Efeito inibitório in vitro do composto farnesol frente ao biofilme de Burkholderia pseudomallei

A intrínseca resistência apresentada pela bactéria Burkholderia pseudomallei é um grave empecilho para o tratamento da melioidose. Muitas pesquisas focam na busca de adjuvantes que aumentem a sensibilidade desta bactéria aos antimicrobianos. Nesse contexto, a açãoinibitória do farnesol frente às cepas de B. pseudomallei, na forma planctônica, já foi relatadaem estudo prévio. Diante disso, esse estudo objetivou analisar a atividade in vitro do farnesol contra cepas de B. pseudomallei na forma de biofilme. Aliado à análise da ação do farnesolisoladamente, foi investigada a combinação desse composto com os antimicrobianosamoxicilina, ceftazidima, doxiciclina, imipenem e sulfametoxazol/trimetoprim frente a obiofilme. A sensibilidade foi avaliada por meio do teste de microdiluição em caldo e a leiturada viabilidade celular feita com a resazurina. A concentração inibitória mínima em biofilme(CEMB) para o farnesol foi de 75 a 2400 mM. Ademais, o farnesol reduziu em até 256, 16, 4e 4 vezes os valores de CEMB para ceftazidima, amoxicilina, doxiciclina esulfametoxazol/trimetoprim, respectivamente (P<0.05). Por meio de técnicas de microscopia,tais como óptica, confocal e eletrônica, observou-se que o farnesol foi capaz de causar danosna matriz do biofilme, facilitando assim, a penetração dos antibióticos. Deste modo, opresente estudo mostrou a eficácia do farnesol contra biofilmes de B. pseudomallei e seuefeito potenciador, em especial com ceftazidima, amoxicilina, doxiciclina esulfametoxazol/trimetoprim.

The intrinsic antimicrobial resistance of Burkholderia pseudomallei is a serious challenge tothe treatment of melioidosis. Many studies have searched for adjuvants that increasesusceptibility bacteria to antimicrobials. In this context, the antimicrobial activity of farnesolagainst B. pseudomallei in planktonic growth has been reported. Thus, the aim of this studywas to analyze the in vitro activity of farnesol alone against Burkholderia pseudomalleibiofilms, as well as its combination with the antibacterials amoxicillin, doxycycline,ceftazidime and sulfamethoxazole-trimethoprim. Susceptibility was assessed by the brothmicrodilution test and cell viability was read with the oxidation-reduction indicator dyeresazurin. The interaction between farnesol and antibacterial drugs against B. pseudomalleibiofilms was evaluated through the calculation of the fractional inhibitory concentrationindex. The minimum biofilm erradication concentration (MBEC) for farnesol was 75 to 2400mM. In addition, farnesol significantly reduced the MBEC values for ceftazidime,amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 timesrespectively (P<0.05). Optical, confocal and electronic microscopic analyses of farnesoltreatedB. pseudomallei biofilms demonstrated that this compound damages biofilm matrix,facilitating antimicrobial penetration in the biofilm structure. This study demonstrated theeffectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on theactivity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline andsulfamethoxazole-trimethoprim.

Effect of andrographolide on quorum sensing and relevant virulence genes of Candida albicans / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of andrographolide (AG) on quroum sensing (QS) and relevant virulence genes of Candida albicans.</p><p><b>METHOD</b>Gas-chromatography-mass spectrometry (GC-MS) was applied to detect the changes in the content of farnesol and tyrosol in C. albicans intervened by AG. The real-time quantitative PCR (qRT-PCR) was adopted to inspect the expressions of relevant virulence genes such as CHK1, PBS2 and HOG1 regulated by QS.</p><p><b>RESULT</b>At 2 h after the growth of C. albican, the farnesol and tyrosol secretions reduced, without notable change after intervention with AG. The secretions were highest at 12 h and decreased at 24 h. After the intervention with different concentrations of AG, the farnesol content reduces, whereas tyrosol increased, indicating a dose-dependence, particularly with 1 000 mg x L(-1) AG. qRT-PCR revealed that 1 000 mg x L(-1) AG could down-regulate CHK1 by 2.375, 3.330 and 4.043 times and PBS2 by 2.010, 4.210 and 4.760 times, with no significant change in HOG1.</p><p><b>CONCLUSION</b>AG could inhibit the farnesol secretion, promote the tyrosol secretion and down-regulate QS-related virulence genes CHK1 and PBS2 expressions.</p>

Antibiofilm and Antibacterial Activities of Farnesol and Xylitol as Potential Endodontic Irrigants

