Embryonic Stem Cells

Publications

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DISCOVERY AND VALIDATION OF A URINARY EXOSOME MRNA SIGNATURE FOR THE DIAGNOSIS OF HUMAN KIDNEY TRANSPLANT REJECTION
Background: The traditional biomarkers currently used to monitor a kidney allograft for rejection are late markers of injury and they lack sensitivity and specificity. Allograft biopsies on the other hand, are invasive and costly. The authors describe the discovery and validation of two urinary exosomal mRNA multigene signatures for the diagnosis of acute T cell–mediated and antibody-mediated rejection and chronic, active antibody-mediated rejection in recipients of kidney transplant. Using a clinically validated platform for exosome isolation and analysis, they demonstrated the high stability of urinary exosomes and the reliability of this approach in monitoring patients for allograft rejection. One gene signature for all-cause rejection and another for discriminating T cell–mediated rejection from antibody-mediated rejection showed high predictive performances and offer clinicians the possibility of new tools for monitoring emergence of rejection in kidney allografts.

Rania El Fekih, James Hurley, Vasisht Tadigotla, Areej Alghamdi, Anand Srivastava, Christine Coticchia, John Choi, Hazim Allos, Karim Yatim, Juliano Alhaddad, Siawosh Eskandari, Philip Chu, Albana B. Mihali, Isadora T. Lape, Mauricio P. Lima Filho, Bruno T. Aoyama, Anil Chandraker, Kassem Safa, James F. Markmann, Leonardo V. Riella, Richard N. Formica, Johan Skog and Jamil R. Azzi

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CyTOF-Enabled Analysis Identifies Class-Switched B Cells as the Main Lymphocyte Subset Associated With Disease Relapse in Children With Idiopathic Nephrotic Syndrome
Background: B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid–dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.

Miguel Fribourg1†, Michela Cioni2†, GianMarco Ghiggeri2, Chiara Cantarelli1,3, Jeremy S. Leventhal4, Kelly Budge1, Sofia Bin1, Leonardo V. Riella5, Manuela Colucci6, Marina Vivarelli7, Andrea Angeletti2, Laura Perin8‡ and Paolo Cravedi1*‡

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A Comprehensive Phenotypic and Functional Immune Analysis Unravels Circulating Anti-Phospholipase A2 Receptor Antibody Secreting Cells in Membranous Nephropathy Patients
Background: Primary membranous nephropathy (MN) is characterized by the presence of antipodocyte antibodies, but studies describing phenotypic and functional abnormalities in circulating lymphocytes are limited. After adjusting for multiple testing, plasma cells and regulatory B cells (BREG) were significantly higher (P < 0.05) in MN patients compared with both control groups. The percentages of circulating plasma cells correlated with serum anti-PLA2R antibody levels (P = 0.042) and were associated with disease activity. Ex vivo-expanded PLA2R-specific IgG-producing plasmablasts generated from circulating PLA2R-specific memory B cells (mBCs) correlated with serum anti-PLA2R IgG antibodies (P < 0.001) in MN patients. Tumor necrosis factor-α (TNF-α) was the only significantly increased cytokine in MN patients (P < 0.05), whereas there was no significant difference across study groups in the autoantibody and antiviral antibody repertoire.

Chiara Cantarelli 1 2Marta Jarque 3Andrea Angeletti 4Joaquin Manrique 5Susan Hartzell 1Timothy O'Donnell 6Elliot Merritt 6Uri Laserson 6Laura Perin 7Chiara Donadei 4Lisa Anderson 1Clara Fischman 1Emilie Chan 1Juliana Draibe 3Xavier Fulladosa 3Joan Torras 3Leonardo V Riella 8 9Gaetano La Manna 4Enrico Fiaccadori 2Umberto Maggiore 2Oriol Bestard 3Paolo Cravedi 1

