significativas em IL-ß entre os grupo AT1(-)-L e WT-L (p < 0,05), e em IL-6 e TNF-α os grupos AT1(-)-L apresentaram diferenças significativas (p < 0,001) tanto quando comparado aos grupo WT-L quanto aos grupos AT2(-)-L. Os níveis de IL-10 foram maiores em WT-L (p < 0,01), enquanto os grupos AT2(-) e AT1(-) não apresentaram alterações significativas em relação a essa citocina. Houve diferenças significativas em Angiotensina II entre os grupos AT2(-)-NL e AT2(-)-L (p < 0,01); e em Angiotensina 1-7 entre os grupos AT1(-)-L e AT2(-)-L (p < 0,05). Para TLR2 houve diferenças entre os grupos WT-NL/WT-L (p < 0,05); AT1(-)-NL/AT1(-)-L (p < 0,01) e AT2(-)-NL/AT2(-) - L (p < 0,01). Para o receptor MAS houve diferenças entre os grupos WT-NL/WT-L (p < 0,001) e AT2(-)-NL/AT2(-)-L (p < 0,001), e também em relação ao grupo WT-L/AT1(-)-L (p < 0,001) e AT1(-)-L/AT2(-)-L (p < 0,001). Para a expressão dos peptídeos ECA e ECA2, houve diferença estatística apenas para ECA entre os tipos de grupos WT-NL/WT-L (p < 0,001). Conclusão: Os animais do grupo AT1(-) apresentaram menor inflamação que as demais linhagens doentes, assim como uma menor expressão do receptor Mas e Ang 1-7. Além disso os animais dos grupos WT e AT2(-) demonstraram resultados próximos em diversas análises, evidenciando que o bloqueio do receptor AT1, sobre os efeitos moleculares, é mais positiva (AU).

The renin angiotensin system (RAS) is of great importance for water balance and regulation of blood pressure in the body, in addition to being associated with the stimulation of proinflammatory pathways. Its main peptide is angiotensin II, which interacts mainly with type 1 (AT1) and type 2 (AT2) receptors. An interrelationship was found between cardiovascular diseases and periodontitis. This study aimed to evaluate the molecular aspects in mice subjected to an experimental model of periodontal disease, observing the influence of Ang II type 1 (AT1(-)) and Ang II type 2 (AT2(-)) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a ligature with 5.0 nylon thread around the upper left second molar of AT1(-), AT2(-) and wild-type (WT) knockout mice, subdivided into 2 groups for each strain: without ligation and ligation, totaling six groups: three controls and three experimental. After 15 days of disease induction, the animals were euthanized. In order to evaluate whether genetic variations would have an influence on periodontal disease, analyzes of cytokines were carried out, peptides and enzymes were analyzed from gingival tissues by ELISA and RT-PCR. Results: WT and AT2(-) animals showed similar results in relation to the cytokines IL-1ß, IL-6, TNF-α, with increased levels compared to healthy ones (p < 0.001). There were significant differences in IL-ß between the AT1(-)-L and WT-L groups (p < 0.05), and in IL-6 and TNF-α the AT1(-)-L groups showed significant differences (p < 0.001) both when compared to the WT-L and AT2(-)-L groups. IL-10 levels were higher in WT-L (p < 0.01), while the AT2(-) and AT1(-) groups did not show significant changes in relation to this cytokine. There were significant differences in Angiotensin II between the AT2(-)-NL and AT2(-)-L groups (p < 0.01); and in Angiotensin 1-7 between the AT1(-)-L and AT2(-)-L groups (p < 0.05). For TLR2 there were differences between the WT-NL/WT-L groups (p < 0.05); AT1(-)-NL/AT1(-)-L (p < 0.01) and AT2(-)-NL/AT2(-)-L (p < 0.01). For the MAS receptor there were differences between the WT-NL/WT-L (p < 0.001) and AT2(-)-NL/AT2(- )-L (p < 0.001) groups, and also in relation to the WT-L group /AT1(-)-L (p < 0.001) and AT1(-)-L/AT2(-)-L (p < 0.001). For the expression of ACE and ACE2 peptides, there was a statistical difference only for ACE between the types of WT-NL/WT-L groups (p < 0.001). Conclusion: The animals in the AT1(-) group showed less inflammation than the other diseased lines, as well as a lower expression of the Mas and Ang 1-7 receptor. Furthermore, animals from the WT and AT2(-) groups demonstrated similar results in several analyses, showing that the blockade of the AT1 receptor, on molecular effects, is more positive (AU).

Asociación del resultado auditivo final, en pacientes con hipoacusia súbita, a la respuesta a glucocorticoides in vitro / Association of the final hearing result, in patients with sudden hearing loss, to the response to glucocorticoids in vitro

