Bioprospecting, Synergistic Antifungal and Toxicological Aspects of the Hydroxychalcones and Their Association with Azole Derivates against Candida spp. for Treating Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) remains a prevalent fungal disease, characterized by challenges, such as increased fungal resistance, side effects of current treatments, and the rising prevalence of non-albicans Candida spp. naturally more resistant. This study aimed to propose a novel therapeutic approach by investigating the antifungal properties and toxicity of 2-hydroxychalcone (2-HC) and 3'-hydroxychalcone (3'-HC), both alone and in combination with fluconazole (FCZ) and clotrimazole (CTZ). A lipid carrier (LC) was also developed to deliver these molecules. The study evaluated in vitro anti-Candida activity against five Candida species and assessed cytotoxicity in the C33-A cell line. The safety and therapeutic efficacy of in vivo were tested using an alternative animal model, Galleria mellonella. The results showed antifungal activity of 2-HC and 3'-HC, ranging from 7.8 to 31.2 as fungistatic and 15.6 to 125.0 mg/L as fungicide effect, with cell viability above 80% from a concentration of 9.3 mg/L (2-HC). Synergistic and partially synergistic interactions of these chalcones with FCZ and CTZ demonstrated significant improvement in antifungal activity, with MIC values ranging from 0.06 to 62.5 mg/L. Some combinations reduced cytotoxicity, achieving 100% cell viability in many interactions. Additionally, two LCs with suitable properties for intravaginal application were developed. These formulations demonstrated promising therapeutic efficacy and low toxicity in Galleria mellonella assays. These results suggest the potential of this approach in developing new therapies for VVC.

Polypeptides Targeting Paracoccidioides brasiliensis Drk1.

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.

Alternative Non-Mammalian Animal and Cellular Methods for the Study of Host-Fungal Interactions.

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.

Alginate-Based 3D A549 Cell Culture Model to Study Paracoccidioides Infection.

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.

Nanotechnology-Based Approaches for Voriconazole Delivery Applied to Invasive Fungal Infections.

Invasive fungal infections increase mortality and morbidity rates worldwide. The treatment of these infections is still limited due to the low bioavailability and toxicity, requiring therapeutic monitoring, especially in the most severe cases. Voriconazole is an azole widely used to treat invasive aspergillosis, other hyaline molds, many dematiaceous molds, Candida spp., including those resistant to fluconazole, and for infections caused by endemic mycoses, in addition to those that occur in the central nervous system. However, despite its broad activity, using voriconazole has limitations related to its non-linear pharmacokinetics, leading to supratherapeutic doses and increased toxicity according to individual polymorphisms during its metabolism. In this sense, nanotechnology-based drug delivery systems have successfully improved the physicochemical and biological aspects of different classes of drugs, including antifungals. In this review, we highlighted recent work that has applied nanotechnology to deliver voriconazole. These systems allowed increased permeation and deposition of voriconazole in target tissues from a controlled and sustained release in different routes of administration such as ocular, pulmonary, oral, topical, and parenteral. Thus, nanotechnology application aiming to delivery voriconazole becomes a more effective and safer therapeutic alternative in the treatment of fungal infections.

Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

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.