Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.     

Differentially expressed proteins in the interaction of Paracoccidioides lutzii with human monocytes

BackgroundFungi of the genus Paracoccidioides are the etiological agents of paracoccidioidomycosis, a highly prevalent mycosis in Latin America. Infection in humans occurs by the inhalation of conidia, which later revert to the form of yeast. In this context, macrophages are positioned as an important line of defense, assisting in the recognition and presentation of antigens, as well as producing reactive oxygen species that inhibit fungal spreading.AimsThe objective of this study was to identify differentially expressed proteins during the interaction between Paracoccidioides lutzii Pb01 strain and human U937 monocytes.MethodsTwo-dimensional electrophoresis, combined with mass spectrometry, was used to evaluate the differential proteomic profiles of the fungus P. lutzii (Pb01) interacting with U937 monocytes.ResultsIt was possible to identify 25 proteins differentially expressed by Pb01 alone and after interacting with U937 monocytes. Most of these proteins are directly associated with fungal metabolism for energy generation, such as glyceraldehyde-3-phosphate dehydrogenase, and intracellular adaptation to monocytes. Antioxidant proteins involved in the response to oxidative stress, such as peroxiredoxin, cytochrome, and peroxidase, were expressed in greater quantity in the interaction with monocytes, suggesting their association with survival mechanisms inside phagocytic cells. We also identified 12 proteins differentially expressed in monocytes before and after the interaction with the fungus; proteins involved in the reorganization of the cytoskeleton, such as vimentin, and proteins involved in the response to oxidative stress, such as glioxalase 1, were identified.ConclusionsThe results of this proteomic study of a P. lutzii isolate are novel, mimicking in vitro what occurs in human infections. In addition, the proteins identified may aid to understand fungal–monocyte interactions and the pathogenesis of paracoccidioidomycosis.     

Antiproliferative activity and QSAR study of copper(II) mixed chelate [Cu(N-N)(acetylacetonato)]NO3 and [Cu(N-N)(glycinato)]NO3 complexes, (Casiopeínas)

Mixed chelate copper(II) complexes patented and mark title registered as Casiopeínas^® are antineoplastic agents with general formulas [Cu(N-N)(α-l-amino acidato)]NO₃ and [Cu(N-N)(O-O)]NO₃, where the N-N donor is an aromatic substituted diimine (1,10-phenanthroline (phen) or 2,2′-bipyridine (bpy)) and the O-O donor is acetylacetonate (acac) or salicylaldehydate (salal). In the present work, the series of complexes [Cu(N-N)(acac)]NO₃ and [Cu(N-N)(gly)]NO₃ with several substituents on the diimine ligand were selected to perform a quantitative structure-activity relationship (QSAR) study. Two main analysis were performed: (1) the study of the influence of the substituents on diimine ligand on physicochemical properties such as half-wave potential (E_1/2) and their relationship with medial lethal dose (LD50) or medial inhibitory concentration (IC50) on several tumor cell lines and (2) the study of the influence of the secondary ligand when acac is changed for glycinate (gly). Results showed that the presence of the central fused aromatic ring in the phen containing complexes is necessary to preserve the antiproliferative activity. The QSAR equations showed a strong relationship between the IC50 and E_1/2; the most active complexes are the weaker oxidants. The change of secondary ligand from acac to gly has less influence on biological activity than the changes on the diimine ligand.