Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different ...biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO
) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO
beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO
beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.
Leishmania spp. is the aetiologic agent of leishmaniasis, a disease endemic in several developing countries. The parasite expresses and secretes several virulence factors that subvert the macrophage ...function and immune response. Extracellular vesicles (EVs) can carry molecules of the parasites that show immunomodulatory effects on macrophage activation and disease progression. In the present work, we detected a significantly higher expression of lpg3 and gp63 genes in Leishmania amazonensis promastigotes recovered after successive experimental infections (IVD-P) compared to those cultured for a long period (LT-P). In addition, we observed a significantly higher percentage of infection and internalized parasites in groups of macrophages infected with IVD-P. Macrophages previously treated with EVs from LT-P showed higher percentages of infection and production of inflammatory cytokines after the parasite challenge compared to the untreated ones. However, macrophages infected with parasites and treated with EVs did not reduce the parasite load. In addition, no synergistic effects were observed in the infected macrophages treated with EVs and reference drugs. In conclusion, parasites cultured for a long period in vitro and recovered from animals’ infections, differently affected the macrophage response. Furthermore, EVs produced by these parasites affected the macrophage response in the early infection of these cells.
We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples
Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was ...extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis.
Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed.
Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.
Glioblastoma (GBM) is an incurable disease ranked among the deadliest solid cancers worldwide. A better understanding on the molecular aspects of this malignancy could contribute to the development ...of new treatment strategies and help to improve survival rates. Previously, our group had shown that GBM patients expressing the cancer/testis antigen
Opa Interacting Protein 5
(
OIP5
) present a longer survival period than the
OIP5
-negative group. The main goal of this study was to evaluate the
OIP5
contribution to GBM tumorigenesis and assess the role of
OIP5
in GBM cell response to lomustine, an alkylating agent used in the treatment of this malignancy. So, the effect of
OIP5
knockdown was evaluated in A172 and T98G GBM cell lines. Our results demonstrated that downregulation of the OIP5 stimulates glioma cell viability and inhibits cell death-induced necrosis prompted by lomustine. In conclusion, our data shows that OIP5 expression in GBM cells seems to be able to enhance lomustine cytotoxic effects, reinforcing that this gene is a potential therapeutic target and putative molecular biomarker for treatment response in GBM.
The histamine receptors (HRs) are members of G‐protein‐coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H3R and H4R have been explored as targets for drug ...discovery, including in the search for dual‐acting H3R/H4R ligands. The H4R, the most recent histamine receptor, is a promising target for novel anti‐inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1‐(2,3‐dihydro‐1‐benzofuran‐2‐yl)methylpiperazines were synthesized and evaluated in competitive binding assays as H3R/H4R ligands herein. The results showed the compounds presented affinity (Ki) for H3R/H4R in micromolar range, and they are more selective to H3R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl‐substituted compound LINS01005 has shown the higher affinity of the set for H4R, but no considerable selectivity toward this receptor over H3R. LINS01005 showed interesting anti‐inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX‐2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti‐inflammatory agents.
Histamine H3R and H4R receptors have been explored as targets for drug discovery. We present herein four non‐cytotoxic compounds with moderate affinity for these targets. The compound LINS01005 has shown promising anti‐inflammatory activity in murine asthma model.
The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H
R and H
R have been explored as targets for drug ...discovery, including in the search for dual-acting H
R/H
R ligands. The H
R, the most recent histamine receptor, is a promising target for novel anti-inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1-(2,3-dihydro-1-benzofuran-2-yl)methylpiperazines were synthesized and evaluated in competitive binding assays as H
R/H
R ligands herein. The results showed the compounds presented affinity (K
) for H
R/H
R in micromolar range, and they are more selective to H
R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl-substituted compound LINS01005 has shown the higher affinity of the set for H
R, but no considerable selectivity toward this receptor over H
R. LINS01005 showed interesting anti-inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX-2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti-inflammatory agents.
The present study pretends to evaluate the in vivo efficacy of the crude chloroform bark extract of Helietta apiculata, then the activity will be compared with the reference drug, benznidazole, in ...acute Trypanosoma cruzi infected mice when administered by oral route. The chloroformic extract of Helieta apiculata was administered by oral route at 5, 10 and 50 mg/kg daily for two weeks. This study has shown a moderate efficacy of the H. apiculata bark extract in reducing T. cruzi parasitaemia in 42 to 54% after a monitoring of 60 days post-infection and when compared with control groups. Concerning mice mortality, only two only two mice died, one from the control group and the other one from the group threated with 10 mg of the chlorofom extract of H. apiculata, suggesting the potential of H. apiculta extracts as a safe and inexpensive treatment of Chagas disease.
