Recovery of bioactive compounds from an agro-industrial waste: extraction, microencapsulation, and characterization of jaboticaba(Myrciaria cauliflora Berg) pomace as a source of antioxidant.

This study aimed to evaluate the extraction of bioactive compounds from jaboticaba pomace, produce microcapsules by spray dryer technique, and characterize antioxidant compounds. A factorial experimental design was used in the extraction step. Maltodextrin (DE 10) was used as an encapsulating agent, in a ratio of 1: 1 (w/w), in the microencapsulation process. It was observed the increase of all bioactive compounds analyses comparing jaboticaba pomace with the extract. ATR-FTIR spectroscopy showed a vibrational stretching aromatic ring (1718 - 1731 cm-1) typical for anthocyanins. The Gaussian deconvolution presented extract peak area 7.56% higher than pomace. The encapsulating agent protected anthocyanins during the drying process. Microencapsulation of bioactive compounds from jaboticaba pomace can be useful for food applications whereas they are a rich source of antioxidant compounds. Moreover, the use of agro-industrial waste is promising linked to the use of clean technology as water as an antioxidant extractor.

Peptidorhamnomannan from Lomentospora prolificans modulates the inflammatory response in macrophages infected with Candida albicans.

BACKGROUND: Peptidorhamnomannan is a glycoconjugate that consists of a peptide chain substituted by O- and N-linked glycans, present on the cell surface of Lomentospora prolificans, a saprophytic fungus which is widely distributed in regions with temperate climates. O-linked oligosaccharides from peptidorhamnomannan isolated from Lomentospora prolificans conidia are recognized by macrophages mediating macrophage - conidia interaction. In this work, peptidorhamnomannan was isolated from L. prolificans mycelium cell wall and its role in macrophage - Candida albicans interaction was evaluated. RESULTS: Purified peptidorhamnomannan inhibits the reactivity of rabbit immune sera to mycelial and conidia forms of L. prolificans, indicating that this glycoconjugate is exposed on the fungal surface and can mediate interaction with host immune cells. We demonstrated that peptidorhamnomannan leads to TNF-α production in J774 macrophages for 1, 2 and 3 h of incubation, suggesting that this glycoconjugate may have a beneficial role in the response to fungal infections. In order to confirm this possibility, the effect of peptidorhamnomannan on the macrophage - C. albicans interaction was evaluated. Macrophages treated with peptidorhamnomannan led to a lower fungal survival, suggesting that peptidorhamnomannan induces an increased fungicidal activity in macrophages. Furthermore, TNF-α levels were measured in supernatants after macrophage - C. albicans interaction for 1, 2 and 3 h. Peptidorhamnomannan treatment led to a higher TNF-α production at the beginning of the interaction. However, the release of TNF-α was not maintained after 1 h of incubation. Besides, peptidorhamnomannan did not show any inhibitory or fungicidal effect in C. albicans when used at 100 µg/ml but it was able to kill C. albicans at a concentration of 400 µg/ml. CONCLUSION: We suggest that peptidorhamnomannan acts as a molecular pattern on the invading pathogen, promotes TNF-α production and, thus, increases macrophage fungicidal activity against Candida albicans.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

Targeting the P10 Peptide in Maturing Dendritic Cells via the DEC205 Receptor In Vivo: A New Therapeutic Strategy against Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus, which is the most frequent endemic systemic mycosis in many Latin American countries, where ~10 million people are believed to be infected. In Brazil, it is ranked as the tenth most common cause of death among chronic infectious diseases. Hence, vaccines are in development to combat this insidious pathogen. It is likely that effective vaccines will need to elicit strong T cell-mediated immune responses composed of IFNγ secreting CD4+ helper and CD8+ cytolytic T lymphocytes. To induce such responses, it would be valuable to harness the dendritic cell (DC) system of antigen-presenting cells. To assess the potential of targeting P10, which is a peptide derived from gp43 secreted by the fungus, directly to DCs, we cloned the P10 sequence in fusion with a monoclonal antibody to the DEC205 receptor, an endocytic receptor that is abundant on DCs in lymphoid tissues. We verified that a single injection of the αDEC/P10 antibody caused DCs to produce a large amount of IFNγ. Administration of the chimeric antibody to mice resulted in a significant increase in the levels of IFN-γ and IL-4 in lung tissue relative to control animals. In therapeutic assays, mice pretreated with αDEC/P10 had significantly lower fungal burdens compared to control infected mice, and the architecture of the pulmonary tissues of αDEC/P10 chimera-treated mice was largely normal. Altogether, the results obtained so far indicate that targeting P10 through a αDEC/P10 chimeric antibody in the presence of polyriboinosinic: polyribocytidylic acid is a promising strategy in vaccination and therapeutic protocols to combat PCM.

Comparative Genomics of Sibling Species of Fonsecaea Associated with Human Chromoblastomycosis.

Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.

Increase in virulence of Sporothrix brasiliensis over five years in a patient with chronic disseminated sporotrichosis.

The metropolitan region of Rio de Janeiro is hyperendemic for cat-associated sporotrichosis. This study aimed to assess the virulence of serial Sporothrix isolates from a 61-year-old male patient with chronic, destructive disseminated sporotrichosis. Five Sporothrix isolates were cultured from skin exudates and bone samples over a 5-year period, and all were molecularly identified as Sporothrix brasiliensis. The final isolate was significantly more virulent in Galleria mellonella larvae compared to earlier isolates. We conclude that S. brasiliensis has the capacity to increase in virulence in vivo. This finding is significant to clinicians caring for individuals with S. brasiliensis disease and it suggests that further studies are needed to identify the mechanisms underlying pathogenicity enhancement during chronic disease.

Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.