Peripheral Vesicles Research Articles

Changes in the Release of Endothelial Extracellular Vesicles CD144+, CCR6+, and CXCR3+ in Individuals with Acute Myocardial Infarction.

Acute myocardial infarction (AMI) results from vulnerable plaque rupture, causing ischemic cardiomyocyte necrosis and intense inflammation. Paradoxically, this inflammation releases factors that aid heart repair. Recent findings suggest a role for extracellular vesicles (EVs) in intercellular communication during post-AMI cardiac repair. However, EVs' tissue origin and chemokine profile in the blood of patients with AMI remains unclear. This study characterized the tissue origin and chemokine receptor profile of EVs in the coronary and peripheral blood of patients with AMI. The results reveal that vesicles isolated from coronary and peripheral blood plasma are enriched in tetraspanin (CD9) and express CD81+, CD90+, and CD144+. The vesicle size ranged between 145 and 162 nm, with the control group exhibiting smaller vesicles (D10) than the AMI group. Furthermore, all vesicles expressed CCR6 and CXCR3, whereas a small percentage expressed CCR4. In addition, a decrease in CXCR3 and CCR6 expression was observed in coronary and peripheral AMI blood vesicles compared with the control; however, no difference was found between AMI coronary and AMI peripheral blood vesicles. In conclusion, our study demonstrates, for the first time, changes in the number of extracellular vesicles expressing CD144+, CXCR3, and CCR6 in the peripheral circulation of patients with AMI. Extracellular vesicles present in the circulation of patients with AMI hold excellent promise as a potential diagnostic tool.

Diagnostic value of microRNA-200 expression in peripheral blood-derived extracellular vesicles in early-stage non-small cell lung cancer

ObjectiveThis study assessed the diagnostic value of microRNA-200 (miR-200) expression in peripheral blood-derived extracellular vesicles (EVs) in early-stage non-small cell lung cancer (NSCLC).MethodsThis study retrospectively analyzed 100 healthy volunteers (the control group) receiving physical examinations, 168 early-stage NSCLC patients (the NSCLC group), and 128 patients with benign lung nodules (the benign group). The basic and clinical data of participants were obtained, including age, sex, smoking history, carbohydrate antigen 242 (CA242), carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), forced expiratory volume in 1 s, maximal voluntary ventilation, forced vital capacity, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and miR-200 expression. The correlation of miR-200 expression in peripheral blood-derived EVs with CA242, CEA, and CA199 was analyzed, and the diagnostic value of peripheral blood-derived EV miR-200 for early-stage NSCLC was assessed. The risk factors of early-stage NSCLC development were also determined.ResultsAge, the percentage of patients with smoking history, CA242, CEA, CA199, IL-6, and TNF-α levels, and miR-200 expression in peripheral blood-derived EVs were significantly higher in the NSCLC group than in the benign and control groups. Lung disease patients with high miR-200 expression in peripheral blood-derived EVs comprised a higher percentage of patients with smoking history and mixed lesions and had higher CA242, CEA, CA199, and TNF-α levels than those with low miR-200 expression in peripheral blood-derived EVs. In lung diseases, miR-200 expression in peripheral blood-derived EVs was significantly and positively correlated with CA242, CEA, and CA199. Peripheral blood-derived EV miR-200 combined with CA242, CEA and CA199 had higher diagnostic value (area under the curve = 0.942) than single detection, along with higher specificity, and high expression of peripheral blood-derived EV miR-200 was an independent risk factor for early-stage NSCLC.ConclusionPeripheral blood-derived EV miR-200 expression in patients with lung diseases is closely correlated with CA242, CEA, and CA199, and high expression of peripheral blood-derived EV miR-200 is an independent risk factor for early-stage NSCLC and is of high clinical diagnostic value for early-stage NSCLC.

