Browsing by Subject "Extracellular vesicles"

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Open Access
Characterization of functional and molecular properties of circulating extracellular vesicles of childhood idiopathic nephrotic syndrome patients
(2021-10) Eroğlu, Fehime Kara
Nephrotic syndrome (NS) is one of the most common causes of glomerular disease in children and is characterized by the triad of proteinuria, hypoalbuminemia, and edema. The major molecular event in the pathogenesis of NS is the disruption of the glomerular filtration barrier, which is primarily driven by podocyte injury. The most common clinical presentation of NS in children is steroid-sensitive nephrotic syndrome (SSNS), characterized by complete remission within 4 weeks of steroid therapy and no apparent glomerular change in the light microscopic evaluation of kidney biopsies, thereby named as Minimal Change Disease (MCD). Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and ingenuity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.
Open Access
Characterization of innate and adaptive immune responses of two rare primary immune deficiencies: CTPS1 and CD55
(2019-09) Kaya, Göksu Gökberk
Primary Immune deficiencies (PIDs) are disorders of immune system caused by mutated genes. There are approximately 350 different disorders and each day novel ones are being defined. They can be categorized based on part of immune system harboring mutation; that is, they can be divided into disorders of innate and adaptive immune system. Each of them represents itself distinctly. In that perspective, studies based on characterization of PIDs enable us to comprehend how immune system works. Herein, we characterized innate and adaptive immune responses of two rare immune deficiencies: CTPS1 and CD55 which are novel examples of disorders of adaptive and innate immune system, respectively. CTPS1 is an enzyme functioning in de novo synthesis of nucleotide, CTP. Defective CTPS1 enzyme impairs lymphocytes to proliferate, however, other aspects of this deficiency still remain elusive. Since patients are prone to viral infections, we first explored functionality of cytotoxic T-cells through assessing STAT1 phosphorylation levels and expression of activation markers. Even though flow cytometry analyses revealed that CTPS1 deficient CD8+ T-cells had normal phospho-STAT1 levels, degranulation marker confined to surface of CD8+ Tcells were found to be elevated. Next, we investigated CD4+ T-cells with cytokines that are crucial for differentiation and fate. We detected that patient CD4+ T-cells had low phospho-STAT3 and phospho-STAT5 levels. Then, we checked the cytokine production profiles of CD4+ T-cells. Data indicated that percentages of IL-17a and IL 10 secreting cells are reduced in patient whereas Th1 and Th2 signatures were similar to healthy controls. Moreover, IFN-a levels of PBMCs upon TLR3, TLR7 and TLR9 ligand stimulations were found to be similar to healthy responses. Notably, patient had slightly reduced TLR7 and IFI16-STING mediated type II IFN secretion. We further showed that CTPS1 PBMCs had normal IL-12 levels, implying that the reduction in IFN-g was not due to either dysfunction of innate immune cells or by aberrant APC function. Surprisingly, patient PBMCs had higher number of granulocytes and flow cytometry analyses revealed that these granulocytes were CD14- CD15+ low-density granulocytes. This prompted us to assess the NETotic tendencies of CTPS1 neutrophils and we observed via microscopic and spectrofluorometric investigations that they underwent spontaneous NETosis. In the second part of this study, we worked with CD55 deficient patient PBMCs. CD55 is a complement regulatory protein and it inhibits formation of C3-convertase in classical and alternative complement pathways. Thus, patients suffer from aberrant complement activation in its absence as well as severe bowel inflammation and recurrent infections along with nutrient loss leading to malnutrition and growth deprivation. Eculizumab therapy was initiated to these patients in order to neutralize their pathologic C5 levels. We attempted to investigate effect of CD55 deficiency on PRR-complement cross-talk, recurrent infections and checked the contribution of Eculizumab therapy to their immune status. PBMCs of 4 patients, i) before (BT) and ii) after (AT) a single dose of Eculizumab administration were isolated. BT PBMCs had significantly reduced IFN-a and IP-10 secretions upon endosomal (TLR3, TLR7 and TLR9) TLRs and nucleic acid sensors (STING, DAI, RIG-I & MAVS) stimulations. Moreover, single Eculizumab therapy did not alter this innate immune dysfunction. Furthermore, we assessed levels of TNF-a, IL-6 productions from PBMCs stimulated with same ligands and observed that IL-6 but not TNF-a was reduced after PRR stimulations. Next, immunomodulatory effects of CD55 EVs before and after Eculizumab therapy was sought. ELISA results demonstrated that AT EV incubation on CD55-/- PBMCs lead to reduced TNF-a, IL-1b and IFN-g production. Meanwhile, AT EVs increased IL-10 production from patient PBMCs. Lastly, we assessed cytokine levels after healthy PBMCs were incubated with BT and AT EVs and found that PBMCs that incubated with BT EVs had elevated levels of IP-10 cytokine. When taken together, progression of PIDs might have been contributed by extracellular vesicles.
Open Access
Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes
(American Physiological Society, 2021-11-09) Kara Eroğlu, Fehime; Yazar, Volkan; Guler, Ulku; Yıldırım, Muzaffer; Yıldırım, Tuğçe; Gungor, Tulin; Celikkaya, Evra; Karakaya, Deniz; Turay, Nilsu; Ciftci Dede, Eda; Korkusuz, Petek; Salih, Bekir; Bulbul, Mehmet; Gürsel, İhsan
Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.
Open Access
Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behçet's disease severity
(Taylor and Francis, 2017-02) Kahraman, T.; Gucluler, G.; Simsek, I.; Yagci, F. C.; Yildirim, M.; Ozen, C.; Dinc, A.; Gursel, M.; Ikromzoda, L.; Sutlu, T.; Gay, S.; Gursel, I.
Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis.
Open Access
Development of a combination vaccine against N. Meningitidis and SARS-CoV-2 and analysis of MIS-C plasma and extracellular vesicles in relation to disease severity in pediatric patients
(2024-12) Yıldırım, Tuğçe Canavar
Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). Serogroup B remains the leading cause of IMD, representing 62% of documented serogroup cases overall, and is the most prevalent across all age groups under 65. In Türkiye, Serogroups B and W are responsible for most cases, accounting for over 75%. IMD has a case fatality rate of 10%, and 10% to 20% of survivors experience lifelong, disabling complications. Coronavirus disease 2019 (COVID-19), caused by the highly contagious SARS-CoV-2 virus, has had a devastating impact globally, leading to over 7 million deaths. It has become the most significant global health crisis since the influenza pandemic in 1918. Since the introduction of the first COVID-19 vaccine in the U.S., it is estimated that over 18 million hospitalizations and 3 million deaths have been prevented. Like flu vaccines, COVID-19 vaccines are expected to be introduced seasonally. In this thesis, we aim to develop a combination vaccine for populations where the prevalence of both COVID-19 and meningitis presents significant health risks. Our previous data indicated that our OMV-based bivalent vaccine, targeting B and W serogroups of Neisseria meningitidis, generates a broader humoral response and exhibits strong bactericidal activity. Similarly, our lab has developed a virus-like particle-based SARS-CoV-2 vaccine incorporating all four virus structural proteins, eliciting both humoral and cell-mediated immunity. Herein, we combined these two platforms and assessed their respective protective potencies in mice. Data revealed that combining the OMV and VLP vaccines with Alum adjuvant produced the most robust anti-meningococcal and anti-SARS-CoV-2 responses. The second part of the study focused on uncovering the immune parameters present in the plasma of MIS-C patients and investigating the role of extracellular vesicles in influencing disease severity. The data revealed that plasma from MIS-C patients contained significantly elevated levels of proinflammatory cytokines, including TNF-α, IFN-γ, IL-6, and IL-17. We also investigated the pathological role of extracellular vesicles (EVs) in MIS-C. Our findings showed that patient-derived EVs induced significantly higher levels of IRF9 in the THP1-dual reporter cell line. Contrary to our expectations, however, we did not observe EV-specific NF-κB induction in the THP1 cells.
