In vitro evaluation of human intravenous immunoglobulin in combination with antimicrobials and human serum against multidrug-resistant isolates of Acinetobacter baumannii.

The high incidence of multidrug-resistant (MDR) Acinetobacter baumannii has been a challenge for health worldwide, due to the reduction of therapeutic options, making the use of antimicrobial combinations necessary for the treatment, such as meropenem, amikacin, and colistin. Antibodies against bacterial species, mainly immunoglobulins G (IgG), are produced for acting as effector mechanisms (neutralization, opsonization, phagocytosis, and complement system activation). Some studies have demonstrated promising results of IgG in combination with antimicrobial preparations against bacterial infections, in which the direct action of IgG has restored the immune system balance. Serious problem caused by the increase of MDR A. baumannii isolates results in a constant search for therapeutic alternatives to defeat these infections. However, this study aims to verify in vitro the phagocytosis rate of the A. baumannii-infected human monocytes, as well as to analyze possible morphological changes induced by intravenous immunoglobulin G (IVIG) with human serum in association with antimicrobials. The phagocytosis rate and bacterial cell binding capacity of IVIG were determined for two A. baumannii isolates submitted to 4 mg/mL of human IVIG alone and in combination with different sub-minimum inhibitory concentrations (sub-MICs) of meropenem, amikacin, and colistin and processed for indirect immunofluorescence. Subsequently, these isolates were resubmitted and coupled with human serum and processed for scanning electron microscopy. There was no statistical difference for phagocytosis rates in the isolates tested. Bacterial isolates showed alterations in cell morphology when exposed to IVIG/human serum alone and in combination with antimicrobials such as alteration in shape, wrinkling, membrane depression, and especially cell rupture with extravasation of cytoplasmic material. The isolates visually differed in the IVIG binding to the bacterial cell, with higher fluorescence intensity, which corresponds to the highest IVIG binding, in the isolate more sensitive to meropenem, amikacin, and colistin. No differences between treatments were observed in the IVIG binding to the bacterial cell. The combined action of IVIG with meropenem, amikacin, and colistin against A. baumannii MDR isolates induced several bacterial cell damages. And when associated with human serum, a massive destruction of cells can be observed. These results may suggest the analysis of the use of IgG preparations for the treatment of A. baumannii MDR infections.

The endoplasmic reticulum of trypanosomatids: An unrevealed road for chemotherapy.

The endoplasmic reticulum (ER) of higher eukaryotic cells forms an intricate membranous network that serves as the main processing facility for folding and assembling of secreted and membrane proteins. The ER is a highly dynamic organelle that interacts with other intracellular structures, as well as endosymbiotic pathogenic and non-pathogenic microorganisms. A strict ER quality control (ERQC) must work to ensure that proteins entering the ER are folded and processed correctly. Unfolded or misfolded proteins are usually identified, selected, and addressed to Endoplasmic Reticulum-Associated Degradation (ERAD) complex. Conversely, when there is a large demand for secreted proteins or ER imbalance, the accumulation of unfolded or misfolded proteins activates the Unfold Protein Response (UPR) to restore the ER homeostasis or, in the case of persistent ER stress, induces the cell death. Pathogenic trypanosomatids, such as Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp are the etiological agents of important neglected diseases. These protozoans have a complex life cycle alternating between vertebrate and invertebrate hosts. The ER of trypanosomatids, like those found in higher eukaryotes, is also specialized for secretion, and depends on the ERAD and non-canonical UPR to deal with the ER stress. Here, we reviewed the basic aspects of ER biology, organization, and quality control in trypanosomatids. We also focused on the unusual way by which T. cruzi, T. brucei, and Leishmania spp. respond to ER stress, emphasizing how these parasites' ER-unrevealed roads might be an attractive target for chemotherapy.

The nanotoxicity assessment of cube-like iron nitride magnetic nanoparticles at the organismal level of nematode Caenorhabditis elegans.

