Common virulence factors between Histoplasma and Paracoccidioides: Recognition of Hsp60 and Enolase by CR3 and plasmin receptors in host cells.

Over the last two decades, the incidence of Invasive Fungal Infections (IFIs) globally has risen, posing a considerable challenge despite available antifungal therapies. Addressing this, the World Health Organization (WHO) prioritized research on specific fungi, notably Histoplasma spp. and Paracoccidioides spp. These dimorphic fungi have a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. Inhalation of conidia and mycelial fragments initiates the infection, crucially transforming into the yeast form within the host, influenced by factors like temperature, host immunity, and hormonal status. Survival and multiplication within alveolar macrophages are crucial for disease progression, where innate immune responses play a pivotal role in overcoming physical barriers. The transition to pathogenic yeast, triggered by increased temperature, involves yeast phase-specific gene expression, closely linked to infection establishment and pathogenicity. Cell adhesion mechanisms during host-pathogen interactions are intricately linked to fungal virulence, which is critical for tissue colonization and disease development. Yeast replication within macrophages leads to their rupture, aiding pathogen dissemination. Immune cells, especially macrophages, dendritic cells, and neutrophils, are key players during infection control, with macrophages crucial for defense, tissue integrity, and pathogen elimination. Recognition of common virulence molecules such as heat- shock protein-60 (Hsp60) and enolase by pattern recognition receptors (PRRs), mainly via the complement receptor 3 (CR3) and plasmin receptor pathways, respectively, could be pivotal in host-pathogen interactions for Histoplasma spp. and Paracoccidioides spp., influencing adhesion, phagocytosis, and inflammatory regulation. This review provides a comprehensive overview of the dynamic of these two IFIs between host and pathogen. Further research into these fungi's virulence factors promises insights into pathogenic mechanisms, potentially guiding the development of effective treatment strategies.

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.

Influence of Zinc on Histoplasma capsulatum Planktonic and Biofilm Cells.

Histoplasma capsulatum causes a fungal respiratory disease. Some studies suggest that the fungus requires zinc to consolidate the infection. This study aimed to investigate the influence of zinc and the metal chelator TPEN on the growth of Histoplasma in planktonic and biofilm forms. The results showed that zinc increased the metabolic activity, cell density, and cell viability of planktonic growth. Similarly, there was an increase in biofilm metabolic activity but no increase in biomass or extracellular matrix production. N'-N,N,N,N-tetrakis-2-pyridylmethylethane-1,2 diamine (TPEN) dramatically reduced the same parameters in the planktonic form and resulted in a decrease in metabolic activity, biomass, and extracellular matrix production for the biofilm form. Therefore, the unprecedented observations in this study highlight the importance of zinc ions for the growth, development, and proliferation of H. capsulatum cells and provide new insights into the role of metal ions for biofilm formation in the dimorphic fungus Histoplasma, which could be a potential therapeutic strategy.

Molecular detection of Histoplasma capsulatum in organ samples from bats randomly captured in urban areas of Araraquara, São Paulo state, Brazil.

The mycosis histoplasmosis is also considered a zoonosis that affects humans and other mammalian species worldwide. Among the wild mammals predisposed to be infected with the etiologic agent of histoplasmosis, bats are relevant because they are reservoir of Histoplasma species, and they play a fundamental role in maintaining and spreading fungal propagules in the environments since the infective mycelial phase of Histoplasma grows in their accumulated guano. In this study, we detected the fungal presence in organ samples of bats randomly captured in urban areas of Araraquara City, São Paulo, Brazil. Fungal detection was performed using a nested polymerase chain reaction to amplify a molecular marker (Hcp100) unique to H. capsulatum, which revealed the pathogen presence in organ samples from 15 out of 37 captured bats, indicating 40.5% of infection. Out of 22 Hcp100-amplicons generated, 41% corresponded to lung and trachea samples and 59% to spleen, liver, and kidney samples. Data from these last three organs suggest that bats develop disseminated infections. Considering that infected bats create environments with a high risk of infection, it is important to register the percentage of infected bats living in urban areas to avoid risks of infection to humans, domestic animals, and wildlife.

Paracoccidioides spp.: the structural characterization of extracellular matrix, expression of glucan synthesis and associated genes and adhesins during biofilm formation.

The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.

Exploring the Complexity of the Interaction between T. rubrum and S. aureus/S. epidermidis in the Formation of Polymicrobial Biofilms.

