Galleria mellonella is a well-accepted insect model for the study of pathogen-host interactions and antimicrobial compounds. The main advantages of this model include the low cost of maintenance, the fast life cycle, the possibility of using a large number of caterpillars and the innate immune system, which is evolutionarily conserved relative to mammals. Because of these advantages, different research groups have been working to implement the rearing of G. mellonella in laboratory conditions. This protocol describes our experience in the rearing of G. mellonella caterpillars for experimental infection models and the influence of different artificial diets on developmental and physiological parameters. Here, we suggest a diet composition that benefits the life cycle of G. mellonella by accelerating the larval phase length and increasing the caterpillar weight. This diet also stimulated the immune system of G. mellonella by increasing the hemolymph volume and hemocyte concentration. In addition, our rearing protocol generated caterpillars that are more resistant to infection by Staphylococcus aureus, Escherichia coli and Candida albicans. A standard G. mellonella rearing protocol is fundamental to minimize external influences on the results, and this simple and easy protocol can support researchers starting to rear G. mellonella.
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.
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 10 6 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 ≤ 0.05).
This study evaluated the prophylactic effects of the live or heat-killed probiotic strain Lactobacillus rhamnosus ATCC 7469 in Galleria mellonella, inoculated with Staphylococcus aureus or Escherichia coli. L. rhamnosus suspension was prepared and a part of it was autoclaved to obtain heat-killed lactobacilli. The larvae were inoculated of these suspensions and pathogenic. The survival of the larvae was observed during 7 days and after 24 h of inoculation haemocytes counted, melanization and nitric oxide production were analyzed. Larvae survival rate increased in the group inoculated with heat-killed L. rhamnosus, however, with no statistical difference. There was a significant increase in total haemocyte counts in all test groups. Haemolymph melanization and nitric oxide production were higher in the group inoculated with L. rhamnosus and infected with S. aureus. It was concluded that, in this model of infection, heat-killed L. rhamnosus ATCC 7469 promoted greater protection in Galleria mellonella infected with S. aureus or E. coli.
Objective: Achyrocline satureioides “A.satureioides” is a subshrub, widely distributed in South America because of its medicinal properties. Although it is widely used in folk medicine, there is still no approval for its therapeutic use, and its biocompatibility is little explored. This study aimed to evaluate its cytotoxicity and genotoxicity over human gingival fibroblasts (FMM-1). Methodology: Ten different concentration of the glycolic extract of A.satureioides were tested for 5min and 24h of contact with the cells to evaluate its cytotoxicity using the MTT colorimetric assay and to evaluate its genotoxicity using the micronucleus assay. Data were analyzed with one-way ANOVA and Tukey test with a significance level (α=0.05). Results: All tested concentrations of the extract presented cell viability more than 70% and has no significant difference of the control group after 5min and 24h. However, for 5min the 100 mg/mL was cytotoxic and for 24h the 1.56 mg/mL stimulated cell proliferation. For genotoxicity analysis, only the concentration 6.25 mg/mL showed results similar to the control of cell culture in the micronucleus count after 5min and 24h. Conclusions: The glycolic extract of A.satureioides doesn’t have cytotoxic and genotoxic effects in concentrations up to 6.25 mg/mL, but in high concentrations it is considered genotoxic.
