techniques-for-analysis-extracellular-vesicles-using-flow

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Blood Systems Research Institute

Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.

The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: results of a ring-study in five laboratories.

BACKGROUND: Transfusion of allogeneic blood products can lead to alloimmunization, impacting success of subsequent transfusions and solid organ transplants. Pathogen reduction using riboflavin and ultravioletâ€‰B (UVB) light has been shown to eliminate the immunogenicity of white blood cells (WBCs) in vitro through down regulation of surface adhesion molecules, effectively blocking cell-cell conjugation and direct presentation. We sought to determine if this loss of immunogenicity is extended in vivo where indirect presentation of allogeneic antigens can occur. STUDY DESIGN AND METHODS: BALB/cJ mice were transfused with either untreated or riboflavin and UVB-treated C57Bl/6J platelet-rich plasma (PRP) containing WBCs. Circulating alloantibody and allospecific splenocyte cytokine responses were measured. RESULTS: Pathogen reduction of allogeneic WBC-enriched PRP using riboflavin and UVB light before transfusion prevented alloimmunization, with a loss of both alloantibody generation and priming of secondary cytokine responses ex vivo. When mice given treated transfusions were subsequently given untreated transfusions, they produced normal levels of alloantibodies but had reduced secondary cytokine responses ex vivo. This immune modulation was antigen specific and was dependent on the presence of WBCs in the treated product. CONCLUSIONS: UVB plus riboflavin treatment of WBC-enriched PRP effectively blocks alloimmunization and modulates immune responses to subsequent exposures.

Immune modulation and lack of alloimmunization following transfusion with pathogen-reduced platelets in mice.

FOND : Transfusion de produits sanguins allog&eacute;niques peut conduire &agrave; l&#39;allo-immunisation, ayant une incidence sur la r&eacute;ussite des transfusions ult&eacute;rieures et transplantation d&#39;organes solides. R&eacute;duction des germes pathog&egrave;nes &agrave; l&#39;aide de riboflavine et rayonnement ultraviolet B (UVB) s&#39;est av&eacute;r&eacute;e d&#39;&eacute;liminer l&#39;immunog&eacute;nicit&eacute; des globules blancs (leucocytes) in vitro par down r&eacute;gulation des mol&eacute;cules d&#39;adh&eacute;rence surface, bloquant efficacement la conjugaison de cellule-cellule et la pr&eacute;sentation directe. Nous avons cherch&eacute; &agrave; d&eacute;terminer si cette perte de l&#39;immunog&eacute;nicit&eacute; est &eacute;tendue in vivo o&ugrave; indirecte pr&eacute;sentation des antig&egrave;nes allog&eacute;niques peut se produire. CONCEPTION des &eacute;tudes et m&eacute;thodes : Les souris BALB/cJ ont &eacute;t&eacute; transfus&eacute;es avec ou sans traitement ou riboflavine et impr&eacute;gn&eacute;es d&#39;UVB C57Bl/6J de plasma riche en plaquettes (PRP) contenant des globules blancs. r&eacute;ponses de cytokine de spl&eacute;nocytes alloanticorps et &eacute;tendu en circulation ont &eacute;t&eacute; mesur&eacute;s. R&Eacute;SULTATS : R&eacute;duction des germes pathog&egrave;nes d&#39;allog&eacute;nique PRP WBC-enrichi &agrave; l&#39;aide de riboflavine et UVB l&eacute;ger avant la transfusion a emp&ecirc;ch&eacute; allo-immunisation, avec une perte de g&eacute;n&eacute;ration d&#39;alloanticorps fois et amor&ccedil;age des cytokines secondaire ex vivo. Lorsque les souris ayant re&ccedil;u des transfusions de trait&eacute;s ont &eacute;t&eacute; par la suite donn&eacute; des transfusions non trait&eacute;es, elles produisent des niveaux normaux d&#39;allo-anticorps mais avaient r&eacute;duit les cytokines secondaire ex vivo. Cette modulation du syst&egrave;me immunitaire &eacute;tait antig&egrave;ne sp&eacute;cifique et d&eacute;pend de la pr&eacute;sence de globules blancs dans le produit trait&eacute;. CONCLUSIONS : Traitement UVB plus riboflavine de PRP WBC enrichi efficacement bloque l&#39;allo-immunisation et module les r&eacute;ponses immunitaires aux expositions subs&eacute;quentes.