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REGULATORY T CELLS ENGINEERED WITH TCR SIGNALING–RESPONSIVE IL-2 NANOGELS SUPPRESS ALLOIMMUNITY IN SITES OF ANTIGEN ENCOUNTER
Background: Adoptive cell transfer of ex vivo expanded regulatory T cells (Tregs) has shown immense potential in animal models of auto- and alloimmunity. However, the effective translation of such Treg therapies to the clinic has been slow. Because Treg homeostasis is known to require continuous T cell receptor (TCR) ligation and exogenous interleukin-2 (IL-2), some investigators have explored the use of low-dose IL-2 injections to increase endogenous Treg responses. Systemic IL-2 immunotherapy, however, can also lead to the activation of cytotoxic T lymphocytes and natural killer cells, causing adverse therapeutic outcomes. Here, we describe a drug delivery platform, which can be engineered to autostimulate Tregs with IL-2 in response to TCR-dependent activation, and thus activate these cells in sites of antigen encounter. To this end, protein nanogels (NGs) were synthesized with cleavable bis(N-hydroxysuccinimide) cross-linkers and IL-2/Fc fusion (IL-2) proteins to form particles that release IL-2 under reducing conditions, as found at the surface of T cells receiving stimulation through the TCR. Tregs surface-conjugated with IL-2 NGs were found to have preferential, allograft-protective effects relative to unmodified Tregs or Tregs stimulated with systemic IL-2. We demonstrate that murine and human NG–modified Tregs carrying an IL-2 cargo perform better than conventional Tregs in suppressing alloimmunity in murine and humanized mouse allotransplantation models. In all, the technology presented in this study has the potential to improve Treg transfer therapy by enabling the regulated spatiotemporal provision of IL-2 to antigen-primed Tregs.
Siawosh K EskandariIna SulkajMariane B MeloNa LiHazim AllosJuliano B AlhaddadBranislav KollarThiago J BorgesArach S EskandariMax A ZinterSongjie CaiJean Pierre AssakerJohn Y ChoiBasmah S Al DulaijanAmr MansouriYousef HaikBakhos A TannousWillem J van SonHenri G D LeuveninkBohdan PomahacLeonardo V Riella , Li TangMarc A J SeelenDarrell J IrvineJamil R Azzi

Science translational medicine vol. 12,569 (2020): eaaw4744. doi:10.1126/scitranslmed.aaw4744

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HUMAN REGULATORY T CELLS UNDERGO SELF-INFLICTED DAMAGE VIA GRANZYME PATHWAYS UPON ACTIVATION
Background: Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show – by using cytometry by time of flight (CYTOF) – an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non-GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.

Esilida Sula Karreci, Siawosh K. Eskandari, Farokh Dotiwala, Sujit K. Routray, Ahmed T. Kurdi, Jean Pierre Assaker, Pavlo Luckyanchykov, Albana B. Mihali, Omar Maarouf, Thiago J. Borges, Abdullah Alkhudhayri, Kruti R. Patel, Amr Radwan, Irene Ghobrial, Martina McGrath, Anil Chandraker, Leonardo V. Riella, Wassim Elyaman, Reza Abdi, Judy Lieberman, and Jamil Azzi1

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INTEGRATED KIDNEY EXOSOME ANALYSIS FOR THE DETECTION OF KIDNEY TRANSPLANT REJECTION

Background: Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Jongmin Park, Hsing-Ying Lin, Jean Pierre Assaker, Sangmoo Jeong, Chen-Han Huang, A Kurdi, Kyungheon Lee, Kyle Fraser, Changwook Min, Siawosh Eskandari, Sujit Routray, Bakhos Tannous, Reza Abdi, Leonardo Riella, Anil Chandraker, Cesar M Castro, Ralph Weissleder, Hakho Lee, Jamil R Azzi 