Introducción: La hipoacusia súbita (HS) es poco frecuente y su etiopatogenia no está definida. La terapia con corticoides es de elección en base a recomendaciones de expertos por sus efectos teóricos y no en base a utilidad clínica demostrada. Objetivo: Evaluar si existe correlación entre el resultado auditivo final, de pacientes con HS tratados con corticoides, y la respuesta in vitro de sus leucocitos a corticoides, medida como diferencias en la expresión relativa de genes blanco del receptor de glucocorticoides. Material y Método: Estudio de casos (recuperación total) y controles (no recuperados) de pacientes con HS tratados con corticoides en el Hospital Clínico de la Universidad de Chile, durante 2017-2019. Se obtuvo DNA que fue almacenado en el Biobanco de Tejidos y Fluidos de la Universidad de Chile (BTUCH). Se purificaron y cultivaron leucocitos mononucleares de sangre periférica, expuestos in vitro a hidrocortisona. Se determinó la diferencia en la expresión relativa de genes blanco (IGFBP1, CAT, HSD17B12, APOA2), por Q-RTPCR, entre ambos grupos. Resultados: Se reclutaron 35 pacientes; se incluyeron para análisis 23: 11 casos y 12 controles, con edad promedio de 54,9 y 50,8 años respectivamente, distribución homogénea de sexo. No hubo diferencia estadísticamente significativa en la expresión relativa de los genes blanco, a la exposición in vitro a corticoides, entre ambos grupos. Conclusión: En nuestro estudio, modelo, y sistema de evaluación no se evidenciaron efectos de los corticoides. No podemos descartar que, con un número mayor de pacientes, otros genes blanco u otros protocolos de estudio podrían detectarse diferencias.

Introduction: Sudden hearing loss (SHL) is rare and its etiopathogenesis is still not clear. Corticosteroid therapy is of choice based on expert recommendations due to its theoretical effects and no based on proved clinical efficacy. Objectives: To assess whether there is a correlation between the final auditory outcome of patients with SHL treated with corticosteroids and the in vitro response of their leukocytes to corticosteroids, measured as differences in the relative expression of glucocorticoid receptor target genes. Material and Method: Case-control (total recovery and not recovered respectively) study of patients with SHL treated with corticosteroids at Clinical Hospital Universidad de Chile between 2017 and 2019. DNA was obtained and stored in the Biobanco de Tejidos y Fluidos de la Universidad de Chile (BTUCH). Peripheral blood mononuclear leukocytes were purified and cultured and then exposed to hydrocortisone. The difference in the relative expression of target genes (GFBP1, CAT, HSD17B12, APOA2), by Q-RTPCR was determined. Results: Thirty-five patients were recruited, 24 were included for the analysis: 11 cases and 12 controls, with and average age of 54.9 and 50.,8 years respectively, homogeneous sex distribution. There was no statistically significant difference in the relative expression of the target genes, upon in vitro exposure to corticosteroids, between both groups. Conclusion: In our study, model and evaluation system, no effects of corticosteroids were evidenced. With a larger number of patients, other target genes or other study protocols, we cannot rule out that differences could be detected.

Small Molecules Accelerate Skin Wound Healing: Shikonin Efficacy and Mechanism of Action in Mice / Las Moléculas Pequeñas Aceleran la Curación de Heridas en la Piel: Eficacia de Shikonin y Mecanismo de Acción en Ratones

SUMMARY: The objective of this study was to investigate the therapeutic wound healing potential and molecular mechanisms of shikonin as small molecules in vitro. A mouse burn model was used to explore the potential therapeutic effect of shikonin; we traced proliferating cells in vivo to locate the active area of skin cell proliferation. Through the results of conventional pathological staining, we found that shikonin has a good effect on the treatment of burned skin and promoted the normal distribution of skin keratin at the damaged site. At the same time, shikonin also promoted the proliferation of skin cells at the damaged site; importantly, we found a significant increase in the number of fibroblasts at the damaged site treated with shikonin. Most importantly, shikonin promotes fibroblasts to repair skin wounds by regulating the PI3K/AKT signaling pathway. This study shows that shikonin can effectively promote the proliferation of skin cell, and local injection of fibroblasts in burned skin can play a certain therapeutic role.

El objetivo de este trabajo fue investigar el potencial terapéutico de cicatrización de heridas y los mecanismos moleculares de la shikonina como moléculas pequeñas in vitro. Se utilizó un modelo de quemaduras en ratones para explorar el posible efecto terapéutico de la shikonina; Rastreamos las células en proliferación in vivo para localizar el área activa de proliferación de células de la piel. A través de los resultados de la tinción para patología convencional, encontramos que la shikonina tiene un buen efecto en el tratamiento de la piel quemada y promueve la distribución normal de la queratina de la piel en el sitio dañado. Al mismo tiempo, la shikonina también promovió la proliferación de células de la piel en el sitio dañado. Es importante destacar que encontramos un aumento significativo en la cantidad de fibroblastos en el sitio dañado tratado con shikonina. Lo más importante es que la shikonina promueve la función reparadora de fibroblastos en las heridas de la piel regulando la vía de señalización PI3K/ AKT. Este estudio muestra que la shikonina puede promover eficazmente la proliferación de células de la piel y que la inyección local de fibroblastos en la piel quemada puede desempeñar un cierto papel terapéutico.

Endotoxin test by recombinant Factor C for 0.9% sodium chloride injection

Endotoxin contamination is a threat to the safety of pharmaceutical products, especially parenteral drugs. Any sterile and/or pyrogen-free pharmaceutical product requires regulatory specifications to ensure safe patient use. This study covers the performance evaluation study of an endotoxin quantitation commercial kit by recombinant Factor C (rFC), Endozyme II® Go, for 0.9% sodium chloride injection. The samples were spiked with endotoxin solutions between 0.0005 and 10 EU/mL and tested by the rFC kit to evaluate precision, accuracy, detection and quantification limits, linearity, and robustness. Each of the six points was assayed at least five times.The relative standard deviation for precision testing ranged from 1.9 to 8.3%. The recovery accuracy values of endotoxin were between 61% and 125% for the range from 0.005 to 10 EU/mL. The results demonstrated that the rFC method allows endotoxin quantification with accuracy, precision, specificity, and linearity for the range of 0.005 and 10 EU/mL for 0.9% sodium chloride injection. (AU)