Modulation du syst&egrave;me immunitaire et l&#39;absence d&#39;allo-immunisation apr&egrave;s transfusion avec plaquettes pathog&egrave;ne-r&eacute;duit chez la souris.

SFONDO: Trasfusione di prodotti ematici allogenici pu&ograve; portare ad alloimmunizzazione, che incidono il successo delle successive trasfusioni e trapianti di organi solidi. Riduzione del patogeno mediante riboflavina e raggi ultravioletti B (UVB) ha dimostrato di eliminare l&#39;immunogenicit&agrave; delle cellule bianche del sangue (globuli bianchi) in vitro attraverso la regolazione delle molecole di adesione superficiale, bloccando efficacemente coniugazione cellula-cellula e presentazione diretta verso il basso. Abbiamo cercato di determinare se questa perdita di immunogenicit&agrave; &egrave; esteso in vivo dove presentazione indiretta degli antigeni allogenici pu&ograve; verificarsi. STUDIO di progettazione e metodi: Topi BALB/cJ sono stati trasfusi con o non trattata o riboflavina e UVB-trattati C57Bl/6J platelet-rich plasma (PRP) contenenti globuli bianchi. circolanti alloanticorpo e allospecific splenocyte cytokine risposte sono state misurate. RISULTATI: Riduzione del patogeno di allogenici PRP WBC-arricchito mediante riboflavina e UVB luce prima trasfusione impedito alloimmunizzazione, con una perdita di generazione alloanticorpo sia e innesco di cytokine secondaria risposte ex vivo. Quando topi dati trasfusioni trattati sono stati successivamente dato trasfusioni non trattati, hanno prodotto livelli normali di alloanticorpi ma aveva ridotto secondario cytokine risposte ex vivo. Questa modulazione immunitaria antigene specifico e fu dipendente dalla presenza di globuli bianchi nel prodotto trattato. CONCLUSIONI: Trattamento UVB e riboflavina di PRP arricchito WBC efficacemente blocca alloimmunizzazione e modula la risposta immunitaria ad esposizioni successive.

Modulazione immunitaria e mancanza di alloimmunizzazione seguito trasfusione con piastrine patogeno-ridotta nei topi.

Extracellular vesicles (EVs) are small, double membrane vesicles derived from leukocytes, platelets, and cells of other tissues under physiological or pathological conditions. Generation of EVs in stored blood is thought to be associated with adverse effects and potentially immunosuppression in blood transfusion recipients. We measured the quantity and cells of origin for EVs isolated from stored red blood cell (RBC) units and tested whether they had any effects on T-cell-mediated immune responses. Mixing peripheral blood mononuclear cells (PBMCs) with EVs resulted in secretion of proinflammatory cytokines and chemokines and increased survival of unstimulated PBMCs. EVs augmented mitogen-induced CD4(+) and CD8(+) T-cell proliferation in an antigen-presenting cell (APC)-dependent manner. We demonstrated that EVs interacted primarily with monocytes and induced proinflammatory cytokine secretion. We also showed that the exosome fraction of EVs and not larger microvesicles was responsible for induction of TNF-α production by monocytes. Furthermore, blockade of CD40 or CD40L accessory molecules largely neutralized the EV augmentation of T-cell responses, implying a role for cell-cell interaction between T cells and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC units increase the potency of APCs and boost mitogen-driven T-cell proliferative responses.

Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro.

More than a decade after West Nile virus (WNV) entered North America, and despite a significant increase in reported cases during the 2012 and 2013 seasons, no treatment or vaccine for humans is available. Although antiviral T cells contribute to the control of WNV, little is known about their regulation during acute infection. We analyzed the expression of Tim-3 and PD-1, two recently identified T cell negative immune checkpoint receptors, over the course of WNV infection. Symptomatic WNV+ donors exhibited higher frequencies of Tim-3+ cells than asymptomatic subjects within naïve/early differentiated CD28+/-CD57-CD4+ and differentiated CD28-CD57-CD8+ T cells. Our study links Tim-3-expression on T cells during acute WNV infection with the development of symptomatic disease, suggesting Tim-3 and its ligands could be targeted therapeutically to alter anti-WNV immunity and improve disease outcome.

Heather Inglis

Blood Systems Research Institute

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry