Didactical characteristics of Dutch websites about kidney transplantation targeted for kidney patients and living donors: An exploratory study.

Objective: The aim was to explore the origin, content topics, teaching modes (instruction, interaction, and assessment), and corresponding social-epistemological dimensions (choices in knowledge transfer vs. knowledge building, and individual vs. group learning) of web-based information on kidney transplantation targeted for patients and living donors. Methods: Dutch websites on kidney transplantation were retrieved using the search engine Google.nl. From 24 websites, 250 webpages were examined on origin, content topics, teaching modes, and corresponding social-epistemological dimensions. Results: The majority of the websites had a professional organization as origin (20/24). The number and distribution of content topics varied among the websites. Of the 16 different teaching modes found, 11 were instructional, 4 were interactional, and 1 assessment mode was found. The websites offered almost exclusively teaching modes on individual and passive learning, whereas group learning and interactive knowledge building was hardly encountered. Conclusion: The diversity in teaching modes and social-epistemological dimensions of Dutch websites on kidney transplantation targeted for patients and living donors is limited. The websites only provided a partial view on kidney transplantation; information regarding contact with others, e.g. support, was limited. A more balanced availability of teaching approaches and content topics is desirable to fit with the didactical goals to make well-considered health decisions.

Development and application of a massive open online course to deliver innovative transplant education.

Massive Open Online Courses (MOOCs) offer an entirely new course concept for delivering content and engaging learners. This method of teaching has huge potential for the field of transplant education. In this study we describe the development and implementation of the MOOC "Clinical Kidney, Pancreas and Islet Transplantation". Three and a half years after the introduction of the course, the learning demographics have been analyzed. The majority of learners were from Europe, North America and Asia. The course has been offered at several different stages of education at Leiden University Medical Center from undergraduate to continuous medical education. The level of engagement with the content was associated with the background and motivations of the learners. 74% had a bachelor's degree or higher. 48% of the undergraduate students participated in other content than instructed. Learners reported having liked the design of the course. Personal growth was the main motivation for 93% of worldwide learners. 69% considered the content of the MOOC to be relevant to their job. In general student's intentions focused more on reasons of personal growth, general interest, and relevance to school or degree program. Overall the integration of the MOOC in different settings of formal transplant education offered an added value to the on-campus program.

Topics, Delivery Modes, and Social-Epistemological Dimensions of Web-Based Information for Patients Undergoing Renal Transplant and Living Donors During the COVID-19 Pandemic: Content Analysis.

BACKGROUND: The COVID-19 pandemic has markedly affected renal transplant care. During this time of social distancing, limited in-person visits, and uncertainty, patients and donors are relying more than ever on telemedicine and web-based information. Several factors can influence patients' understanding of web-based information, such as delivery modes (instruction, interaction, and assessment) and social-epistemological dimensions (choices in interactive knowledge building). OBJECTIVE: The aim of this study was to systemically evaluate the content, delivery modes, and social-epistemological dimensions of web-based information on COVID-19 and renal transplantation at time of the pandemic. METHODS: Multiple keyword combinations were used to retrieve websites on COVID-19 and renal transplantation using the search engines Google.com and Google.nl. From 14 different websites, 30 webpages were examined to determine their organizational sources, topics, delivery modes, and social-epistemological dimensions. RESULTS: The variety of topics and delivery modes was limited. A total of 13 different delivery modes were encountered, of which 8 (62%) were instructional and 5 (38%) were interactional; no assessment delivery modes were observed. No website offered all available delivery modes. The majority of delivery modes (8/13, 62%) focused on individual and passive learning, whereas group learning and active construction of knowledge were rarely encountered. CONCLUSIONS: By taking interactive knowledge transfer into account, the educational quality of eHealth for transplant care could increase, especially in times of crisis when rapid knowledge transfer is needed.