A contaminação por endotoxinas é uma ameaça à segurança dos produtos farmacêuticos, especialmente dos medicamentos parenterais. Qualquer produto farmacêutico estéril e/ou livre de pirogênios requer especificações regulatórias para garantir a segurança de uso para o paciente. Este estudo abrange o estudo de avaliação de desempenho empregando o kit comercial Endozyme II® Go para quantificação de endotoxina, por Fator C recombinante (FCr), em amostras de cloreto de sódio 0,9% para uso parenteral. As amostras foram fortificadas com cinco concentrações distintas de soluções de endotoxina na faixa entre 0,0005 e 10 UE/mL. Cada um dos cinco níveis foi testado pelo menos cinco vezes para avaliação dos critérios de precisão, exatidão, limites de detecção e quantificação, linearidade e robustez. O desvio padrão relativo para os testes de precisão variou de 1,9 a 8,3%. Os valores de recuperação de endotoxina para o parâmetro exatidão estiveram compreendidos entre 61% e 125%. Os resultados demonstraram que o método por FCr permite a quantificação de endotoxinas com exatidão, precisão, especificidade e linearidade para a faixa de 0,005 e 10 UE/mL em amostras de cloreto de sódio 0,9% para uso parenteral. (AU)

Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp / Atividade antifúngica de 2-cloro-N-fenilacetamida: uma nova molécula com atividade fungicida e anti-biofilme contra Candida spp. resistentes a fluconazol

In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

No atual contexto de patógenos fúngicos resistentes emergentes tais como Candida albicans e Candida parapsilosis, a descoberta de novos agentes antifúngicos é uma questão urgente. Esta pesquisa teve como objetivo avaliar o potencial antifúngico da 2-cloro-N-fenilacetamida contra cepas clínicas de C. albicans e C. parapsilosis resistentes a fluconazol. A atividade antifúngica da substância foi avaliada in vitro através da determinação da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM), ruptura e inibição da formação de biofilme, ensaios de sorbitol e ergosterol, e associação entre esta molécula e antifúngicos comuns, anfotericina B e fluconazol. O produto teste inibiu todas as cepas de C. albicans e C. parapsilosis, com uma CIM variando de 128 a 256 µg.mL-1, e uma CFM de 512-1,024 µg.mL-1. Também inibiu até 92% da formação de biofilme e causou a ruptura de até 87% de biofilme pré-formado. A 2-cloro-N-fenilacetamida não promoveu atividade antifúngica pela ligação ao ergosterol da membrana celular fúngica, tampouco danificou a parede celular. Antagonismo foi observado ao combinar esta substância com anfotericina B e fluconazol. A substância exibiu atividade antifúngica significativa ao inibir tanto as células planctônicas quanto o biofilme das cepas resistentes ao fluconazol. Sua combinação com outros antifúngicos deve ser evitada e seu mecanismo de ação deve ser estabelecido.

Effect of Toothpaste Fluoride Concentration on Color Stability of Resin Composites: An In Vitro Study

ABSTRACT Objective: To investigate the effects of two different dentifrice fluoride concentrations on the color stability of the composite. Material and Methods: Twenty-seven specimens (2×4×5 mm) each of microfilled (Gradia, GC, Japan) and nanohybrid (Grandio, VOCO, Germany) composites were prepared. The specimens were randomly divided into six groups (control, Fluoflor caries protection toothpaste with 1450ppm Fluoride (EXW, France), and Fluoflor kids toothpaste with 500ppm Fluoride (EXW, France) (n = 9). The specimens were immersed in a mixture of artificial saliva and toothpaste in a ratio of 1:3 and applied for 60 seconds every 12 hours for 42 days. The control samples were incubated in artificial saliva at 37°C. Primary and secondary color measurements were performed using color parameters (L∗a∗b) with a spectrophotoshade (MHT Optic Research AG, Niederhasli, Switzerland). Data were analyzed using a two-way analysis of variance at a significance level of 0.05. Results: According to the two-way ANOVA analysis, there was no significant difference in color change between the composites and no difference in the level of discoloration between different fluoride concentrations(p>0.05). Also, None of the dentifrices caused clinically significant color changes(∆E˂3.3). Conclusion: No clinically unacceptable color changes were observed in the microfilled and nanofilled composites with different concentrations of fluoride toothpaste.

Assessment of Efficiency of Bioactive Glass, Self-Assembling Peptide, and Ozone Remineralising Agents on Artificial Carious Lesion

ABSTRACT Objective: To assess the efficacy of bioactive glass, self-assembling peptide, and ozone-remineralizing agents on the artificial carious lesion. Material and Methods: On the extracted 60 premolar teeth, an artificial carious lesion/demineralization was created. Later, the remineralization of demineralized teeth was done with respective remineralizing agents (Group A: Calcium sodium phosphosilicate (bioactive glass), Group B: Self-assembling peptide, Group C: Ozone remineralizing agents and Group D (Control): De ionized water. The degree of demineralization and remineralization were evaluated using the Vickers Hardness Number. Results: There was a decrease in microhardness from baseline to demineralization in all the groups, and this reduction was found to be statistically considerable. After the remineralization of demineralized samples with respective remineralizing agents, there was an increase in microhardness of 312.38, 276.67, and 254.42 in groups A, B, and C, respectively. In contrast, in Group D, there were no changes. Conclusion: Bioactive glass and self-assembling peptides had higher remineralizing capacities, which can be used to treat early carious lesions.

