Márcio Rodrigues

Melanin from Fonsecaea pedrosoi Induces Production of Human Antifungal Antibodies and Enhances the Antimicrobial Efficacy of Phagocytes

ABSTRACT Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients’ sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients’ lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.

Extracellular vesicle-mediated export of fungal RNA

Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. In this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. The EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

ABSTRACT We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Biogenesis of extracellular vesicles in yeast: Many questions with few answers

The cellular events required for unconventional protein secretion in eukaryotic pathogens are beginning to be revealed. In fungi, extracellular release of proteins involves passage through the cell wall by mechanisms that are poorly understood. In recent years, several studies demonstrated that yeast cells produce vesicles that traverse the cell wall to release a wide range of cellular components into the extracellular space. These studies suggested that extracellular vesicle release involves components of both conventional and unconventional secretory pathways, although the precise mechanisms required for this process are still unknown. We discuss here cellular events that are candidates for regulating this interesting but elusive event in the biology of yeast cells.

A critical review of microextraction by packed sorbent as a sample preparation approach in drug bioanalysis

Sample preparation is widely accepted as the most labor-intensive and error-prone part of the bioanalytical process. The recent advances in this field have been focused on the miniaturization and integration of sample preparation online with analytical instrumentation, in order to reduce laboratory workload and increase analytical performance. From this perspective, microextraction by packed sorbent (MEPS) has emerged in the last few years as a powerful sample preparation approach suitable to be easily automated with liquid and gas chromatographic systems applied in a variety of bioanalytical areas (pharmaceutical, clinical, toxicological, environmental and food research). This paper aims to provide an overview and a critical discussion of recent bioanalytical methods reported in literature based on MEPS, with special emphasis on those developed for the quantification of therapeutic drugs and/or metabolites in biological samples. The advantages and some limitations of MEPS, as well as its comparison with other extraction techniques, are also addressed herein.

Liquid chromatographic assay based on microextraction by packed sorbent for therapeutic drug monitoring of carbamazepine, lamotrigine, oxcarbazepine, phenobarbital, phenytoin and the active metabolites carbamazepine-10,11-epoxide and licarbazepine

A new, sensitive and fast high-performance liquid chromatography-diode-array detection assay based on microextraction by packed sorbent (MEPS/HPLC-DAD) is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in human plasma. Chromatographic separation of analytes and ketoprofen, used as internal standard (IS), was achieved in less than 15min on a C18-column, at 35°C, using acetonitrile (6%) and a mixture (94%) of water-methanol-triethylamine (73.2:26.5:0.3, v/v/v; pH 6.5) pumped at 1mL/min. The analytes and IS were detected at 215, 237 or 280nm. The method showed to be selective, accurate [bias ±14.8% (or ±17.8% in the lower limit of quantification)], precise [coefficient variation ≤9.7% (or ≤17.7% in the lower limit of quantification)] and linear (r(2)≥0.9946) over the concentration ranges of 0.1-15μg/mL for CBZ; 0.1-20μg/mL for LTG; 0.1-5μg/mL for OXC and CBZ-E; 0.2-40μg/mL for PB; 0.3-30μg/mL for PHT; and 0.4-40μg/mL for LIC. The absolute extraction recovery of the analytes ranged from 57.8 to 98.1% and their stability was demonstrated in the studied conditions. This MEPS/HPLC-DAD assay was successfully applied to real plasma samples from patients, revealing to be a cost-effective tool for routine therapeutic drug monitoring of CBZ, LTG, OXC, PB and/or PHT.

Binding of Glucuronoxylomannan to the CD14 Receptor in Human A549 Alveolar Cells Induces Interleukin-8 Production

ABSTRACT Glucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans. GXM receptors have been characterized in phagocytes and endothelial cells, but epithelial molecules recognizing the polysaccharide remain unknown. In the current study, we demonstrate that GXM binds to the CD14 receptor in human type II alveolar epithelial cells, resulting in the production of the proinflammatory chemokine interleukin-8.

