Efeitos do Lactobacillus rhamnosus e seus produtos sobre a liberação de mediadores pró e anti-inflamatórios em macrófagos de Camundongo

De acordo com a literatura, o uso frequente de probióticos em humanos favorece a modulação do sistema imunológico, por mecanismos ainda não totalmente definidos. O objetivo do presente estudo foi avaliar a capacidade de Lactobacillus rhamnosus ou seus produtos induzirem a liberação de mediadores pró e anti-inflamatórios (TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-12 e óxido nítrico) por macrófagos de camundongo (RAW 264.7). Para tanto, foram realizadas 3 preparações do microrganismo: SpL: suspensão de bactérias vivas (5 x 107 UFC/mL), SpLA: suspensão de bactérias autoclavadas a 121ºC por 15 min (5 x 107 células/mL); e SnLA: sobrenadante da suspensão anterior (SpLA) após centrifugação. As células (macrófagos RAW 264.7) foram cultivadas (37ºC, 5% de CO2) em três diferentes situações: na presença de SpL, SpLA ou SnLA. Após 2 h e 30 min de cultivo, o meio de cultura foi trocado e as células foram cultivadas por mais 16 h. Em seguida, os sobrenadantes foram removidos e estocados em freezer (-80ºC) para posterior análise. Como controle positivo foi utilizado LPS de Escherichia coli (10 EU/mL) e como controle negativo solução fisiológica. A quantificação de citocinas foi realizada pelo teste imunoenzimático (ELISA) utilizando anticorpos específicos. A produção de óxido nítrico foi determinada indiretamente pela concentração de nitrito detectada pelo reagente de Griess. Os resultados foram analisados estatisticamente, pela análise de variância ANOVA, com nível de significância de 5%, e pelo teste de Tukey. As suspensões SpL e SpLA foram capazes de induzir produção significativamente superior de TNF-α, IL-6, IL-10 e óxido nítrico em relação ao controle negativo (p 0.05). It was not observed production of IL-4 and IL-1β, by any of the suspensions analyzed. In relation to... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Metodologia da criação de Galleria mellonella para uso como modelo de infecção e efeitos de Lactobacillus rhamnosus inativado pelo calor in vivo e in vitro, desafiados por Staphylococcus aureus e Escherichia coli

Galleria mellonella é utilizada para estudar a virulência de micro-organismos e a potência de antimicrobianos. Este estudo buscou estabelecer criação de lagartas utilizadas em ensaios in vivo e avaliar o efeito do probiótico Lactobacillus rhamnosus ATCC 7469, inativado pelo calor, no modelo e in vitro. Os objetivos foram: a) desenvolver uma metodologia de criação de G. mellonella, avaliando quatro dietas diferentes sobre crescimento larval, volume da hemolinfa, quantidade de hemócitos e resposta à infecção por meio da curva de sobrevivência b) avaliar os efeitos de L. rhamnosus sobre G. mellonella analisando: curva de sobrevivência; contagem de hemócitos, melanização da hemolinfa, produção de óxido nítrico na hemolinfa e os efeitos de L. rhamnosus sobre macrófagos RAW 264.7 desafiados por S. aureus ou E. coli, analisando o perfil de indução de citocinas e óxido nítrico. Os resultados foram analisados estatisticamente (ANOVA e Tukey, 5%) e a curva de morte e estimativa das diferenças na sobrevivência foram determinadas por Log-rank (Mantel-Cox, 5%). As rações a base de fubá e pólen apresentaram os melhores resultados, sendo semelhantes entre si e diferentes das demais rações (p < 0,05), sendo a ração a base de fubá escolhida por apresentar resultados semelhante ao pólen e menor custo. Os resultados in vivo demontraram diminuição na mortalidade das lagartas no grupo com inoculação de L. rhamnosus, entretanto, sem diferença estatística. Houve aumento na contagem de hemócitos quando G. mellonella foi inoculada com S. aureus e E. coli, com ou sem inoculação de L. rhamnosus, além de haver melanização da hemolinfa, demonstradno que o L. rhamnosus melhorou a resposta de G. mellonella quando desafiada por bactérias. Os resultados in vitro demonstram que L. rhamnosus induziu alta produçaõ de TNF-α, igualmente aos demais grupos (p ≤ 0,05), não havendo produção de IL-1β e IL-6 no grupo estimulado apenas por L. rhamnosus. Nos grupos que receberam o segundo estímulo ou apenas o contato com S. aureus ou E. coli, houve produção de IL-1β, IL-6 e IL-10. As maiores produções de óxido nítrico foram observadas nos grupos estimulados com S. aureus. Houve diminuição na contagem de UFC/mL de S. aureus e E. coli, quando os macrófagos foram estimulados com lactobcilos. Isso sugere que L. rhamnosus inativado foi capaz de modular produção de citocinas e óxido nítrico quando as células foram desafiadas por S. aureus e E. coli, além de diminuir a contagem de CFU/mL, sugerindo melhorar a capacidade fagocitária dos macrófagos.Galleria mellonella is used to study microorganisms virulence and antimicrobial power. This study aimed to standardize the creation of worms used in In vivo assays and evaluate the effect of heat-killed probiotic, Lactobacillus rhamnosus ATCC 7469, on this model and in in vitro studies. The objectives were: a) developing a methodology for breeding G. mellonella with four different diets influence on larval growth, hemolymph volume, quantity of hemocytes and infection response by means of survival curve; b) evaluating the effects of L. rhamnosus on G. mellonella by means of the following analyzes: survival curve, hemocytes counting, hemolymph melanization, nitric oxide release, and the effects of L. rhamnosus on macrophages RAW 264.7 challenged by S. aureus or E. coli by means of cytokines and nitric oxide production. Results were statistically analyzed (ANOVA and Tukey, 5%). Death curve and estimation of differences in survival were determined by Log-rank (MantelCox, 5%). Cornmeal and pollen-based rations showed the best results, being similar to each other and different from the other ones (p <0.05).Cornmeal ration was chosen since it presents results similar to pollen and lower cost. In vivo results showed reduction in mortality of caterpillars in the group inoculated with L. rhamnosus, with no statistical difference. Hemocyte counting increased when G. mellonella was inoculated with S. aureus and E. coli, with or without inoculation of L. rhamnosus, add to that hemolymph melanization, showing that L. rhamnosus improved G. mellonella response challenged by bacteria. In vitro results show that L. rhamnosus induced high production of TNF-α, like other groups (p = 0.05), with no production of IL-1β and IL-6 in the group stimulated only by L. rhamnosus. The groups which received only the second stimulus or only the contact with S. aureus or E. coli, there was IL-1β, IL-6 and IL-10 production. The highest nitric oxide production was observed in the groups challenged with S. aureus. S. aureus and E. coli CFU/mL counting decreased when macrophages were stimulated with lactobacilli. This suggests that heat-killed L. rhamnosus was capable of modulating cytokine and nitric oxide production when cells were challenged with S. aureus and E. coli and reduce the CFU/mL counting, suggesting that there was enhancement in the phagocytic capacity of macrophages