Effect of Water and Chlorhexidine with Different Pressures of Oral Irrigation Device on the Surface Roughness and Topography of Giomer

Abstract Objective: To evaluate the effect of different pressures of an oral irrigation device (OID) and the irrigation solution type on the surface roughness of the giomer restorative material. Material and Methods: In this in vitro study, disk-shaped giomer samples were fabricated and assigned to 5 groups (n=23): Group 1, storage in distilled water (control); Group 2, OID #7 pressure/ water; Group 3, OID #10 pressure/ water; Group 4, OID #7 pressure/ 0.05% CHX; Group 5, OID #10 pressure/ 0.05% CHX. The samples' treatment simulated a one-year application of OID. Surface roughness (Ra) and topography of the giomer were evaluated using profilometry and scanning electron microscopy. The data were analyzed with Paired t-test, Tukey, and ANOVA tests (α=0.05). Results: The Ra of the samples increased significantly after treatment with OID (p<0.001). The roughness increase in groups with a pressure of 10 was higher than those with a pressure of 7 (p<0.001). The effect of pressure on surface changes was significant (p<0.001). However, the solution type and the cumulative effect of these two factors were insignificant (p=0.08 and p=0.43, respectively). Conclusion: Oral irrigation device with both solutions significantly increased the surface roughness and topographic changes of the giomer. The severity of these changes was related to the device's pressure.

Regulação da Septina 9 na síndrome do desconforto respiratório agudo associado à malária / The role of Septin 9 in regulating vascular permeability in malarial respiratory distress syndrome

A malária é um problema mundial de saúde, com 249 milhões de casos de infecção, ocasionando 608 mil mortes no ano de 2022. Causada pelo gênero Plasmodium, são cinco principais espécies causadoras da malária no ser humano, o Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium vivax e Plasmodium falciparum sendo os dois últimos responsáveis pelo maior número de casos clínicos e mortes ao redor do mundo, transmitida pelo mosquito fêmea do gênero Anopheles durante o repasto sanguíneo. Sabe-se ainda que eritrócitos infectados por Plasmodium berghei ANKA causam alteração no citoesqueleto de actina, consequentemente levando a hiperpermeabiliade da barreira endotelial. Em experimentos in vitro, a imunofluorescência, foi observada alteração do citoesqueleto de actina em células estimuladas com eritrócitos parasitados por PbA (EP), em contrapartida, aquelas não estimuladas (NE) e estimuladas com eritrócitos não parasitados por PbA (EnP), não mostraram alterações no mesmo. Nos experimentos in vivo, ao observar dados coletados, sendo estes respiratórios (penh e frequência respiratória) e parasitemia coletados no 7º DPI, foi observado um mesmo padrão entre o experimento 1 e o experimento 2. Os animais infectados com 106 de eritrócitos infectados, foram alocados em dois grupos, sendo eles hiperparasitemia (HP) ou síndrome do desconforto respiratório agudo-associado a malária (SDRA/SDRA-MA) e comparados àqueles não infectados (NI). Os animais NI, não apresentam parasitemia, em contrapartida, os animais SDRA, tem maior parasitemia que os HP, visto que estes têm aumento em sua parasitemia após o 12º DPI, e assim seguem aumentando gradativamente até levar os animais a óbito.O penh tem o mesmo padrão que a parasitemia, os NI com penh mais baixa que os HPs e os SDRA, sendo dentre estes, o grupo SDRA o mais elevado. A frequência respiratória, por sua vez se apresenta mais elevada no grupo NI, sendo o grupo SDRAmenor que o HP, um achado tido como normal, visto que os pulmões de animais com SDRA sofrem maior dano que os HPs. Apesar de não apresentar um valor significativo, as imagens de gel SDS-PAGE (WB) mostram maior concentração da Septina 9 nos animais com SDRA em comparação com os HPs e com os NIs. O mesmo é observado na qRT-PCR, mesmo sem significância estatística, o valor mostrado nos gráficos temmaior concentração nos SDRA. Assim, a Septina 9 está presente nas CEPP, e, mesmo sem significância estatística, da mesma forma que está presente nas amostras de tecido pulmonar utilizadas no WB e qT-PCR. É hipotetizado ainda que esta proteína pode ser ativada e assim sofrer alteração em sua localização intracelular

Malaria is a global health problem, with 249 million cases of infection, causing 608 thousand deaths in the year 2022. Caused by the genus Plasmodium, there are five main species that cause malaria in humans, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium vivax and Plasmodium falciparum, the last two being responsible for the largest number of clinical cases and deaths around the world, transmitted by the female mosquito of the genus Anopheles during blood meal. It is also known that erythrocytes infected by Plasmodium berghei ANKA (PbA) cause changes in the actin cytoskeleton, consequently leading to hyperpermeability of the endothelial barrier. In in vitro experiments, immunofluorescence, changes in the actin cytoskeleton were observed in cells stimulated with erythrocytes parasitized by PbA (EP), in contrast, those not stimulated (NE) and stimulated with erythrocytes not parasitized by PbA (EnP), did not show changes the same. In the in vivo experiments, when observing collected data, these being respiratory (penh and respiratory frequency) and parasitemia collected on the 7th DPI, the same pattern was observed between experiment 1 and experiment 2. Animals infected with 106 infected erythrocytes were allocated into two groups, namely hyperparasitemia (HP) or malaria-associated acute respiratory distress syndrome (ARDS/ARDS-MA) and compared to those not infected (NI). NI animals do not present parasitemia, on the other hand, ARDS animals have greater parasitemia than HP animals, as the latter have an increase in their parasitemia after the 12th DPI, and thus continue to gradually increase until the animals die. the same pattern as parasitemia, NI with lower penh than HPs and ARDS, among these, the ARDS group being the highest. The respiratory rate, in turn, is higher in the NI group, with the ARDS group being lower than the HP, a finding considered normal, given that the lungs of animals with ARDS suffer greater damage than the HPs. Despite not showing a significant value, SDS-PAGE (WB) gel images show a higher concentration of Septin 9 in animals with ARDS compared to HPs and NIs. The same is observed in qRT-PCR, even without statistical significance, the value shown in the graphs has a higher concentration in ARDS. Thus, Septin 9 is present in CEPP, and, even without statistical significance, in the same way that it is present in lung tissue samples used in WB and qT-PCR. It is also hypothesized that this protein can be activated and thus undergo changes in its intracellular location

