Search Results (25)

Didelphis aurita is a widely distributed neotropical marsupial frequently found in peri-urban environments and known to harbor various pathogens, including hemoparasites of the genus Hepatozoon. However, the systemic physiological responses of naturally infected individuals remain poorly understood. This study aimed to characterize the serum proteomic profile of Didelphis aurita naturally infected with Hepatozoon sp., providing insights into host–parasite interactions and potential biomarkers of infection. Serum samples were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), followed by functional annotation based on Gene Ontology and KEGG pathway enrichment. A total of 67 proteins were identified, 33 of which were exclusive to infected animals. The most abundant proteins included albumin, hemoglobin subunits, and venom metalloproteinase inhibitors (DM43 and DM64). Functional enrichment revealed significant involvement in complement and coagulation cascades, protease inhibition, antioxidant defense, and extracellular vesicle localization. Key proteins such as fibrinogen, plasminogen, antithrombin, SERPIN family members, vitronectin, and fibronectin suggest an integrated host response involving hemostasis, inflammation control, and tissue remodeling. This is the first report of the serum proteome of Didelphis aurita naturally infected with Hepatozoon sp. Despite the absence of protein validation, the findings provide novel insights into marsupial immunophysiology and offer a foundation for future biomarker research and ecoimmunological surveillance in synanthropic species. Full article

Our understanding of the molecular mechanisms undergirding artemisinin (ART) resistance in Plasmodium falciparum is currently based on two organizing principles: reduced hemoglobin trafficking into the digestive food vacuole, resulting in lower levels of activated ART, and increased tolerance to ART-induced oxidative stress in the infected erythrocyte. We had previously proposed an extracellular vesicle (EV) export model of ART resistance in P. falciparum. This model predicts that EV abundance will be altered by ART exposure and that the peptide cargo of EVs from the ART-exposed condition will be enriched with aggregation-prone peptides. We tested the predictions of the EV export hypothesis in this study using in vitro culture assays of an ART-resistant transgenic line engineered on a 3D7 background (R561H) and a 3D7 knock-out line (PfVps60KO) with deficient EV production phenotype. EV enrichment was obtained from in vitro parasite culture supernatants via a series of ultracentrifugation and filtration steps, followed by size exclusion chromatography. A quality check on EVs was performed using dynamic light scattering. Liquid chromatography with tandem mass spectrometry was used to determine the proteome cargo from extracted EVs, and parasite peptides were queried for aggregation-prone tendency using open-access software. We report that dihydroartemisinin (DHA) exposure was positively correlated with EV abundance (coefficient estimate = 1038.58, confidence interval of 194.86–1882.30, and p-value = 0.018) and suggests that EV biogenesis is part of the parasite’s response to DHA/ART. Furthermore, our findings suggest the expression of a non-constitutive DHA-induced alternate EV biogenesis pathway as the PfVps60KO was observed to produce the highest number of EVs under DHA exposure. Finally, we show that EVs from both ART-susceptible and resistant parasites under DHA exposure carry a cargo of Chorein N-terminal domain-containing protein (PF3D7_1021700) with a high aggregation-prone index (prion-like domain [PrLD] score = 26.5) out of nine identified parasite peptides. The former of these findings is in concordance with the EV export hypothesis, which posits that the removal of DHA/ART-induced aggregated and/or misfolded peptides is critical to the parasite’s survival under DHA/ART exposure. This observation further implicates EVs in the development of the ART-resistant phenotype. However, the finding of one aggregation-prone peptide out of the nine parasite proteins in the EV cargo does not sufficiently support the EV export hypothesis. Future replicates of this study and further interrogations of the EV export hypothesis are needed. Full article