Produtos naturais, como extratos glicólicos de plantas, são importantes para a aplicação clínica na área da saúde, como em enxaguatórios bucais, cremes dentais e irrigação intracanal. Assim é necessário realizar estudos de citotoxicidade desses extratos glicólicos. O presente estudo buscou avaliar a atividade citotóxica dos extratos glicólicos de Cynara scolymus L. (alcachofra), Myracrodruom urundeuva Allem. (aroeira-do-sertão-do-sertão) e Camellia sinensis (L.) Kuntze (chá verde) em macrófagos de camundongo (RAW 264.7) pelo teste de atividade metabólica MTT. Para tanto, as células foram distribuídas em microplacas de 96 poços e foram expostas a 11 diluições seriadas de cada extrato (200 mg/mL,100 mg/mL, 50 mg/mL, 25 mg/mL, 12,5 mg/mL, 6,25 mg/mL, 3,13 mg/mL, 1,56 mg/mL, 0,78 mg/mL, 0,39 mg/mL e 0,20 mg/mL), sendo n=8 para cada diluição. Após o tempo de contato de 5 minutos e 24 horas, foi avaliada a viabilidade celular utilizando o teste MTT. Diante destes resultados, no tempo de exposição de 5 minutos com os extratos, em ordem crescente de redução da viabilidade celular, seguiram-se o chá verde, com aumento da viabilidade celular, a aroeira-do-sertão e o extrato de alcachofra. Por meio do MTT dos três extratos por tempo de exposição de 24 horas, observou-se que o extrato de alcachofra apresentou maior toxicidade, seguido do extrato de chá verde e aroeira-do-sertão. A análise estatística foi realizada por ANOVA e teste de Tukey, com significância de 5%. Conclusões: Entre os extratos, o chá verde com 12,5mg/mL, com interessantes 5 minutos e 24 horas, despertou atenção, pois foi a maior concentração e não apresentou citotoxicidade para os macrófagos, assim como a alcachofra que foi a mais citotóxica para os macrófagos, em ambos os tempos (5 min e 24 h).
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) ABSTRACT 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 1096 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. RESUMOExtratos de plantas podem ser fonte de diversas atividades biológicas. Dentre essas, a ação antiinflamatória se apresenta como uma das características de interesse para enxaguantes bucais, pastas dentais e medicações intracanais. O objetivo deste estudo foi avaliar a atividade citotóxica e antiinflamatória do extrato glicólico de Camellia sinensis (L.) Kuntze (chá verde) em macrófagos de camundongos (RAW 264.7). A atividade citotóxica foi medida pela atividade metabólica do teste de MTT, os macrófagos foram distribuídos em 96 poços e expostos a 11 diluições seriadas de cada extrato (200 mg / mL a 0,20 mg / mL). Após 5 min e 24 h de contato, a viabilidade celular foi avaliada. Então, o extrato nas concentrações de 3,13mg/mL e 12,5 mg / mL , foram escolhidas para verificar a atividade anti-inflamatória. Para isso, após a exposição do extrato por 5 minutos ou 24 horas, os sobrenadantes das células RAW 264.7 foram coletados para quantificação das citocinas pró-inflamatórias IL-1β e TNF-α pelo teste imunoenzimático ELISA...
Objective: The aim of this study was evaluate the effect of Bacillus subtilis on Candida albicans biofilm formation and filamentation by evaluating the gene expression of ALS3, HWP1, BCR1, EFG1 and TEC1. Material and Methods: Mixed (C. albicans / B.subtilis) and monotypic biofilms were cultured in plates at 37°C for 48 h under shaking for counting viable cells (CFU / mL) and analysis of gene expression by real-time PCR. The C. albicans filamentation assay was performed in medium containing 10% fetal bovine serum at 37°C for 6 hours. Data was analysed by t-Student and Mann– Whitney tests. Results: B. subtilis reduced the biofilm formation of C. albicans in 1 log when cultured in the same environment (p<0.0001). In addition, it significantly reduced the yeast -hypha transition affecting the morphology of C. albicans. Among all of the analyzed genes, the ALS3 and HWP1 genes were the most affected, achieving 111.1- and 333.3- fold decreases in the C. albicans biofilms associated with B. subtilis, respectively. Conclusion: B. subtilis reduced the biofilm formation and filamentation of C. albicans by negatively regulating the ALS3, HWP1, BCR1, EFG1 and TEC1 genes that are essential for the production of biofilm and hyphae.KeywordsBacillus subtilis; Candida albicans; Biofilm; Filamentation; Gene expression.
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