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MICRONEEDLE-BASED LOCAL DELIVERY OF CCL22 AND IL-2 ENRICHES TREG HOMING TO THE SKIN ALLOGRAFT AND ENABLES TEMPORAL MONITORING OF IMMUNOTHERAPY EFFICACY
Background: Skin allografts only serve as temporary dressing for patients suffering major burns due to their high immunogenicity and rejection by the immune system, requiring systemic immunosuppressive therapies that lead to deleterious side effects. Microneedle arrays composed of hyaluronic acid (HA) and placed on skin allografts can locally deliver immunomodulators and simultaneously sample immune cells in interstitial fluid to monitor the response to the therapy. The cells can be retrieved from the microneedles for downstream analysis by degrading the HA using a reducing agent. Using an allogeneic skin transplantation model, it is shown that the microneedle-mediated local delivery of the chemokine CCL22 (to attract Tregs) and the cytokine IL-2 (to promote their expansion) increases the local immune suppression in the allograft. Moreover, immune cell population in the allograft correlates with that seen in the microneedles. The delivery and sampling functions of the microneedle arrays can help regulate the immune system locally, without inducing systemic immune suppression, and facilitate the monitoring of the response to the therapy following skin transplantation.
Núria Puigmal, Pere Dosta, Zhabiz Solhjou, Karim Yatim, Cynthia Ramírez, John Y. Choi, Juliano B. Alhaddad, Ana Paula Cosme, Jamil Azzi,* and Natalie Artzi*

Adv. Funct. Mater. 2021, 31, 2100128. https://doi.org/10.1002/adfm.202100128

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DONOR MYELOID DERIVED SUPPRESSOR CELLS (MDSCS) PROLONG ALLOGENEIC CARDIAC GRAFT SURVIVAL THROUGH PROGRAMMING OF RECIPIENT MYELOID CELLS IN VIVO
Background: Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2Kb, I-Ab) recipients pre-treated with BALB/c MDSCs were transplanted with either donor-type (BALB/c, H2Kd, I-Ad) or third-party (C3H, H2Kk, I-Ak) cardiac grafts. Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b+Gr1+ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b+Gr1+ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient’s endogenous MDSCs.

Songjie CaiJohn Y. ChoiThiago J. BorgesHengcheng ZhangJi MiaoTakaharu IchimuraXiaofei LiSimiao XuPhilip ChuSiawosh K. EskandariHazim AllosJuliano B. AlhaddadSaif A. MuhsinKarim YatimLeonardo V. RiellaPeter T. SageAnil K. Chandraker & Jamil R. Azzi 

Sci Rep 10, 14249 (2020). https://doi.org/10.1038/s41598-020-71289-z

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REGULATORY CD8 T CELLS THAT RECOGNIZE QA-1 EXPRESSED BY CD4 T-HELPER CELLS INHIBIT REJECTION OF HEART ALLOGRAFTS
Background: Induction of longstanding immunologic tolerance is essential for the survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.

John Y ChoiSiawosh K EskandariSongjie CaiIna SulkajJean Pierre AssakerHazim AllosJuliano AlHaddadSaif A MuhsinEman AlhussainAmr MansouriMelissa Y YeungMarc A J SeelenHye-Jung KimHarvey CantorJamil R Azzi

Proc Natl Acad Sci U S A. 2020 Mar 17;117(11):6042-6046. doi: 10.1073/pnas.1918950117.

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SWITCHING RENAL TRANSPLANT RECIPIENTS TO BELATACEPT THERAPY: RESULTS OF A REAL-LIFE GRADUAL CONVERSION PROTOCOL
Background: Conversion to belatacept immunosuppression is a therapeutic option for renal-transplant recipients with calcineurin inhibitors (CNI) toxicity, but it associates with high risk of acute rejection. Gradual conversion and serial immune monitoring with urinary chemokine CXCL9 may allow increasing safety of this maneuver. We converted kidney transplant recipients with signs of toxicity to CNI or other immunosuppressive drugs to belatacept over a 2-month period. We monitored renal function, metabolic profile, and circulating lymphocyte subsets. We also quantified urinary CXCL9 over a 12-month follow-up period. Between September 2016 and March 2017, 35 patients were successfully switched to belatacept immunosuppression at 3.3 (1.3-7.2) years after transplant. Two patients had a reversible rise in serum creatinine, associated with acute rejection in one case. Urinary CXCL9 increased before serum creatinine. After conversion, blood pressure and HbA1c significantly declined while eGFR and proteinuria remained stable. The percentage of circulating effector T cells and memory B cells significantly declined. Conversion from CNI to belatacept, in this setting, was feasible and safe, provided it was performed over a 2-month time-period. Monitoring urinary CXCL9 may further increase safety through earlier identification of patients at risk for acute rejection. The procedure associates with improved blood pressure, metabolic profile, and reduced circulating effector T and B cells.

