scholarly journals Affinity enrichment of extracellular vesicles from plasma reveals mRNA changes associated with acute ischemic stroke

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Harshani Wijerathne ◽  
Malgorzata A. Witek ◽  
Joshua M. Jackson ◽  
Virginia Brown ◽  
Mateusz L. Hupert ◽  
...  
Keyword(s):  
T Cells ◽  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Extracellular Vesicles ◽  
Microfluidic Device ◽  
Digital Pcr ◽  
Affinity Enrichment ◽  
Healthy Donors ◽  
In Vitro Diagnostic

Abstract Currently there is no in vitro diagnostic test for acute ischemic stroke (AIS), yet rapid diagnosis is crucial for effective thrombolytic treatment. We previously demonstrated the utility of CD8(+) T-cells’ mRNA expression for AIS detection; however extracellular vesicles (EVs) were not evaluated as a source of mRNA for AIS testing. We now report a microfluidic device for the rapid and efficient affinity-enrichment of CD8(+) EVs and subsequent EV’s mRNA analysis using droplet digital PCR (ddPCR). The microfluidic device contains a dense array of micropillars modified with anti-CD8α monoclonal antibodies that enriched 158 ± 10 nm sized EVs at 4.3 ± 2.1 × 109 particles/100 µL of plasma. Analysis of mRNA from CD8(+) EVs and their parental T-cells revealed correlation in the expression for AIS-specific genes in both cell lines and healthy donors. In a blinded study, 80% test positivity for AIS patients and controls was revealed with a total analysis time of 3.7 h.

scholarly journals Incorporation of α2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 347
Author(s):  
Zsuzsa Bagoly ◽  
Barbara Baráth ◽  
Rita Orbán-Kálmándi ◽  
István Szegedi ◽  
Réka Bogáti ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Intracranial Hemorrhage ◽  
Intravenous Thrombolysis ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Stroke Patients ◽  
Plasmin Inhibitor ◽  
Fibrin Clots

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

scholarly journals Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Silvia R. Vitale ◽  
Jean A. Helmijr ◽  
Marjolein Gerritsen ◽  
Hicret Coban ◽  
Lisanne F. van Dessel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Extracellular Vesicles ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Solid Cancer ◽  
Expression Levels ◽  
Healthy Donors ◽  
Spin Column ◽  
High Concordance ◽  
Tumor Dna

Abstract Background Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

In vitro and in silico modeling of endovascular stroke treatments for acute ischemic stroke

2021 ◽  
pp. 110693
Author(s):  
Giulia Luraghi ◽  
Rachel M. E. Cahalane ◽  
Emma van de Ven ◽  
Serena Overschie ◽  
Frank J. H. Gijsen ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
In Silico ◽  
In Silico Modeling

scholarly journals Cerebral endothelial cell-derived small extracellular vesicles enhance neurovascular function and neurological recovery in rat acute ischemic stroke models of mechanical thrombectomy and embolic stroke treatment with tPA

2021 ◽  
pp. 0271678X2199298
Author(s):  
Chao Li ◽  
Chunyang Wang ◽  
Yi Zhang ◽  
Owais K Alsrouji ◽  
Alex B Chebl ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Extracellular Vesicles ◽  
Mechanical Thrombectomy ◽  
Infarct Volume ◽  
Artery Occlusion ◽  
Therapeutic Effects ◽  
Vessel Occlusion ◽  
Healthy Human ◽  
Stroke Treatment

Treatment of patients with cerebral large vessel occlusion with thrombectomy and tissue plasminogen activator (tPA) leads to incomplete reperfusion. Using rat models of embolic and transient middle cerebral artery occlusion (eMCAO and tMCAO), we investigated the effect on stroke outcomes of small extracellular vesicles (sEVs) derived from rat cerebral endothelial cells (CEC-sEVs) in combination with tPA (CEC-sEVs/tPA) as a treatment of eMCAO and tMCAO in rat. The effect of sEVs derived from clots acquired from patients who had undergone mechanical thrombectomy on healthy human CEC permeability was also evaluated. CEC-sEVs/tPA administered 4 h after eMCAO reduced infarct volume by ∼36%, increased recanalization of the occluded MCA, enhanced cerebral blood flow (CBF), and reduced blood-brain barrier (BBB) leakage. Treatment with CEC-sEVs given upon reperfusion after 2 h tMCAO significantly reduced infarct volume by ∼43%, and neurological outcomes were improved in both CEC-sEVs treated models. CEC-sEVs/tPA reduced a network of microRNAs (miRs) and proteins that mediate thrombosis, coagulation, and inflammation. Patient-clot derived sEVs increased CEC permeability, which was reduced by CEC-sEVs. CEC-sEV mediated suppression of a network of pro-thrombotic, -coagulant, and -inflammatory miRs and proteins likely contribute to therapeutic effects. Thus, CEC-sEVs have a therapeutic effect on acute ischemic stroke by reducing neurovascular damage.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

Abstract WP430: Endothelial Cell Collection from Ipsilateral Middle Cerebral Artery in Acute Ischemic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Sunil A Sheth ◽  
Abhishek Verma ◽  
David S Liebeskind ◽  
Jeffrey L Saver ◽  
Gary Duckwiler ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Endothelial Cell ◽  
Middle Cerebral Artery ◽  
Acute Ischemic Stroke ◽  
Cerebral Artery ◽  
Critical Role ◽  
Early Time ◽  
Stent Retriever ◽  
Positive Controls

Introduction: The cerebrovascular endothelium plays a critical role in the pathogenesis of and response to acute ischemic stroke (AIS). To date, techniques to study its function have relied on animal and in vitro models. A robust method of endothelial cell (EC) capture in patients with AIS at early time points, from within the ischemic region, could greatly advance our understanding of cerebrovascular injury. Method: Patients undergoing thrombectomy for middle cerebral artery occlusion (MCA) within 8 hours of onset were offered enrollment if the pass of their stent-retriever device occurred directly into a distal access catheter in the proximal M1 segment, limiting exposure of the device to only the MCA. After retrieval, ECs adherent to the devices were retrieved and stained for EC (CD31) and leukocyte (CD45) markers. EC identity and yield were confirmed by flow cytometry with simultaneous immuno-fluorescence microscopy. Cultured human ECs were used as positive controls. The EC fraction was defined as CD31 + CD45 - with size and morphological features consistent with the positive controls. Results: ECs from stent-retriever devices (n=3) were collected and pooled. Approximately 8% of the collected cells represented ECs. EC collected from the stent-retrievers demonstrated highly similar shape, morphology and antibody staining patterns compared to the positive controls (Figure). Conclusions: Here we provide the first demonstration of a rapid post-thrombectomy method for reliable harvesting of cerebral ECs in humans. The ability to capture these cells in patients with AIS within hours of symptom onset opens many avenues of exploration for determining the role of ECs in AIS.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Abstract Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

scholarly journals T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Daniele Mennonna ◽  
Cristina Maccalli ◽  
Michele C Romano ◽  
Claudio Garavaglia ◽  
Filippo Capocefalo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Epitope ◽  
Patient Specific ◽  
Cancer Genes ◽  
Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

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