Dermatophytes associated with bacteria can lead to severe, difficult-to-treat infections and contribute to chronic infections. Trichophyton rubrum, Staphylococcus aureus, and Staphylococcus epidermidis can form biofilms influenced by nutrient availability. This study investigated biofilm formation by these species by utilizing diverse culture media and different time points. These biofilms were studied through scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), biomass, metabolic activity, and colony-forming units (CFUs). The results revealed that mixed biofilms exhibited high biomass and metabolic activity when cultivated in the brain heart infusion (BHI) medium. Both bacterial species formed mature biofilms with T. rubrum within 72 h, irrespective of media. The timing of bacterial inoculation was pivotal in influencing biomass and metabolic activity. T. rubrum's development within mixed biofilms depended on bacterial addition timing, while pre-adhesion influenced fungal growth. Bacterial communities prevailed initially, while fungi dominated later in the mixed biofilms. CLSM revealed 363 µm thick T. rubrum biofilms with septate, well-developed hyphae; S. aureus (177 µm) and S. epidermidis (178 µm) biofilms showed primarily cocci. Mixed biofilms matched T. rubrum's thickness when associated with S. epidermidis (369 µm), with few hyphae initially. Understanding T. rubrum and Staphylococcal interactions in biofilms advances antimicrobial resistance and disease progression knowledge.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Amphotericin B-loaded natural latex dressing for treating Candida albicans wound infections using Galleria mellonella model.

Amphotericin B (AmB) is the gold standard for antifungal drugs. However, AmB systemic administration is restricted because of its side effects. Here, we report AmB loaded in natural rubber latex (NRL), a sustained delivery system with low toxicity, which stimulates angiogenesis, cell adhesion and accelerates wound healing. Physicochemical characterizations showed that AmB did not bind chemically to the polymeric matrix. Electronic and topographical images showed small crystalline aggregates from AmB crystals on the polymer surface. About 56.6% of AmB was released by the NRL in 120 h. However, 33.6% of this antifungal was delivered in the first 24 h due to the presence of AmB on the polymer surface. The biomaterial's excellent hemo- and cytocompatibility with erythrocytes and human dermal fibroblasts (HDF) confirmed its safety for dermal wound application. Antifungal assay against Candida albicans showed that AmB-NRL presented a dose-dependent behavior with an inhibition halo of 30.0 ± 1.0 mm. Galleria mellonella was employed as an in vivo model for C. albicans infection. Survival rates of 60% were observed following the injection of AmB (0.5 mg.mL-1) in G. mellonella larvae infected by C. albicans. Likewise, AmB-NRL (0.5 mg.mL-1) presented survival rates of 40%, inferring antifungal activity against fungus. Thus, NRL adequately acts as an AmB-sustained release matrix, which is an exciting approach, since this antifungal is toxic at high concentrations. Our findings suggest that AmB-NRL is an efficient, safe, and reasonably priced ($0.15) dressing for the treatment of cutaneous fungal infections.

Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Unusual Differences in the Pulmonary Histopathology of Mice after Intranasal Infection with Mycelial Propagules of Histoplasma capsulatum Strains Classified as LAm A2 and NAm 2 Phylogenetic Species.

The ascomycete Histoplasma capsulatum is the causative agent of systemic respiratory mycosis histoplasmosis, which sometimes develops acute disseminated or chronic clinical forms, with the latter usually associated with granuloma formation. The present report shows differential histopathological changes in the pulmonary inflammatory response of mice infected intranasally with the mycelial morphotype of H. capsulatum strains with distinct genotypes, EH-46 and G-217B, classified as LAm A2 and NAm 2 phylogenetic species, respectively. Infected male BALB/c mice were sacrificed at different postinfection times, and their serial lung sections were stained with periodic acid-Schiff and analyzed via microscopy. In mice infected with the LAm A2 strain, the results showed progressive changes in the inflammatory infiltrate of the lung parenchyma during the first hours and days postinfection as well as granulomas with macrophages containing intracellular yeast cells, which prevailed at 14 and 21 days postinfection. Bronchiolar-associated lymphoid tissue was induced in mice infected with both strains, primarily in mice infected with the NAm 2 strain. Several lung sections from mice infected with the LAm A2 strain showed PAS-positive yeast cells aggregated in a perinuclear crown-like arrangement in macrophages from 3 h to 21 days postinfection. These findings highlight differences in the host pulmonary inflammatory response associated with distinct H. capsulatum species.

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.

Study of IsCT analogue peptide against Candida albicans and toxicity/teratogenicity in zebrafish embryos (Danio rerio).

