The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce ...their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.
Biodistribution scintigraphic images of nanoparticles labeled with the radioisotope technetium 99: amphotericin loaded nanoparticles (99mTc-NANO-D-AMB) or free DMSA after 1 and 8 h of intravenously injection. Display omitted
•Amphotericin B is a widely used antifungal, but toxicity limits its use.•NANO-D-AMB was proposed to optimize efficacy and reduce toxicity of this drug.•NANO-D-AMB had similar antifungal efficacy than AMBISOME®.•NANO-D-AMB had lower toxicity compared to D-AMB in vivo and in vitro.•NANO-D-AMB improves drug delivery and might be a potential alternative to D-AMB.
Objectives The present study reports on the preparation and testing of a desoxycholate amphotericin B (D-AMB) sustained delivery system based on poly(lactic-co-glycolic acid) (PLGA) and ...dimercaptosuccinic acid (DMSA) polymeric blends (Nano-D-AMB) aimed at reducing the number of AMB administrations required to treat mycosis. Methods BALB/c mice were infected with the yeast Paracoccidioides brasiliensis intravenously to mimic the chronic form of paracoccidioidomycosis. At 30 days post-infection, the animals were treated with Nano-D-AMB 6 mg/kg of encapsulated D-AMB, intraperitoneally (ip), interval of 72 h or D-AMB (2 mg/kg, ip, interval of 24 h). Drug efficacy was investigated by the fungal burden recovery from tissues. Toxicity was assessed by renal and hepatic biochemical parameters, physical appearance of the animals and haematological investigation. The control groups used were non-infected and the infected mice mock treated with PBS. Results Nano-D-AMB presented results comparable to free D-AMB, with a marked antifungal efficacy. The Nano-D-AMB-treated group presented lower loss of body weight and absence of stress sign (piloerection and hypotrichosis) observed after D-AMB treatment. No renal blood urea nitrogen (BUN), creatinine or hepatic (pyruvic and oxalacetic glutamic transaminases) biochemical abnormalities were found. The micronucleus assay showed no significant differences in both the micronucleus frequency and percentage of polychromatic erythrocytes for Nano-D-AMB, indicating the absence of genotoxicity and cytotoxic effects. Conclusions The D-AMB-coated PLGA–DMSA nanoparticle showed antifungal efficacy, fewer undesirable effects and a favourable extended dosing interval. Nano-D-AMB comprises an AMB formulation able to lessen the number of drug administrations. Further studies would elucidate whether Nano-D-AMB would be useful to treat systemic fungal infections such as paracoccidioidomycosis, candidiasis, aspergillosis and cryptococcosis.
Highlights • The tested S19 and RB51 commercial vaccines proved to be genetically homogeneous among themselves. • All batches showed genotypes identical to S19 and RB51 reference strains, except ...batches from one S19 and one RB51 producers. • MLVA16 genotypes of S19 and RB51 strains were stable through 10 in vitro serial passages. • MLVA16 should be included among official tests required for approval of vaccines against brucellosis.
The aim of this study was to silence Ether à go-go 1 (EAG1) in glioma cells by RNAi in order to further analyze whether silencing this channel would improve injury caused by interferon gamma (IFN-γ).
...EAG1 silencing by the siRNAs EAG1hum_287 and EAG1hum_1727 (sequence targets 5'-GGCCTATTGTGTACAGCAATT-3' and 5'-GGGACTTCCTGAAGCTCTATT-3', respectively) was determined by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability was measured by the 3-(4,5)-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. U-138MG glioma cells were injured by IFN-γ (25 ng/ml, 24 h) with or without the RNAi for EAG1 by a non-viral vector (pKV10.1-3, 0.2 μg).
EAG1hum_287 and EAG1hum_1727 caused 0.46- and 0.52-fold decrease in EAG1 mRNA content, respectively. RNAi for EAG1 by pKv10.1-3 strengthened the reduction in cell viability caused by IFN-γ (11.4% versus 40.4%, p<0.05).
The present study reinforces the notion that EAG1 has a role in glioma biology, suggesting that this channel is a relevant player preserving the cell viability during IFN-γ injury.