Exploring Peripheral Blood-Derived Extracellular Vesicles as Biomarkers: Implications for Chronic Chagas Disease with Viral Infection or Transplantation.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encapsulate cell-specific cargo, rendering them promising biomarkers for diverse diseases. Chagas disease, caused by the parasite Trypanosoma cruzi, poses a significant global health burden, transcending its initial epicenter in Latin America to affect individuals in Europe, Asia, and North America. In this study, we aimed to characterize circulating EVs derived from patients with chronic Chagas disease (CCD) experiencing a reactivation of acute symptoms. Blood samples collected in EDTA were processed to isolate plasma and subsequently subjected to ultracentrifugation for particle isolation and purification. The EVs were characterized using a nanoparticle tracking analysis and enzyme-linked immunosorbent assay (ELISA). Our findings revealed distinctive differences in the size, concentration, and composition of EVs between immunosuppressed patients and those with CCD. Importantly, these EVs play a critical role in the pathophysiology of Chagas disease and demonstrate significant potential as biomarkers in the chronic phase of the disease. Overall, our findings support the potential utility of the CL-ELISA assay as a specific sensitive tool for detecting circulating EVs in chronic Chagasic patients, particularly those with recurrent infection following an immunosuppressive treatment or with concurrent HIV and Chagas disease. Further investigations are warranted to identify and validate the specific antigens or biomarkers responsible for the observed reactivity in these patient groups, which may have implications for diagnosis, the monitoring of treatment, and prognosis.

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis, ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogeneous surface charge allows multi-omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Dysregulation of MicroRNAs Derived from Plasma Extracellular Vesicles in Schizoaffective Disorder.

The association between peripheral blood extracellular vesicles (EVs)-derived miRNAs (EVs-miRNAs) and neuropsychiatric diseases has been extensively studied. However, it remains largely unclear about the expression profile of EVs-miRNAs in schizoaffective disorder (SAD) patients. In our study, we isolated the EVs from plasma samples of patients and healthy controls, and then analyzed the expression profiles of EVs-miRNAs through small RNA sequencing. Our results identified 32 differentially expressed (DE) miRNAs (25 upregulated and 7 downregulated) in SAD patients. A module containing 42 miRNAs closely related to SAD was identified by weighted gene co-expression network analysis (WGCNA), among which has-miR-15b-5p, has-miR-301a-3p, has-miR-342-3p, has-miR-219b-5p, and has-miR-145-5p were identified as hub miRNAs. The enrichment analysis showed that the target genes of these 42 miRNAs were significantly enriched in multiple pathways related to neuropathology and located at synapses. A total of 6 DE miRNAs (has-miR-7-5p, has-miR-144-3p, has-miR-155-5p, has-miR-342-3p, has-miR-342-5p, and has-miR-487b-3p) associated with SAD were selected for qRT-PCR verification. The level of has-miR-342-3p in SAD patients was downregulated, and hsa-miR-155-5p was upregulated. Our findings support the hypothesis that dysregulation of EVs-miRNAs in plasma might be involved in the underlying neuropathology of SAD through several biological pathways and provide important preliminary evidence supporting the use of EVs-miRNAs as potential novel biomarkers in SAD.

Aβ1-42-containing platelet-derived extracellular vesicle is associated with cognitive decline in Parkinson's disease.

Cortical amyloid deposition is a common observation in Parkinson's disease dementia (PDD) patients. Aβ1-42 is linked to a more rapid progression of dementia. Platelets, which degranulate upon activation, are a primary source of Aβ. It has been repeatedly reported that peripheral extracellular vesicles (EVs) can partially reach the central nervous system. Thus, we speculate that activated platelet-derived Aβ1-42-containing EVs (PEV-Aβ1-42) play a crucial role in the cognitive decline of PD patients. The study included 189 participants: 66 with non-dementia PD, 73 with PDD, and 50 healthy controls. All participants underwent blood collection and clinical assessments. Twenty PD patients underwent re-examination and repeated blood collection 14 months later. A nano-scale flow cytometry assay was used to detect PEVs and PEV-Aβ1-42 using fluorescence-labeled CD62P and Aβ1-42 antibodies. Parkinson's disease dementia patients had higher PEV-Aβ1-42 concentrations than healthy controls (p = 0.028). The ratio of PEV-Aβ1-42 to PEV was significantly higher in PDD patients compared to those in non-dementia PD and healthy controls (p PD-ND < 0.001, p HC = 0.041). The PEV-Aβ1-42/PEV ratio appears to influence the odds of developing dementia (OR = 1.76, p < 0.001). The change in the PEV-Aβ1-42/PEV ratio was also correlated with cognitive decline over 14 months (r = -0.447, p < 0.05). The plasma PEV-Aβ1-42/PEV ratio may serve as a diagnostic and prognostic biomarker for PDD patients.

