Mechanism of Antifungal Activity by 5-Aminoimidazole-4-Carbohydrazonamide Derivatives against Candida albicans and Candida krusei

Systemic mycoses are one major cause of morbidity/mortality among immunocompromised/debilitated individuals. Studying the mechanism of action is a strategy to develop safer/potent antifungals, warning resistance emergence. The major goal of this study was to elucidate the mechanism of action of three (Z)-5-amino-N’-aryl-1-methyl-1H-imidazole-4-carbohydrazonamides (2h, 2k, 2l) that had previously demonstrated strong antifungal activity against Candida krusei and C. albicans ATCC strains. Activity was confirmed against clinical isolates, susceptible or resistant to fluconazole by broth microdilution assay. Ergosterol content (HPLC-DAD), mitochondrial dehydrogenase activity (MTT), reactive oxygen species (ROS) generation (flow cytometry), germ tube inhibition and drug interaction were evaluated. None of the compounds inhibited ergosterol synthesis. Ascorbic acid reduced the antifungal effect of compounds and significantly decreased ROS production. The metabolic viability of C. krusei was significantly reduced for values of 2MIC. Compounds 2h and 2k caused a significant increase in ROS production for MIC values while for 2l a significant increase was only observed for concentrations above MIC. ROS production seems to be involved in antifungal activity and the higher activity against C. krusei versus C. albicans may be related to their unequal sensitivity to different ROS. No synergism with fluconazole or amphotericin was observed, but the association of 2h with fluconazole might be valuable due to the significant inhibition of the dimorphic transition, a C. albicans virulence mechanism.

Effects of Albumin Adsorption on Cell Adhesion in Hydroxyapatite Modified Surfaces

Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p<0.001). Although the BSA adsorption didn`t affect cell adhesion, the results obtained in fluorescence quantification suggests that de dense surface was best for cellular adhesion and spread than the porous one. We conclude that differences in the topography of a biomaterial can directly influence their ability to adsorb proteins, while the dense surface was more favorable for both the adhesion and the spreading of pre-osteoblasts.

Preparation of Tween 80-Zn/Al-Levodopa-Layered Double Hydroxides Nanocomposite for Drug Delivery System

We incorporated anti-Parkinsonian drug, levodopa (dopa), in Zn/Al-LDH by coprecipitation method to form dopa-LDH nanocomposite. Further coating of Tween-80 on the external surfaces of dopa-LDH nanocomposite was achieved through the oxygen of C=O group of Tween-80 with the layer of dopa-LDH nanocomposite. The final product is called Tween-dopa-LDH nanocomposite. The X-ray diffraction indicates that the Tween-dopa-LDH nanocomposite was formed by aggregation structure. From the TGA data, the Tween-80 loading on the surface of LDH and dopa-LDH was 8.6 and 7.4%, respectively. The effect of coating process on the dopa release from Tween-dopa-LDH nanocomposite was also studied. The release from Tween-dopa-LDH nanocomposite shows slower release compared to the release of the drug from dopa-LDH nanocomposite as done previously in our study, presumably due to the retarding shielding effect. The cell viability study using PC12 showed improved viability with Tween-80 coating on dopa-LDH nanocomposite as studied by mitochondrial dehydrogenase activity (MTT assay).

Growth Inhibition of Candida albicans in the Presence of Antiserum Elicited in Rabbits by Mannan-Protein Conjugate

Antifungal properties of rabbit antiserum prepared by immunization are reported. The immunization was done by a chemically prepared conjugate consisting of Candida albicans (serotype A) surface mannan and human serum albumin. Addition of rabbit antiserum to d-glucose medium inoculated with C. albicans effectively inhibited its growth. Moreover, C. albicans cells treated with rabbit antiserum revealed the entire loss of viability (expressed as decreased mitochondrial dehydrogenase activity). No growth of treated cells on an agar plate was observed. The results confirmed that the mannan-protein conjugate could be considered as an effective component of perspective vaccine.

In Vitro Apoptotic Bioactivity of Flavonoids from Astragalus Verrucosus Moris

Six aglycone flavonoids and their corresponding glycosides: genistein and genistin, quercetin and rutin, apigenin and apigenin 7-O-β-D-(6- p-coumaroyl) glucoside, as well as the aglycone daidzein isolated from Astragalus verrucosus Moris, were tested for their apoptosis-inducing potential. In vitro techniques that monitor bioactivity through morphological and biochemical changes were carried out on HCT116 (human colon carcinoma) and MCF7 (human Caucasian breast adenocarcinoma) cancer cell lines. Dose-dependent cytotoxic effects were monitored through changes in mitochondrial dehydrogenase activity using the standard MTT assay. The median inhibitory concentration (GI50) determined from the dose-response curves showed that the aglycones apigenin and quercetin were the most bioactive (low GI50), whilst daidzein and genistein, which had not been previously tested on these cell lines, showed a smaller cytotoxic effect (high GI50). The remaining flavonoids, mostly glycosides, showed negligible cytotoxicity. Morphological changes were monitored by microscopic observation with a photographic record. Results showed important hallmarks of apoptosis, including cell rounding with reduction of cell volume, small condensed nuclei, membrane blebbing and formation of apoptotic bodies.

Endothelial Cell-Derived Microparticles in the Pathogenesis of Chemotherapy-Associated Hypercoagulability.

Background: Cisplatin-based chemotherapy is a risk factor of venous thromboembolism in cancer patients. The underlying pathogenesis remains unclear. We hypothesized an apoptotic effect of cisplatin on endothelial cells (EC) inducing a release of small membrane vesicles, so-called microparticles (MP) which are known to cause hemostasis activation. Objectives: To quantify the release of MP from EC following administration of cisplatin and to investigate MP-associated procoagulant mechanisms. Methods: Two EC lines (HUVEC, HMVEC-L) were exposed to cisplatin (1, 2.5, 5, 10, and 20 μM) for up to 120 h. Cell viability was assessed by quantification of mitochondrial dehydrogenase activity, counts and procoagulant activity of MP were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen levels were determined by ELISA. Results: EC viability decreased in a dose- and time-dependent manner and was accompanied by an increasing release of MP into culture media (maximum: HUVEC + 544%; HMVEC-L + 1738%). In parallel, procoagulant activity of media increased by up to 150% (HUVEC) and 493% (HMVEC-L), respectively. The procoagulant activity was almost abolished by annexin V but was not suppressed by a monoclonal TF-antibody. TF antigen levels on MP were persistently low even at high cisplatin concentrations. Conclusion: At pharmacologically relevant concentrations, cisplatin induced a marked release of procoagulant MP from EC. Negatively charged phospholipids but not TF on MP were decisive for total thrombin generation. Further studies are warranted to investigate the cisplatin-induced release of EC-derived MP in vivo.