Open Access
The development of prophylactic and therapeutic vaccine using cell derived extracellular vesicles
(2022-08) Yıldırım, Muzaffer
The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded cell-line derived and tumor derived exosomes as a prophylactic and therapeutic carrier against cancer treatment. Exosomes with a size between 30 to 150 nm are small extracellular vesicles secreted by all types of mammalian cells. They mediate a novel mode of intercellular communication through their bioactive cargos such as lipids, nucleic acids, metabolites, and proteins, which can be delivered to the target cells. Exosomes have successfully served as immunotherapeutic nanocarriers in cancer treatment using their natural delivery capabilities. Furthermore, they are attractive as a delivery system because of their stability in circulation, biocompatibility, and low toxicity. In the first part of this thesis, we used exosomes as a nanocarrier system to develop cancer vaccines in a therapeutic murine melanoma cancer treatment. We show that lyophilization of exosomes together with the CpG ODN, model antigen OVA and lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution is successfully accomplished. We analyzed the effect of the lyophilization on a cell line-derived exosomes and we characterized the exosomes by using bead-based technique via flow cytometry, qNano, Scaning electron microscopy, and western blotting. We showed that lyophilization does not harm exosomes’ vesicular integrity and fundamental biological features. Furthermore, we tested the biodistribution and activating capacity of encapsulated exosomes in mouse PEC, mesenteric lymph node, and spleen cells. We found out that loaded exosomes are mostly taken up by antigen-presenting cells. Also, we showed that loading CpG ODN into exosomes significantly improves APC activation markers in macrophages, B cells, and DCs and induced significantly higher IFNγ production from mouse mLN and splenocytes. Finally, we tested the therapeutic utility of the CpG ODN, OVA and αGC encapsulating exosome in the B16F10-OVA melanoma tumor-bearing mice. We found out that therapeutic vaccination with triple (CpG ODN, OVA, and αGC) ligand encapsulating exosomes suppressed the progression of established melanoma tumors in mice. Moreover, our triple ligand loaded exosomes triggered Th-1 biased anti-IgG OVA immunity and converted immune cells in tumor microenvironment to the tumor-suppressing phenotype. In the second part of this thesis, we used tumor-derived exosomes (TEXs) as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells, TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). We showed that engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic breast cancer model, we found out that administration of 4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regressed tumor growth in 4T1 tumor-bearing mice. As a result, this work implicated that an exosome based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. The last part of this thesis, we studied the therapeutic potential of cell line-derived exosomes loaded with superparamagnetic iron oxide nanoparticles(SPION) and immunostimulatory ligands. We showed that loading SPION enhanced the in vitro delivery of exosomes within immune cells. Also, we found out that spleen cells incubated with exosomes encapsulating with SPION and CpG ODN induced significantly higher levels of IL-12 and IFNγ. Finally, we tested our exosomal vaccine candidate in human hepatocellular carcinoma tumor model in athymic mice. We showed that TLR3 and TLR9 ligands encapsulated with SPION loaded exosomes induced pronounced innate immune activation and regressed tumors and improve survival rate of treated mice.
Open Access
Immunotherapeutic action of extracellular vesicles and effects of TLR signaling to immune dysfunction of SCI patients
(2017-03) Güçlüler, Gözde
The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded exosomes as prophylactic or therapeutic carriers against disease treatment. Exosomes are secreted from all cells and could be purified from all bodily fluids; however, engineering of exosomes to carry specific ligands post-purification is a daunting task. Herein, we show that lyophilization of exosomes together with the biological cargo alone or in combination of CpG ODN motifs, model protein antigen ovalbumin or lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution successfully internalizes these cargos within exosomes. Furthermore, the bioactivity of the loaded ligand(s) surpasses the unloaded free ligand activities. When tested in vivo, exosome incorporated ligand(s) proved to be significantly effective against model tumors such as E.G7 thymoma or established melanoma models. The mechanism behind this elevated immune activity is the ability of exosomes to be delivered to target cells and boost immune antigen dependent immune activation. Our in vitro findings revealed that encapsulation of CpG ODN into exosomes enhances immunostimulatory activity of CpG ODN than free form as evidenced by superior levels of cytokines like IL6, IL12 and Type-I and II interferons. This magnified immune activity might be partly due the increased APC activation observed as elevated CD86/MHCII surface marker expression. Immunization of C57/Bl6 mice with exosomal CpG ODN plus OVA induced strong Th1-biased anti-OVA response. Following thymoma induction in naive and OVAimmunized animals, >85% of exosomal vaccine treated mice cleared tumors whereas almost all naive animals were positive for tumor. This data suggests that CpG ODN encapsulation into exosomes improve immunostimulatory activity, provide better anti-OVA immunity thereby contribute effective tumor clearance in mice. A second aim of this thesis was to establish