It has been suggested that postsynaptic 5-HT1A receptors in the hippocampus, innervated by 5-HT neurons localized in the median raphe nucleus, mediate adaptive or coping responses to aversive events ...and that dysfunction of this system is related to symptoms of depression. To test this hypothesis we investigated the expression of c-fos mRNA in animals submitted to immobilization stress. The results showed that c-fos mRNA expression is significantly increased in the dentate gyrus and CA1-CA3 regions of the hippocampus after 30 min of forced restraint, suggesting that this structure is activated during stress. To investigate the role of 5-HT neurotransmission in the hippocampus on adaptation to aversive events we immobilized rats for 2 h and tested them 24 h later in an elevated plus-maze. Our results showed that the previous restraint period decreases exploration of open arms in the maze. This effect was reversed by bilateral microinjection of zimelidine (20 and 100 nmol), a 5-HT re-uptake blocker, or 8-OH-DPAT (3 nmol), a 5-HT1A agonist, into the dorsal hippocampus immediately after restraint. These results are compatible with the idea that postsynaptic 5-HT1A receptors located in the hippocampus participate in the development of tolerance to aversive events.
We examined cloacal samples from poultry raised on nonintensive production farms in Brazil for the presence of vancomycin-resistant enterococci. No VanA- or VanB-containing enterococci were ...identified in a total of 200 cloacal swabs. The most prevalent species were Enterococcus gallinarum (vanC1; 13.0%) and E. casseliflavus (vanC2/3; 5.5%).
We studied the genetic relationships between vancomycin-susceptible (n = 11) and -resistant Enterococcus faecium (VRE, n = 20) recovered from Brazil using a multilocus sequence typing (MLST) scheme. ...Grouping of allelic profiles revealed six clusters of related sequence types (STs) that differ in no more than two of the seven alleles. Of these, one cluster harbored 16 of the 20 isolates recovered during the first VRE outbreak in Brazil. The ampicillin and gentamicin resistance profiles were stable in the isolates that clustered within the groups I-III. Comparison with the allelic profiles of 139 E. faecium from different geographical regions and origins found in the international database http://www.mlst.net revealed that the Brazilian outbreak clone did not cluster in the previously named complex-17. This genetic complex contains hospital epidemic and clinical isolates recovered from different countries and continents. Twenty two of the 31 Brazilian isolates, including the VRE outbreak clone, clustered apart from the E. faecium isolates from the database, suggesting that these Brazilian isolates have a distinct evolutionary history.
To investigate the effects of stress on c-fos mRNA expression, rats were submitted to forced immobilization for 15, 30, 60 or 120 min before sacrifice. In situ hybridization was performed on sections ...containing the dorsal hippocampus with a 32P-labelled 50-base oligonucleotide probe (10(7)-10(9) cpm/micrograms) complementary to nucleotides 370-319 of rat c-fos. Forced restraint induced a time-dependent increase in c-fos mRNA expression which was most pronounced in the dentate gyrus and CA1-CA3 regions of the hippocampal formation, and which peaked after 30 min of immobilization (72.7 +/- 1.0 vs 24.1 +/- 0.8 cpm/mm2 in unrestrained animals). A positive but weaker signal was also detected in the amygdala, pyriform cortex and other parts of the cerebral cortex and habenulae. These results suggest that the hippocampal formation is activated during stress.
To investigate the effects of stressful stimuli on pineal gland activity, male Wistar albino rats (200-250 g, 2-4 per group) were submitted to 30 min of forced immobilization or to unilateral ...vibrissotomy 30 min before sacrifice. In situ hybridization was performed with a 35S-labelled 50-base oligonucleotide probe complementary to nucleotides 270-319 of rat c-fos on sections containing the pineal gland. Autoradiograms were quantified using a JAVA microdensitometer. Stressful stimuli induced a significant increase in the expression of c-fos mRNA in the pineal gland (restraint = 144.3 +/- 14.4 cpm/mm2; hemivibrissotomy = 206.7 +/- 29.5 cpm/mm2) as compared to no restraint animals (30.6 +/- 5.1 cpm/mm2), animals displaying tonic-clonic seizures after an ip (64 mg/kg) injection of pentylenetetrazole (34.0 +/- 4.7 cpm/mm2), or competition (70.6 +/- 11.4 cpm/mm2) and RNAase-treated (52.7 +/- 9.1 cpm/mm2) controls. These results raise the possibility that stressful stimuli may interfere with pineal gland function.
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