Paraprobiotic Enterococcus faecalis EC-12 prevents the development of irinotecan-induced intestinal mucositis in mice.

AIMS: This study tested the protective effect of purified paraprobiotic Enterococcus faecalis (EC-12) and an E. faecalis-based formulation (Med LanS) on irinotecan-induced intestinal mucositis murine model. MAIN METHODS: C57BL/6 male mice received saline, irinotecan (75 mg/Kg, i.p.), EC-12 (0.3, 1, or 3 × 107 CFU/Kg, p.o.) + irinotecan or Med Lan-S (3 × 107 CFU/Kg, p.o.) + irinotecan. Body mass variation was assessed daily, and blood samples were collected for evaluating bacteremia and leukocyte count. The ileum was harvested for myeloperoxidase assay, histopathology, quantitative PCR, and immunofluorescence for macrophages (F4/80), TLR4, and IL-18 binding protein (IL-18BP). KEY FINDINGS: The best therapeutic strategy was EC-12 administration at 3 × 107 CFU/Kg, starting 1 week before irinotecan. EC-12 and Med Lan-S did not prevent the irinotecan-induced body mass loss or leukopenia but attenuated the neutrophil infiltration in the intestine and increased the villus/crypt ratio (P < 0.05). Additionally, EC-12 and Med Lan-S reduced the mRNA expression of Cldn-2, Ocln, and Tlr4 versus the irinotecan group (P < 0.05). Irinotecan also augmented the expression of Il-18, IL-18BP, the immunofluorescence of F4/80, and TLR4, while only EC-12 prevented the expression of all these markers. Remarkably, EC-12 and Med Lan inhibited the irinotecan-induced bacterial translocation to the blood. SIGNIFICANCE: Paraprobiotic E. faecalis EC-12 prevents the development of intestinal mucositis by downregulating the inflammatory response. Med Lan-S also protects from mucositis. Possibly, the complexity of the formulation accounts for an innate immune-driven protective mechanism.

TLR4 deficiency upregulates TLR9 expression and enhances irinotecan-related intestinal mucositis and late-onset diarrhoea.

BACKGROUND AND PURPOSE: Severe diarrhoea, a common gastrointestinal manifestation of anticancer treatment with irinotecan, might involve single nucleotide polymorphisms (SNPs) of toll-like receptors (TLRs), described as critical bacterial sensors in the gut. Here, colorectal cancer patients carrying missense TLR4 A896G (rs4986790) or C1,196T (rs4986791) SNPs and Tlr4 knockout (Tlr4-/-) mice were given irinotecan to investigate the severity of the induced diarrhoea. EXPERIMENTAL APPROACH: Forty-six patients treated with irinotecan-based regimens had diarrhoea severity analysed according to TLR4 genotypes. In the experimental setting, wild-type (WT) or Tlr4-/- mice were given irinotecan (45 or 75 mg·kg-1 , i.p.) or saline (3 ml·kg-1 ). Diarrhoea severity was evaluated by measuring intestinal injury and inflammatory markers expression after animals were killed. KEY RESULTS: All patients with TLR4 SNPs chemotherapy-treated presented diarrhoea, whereas gastrointestinal toxicity was observed in 50% of the wild homozygous individuals. Mice injected with irinotecan presented systemic bacterial translocation and increased TLR4 immunostaining in the intestine. In line with the clinical findings, Tlr4 gene deficiency enhanced irinotecan-related diarrhoea and TLR9 expression in mice. An increased myeloperoxidase activity and Il-18 expression along with IL-10 decreased production in Tlr4-/- mice also indicated an intensified intestinal damage and inflammatory response. CONCLUSION AND IMPLICATIONS: TLR4 deficiency upregulates TLR9 expression and enhances intestinal damage and the severity of late-onset diarrhoea during irinotecan-based treatment. Identifying patients genetically predisposed to chemotherapy-associated diarrhoea is a strategy toward precision medicine.

SN-38, the active metabolite of irinotecan, inhibits the acute inflammatory response by targeting toll-like receptor 4.

PURPOSE: Anticancer-drug efficacy seems to involve the direct interaction with host immune cells. Although topoisomerase I (Top I) inhibitors have been suggested to block LPS-evoked inflammation, the interaction between these drugs and toll-like receptor 4 (TLR4) is unaddressed. METHODS: SN-38, the active metabolite of the Top I inhibitor irinotecan, and TLR4 interaction was assessed using the in vitro luciferase nuclear factor-κB reporter assay, neutrophil migration to murine air-pouch, in silico simulation, and the thermal shift assay (TSA). Topotecan was used as a positive anti-inflammatory control. RESULTS: Non-cytotoxic concentrations of SN-38 attenuated LPS (a TLR4 agonist)-driven cell activation without affecting peptidoglycan (a TLR2 agonist)-activating response. Similarly, topotecan also prevented LPS-induced inflammation. Conversely, increasing concentrations of LPS reversed the SN-38 inhibitory effect. In addition, SN-38 abrogated LPS-dependent neutrophil migration and reduced TNF-α, IL-6, and keratinocyte chemoattractant levels in the air-pouch model, but failed to inhibit zymosan (a TLR2 agonist)-induced cell migration. A two-step molecular docking analysis indicated two potential binding sites for the SN-38 in the MD-2/TLR4 complex, the hydrophobic MD-2 pocket (binding energy of - 8.1 kcal/mol) and the rim of the same molecule (- 6.9 kcal/mol). The topotecan also bound to the MD-2 pocket. In addition, not only the lactone forms, but also the carboxylate conformations of both Top I inhibitors interacted with the MD-2 molecule. Furthermore, the TSA suggested the interaction of SN-38 with MD-2. CONCLUSIONS: Therefore, SN-38 inhibits acute inflammation by blocking LPS-driven TLR4 signaling. This mechanism seems to be shared by other Top I inhibitors.