Objectives: We aimed to assess the relationship among circulating extracellular vesicles (EVs), hypoxia-related proteins, and the conventional risk factors of life-threatening coronary artery disease (CAD) to find more precise novel biomarkers. Methods: Patients were categorized based on coronary CT angiography. Patients with a Segment Involvement Score > 5 were identified as CAD patients. Individuals with a Segment Involvement Score < 5 were considered control subjects. The characterization of EVs and analysis of the plasma concentration of growth differentiation factor-15 were performed using multicolor or bead-based flow cytometry. The plasma protein levels of glycogen phosphorylase, muscle form, clusterin, and carboxypeptidase N subunit 1 were determined using an enzyme-linked immunosorbent assay. Multiple logistic regression was used to determine the association of the biomarkers with the CAD outcome after accounting for established risk factors. The analysis was built in three steps: first, we included the basic clinical and laboratory variables (Model 1), then we integrated the plasma protein values (Model 2), and finally, we complemented it with the circulating EV pattern (Model 3). To assess the discrimination value of the models, an area under (AUC) the receiver operating curve was calculated and compared across the three models. Results: The area under the curve (AUC) values were 0.68, 0.77, and 0.84 in Models 1, 2, and 3, respectively. The variables with the greatest impact on the AUC values were hemoglobin (0.2 (0.16–0.26)) in Model 1, carboxypeptidase N subunit 1 (0.12 (0.09–0.14)) in Model 2, and circulating CD41+/CD61+ EVs (0.31 (0.15–0.5)) in Model 3. A correlation analysis showed a significant impact of circulating CD41+/CD61+ platelet-derived EVs (p = 0.03, r = −0.4176) in Model 3. Conclusions: Based on our results, the circulating EV profile can be used as a supportive biomarker, along with the conventional laboratory markers of CAD, and it enables a more sensitive, non-invasive diagnostic analysis of CAD. Full article

Despite the understanding of the coronavirus disease-19 (COVID-19), the role of salivary extracellular vesicles (sEVs) in COVID-19 remains unclear. Exploring the proteomic cargo of sEVs could prove valuable for diagnostic and prognostic purposes in assessing COVID-19. The proteomic cargo of sEVs from COVID-19(+) subjects and their healthy close contacts (HCC) was explored. sEVs were isolated by ultracentrifugation from unstimulated saliva samples, and subsequently characterized through nanoparticle tracking, transmission electron microscopy, and Western blot analyses. The proteomic cargo of sEVs was processed by LC-MS/MS. sEVs were morphologically compatible with EVs, with the presence of Syntenin-1 and CD81 EV markers. The sEV pellet showed 1417 proteins: 1288 in COVID-19(+) cases and 1382 in HCC. In total, 124 proteins were differentially expressed in sEVs from COVID-19(+) subjects. “Coronavirus-disease response”, “complement and coagulation cascades”, and “PMN extracellular trap formation” were the most enriched KEGG pathways in COVID-19(+) cases. The most represented biological processes were “Hemoglobin and haptoglobin binding” and “oxygen carrier activity”, and the best-denoted molecular functions were “regulated exocytosis and secretion” and “leucocyte and PMN mediated immunity”. sEV proteomic cargo in COVID-19(+) suggests activity related to immune response processes, oxygen transport, and antioxidant mechanisms. In contrast, in HCC, sEV signature profiles are mainly associated with epithelial homeostasis. Full article

We previously reported that granulocytic myeloid-derived suppressor cells (G-MDSCs) suppressed T-cell activation and attenuated bone marrow failure (BMF) in a minor histocompatibility (minor-H) antigen mismatched murine aplastic anemia (AA) model. In the current study, we tested the hypothesis that exosomes, a subset of extracellular vesicles, are responsible at least partially for G-MDSCs’ therapeutic efficacy. Indeed, exosomes isolated from GMDSCs (G-MDSC-exos) suppressed CD4⁺ and CD8⁺ T-cell proliferation in vitro and mildly attenuated immune BMF in the minor-H mismatched AA model. G-MDSC-exos treatment significantly increased red blood cells, hemoglobin, and total bone marrow (BM) cells, and moderately reduced BM CD8⁺ T cells. G-MDSC-exos’ effects were associated with upregulations in an array of lymphocyte-suppression-related miRNAs such as hsa-miR-142-5p, miR-19a-3p, and miR-19b-3p in both BM CD4⁺ and CD8⁺ T cells. We concluded that G-MDSC-exos attenuate immune BMF via modulating the delivery of immunosuppressive miRNAs into activated T lymphocytes. Full article

