Ludmila Ferreira Gouveia

Pyridine-derived thiosemicarbazones and their tin(IV) complexes with antifungal activity against Candida spp.

[(n-Bu)Sn(2Ac4oClPh)Cl2] (1), [(n-Bu)Sn(2Ac4oFPh)Cl2] (2), [(n-Bu)Sn(2Ac4oNO2Ph)Cl2] (3), [(n-Bu)Sn(2Bz4oClPh)Cl2] (4), [(n-Bu)Sn(2Bz4oFPh)Cl2] (5) and [(n-Bu)Sn(2Bz4oNO2Ph)Cl2] (6) were obtained by reacting [(n-Bu)SnCl3] with 2-acetylpyridine-N4-orthochlorophenyl thiosemicarbazone (H2Ac4oClPh), 2-acetylpyridine-N4-orthofluorphenyl thiosemicarbazone (H2Ac4oFPh), 2-acetylpyridine-N4-orthonitrophenyl thiosemicarbazone (H2Ac4oNO2Ph), and with the corresponding 2-benzoylpyridine-derived thiosemicarbazones (H2Bz4oClPh, H2ABz4oFPh and H2Bz4oNO2Ph). The antifungal activity of the studied compounds was evaluated against several Candida species. Upon coordination of H2Bz4oNO2Ph to tin in complex (6) the antifungal activity increased three times against Candida albicans and Candida krusei and six times against Candida glabrata and Candida parapsilosis. The minimum inhibitory concentration (MIC) values of H2Ac4oNO2Ph and its complex (3) against C. albicans, C. parapsilosis and C. glabrata are similar to that of fluconazole. All studied compounds were more active than fluconazole against C. krusei.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death

OBJECTIVES Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. METHODS We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. RESULTS The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. CONCLUSIONS Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.

Dynamic interaction between fluconazole and amphotericin B against Cryptococcus gattii

ABSTRACT Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii. Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii.

Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Photodynamic therapy efficiently controls dermatophytosis caused by Trichophyton rubrum in a murine model

DEAR EDITOR, Dermatophytes are filamentous fungi that use keratin to colonize the host, causing superficial infections called dermatophytoses. Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum spp. and Epidermophyton spp. are important aetiological agents worldwide. A few drugs, including azoles (itraconazole), allylamines (terbinafine) and hydroxypiridones [ciclopirox olamine (CPX)], are available for the treatment of dermatophytoses, but the widespread use of antifungals has been linked to the emergence of resistant strains of T. rubrum. This problem may jeopardize treatment and thereby justifies the search for new therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer and a nonthermal light source to induce a reaction that results in cell death. In the presence of molecular oxygen, aPDT triggers the production of reactive oxygen (ROS) and reactive nitrogen species, which have short lifetimes and limited diffusion through the tissue, explaining the localized damage induced by aPDT. Previously, we have shown that T. rubrum is susceptible to photodynamic inhibition using toluidine blue (TBO) and light-emitting diode (LED) at 630 nm; however, there is a lack of studies regarding its efficacy in vivo. Therefore, the aim of this study was to evaluate the effects of aPDT, using TBO and LED, in controlling T. rubrum infection in a murine model of dermatophytosis. In this study, C57BL/6 mice were infected with 1 9 10 conidia per animal of T. rubrum American Type Culture Collection 28189 and treated with aPDT daily [using TBO 0 2% in a gel formulation (Sigma-Aldrich, St Louis, MO, U.S.A.) and 630-nm LED (Fisioled; MMoptics, S~ao Paulo, Brazil) at a dose of 42 J cm] or CPX [10 mg g 1 (dose of 0 65 mg 1 per mouse); Medley, S~ao Paulo, Brazil] for 48 h, over a period of 7 days (Comiss~ao de Etica no Uso de Animals/Universidade Federal de Minas Gerais ethics protocol approval no. 040/ 2011). Histopathology of the skin, fungal burden in potato agar, and myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity were evaluated. The influence of aPDT on the viability of intraperitoneal macrophages, intracellular formation of hyphae and oxidative burst (ROS) was determined. For this purpose, TBO (10 lg mL ) and LED (48 J cm) were used. The groups of mice and macrophages used in this work are described in Table 1. Histopathological analyses showed that aPDT reduced the signs of dermatitis and recovered tissue architecture (Fig. 1c, g,i) compared with the infected and nontreated group (Fig. 1b,f,i). Interestingly, fungal cells were found mainly in hair follicles, corroborating the keratinophilic aspect of T. rubrum (Fig. 1f–h). Indeed, aPDT significantly reduced the fungal burden by 87% compared with the untreated group (P < 0 01) and by 64% compared with the CPX group (P < 0 05) (Fig. 1d,f–h,j). LED or TBO alone did not reduce the fungal burden (Fig. 1j). In addition, aPDT significantly

Antigenic response to CT-P13 and infliximab originator in inflammatory bowel disease patients shows similar epitope recognition

To test the cross‐immunogenicity of anti‐CT‐P13 IBD patients’ sera to CT‐P13/infliximab originator and the comparative antigenicity evoked by CT‐P13/infliximab originator sera.

Butenafine and analogues: An expeditious synthesis and cytotoxicity and antifungal activities

Graphical abstract