The Effect of Restoration Polymerization and Residual Dentine Thickness on Thermal Changes of Pulp Chamber of Immature Permanent Teeth

ABSTRACT Objective: To evaluate the pulpal temperature changes due to the polymerisation of resin and glass ionomer-based materials in dentine thicknesses in immature permanent teeth with open apices. Material and Methods: Forty extracted sound human third molar teeth with open apices were included. The width of the cavities prepared on the occlusal surface was 4×5 mm. The depth was 2 mm in the resin groups. 4 mm in the groups in which glass ionomer liner was applied before composite restoration. The coronal parts of the samples were then placed on an acrylic plate with three gaps for feeding-extraction needles and the thermocouple. The temperature changes were recorded. The data was analyzed by SPSS. Statistical significance was accepted as p<0.05. Results: The temperature increase in the group of 1 mm remaining dentin thickness revealed higher results than the values detected from the 2 mm group (1.01 °C) (p=0.00). The mean values (1.49 °C, 1mm) of temperature changes in only glass ionomer applied group were lower than the avarage values (2.210°C, 1mm) determined in the polymerization process of resin composites with light-emitting diode devices. Conclusion: In a remaining dentin thickness of 1 mm in teeth with open apices, using a glass ionomer liner might be a useful effort for protecting the pulp from the heat generated by polymerisation devices.

Accuracy of Working Length Determination - Electronic Apex Locator Versus Cone Beam Computed Tomography

ABSTRACT Objective: To compare the accuracy of working length determination between cone beam computed tomography (CBCT) and electronic apex locator by measuring the actual working length of teeth. Material and Methods: A total of 150 single-rooted tooth assessed by radiograph undergoing root canal therapy were selected. The process was repeated to obtain a buccolingual and mesiodistal section of all teeth. The measurement line was considered from the reference occlusal plane following the center of the canal to the terminus. All information regarding the accuracy of cone-beam computed tomography and apex locator was noted in a pre-designed proforma. Results: CBCT consistently demonstrated high accuracy across all tooth types in both jaws. The electronic apex locator exhibited varying precision, with greater accuracy observed in the mandible. Statistical analyses revealed significant differences in electronic apex locator accuracy among tooth types in the maxilla (p=0.042), emphasizing the importance of specific clinical considerations. Conclusion: Cone beam computed tomography emerges as a reliable diagnostic tool for accurate working length determination, especially in complex cases, while the electronic apex locator remains valuable with careful consideration of potential variations in accuracy. An individualized approach, considering tooth type, jaw location, and clinical context, is crucial for precise working length determination in endodontic practice.

Hymenaea rubriflora Ducke stem bark extract has vasorelaxant and contractile inhibition capacity

Abstract We investigated the vasodilatory effects of Hymenaea rubriflora Ducke stem bark extract (HRHAc). Vascular reactivity of the aortic rings of Wistar rats was tested by in vitro cumulative doses (0.1 - 729 µg/mL). Rats (n=5) were treated with 25 (G25), 50 (G50) and 100 (G100) mg/ kg of HR-HAc or saline (control group - CG) for four weeks. An in vitro assay resulted in dose-dependent relaxation of the aortic rings with functional endothelium, which was inhibited in the presence of L-NAME. Rings of the treated animals increased acetylcholine relaxing potency at all doses, with a greater effect on G50 (pD2 = 7.8±0.1, Emax = 95.6±1.1) and a decreased contractile potency to phenylephrine in G25 (pD2 = 6.9±0.06, Emax = 61.5±6.0%) and G50 (pD2= 6.6±0.06, Emax = 71.0±8.5%) when compared to the CG in the presence and absence of endothelium (pD2= 6.4± 0.1, 6.4±0.1 and 6.9±0.1, respectively). Cumulative doses of nitroprusside resulted in increased relaxing potency in all treated groups and maintained Emax at 100%. It is concluded that HR-HAc has vasorelaxant capacity and inhibitory vascular contraction activity applied either directly to aortic rings or after treatment with in vivo supplementation, which places this extract as a potential nutraceutical or pharmacological agent for treating diseases associated with vascular dysfunction.