First liquid chromatographic method for the simultaneous determination of amiodarone and desethylamiodarone in human plasma using microextraction by packed sorbent (MEPS) as sample preparation procedure

For the first time a simple and fast high-performance liquid chromatography (HPLC) method using a novel sample preparation procedure based on microextraction by packed sorbent (MEPS) was developed and validated for the determination of amiodarone (AM) and its main metabolite desethylamiodarone (DEA) in human plasma. Chromatographic separation of the analytes (AM and DEA) and tamoxifen, used as internal standard (IS), was achieved within less than 5min on a LiChroCART Purospher(®) Star C(18) column (55mm×4mm, 3μm). The mobile phase consisting of 50mM phosphate buffer with 0.1% formic acid (pH 3.1)/methanol/acetonitrile (45:5:50, v/v/v) was pumped isocratically at a flow rate of 1.2mL/min. The detection was carried out at 254nm. Calibration curves were linear (r(2)≥0.9976) in the ranges of 0.1-10μg/mL for AM and DEA. The limits of quantification were established at 0.1μg/mL for AM and DEA. The overall imprecision did not exceed 6.67% and inaccuracy was within ±9.84%. The overall mean recovery of AM and DEA ranged from 58.6% to 68.2%. Neither endogenous nor tested exogenous compounds were found to interfere at retention times of the analytes (AM and DEA) and IS. This new MEPS/HPLC method was also applied to real samples obtained from polymedicated patients receiving AM therapy. Thus, this bioanalytical method seems to be a useful tool for therapeutic drug monitoring of patients under AM treatment and also to support other clinical pharmacokinetic-based studies involving this drug, such as bioavailability/bioequivalence studies.

Huperzine A from Huperzia serrata: a review of its sources, chemistry, pharmacology and toxicology

The use and popularity of herbal medicines has been increasing worldwide. In fact, today, the traditional Chinese medicine offers a vast repertory for pharmaceutical research, as is the case of Huperzia serrata, a member of Huperziaceae family. This review reports the Lycopodium alkaloids that have been isolated from this plant. However, it was mainly focused on the huperzine A (HupA), a promising therapeutic option in several acute and chronic disorders. The major therapeutic interest described for HupA has been directed to the treatment of acetylcholine-deficit dementia, including Alzheimer’s disease. However, HupA was also shown to be effective on cerebrovascular dementia and other neurodegenerative disorders with an ischemic component, as well as on other kind of cognitive impairments; the value of HupA on myasthenia gravis, organophosphate poisoning and schizophrenia has also been described. In addition, many other pharmacological properties have been ascribed to HupA, namely its anti-inflammatory, antinociceptive and anticonvulsant properties, which was recently identified, promoting a growing interest on HupA research. Furthermore, its particular chemical structure and the fact that HupA is well tolerated in humans, even at doses well above those clinically required, along with its favorable pharmacokinetics, also boosted an intense research in the pharmaceutical industry. Therefore, several HupA-related features are addressed in this review, including not only its therapeutic properties, but also its chemistry, biological and chemical sources, structure–activity relationship, pharmacokinetics and toxicology, which are discussed in detail covering the literature published from 1962 to 2014.

A Rapid HPLC Method for the Simultaneous Determination of Amiodarone and its Major Metabolite in Rat Plasma and Tissues: A Useful Tool for Pharmacokinetic Studies

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed and validated in rat plasma and tissue (heart, liver, kidney and lung) homogenates for the determination of amiodarone and its primary metabolite (desethylamiodarone), using tamoxifen as internal standard. Chromatographic separation was achieved within less than 5 min on a LiChroCART Purospher Star C18 column (55 × 4 mm, 3 µm). The mobile phase, consisting of phosphate buffer (50 mM) with 0.1% formic acid (pH 3.1)-methanol-acetonitrile (45:5:50, v/v/v), was pumped isocratically at a flow rate of 1.2 mL/min. The detection was conducted at 254 nm for all compounds. Calibration curves were linear (r(2) ≥ 0.995) in the range of 0.1-15 µg/mL for amiodarone and desethylamiodarone. The limits of quantification were established at 0.1 µg/mL for both analytes. The overall data of precision and accuracy were in accordance with international guidelines for bioanalytical method validation. Amiodarone and desethylamiodarone were extracted from rat matrices by a liquid-liquid extraction procedure and the mean recovery ranged from 59.9 to 97.6%. This novel HPLC method allows the fast and reliable determination of amiodarone and desethylamiodarone from several rat matrices (plasma, liver, kidneys, lungs and heart) and was successfully applied in a preliminary pharmacokinetic study.