ZDHHC9 Regulates Cardiomyocyte Rab3a Activity and Atrial Natriuretic Peptide Secretion Through Palmitoylation of Rab3gap1

Production and release of natriuretic peptides by the stressed heart reduce cardiac workload by promoting vasodilation, natriuresis, and diuresis, which has been leveraged in the recent development of novel heart-failure pharmacotherapies, yet the mechanisms regulating cardiomyocyte exocytosis and natriuretic peptide release remain ill defined. We found that the Golgi S-acyltransferase zDHHC9 palmitoylates Rab3gap1 resulting in its spatial segregation from Rab3a, elevation of Rab3a-GTP levels, formation of Rab3a-positive peripheral vesicles, and impairment of exocytosis that limits atrial natriuretic peptide release. This novel pathway potentially can be exploited for targeting natriuretic peptide signaling in the treatment of heart failure.

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada

BackgroundGiardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space.ResultsHere, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified.ConclusionsTaken together, this provides the first comprehensive nanometric view of Giardia’s endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

Intracellular trafficking of antigen in B lymphocytes

B cells are an essential part of the adaptive immune system by providing the humoral arm of the response, and specific and long-lasting protection against a pathogens. Successful antigen internalization, intracellular trafficking, processing and presentation are critical for full B cell activation and high-affinity antibody production. Despite the fundamental importance of this pathway for the immune response, the mechanisms underlying antigen trafficking for successful processing and MHCII presentation remain unclear. By using confocal and super-resolution microscopy, we followed the traffic of internalized antigen along the endosomal pathway in B cells. To identify different vesicle populations, we took advantage of different Rab GTPases as well-known markers for early-, late-and recycling-endosomes. After internalization, antigen traffics from early-to late-endosomes and typically reaches a dense endosomal compartment located in the perinuclear region. This compartment is rich in lysosomal markers and Cathepsin-S, as well as MHCII, suggesting antigen loading compartment. Interestingly however, when we investigated the early timepoints after activation in detail, we found the antigen in peripheral endosomes with atypical features combining both early (EEA1+, Rab5+) and late (LAMP1+) markers. These endosomes also showed colocalization with Rab11, a marker of recycling-endosomes, and MHCII, suggesting that already these early peripheral vesicles could degrade antigen and load peptides on MHCII for presentation. In support of their role in antigen processing, these vesicles also contained Cathepsin-S and showed hydrolysis of antigen labeled with DQ, a dye that only fluoresces upon hydrolysis. We postulate that these early vesicles could play a role in fast antigen processing, or could promote presentation of different peptide repertoire as compared to the central, perhaps more mature, perinuclear compartment. Further studies are needed to unravel how these pathways are regulated and how their balance affects T cell help and thereby B cell fate and production of high-affinity antibodies.

Adjuvant crizotinib in high-risk uveal melanoma following definitive therapy.

