Generation Of Extracellular Vesicles Research Articles

One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using "Photoclick" Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori.

Surface bioconjugation of antimicrobial peptides (AMP) onto nanoparticles (AMP-NP) is a complex, multistep, and time-consuming task. Herein, a microfluidic system for the one-pot production of AMP-NP was developed. Norbornene-modified chitosan was used for NP production (NorChit-NP), and thiolated-AMP was grafted on their surface via thiol-norbornene "photoclick" chemistry over exposure of two parallel UV LEDs. The MSI-78A was the AMP selected due to its high activity against a high priority (level 2) antibiotic-resistant gastric pathogen: Helicobacter pylori (H. pylori). AMP-NP (113 ± 43 nm; zeta potential 14.3 ± 7 mV) were stable in gastric settings without a cross-linker (up to 5 days in pH 1.2) and bactericidal against two highly pathogenic H. pylori strains (1011 NP/mL with 96 μg/mL MSI-78A). Eradication was faster for H. pylori 26695 (30 min) than for H. pylori J99 (24 h), which was explained by the lower minimum bactericidal concentration of soluble MSI-78A for H. pylori 26695 (32 μg/mL) than for H. pylori J99 (128 μg/mL). AMP-NP was bactericidal by inducing H. pylori cell membrane alterations, intracellular reorganization, generation of extracellular vesicles, and leakage of cytoplasmic contents (transmission electron microscopy). Moreover, NP were not cytotoxic against two gastric cell lines (AGS and MKN74, ATCC) at bactericidal concentrations. Overall, the designed microfluidic setup is a greener, simpler, and faster approach than the conventional methods to obtain AMP-NP. This technology can be further explored for the bioconjugation of other thiolated-compounds.

Pravastatin reduces plasma levels of extracellular vesicles in pregnancies at high risk of term preeclampsia.

Introduction: Elevated plasma levels of extracellular vesicles have been associated with impaired placentation, angiogenesis imbalance, intravascular inflammation, and endothelial dysfunction in women with preeclampsia, thus suggesting that circulating vesicles may be a good therapeutic target for the treatment of the disease. Recently, statins have been considered a potential treatment for the prevention of preeclampsia because of their pleiotropic effects, including the improvement of endothelial dysfunction and inhibition of inflammatory responses. However, the effects of these drugs on circulating vesicles concentration in women at risk of preeclampsia have not been established. Herein, we aimed to assess the effects of pravastatin on circulating extracellular vesicle generation in women at high risk of term preeclampsia. Methods: In a sample of 68 singleton pregnant women participating in the multicenter, double-blind, placebo-controlled STATIN trial (Nº EducraCT 2016-005206-19 ISRCTN), 35 women received a placebo and 33 women received a 20mg/day dose of pravastatin for approximately 3weeks (from 35 to 37weeks of gestation until delivery). Large extracellular vesicles were characterized and quantified by flow cytometry using annexin V and cell-specific antibodies directed against platelet, endothelial, leukocyte, and syncytiotrophoblast cell surface markers. Results: In women who received the placebo, a significant increase in the plasma levels of large extracellular vesicles from platelets (34%, p < 0.01), leukocytes (33%, p < 0.01), monocytes (60%, p < 0.01), endothelial cells (40%, p < 0.05), and syncytiotrophoblast cells (22%, p < 0.05) were observed. However, treatment with pravastatin significantly reduced the plasma levels of large extracellular vesicles from platelets (42%, p < 0.001), leukocytes (25%, p < 0.001), monocytes (61%, p < 0.001), endothelial cells (69%, p < 0.001), activated endothelial cells (55%, p < 0.001), and syncytiotrophoblast cells (44%, p < 0.001). Discussion: These results indicate that pravastatin reduces the levels of activated cell-derived membrane vesicles from the maternal vasculature, blood, and placental syncytiotrophoblast of women at high risk of term preeclampsia, suggesting that this statin may be beneficial in reducing endothelial dysfunction and pro-inflammatory and pro-coagulatory state characteristics of the disease.