Magnetic nanoparticles (NPs) are suitable candidates for various medical and biological applications, despite some concerns that they may have negative impacts on human health. In this study, the toxicity effects of magnetic NPs consisting of α"-Fe16N2 captured and bioaccumulated by the nematode Caenorhabditis elegans (C. elegans) in the early larval stage are evaluated. The choice of α"-Fe16N2 NPs is based on their good structural stability when stored in saline solution and high magnetic performance. The uptake and bioaccumulation of α"-Fe16N2 NPs in intestinal cells of C. elegans was evidenced by transmission electron microscopy. After exposure to NPs up to 40 mg mL-1, C. elegans larval development, survival, feeding behavior, defecation cycles, movement and reproduction were monitored. C. elegans survival and other monitored behavioral evolutions do not show significant changes, except for a slight statistical reduction in the reproductive profile. Therefore, the present results are promising and very encouraging for investigations of applications of α"-Fe16N2 NPs in the biomedical area.

Lignin from Morinda citrifolia leaves: Physical and chemical characterization, in vitro evaluation of antioxidant, cytotoxic, antiparasitic and ultrastructural activities.

In this work, we investigated in vitro the antioxidant, cytotoxic and anti-leishmanial activities of a lignin extracted from the leaves of Morinda citrifolia. Initially, an analysis of the composition of the sheets was performed, then the lignin was obtained by alkaline delignification and characterized by different techniques: elemental analysis, FT-R, UV-vis, HSQC-NMR, thermal analysis, Py-GC/MS and by GPC. The results showed that the leaves had in their composition cellulose (31.29%), hemicellulose (25.01%), lignin (18.34%), extractives (14.39%) and ash (10.03%). The lignin extraction yield was 89.8%. The lignin obtained is of the GSH type with the following contents 79.39%, 13.58% and 7.03% respectively. Furthermore, it is low molecular weight and thermally stable. It had a phenolic content of 93.3 mg GAE/g and low antioxidant activity. In macrophage cytotoxicity assays, it presented a CC50 of 31.0 µg/mL, showing less toxicity than amphotericin B. In assays against the promastigote forms of Leishmania amazonensis, lignin presented an IC50 of 29.56 µg/mL, a less effective concentration than amphotericin B (IC50 = 0.14 µg/mL). However, it was able to promote inhibition of the parasites, a fact confirmed by structural changes. These findings reinforce that M. citrifolia lignin is a promising macromolecule for use as an antiparasitic and antioxidant agent.

Ultrastructural changes caused by the combination of intravenous immunoglobulin with meropenem, amikacin and colistin in multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen associated with increased morbidity and mortality in Healthcare-associated infections (HAI). Combination antimicrobial therapy, meropenem, amikacin and colistin, has been used as an alternative in multidrug-resistant (MDR) A. baumannii infections due to reduced treatment options. However, these combinations are not always effective and exhibit high toxicity. Empiric therapy of intravenous immunoglobulin (IVIG) associated with antimicrobials has shown promising results in bacterial infections, considering the immunomodulatory action of IVIG. Thus, the aim of this study was to determine the combined antimicrobial action and to describe the ultrastructural changes caused in ten MDR A. baumannii isolates submitted to IVIG alone and in combination with colistin, meropenem and amikacin. Minimum Inhibitory Concentration (MIC) of antimicrobials and checkerboard were determined. Isolates were submitted to 4 mg/mL of IVIG alone and in combination with different synergistic sub-MIC of antimicrobials tested, and processed for scanning electron microscopy. Nine bacterial isolates showed meropenem-resistant, two isolates had colistin-intermediate, and four isolates were considered intermediate to amikacin. Synergism in five isolates for meropenem/amikacin and meropenem/colistin were observed. Bacterial cells submitted to IVIG and meropenem, amikacin and colistin presented several ultrastructural changes, such as cell elongation and rupture, membrane roughness, incomplete cell division, cell surface "bubbles" and "depression". A. baumannii isolates presented high resistance to meropenem and synergism among evaluated antimicrobials. In addition, it was possible to verify in vitro that IVIG associated with meropenem, amikacin and colistin is a promising alternative for MDR A. baumannii infections. Thus, these data support the continued empirical use and stimulate in vivo analyzes with IVIG to search for new therapeutic options for HAI.

Thiophene-thiosemicarbazone derivative (L10) exerts antifungal activity mediated by oxidative stress and apoptosis in C. albicans.