IntroductionApproximately 40% of patients with uveal melanoma (UM) will develop metastatic disease. Tumors measuring at least 12mm in basal diameter with a class 2 signature, as defined by a widely used gene expression-profiling test, are associated with significantly higher risk of metastasis, with a median time to recurrence of 32 months. No therapy has been shown to reduce this risk.Materials and MethodsThis was a single-arm, multicenter study in patients with high-risk UM who received definitive treatment of primary disease and had no evidence of metastasis. Patients were consecutively enrolled to receive 12 four-week cycles of adjuvant crizotinib at a starting dose of 250mg twice daily and were subsequently monitored for 36 months. The primary outcome of this study was to assess recurrence-free survival (RFS) of patients with high-risk UM who received adjuvant crizotinib.Results34 patients enrolled and received at least one dose of crizotinib. Two patients were unevaluable due to early withdrawal and loss to follow-up, leaving 32 patients evaluable for efficacy. Eight patients (25%) did not complete the planned 48-week course of treatment due to disease recurrence (n=5) or toxicity (n=3). All patients experienced at least one adverse event (AE), with 11/34 (32%) experiencing a Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or 4 AE. After a median duration of follow up of 47.1 months, 21 patients developed distant recurrent disease. The median RFS was 34.9 months (95% CI (Confidence Interval), 23-55 months), with a 32-month recurrence rate of 50% (95% CI, 33-67%). Analysis of protein contents from peripheral blood extracellular vesicles in a subset of patient samples from baseline, on-treatment, and off-treatment, revealed a change in protein content associated with crizotinib exposure, however without a clear association with disease outcome.ConclusionsThe use of adjuvant crizotinib in patients with high-risk UM did not result in improved RFS when compared to historical controls. Analysis of blood extracellular vesicles revealed changes in protein content associated with treatment, raising the possibility of future use as a biomarker. Further investigation of adjuvant treatment options are necessary for this challenging disease.

Itchy Vesicular Rash.

A 38-year-old woman presented with a rash of 5 days’ duration that initially appeared on the wrists after playing with her kitten, with subsequent involvement of the chest, back, abdomen, and upper and lower extremities. Physical examination revealed multiple annular plaques with raised erythematous borders, rare peripheral vesicles, and superficial central scaling.

A relapse of pemphigus vulgaris in pemphigus herpetiformis or a phenotypic “switch”

Sir, Pemphigus herpetiformis (PH) was originally described by Jablonska et al. in 1975. Clinically, PH presents itself as a herpetiform dermatitis with immunopathological characteristics of pemphigus [1,2]. We report an exceptional case of typical pemphigus vulgaris (PV) relapsing after 36 years in PH. A 65-year-old patient, followed for PV for 36 years and treated with corticosteroid therapy with a remission for more than thirty years, consulted for pruriginous lesions evolving for the previous eight months. A dermatological examination revealed urticariform pruriginous ring lesions surmounted by small peripheral vesicles spread throughout the body (Fig. 1), sparing the mucous membranes, and without Nikolsky’s sign. After two non-specific skin biopsies, the histological examination revealed an intraepidermal bubble with acantholytic cells and eosinophilic spongiosis (Figs. 2a and 2b). Direct immunofluorescence confirmed the diagnosis of pemphigus and indirect immunofluorescence was at the upper limit. The diagnosis of a PV relapse in PH was retained and a dapsone-based treatment was initiated at a dose of 150 mg/day and stopped seven days later when met with hemolytic anemia. Oral corticosteroid therapy involving prednisone at a dose of 1 mg/kg/day was initiated but, given the persistence of the pruritus, the decision was to combine methotrexate at a dose of 12.5 mg/week. A good evolution and a decline within eight months were observed. An improved pruritus and the disappearance of the skin lesions were achieved after one month of treatment. PV and PH are two different anatomical and clinical entities of the autoimmune disease pemphigus, with distinct clinical, histopathological, and immunopathological characteristics [1,2]. Our observation documents a complete phenotypic “switch” of pemphigus with a transition from PV to PH both clinically, histologically, and immunologically. Several rare cases of PV switching to superficial pemphigus (SP) (“phenotypic switch”) have, since 1991, been reported, with a higher frequency this direction than otherwise; the transition period varies from six months to twenty years [3]. To the best of our knowledge, no case has been described of a progression from PV to PH. Having observed one firsthand, we are first to describe the case of a complete phenotypic switch from PV to PH. The mechanism of such a transition remains poorly understood and is often observed during a relapse. Some authors suggest that the effect of immunosuppressants on the desmoglein DSG3 more marked than on DSG1 could explain the relapse of PS in PH [3,4]. Future studies on the immunological factors and predictors of PV relapses after the discontinuation of treatment would be useful to better understand the mechanisms of a relapse in pemphigus, with or without a phenotypic transition.

