The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires ...thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.
A unifying hypothesis for the genesis of cerebral malaria proposes that parasite antigens (released by replication in blood, surface molecules on parasitized erythrocytes, or merozoites) activate ...platelets that, in turn, contribute to the activation of the inflammatory response and increased levels of endothelial cell adhesion molecules (eCAMs). Increased levels of eCAMs result in further parasitized-erythrocyte sequestration and marked local inflammation that might disrupt the brain microvasculature, which cannot be repaired by the hemostasis system because of its procoagulant state. Disruption of the brain microvasculature can result in vascular leak and/or hemorrhaging into the brain; similar processes can occur in other vascular beds, including the lung. The blockage of functional capillaries by parasitized and/or unparasitized erythrocytes with decreased deformability or rosettes is also a key interaction between hemostasis and mechanical obstruction leading to pathogenesis. The events resulting in the development of cerebral malaria complications are multi-factorial, encompassing a dynamic interaction between three processes, thereby explaining the complexity of this deadly syndrome.
Extracellular vesicles (EVs) have been broadly studied in malaria for nearly a decade. These vesicles carry various functional biomolecules including RNA families such as microRNAs (miRNA). These ...EVs-derived microRNAs have numerous roles in host-parasite interactions and are considered promising biomarkers for disease severity. However, this field lacks clinical studies of malaria-infected samples. In this study, EV specific miRNAs were isolated from the plasma of patients from Thailand infected with Plasmodium vivax and Plasmodium falciparum. In addition, it is postulated that these miRNAs were differentially expressed in these groups of patients and had a role in disease onset through the regulation of specific target genes.
EVs were purified from the plasma of Thai P. vivax-infected patients (n = 19), P. falciparum-infected patients (n = 18) and uninfected individuals (n = 20). EV-derived miRNAs were then prepared and abundance of hsa-miR-15b-5p, hsa-miR-16-5p, hsa-let-7a-5p and hsa-miR-150-5p was assessed in these samples. Quantitative polymerase chain reaction was performed, and relative expression of each miRNA was calculated using hsa-miR-451a as endogenous control. Then, the targets of up-regulated miRNAs and relevant pathways were predicted by using bioinformatics. Receiver Operating Characteristic with Area under the Curve (AUC) was then calculated to assess their diagnostic potential.
The relative expression of hsa-miR-150-5p and hsa-miR-15b-5p was higher in P. vivax-infected patients compared to uninfected individuals, but hsa-let-7a-5p was up-regulated in both P. vivax-infected patients and P. falciparum-infected patients. Bioinformatic analysis revealed that these miRNAs might regulate genes involved in the malaria pathway including the adherens junction and the transforming growth factor-β pathways. All up-regulated miRNAs could potentially be used as disease biomarkers as determined by AUC; however, the sensitivity and specificity require further investigation.
An upregulation of hsa-miR-150-5p and hsa-miR-15b-5p was observed in P. vivax-infected patients while hsa-let-7a-5p was up-regulated in both P. vivax-infected and P. falciparum-infected patients. These findings will require further validation in larger cohort groups of malaria patients to fully understand the contribution of these EVs miRNAs to malaria detection and biology.
Cerebral malaria (CM) and severe anemia (SA) are the most severe complications of Plasmodium falciparum infections. Although increased release of endothelial microparticles (MP) correlates with ...malaria severity, the full extent of vascular cell vesiculation remains unknown. Here, we characterize the pattern of cell-specific MP in patients with severe malaria. We tested the hypothesis that systemic vascular activation contributes to CM by examining origins and levels of plasma MP in relation to clinical syndromes, disease severity and outcome. Patients recruited in Douala, Cameroon, were assigned to clinical groups following WHO criteria. MP quantitation and phenotyping were carried out using cell-specific markers by flow cytometry using antibodies recognizing cell-specific surface markers. Platelet, erythrocytic, endothelial and leukocytic MP levels were elevated in patients with cerebral dysfunctions and returned to normal by discharge. In CM patients, platelet MP were the most abundant and their levels significantly correlated with coma depth and thrombocytopenia. This study shows for the first time a widespread enhancement of vesiculation in the vascular compartment appears to be a feature of CM but not of SA. Our data underpin the role of MP as a biomarker of neurological involvement in severe malaria. Therefore, intervention to block MP production in severe malaria may provide a new therapeutic pathway.
Abstract In this study we have developed Ca3 ZrSi2 O9 (Baghdadite) ceramics by incorporating Zirconium in Ca–Si system and determined their biological properties. Ca3 ZrSi2 O9 ceramics possess ...apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca3 ZrSi2 O9 ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO3 , Ca3 ZrSi2 O9 ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-κB ligand and osteoprotegerin). Ca3 ZrSi2 O9 ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of αv β3 integrin consistent with functional activity. Osteoclasts cultured on Ca3 ZrSi2 O9 expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-κB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca3 ZrSi2 O9 supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca3 ZrSi2 O9 ceramics have the potential for applications in bone tissue regeneration.
