Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

Redução do marcador inflamatório TNF-α após consumo de semente de linhaça por coelhos hipercolesterolemicos / Decrease in the inflammatory marker TNF-a after comsumption of flaxseed by hypercholesterolemic rabbits

Background: Functional foods such as flaxseed have been commonly consumed to prevent atherosclerosis. Objectives: To assess the effects of flaxseed in atherogenesis in rabbits consuming a high-cholesterol diet. Methods: Thirty male albino rabbits were randomized to three groups based on a 12-week dietary treatment: control group (G1), standard diet; high-cholesterol diet (G2), standard diet plus 0.25% cholesterol from lyophilized eggs; and high-cholesterol plus flaxseed (G3), similar diet as G2 plus flaxseed. Biochemical (total cholesterol [TC], high-density lipoprotein [HDL-C], low-density lipoprotein cholesterol [LDL-C], and triglycerides) and immunohistochemical (intercellular adhesion molecule 1 [ICAM-1] and tumor necrosis factor alpha [TNF- α ]) analyses were performed in all groups. P values < 0.05 indicated statistical significance. Results: At 12 weeks, serum TC levels increased significantly in G2 and G3 compared with G1. Serum LDL-C levels were higher in group G2, and the increase in group G3 was approximately six times lower than that in G2. HDL-C levels increased in all groups, with the highest increase observed in G2. Triglycerides levels in G3 decreased by ~70% and differed significantly in G1 and G3 (p = 0.034) and G2 and G3 (p = 0.015). ICAM-1 levels increased only in aortic segment 4 in G3. TNF- α levels in G3 were similar to those in the control group, while the levels in G2 were greater than twice as those in the control group (p < 0.05). Conclusions: The group fed with a functional diet (flaxseed) showed decreased development of atherosclerosis, reduced serum triglycerides levels, and lower TNF- α levels on immunohistochemistry

Fundamentos: Alimentos funcionais, como a linhaça, têm sido consumidos com frequência para prevenção da aterosclerose. Objetivos: Avaliar os efeitos da linhaça sobre a aterogênese em coelhos submetidos a uma dieta rica em colesterol. Métodos: Trinta coelhos albinos machos foram randomizados em três grupos com base em um tratamento dietético por 12 semanas: grupo controle (G1), dieta padrão; dieta rica em colesterol (G2), dieta padrão mais 0,25% de colesterol proveniente de ovos liofilizados; e dieta rica em colesterol mais linhaça (G3), dieta semelhante à do G2 adicionada de linhaça. Análise bioquímica (colesterol total [CT], lipoproteína de alta densidade [HDL-colesterol], lipoproteína de baixa densidade [LDL-colesterol] e triglicérides) e imunohistoquímica (molécula de adesão intercelular 1 [ICAM-1] e fator de necrose tumoral alfa [TNF- α ]) foram realizadas em todos os grupos. Valores de p < 0,05 indicaram significância estatística. Resultados: Às 12 semanas, os níveis séricos de CT aumentaram significativamente nos grupos G2 e G3 em comparação com o G1. Os níveis séricos de LDL-colesterol foram mais altos no grupo G2, e o aumento no grupo G3 foi cerca de seis vezes menor do que no G2. Os níveis de HDL-colesterol aumentaram em todos os grupos, com o maior aumento observado no G2. Os níveis de triglicérides no G3 reduziram em ~70% e diferiram significativamente entre o G1 e G3 (p = 0,034) e G2 e G3 (p = 0,015). Níveis de ICAM-1 aumentaram apenas no segmento aórtico 4 no G3. Os níveis de TNF- α no grupo G3 foram semelhantes aos do grupo controle, enquanto os níveis no G2 foram maiores do que o dobro em relação aos do grupo controle (p < 0,05). Conclusões: O grupo alimentado com uma dieta funcional (linhaça) mostrou redução no desenvolvimento de aterosclerose, níveis séricos mais baixos de triglicérides e níveis mais baixos de TNF- α à imunohistoquímica

Quantificação da angiogênese induzida por tumor em membrana corioalantóica de embrião de galinha / Quantification of tumor-induced angiogenesis on chicken embryo chorioallantoic membrane