Purpose: Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Extracellular vesicles (EVs) have been extensively studied as a biomarker to monitor disease in patients. The ...study of new biomarkers in melanoma patients could prevent metastasis by earlier diagnosis. In this study, we determined the proteomic profile of EVs isolated from aqueous humor (AH), vitreous humor (VH), and plasma from UM patients in comparison with cancer-free control patients.
Methods: AH, VH and plasma were collected from seven patients with UM after enucleation; AH and plasma were collected from seven cancer-free patients with cataract (CAT; control group). EVs were isolated using the membrane-based affinity binding column method. Nanoparticle tracking analysis (NTA) was performed to determine the size and concentration of EVs. EV markers, CD63 and TSG101, were assessed by immunoblotting, and the EV proteome was characterized by mass spectrometry.
Results: Mean EV concentration was higher in all analytes of UM patients compared to those in the CAT group. In the UM cohort, the mean concentration of EVs was significantly lower in AH and plasma than in VH. In contrast, the mean size and size distribution of EVs was invariably identical in all analyzed analytes and in both studied groups (UM vs. CAT). Mass spectrometry analyses from the different analytes from UM patients showed the presence of EV markers.
Conclusion: EVs isolated from AH, VH, and plasma from patients with UM showed consistent profiles and support the use of blood to monitor UM patients as a noninvasive liquid biopsy.
Context: Extracellular vesicles (EVs) are related to the dissemination of the pathogen and to the regulation of the host immune system in infectious diseases. However, the role of EVs in Ocular ...toxoplasmosis remains unclear. Aims: The goal of this study was to identify and characterize the concentration and size of EVs in the aqueous humor (AH) and plasma of patients with ocular toxoplasmosis (OT) compared to other types of uveitis (OTU) and cataract. Settings and Design: AH and plasma were collected from six patients with active OT, six patients with OTU, and six patients with cataract. All patients were also assessed clinically. Subjects and Methods: EVs were isolated using the membrane affinity column method. Nanoparticle Tracking Analysis (NTA) was performed to determine the size and concentration of EVs. Statistical Analysis Used: The ANOVA test was used to determine statistical difference. P < 0.05 was considered statistically significant. Results: EV was present in the AH of different types of uveitis as well as in patients with cataract. The concentration of EV in AH was significantly lower in OT and OTU compared to cataract (P = 0.03). However, in the plasma, all groups presented a similar concentration of EV. The size of EV was the same in the AH and plasma among the three groups. Conclusion: This initial study successfully identified and characterized EVs in the AH and plasma from patients with OT as well as OTU and cataract. Further studies are necessary to better understand the role of EVs in different ocular pathologies.
Background: The infection by the protozoan Trypanosoma cruzi, a parasite that cause Chagas' disease, modulates host innate immune response. The objective of our study was to determine if during ...infection, or after incubation of human macrophages with parasite extracellular vesicles (EV), these cells released EVs with immunomodulatory activity or with the capacity to affect cell invasion by the parasite. Methods: We employed THP-1 lineage which were differentiated by the addition of Phorbol-12-myristate-13-acetate (PMA) for 24 h. The cells were either directly infected with the parasites, or incubated with parasite EVs, or EVs isolated from non-infected cells. mRNA was extracted from the cells and submitted to NGS transcriptome analysis. The released EVs from macrophages submitted to the different treatments were analysed by scanning electronic microscope and NTA or used to treat TLR2- or TLR4-transfected CHO cells, and these receptors' activation is measured by expression of CD25. Results: We detected 106 genes with increased expression, 15 of them related to the host pro-inflammatory response in infected cells or cells treated with parasite EVs compared to control macrophages. Concomitantly, the incubation of cells for 1, 6 and 24 h with the parasites produced an increased release of EVs from the plasma membrane of THP-1 cells. These EVs had similar size of EVs released from non-infected macrophages but only the EVs produced by infected cells, or cells incubated with parasite EVs induced preferentially TLR2 than TLR4 activation on CHO reporter cells. The TLR2 CHO cells become also more infected than TLR4 or non-transfected cells when incubated with similar amounts of EVs. Summary/conclusion: Either infection or the presence of T. cruzi EVs induce the inflammatory responses in macrophages and the release of EVs that can modulate the innate host response through activation mainly of TLR2. These EVs also promote an increased infection. These results demonstrate an orchestration of signalling response by the release of EVs from the parasite and host cells.
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