Aim: An IsCT analogue peptide (PepM3) was designed based on structural studies of wasp mastoparans and tested against Candida albicans. Its effects on fungal cell membranes and toxicity were evaluated. Materials & methods: Antifungal activity was analyzed using a microdilution susceptibility test. Toxicity was assessed using human skin keratinocytes (HaCaT) and zebrafish embryos. Results: PepM3 demonstrated activity against C. albicans and a synergistic effect with amphotericin B. The peptide presented fungicidal action with damage to the fungal cell membrane, low toxicity in HaCat cells and was nonteratogenic in zebrafish embryos. Conclusion: Evaluating structural modifications is essential for the development of new agents with potential activity against fungal pathogens and for the reduction of toxic and teratogenic effects.

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.

Membranolytic Activity Profile of Nonyl 3,4-Dihydroxybenzoate: A New Anti-Biofilm Compound for the Treatment of Dermatophytosis.

The ability of dermatophytes to live in communities and resist antifungal drugs may explain treatment recurrence, especially in onychomycosis. Therefore, new molecules with reduced toxicity that target dermatophyte biofilms should be investigated. This study evaluated nonyl 3,4-dihydroxybenzoate (nonyl) susceptibility and mechanism of action on planktonic cells and biofilms of T. rubrum and T. mentagrophytes. Metabolic activities, ergosterol, and reactive oxygen species (ROS) were quantified, and the expression of genes encoding ergosterol was determined by real-time PCR. The effects on the biofilm structure were visualized using confocal electron microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). T. rubrum and T. mentagrophytes biofilms were susceptible to nonyl and resistant to fluconazole, griseofulvin (all strains), and terbinafine (two strains). The SEM results revealed that nonyl groups seriously damaged the biofilms, whereas synthetic drugs caused little or no damage and, in some cases, stimulated the development of resistance structures. Confocal microscopy showed a drastic reduction in biofilm thickness, and transmission electron microscopy results indicated that the compound promoted the derangement and formation of pores in the plasma membrane. Biochemical and molecular assays indicated that fungal membrane ergosterol is a nonyl target. These findings show that nonyl 3,4-dihydroxybenzoate is a promising antifungal compound.

Biocompatible anti-aging face mask prepared with curcumin and natural rubber with antioxidant properties.

Natural rubber latex (NRL) is a biopolymer widely used in biomedical applications. In this work, we propose an innovative cosmetic face mask, combining the NRL's biological properties with curcumin (CURC), which has a high level of antioxidant activity (AA) to provide anti-aging benefits. Chemical, mechanical and morphological characterizations were performed. The CURC released by the NRL was evaluated by permeation in Franz cells. Cytotoxicity and hemolytic activity assays were performed to assess safety. The findings showed that the biological properties of CURC were preserved after loading in the NRL. About 44.2 % of CURC was released within the first six hours, and in vitro permeation showed that 9.36 % ± 0.65 was permeated over 24h. CURC-NRL was associated with a metabolic activity higher than 70 % in 3 T3 fibroblasts, cell viability ≥95 % in human dermal fibroblasts, and a hemolytic rate ≤ 2.24 % after 24 h. Furthermore, CURC-NRL maintained the mechanical characteristics (range suitable) for human skin application. We observed that CURC-NRL preserved ~20 % antioxidant activity from curcumin-free after loading in the NRL. Our results suggest that CURC-NRL has the potential to be used in the cosmetics industry, and the experimental methodology utilized in this study can be applied to different kinds of face masks.

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.

Biological therapy in elderly patients with acute myeloid leukemia.

INTRODUCTION: The introduction of target molecules and immunological therapies is changing the treatment landscape of acute myeloid leukemia (AML). AREAS COVERED: We recapitulate the biological therapies that can be employed in the treatment of elderly patients with AML. Alongside small molecules inhibitors that target specific gene mutations, antibodies, tumor microenvironment modulators, and cellular therapies are being developed for the cure of the disease. Here, we report the biological activities, the efficacy and toxicities of humanized antibodies and antibody-drug conjugates that targets surface antigens as CD33 (gemtuzumab ozogamicine) or CD123 (pivekimab sunirine). We further explore mechanisms and effectiveness of medications that modify the microenvironment, such as glasdegib, or that harness the immune system against leukemia, such as CD47 antibody magrolimab, PD1/PDL1 inhibitors pembrolizumab and nivolumab, TIM3 inhibitor sabatolimab, T-cell and NK-cell engagers. Cellular therapies are considered, even if a large trial is still pending for the feasibility of the approach. In this scenario, a brief overview of the mechanism of action of target agents is provided, particularly with respect to their biological mechanisms. EXPERT OPINION: Overall, this therapeutic armamentarium will constitute the basis for multimodal and personalized combinations that, in the idea of precision medicine, will enormously benefit elderly AML patients.

Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures.

Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis-apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.