Transitions in frailty state 12 months after kidney transplantation: a prospective cohort study.

BACKGROUND: Frailty is associated with several unfavorable outcomes after kidney transplantation (KTx). However, limited information is available regarding the transitions in frailty state and its components after KTx. This study aimed to evaluate the transitions in physical frailty phenotype and its components over a period of 12 months after KTx. METHODS: In this prospective single-center cohort study, we measured physical frailty phenotype (PFP) and its components at the time of admission for KTx and 12 months after KTx. The evaluation includes five components: weakness (grip strength analysed by sex and body mass index quartiles), physical activity (kcals/week based on the Minnesota Leisure Time Physical Activity questionnaire), exhaustion (self-report using the Center for Epidemiological Studies Depression Scale), gait speed (time taken to walk 15 feet based on sex and height-specific cutoff), and unintentional weight loss (self-report of unintentional weight loss > 10 lbs in the last year). The exhaustion and physical activity components are validated in the Brazilian population. Each component is scored as 0 or 1 according to its presence or absence, and a PFP score of 3-5 defines frailty, 2 is intermediate, and 0-1 is rated as non-frail. We used the McNemar and Wilcoxon test to compare physical frailty phenotype and its components between the two periods. RESULTS: Among 87 patients included in the study, 16.1% were classified as frail, 20.7% as intermediately frail, and 63.2% as non-frail. Sixty-four patients were included in the analysis to evaluate transitions in frailty. At the time of admission for KTx, 15.6% of patients were defined as frail compared to 4.7% of patients at 12 months after KTx (p = 0.023). Among the physical frailty phenotype components, the proportion of patients who scored in the weight loss category 12 months after KTx was significantly lower than that at the time of KTx (6.3% vs 34.4%, p < 0.001). CONCLUSIONS: There was a 69.9% reduction in the prevalence of frail patients at the end of the 12-month follow-up after KTx. Among the components of frailty, weight loss showed a significant improvement.

Down-regulation of TUFM impairs host cell interaction and virulence by Paracoccidioides brasiliensis.

The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.

Preliminary evaluation of circulating microRNAs as potential biomarkers in paracoccidioidomycosis.

MicroRNAs (miRNAs) are small RNAs (length, 19-24 nucleotides) that regulate gene expression by either mRNA degradation or translational inhibition of proteins. Circulating miRNAs, which are extremely stable and protected from RNAse-mediated degradation in body fluids, have appeared as candidate biomarkers for numerous diseases. However, little is known about circulating miRNAs in fungal infections. Paracoccidioidomycosis (PCM) is caused by the Paracoccidioides species, and is endemic in Central and South America, with predominance in adult male workers from rural areas. The current study aimed to identify a serum miRNA expression profile that could serve as a novel diagnostic biomarker for PCM. Total RNA was isolated and the levels of circulating miRNAs were compared between patients with PCM and healthy control subjects using reverse transcription-quantitative polymerase chain reaction. Bioinformatic analysis was used to evaluate the potential roles of these miRNAs in PCM. Eight miRNAs were differentially expressed in serum samples from patients with PCM. These miRNAs are associated with apoptosis and immune response. The identified miRNAs facilitate with understanding the regulatory mechanisms involved in the host-parasite interaction of PCM. Furthermore, considering that the diagnosis of PCM presents difficulties, these miRNAs may serve as novel biomarkers for this disease.

Surface-expressed enolase contributes to the adhesion of Paracoccidioides brasiliensis to host cells.

Paracoccidioidomycosis is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization, and to study the P. brasiliensis enolase amino acid sequence. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. The analysis of the amino acid sequence revealed an internal plasminogen-binding motif ((254)FYKADEKKY(262)), which is conserved in most organisms and described as an important interaction site of the enolase with the host cell surface. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis.

Comparative transcriptome analysis of Paracoccidioides brasiliensis during in vitro adhesion to type I collagen and fibronectin: identification of potential adhesins.

Paracoccidioidomycosis is caused by the dimorphic fungus Paracoccidioides brasiliensis. The extracellular matrix (ECM) plays an important role in regulation of cell adhesion, differentiation, migration and proliferation of cells. An in vitro binding assay of P. brasiliensis yeast cells adhering to type I collagen and fibronectin was performed in order to identify novel adhesins. Representational difference analysis (RDA) was employed to identify genes upregulated under adhesion-inducing conditions. Expressed sequence tags (ESTs) from cDNA libraries generated by the RDA technique were analyzed. Genes related to functional categories, such as metabolism, transcription, energy, protein synthesis and fate, cellular transport and biogenesis of cellular components were upregulated. Transcripts encoding the P. brasiliensis protein enolase (PbEno) and the high-affinity cooper transporter (PbCtr3) were identified and further characterized. The recombinant enolase (rPbEno) and a synthetic peptide designed for PbCtr3 were obtained and demonstrated to be able to bind ECM components. Immunofluorescence assays demonstrated that rPbEno specifically binds to the macrophage surface, reinforcing the role of this molecule in the P. brasiliensis interaction with host cells. In addition, upregulation of selected genes was demonstrated by qRT-PCR. In synthesis, the strategy can be useful in characterization of potential P. brasiliensis adhesins.

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin.

BACKGROUND: The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. RESULTS: Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS) is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. CONCLUSION: These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.

Interactions of Paracoccidioides brasiliensis with host cells: recent advances.

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective antifungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.