Reactive oxygen species (ROS) cause cell damage and death. To reverse these effects, cells produce substances such as reduced glutathione (GSH) that serve as substrates for antioxidant enzymes. One way to combat microbial resistance includes nullifying the effect of glutathione in microbial cells, causing them to die from oxidative stress. The compound 2-((5-nitrothiophen-2-yl)methylene)-N-(pyridin-3-yl) hydrazine carbothioamide (L10) is a new thiophene-thiosemicarbazone derivative with promising antifungal activity. The aim of this study was to evaluate its mechanism of action against Candida albicans using assays that evaluate its effects on redox balance. Treatment with L10 promoted significant changes in the minimum inhibitory concentration (MIC) values in ascorbic acid and GSH protection tests, the latter increasing up to 64-fold of the MIC. Using nuclear magnetic resonance, we demonstrated interaction of L10 and GSH. At concentrations of 4.0 and 8.0 µg/mL, significant changes were observed in ROS production and mitochondrial membrane potential. The cell death profile showed characteristics of initial apoptosis at inhibitory concentrations (4.0 µg/mL). Transmission electron microscopy data corroborated these results and indicated signs of apoptosis, damage to plasma and nuclear membranes, and to mitochondria. Taken together, these results suggest a possible mechanism of action for L10 antifungal activity, involving changes in cellular redox balance, ROS production, and apoptosis-compatible cellular changes.

Anti-Staphylococcus aureus action of three Caatinga fruits evaluated by electron microscopy.

This study evaluated the antibacterial activity of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruit extracts against clinical strains of Staphylococcus aureus. The samples were active for all S. aureus strains (minimum inhibitory concentration: 0.38-3.13 mg mL⁻¹), including the multiresistant strain. The morphological changes suggested the cell wall as the main action target. The treated-cells also lose their ability to form aggregates. The analysis suggests cell wall impairment, which causes the loss of viability and death. This study showed for the first time the morphologic alterations involved in the anti-S. aureus action of fruits of A. colubrina, L. ferrea and P. moniliformis. These findings indicated that these fruit extracts are sources of bioactive compounds that can be used as antibacterial agents.

Avaliação da atividade biológica de adutos derivados da reação morita-baylis-hillman sobre Trypanosoma cruzi: uma abordagem molecular, citoquímica e ultraestrutural / Biological activity evaluation of morita-baylis-hillman adducts against Trypanosoma cruzi: a molecular, cytochemical and ultrastructural approach

A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é um importante problema de saúde pública na América Latina, com cerca de 7,6 milhões de pessoas infectadas. O benznidazol e o nifurtimox são os únicos fármacos disponíveis para o tratamento, e além de apresentarem sérios efeitos colaterais, a sua eficácia na fase crônica da doença ainda é controversa. Neste sentido, nós sintetizamos o aduto Morita-Baylis-Hillman 3-hidroxi-2-metileno-3-(4-nitrofenilpropanonitrilo) (MBHA 3) e avaliamos sua atividade biológica sobre o T. cruzi. O MBHA 3 inibiu fortemente o crescimento das formas epimastigotas, com IC(50) / 72h de 28,5 µM e causou intensa lise de tripomastigotas, com IC(50)/ 24h de 25,5 µM. A análise ultraestrutural mostrou alterações morfológicas significantes, como arredondamento do corpo celular e desorganização intracelular. Alterações indicativas de apoptose, autofagia ou necrose também foram observadas nas células mais afetadas. A fim de melhor compreender o mecanismo envolvido na morte celular induzida pelo composto, nós utilizamos a microscopia confocal e a citometria de fluxo aliadas a sondas fluorescentes, como anexina-V (AV)/ iodeto de propídio (IP); Calceína-AM (CA)/ homodímero de etídio (HE); laranja de acridina (LA) e rodamina 123. O tratamento com 6 e 12 µg/ mL revelou uma alta porcentagem de células viáveis pela CA/ HE, mas também induziu alterações mitocondriais e aumento da marcação com a LA, sugerindo que um processo de morte celular programada (MCP) por autofagia poderia estar ocorrendo. Por outro lado, o tratamento com 24 µg/ mL levou à perda de viabilidade celular com danos excessivos sobre a membrana plasmática e mitocondrial e fragmentação inespecífica do DNA. Em conclusão, nossos achados sugerem que o MBHA 3, em alta concentração, induz MCP por necrose em T. cruzi.