Live and Heat-Killed Lactobacillus rhamnosus ATCC 7469 May Induce Modulatory Cytokines Profiles on Macrophages RAW 264.7

This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P<0.05). SHKLR also significantly increased the production of TNF-α and IL-10 (P<0.05) but not IL-6 (P>0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1β or significant levels of IL-4 and IL-12 (P>0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses

Atividade citotóxica e anti-inflamatória do extrato glicólico de Camellia sinensis (L.) Kuntze em macrófagos (RAW 264.7) estimulados por LPS / Cytotoxic and antiinflamatory activity of the glycolic extract of Camellia sinensis (L.) Kuntze in LPS-stimulated machrophages (RAW 264.7)

Plant extracts can be a source of diverse biologic activities, as anti-inflammatory action, which is an interesting characteristic for mouthwashes, toothpastes and intracanal medication. This study aimed to evaluate cytotoxic and anti-inflammatory activity of Camellia sinensis (L.) Kuntze (green tea) glycolic extract in mouse macrophages (RAW 264.7).  Cytotoxic activity was measured by the metabolic activity of MTT test, macrophages were distributed in 96 wells and exposed to 11 serial dilutions of each extract (200 mg/mL to 0.20 mg/mL). After 5 min and 24 h of contact, cell viability was assessed. Then, the extract at concentrations of 3.13 mg / mL and 12.5 mg / mL, were chosen to verify antiinflammatory activity. For this, after exposure time of 5 min or 24 h to the extract, supernatants of LPS-stimulated RAW 264.7 cultures were collected to quantify pro-inflammatory cytokines IL-1β and TNF-α by immunoenzymatic test (ELISA). The results were evaluated with statistical analyses (ANOVA and Turkey test), with p ≤ 0.05. Results: In MTT test green tea promoted increase cell viability in all concentrations at 5 min. The cell viability was greater than 100% in concentration of 0,20mg/mL to 12,5 mg/mL, at the time of exposure of 24h. The concentration of 12.5 mg/mL was the highest concentration less cytotoxic. The extract showed anti-inflammatory potential evidenced by the production decrease of IL-1β and TNF-α with better results at a concentration of 12.5 mg/mL for both exposure times. These results indicate promising immunomodulatory features of green tea. Therefore, this plant extract showed to be an interesting alternative to be inserted in medical or oral products or even as a source of active compounds

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p&#8804;0.05)