The role of lysosomes in metabolic and autoimmune diseases.

Lysosomes are catabolic organelles that contribute to the degradation of intracellular constituents through autophagy and of extracellular components through endocytosis, phagocytosis and macropinocytosis. They also have roles in secretory mechanisms, the generation of extracellular vesicles and certain cell death pathways. These functions make lysosomes central organelles in cell homeostasis, metabolic regulation and responses to environment changes including nutrient stresses, endoplasmic reticulum stress and defects in proteostasis. Lysosomes also have important roles in inflammation, antigen presentation and the maintenance of long-lived immune cells. Their functions are tightly regulated by transcriptional modulation via TFEB and TFE3,as well as bymajor signalling pathways that lead to activation of mTORC1 and mTORC2, lysosome motility and fusion with other compartments. Lysosome dysfunction and alterations in autophagy processes have been identified in a wide variety of diseases, including autoimmune, metabolic and kidney diseases. Deregulation of autophagy can contribute to inflammation, and lysosomal defects in immune cells and/or kidney cells have been reported in inflammatory and autoimmune pathologies with kidney involvement. Defects in lysosomal activity have also been identified in several pathologies with disturbances in proteostasis, including autoimmune and metabolic diseases such as Parkinson disease, diabetes mellitus and lysosomal storage diseases. Targeting lysosomes is therefore a potential therapeutic strategy to regulate inflammation and metabolism in a variety of pathologies.

Live Imaging of Pyroptosis in Primary Murine Macrophages.

Inflammasomes are the ultimate weapon of the macrophage immune arsenal. Inflammasome signalling in macrophages triggers pyroptosis, a lytic cell death pathway that facilitates inflammation-driven pathogen clearance. Imaging-based approaches to investigating cell death have proven useful, revealing cellular remodelling events such as the generation of extracellular vesicles, and continuing to uncover important structural changes in cells involved in inflammatory signalling. Pyroptosis has proved extremely challenging to image, because its lytic nature is incompatible with many well-established imaging approaches employed for other, non-lytic pathways. The complexities of ectopically expressing fluorescent constructs in primary macrophages and the sensitivity of such proteins to drug-based probes compound this difficulty. We and others have demonstrated key differences in pyroptosis induced by canonical versus noncanonical inflammasomes that delineate functional differences between these signalling pathways. Here, we describe a live imaging approach to study and compare canonical versus noncanonical inflammasome signalling and pyroptotic architecture in primary murine macrophages.

SI: PAF: Topical application of gemcitabine generates microvesicle particles in human and murine skin.

Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Our recent studies demonstrated that exposure to pro-oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet-activating factor-receptor (PAFR), a G-protein coupled receptor present on various cell types, and acid sphingomyelinase (aSMase), an enzyme required for MVP biogenesis. Based upon this premise, we tested the hypothesis that topical application of gemcitabine will induce MVP generation in human and murine skin. Our ex vivo studies using human skin explants demonstrate that gemcitabine treatment results in MVP generation in a dose-dependent manner in a process blocked by PAFR antagonist and aSMase inhibitor. Importantly, gemcitabine-induced MVPs carry PAFR agonists. To confirm the mechanisms, we employed PAFR-expressing and deficient (Ptafr-/- ) mouse models as well as mice deficient in aSMase enzyme (Spmd1-/- ). Similar to the findings using pharmacologic tools, genetic-based approaches demonstrate that gemcitabine-induced MVP release in WT mice was blunted in Ptafr-/- and Spmd1-/- mice. These findings demonstrate a novel mechanism by which local chemotherapy can generate bioactive components as a bystander effect in a process that is dependent upon the PAFR-aSMase pathway.

588 Topical application of gemcitabine chemotherapy generates microvesicle particles in a platelet-activating factor-receptor- and acid sphingomyelinase-dependent manner

Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Given our ongoing studies that exposure to pro-oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet-activating factor-receptor (PAFR), a G-protein coupled receptor present on various cell types, and acid sphingomyelinase (ASMase), an enzyme required for MVP biogenesis.

