Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a–better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Several species of Acanthamoeba genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target tissues have been documented, evidencing the relevant role of contact-dependent mechanisms. With the purpose of describing the pathogenic processes carried out by these protozoans more precisely, we considered it important to determine the emission of extracellular vesicles (EVs) as part of the contact-independent pathogenicity mechanisms of A. culbertsoni, a highly pathogenic strain. Through transmission electronic microscopy (TEM) and nanoparticle tracking analysis (NTA), EVs were characterized. EVs showed lipid membrane and a size between 60 and 855 nm. The secretion of large vesicles was corroborated by confocal and TEM microscopy. The SDS-PAGE of EVs showed proteins of 45 to 200 kDa. Antigenic recognition was determined by Western Blot, and the internalization of EVs by trophozoites was observed through Dil-labeled EVs. In addition, some EVs biological characteristics were determined, such as proteolytic, hemolytic and COX activity. Furthermore, we highlighted the presence of leishmanolysin in trophozites and EVs. These results suggest that EVs are part of a contact-independent mechanism, which, together with contact-dependent ones, allow for a better understanding of the pathogenicity carried out by Acanthamoeba culbertsoni.

Extracellular vesicles (EVs) can transport molecules that combat viruses, such as RNA against SARS-CoV-2. Bacterial coinfections can help establish certain viruses and worsen diseases. Thus, we designed a model to induce the secretion of polydisperse EVs shown with SARS-CoV-2 and bacterial coinfection using macrophages and E. coli fractions as in vitro inducers. We obtained short and large macrophage EVs. The E. coli fraction was designated as SDS-soluble bacterial membrane fraction and its associated proteins (SDS-SBMF). The proteins were identified using a mass spectrometer. SDS-SBMF contained mainly OmpF, OmpA, OmpC, OmpX, and lpp. The SDS-SBMF macrophages induced the secretion of polydisperse EVs at 30 min, reaching optimal secretion at 120 min, as observed via scanning electron microscopy and confocal microscopy. Macrophage EVs contained mainly HSP7C, actin, apolipoprotein, GAPDH, annexin A5, PKM, moesin, and cofilin. We observed an increase in EVs in the bloodstream of patients with SARS-CoV-2 and bacterial coinfection, in addition to the presence of SARS-CoV-2 genes (E, ORF) in EVs. This in vitro method for inducing EVs has the potential to be used to obtain larger samples for study and for the detection of diagnostic and prognostic biomarkers of different diseases.

Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during encystment and adhesion. Trophozoites were continuously exposed to each drug and analyzed for adhesion, migration on host-derived discontinuous brain micropatterns, encystment efficiency, and parasite-induced cytoskeletal remodeling in MDCK epithelial cells. In silico docking was performed to assess potential drug–protein interactions. Drug exposure reduced adhesion with maximal inhibition at 60 min. After 1 h, migration decreased by 49%, 64%, and 38%, and encystment was reduced by 50%, 85%, and up to 90%, respectively, in cultures treated with acetylsalicylic acid, ibuprofen, and diclofenac. Co-incubation with untreated trophozoites lowered actin fluorescence to approximately 50%, whereas drug-treated co-cultures preserved fluorescence near control levels. Colocalization analysis showed increased spatial overlap between gp63-like protein and F-actin in cysts (~40%) and migrating trophozoites (~20%) compared with non-stimulated forms (~3.8%). Collectively, these findings suggest that NSAID-sensitive pathways influence host interaction, migration, and encystment in A. castellanii and allow for the proposal of gp63-like protein as a putative molecular component of the NSAIDs sensitive pathways.

Histiocytic sarcoma (HS) is an aggressive hematological malignancy whose transformed cells exhibit morphological and immunophenotypic characteristics similar to macrophages, and arises de novo or as part of a clonal ‘evolution’ of other pre-existing hematological neoplasms. This study investigates the potential use of the J774A.1 cell line (a cell line derived from murine tumor cells, commonly used in macrophage research) as a research model to study the role of polydisperse extracellular vesicles (PEVs) secreted by the HS cells, considering that bacterial infections are common in patients with cancer, including HS. The influences of bacterial components on tumor progression are still not fully understood. We stimulated the J774A.1 cell line in vitro with a fraction of E. coli, and our results show that the bacterial stimulation increases the secretion of PEVs by these cells. Comparative results of J774A.1 cells with PEVs using confocal and scanning electron microscopy with micrographic reports of HS histological slides (from several cited mammal species, including humans) suggest a possible relationship of large PEVs with marks, footprints, or traces of possible large PEVs disrupted in the HS of these reports. A subsequent proteomic analysis of these PEVs revealed a diverse subcellular origin of their components, such as proteins including: Triosephosphate isomerase (TPI), Heat shock cognate 71 kDa, Apolipoprotein A-1, Rho GDP-dissociation inhibitor 1, GAPDH, Galectin, Moesin, globular Actin, and Annexin. These results highlight the importance of studying the interplay between HS, other hematological cancers, and bacterial infections to better understand the progression of this cancer, identify new therapeutic targets, and emphasize the importance of preventing bacterial infections in cancer patients. Furthermore, the results demonstrate the potential use of the stimulated J774A.1 cell line for research on HS-related PEVs.

Extracellular vesicles (EVs) are lipid bilayer-bound structures capable of transporting molecular markers from their cell of origin and are secreted by multiple cell types, including malignant cells. EVs have emerged as promising tools for developing less invasive diagnostic approaches. In B-cell acute lymphoblastic leukemia (B-ALL), immunophenotypic characterization of extracellular vesicle-enriched populations (EVEPs) in peripheral blood (PB) may provide complementary information for disease detection and monitoring. This exploratory study aimed to characterize EVEPs obtained from PB and bone marrow (BM) of adult patients with B-ALL and to compare them with the clinical immunophenotype (CIP). A total of 12 individuals were initially recruited (eight with B-ALL, one with T-ALL, and three healthy controls). The study focused on the eight B-ALL patients and three controls, while the T-ALL sample was used as a specificity control. EVEPs were isolated by differential centrifugation and analyzed by flow cytometry and confocal microscopy, primarily evaluating CD3 and CD19 expression. EVEPs derived from PB samples of patients with B-ALL showed a higher percentage of marker-positive events by flow cytometry (CD45, CD34, CD19, CD20, and CD10), consistent with the leukemic phenotype identified in the CIP. Additionally, CD3⁺CD19⁺ EVEPs were occasionally detected. These findings suggest that EVEPs partially reflect the leukemic immunophenotype and may serve as a complementary source of biological information. The detection of CD3⁺CD19⁺ events highlights complex cellular interactions within the leukemic niche and warrants further investigation.