The Importance of Dosing, Timing, and (in)Activation of Adipose Tissue-Derived Mesenchymal Stromal Cells on Their Immunomodulatory Effects.

Mesenchymal stromal cells (MSCs) are attractive candidates for immunomodulatory cell therapy. However, it remains unknown how far therapeutic efficacy and potency are dependent on the dosage and activity of the MSCs. We previously observed that infusion of MSCs leads to rapid and transient changes in cytokine expression in blood, lung, and liver. In the present study, increasing doses of syngeneic adipose tissue-derived MSCs were infused in healthy mice and systemic changes in G-CSF, IL6, IL-10, and CXCL5 were detected 2 h after administration of 3 × 105 MSCs per animal, but not at lower doses. In lung and liver tissue, dose-dependent effects of MSCs on cytokine mRNA expression levels were detected from doses as low as 3 × 103 MSCs. Infusion of secretome-deficient or IFNγ-activated MSCs in healthy mice had similar effects on systemic cytokine levels as control MSCs, suggesting that in vivo at least the initial systemic effect of MSC administration is independent of the level of activity of MSCs, but depends on the response of host cells to MSCs. The results of this study reveal a rapid dose-dependent effect of MSCs and stress the important role of host cells in MSC treatment. This knowledge contributes to the design of rational MSC trials and to the quest for clinical efficacy of MSC therapy.

The Effects of an IL-21 Receptor Antagonist on the Alloimmune Response in a Humanized Mouse Skin Transplant Model.

BACKGROUND: Interleukin 21 (IL-21) is involved in regulating the expansion and effector function of a broad range of leukocytes, including T cells and B cells. In transplantation, the exact role of IL-21 in the process of allograft rejection is unknown. To further explore this, the aim of this study is to test the effect of an IL-21 receptor (IL-21R) blocking antibody on the early phase of allograft rejection in a humanized skin transplantation model in mice reconstituted with human T and B cells. METHODS: Immunodeficient Balb/c IL2rγRag2 mice were transplanted with human skin followed by adoptive transfer of human allogeneic splenocytes. Control animals were treated with a phosphate buffered saline vehicle while the other group was treated with a humanized anti-IL-21R antibody (αIL-21R). RESULTS: In the phosphate buffered saline-treated animals, human skin allografts were infiltrated with lymphocytes and developed a thickened epidermis with increased expression of the inflammatory markers Keratin 17 (Ker17) and Ki67. In mice treated with αIL-21R, these signs of allograft reactivity were significantly reduced. Concordantly, STAT3 phosphorylation was inhibited in this group. Of note, treatment with αIL-21R attenuated the process of T and B cell reconstitution after adoptive cellular transfer. CONCLUSIONS: These findings demonstrate that blockade of IL-21 signaling can delay allograft rejection in a humanized skin transplantation model.

Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice.

Defects in Fas function correlate with susceptibility to systemic autoimmune diseases like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE). C57BL/6 lpr (B6/lpr) mice are used as an animal model of ALPS and develop a mild SLE phenotype. Involvement of interleukin-17A (IL-17A) has been suggested in both phenotypes. Since IL-17 receptor A is part of the signaling pathway of many IL-17 family members we investigated the role of IL-17 receptor signaling in disease development in mice with a B6/lpr background. B6/lpr mice were crossed with IL-17 receptor A deficient (IL-17RA KO) mice and followed over time for disease development. IL-17RA KO/lpr mice presented with significantly enhanced lymphoproliferation compared with B6/lpr mice, which was characterized by dramatic lymphadenomegaly/splenomegaly and increased lymphocyte numbers, expansion of double-negative (DN) T-cells and enhanced plasma cell formation. However, the SLE phenotype was not enhanced, as anti-nuclear antibody (ANA) titers and induction of glomerulonephritis were not different. In contrast, levels of High Mobility Group Box 1 (HMGB1) and anti-HMGB1 autoantibodies were significantly increased in IL-17RA KO/lpr mice compared to B6/lpr mice. These data show that lack of IL-17RA signaling aggravates the lymphoproliferative phenotype in B6/lpr mice but does not affect the SLE phenotype.