Modelos avançados de pele humana 3D in vitro: epiderme bioimpressa e pele tricamada contendo hipoderme para avaliação de segurança e mimético de dermatite atópica nocaute de IL-13Rα2 para estudo da função barreira / Advanced in vitro 3D human skin models: bioprinted epidermis and trilayer skin containing hypodermis for safety assessment and atopic dermatitis mimetic with IL-13Rα2 knockout for barrier function study

A reconstrução de modelos avançados de pele tridimensional in vitro, que corresponda de forma mais fidedigna ao complexo microambiente da pele humana, depende da utilização de inovações tecnológicas e da adição de novos tipos celulares representativos da pele humana. Desta maneira, estes miméticos fornecem uma plataforma de alta relevância para estudos de fisiopatologia da pele, além de propiciar um sistema para a avaliação da segurança e eficácia de cosméticos e medicamentos alternativo ao uso de animais. Dessa maneira, o Capítulo I compara a performance de uma epiderme reconstruída humana (RHE) bioimpressa com a manual utilizando o teste in vitro de irritação cutânea descrito no guia OCDE número 439. Nossos resultados demonstram que ambos os modelos de pele exibiram morfologia estratificada e a função barreira epidérmica equivalente aos modelos validados. Nos testes de irritação in vitro, ambos modelos distinguiram corretamente as substâncias de referência, classificadas entre irritantes ou não-irritantes de acordo com o limiar de viabilidade de 50%. Esse resultado indica que a bioimpressora poderia ser de grande utilidade para a automação da reconstrução de modelos epidérmicos. O tecido hipodérmico possui importante papel na homeostase da pele humana. O Capítulo II aborda a reconstrução de uma pele tricamada, contendo a camada hipodérmica, além da epiderme e derme. Usando esferoides de adipócitos diferenciados in vitro, um modelo de pele tricamada em matriz de colágeno foi construído. Ao comparar este com a pele bicamada obtivemos maior expressão de loricrina e involucrina no modelo tricamada, indicando um potencial para maior função barreira, além de maior expressão de PPAR-γ. Testes de função barreira através da resistividade elétrica não demonstraram diferenças entre os modelos, mas a aplicação de SDS a 5 mg/ml por 18 horas induziu o aumento da viabilidade na pele tricamada. Além disso, após a aplicação de SDS a 2,5% para induzir uma irritação aguda, seguida de recuperação por 42h, obtivemos maior viabilidade na pele tricamada, indicando melhor recuperação pós-lesão irritativa induzida. A pele tricamada é promissora para estudos do metabolismo da pele humana e recuperação de lesões. A dermatite atópica (DA) é uma doença eczematosa de pele caracterizada por inflamação do tipo Th2 e alteração da barreira epidérmica. IL-13 e IL-4 são centrais no comprometimento da barreira epidérmica na DA. Entre os receptores de IL-13 em queratinócitos, o receptor IL-13Rα2, tem um papel controverso na alteração da barreira cutânea. O objetivo do Capítulo III foi estudar a deleção da expressão de IL-13Rα2 em RHE, que foram expostas a IL-4 e IL-13, e avaliadas conforme a expressão dos receptores e de proteínas alteradas na DA. As epidermes com knockout em IL-13Rα2 apresentaram redução da expressão de NELL2 (p<0,0021), tipicamente aumentadas na DA. Além disso, houve redução da expressão do receptor do IL-2Rγ. Assim, um possível papel de exacerbação da DA do receptor IL-13Rα2 deve ser estudado mais extensamente para ser caracterizado

The reconstruction of advanced three-dimensional in vitro skin models, which more reliably correspond to the complex microenvironment of human skin, depends on the use of technological innovations and the addition of new cell types representative of human skin.In this way, these mimetics provide a highly relevant platform for studies of skin pathophysiology, in addition to providing a system for evaluating the safety and efficacy of cosmetics and medicines alternative to animal use. In this way, Chapter I compares the performance of a bioprinted human reconstructed epidermis (RHE) with a manual one using the in vitro skin irritation test described in OECD guide number 439. Our results demonstrate that both skin models exhibited stratified morphology and the epidermal barrier function equivalent to validated models. In in vitro irritation tests, both models correctly distinguished the reference substances, classified as irritating or non-irritating according to the viability threshold of 50%. This result indicates that the bioprinter could be of great use for automating the reconstruction of epidermal models Hypodermic tissue plays an important role in the homeostasis of human skin. Chapter II addresses the reconstruction of a three-layer skin, containing the hypodermic layer, in addition to the epidermis and dermis. Using in vitro differentiated adipocyte spheroids, a trilayer skin model in collagen matrix was constructed. When comparing this with bilayer skin, we obtained greater expression of loricrin and involucrin in the trilayer model, indicating a potential for greater barrier function, in addition to greater expression of PPAR-γ . Barrier function tests using electrical resistivity did not demonstrate differences between the models, but the application of SDS at 5 mg/ml for 18 hours induced an increase in viability in the three-layer skin. Furthermore, after applying 2.5% SDS to induce acute irritation, followed by recovery for 42 hours, we obtained greater viability in the three-layer skin, indicating better recovery after induced irritant injury. Trilayer skin holds promise for studies of human skin metabolism and injury recovery. Atopic dermatitis (AD) is an eczematous skin disease characterized by Th2-type inflammation and alteration of the epidermal barrier. IL-13 and IL-4 are central to the impairment of the epidermal barrier in AD. Among the IL-13 receptors on keratinocytes, the IL-13Rα2 receptor has a controversial role in altering the skin barrier. The objective of Chapter III was to study the deletion of IL-13Rα2 expression in RHE, which were exposed to IL-4 and IL-13, and evaluated according to the expression of receptors and proteins altered in AD. Epidermis with IL-13Rα2 knockout showed reduced NELL2 expression (p<0.0021), typically increased in AD. Furthermore, there was a reduction in the expression of the IL-2Rγ receptor. Therefore, a possible AD exacerbation role of the IL-13Rα2 receptor should be studied more extensively to be characterized

Interação de cepas de Lactobacillus crispatus com Streptococcus agalactiae em culturas celulares mimetizando o ambiente vaginal / Interaction between Lactobacillus crispatus and Streptococcus agalactiae strains in cell cultures mimicking the vaginal environment

