Biology subjects

da Silva, T. A.

Publications and source records attributed to da Silva, T. A..

5 recordsLinked to original sources

bioRxiv2024

A novel Mannan-specific chimeric antigen receptor M-CAR redirects T cells to interact with Candida spp. hyphae and Rhizopus oryzae spores

Invasive fungal infections (IFIs) are responsible for elevated rates of morbidity and mortality, causing around of 1.5 million deaths annually worldwide. One of the main causative agents of IFIs is Candida albicans, and non-albicans Candida species have emerged as a spreading global public health concernment. Furthermore, COVID-19 has contributed to a boost in the incidence of IFIs, such as mucormycosis, in which Rhizopus oryzae is the most prevalent causative agent. The effector host immune response against IFIs depends on the activity of T cells, which are susceptible to the regulatory effects triggered by fungal virulence factors. The fungal cell wall plays a crucial role as a virulence factor, and its remodeling compromises the development of a specific T-cell response. The redirection of Jurkat T cells to target Candida spp. by recognizing targets expressed on the fungal cell wall can be facilitated using chimeric antigen receptor (CAR) technology. This study generated an M-CAR that contains an scFv with specificity to -1,6 mannose backbone of fungal mannan, and the expression of M-CAR on the surface of modified Jurkat cells triggered a strong activation against Candida albicans (hyphae form), Candida tropicalis (hyphae form), Candida parapsilosis (pseudohyphal form), and Candida glabrata (yeast form). Moreover, M-CAR Jurkat cells recognized Rhizopus oryzae spores, which induced high expression of cell activation markers. Thus, a novel Mannan-specific CAR enabled strong signal transduction in modified Jurkat cells in the presence of Candida spp. or R. oryzae.

bioengineering↗

bioRxiv2024

Enhanced antifungal activity of NK-92 cells against Candida albicans mediated by a mannan-specific chimeric antigen receptor

Chimeric antigen receptors (CARs) offer promising prospects for innovative cell-based therapies against invasive fungal infections such as invasive candidiasis. Here, we have developed four CARs targeting Candida albicans with distinct single-chain variable fragments (scFvs): scFv3-CAR, scFv5-CAR, scFv12-CAR, and scFv{kappa}3-1-CAR. In T cells, scFv5-CAR induced IL-2 expression in response to C. albicans hyphae, while scFv3-CAR and scFv12-CAR did not mediate cell activation against C. albicans. Notably, scFv{kappa}3-1-CAR mediated the strongest cell activation against C. albicans yeast, hyphae, and other clinically relevant Candida species. scFv{kappa}3-1-CAR-NK-92 cells exhibited elevated IFN-{gamma} and CD107a expression, reducing C. albicans viability. NOD scid gamma (NSG) mice treated with scFv{kappa}3-1-CAR-NK-92 cells had reduced C. albicans burden in the kidney 24 hours postinfection. We showed that scFv{kappa}3-1-CAR targets C. albicans mannan but no other glycans in glycan microarray screening analyses. These findings reveal the scFv{kappa}3-1-CAR potential as a therapeutic strategy for treating Candida spp. by modifying peripheral blood mononuclear cells. ImportanceRecent studies on novel immunotherapies, including chimeric antigen receptor (CAR)-T cells, have shown promising results in preclinical models against invasive fungal infections (IFIs). However, the application of CAR technology in natural killer (NK) cells for treating IFIs remains unexplored. NK cells play a key role in early fungal clearance due to their antifungal activity mediated by granzymes, perforins, and the secretion of proinflammatory cytokines. This study is the first to demonstrate the feasibility and efficacy of CAR-modified NK cells targeting Candida spp. We provided proof-of-concept data showing that CAR-expressing NK cells exhibit enhanced activation and antifungal effects against clinically relevant Candida species by targeting mannan in the fungal cell wall. These findings are significant as they open new avenues for developing CAR-NK-based therapies to treat invasive candidiasis - a severe infection with limited treatment options and high mortality rates, particularly in immunocompromised patients.

bioengineering↗

bioRxiv2022

Paracoccin impacts on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs by modifying the expression of genes related to remodeling of the yeasts cell wall

The study of the paracoccin lectin (PCN) has provided knowledge about its role in the biology of Paracoccidioides brasiliensis and in the pathogenesis of paracoccidioidomycosis (PCM). In this context, PCN has proved to be a promising immunomodulatory agent for the exploration of vaccine target molecules and/or for diagnostic or therapeutic purposes. Previous investigations allowed establishing PCN as a factor of fungal virulence. However, the effect PCN exerts on the yeasts resistance to antifungal pharmacological agents used to treat human PCM are not known. Therefore, this work characterizes the role of PCN functional duality on virulence and susceptibility of P. brasiliensis to antifungal drugs. We show that the PCN overexpression increases the virulence of P. brasiliensis yeasts in an alternative model of infection, induces high susceptibility in vitro and in vivo of P. brasiliensis yeasts to antifungal therapy, and impact reducing relative mRNA expression of genes encoding proteins related to cell wall degradation. Conversely, PCN silencing minimized the yeasts virulence in Galleria mellonella, correlates with the lowest susceptibility to treatment with antifungal agent in vivo and impact differently from the PCN overexpression on the relative expression of markers related to P. brasiliensis yeasts cell wall remodelling. Our study demonstrates the impact of endogenous PCN on the P. brasiliensis yeasts virulence vs. susceptibility to antifungal drugs, the fungal biology, and the relationship of the yeasts-host cells.

microbiology↗

bioRxiv2022

ArtinM Cytotoxicity in B cells derived from Non-Hodgkins Lymphoma is regulated by CD45 phosphatase activity and Src family kinases

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3{gamma} N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkins lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cells incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell lines sourced from non-Hodgkins lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

cell biology↗

bioRxiv2019

Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR+ T-cell therapy

The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: C. gattii and C. neoformans. To establish invasive human disease, inhaled Cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infections mortality rate ranges from 20% to 70%. HIV+/AIDS patients are highly susceptible to Cryptococcus infection. Therefore, we hypothesized that CD8+ T cells could be redirected to target glucuronoxylomannan (GXM), a sugar present in the Cryptococcus species capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-{varsigma} and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined using bright-field microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed, while no clusters were present in the control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry using a GXM-specific antibody. The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected with NoDNA T cells. Furthermore, when GXMR-CAR+ T cells were administered to immunocompromised NSG mice infected with C. neoformans their C. neoformans burden was significantly lower than mock-transduced control T cell treated mice as shown via immunofluorescence using an anti-GXM antibody and Gomori methenamine-silver (GMS) staining of Titan cells in lung tissue. Thus, these findings demonstrated the effectiveness of GXMR-CAR+ T-cell therapy for cryptococcosis in a murine model.\n\nAuthor summaryCryptococcus gattii infects both immunocompetent and immunodeficient patients such as those with HIV/AIDS, while C. neoformans usually infects only immunocompromised patients. Every year, almost one million HIV/AIDS patients suffer from Cryptococcus fungal co-infection. At present, no curative treatment is available to treat cryptococcosis in chronic HIV/AIDS patients. The objective of this research was to develop novel \"Bioengineered\" Cryptococcus specific chimeric antigen receptor (CAR) CD8+ T-cells to target and kill Cryptococcus. By using a culture model, we demonstrated that the Cryptococcus specific CAR T cells were able to bind to the yeast form of C. neoformans. Using a mouse model of Cryptococcus, the Cryptococcus specific CAR treated group showed a significant reduction of fungal burden in lung tissue when compared to the control group. This gives new hope to HIV/AIDS patients suffering from cryptococcal infections.

immunology↗