A novel prothrombotic role of proprotein convertase subtilisin kexin 9: the generation of procoagulant extracellular vesicles by human mononuclear cells.

BackgroundProprotein convertase subtilisin kexin 9 (PCSK9) is a serin protease synthesized mainly in the liver that binds the receptor of low-density lipoprotein and promotes its degradation in lysosomes. PCSK9 is considered a promising target for the development of new therapies for the treatment of hypercholesterolemia and related cardiovascular diseases. Extracellular vesicles represent a heterogeneous population of vesicles, ranging in size between 0.05 and 1 μm involved in numerous pathophysiological processes, including blood coagulation. We investigated whether PCSK9 stimulation induces the release of procoagulant extracellular vesicles from human mononuclear cells (PBMCs) and THP-1 cells.Methods and resultsPBMCs and THP-1 cells were stimulated whit PCSK9, the generation of EV was assessed by the prothrombinase assay and by cytofluorimetric analysis. EV-associated tissue factor activity was assessed by a one-stage clotting assay. PCSK9 induced an increase in extracellular generation by PBMCs and THP-1 cells as well as an increase in extracellular vesicle-associated tissue factor. Pre-treatment with inhibitors of the toll like receptor, TLR4 (C34), and of NF-κB signaling (BAY 11-7082), downregulated PCSK9-induced extracellular vesicle generation and of extracellular- bound tissue factor. Similar effect was obtained by an anti-PCSK9 human-monoclonal antibody.ConclusionsPCSK9-mediated generation of procoagulant EV could contribute to increase the prothrombotic status in patients with cardiovascular diseases.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Monensin Enhanced Generation of Extracellular Vesicles as Transfersomes for Promoting Tumor Penetration of Pyropheophorbide-a from Fusogenic Liposome

The current state of antitumor nanomedicines is severely restricted by poor penetration in solid tumors. It is indicated that extracellular vesicles (EVs) secreted by tumor cells can mediate the intercellular transport of antitumor drug molecules in the tumor microenvironment. However, the inefficient generation of EVs inhibits the application of this approach. Herein, we construct an EV-mediated self-propelled liposome containing monensin as the EV secretion stimulant and photosensitizer pyropheophorbide-a (PPa) as a therapeutic agent. Monensin and PPa are first transferred to the tumor plasma membrane with the help of membrane fusogenic liposomes. By hitchhiking EVs secreted by the outer tumor cells, both drugs are layer-by-layer transferred into the deep region of a solid tumor. Particularly, monensin, serving as a sustainable booster, significantly amplifies the EV-mediated PPa penetration by stimulating EV production. Our results show that this endogenous EV-driven nanoplatform leads to deep tumor penetration and enhanced phototherapeutic efficacy.

Ultraviolet B Irradiation Alters the Level and miR Contents of Exosomes Released by Keratinocytes in Diabetic Condition.

Ultraviolet B (UVB) stimulates the generation of extracellular vesicles, which elicit systemic effects. Here, we studied whether UVB affects the release and microRNA (miR) content of keratinocyte exosomes (EXs) in diabetic conditions. In vitro, we examined the UVB effects on affecting EX release from keratinocyte HaCaT cells (HaCaT‐EX) pretreated with high glucose. HaCaT‐EX functions were evaluated on Schwann cells (SCs). In vivo, UVB‐induced miR change in skin EXs of diabetic db/db mice was analyzed. The miRs of interest were validated in HaCaT‐EXs. We found that: (1) UVB promoted HaCaT‐EX generation in dose‐ and time‐dependent manners; 100 and 1800 J m−2 of UVB had the most prominent effect and were selected as effective low‐ and high‐fluence UVB in vitro. (2) A total of 13 miRs were differentially expressed >3‐fold in skin EXs in UVB‐treated db/db mice; miR‐126 was the most up‐regulated by low‐fluence UVB. (3) Functional studies revealed that the SC viability was improved by low‐fluence UVB HaCaT‐EXs, while worsened by high‐fluence UVB HaCaT‐EXs. (4) MiR‐126 inhibitor attenuated the effects induced by low‐fluence UVB HaCaT‐EXs. Our data have demonstrated that low‐ and high‐fluence UVBs promote HaCaT‐EX generation but differentially affect exosomal miR levels and functions under diabetic conditions.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