Angiogênese é um processo de surgimento de novos microvasos provenientes de vasos sanguíneos já existentes. O desenvolvimento tumoral e o processo de metástase são dependentes de angiogênese, pois o tumor em crescimento necessita de uma rede capilar que forneça nutrientes e oxigênio. A membrana corioalantóica de embrião de galinha (CAM) é um modelo experimental in vivo que oferece muitas vantagens, como a alta vascularização natural e alta taxa de reprodução, além de ser um modelo simples e de baixo custo. A CAM é composta por proteínas de matriz extracelular, que mimetizam o ambiente fisiológico de células cancerosas. A etapa de contagem do número total de vasos permite a determinação dos efeitos dos estímulos pró ou anti angiogênicos, portanto a padronização de um método eficaz é necessário. O presente estudo teve como objetivo geral avaliar o potencial angiogênico de células de uma linhagem de adenocarcinoma de cólon humano (HT29) e propor um método para quantificação da angiogênese induzida por células tumorais na CAM. Os embriões foram mantidos em sistema ex ovo. No oitavo dia, foram adicionados sobre a CAM, implantes de colágeno contendo células tumorais em diferentes concentrações. No décimo primeiro dia foi feito o registro fotográfico utilizando microscópio estereoscópico e foram determinados quatro scores para a quantificação e caracterização dos vasos, considerando-se se seccionavam o implante e também seu grau de ramificação. A contagem dos vasos, feita em uma área específica ao redor do implante, foi realizada após edição das imagens pelo programa Image Pro Plus. Os resultados mostraram aumento significativo do número de vasos que não seccionavam o implante para aqueles contendo 3 x 104 e 6 x 104 células. Pode-se concluir que a metodologia de contagem dos vasos, utilizando registros fotográficos e edições de imagens, é eficaz. Demonstrou-se que as células HT29 induzem a uma alteração no padrão de crescimento de novos vasos quando depositada sobre a CAM em implantes de colágeno e podem ser utilizadas como modelo experimental para se investigar o efeito de diferentes compostos sobre a angiogênese induzida por tumor.

Angiogenesis is a process of sprouting of new microvessels from existing blood vessels. The tumoral development and the metastasis process are angiogenesis dependent, because the growing tumor needs a capillaries network that provides nutrients and oxygen. The chicken chorioallantoic membrane (CAM) is an experimental in vivo model which offer many advantages, such as the high natural vascularization and high reproducibility, besides the simplicity and low cost. The CAM contains extracellular matrix proteins, which mimics the physiological cancer cell environment. The counting of the total number of vessels allows a determination of pro- and anti-angiogenic effects of different stimulus, therefore patterning an effective method is necessary. The general goal of the present study was to evaluate the angiogenic potential of a human colon adenocarcinoma cell line (HT29) and propose a method to quantify angiogenesis induced by cancer cells on the CAM. Embryos were cultivated in an ex ovo system. At the eighth day, collagen implants containing cancer cells in different concentrations were added on the top of CAM. At the eleventh day, the photographic records were made by using stereoscope microscope and were determined four scores for vessels quantification and characterization. Vessels counting were done in a specific area around the implant, and edition of the captured images were done using Image Pro Plus software. Our results showed a significant increase in vessels that do not section the implant. It was demonstrated that HT29 cells induce a change in the pattern of growth of new blood vessels when placed on CAM into collagen implants and can be used as an experimental model for investigating the effect of different compounds on tumor-induced angiogenesis.

Influence of glyphosate in planktonic and biofilm growth of Pseudomonas aeruginosa

This study evaluated the impact of different concentrations of glyphosate (Rondup®) on planktonic and biofilm growth of P. aeruginosa. Aerobic and anaerobic cultures of P. aeruginosa ATCC®15442 inoculated in MHB + glyphosate (0.845 ppm, 1.690 ppm, 8.45 ppm, 16.90 ppm, 84.50 ppm, 169 ppm, 845 ppm, and 1690 ppm) and cultured in normoxia and anoxia, following their OD560nm every hour for 24 h. Biofilms of adapted cells were formed in the presence of glyphosate (0.845 to 1690 ppm) in normoxia and anoxia for 36 h. Glyphosate at concentrations higher than 84.5 ppm reduces the cell density of planktonic aerobic cultures (p < 0.05). However, these same concentrations favor the planktonic anaerobic growth (p < 0.05). On the other hand, the herbicide favors a slight growth of biofilms in a concentration-dependent manner up to 84.5 ppm (p > 0.05), and more pronounced over 169 ppm. Anaerobic biofilms have their growth more readily favored (p < 0.05), regardless of concentration. In a concentration-dependent manner, glyphosate interferes with the growth ability of P. aeruginosa ATCC®15442.

Enhanced susceptibility of Candida albicans to chlorhexidine under anoxia

Aim: Periodontal pockets can be colonized not only by bacteria, but also by Candida albicans. However, its role in periodontitis is unknown. This study evaluated the inhibitory performance of chlorhexidine digluconate under normoxic and anoxic conditions against 16 strains of C. albicans from periodontal pockets and other 20 from the oral mucosa. Methods: Strains were grown in normoxia and anoxia to adapt themselves to the different atmospheric conditions. Microdilution-based assays were carried out to determine the minimum concentrations of chlorhexidine that may restrain the conditioned candidal strains, in normoxia (normoxic MIC) and anoxia (anoxic MIC). The Mann-Whitney U test was used to evaluate the antimicrobial effect of chlorhexidine on C. albicans under normoxic and anoxic conditions (α = 0.05). Results: The normoxic MIC of chlorhexidine varied broadly from 150 to 1200 µg/mL, whereas its anoxic MIC varied narrower from 2.34 to 37.5 µg/mL. Regarding the origins of strains, no statistically significant differences (p > 0.05) were found. Conclusions: These results indicate that anoxic environmental conditions, compatible with periodontal pockets, tend to enhance C. albicans susceptibility to chlorhexidine.