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In the study of fungal pathogenesis, alternative methods have gained prominence due to recent global legislation restricting the use of mammalian animals in research. The principle of the 3 Rs (replacement, reduction, and refinement) is integrated into regulations and guidelines governing animal experimentation in nearly all countries. This principle advocates substituting vertebrate animals with other invertebrate organisms, embryos, microorganisms, or cell cultures. This review addresses host-fungus interactions by employing three-dimensional (3D) cultures, which offer more faithful replication of the in vivo environment, and by utilizing alternative animal models to replace traditional mammals. Among these alternative models, species like Caenorhabditis elegans and Danio rerio share approximately 75% of their genes with humans. Furthermore, models such as Galleria mellonella and Tenebrio molitor demonstrate similarities in their innate immune systems as well as anatomical and physiological barriers, resembling those found in mammalian organisms.

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A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host-pathogen interactions.

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Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

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BACKGROUND: Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that spreads rapidly, reaching pandemic status, causing the collapse of numerous health systems, and a strong economic and social impact. The treatment so far has not been well established and there are several clinical trials testing known drugs that have antiviral activity, due to the urgency that the global situation imposes. Drugs with specific mechanisms of action can take years to be discovered, while vaccines may also take a long time to be widely distributed while new virus variants emerge. Thus, drug repositioning has been shown to be a good strategy for defining new therapeutic approaches. Studies of the effect of enriched heparin in the replication of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) in vitro assays justify the advance for clinical tests. METHODS AND ANALYSIS: A phase I/II triple-blind parallel clinical trial will be conducted. Fifty participants with radiological diagnosis of grade IIA pneumonia will be selected, which will be allocated in 2 arms. Participants allocated in Group 1 (placebo) will receive nebulized 0.9% saline. Participants allocated in Group 2 (intervention) will receive nebulized enriched heparin (2.5 mg/mL 0.9% saline). Both groups will receive the respective solutions on a 4/4 hour basis, for 7 days. The main outcomes of interest will be safety (absence of serious adverse events) and efficacy (measured by the viral load).Protocols will be filled on a daily basis, ranging from day 0 (diagnosis) until day 8.

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Assuntos

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The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 107 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins ITGAV, ITGA5, ITGB1, ITGB3, and ITGB5. Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of ITGAV, ITGA5, ITGB3, ITGB5 genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies.

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Venous thromboembolism (VTE) is a serious clinical condition which early and accurate diagnosis may contribute to the reduction of associated morbidity and mortality. VTE occurs when a blood clot (thrombus) blocks the vein blood flow causing deep vein thrombosis (DVT) and, when it migrates to the lungs, it may clog the pulmonary arteries characterizing pulmonary embolism (PE). Analysis using fibrin degradation products or D-dimer and coagulation factor VIII may assist early diagnosis of VTE. Thus, two immunosensors were built using layer-by-layer (LbL) films technique, one containing the anti-D-dimer immobilized on polyethylene imine (PEI) and another the anti-FVIII on silk fibroin (SF). Immunosensor response, the antigen-antibody specific interaction, was investigated using cyclic voltammetry. When immunosensors, PEI/anti-D-dimer and SF/anti-FVIII, were exposed to antigens, D-dimer and Factor VIII, the voltammograms area and current were significantly increased with increasing specific antigen concentration. The specific interaction was confirmed with control experiments, electrodes containing only PEI or SF, that no significant changes in the voltammogram responses were observed and principal component analysis confirmed these results. The films formation and response were verified using scanning electronic microscopy (SEM). The developed immunosensor seems to be a promising and effective early complementary exam to assist in the VTE diagnosis, through the combined response of two biomarkers very sensible.

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As an emerging global health challenge, COVID-19 requires international knowledge to reach novel possible therapeutic strategies, especially for intensive-care patients. During the early stages of infection, pneumocytes II are the primary infected cells, harming the respiratory system. We have previous evidence in murine models that MSc's secretome can be used to treat pulmonary injuries induced with bleomycin, due to its content: growth factors, extracellular vesicles, and exosomes. We hypothesize and strongly recommend MSc secretome testing and production, in xenofree conditions, to be used as an alternative approach in SARS-Cov-2 patients in critical conditions.

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The original article [1] contained a minor error regarding the mean diameter of the alginate microcapsules described in relation to Fig. 4 in the Results section. The microcapsules had an actual mean diameter of 3000 µm instead of 1000 µm as mistakenly mentioned in the original article.

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We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

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