This study investigated the antibiofilm and antibacterial effects of farnesol and xylitol in a series of experiments in order to evaluate their potential use as root canal irrigants. The following substances were tested: 0.2% farnesol; 5% and 20% xylitol; 0.2% farnesol plus 20% xylitol; and saline (control). For comparison with an established endodontic irrigant, 2.5% NaOCl was included in each test. Three experiments were conducted: the crystal violet assay, to evaluate the effects on the biofilm biomass; the dentin disinfection test, to evaluate the effects on bacterial viability in biofilms; and the root canal disinfection test, to simulate the use in the root canal environment. Farnesol was the most effective substance in reducing the biofilm biomass, followed by 20% xylitol. All substances affected bacterial viability in biofilms; farnesol showed the best results followed by the farnesol/xylitol combination. Irrigation with all substances significantly reduced the bacterial load (p<0.001), but only the farnesol/xylitol combination was significantly more effective than saline (p=0.02). NaOCl was more effective than any other substance tested in the three experiments (p<0.001). The findings demonstrated that farnesol affected both the biofilm biomass and the viability of cells in the biofilm, while 20% xylitol affected only the biofilm biomass. Although not more effective than NaOCl, the combination of these two antibiofilm substances has potential to be used in endodontics in certain situations.

Este estudo investigou os efeitos antibiofilme e antibacteriano de farnesol e xilitol em uma série de experimentos para avaliar seu uso potencial como irrigante de canais radiculares. As seguintes substâncias foram testadas: farnesol a 0,2%; xilitol a 5% e 20%; farnesol a 0,2% combinado com xilitol a 20%; e solução salina (controle). NaOCl foi testado para comparação. Três experimentos foram conduzidos: o teste do cristal violeta para avaliar os efeitos sobre a biomassa de biofilme, o teste da desinfecção de fragmentos de dentina para avaliar os efeitos na viabilidade bacteriana nos biofilmes e o teste da desinfecção de canal radicular para simular o uso no ambiente do canal radicular. Farnesol foi o mais eficaz, seguido por xylitol a 20%. Todas as substâncias afetaram a viabilidade bacteriana nos biofilmes; farnesol mostrou os melhores resultados, seguido pela combinação farnesol/xilitol. A irrigação com todas as substâncias reduziu significativamente a carga bacteriana (p<0,001), mas somente a combinação farnesol/xilitol foi significativamente mais eficaz que a solução salina (p=0,02). NaOCl foi mais eficaz que qualquer outra substância testada nos três experimentos (p<0,001). Os achados demonstraram que farnesol afetou tanto a biomassa de biofilme quanto a viabilidade das células no biofilme, enquanto que xilitol a 20% afetou a biomassa de biofilme. Embora não mais eficazes que NaOCl, combinações dessas duas substâncias antibiofilmes têm o potencial de ser usadas na Endodontia, em determinadas situações.

Biofilm biomass disruption by natural substances with potential for endodontic use

This study evaluated the in vitro effects of four natural substances on the biomass of bacterial biofilms to assess their potential use as root canal irrigants. The following substances and their combinations were tested: 0.2% farnesol; 5% xylitol; 20% xylitol; 0.2% farnesol and 5% xylitol; 0.2% farnesol, 5% xylitol, and 0.1% lactoferrin; 5% xylitol and 0.1% lactoferrin; and 20 mM salicylic acid. The crystal violet assay was used to evaluate the effects of these substances on the biomass of biofilms formed by Enterococcus faecalis and Staphylococcus epidermidis. All substances except for 20 mM salicylic acid and 20% xylitol reduced biofilm mass when compared to controls. The combination of farnesol and xylitol was the most effective agent against E. faecalis ATCC 29212 (p < 0.05). Farnesol combined with xylitol and lactoferrin was the most effective against biofilms of the endodontic strain of E. faecalis MB35 (p < 0.05). Similarly, combinations involving farnesol, xylitol, and lactoferrin reduced the biomass of S. epidermidis biofilms. In general, farnesol, xylitol, and lactoferrin or farnesol and xylitol reduced biofilm biomass most effectively. Therefore, it was concluded that combinations of antibiofilm substances have potential use in endodontic treatment to combat biofilms.

Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms / 国际口腔科学杂志·英文版

Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt-farnesol (1 mmol x L(-1)) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P < 0.05). Moreover, topical applications of 1 mmol x L(-1) tt-farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed-species biofilms after introduction of 1% sucrose. S. oralis (a non-cariogenic organism) became the major species after treatments with tt-farnesol, whereas vehicle-treated biofilms contained mostly S. mutans (>90% of total bacterial population). However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt-farnesol may affect the competitiveness of S. mutans in a mixed-species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti-biofilm/anti-caries chemotherapeutic strategies.