that it is feasible to load exosomes with more than two ligands. Next, invariant natural killer T (iNKT) cell ligand αGC was included within exosomes as the third element next to OVA and CpG ODN. Initial in vivo studies revealed that exosomes containing αGC were significantly more potent in inducing antigen dependent immune responses in comparison to free form of CpG ODN, OVA and αGC. In therapeutic tumor vaccine model, two exosome injections (@d: 9 and d: 15) were done to B16-OVA tumor bearing animals and tumor regression was followed. Mice that had triple exosomal ligands significantly reduced tumors compared to mice treated with non-exosomal ligands. This study confirmed that exosomes with triple ligands could be effectively control established tumor development. In this thesis, the elucidation of the involvement of extracellular vesicles (EVs) on the pathogenesis of autoimmune/autoinflammatory diseases was studied. The underlying mechanism in BD pathogenesis is still unclear. We found that one of the human cathelicidin group members, antimicrobial peptide LL37 along with EVs were elevated in active BD patients` plasmas. Strikingly, majority of plasma LL37 was associated with circulating EVs. We found that there was a strong correlation between i) LL37 level, ii) EV #/ml plasma and iii) cytokine production. In the last part of this thesis, one of the possible mechanisms of immune dysfunction contributing to severe neurological deterioration of chronic spinal cord injured (SCI) patients was unearthed. We aimed to investigate whether there is a correlation between susceptibility to infections of chronic SCI patients within the context of impaired innate recognition of pathogen associated molecular patterns (PAMPs). Our data implicated that although there was no dysfunction of B cell, or CD4+ Treg activity, but sensing TLR7 and TLR9 ligands by monocytes and pDCs were ablated in patients with SCI, leading to lower IFNγ and IP10 production along with costimulatory molecule expression, that could explain the immunological dysfunction in patient with SCI contributing to persistent complications.
Open Access
Label-free identification of exosomes using raman spectroscopy and machine learning
(Wiley-VCH Verlag GmbH & Co. KGaA, 2023-01-15) Parlatan, U.; Ozen, M.O.; Kecoglu, I.; Koyuncu, B.; Torun, H.; Khalafkhany, D.; Loc, I.; Ogut, M.G.; Inci, Fatih; Akin, D.; Solaroglu, I.; Ozoren, N.; Unlu, M. B.; Demirci, U.
Exosomes, nano-sized extracellular vesicles (EVs) secreted from cells, carry various cargo molecules reflecting their cells of origin. As EV content, structure, and size are highly heterogeneous, their classification via cargo molecules by determining their origin is challenging. Here, a method is presented combining surface-enhanced Raman spectroscopy (SERS) with machine learning algorithms to employ the classification of EVs derived from five different cell lines to reveal their cellular origins. Using an artificial neural network algorithm, it is shown that the label-free Raman spectroscopy method's prediction ratio correlates with the ratio of HT-1080 exosomes in the mixture. This machine learning-assisted SERS method enables a new direction through label-free investigation of EV preparations by differentiating cancer cell-derived exosomes from those of healthy. This approach will potentially open up new avenues of research for early detection and monitoring of various diseases, including cancer.
Open Access
Mesenchymal stem cell derived extracellular vesicles: promising immunomodulators against autoimmune, autoinflammatory disorders and SARS-CoV-2 infection
(Scientific and Technical Research Council of Turkey, 2020) Bulut, Özlem; Gürsel, İhsan
Discovery of novel and broad-acting immunomodulators is of critical importance for the prevention and treatment of disorders occurring due to overexuberant immune responseincluding SARS-CoV-2 triggered cytokine storm leading to lung pathology and mortality during the ongoing viral pandemic. Mesenchymal stem/stromal cells (MSCs), highly regarded for their regenerative capacities, also possessesremarkable immunoregulatory functions affecting all types of innate and adaptive immune cells. Owing to that, MSCs have been heavily investigated in clinic for the treatment of autoimmune and inflammatory diseases along with transplant rejection. Extensive research in the last decaderevealed that MSCs carry out most of their functions through paracrine factors which are soluble mediators and extracellular vesicles (EVs). EVs, including exosomes and microvesicles, are an efficient way of intercellular communication due to their unique ability to carry biological messages such as transcription factors, growth factors, cytokines, mRNAs and miRNAs over long distances. EVs originate through direct budding of the cell membrane or the endosomal secretion pathway and they consist of the cytosolic and membrane components of their parent cell. Therefore, they are able to mimic the characteristics of the parent cell, affecting the target cells upon binding or internalization. EVs secreted by MSCs are emerging as a cell-free alternative to MSC-based therapies. MSC EVs are being tested in preclinical and clinical settings where they exhibit exceptional immunosuppressivecapacity. They regulate the migration, proliferation, activation and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory response. Being as effective immunomodulators as their parent cells, MSC EVs are also preferable over MSC-based therapies due to their lower risk of immunogenicity, tumorigenicity and overall superior safety. In this review, we present the outcomes of preclinical and clinical studies utilizing MSC EVs as therapeutic agents for the treatment of a wide variety of immunological disorders.