Background: Hepatopulmonary syndrome (HPS) is a pulmonary vasculature complication in the setting of liver disease that is characterized by pathological vasodilation resulting in arterial oxygenation defects. We investigated the role of extracellular vesicles (EV) in cirrhosis patients with HPS, as well as the functional effect of EV administration in a common bile duct ligation (CBDL) HPS mouse model. Methods: A total of 113 cirrhosis patients were studied: 42 (Gr. A) with HPS and 71 (Gr. B) without HPS, as well as 22 healthy controls. Plasma levels of EV associated with endothelial cells, epithelial cells, and hepatocytes were measured. The cytokine cargoes were estimated using ELISA. The effect of EV administered intranasally in the CBDL mouse model was investigated for its functional effect in vascular remodeling and inflammation. Results: We found endothelial cells (EC) associated EV (EC-EV) were elevated in cirrhosis patients with and without HPS (p < 0.001) than controls. EC-EV levels were higher in HPS patients (p = 0.004) than in those without HPS. The epithelial cell EVs were significantly high in cirrhosis patients than controls (p < 0.001) but no changes found in patients with HPS than without. There was a progressive increase in EC-EV levels from mild to severe intrapulmonary shunting in HPS patients (p = 0.02 mild vs. severe), and we were able to predict severe HPS with an AUROC of 0.85; p < 0.001. An inverse correlation of EC-EVs was found with hemoglobin (r = −0.24; p = 0.031) and PaO2 (r = 0.690; p = 0.01) and a direct correlation with MELD (r = 0.32; p = 0.014). Further, both TNF-α (p = 0.001) and IL-1β (p = 0.021) as cargo levels were significantly elevated inside the EVs of HPS patients than without HPS. Interestingly, upon administration of intranasal EVs, there was a significant decrease in Evans blue accumulation and lung wet–dry ratio (p = 0.042; 0.038). A significant reduction was also noticed in inflammation and cholestasis. Conclusion: High levels of plasma EC-EV levels were found in patients with HPS with elevated pro-inflammatory cytokine cargoes. EC-EVs were indicative of severe HPS condition. In the CBDL HPS model, we were able to prove the beneficial effects of improving vascular tone, inflammation, and liver pathogenesis. Full article

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman’s assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca²⁺-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis. Full article

Chlamydia trachomatis is the bacterial pathogen that causes most cases of sexually transmitted diseases annually. To combat the global spread of asymptomatic infection, development of effective (mucosal) vaccines that offer both systemic and local immune responses is considered a high priority. In this study, we explored the expression of C. trachomatis full-length (FL) PmpD, as well as truncated PmpD passenger constructs fused to a “display” autotransporter (AT) hemoglobin protease (HbpD) and studied their inclusion into outer membrane vesicles (OMVs) of Escherichia coli and Salmonella Typhimurium. OMVs are considered safe vaccine vectors well-suited for mucosal delivery. By using E. coli AT HbpD-fusions of chimeric constructs we improved surface display and successfully generated Salmonella OMVs decorated with a secreted and immunogenic PmpD passenger fragment (aa68-629) to 13% of the total protein content. Next, we investigated whether a similar chimeric surface display strategy could be applied to other AT antigens, i.e., secreted fragments of Prn (aa35-350) of Bordetella pertussis and VacA (aa65-377) of Helicobacter pylori. The data provided information on the complexity of heterologous expression of AT antigens at the OMV surface and suggested that optimal expression strategies should be developed on an antigen-to-antigen basis. Full article