A microbiota vaginal é dominada pelo gênero Lactobacillus, mantendo um pH ácido crucial para a saúde. Alterações hormonais e a menopausa podem impactar essa microbiota. Estreptococos do Grupo B (EGB) são associados a infecções neonatais e podem colonizar a microbiota vaginal. A interação entre Lactobacillus e EGB é complexa, com resultados conflitantes em estudos in vitro e in vivo. O uso de probióticos contendo Lactobacillus pode ter benefícios, como alterar a positividade para EGB em um grupo que recebeu o tratamento. Contudo a replicabilidade deste resultado é limitada, e os mecanismos envolvidos nessa interação ainda são pouco elucidados. Portanto o objetivo desse estudo foi caracterizar cepas de EGB, avaliar sua interação com L. crispatus em diversas condições. Neste estudo foram selecionadas seis cepas, pertencentes aos sorotipos Ia, II, III e V. Essas cepas foram caracterizadas de acordo com o sorotipo capsular, presença de genes de virulência (hialuronidase, ß-citolisina/hemolisina, e ilhas de pili 1, 2a e 2b), resistência a antimicrobianos (penicilina, cefepima, vancomicina, eritromicina e clindamicina), curva de crescimento, Restriction Fragment Length Polymorphism (RFLP) e Multi-Locus Sequence Typing (MLST). O resultado de MLST identificou os sequence types ST1, ST23 ST28 e ST182, e os clonal complexes CC1, CC19 e CC23. Cepas possuem a maior parte dos genes de virulência testados. Somente uma cepa apresentou alguma resistência, no caso à eritromicina e clindamicina. Resultados que estão de acordo com a literatura como os principais descritos em isolados clínicos. Os ensaios de interação realizados foram: crescimento em co-cultura, formação de biofilme, ensaios de adesão e exclusão em cultura celular e por fim, análise do perfil proteico. Alguns efeitos de L. crispatus no EGB observados foram aumento na formação de biofilme de EGB e diminuição da sua proliferação quando em co-cultura. Contudo esses resultados variaram de acordo com a cepa de EGB. Nos ensaios de competição em cultura celular, a presença de L. crispatus levou à diminuição da adesão em uma cepa pertencente ao sorotipo III, e aumento de adesão em uma cepa pertencente ao sorotipo V. Quando a cultura de células recebeu tratamento prévio de L. crispatus, nos ensaios de exclusão, houve menor adesão de EGB. Contudo todos os efeitos observados são cepa-dependente; não foram encontradas associações entre o comportamento das cepas e seu sorotipo capsular ou fenótipo MLST. Estudos futuros podem elucidar as respostas ativadas na presença de microrganismos comensais

The vaginal microbiota is dominated by the genus Lactobacillus, which helps to maintain a crucial acidic pH for health. Hormonal changes and menopause can impact this microbiota. Group B Streptococcus (GBS) are associated with neonatal infections and can colonize the vaginal microbiota. The interaction between Lactobacillus and GBS is complex, with conflicting results in in vitro and in vivo studies. The use of probiotics containing Lactobacillus may have benefits, such as altering GBS positivity in a treated group. However, the replicability of this result is limited, and the mechanisms involved in this interaction are still poorly understood. The use of probiotics containing Lactobacillus may have some benefits, but further research is needed. These strains were characterized based on capsular serotype, presence of virulence genes (hyaluronidase, ß-cytolysin/hemolysin, and pili islands 1, 2a, and 2b), antimicrobial resistance (penicillin, cefepime, vancomycin, erythromycin, and clindamycin), growth curve, Restriction Fragment Length Polymorphism (RFLP), and Multi-Locus Sequence Typing (MLST) Therefore, the aim of this study was to characterize GBS strains, evaluate their interaction with L. crispatus under various conditions. Six strains were selected for this study, belonging to serotypes Ia, II, III, and V. MLST results identified sequence types ST1, ST23, ST28, and ST182, and clonal complexes CC1, CC19, and CC23. Most strains possessed the tested virulence genes, with only one strain showing resistance to erythromycin and clindamycin. These results are in line with the literature. Interaction assays included co-culture growth, biofilm formation, adhesion and exclusion assays in cell culture, and, finally, protein profile analysis. Some observed effects of L. crispatus on GBS included an increase in GBS biofilm formation and a decrease in GBS proliferation during co-culture. However, these results varied according to the GBS strain. GBS interaction with L. crispatus resulted in increased biofilm formation and decreased proliferation in co-culture. In cell culture competition assays, the presence of L. crispatus led to decreased adhesion in one GBS strain belonging to serotype III and increased adhesion in one strain belonging to serotype V. When cell culture received pre-treatment with L. crispatus, exclusion assays showed lower GBS adhesion. However, all observed effects are strain-dependent; no associations were found between strain behavior and capsular serotype or MLST phenotype. Future studies may elucidate the responses activated in the presence of commensal microorganisms

Novas formulações de Anexina A1 como estratégia terapêutica para a colite experimental / New formulations of Annexin A1 as a therapeutic strategy to experimental colitis