New insights into B cells as antigen presenting cells

In addition to their role as antibody producing cells, B cells make a critical contribution to adaptive immune responses by functioning as professional antigen-presenting cells (APC). Distinctive features of B cells as APC include the expression of the B cell receptor (BCR) for antigen and regulated expression of HLA-DO. Here, we discuss recent progress in investigation of B cells as APC. We start with an update on the canonical MHC class II antigen presentation pathway in B cells and alternative pathways, including generation of extracellular vesicles. Turning to APC function, we highlight the roles of B cells as thymic APC, as APC for T follicular helper (TFH), as APC for CD4 memory T cells and as presenters of idiotypic BCR determinants. We also note recent examples that link B cell Ag-presentation to disease. Emerging evidence indicates that, in addition to unique features of B cells compared to other professional APC, there is appreciable heterogeneity among B cells, arising from, for example, B cell activation state or the microenvironment.

Cell-Released Magnetic Vesicles Capturing Metabolic Labeled Rare Circulating Tumor Cells Based on Bioorthogonal Chemistry.

Capture of circulating tumor cells (CTCs) with high efficiency and high purity holds great value for potential clinical applications. Besides the existing problems of contamination from blood cells and plasma proteins, unknown/down-regulated expression of targeting markers (e.g., antigen, receptor, etc.) of CTCs have questioned the reliability and general applicability of current CTCs capture methodologies based on immune/aptamer-affinity. Herein, a cell-engineered strategy is designed to break down such barriers by employing the cell metabolism as the leading force to solve key problems. Generally, through an extracellular vesicle generation way, the cell-released magnetic vesicles inherited parent cellular membrane characteristics are produced, and then functionalized with dibenzoazacyclooctyne to target and isolate the metabolic labeled rare CTCs. This strategy offers good reliability and broader possibilities to capture different types of tumor cells, as proven by the capture efficiency above 84% and 82% for A549 and HepG2 cell lines as well as an extremely low detection limitation of 5 cells. Moreover, it enabled high purity enrichment of CTCs from 1mL blood samples of tumor-bearing mice, only ≈5-757 white blood cells are non-specific caught, ignoring the potential phenotypic fluctuation associated with the cancer progression.

High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells

Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients.

High-efficiency unassisted transfection of platelets with naked double-stranded miRNAs modulates signal-activated translation and platelet function

We sought novel approaches to improve transfection efficiencies of microRNAs (miRNAs) in platelets, and to apply these approaches to investigate the roles of miRNAs in regulating signal-activated protein translation and functional effects. We found that ex vivo human platelets support gymnosis––-internalization of ectopic miRNAs following co-incubation in the absence of conventional transfection reagents or schemes---and subsequently incorporate transfected miRNA into ARGONAUTE2 (AGO2)-based RNA-induced silencing complexes (RISC). Thrombin/fibrinogen stimulation activated translation of miR-223-3p target SEPTIN2, which was suppressed by miR-223-3p transfection in an AGO2/RISC-dependent manner. Thrombin/fibrinogen-induced exosome and microvesicle generation was inhibited by miR-223-3p transfection, and this effect was reversed with a RISC inhibitor. Platelet gymnosis of naked miRNAs appeared to be mediated in part by endocytic pathways including clathrin-dependent and fluid-phase endocytosis and caveolae. These results demonstrate the ability of ex vivo platelets to internalize ectopic miRNAs by unassisted transfection, and utilize them to modulate signal-activated translation and platelet function. Our results identify new roles for miR-223-3p in extracellular vesicle generation in stimulated platelets. High-efficiency gymnotic transfection of miRNAs in ex vivo platelets may be a broadly useful tool for exploring molecular genetic regulation of platelet function.