A Role of Staphyococcus aureus, Interleukin-18, Nerve Growth Factor and Semaphorin 3A, an Axon Guidance Molecule, in Pathogenesis and Treatment of Atopic Dermatitis

Staphylococcus aureus (SA) is usually present not only in the skin lesions of atopic dermatitis (AD) but also in the atopic dry skin. SA discharges various toxins and enzymes that injure the skin, results in activation of epidermal keratinocytes, which produce and release IL-18. IL-18 that induces the super Th1 cells secreting IFN-gamma and IL-13 is supposed to be involved in development of AD and its pathogenesis. Indeed, the number of SA colonies on the skin surface and the serum IL-18 levels in patients with AD significantly correlated with the skin scores of AD lesions. Also, there is strong positive correlation between the skin scores and serum IL-18 levels in DS-Nh mice (P<0.0001, r=0.64), which develop considerable AD-like legions when they are housed under conventional conditions, but develop skin legions with less severity and less frequency under specific pathogens free (SPF) conditions. Therefore, they are well-known as model mice of AD, in which SA is presumed to be critical factor for the development of AD lesions. Also, theses DS-Nh mice pretreated with Cy developed more remarkable AD-like lesions in comparison with non-treated ones. The levels of INF-r and IL-13 in the supernatants of the lymph node cell cultures stimulated with staphylococcal enterotoxin B (SEB) or ConA were increased in the Cy-treated mice, although the serum levels of total IgE were not. In this experiment, we revealed that Cy-treated mice, to which CD25 +CD4 + reguratory T cells taken from non-treated ones had been transferred, developed the AD-like legions with less severity and less number of SA colonies on the skin surface. Therefore, it is presumed that CD25 +CD4 + reguratory T cells might be involved in the suppression of super Th1 cells which are induced by IL-18 and are involved in the development of AD-like lesions rather than IgE production. The efficient induction of CD25 +CD4 + reguratory T cells is expected for the new type of treatment of AD. We also found that farnesol (F) and xylitol (X) synergistically inhibited biofilm formation by SA, and indeed the ratio of SA in total bacteria at sites to which the FX cream containing F and X had been applied was significantly decreased 1 week later, accompanied with improvement of AD, when compared with that before application and at placebo sites. Therefore, the FX cream is a useful skin-care agent for atopic dry skin colonized by SA. The nerve growth factor (NGF) in the horny layer (the horn NGF) of skin lesions on the cubital fossa was collected by tape stripping and measured using ELISA in AD patients before and after 2 and 4 weeks treatments. Simultaneously, the itch and eruptions on the whole body and on the lesions, in which the horn NGF was measured, were recorded, and also the peripheral blood eosinophil count, serum LDH level and serum total IgE level were examined. The level of NGF was significantly higher in AD patients than in healthy controls, correlated with the severity of itch, erythema, scale/xerosis, the eosinophil count and LDH level, and also significantly decreased after treatments with olopatadine and/or steroid ointment for 2 and 4 weeks. Therefore, the measurement of the NGF by this harmless method seems to be useful to assess the severity of AD and the therapeutic effects on AD. In AD patients, C-fiber in the epidermis increase and sprout, inducing hypersensitivity, which is considered to aggravate the disease. Semaphorin 3A (Sema3A), an axon guidance molecule, is a potent inhibitor of neurite outgrowth of sensory neurons. We administered recombinant Sema3A intracutaneously into the skin lesions of NC/Nga mice, an animal model of AD, and investigated the effect of Sema3A on the skin lesions and their itch. Sema3A dose-dependently improved skin lesions and attenuated the scratching behavior in NC/Nga mice. Histological examinations revealed a decrease in the epidermal thickness, the density of invasive nerve fibers in the epidermis, inflammatory infiltrate including mast cells and CD4 +T cells, and the production of IL-4 in the Sema3A-treated lesions. Because the interruption of the itch-scratch cycle likely contributes to the improvement of the AD-like lesions, Sema3A is expected to become a promising treatment of patients with refractory AD.

