Late antibody-mediated rejection (ABMR) triggered by donor-specific antibodies (DSA) is a cardinal cause of kidney allograft dysfunction and loss. Diagnostic criteria for this rejection type are well established, but effective treatment remains a major challenge. Recent randomized controlled trials (RCT) have failed to demonstrate the efficacy of widely used therapies, such as rituximab plus intravenous immunoglobulin or proteasome inhibition (bortezomib), reinforcing a great need for new therapeutic concepts. One promising target in this context may be interleukin-6 (IL-6), a pleiotropic cytokine known to play an important role in inflammation and adaptive immunity.

Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency.

Peritonitis remains a major cause of morbidity and mortality during chronic peritoneal dialysis (PD). Glucose-based PD fluids reduce immunological defenses in the peritoneal cavity. Low concentrations of peritoneal extracellular glutamine during PD may contribute to this immune deficit. For these reasons we have developed a clinical assay to measure the function of the immune-competent cells in PD effluent from PD patients. We then applied this assay to test the impact on peritoneal immune-competence of PD fluid supplementation with alanyl-glutamine (AlaGln) in 6 patients in an open-label, randomized, crossover pilot trial (EudraCT 2012-004004-36), and related the functional results to transcriptome changes in PD effluent cells. Ex-vivo stimulation of PD effluent peritoneal cells increased release of interleukin (IL) 6 and tumor necrosis factor (TNF) α. Both IL-6 and TNF-α were lower at 1 h than at 4 h of the peritoneal equilibration test but the reductions in cytokine release were attenuated in AlaGln-supplemented samples. AlaGln-supplemented samples exhibited priming of IL-6-related pathways and downregulation of TNF-α upstream elements. Results from measurement of cytokine release and transcriptome analysis in this pilot clinical study support the conclusion that suppression of PD effluent cell immune function in human subjects by standard PD fluid is attenuated by AlaGln supplementation.

Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

Extracorporeal circulation during major cardiac surgery triggers a systemic inflammatory response affecting the clinical course and outcome. Recently, extracellular vesicle (EV) research has shed light onto a novel cellular communication network during inflammation. Hemoadsorption (HA) systems have shown divergent results in modulating the systemic inflammatory response during cardiopulmonary bypass (CPB) surgery. To date, the effect of HA on circulating microvesicles (MVs) in patients undergoing CPB surgery is unknown.

The gut microbiota plays a pivotal role in the conversion of dietary flavonoids, which can affect their bioavailability and bioactivity and thereby their health-promoting properties. The ability of flavonoids to metabolically-activate the microbiota has, however, not been systematically evaluated. In the present study, we used a fluorescence-based single-cell activity measure [biorthogonal non-canonical ammino acid-tagging (BONCAT)] combined with fluorescence activated cell sorting (FACS) to determine which microorganisms are metabolically-active after amendment of the flavonoid rutin. We performed anaerobic incubations of human fecal microbiota amended with rutin and in the presence of the cellular activity marker L-azidohomoalanine (AHA) to detect metabolically-active cells. We found that 7.3% of cells in the gut microbiota were active after a 6 h incubation and 26.9% after 24 h. We then sorted BONCAT-positive cells and observed an enrichment of Lachnospiraceae (Lachnoclostridium and Eisenbergiella), Enterobacteriaceae, Tannerellaceae, and Erysipelotrichaceae species in the rutin-responsive fraction of the microbiota. There was marked inter-individual variability in the appearance of rutin conversion products after incubation with rutin. Consistent with this, there was substantial variability in the abundance of rutin-responsive microbiota among different individuals. Specifically, we observed that Enterobacteriaceae were associated with conversion of rutin into quercetin-3-glucoside (Q-glc) and Lachnospiraceae were associated with quercetin (Q) production. This suggests that individual microbiotas differ in their ability to metabolize rutin and utilize different conversion pathways.

The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity.

Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification-ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and-most importantly-with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.

A replication defective influenza A vaccine virus (delNS1 virus) was developed. Its attenuation is due to potent stimulation of the innate immune system by the virus. Since the innate immune system can also target cancer cells, we reasoned that delNS1 virus induced immune-stimulation should also lead to the induction of innate cytotoxic effects towards cancer cells.

The p21-activated kinases (PAKs) are key nodes in oncogenic signalling pathways controlling growth, survival, and motility of cancer cells. Their activity is increased in many human cancers and is associated with poor prognosis. To date, PAK deregulation has mainly been studied in solid tumours, where PAK1 and PAK4 are the main isoforms deregulated. We show that PAK1 and PAK2 are the critical isoforms in a BCR/ABL1+ haematopoietic malignancy. In suspension, leukaemic cells deficient for PAK1 and PAK2 undergo apoptosis, while the loss of either protein is well tolerated. Transfer of medium conditioned by shPAK2- but not shPAK1-expressing leukaemic cells interferes with endothelial cell growth. We found that leukaemic cells produce exosomes containing PAK2. Transfer of isolated exosomes supports endothelial cell proliferation. In parallel, we found that leukaemic cells explicitly require PAK2 to grow towards an extracellular matrix. PAK2-deficient cells fail to form colonies in methylcellulose and to induce lymphomas in vivo. PAK2 might therefore be the critical isoform in leukaemic cells by controlling tumour growth in a dual manner: vascularization via exosome-mediated transfer to endothelial cells and remodelling of the extracellular matrix. This finding suggests that the PAK2 isoform represents a promising target for the treatment of haematological diseases.

Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA.

The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.

The product of the ecotropic virus integration site 1 (EVI1) gene, whose overexpression is associated with a poor prognosis in myeloid leukemias and some epithelial tumors, regulates gene transcription both through direct DNA binding and through modulation of the activity of other sequence specific transcription factors. Previous results from our laboratory have shown that EVI1 influenced transcription regulation in response to the myeloid differentiation inducing agent, all-trans retinoic acid (ATRA), in a dual manner: it enhanced ATRA induced transcription of the RARβ gene, but repressed the ATRA induction of the EVI1 gene itself. In the present study, we asked whether EVI1 would modulate the ATRA regulation of a larger number of genes, as well as biological responses to this agent, in human myeloid cells. U937 and HL-60 cells ectopically expressing EVI1 through retroviral transduction were subjected to microarray based gene expression analysis, and to assays measuring cellular proliferation, differentiation, and apoptosis. These experiments showed that EVI1 modulated the ATRA response of several dozens of genes, and in fact reinforced it in the vast majority of cases. A particularly strong synergy between EVI1 and ATRA was observed for GDF15, which codes for a member of the TGF-β superfamily of cytokines. In line with the gene expression results, EVI1 enhanced cell cycle arrest, differentiation, and apoptosis in response to ATRA, and knockdown of GDF15 counteracted some of these effects. The potential clinical implications of these findings are discussed.

Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplaninhigh and podoplaninlow cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed differences in genes specific for signaling and vascular tissue. Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis in vivo.

Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Pancreatic islets of Langerhans contain several specialized endocrine cell types, which are commonly identified by the expression of single marker genes. However, the established marker genes cannot capture the complete spectrum of cellular heterogeneity in human pancreatic islets, and existing bulk transcriptome datasets provide averages across several cell populations. To dissect the cellular composition of the human pancreatic islet and to establish transcriptomes for all major cell types, we performed single-cell RNA sequencing on 70 cells sorted from human primary tissue. We used this dataset to validate previously described marker genes at the single-cell level and to identify specifically expressed transcription factors for all islet cell subtypes. All data are available for browsing and download, thus establishing a useful resource of single-cell expression profiles for endocrine cells in human pancreatic islets.

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14++CD16+) monocytes in whole blood.

Chronic inflammation impairs insulin secretion and sensitivity. β-cell dedifferentiation has recently been proposed as a mechanism underlying β-cell failure in T2D. Yet the effect of inflammation on β-cell identity in T2D has not been studied. Therefore, we investigated whether pro-inflammatory cytokines induce β-cell dedifferentiation and whether anti-inflammatory treatments improve insulin secretion via β-cell redifferentiation. We observed that IL-1β, IL-6 and TNFα promote β-cell dedifferentiation in cultured human and mouse islets, with IL-1β being the most potent one of them. In particular, β-cell identity maintaining transcription factor Foxo1 was downregulated upon IL-1β exposure. In vivo, anti-IL-1β, anti-TNFα or NF-kB inhibiting sodium salicylate treatment improved insulin secretion of isolated islets. However, only TNFα antagonism partially prevented the loss of β-cell identity gene expression. Finally, the combination of IL-1β and TNFα antagonism improved insulin secretion of ex vivo isolated islets in a synergistic manner. Thus, while inflammation triggered β-cell dedifferentiation and dysfunction in vitro, this mechanism seems to be only partly responsible for the observed in vivo improvements in insulin secretion.

Human monocytes include CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical) subsets with divergent roles in immune regulation and inflammation. Since the functional characterization of monocyte subsets is most commonly performed using isolated monocytes, we investigated the influence of different monocyte isolation protocols on the relative abundance of monocyte subsets. Using flow cytometric subset characterization directly in whole blood as a reference, we found that monocyte isolation by enrichment of peripheral blood mononuclear cells and subsequent depletion of non-monocytes by magnetic labeling did not alter the distribution of monocyte subsets. Particularly, we failed to detect a loss of CD16+ subsets upon monocyte isolation, although one of the negative depletion protocols used contained an anti-CD16 antibody to label granulocytes. Overnight storage of isolated monocytes induced a significant repartition of monocyte subsets towards CD14++CD16+ intermediate monocytes, which was barely seen in stored whole blood. We identified intermediate monocytes as main binding partners of platelet-derived extracellular vesicles (EVs) and propose that residual platelets contained in isolated monocyte preparations release EVs that induce the expression of the IgG receptor FcγRIII (CD16) on monocytes.

As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.