Paolo MalvezziClara FischmanGuillaume RigaultMarie Christine JacobTatiana RaskovalovaThomas JouveBénédicte Janbon , Lionel Rostaing , Paolo Cravedi 

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Extracellular vesicles derived from patients with antibody-mediated rejection induce tubular senescence and endothelial to mesenchymaltransition in renal cells
Background: Extracellular vesicles (EV) are emerging mediators in several diseases. However, their role in the pathophysiology of antibody-mediated allograft rejection (AMR) has been poorly investigated. Here, we investigated the role of EV isolated from AMR patients in inducing tubular senescence and endothelial to mesenchymal transition (EndMT) and analyzed their miRNA expression profile. By multiplex bead flow cytometry, we characterized the immunophenotype of plasma AMR-derived EV and found a prevalent platelet and endothelial cell origin. In vitro, AMR-derived EV induced tubular senescence by upregulating SA-β Gal and CDKN1A mRNA. Furthermore, AMR-derived EV induced EndMT. The occurrence of tubular senescence and EndMT was confirmed by analysis of renal biopsies from the same AMR patients. Moreover, AMR-derived EV induced C3 gene upregulation and CFH downregulation in tubular epithelial cells, with C4d deposition on endothelial cells. Interestingly, RNase-mediated digestion of EV cargo completely abrogated tubular senescence and EndMT. By microarray analysis, miR-604, miR-515-3p, miR-let-7d-5p, and miR-590-3p were significantly upregulated in EV from AMR group compared with transplant controls, whereas miR-24-3p and miR-29a-3p were downregulated. Therefore, EV-associated miRNA could act as active player in AMR pathogenesis, unraveling potential mechanisms of accelerated graft senescence, complement activation and early fibrosis that might lead to new therapeutic intervention
Franzin R, Stasi A, Sallustio F, Bruno S, Merlotti G, Quaglia M, Grandaliano G, Pontrelli P, Thurman JM, Camussi G, Stallone G, Cantaluppi V, Gesualdo L, Castellano G.
Am J Transplant. 2022 May 18. doi: 10.1111/ajt.17097.
Epub ahead of print. PMID: 35583104

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Stem Cell-Derived Extracellular Vesicles as Potential Therapeutic Approach for Acute Kidney Injury
Background: Acute kidney injury is a frequent complication of hospitalized patients and significantly increases morbidity and mortality, worsening costs and length of hospital stay. Despite this impact on healthcare system, treatment still remains only supportive (dialysis). Stem cell-derived extracellular vesicles are a promising option as they recapitulate stem cells properties, overcoming safety issues related to risks or rejection or aberrant differentiation. A growing body of evidence based on pre-clinical studies suggests that extracellular vesicles may be effective to treat acute kidney injury and to limit fibrosis through direct interference with pathogenic mechanisms of vascular and tubular epithelial cell damage. We herein analyze the state-of-the-art knowledge of therapeutic approaches with stem cell-derived extracellular vesicles for different forms of acute kidney injury (toxic, ischemic or septic) dissecting their cytoprotective, regenerative and immunomodulatory properties. We also analyze the potential impact of extracellular vesicles on the mechanisms of transition from acute kidney injury to chronic kidney disease, with a focus on the pivotal role of the inhibition of complement cascade in this setting. Despite some technical limits, nowadays the development of therapies based on stem cell-derived extracellular vesicles holds promise as a new frontier to limit acute kidney injury onset and progression.