A Anexina A1 (AnxA1) é uma proteína de 37 kDa que controla o desenvolvimento da reação inflamatória inata, e favorece a eferocitose e o reparo tecidual. Em doenças inflamatórias intestinais (DIIs), tanto a AnxA1 endógena, como a sintética e o peptídeo sintético mimético ao N-terminal da proteína (Ac2-26) inibem o desenvolvimento de doença e induzem a cicatrização. O presente projeto teve o objetivo de obter novas formulações para carrear a AnxA1 recombinante (rAnxA1) ou o Ac2-26 e testar suas eficácias no modelo de colite experimental induzida pelo dextram sulfato de sódio (DSS, 0-6 dias) em camundongos C57Bl/6 machos. A rAnxA1 foi funcionalizada em nanocápsulas de núcleo lipídico de parede múltipla (MLNC) pela ligação Zn2+, com alta eficência de incorporação (92%) e adminsitrada pelas vias oral, intravenosa ou intraperitoneal durante a fase latente da doença (6º-9º dia). Somente o tratamento intraperitoneal com MLNC-AnxA1 (12,5 µg/mL) reduziu significativamente os sinais clínicos da doença, restaurou a integridade da estrutura colônica e a proliferação celular, bem como aumentou expressão de junções celulares da barreira intestinal. Ainda, MLNC-AnxA1 induziu a polarização de macrófagos para o fenótipo M2 in vivo no tecido inflamado e in vitro após estímulo com lipopolissacarídeos (LPS) bacteriano. Na tentativa de obter uma formulação terapêutica com atividade por vial oral, o peptídeo Ac2-26 foi incorporado em sílica mesoporosa ordenada SBA-15 e revestidos com Eudragit® L30-D55. A incorporação do peptídeo foi efetiva (88%) e a administração oral de Eudragit-SBA15-Ac2-26 (6º-9º dia; 200 µg/camundongo; 8 mg/kg) reduziu significativamente os sintomas clínicos e inflamação. De fato, ensaios de PET-SCAN mostraram que o SBA-15 permaneceu no intestino por até 16 horas após a administração e promoveu a liberação do peptídeo no intestino inflamado. Em cultura celular de epitélio (Caco-2), Eudragit-SBA15-Ac2-26 favoreceu a internalização de Ac2-26. Em conjunto, as duas estratégias expermentais de entrega do rAnxA1 ou Ac2-26 foram eficientes e os resultados obtidos sugerem que mais estudos devem ser realizados para a confirmação das estratégias de tratamento. Com o intuito de buscar ferramentas para ampliar estes estudos, durante estágio BEPE foram realizados estudos em cultura de células epiteliais baseado em células-tronco adultas diferenciadas in vitro. Os resultados mostraram que rAnxA1 ou Ac2-26 protegeram a integridade epitelial após desafio com LPS, pela regulação positiva da expressão das junções oclusivas e aderentes e redução da expressão de claudina-2, responsável pelo aumento da permeabilidade intercelular; pela modulação negativa decitocinas pró-inflamatórias CXCL-1 e MCP-1, e positiva de citocina antiinflamatória IL-10. Desta forma, padronizamos um novo modelo de cultura celular ainda não testada para a AnxA1 ou Ac2-26, que poderá ser empregada para desvendar os mecanismos da MLNC-AnxA1 e do Eudragit-SBA15-Ac2-26

Annexin Al (AnxA1) is a 37 kDa protein that controls the development of the innate inflammatory reaction, and favors efferocytosis and tissue repair. In inflammatory bowel diseases (IBDs), both endogenous and synthetic AnxA1 and the synthetic peptide mimetic to the N-terminal of the protein (Ac2-26) inhibit the development of disease and induce healing. The present project aimed to obtain new formulations to carry recombinant AnxA1 (rAnxA1) or Ac2-26 and test their efficacy in the experimental colitis model induced by dextram sodium sulfate (DSS, 0-6 days) in C57Bl/6 mice. rAnxA1 was functionalized into multiwall lipid core nanocapsules (MLNC) by Zn2+ binding, with high incorporation efficiency (92%) and administered orally, intravenously or intraperitoneally during the latent phase of the disease (6º-9º day). Only intraperitoneal treatment with MLNC-AnxA1 (12.5 µg/mL) significantly reduced the clinical signs of the disease, restored the integrity of the colonic structure and cell proliferation, as well as increased the expression of intestinal barrier cell junctions. Furthermore, MLNC-AnxA1 induced macrophage polarization to the M2 phenotype in vivo in inflamed tissue and in vitro after stimulation with bacterial lipopolysaccharides (LPS). In an attempt to obtain a therapeutic formulation with oral activity, the Ac2-26 peptide was incorporated into ordered mesoporous silica SBA-15 and coated with Eudragit® L30-D55. Peptide incorporation was effective (88%) and oral administration of Eudragit-SBA15-Ac2-26 (6º-9º day; 200 µg/mice; 8 mg/kg) significantly reduced clinical symptoms and inflammation. PET-SCAN assays showed SBA-15 remained in the intestine for up to 16 hours after administration and promoted the release of the peptide in the inflamed intestine. In epithelial cell culture (Caco-2), SBA15-Ac2-26 favored the internalization of Ac2-26. Taken together, the two experimental delivery strategies for rAnxA1 or Ac2-26 were efficient and the results obtained suggest that more studies should be carried out to confirm the treatment strategies. In order to seek tools to expand these studies, during the BEPE internship, studies were carried out in epithelial cell cultures based on adult stem cells differentiated in vitro. The results showed rAnxA1 or Ac2-26 protected epithelial integrity after challenge with LPS, by upregulating the expression of tight and adherens junctions and reducing the expression of claudin-2, responsible for increasing intercellular permeability; by negative modulation of pro-inflammatory cytokines CXCL-1 and MCP-1, and positive modulation of anti-inflammatory cytokine IL-10. In this way, we standardized a new cell culture model that has not yet been tested for AnxA1 or Ac2-26, which could be used to unravel the mechanisms of MLNC-AnxA1 and Eudragit-SBA15-Ac2-26