Prospects and challenges in using neuronal extracellular vesicles in biomarker research.

Extracellular vesicles (EVs) hold promise as a source of disease biomarkers. The diverse molecular cargo of EVs can potentially indicate the status of their tissue of origin, even against the complex background of whole plasma. The main tools currently available for assessing biomarkers of brain health include brain imaging and analysis of the cerebrospinal fluid of patients. Given the costs and difficulties associated with these methods, isolation of EVs of neuronal origin (NEVs) from the blood is an attractive approach to identify brain-specific biomarkers. This perspective describes current key challenges in EV- and NEV-based biomarker research. These include the relative low abundance of EVs, the lack of validated isolation methods, and the difficult search for an adequate target for immunocapturing NEVs. We discuss that these challenges must be addressed before NEVs can fulfill their potential for biomarker research. HIGHLIGHTS: NEVs are promising sources of biomarkers for brain disorders. Immunocapturing NEVs from complex biofluids presents several challenges. The choice of surface target for capture will determine NEV yield. Contamination by non-EV sources is relevant for biomarkers at low concentrations.

Structure-Based Optimization of Quinazolines as Cruzain and TbrCATL Inhibitors.

The cysteine proteases, cruzain and TbrCATL (rhodesain), are therapeutic targets for Chagas disease and Human African Trypanosomiasis, respectively. Among the known inhibitors for these proteases, we have described N4-benzyl-N2-phenylquinazoline-2,4-diamine (compound 7 in the original publication, 1a in this study), as a competitive cruzain inhibitor (Ki = 1.4 µM). Here, we describe the synthesis and biological evaluation of 22 analogs of 1a, containing modifications in the quinazoline core, and in the substituents in positions 2 and 4 of this ring. The analogs demonstrate low micromolar inhibition of the target proteases and cidal activity against Trypanosoma cruzi with up to two log selectivity indices in counterscreens with myoblasts. Fourteen compounds were active against Trypanosoma brucei at low to mid micromolar concentrations. During the optimization of 1a, structure-based design and prediction of physicochemical properties were employed to maintain potency against the enzymes while removing colloidal aggregator characteristics observed for some molecules in this series.

Candida albicans Clinical Isolates from a Southwest Brazilian Tertiary Hospital Exhibit MFS-mediated Azole Resistance Profile.

Candida albicans is the most frequent fungal species that causes infections in humans. Fluconazole is the main antifungal used to treat Candida infections, and its prolonged and indiscriminate use for the last decades are the most established causes which originated resistant strains. Fungal drug resistance is associated to alterations in ERG11 gene and overexpression of multidrug resistance (MDR) transporters belonging to two families: ATP-binding cassette (ABC) and Major Facilitator Superfamily (MFS). To evaluate the role of MFS transporters in azoles resistance of C. albicans clinical strains, this study aimed to analyze four Candida albicans clinical isolates from the University Hospital in Juiz de Fora (Minas Gerais/Brazil), selected in our previous study as they were unaffected by FK506, an ABC pumps inhibitor. In a primary investigation on MFS proteins overexpression, the extrusion of fluorescent substrates (rhodamine 6G and nile red) was analyzed by fluorescence microscopy and flow cytometry. Results suggest participation of MFS transporters in azole resistance of C. albicans isolates and indicate the existence of secondary resistance mechanisms. Therefore, this study contributes to the information about Candida albicans infections in Brazil and reinforces the importance of epidemiological studies focusing on an improved understanding of the disease and further resistance reversion.

Synthesis and structure-activity relationship studies of cruzain and rhodesain inhibitors.

Chagas disease and Human African trypanosomiasis (HAT) are important public health issues in Latin American and sub-Saharan African countries, respectively, and are responsible for a significant number of deaths. The drugs currently used to treat Chagas disease and HAT present efficacy, toxicity, and/or resistance issues; thus, there is a clear need for the discovery of novel targets and drug candidates to combat these diseases. In recent years, much effort has been made to find inhibitors of cruzain and rhodesain, which are promising targets for the design of novel trypanocidal compounds, since they are essential for parasite survival. Many reviews covering the design of novel cruzain and rhodesain inhibitors have been published; however, none have focused on the chemistry of the inhibitors. Thus, in the present work we reviewed the synthetic strategies and routes for the preparation of relevant classes of cruzain and rhodesain inhibitors. Perhaps the most important are the vinyl sulfone derivatives, and a very efficient synthetic strategy based on the Horner-Wadsworth-Emmons reaction was developed to yield these compounds. Modern approaches such as the asymmetric addition of substituted ethynyllithium to N-sulfinyl ketimines were used to produce the chiral alkynes that were employed in the preparation of important chiral triazole derivatives (potent cruzain inhibitors) and chiral HPLC resolution was used for the preparation of enantiopure 3-bromoisoxazoline derivatives (rhodesain inhibitors). Moreover, we also highlight the most important activity results and updated SAR results.