Reduced deformability of stored red blood cells is associated with generation of extracellular vesicles

Throughout storage, red blood cells (RBCs) undergo detrimental changes in viability and their ability to effectively transport oxygen. RBC storage lesions are mediated, in part, by a progressive loss of cell deformability, and associated with the release of extracellular vesicles (EVs). Accumulation of EVs during the storage of RBCs correlates with a decrease in RBC surface area to volume ratio. Similarly, the loss of RBC-deformability is associated with loss of RBC surface area to volume ratio. In this study we thus tested whether loss of RBC-deformability is associated with increased RBC-EV production during blood storage.EVs obtained by differential centrifugation of stored RBCs (non-leukoreduced non-irradiated or leukoreduced γ-irradiated RBCs stored 35 or 28 days respectively) were enumerated by high-sensitivity flow cytometry. RBC deformability was quantified, using a cell-flow-properties-analyzer, by measuring the median cell elongation ratio (MER) and percentage of low and high deformable cells in the population (%, LDFC, and HDFC, respectively).The number of EVs was inversely correlated with the MER and positively correlated with the %LDFC with both measures showing highly significant logarithmic dependence with EV levels in stored RBCs. Considering how highly deformable cells did not correlate with EV formation as compared with low deformable RBCs we propose that the formation of EVs is a key factor leading to increased RBC-rigidity.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process.

Role of Mac-1 integrin in generation of extracellular vesicles with antibacterial capacity from neutrophilic granulocytes

ABSTRACT Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are “tailor-made” with different composition and functional properties depending on the environmental circumstances.

Plasmodium helical interspersed subtelomeric family—an enigmatic piece of the Plasmodium biology puzzle

Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family 'Plasmodium Helical Interspersed Subtelomeric' (PHIST) has gained attention for its significant roles in parasite biology. Here, we have collected and analysed available information on PHIST members to enhance understanding of their functions, varied localization and structure-function correlation. Functional diversity of PHIST proteins is highlighted by their involvement in PfEMP1 (Pf erythrocyte membrane protein 1) expression, trafficking and switching. This family also contributes to cytoadherence, gametocytogenesis, host cell modification and generation of extracellular vesicles. While the PHIST domain forms the hallmark of this family, existence and functions of additional domains (LyMP, TIGR01639) and the MEC motif underscores its diversity further. Since specific PHIST proteins seem to form pairs with PfEMP1 members, we have used in silico tools to predict such potential partners in Pf. This information and our analysis of structural data on a PHIST member provide important insights into their functioning. This review overall enables readers to view the PHIST family comprehensively, while highlighting key knowledge gaps in the field.

T Cell Microvilli: Sensors or Senders?

Communication between cells is essential for multicellular life. During cognate immune interactions, T cells communicate with antigen-presenting cells (APC) via direct cell–cell contact or the release of molecules and vesicles containing T cell messages. A wide variety of mechanisms have been reported and among them a process called “trogocytosis” has traditionally been thought to be the fastest way to directly transfer membrane portions containing intact proteins from one cell to another; however, the mechanism is unverified. Trogocytosis has been distinguished from the generation of extracellular vesicles (EVs), a term that encompasses exosomes and microvesicles, as EVs are released via a contact-independent manner and are suggested to potentially send molecular messages over a distance. However, some previous reports regarding EVs in T cells may be misleading in terms of explaining their cellular origins. In addition, there is little evidence on how EVs are generated from T cells in vivo and function to regulate complex immune responses. A recent work demonstrated that T cell microvilli—thin and finger-like membrane protrusions—are highly fragile and easily separated as membrane particles by trogocytosis, forming a new class of EVs. Surprisingly, released T cell microvilli-derived particles act as vectors, transmitting T cell messages to cognate APCs. This review focuses on how T cell microvilli vesicles are connected with immune regulation mechanisms discovered previously.