Open Access
Regulation of human monocyte differentiation into m1- and m2-like macrophages
(2016-05) Bayık, Defne
Myeloid-derived suppressor cells (MDSC) play a key role in down-regulating activated T and NK cells. MDSC are emerging as targets for cancer immunotherapy since they protect tumor cells from immune elimination. We previously showed that the TLR7/8 agonist R848 and the TLR2/1 dual agonist PAM3 had opposite effect on the maturation of human monocytic MDSC (mMDSC). While the former triggered them to differentiation in M1-like macrophages with pro-inflammatory/anti-tumoricidal capacity, the latter generated immunosuppressive M2-like macrophages. This work seeks to identify the soluble factors that regulate the differentiation of mMDSC into macrophages. Our studies reveal that TNFα and M-CSF are essential for mMDSC to mature into functional M1- and M2-like macrophages, respectively. IL-6 and IL-10 play secondary roles but when used in combination with TNFα or M-CSF exceed the effects of TLR agonists. Understanding the response of mMDSC to cytokines should help efforts to direct the mMDSC maturation to therapeutic benefit. The finding that PAM3 could induce human mMDSC to mature into M2-like macrophage triggered us to study the effect of this TLR agonist on other monocyte populations. Our findings reveal that PAM3 was unique among TLR agonists in generating M2-like macrophages. We compared the polarizing activity of PAM3 to that of M-CSF. PAM3 was slightly less efficient than M-CSF in driving maturation of HLA-DR+ monocytes based on phenotypic characterization and phagocytic ability. Yet macrophages generated by PAM3 or M-CSF were equally capable of suppressing T cell proliferation. Analysis of gene regulatory networks by microarray and subsequent validation of the pathways identified by using specific inhibitors defined the NF-κB – COX-2 axis as playing a primary role. However, PAM3 also induced monocyte differentiation via an IL-6-dependent pathway that was largely absent from M-CSF driven cultures. Our findings clarified the pathways by which immunosuppressive M2-like macrophage arise from human monocytes and identify PAM3 as a potential therapeutic modulator of monocyte differentiation in patients with autoimmune disease. Extracellular vesicles (EV) are a heterogeneous population of biological nanoscaled particles that serve as vectors to enhance intercellular communication. In addition to this physiological role evidence indicates that EV can be harnessed as therapeutic agents for cancer. The major limitation to EV-based therapeutics is their rapid clearance by the reticuloendothelial system (RES). To overcome this problem, we sought to reduce macrophage uptake of EV by blocking scavenger receptors. In vitro results using human and murine cells suggests that inhibiting class A scavenger receptors selectively impairs EV uptake by monocytes and macrophages. In vivo studies document reduced liver accumulation and enhanced plasma circulation of i.v. injected EV after such blockade. These findings provide a strategy for reducing EV uptake by the RES thereby increasing their targeting and activity.
Embargo
Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet
(Elsevier Ltd, 2023-12-28) Lucena, M. I.; Villanueva-Paz, M.; Alvarez-Alvarez, I.; Aithal, G. P; Bjornsson, E. S.; Cakan-Akdogan, G.; Cubero, F. J.; Esteves, F.; Falcon-Perez, J. M.; Fromenty, B.; Garcia-Ruiz, C.; Grove, J. I.; Konu, Özlen; Kranendonk, M.; Kullak-Ublick, G. A.; Miranda, J. P.; Remesal-Doblado, A.; Sancho-Bru, P.; Nelsons, L.; Andrade, R. J.; Daly, A. K.; Fernandez-Checa, J. C.