Immunomodulation By Therapeutic Mesenchymal Stromal Cells (MSC) Is Triggered Through Phagocytosis of MSC By Monocytic Cells.

Mesenchymal stem or stromal cells (MSC) are under investigation as a potential immunotherapy. MSC are usually administered via intravenous infusion, after which they are trapped in the lungs and die and disappear within a day. The fate of MSC after their disappearance from the lungs is unknown and it is unclear how MSC realize their immunomodulatory effects in their short lifespan. We examined immunological mechanisms determining the fate of infused MSC and the immunomodulatory response associated with it. Tracking viable and dead human umbilical cord MSC (ucMSC) in mice using Qtracker beads (contained in viable cells) and Hoechst33342 (staining all cells) revealed that viable ucMSC were present in the lungs immediately after infusion. Twenty-four hours later, the majority of ucMSC were dead and found in the lungs and liver where they were contained in monocytic cells of predominantly non-classical Ly6Clow phenotype. Monocytes containing ucMSC were also detected systemically. In vitro experiments confirmed that human CD14++ /CD16- classical monocytes polarized toward a non-classical CD14++ CD16+ CD206+ phenotype after phagocytosis of ucMSC and expressed programmed death ligand-1 and IL-10, while TNF-α was reduced. ucMSC-primed monocytes induced Foxp3+ regulatory T cell formation in mixed lymphocyte reactions. These results demonstrate that infused MSC are rapidly phagocytosed by monocytes, which subsequently migrate from the lungs to other body sites. Phagocytosis of ucMSC induces phenotypical and functional changes in monocytes, which subsequently modulate cells of the adaptive immune system. It can be concluded that monocytes play a crucial role in mediating, distributing, and transferring the immunomodulatory effect of MSC. Stem Cells 2018;36:602-615.

Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes.

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4+ and CD8+ T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14+CD16+ monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of anti-inflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders.

Inflammatory Conditions Dictate the Effect of Mesenchymal Stem or Stromal Cells on B Cell Function.

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43- selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC.

Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.

BACKGROUND: Mesenchymal stromal cells (MSC) possess immunomodulatory properties and low immunogenicity, both crucial properties for their development into an effective cellular immunotherapy. They have shown benefit in clinical trials targeting liver diseases; however the efficacy of MSC therapy will benefit from improvement of the immunomodulatory and immunogenic properties of MSC. METHODS: MSC derived from human umbilical cords (ucMSC) were treated for 3 days in vitro with various inflammatory factors, interleukins, vitamins and serum deprivation. Their immunogenicity and immunomodulatory capacity were examined by gene-expression analysis, surface-marker expressions, IDO activity, PGE2 secretion and inhibition of T cell proliferation and IFNγ production. Furthermore, their activation of NK cell cytotoxicity was investigated via CD107a expression on NK cells. The immunomodulatory capacity, biodistribution and survival of pre-treated ucMSC were investigated in a CCl4-induced liver disease mouse model. In addition, capacity of pre-treated MSC to ameliorate liver inflammation was examined in an ex vivo liver inflammation co-culture model. RESULTS: IFN-γ and a multiple cytokine cocktail (MC) consisting of IFN-γ, TGFß and retinoic acid upregulated the expression of immunomodulatory factor PD-L1 and IDO activity. Subsequently, both treatments enhanced the capacity of ucMSC to inhibit CD4 and CD8 T cell proliferation and IFN-γ production. The susceptibility of ucMSC for NK cell lysis was decreased by IFN-ß, TGFß and MC treatment. In vivo, no immunomodulation was observed by the ucMSC. Four hours after intravenous infusion in mice with CCl4-induced inflammatory liver injury, the majority of ucMSC were trapped in the lungs. Rapid clearance of ucMSC(VitB6), ucMSC(Starv + VitB6) and ucMSC(MC) and altered bio-distribution of ucMSC(TGFß) compared to untreated ucMSC was observed. In the ex vivo co-culture system with inflammatory liver slices ucMSC(MC) showed significantly enhanced modulatory capacity compared to untreated ucMSC. CONCLUSIONS: The present study demonstrates the responsiveness of ucMSC to in vitro optimisation treatment. The observed improvements in immunomodulatory capacity as well as immunogenicity after MC treatment may improve the efficacy of ucMSC as immunotherapy targeted towards liver inflammation.