Quaglia M, Merlotti G, Colombatto A, Bruno S, Stasi A, Franzin R, Castellano G, Grossini E, Fanelli V, Cantaluppi V.
Front Immunol. 2022 Mar 10;13:849891. doi: 10.3389/fimmu.2022.849891. PMID: 35359949; PMCID: PMC8960117.

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Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury
Background: Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.

Medica D, Franzin R, Stasi A, Castellano G, Migliori M, Panichi V, Figliolini F, Gesualdo L, Camussi G, Cantaluppi V.
Cells. 2021 Jul 2;10(7):1675. doi: 10.3390/cells10071675. PMID: 34359843; PMCID: PMC8304261.

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Emerging biomarkers of delayed graft function in kidney transplantation
Background: Delayed Graft Function (DGF) is one of the most common early complications in kidney transplantation, associated with poor graft outcomes, prolonged post-operative hospitalization and higher rejection rates. Given the severe shortage of high-quality organs for transplantation, DGF incidence is expected to raise in the next years because of the use of nonstandard kidneys from Extended Criteria Donors (ECD) and from Donors after Circulatory Death (DCD). Alongside conventional methods for the evaluation of renal allograft [e.g. serum creatinine Glomerular Filtration Rate (GFR), needle biopsy], recent advancements in omics technologies, including proteomics, metabolomics and transcriptomics, may allow to discover novel biomarkers associated with DGF occurrence, in order to identify early preclinical signs of renal dysfunction and to improve the quality of graft management. Here, we gather contributions from basic scientists and clinical researchers to describe new omics studies in renal transplantation, reporting the emerging biomarkers of DGF that may implement and improve conventional approaches.

Mezzolla V, Pontrelli P, Fiorentino M, Stasi A, Pesce F, Franzin R, Rascio F, Grandaliano G, Stallone G, Infante B, Gesualdo L, Castellano G
Transplant Rev (Orlando). 2021 Dec;35(4):100629. doi: 10.1016/j.trre.2021.100629. Epub 2021 May 26. PMID: 34118742.

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Extracellular Vesicles as Mediators of Cellular Crosstalk Between Immune System and Kidney Graft
Background: Extracellular vesicles (EVs) are known immune-modulators exerting a critical role in kidney transplantation (KT). EV bioactive cargo includes graft antigens, costimulatory/inhibitory molecules, cytokines, growth factors, and functional microRNAs (miRNAs) that may modulate expression of recipient cell genes. As paracrine factors, neutrophil- and macrophage-derived EVs exert immunosuppressive and immune-stimulating effects on dendritic cells, respectively. Dendritic cell-derived EVs mediate alloantigen spreading and modulate antigen presentation to T lymphocytes. At systemic level, EVs exert pleiotropic effects on complement and coagulation. Depending on their biogenesis, they can amplify complement activation or shed complement inhibitors and prevent cell lysis. Likewise, endothelial- and platelet-derived EVs can exert procoagulant/prothrombotic effects and also promote endothelial survival and angiogenesis after ischemic injury. Kidney endothelial- and tubular-derived EVs play a key role in ischemia-reperfusion injury (IRI) and during the healing process; additionally, they can trigger rejection by inducing both alloimmune and autoimmune responses. Endothelial EVs have procoagulant/pro-inflammatory effects and can release sequestered self-antigens, generating a tissue-specific autoimmunity. Renal tubule-derived EVs shuttle pro-fibrotic mediators (TGF-β and miR-21) to interstitial fibroblasts and modulate neutrophil and T-lymphocyte influx. These processes can lead to peritubular capillary rarefaction and interstitial fibrosis-tubular atrophy. Different EVs, including those from mesenchymal stromal cells (MSCs), have been employed as a therapeutic tool in experimental models of rejection and IRI. These particles protect tubular and endothelial cells (by inhibition of apoptosis and inflammation-fibrogenesis or by inducing autophagy) and stimulate tissue regeneration (by triggering angiogenesis, cell proliferation, and migration). Finally, urinary and serum EVs represent potential biomarkers for delayed graft function (DGF) and acute rejection. In conclusion, EVs sustain an intricate crosstalk between graft tissue and innate/adaptive immune systems. EVs play a major role in allorecognition, IRI, autoimmunity, and alloimmunity and are promising as biomarkers and therapeutic tools in KT.