Efeito Antimicrobiano in Vitro do Extrato Aquoso e Hidroalcoólico das Folhas de Anacardium ocidentale e Mangifera indica / Antimicrobial Effect in Vitro of Aqueous Extract and Hydroalcoholic of Sheets Anacardium occidentale and Mangifera indica

As doenças causadas pelos micro-organismos são seriamente agravadas pela resistência generalizada ocasionada pelo uso indiscriminado dos antimicrobianos. A busca por novos agentes antimicrobianos tornou-se o principal objetivo de muitos grupos de pesquisa. Muitos deles focando seu trabalho no Reino Plantae. Em face da capacidade evolutiva dos micro-organismos e apesar dos antibióticos sintéticos serem uma opção viável, o estudo dos Metabólitos Secundários das Plantas continua sendo um excelente ponto de partida para a descoberta de moléculas de potencial antimicrobiano. Portanto, neste trabalho foi verificado o efeito antimicrobiano do extrato aquoso e hidroalcoólico de folhas de Anacardium occidentale e Mangifera indica frente a bactérias multirresistentes, empregando a técnica de poços por difusão em Agar. O presente estudo demonstrou que o extrato hidroalcoólico apresentou melhor atividade antimicrobiana, quando comparado ao extrato aquoso. Nos ensaios de antibiose foi verificada atividade antibacteriana para espécie de Staphylococcus aureus quando exposto ao extrato aquoso de Anacardium occidentale na concentração de 150 mg.mL-1. Nenhuma das espécies estudadas apresentou inibição do crescimento,quando expostas ao extrato aquoso de Mangifera indica. Os maiores halos de inibição ocorreram com o extrato hidroalcoólico de Anacardium occidentale frente à espécie de Staphylococcus aureus com a formação de um halo de inibição de 10 mm. Alguns processos devem ser considerados, dentre eles o procedimento utilizado para o isolamento e purificação dos Metabólitos Secundários das Plantas, o tipo de amostra estudada e a concentração adotada nos ensaios. Tais resultados sugerem que os produtos vegetais analisados representam um potencial para síntese de moléculas com atividade antibacteriana em processos infecciosos causados por Staphylococcus aureus, porém carece de estudos mais aprofundados que confirmem a hipótese. Outras metodologias poderiam evidenciar outros resultados com as espécies envolvidas, tais como outras formar de obtenção dos extratos e outras concentrações.

Diseases caused by microorganisms are seriously aggravated by widespread resistance caused by the indiscriminate use of antibiotics. The search for new antimicrobial agents became the main goal of a lot of research groups. A lot of them focusing their work on the Kingdom Plantae. Given the evolutionary ability of microorganisms and synthetic antibiotics and despite of being a viable option, the study of Secondary Metabolites of Plants remains an excellent starting point for the discovery of potential antimicrobial molecules. Therefore, this study evaluated the antimicrobial effect of hydroalcoholic and aqueous extracts of leaves of Anacardium occidentale and Mangifera indica against multidrugresistant bacteria using a technique well by diffusion in Agar. This study showed that hydroalcoholic extract showed better antimicrobial activity when compared to aqueous extract. In antibiosis tests antibacterial activity was verified to species of Staphylococcus aureus when exposed to aqueous extract of Anacardium occidentale in the concentration of 150 mg.mL-1. None of the species showed growth inhibition when exposed to aqueous extract of Mangifera indica. The largest zones of inhibition occurred at the hydroalcoholic extract of the species Anacardium occidentale front of Staphylococcus aureus with the formation of an inhibition zone of 10 mm. Some procedures should be considered among them, the procedure used for the isolation and purification of Secondary Metabolites of Plants, the type of sample and the concentration adopted in the testing. These results suggest that plant products analyzed represent a potential for synthesis of molecules with antibacterial activity in infectious processes caused by Staphylococcus aureus, but it needs further study to confirm the hypothesis. Other methodologies could show other results with the species involved, such as other forms of obtaining extracts and other concentrations.

Time-dependent modulation of AMPA receptor phosphorylation and mRNA expression of NMDA receptors and glial glutamate transporters in the rat hippocampus and cerebral cortex in a pilocarpine model of epilepsy.

The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. It has been demonstrated in this model that the phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit is increased 1 h after pilocarpine treatment. Moreover, alterations in the levels of glutamate transporters have been associated with chronic epilepsy in humans. Despite these studies, the profile of these changes has not yet been addressed. We analyzed the protein content and phosphorylation profile of the AMPA receptor GluR1 subunit by western blotting. We also used quantitative real-time polymerase chain reaction to analyze the expression of glial glutamate transporters and the N-methyl-D-aspartate receptor NR1 subunit in the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induced status epilepticus (Pilo-SE) in male adult Wistar rats. Biochemical analysis was performed in the Hip and Ctx at 1, 3, 12 h (acute period), 5 days (latent period), and 50 days (chronic period) after Pilo-SE. Key findings include an increase in the phosphorylation of GluR1-Ser(845) in the Ctx and GluR1-Ser(831) in the Hip at different times during the acute period, and a decrease in the total content of the GluR1 subunit in the Ctx in the latent period. There was a down-regulation of the mRNA expression and protein levels of EAAT1 and EAAT2, and a decrease of the NR1 mRNA expression, in the Ctx during the latent period. Notably, during the chronic period, the EAAT2 mRNA expression and protein levels decreased while the NR1 mRNA levels increased in the Hip. Taken together, our findings suggest a time- and structure-dependent imbalance of glutamatergic transmission in response to Pilo-SE, which might be associated with either epileptogenesis or the seizure threshold in MTLE-HS.