Extracellular vesicles (EVs) are nano-scaled vesicles released from all cell types into extracellular fluids and specifically contain signature molecules of the original cells and tissues, including the placenta. Placenta-derived EVs can be detected in maternal circulation at as early as six weeks of gestation, and their release can be triggered by the oxygen level and glucose concentration. Placental-associated complications such as preeclampsia, fetal growth restriction, and gestational diabetes have alterations in placenta-derived EVs in maternal plasma, and this can be used as a liquid biopsy for the diagnosis, prediction, and monitoring of such pregnancy complications. Alpha-thalassemia major (“homozygous alpha-thalassemia-1”) or hemoglobin Bart’s disease is the most severe form of thalassemia disease, and this condition is lethal for the fetus. Women with Bart’s hydrops fetalis demonstrate signs of placental hypoxia and placentomegaly, thereby placenta-derived EVs provide an opportunity for a non-invasive liquid biopsy of this lethal condition. In this article, we introduced clinical features and current diagnostic markers of Bart’s hydrops fetalis, extensively summarize the characteristics and biology of placenta-derived EVs, and discuss the challenges and opportunities of placenta-derived EVs as part of diagnostic tests for placental complications focusing on Bart’s hydrop fetalis. Full article

The purpose of our study was the obtaining, characterization and biocompatibility estimation of novel carrier systems for diclofenac. Diclofenac is a potent nonsteroidal anti-inflammatory drug with frequent gastrointestinal side effects, impairing the quality of the patient’s life. Original diclofenac-loaded micro-vesicles coated with chitosan were prepared and physico-chemical analyzed. We investigated their in vitro hemocompatibility and in vivo biocompatibility in rats. The animals were treated orally as follows: group 1 (Control): distilled water 0.3 mL/100 g body weight; Group 2 (CHIT): 0.3 mL/100 g body weight 0.5% chitosan solution; Group 3 (DCF): 15 mg/kg body weight diclofenac; Group 4 (DCF-ves): lipid vesicles loaded with diclofenac 15 mg/kg body weight. Blood samples were collected for assessing: red blood cells, hemoglobin, hematocrit and leukocyte formula. A series of specific parameters of the liver and kidney function, some markers of immune defense, as well as the activity of some enzymes involved in oxidative processes, were also investigated. At the end of the experiment, the animals were sacrificed and fragments of liver, kidney and stomach were collected for histopathological examination. No blood hemolysis was evidenced by the in vitro test with the administration of diclofenac vesicles. The animals treated with diclofenac lipid vesicles stabilized with chitosan did not display any notable differences in their hematological and biochemical profile compared to control animals. These data correlated with the histological results, which showed the absence of architectural changes in the examined tissues. Biological in vitro and in vivo evaluation revealed that the microvesicles containing diclofenac are biocompatible, with potential to be used as delivery systems to modify the drug release, thus making them an attractive candidate for biomedical applications. Full article

Hyperglycemia is a trigger for structural alteration of red blood cells (RBCs) and their ability to release extracellular vesicles (EVs). The aim of the study was to elucidate whether glucose control in T2DM patients with concomitant HF and AF affects a circulating number of RBC-derived EVs. We prospectively included 417 T2DM patients with HF, 51 of them had atrial fibrillation and 25 healthy volunteers and 30 T2DM non-HF individuals. Clinical assessment, echocardiography examination and biomarker measures were performed at the baseline of the study. RBC-derived EVs were determined as CD235a+ PS+ particles by flow cytometry. NT-proBNP levels were measured by ELISA. AF patients with glycosylated hemoglobin (HbA1c) < 6.9% had lower levels of CD235a+ PS+ RBC-derived vesicles than those with HbA1c ≥ 7.0%. There were no significant differences in number of CD235a+ PS+ RBC-derived vesicles between patients in entire cohort and in non-AF sub-cohort with HbA1c < 6.9% and HbA1c ≥ 7.0%, respectively. Multivariate linear regression yielded that CD235a+ PS+ RBC-derived vesicles ≥ 545 particles in µL (OR = 1.06; 95% CI = 1.01–1.11, p = 0.044) independently predicted HbA1c ≥ 7.0%. Elevated levels of CD235a+ PS+ RBC-derived EVs independently predicted poor glycaemia control in T2DM patients with HF and AF. Full article