Apolipoprotein E4‐driven effects on inflammatory and neurotrophic factors in peripheral extracellular vesicles from cognitively impaired not demented participants converted to Alzheimer’s disease

AbstractBackgroundIn brain, extracellular vesicles (EVs) play an essential role in neuron‐glia interface and ensure the crosstalk between the brain and the periphery. Only a few studies have demonstrated the apolipoprotein E4 variant (APOE ε4)‐driven dysfunction of EVs pathway and the risk to develop Alzheimer’s disease (AD). To better understand the role of APOE ε4 in pre‐clinical AD, we determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired‐not demented (CIND) participants stratified upon the absence (APOE ε4‐) or the presence (APOE ε4+) of the ε4 allele of APOE.MethodLevels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs by the multiplex Luminex assay and compared to their plasma levels from cognitively normal and CIND participantsResultFor the first time, several neurotrophic and inflammatory markers including LCN‐2, S100B, ANGPTL‐4, NPTX‐2 and α‐synuclein were evidenced in pEVs. Some proteins such as α‐Syn, NPTX‐2 and S100B were enriched in pEVs as compared to plasma. APOE ε4+ was associated with differential regulation of 7 markers and compromised the release of pEVs formed by an endosomal route. The pentraxin‐2/α‐synuclein ratio measured in pEVs was able to predict AD 5 years before the onset among APOE ε4+ CIND individuals. Discussion: Our findings suggest an alteration of the endosomal pathway in APOE ε4+ carriers and that pEVs pentraxin‐2/α‐synuclein ratio could serve as a useful early biomarker for AD susceptibility.ConclusionThe findings reported herein provide a comprehensive insight and enhance our knowledge on the emerging role of ApoE4 in abnormal pEVs cargo proteins processing and the identification of blood‐based biomarkers.

Apolipoprotein E4-driven effects on inflammatory and neurotrophic factors in peripheral extracellular vesicles from cognitively impaired, no dementia participants who converted to Alzheimer's disease.

IntroductionIn brain, extracellular vesicles (EVs) play an essential role in the neuron‐glia interface and ensure the crosstalk between the brain and the periphery. Some studies now link the pathway dysfunction of the EVs to apolipoprotein E gene variant (APOE ε4) and the risk of progression to Alzheimer's disease (AD). To better understand the role of APOE ε4 in pre‐clinical AD, we have determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired, no dementia (CIND) participants stratified upon the absence (APOE ε4‐) or the presence (APOE ε4+ ) of the ε4 allele of APOE.MethodsLevels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs and compared to their plasma levels from cognitively normal and CIND participants.ResultsLevels of neurotrophic and inflammatory markers were reduced in pEVs from APOE ε4+. The pentraxin‐2/α‐synuclein ratio measured in pEVs was able to predict AD 5 years before the onset among APOE ε4+‐CIND individuals.DiscussionOur findings suggest an alteration of the endosomal pathway in APOE ε4+ and that pEVs pentraxin‐2/α‐synuclein ratio could serve as a useful early biomarker for AD susceptibility.

Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Oligomeric α-Syn and SNARE complex proteins in peripheral extracellular vesicles of neural origin are biomarkers for Parkinson's disease

Blood-based biomarkers are needed to be used as easy, reproducible, and non-invasive tools for the diagnosis and prognosis of chronic neurodegenerative disorders including Parkinson's Disease (PD). In PD, aggregated toxic forms of α-Synuclein (α-Syn) accumulate within neurons in the brain and cause neurodegeneration; α-Syn interaction with SNARE proteins also results in synaptic disfunction. We isolated neural derived extravesicles (NDEs) from peripheral blood of 32 PD patients and 40 healthy controls (HC) and measured the concentrations of oligomeric α-Syn and of the presinaptic SNARE complex proteins: STX-1A, VAMP-2 and SNAP-25. Oligomeric α-Syn was significantly augmented whereas STX-1A and VAMP-2 were significantly reduced in NDEs of PD patients compared to HC (p < 0.001 in all cases). ROC curve analyses confirmed the discriminatory ability of NDEs oligomeric α-Syn, STX-1A and VAMP-2 levels to distinguish between PD patients and HC. Oligomeric α-Syn NDEs concentration also positively correlated with disease duration and severity of PD. These results are promising and confirm that NDEs cargoes likely reflect core pathogenic intracellular processes in their originating brain cells and could serve as novel easily accessible bio-markers. Further studies are needed to confirm results and eventually for testing rehabilitation programs and drug treatments effects.