Inactivated Mesenchymal Stem Cells Maintain Immunomodulatory Capacity.

Mesenchymal stem cells (MSC) are studied as a cell therapeutic agent for treatment of various immune diseases. However, therapy with living culture-expanded cells comes with safety concerns. Furthermore, development of effective MSC immunotherapy is hampered by lack of knowledge of the mechanisms of action and the therapeutic components of MSC. Such knowledge allows better identification of diseases that are responsive to MSC treatment, optimization of the MSC product, and development of therapy based on functional components of MSC. To close in on the components that carry the therapeutic immunomodulatory activity of MSC, we generated MSC that were unable to respond to inflammatory signals or secrete immunomodulatory factors, but preserved their cellular integrity [heat-inactivated MSC (HI-MSC)]. Secretome-deficient HI-MSC and control MSC showed the same biodistribution and persistence after infusion in mice with ischemic kidney injury. Both control and HI-MSC induced mild inflammatory responses in healthy mice and dramatic increases in interleukin-10, and reductions in interferon gamma levels in sepsis mice. In vitro experiments showed that opposite to control MSC, HI-MSC lacked the capability to suppress T-cell proliferation or induce regulatory B-cell formation. However, both HI-MSC and control MSC modulated monocyte function in response to lipopolysaccharides. The results of this study demonstrate that, in particular disease models, the immunomodulatory effect of MSC does not depend on their secretome or active cross-talk with immune cells, but on recognition of MSC by monocytic cells. These findings provide a new view on MSC-induced immunomodulation and help identify key components of the therapeutic effects of MSC.

Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.

Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture, and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In this study, we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed interdonor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2 h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and disease-specific conditions may shape MSC after administration.

Efficacy of immunotherapy with mesenchymal stem cells in man: a systematic review.

Mesenchymal stem cells (MSC) are widely studied for their immunomodulatory properties. Data from in vitro and pre-clinical models demonstrate that MSC suppress activated immune cells and ameliorate the severity of experimental immune disease. In complex human studies, the immunomodulatory efficacy of MSC therapy is not well established. We conducted a systematic review of clinical studies which used MSC with the purpose of immunomodulation and included at least 10 patients to investigate the efficacy of MSC therapy. Sixty-two studies comprising 10 different immune disorders were included in the analysis, of which 18 studies represented controlled trials. Although several of the studies reported an amelioration of disease severity, other studies failed to observe a beneficial effect of MSC. The low number of randomized controlled trials, small number of studies per disease category and limited immunological readout parameters made it difficult to draw a definitive conclusion on the efficacy of MSC immune therapy.

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells.

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 µm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.

The life and fate of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are present throughout the body and are thought to play a role in tissue regeneration and control of inflammation. MSC can be easily expanded in vitro and their potential as a therapeutic option for degenerative and inflammatory disease is therefore intensively investigated. Whilst it was initially thought that MSC would replace dysfunctional cells and migrate to sites of injury to interact with inflammatory cells, experimental evidence indicates that the majority of administered MSC get trapped in capillary networks and have a short life span. In this review, we discuss current knowledge on the migratory properties of endogenous and exogenous MSC and confer on how culture-induced modifications of MSC may affect these properties. Finally, we will discuss how, despite their limited survival, administered MSC can bring about their therapeutic effects.