The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4⁺ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4⁺ and CD8⁺ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4⁺ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4⁺ and CD8⁺ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia.

Background The macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregulatory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii. Methods and Results We demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-α in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-α even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control. Conclusion Polarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen.

Concerns over the replicability and reproducibility of published research have grown in many research fields, but empirical data to inform policies are still scarce. Biomedical research in Brazil expanded rapidly over the last three decades, with no systematic assessment of the replicability of its findings. With this in mind, we set up the Brazilian Reproducibility Initiative, a multicenter replication of published experiments from Brazilian science using three common experimental methods: the MTT assay, the reverse transcription polymerase chain reaction (RT-PCR) and the elevated plus maze (EPM). A total of 56 laboratories performed 143 replications of 56 experiments; of these, 90 replications of 45 experiments were considered valid by an independent committee. Replication rates for these experiments varied between 20 and 44% according to five predefined criteria. In median terms, ratios between group means were 58% lower in replications than in original experiments, while coefficients of variation were 82% higher. Effect size decrease was smaller for MTT experiments, original results with less variability and those considered more challenging to replicate, while t values for replications were positively correlated with researcher predictions about replicability, and negatively correlated with the rate of publications by the original article’s last author. Deviations from preregistered protocols were very common in replications, most frequently due to reasons inherent to the experimental model or related to infrastructure and logistics. Our results highlight factors that limit the replicability of results published by researchers in Brazil and suggest ways by which this scenario can be improved.

Chimeric antigen receptors (CARs) offer promising prospects for innovative cell-based therapies against invasive fungal infections such as invasive candidiasis. Here, we have developed 4 CARs targeting Candida albicans with distinct single-chain variable fragments (scFvs): scFv3-CAR, scFv5-CAR, scFv12-CAR, and scFvκ3-1-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, scFvκ3-1-CAR mediated the strongest cell activation against C. albicans yeast, hyphae, and other clinically relevant Candida species. scFvκ3-1-CAR-NK-92 cells exhibited elevated IFN-γ and CD107a expression, reducing C. albicans viability. NOD scid gamma (NSG) mice treated with scFvκ3-1-CAR-NK-92 cells had reduced C. albicans burden in the kidneys 24 hours postinfection. We showed that scFvκ3-1-CAR targets C. albicans mannan but no other glycans in glycan microarray screening analyses. These findings reveal the scFvκ3-1-CAR potential as a therapeutic strategy for treating Candida spp. by modifying peripheral blood mononuclear cells.

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing Toll-like receptor (TLR)2 and cluster of differentiation (CD)14 N-glycans, induces cytokine production, and promotes type 1 T helper (Th1) immunity, a process that plays an assisting role in the combat against fungal infections. We recently demonstrated that ArtinM stimulates CD4⁺ T cells to produce interleukin (IL)-17 through direct interaction with CD3. Here, we further investigated the effects of ArtinM on the production of IL-17 by B cell activation. We showed that ArtinM activates murine B cells, increasing IL-17 and IL-12p40 production. The direct effect of ArtinM was sufficient to induce IL-17 production in B cells, and we did not find differences in the levels of IL-17 between the B cells purified from the wild-type (WT) and knockout (KO) mice for TLR2 or CD14 in the presence of ArtinM. Thus, the effects of ArtinM on splenic B cells through carbohydrate recognition may contribute to Th17 immunity; however, the mechanism involved is not associated with the interaction of ArtinM with TLR2 and CD14. The current work represents a pioneering effort in the understanding of the induction of IL-17 by lectins in B cells.

The low efficacy and side effects associated with antifungal agents have highlighted the importance of developing immunotherapeutic approaches to treat Cryptococcus gattii infection. We developed an immunization strategy that uses selective Dectin-1 agonist as an adjuvant. BALB/c or C57BL/6 mice received curdlan or β-glucan peptide (BGP) before immunization with heat-killed C. gattii, and the mice were infected with viable C. gattii on day 14 post immunization and euthanized 14 days after infection. Adjuvant curdlan restored pulmonary tumor necrosis factor- α (TNF-α) levels, as induced by immunization with heat-killed C. gattii. The average area and relative frequency of C. gattii titan cells in the lungs of curdlan-treated BALB/c mice were reduced. However, this did not reduce the pulmonary fungal burden or decrease the i0,nflammatory infiltrate in the pulmonary parenchyma of BALB/c mice. Conversely, adjuvant curdlan induced high levels of interferon-γ (IFN-γ) and interleukin (IL)-10 and decreased the C. gattii burden in the lungs of C57BL/6 mice, which was not replicated in β-glucan peptide-treated mice. The adjuvant curdlan favors the control of C. gattii infection depending on the immune response profile of the mouse strain. This study will have implications for developing new immunotherapeutic approaches to treat C. gattii infection.