Farnesol inhibits cell proliferation and induces apoptosis after partial hepatectomy in rats / Farnesol inibe a proliferação celular e induz a apoptose após a hepatectomia parcial em ratos

PURPOSE: To study farnesol (FOH) effects on liver regeneration after 70 percent partial hepatectomy (PH) in rats. METHODS: Animals received FOH (25 mg/100 g body weight/day) or corn oil (CO, 0.25 mL/100 g body weight/day, controls). After a 2 week-treatment, all animals were subjected to PH and euthanized at different time points (0 h, 0.5 h, 4 h, 8 h, 18 h and 24 h) after surgery. Hepatic cell proliferation (PCNA positive nuclei) and apoptosis (fluorescence microscopy) were evaluated. RESULTS: Compared to CO treatment, FOH treatment inhibited (p<0.05) cell proliferation at 24h (S phase of the cell cycle) after PH. This was preceded by an induction of apoptosis 0.5 h (p<0.05; G0/G1 transition phase) after surgery. CONCLUSION: The results of the present study suggest that apoptosis induction could be associated with the reduced number of cells at the S phase observed in FOH group. These novel in vivo data reinforce FOH as a promising chemopreventive and therapeutic agent against cancer.

OBJETIVO: Estudar o efeito do farnesol (FOH) durante a regeneração hepática em ratos submetidos à Hepatectomia Parcial (HP) a 70 por cento. MÉTODOS: Os animais foram tratados com FOH (25 mg/100g de peso corpórel/dia) ou óleo de milho (OM, 0,25 mL/100g de peso corpóreo/dia, grupo controle). Depois de 2 semanas de tratamento, todos os animais foram submetidos à HP e eutanaziados em diferentes momentos (0h, 30min., 4h, 8h, 18h, 24h.) após o procedimento cirúrgico. Foi avaliada a proliferação celular (imunohistoquímica para PCNA) e a apoptose (microscopia de fluorescência). RESULTADOS: Em comparação aos animais controles, animais tratados com FOH apresentaram menor (p<0,05) proliferação celular 24h. (fase S do ciclo celular) após a HP. Tal efeito foi precedido de uma indução de apoptose 30min. (p<0,05; transição entre as fases G0/G1 do ciclo celular) após a cirurgia. CONCLUSÃO: Os resultados do presente estudo sugerem que a indução da apoptose pode estar associada com o menor número de células na fase S observadas nos animais tratados com FOH. Essa nova evidência in vivo reforça o farnesol como um promissor agente preventivo e terapêutico contra o câncer.

Phospholipid synthesis, diacylglycerol compartmentation, and apoptosis

Apoptosis is a means by which organisms dispose of unwanted cells without inducing an inflammatory response. Alterations in apoptosis is a common process by which cells become cancerous. Paradoxically, many cancer chemotherapeutics preferentially kill cancer cells by inducing apoptosis. Diacylglycerol is a lipid second messenger that regulates cell growth and apoptosis and is produced during signal transduction by hydrolysis of membrane phospholipids. Protein kinase Cs are a family of diacyglycerol responsive enzymes that are recruited to cellular membranes as a consequence of diacylglycerol production where they phosphorylate specific target proteins responsible for regulating cell growth. In this review, we will first summarize our current understanding of the role of specific proteins kinase C isoforms in the induction of cell growth/apoptosis. Subsequently, we will discuss how insights gained in lipid-mediated regulation of protein kinase Cs promotes our understanding of the role specific family members play in regulating cell growth. Finally, other diacylglycerol binding proteins involved in regulating apoptosis will be discussed

Antiproliferative Effect of Lovastatin on Vascular Smooth Muscle Cell

BACKGROUND AND OBJECTIVES: This study was performed to investigate the antiproliferative effect of lovastatin on vascular smooth muscle cell, especially to determine whether lovastatin induces apoptosis in vascular smooth muscle cell and the products of mevalonate pathway can reverse the antiproliferative effect of lovastatin. METHODS AND MATERIALS: Lovastatin only and lovastatin with one of the products of mevalonate pathway such as isopentenyl adenine, farnesol, mevalonate, cholesterol were added respectively in cultured rat vascular smooth muscle cells stimulated with 10% fetal calf serum. DNA synthesis was measured by tritiated-thymidine incorporation. Cell number was determined by hemocytometric counting. Cells were Giemsa-stained to evaluate morphological changes of apoptosis. Extracted DNA from the cells treated with lovastatin was assessed by gel electrophoresis. RESULTS: 1)Lovastatin inhibited DNA synthesis and cell proliferation in a dose-dependent manner. 2)The inhibitory effects of lovastatin could be reversed almost completely by mevalonate, partially by farnesol. 3)Lovastatin-treated vascular smooth muscle cells showed typical morphological changes of apoptosis. 4)A distinct ladder of DNA bands was visualized by gel electrophoresis of the DNA from the cells treated with lovastatin. CONCLUSION: Mevalonate metabolism is essential for vascular smooth muscle cell proliferation. The antiproliferative effect of lovastatin may result from the induction of apoptosis in vascular smooth muscle cells.