The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose-and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.
Introduction: Candidiasis is an opportunist infection, caused by yeast of the genus Candida, which emerges as one of the main causes of systemic infections in hospitalized patients. Candida albicans is the most common causing agent of these infections. According to the Brazilian Homeopathic Pharmacopeia[1], nosodes are medicines compounded from chemically undefined biological products. Living nosodes are prepared using the etiologic agent of an illness in its infective form, were first developed by Brazilian physician Roberto Costa (RC). Roberto Costa’s research indicated that living nosodes present a higher capability to stimulate the host’s immunological system [2]. Aim: This study aims to evaluate cellular alterations induced in C. albicans yeasts and RAW 264-7 macrophages by Candida albicans RC. Methodology: To prepare Candida albicans RC, one part of C. albicans infective yeast suspension (108 cell/ml) was diluted in 9 parts of sterile distilled water and submitted to 100 mechanical succussions. This process was successively repeated to the potencies of 12x and 30x1. Water 30x was prepared by the same technique, as control. The cell viability of C. albicans previously treated with nosodes in both potencies and respective controls was evaluated using the samples at the concentration of 10% (V/V), in a volume of 1ml, distributed in 1-3 days. The viability of the yeast cells was analyzed by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolic) (5mg/ml) assay [3] and by Propidium Iodide (PI) incorporation methods. Additionally, using macrophages RAW 264-7 as a cell model, Nitric Oxide (NO) production and cell viability were also evaluated. For this, the following protocol of cell treatment was employed: on each experimental day, RAW 264-7 cells were treated 4 times (4 stimuli) with RC nosode 30x at the concentration of 10% (V/V). Results: The nosodes (12x and 30x) did not present cytotoxic effects on macrophage cells (n=1), or on C. albicans yeasts (n=2), as detected by MTT and PI methods. Moreover, no statistically significant differences on NO production were detected among the experimental groups (n=6). Conclusion: Preliminary results of in vitro assays indicate that nosodes (12x and 30x) do not alter mitochondrial activity or cell viability of C. albicans. Similarly, treatment by RC nosodes does not seem to alter NO release and mitochondrial activity of RAW macrophages. New experiments are being performed to confirm these preliminary data.
Background: "Roberto Costa’s Biotherapics" are homeopathic remedies prepared from intact microorganisms which have been proposed for treatment of diseases like influenza. Aim: This study aimed to compare the biochemical effects, in MDCK cells and RAW-264-7 macrophages, of biotherapics prepared from intact influenza virus diluted in water as well as from a sample of the same virus inactivated by ethanol 70% (v / v), both in the homeopathic potencies of 12x and 30x. Water 30x, non-dynamized water and cells without treatment (control cells) were used as control. Methodology: Treatments were performed by incubating MDCK cells with DMEM medium added in a 1:10 ratio for 6 times (3 different aliquots per day) or 18 times (up to 4 aliquots per day) in each experimental situation. Each aliquot was added with an interval of at least 2 hours. After that, the mitochondrial activity of MDCK cells was analyzed by MTT assay. The effects of treatments with intact biotherapics on MDCK cells respiratory parameters were studied using high resolution respirometry (Oroboros Oxygraph-O2K). RAW-264-7 macrophages were treated with intact and inactivated biotherapic 30x (4 treatments, 24 hours) to verify the nitric oxide production. These macrophages were also submitted to MTT assay. Results: Both biotherapic preparations 1x (intact and inactivated virus sample) were analyzed by transmission electronic microscopy. The presence of virus particles was detected when water was used as solvent. The use of ethanol as biotherapic solvent induced complete virus lysis. There was no alteration in cell osmolarity revealed by neutral red assay, when 10% of each test solution was used. Cellular viability analyzed by MTT method increased when MDCK cells were treated with 18 stimuli of inactivated biotherapic 30x when compared to intact biotherapic 30x (p0.05) were detected when these cells were compared to control cells. The maximum respiratory capacity of MDCK cells increased after treatment with 18 stimuli of intact biotherapic 30x when compared to control cells. However, no statistically significant differences (p>0.05) induced by biotherapics in macrophage cells were observed by MTT and nitric oxide assays. Moreover, a reduction in nitric oxide was observed in macrophages treated with dynamized water when compared to control cells. Conclusions: These results indicate that the method of biotherapic compounding (intact or inactivated virus as starting point) can modify the cellular parameters with the tendency to increase cellular response with longer treatments and higher potencies.
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