We demonstrated previously that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) play a critical role in angiogenesis. Here, we examine whether this pro-angiogenic efficacy is enhanced in EVs derived from MSCs overexpressing GATA-4 (MSC^GATA−4). Methods and Results. EVs were isolated from MSC^GATA-4 (EV^GATA-4) and control MSCs transduced with an empty vector (EV^null). EVs from both cell types were of the same size and displayed similar molecular markers. Compared with EV^null, EV^GATA-4 increased both a tube-like structure formation and spheroid-based sprouting of human umbilical vein endothelial cells (HUVECs). The EV^GATA-4 increased the numbers of CD31-positive cells and hemoglobin content inside Matrigel plugs subcutaneously transplanted into mice for 2 weeks. Moreover, EV^GATA-4 encapsulated higher levels of let-7 family miRs compared to EV^null. The transfer of exosomal let-7 miRs into HUVECs was recorded with an accompanied down-regulation of thrombospondin-1 (THBS1) expression, a major endogenous angiogenesis inhibitor. The loss-and-gain of function studies of let-7 miRs showed that let-7f knockdown significantly decreased EV^GATA-4-mediated vascularization inside Matrigel plugs. In contrast, let-7f overexpression promoted HUVEC migration and tube formation. Conclusion. Our results indicate that EVs derived from genetically modified MSCs with GATA-4 overexpression had increased pro-angiogenic capacity due to the delivery of let-7 miRs that targeted THBS1 in endothelial cells. Full article

Sickle cell disease (SCD) occurs when two alleles of mutated hemoglobin (HbS or HbC) are inherited (HbSS and HbSC) rather than one (HbAS or HbAC), which indicates a person carries the sickle cell trait. The high prevalence of these two alleles in Africa have been associated with reduced malaria susceptibility. Recent in vitro research has been shown that microRNAs (miRNAs) miR-451a and let-7i-5p are differentially expressed in HbSS erythrocytes compared to healthy controls (HbAA) and are overexpressed in Plasmodium-infected malaria erythrocytes. However, these miRNAs have not been fully examined in the plasma of people with different sickle hemoglobin genotypes. Plasma circulating miRNAs are commonly encapsulated in extracellular vesicles, such as exosomes, and are thought to play a role in disease development. Circulating exosomal miR-451a and let-7i-5p were quantified from individuals with various hemoglobin genotypes (HbAA, HbAS, HbAC, HbSS, HbSC, and HbCC) with (+) and without (−) malaria. The results showed a higher level of exosomal let-7i-5p and miR-451a in HbSS-. Exosomal let-7i-5p and miR-451a levels were lower in HbSS+ compared to other genotypes. Based on the area under the curve (AUC) of the Receiver Operating Characteristics (ROCs), both exosomal miRNAs may be useful disease biomarkers for SCD with malaria. Finally, miR-451a and let-7i-5p modulate genes involved in inflammation, making them potential biomarkers of pathogenesis for both diseases. Full article

Cisplatin (CDDP) is an essential anti-tumor agent for chemotherapeutic regimens against various types of cancer. However, the progression of nephrotoxicity, which is the main adverse effect of CDDP, leads to discontinuation of CDDP chemotherapy. Therefore, development of a renoprotectant against CDDP-induced nephrotoxicity is crucial. Here, the potential of a carbon monoxide (CO)-loaded hemoglobin-vesicle (CO-HbV) as a renoprotectant for CDDP-induced nephrotoxicity was evaluated for its renoprotective effects against CDDP-induced nephrotoxicity, inhibitory effects on the anti-tumor activity of CDDP, and anti-tumor activity. In healthy mice, after pretreatment with either saline, HbV, or CO-HbV prior to CDDP administration, only the CO-HbV pretreatment group ameliorated the progression of CDDP-induced nephrotoxicity by suppressing apoptosis via caspase-3. In experiments using B16-F10 melanoma cells, the half-maximal inhibitory concentration of CDDP decreased with co-incubation with CO-HbV, owing to the anti-tumor activity of CO. CO-HbV pretreatment had no impact on the anti-tumor activity of CDDP in B16-F10 melanoma cell-bearing mice, which was consistent with the results of the cell experiment. Furthermore, CO-HbV pretreatment improved body growth and survival rates. In conclusion, CO-HbV pretreatment is a potent renoprotectant for CDDP-induced nephrotoxicity, allowing treatment with CDDP to be conducted without failure of cancer treatment. Full article

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here. Full article