In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.
Open Access
Therapeutic and diagnostic applications of extracellular vesicles
(2016-03) Kahraman, Tamer
Extracellular vesicles (EV), consisting of exosomes and microvesicles, are secreted biological nanovesicles and assumed plethora of physiological functions ranging from transport of cargo, regulating distant cell communication, and altering immune response. Accumulating evidence suggests that extracellular vesicles may participate in disease pathogenesis of inflammatory diseases. Moreover, accumulating evidence suggests that EVs are promising nanocarrier capable of modulating immune response. This thesis aims to harness EVs in immunotherapeutic and diagnostic applications. Behçet’s Disease (BD) activity is manifested with sustained, over exuberant immune activation, yet the underlying mechanisms leading to active BD state is poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 and EVs are elevated in BD plasma. Our data suggested that majority of LL37 is associated with EVs. This association drives plasma EVs to immune cells, enhancing pathologic and sustained immune response, thereby leading to aggravating BD pathology. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. Findings of this study could be of clinical interest in the management of BD, implicating that LL37/EV association as one of the major contributors of BD pathogenesis and might be used as a diagnostic readout to stratify the severity of BD patients. EVs, more specifically exosomes, suggested as new tools for biomedical applications such as drug/vaccine carrier vesicles. However, efforts to engineer cells to express desired cargo in/on these secreted exosomes or induce physical complexation with candidate bioactive agents or even use of membrane-breaching techniques such as electroporation to load exosomes with desirable cargo showed limited in vivo performance. Here we developed a mild and simple technique enabling external loading of any type of desired bioactive molecule within exosomes at high yield. Using this approach, we exploited therapeutic potential of exosomes encapsulating CpG ODN together with a protein antigen as a vaccine cancer for preventive tumor therapy. CpG ODN loaded within exosomes displayed pronounced in-vitro activity as evidenced by up to 6-fold higher IL6 and IL12 secretion from splenocytes as well as increased IFNα secretion from pDCs. Exosomes protected CpG ODN from digestion by DNase-I up to 90%. In order to demonstrate improved in-vivo activity, exosomes co-encapsulating CpG ODN and ovalbumin were tested as a potential vaccine vector against EG-7 thymoma. Animals that received Exo(CpG ODN+OVA) vaccine led to a magnified and persistent Th1-biased antiOVA IgG responses that was sufficient to fully protect mice from EG-7 derived tumor challenge even after 24 weeks post-booster injection as opposed to free vaccine combination. Our results suggest that EVs could be of clinical interest in both prognosis and management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. In addition, our studies related with exosomes present a platform that opens a new avenue to personalized cell-free therapeutic intervention and could be developed to harbor other therapeutically important molecules ranging from plasmid to mRNA or si/miRNA for more effective therapeutic modality development in the clinic against debilitating diseases ranging from cancer to infectious diseases
Open Access
The Yin and Yang of exosome isolation methods: conventional practice, microfluidics, and commercial kits
(Elsevier Inc, 2022-01) Shirejini, Saeedreza Zeibi; İnci, Fatih
Exosomes are a subset of extracellular vesicles released from various cells, and they can be found in different bodily fluids. Exosomes have been utilized as biomarkers to diagnose many diseases and to monitor therapy efficiency as they represent the status and origin of the cell, which they are released from. Considering that they co-exist in bodily fluids with other types of particles, their isolation still remains challenging since conventional methods are time-consuming, user-dependent, and result in low isolation yield. This review summarizes the conventional strategies and microfluidic-based methods for exosome isolation along with their strengths and limitations. In particular, microfluidic devices emerge as a promising approach to tackle the existing limitations of conventional methods, and they provide unique features, such as operating with minute volume of samples and rapid process, in order to isolate exosomes with the high yield and the high purity, which make them unprecedented tools for molecular biology and clinical applications in exosome research. This review further elaborates on the existing microfluidic-based exosome isolation methods and denotes their benefits and drawbacks. Herein, we also introduce various commercially available platforms and kits for exosome isolation along with their working principles.