Quaglia M, Dellepiane S, Guglielmetti G, Merlotti G, Castellano G, Cantaluppi V.
Front Immunol. 2020 Feb 27;11:74. doi: 10.3389/fimmu.2020.00074. PMID: 32180768; PMCID: PMC7057849

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Targeting Premature Renal Aging: from Molecular Mechanisms of Cellular Senescence to Senolytic Trials
Background: The biological process of renal aging is characterized by progressive structural and functional deterioration of the kidney leading to end-stage renal disease, requiring renal replacement therapy. Since the discovery of pivotal mechanisms of senescence such as cell cycle arrest, apoptosis inhibition, and the development of a senescence-associated secretory phenotype (SASP), efforts in the understanding of how senescent cells participate in renal physiological and pathological aging have grown exponentially. This has been encouraged by both preclinical studies in animal models with senescent cell clearance or genetic depletion as well as due to evidence coming from the clinical oncologic experience. This review considers the molecular mechanism and pathways that trigger premature renal aging from mitochondrial dysfunction, epigenetic modifications to autophagy, DNA damage repair (DDR), and the involvement of extracellular vesicles. We also discuss the different pharmaceutical approaches to selectively target senescent cells (namely, senolytics) or the development of systemic SASP (called senomorphics) in basic models of CKD and clinical trials. Finally, an overview will be provided on the potential opportunities for their use in renal transplantation during ex vivo machine perfusion to improve the quality of the graft.

 Franzin R, Stasi A, Ranieri E, Netti GS, Cantaluppi V, Gesualdo L, Stallone G, Castellano G
Front Pharmacol. 2021 Apr 29;12:630419. doi: 10.3389/fphar.2021.630419. PMID: 33995028; PMCID: PMC8117359.

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Recent Advances on Biomarkers of Early and Late Kidney Graft Dysfunction
Background: New biomarkers of early and late graft dysfunction are needed in renal transplant to improve management of complications and prolong graft survival. A wide range of potential diagnostic and prognostic biomarkers, measured in different biological fluids (serum, plasma, urine) and in renal tissues, have been proposed for post-transplant delayed graft function (DGF), acute rejection (AR), and chronic allograft dysfunction (CAD). This review investigates old and new potential biomarkers for each of these clinical domains, seeking to underline their limits and strengths. OMICs technology has allowed identifying many candidate biomarkers, providing diagnostic and prognostic information at very early stages of pathological processes, such as AR. Donor-derived cell-free DNA (ddcfDNA) and extracellular vesicles (EVs) are further promising tools. Although most of these biomarkers still need to be validated in multiple independent cohorts and standardized, they are paving the way for substantial advances, such as the possibility of accurately predicting risk of DGF before graft is implanted, of making a "molecular" diagnosis of subclinical rejection even before histological lesions develop, or of dissecting etiology of CAD. Identification of "immunoquiescent" or even tolerant patients to guide minimization of immunosuppressive therapy is another area of active research. The parallel progress in imaging techniques, bioinformatics, and artificial intelligence (AI) is helping to fully exploit the wealth of information provided by biomarkers, leading to improved disease nosology of old entities such as transplant glomerulopathy. Prospective studies are needed to assess whether introduction of these new sets of biomarkers into clinical practice could actually reduce the need for renal biopsy, integrate traditional tools, and ultimately improve graft survival compared to current management.

Quaglia M, Merlotti G, Guglielmetti G, Castellano G, Cantaluppi VInt J Mol
Sci. 2020 Jul 29;21(15):5404. doi: 10.3390/ijms21155404. PMID: 32751357; PMCID: PMC7432796