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions.

Giardia intestinalis colonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) in Giardia, which might relate to the establishment of infection and disease induction. Giardia trophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles during Giardia-host cell interactions.

Therapeutic potential of miR-21 regulation by human peripheral blood derived-small extracellular vesicles in myocardial infarction.

Small extracellular vesicles (sEVs) as natural membranous vesicles are on the frontiers of nanomedical research, due to their ability to deliver therapeutic molecules such as microRNAs (miRNAs). The miRNA-21 (miR-21) is thought to be involved in the initiation and development of myocardial infarction (MI). Here, we examined whether miR-21 regulation using human peripheral blood-derived sEVs (PB-sEVs) could serve as a potential therapeutic strategy for MI. First, we examined miR-21 levels in hypoxic conditions and validated the ability of PB-sEVs to serve as a potential delivery system for miRNAs. Further, bioinformatics analysis and luciferase assay were performed to identify target genes of miR-21 mechanistically. Among numerous target pathways, we focused on nitrogen metabolism, which remains relatively unexplored compared with other possible miR-21-mediated pathways; hence, we aimed to determine novel target genes of miR-21 related to nitrogen metabolism. In hypoxic conditions, the expression of miR-21 was significantly up-regulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The down-regulation of miR-21 expression by PB-sEVs loaded with anti-miR-21 significantly improved survival rates, consistent with the augmentation of cardiac function. However, the up-regulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and down-regulated the mRNA and protein expression of striatin (STRN), which could regulate NOS3 expression. In conclusion, we identified a novel therapeutic strategy to improve cardiac function by regulating the expression of miR-21 with PB-sEVs as an miR-21 or anti-miR-21 delivery vehicle and confirmed the miR-21-associated nitrogen metabolic disorders in MI.

Small RNA fingerprinting of Alzheimer’s disease frontal cortex extracellular vesicles and their comparison with peripheral extracellular vesicles

ABSTRACT Alzheimer’s disease is a progressive neurodegenerative disorder, with the strongest disease-associated changes observed at clinical or end-stage disease. Transcriptomic deregulation of miRNA expression can spread via “horizontal” RNA transfer through extracellular vesicles (EVs) to act in conjunction with proteins, leading to changes in mRNA, which can provide early signals to indicate forthcoming neuropathological changes in the brain. Here, we analysed the small RNA content, in particular, miRNA, contained in brain-derived EVs isolated from the frontal cortex of Alzheimer’s subjects (n = 8) and neurological control subjects (n = 9). Brain-derived EVs were found to contain an upregulation of disease-associated miRNA. RNA species from brain-derived EVs were correlated with miRNA profiles obtained from matching total brain homogenate. These results provide a blueprint into the biological pathways potentially effected during disease that may be assisted by brain-derived EV RNA horizontal transfer.We also correlated the miRNA changes in the brain with those detected in peripheral EVs collected from serum of Alzheimer’s disease patients (n = 23, and healthy controls, n = 23) and revealed a panel of miRNA that could be used as a liquid brain biopsy. Overall, our study provides the first interrogation of the small RNA contents in brain-derived EVs and how they could be used to understand the early pathological changes in Alzheimer’s disease which will benefit the development of an early diagnostic blood test.

B cells rapidly target antigen and surface-derived MHCII into peripheral degradative compartments.

In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to T helper cells (TH cells). While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC). Surprisingly, we detected fast targeting of internalised antigen into peripheral acidic compartments that possessed the hallmarks of the MIIC and also showed degradative capacity. In these vesicles, internalised antigen converged rapidly with membrane-derived MHCII and partially overlapped with cathepsin-S and H2-M, both required for peptide loading. These early compartments appeared heterogenous and atypical as they contained a mixture of both early and late endosomal markers, indicating a specialized endosomal route. Together, our data suggest that, in addition to in the previously reported perinuclear late endosomal MIICs, antigen processing and peptide loading could have already started in these specialized early peripheral acidic vesicles (eMIIC) to support fast peptide-MHCII presentation.