ABSTRACT
Microvesicles (MVs) are involved in cell‐cell interactions, including disease pathogenesis. Nondestructive Fourier‐transform infrared (FTIR) spectra from MVs were assessed as a technique to ...provide new biochemical insights into a LPS‐induced monocyte model of septic shock. FTIR spectroscopy provided a quick method to investigate relative differences in biomolecular content of different MV populations that was complementary to traditional semiquantitative omics approaches, with which it is difficult to provide information on relative changes between classes (proteins, lipids, nucleic acids, carbohydrates) or protein conformations. Time‐dependent changes were detected in biomolecular contents of MVs and in the monocytes from which they were released. Differences in phosphatidylcholine and phosphatidylserine contents were observed in MVs released under stimulation, and higher relative concentrations of RNA and a‐helical structured proteins were present in stimulated MVs compared with MVs from resting cells. FTIR spectra of stimulated monocytes displayed changes that were consistent with those observed in the corresponding MVs they released. LPS‐stimulated monocytes had reduced concentrations of nucleic acids, a‐helical structured proteins, and phosphatidylcholine compared with resting monocytes but had an increase in total lipids. FTIR spectra of MV biomolecular content will be important in shedding new light on the mechanisms of MVs and the different roles they play in physiology and disease pathogenesis.—Lee, J., Wen, B., Carter, E. A., Combes, V., Grau, G. E. R., Lay, P. A. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation. FASEB J. 31, 2817–2827 (2017). www.fasebj.org
Blood and tissue protozoan infections are responsible for an enormous burden in tropical and subtropical regions, even though they can also affect people living in high-income countries, mainly as a ...consequence of migration and travel. These pathologies are responsible for heavy socio-economic issues in endemic countries, where the lack of proper therapeutic interventions and effective vaccine strategies is still hampering their control. Moreover, the pathophysiological mechanisms associated with the establishment, progression and outcome of these infectious diseases are yet to be fully described. Among all the players, extracellular vesicles (EVs) have raised significant interest during the last decades due to their capacity to modulate inter–parasite and host–parasite interactions. In the present manuscript, we will review the state of the art of circulating host-derived EVs in clinical samples or in experimental models of human blood and tissue protozoan diseases (i.e., malaria, leishmaniasis, Chagas disease, human African trypanosomiasis and toxoplasmosis) to gain novel insights into the mechanisms of pathology underlying these conditions and to identify novel potential diagnostic markers.
Cerebral malaria (CM) is a fatal complication of Plasmodium infection, mostly affecting children under the age of five in the sub-Saharan African region. CM pathogenesis remains incompletely ...understood, although sequestered infected red blood cells, inflammatory cells aggregating in the cerebral blood vessels, and the microvesicles (MV) that they release in the circulation, have been implicated. Plasma MV numbers increase in CM patients and in the murine model, where blocking their release, genetically or pharmacologically, protects against brain pathology, suggesting a role of MV in CM neuropathogenesis. In this work, the microRNA (miRNA) cargo of MV is defined for the first time during experimental CM with the overarching hypothesis that this characterization could help understand CM pathogenesis.
The change in abundance of miRNA was studied following infection of CBA mice with Plasmodium berghei ANKA strain (causing experimental CM), and Plasmodium yoelii, which causes severe malaria without cerebral complications, termed non-CM (NCM). miRNA expression was analyzed using microarrays to compare MV from healthy (NI) and CM mice, yielding several miRNA of interest. The differential expression profiles of these selected miRNA (miR-146a, miR-150, miR-193b, miR-205, miR-215, miR-467a, and miR-486) were analyzed in mouse MV, MV-free plasma, and brain tissue by quantitative reverse transcription PCR (RT-qPCR). Two miRNA-miR-146a and miR-193b-were confirmed as differentially abundant in MV from CM mice, compared with NCM and NI mice. These miRNA have been shown to play various roles in inflammation, and their dysregulation during CM may be critical for triggering the neurological syndrome via regulation of their potential downstream targets.
These data suggest that, in the mouse model at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.
In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that ...express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.
ABSTRACT
Drug resistance is a major cause of cancer treatment failure, with multidrug resistance (MDR) being the most serious, whereby cancer cells display cross‐resistance to structurally and ...functionally unrelated drugs. MDR is caused by overexpression of the efflux transporters P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 1 (MRP1). These transporters act to maintain sublethal intracellular drug concentrations within the cancer cell, making the population treatment unresponsive. Recently, we discovered a novel nongenetic basis to MDR whereby microparticles (MPs) transfer P‐gp intercellularly from MDR donor cells to drug‐sensitive recipient cells. MPs isolated from MDR leukemia and breast cancer cells were cocultured with their drug‐sensitive counterparts. P‐gp transfer was assessed by direct immunolabeling, and acquired transcripts and regulatory microRNAs by quantitative real‐time PCR. We show that MDR MPs incorporate nucleic acids; MPs change recipient cells' transcriptional environment to reflect donor MDR phenotype, and distinct pathways exist among cancers of different origin that may be dependent on donor cells' ABCB1 overexpression. We demonstrate that this pathway exists for both hematological and nonhematological malignancies. By conferring MDR and “retemplating” the transcriptional landscape of recipient cells, MPs provide a novel pathway, having implications in the dissemination and acquisition of deleterious traits in clinical oncology.—Jaiswal, R., Gong, J., Sambasivam, S., Combes, V., Mathys, J.‐M., Davey, R., Grau, G. E. R., Bebawy, M. Microparticle‐associated nucleic acids mediate trait dominance in cancer. FASEB J. 26, 420–429 (2012). www.fasebj.org
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