CD86 Expression by Monocytes Influences an Immunomodulatory Profile in Asymptomatic Patients with Chronic Chagas Disease.

In the chronic phase of Chagas disease, 60% of the patients develop the asymptomatic form known as indeterminate (IND). The remaining 30% of the patients develop a life-threatening form in which digestive and/or cardiac (CARD) alterations take place. The mechanisms underlying the development of severe forms of Chagas disease remain poorly understood. It is well known that interactions between immune cells such as monocytes and lymphocytes drive immune responses. Further, the co-stimulatory molecules CD80 and CD86 expressed by monocytes and subsets induce lymphocyte activation, thereby triggering cellular immune response. Here, we revealed, for the first time, the functional-phenotypic profile of monocytes subsets in Chagas disease. Using flow cytometry, we evaluated the effect of in vitro stimulation with Trypanosoma cruzi antigens on the expression of the co-stimulatory molecules CD80 and CD86 in different monocyte subsets of patients with IND and CARD clinical forms of Chagas disease. We also assessed the expression of toll-like receptor (TLR)-2, TLR-4, TLR-9, HLA-DR, IL-10, and IL-12 in the monocyte subsets and of CTLA-4 and CD28, ligands of CD80 and CD86, in T lymphocytes. CD86 expression in all monocyte subsets was higher in IND patients when compared with non-infected (NI) individuals. After stimulation with T. cruzi, these patients also showed a higher frequency of CD4+CTLA-4+ T lymphocytes than NI individuals. We found an association between CD80 and CD28, and between CD86 and CTLA-4 expression, with a high frequency of regulatory T (Treg) cells in IND patients. We proposed that CD86 may be involved in immunoregulation by its association with CTLA-4 in asymptomatic patients. CD86 and CTLA-4 interaction may influence Treg activation, and this could represent a new strategy to control inflammation and tissue damage.

Pan-azole-resistant Candida tropicalis carrying homozygous erg11 mutations at position K143R: a new emerging superbug?

Objectives: Candidaemia is a public health problem mainly in hospitalized individuals worldwide. In Brazil, Candida albicans is the most prevalent species that causes candidaemia, followed by Candida tropicalis and Candida parapsilosis . Few data on the abundance of antifungal resistance are available for Latin America. Methods: We analysed the frequency of azole and echinocandin resistance in Candida isolates ( n = 75) collected between 2012 and 2014 at the University Hospital of Federal University of Juiz de Fora (Brazil). The primary targets erg11 (azoles) and fks1 (echinocandins) were sequenced and modelled at the protein level. Antifungal susceptibility testing was performed according to CLSI (M27-A3 and M27-S4) and according to EUCAST. Results: The three most frequent species were C. albicans (38.0%), C. tropicalis (30.0%) and Candida glabrata (17.0%). Azole resistance was observed in 27.0% of all Candida isolates, while 20.0% of all isolates were echinocandin resistant. A novel mutation in erg11 at location K143R was found to be associated with phenotypically pan-azole-resistant C. tropicalis isolates. This mutation maps near the active binding site of erg11 and is likely to confer pan-azole resistance to C. tropicalis . Conclusions: A novel point mutation (K143R) located in the erg11 gene of C. tropicalis was found in pan-azole-resistant strains. According to our protein homology model, it is very likely that the mutation K143R causes pan-azole resistance in C. tropicalis . Moreover, an up-regulation of ABC transporters was observed, which can add up to a pan-azole-resistant phenotype.

Prevalence and Fluconazole Susceptibility Profile of Candida spp. Clinical Isolates in a Brazilian Tertiary Hospital in Minas Gerais, Brazil.

Candidiasis has become an important concern for clinical practice, especially with the increasing incidence of immunocompromised patients. In this scenario, the development resistance to fluconazole presents a challenge for treating these opportunistic infections. The aim of this study was to evaluate some epidemiology features of Candida infections in a Brazilian University Hospital using data, previously unavailable. We observed that 44% of the 93 clinical isolates tested, belonged to Candida albicans species and 56% belonged to non-Candida albicans species (mainly Candida tropicalis and Candida glabrata). Most strains were isolated from urine samples where C. albicans was predominantly detected. 29 strains presented a fluconazole resistance phenotype and of these, 22 were chemosensitised by FK506, a classical inhibitor of ABC transporters related to azoles resistance. These data suggest the probable role of efflux pumps in this resistance phenotype. Our study highlights the need for developing effective control measures for fungal infections, rational use of antifungal drugs and development of new molecules able to abrogate the active transport of antifungals.