Abstract Background Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. Aim of the study To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. Methods Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. Results Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. Conclusion Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

A novel developed 4D bioprinting technique is used for the manufacturing of human-scale, small-diameter vascular grafts. Accordingly, a bio-ink is synthesized from a hybrid molecule containing sodium alginate (SA) and collagen peptide (COP). Endothelial progenitor cells (EPC) isolated from human whole blood are integrated into the bioartificial vascular graft as an autologous cell source. Likewise, human umbilical vein endothelial cells (HUVEC) are used as experimental standard. The evolving vascular grafts are printed by a customized 4D bioprinter into CaCl2 support medium for rapid cross-linking inducing the temporospatial shaping of the grafts. After culturing for 21 days, histological and ultrastructural analyses of the bioartificial vascular grafts reveal a well-organized matrix with imbedded EPC or HUVEC. Live-3D-cell-imaging and cell viability assays demonstrate a multitude of vital and metabolically active cells. Biomechanics of the grafts are proven to be comparable to human saphenous veins. Coagulation analysis reveals low thrombogenicity and high functional integrity of the vascular grafts. Surgical implantation of the grafts in a perfused cadaver model can be performed effectively and without technical issues. Finally, the current study describes for the first time the 4D bioprinting and characterization of a small-diameter, human-scale vascular graft for putative clinical translation. 4D bioprinting enabled the manufacturing of small-diameter vascular grafts. Accordingly, a novel bio-ink is synthesized containing a hybrid molecule from Sodium Alginate (SA) and Collagen Peptide (COP) and combined with Endothelial Progenitor Cells (EPC) or Human Umbilical Vein Endothelial Cells (HUVEC) respectively. After culturing in the bioreactor for 21 days, the grafts showed characteristics similar to human veins. image

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EV Mreg ) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EV Mreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EV Mreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EV Mreg . Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EV Mreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B–positive cells ( P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 10 6 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B–positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EV Mreg ( P < 0.05 for 3.2 × 10 6 L-EV Mreg /ml). A differential analysis of the effects of Mreg and L-EV Mreg on CD4 + and CD8 + T-cells showed an inhibition of CD4 + T-cells by Mreg ( P < 0.01) and L-EV Mreg ( P < 0.05 for 1.6 × 10 6 L-EV Mreg /ml; P < 0.01 for 3.2 × 10 6 L-EV Mreg /ml). A moderate inhibition of CD8 + T-cells was observed by Mreg ( P < 0.05) and by L-EV Mreg ( P < 0.01 for 1.6 × 10 6 L-EV Mreg /ml and 3.2 × 10 6 L-EV Mreg /ml). PS was restricted to confined regions of the Mreg surface, while L-EV Mreg showed strong signals for PS in the exoplasmic leaflet. L-EV Mreg attenuate CD3/CD28-mediated activation of CD4 + and CD8 + T-cells. L-EV Mreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. Key messages Mreg release large extracellular vesicles (L-EV Mreg ) with an average size of 7.5 µm L-EV Mreg exhibit phosphatidylserine positivity L-EV Mreg suppress CD4 + and CD8 + T-cells L-EV Mreg hold clinical potential in T-cell-related diseases

Background Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function. Aim of this study To characterize the nature of Mreg-derived L-EV (L-EV Mreg ) and to gain insights into their role in wound healing and angiogenesis. Methods Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EV Mreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EV Mreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EV Mreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. Results Mreg and L-EV Mreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EV Mreg regarding their surface marker composition. However, compared to Mreg fewer L-EV Mreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EV Mreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EV Mreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05). Conclusion L-EV Mreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EV Mreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.

Abstract Background Numerous tissue-derived factors have been postulated to be involved in tissue migration of circulating monocytes. The aim of this study was to evaluate whether a defined hypoxic gradient can induce directed migration of naïve human monocytes and to identify responsible autocrine/paracrine factors. Methods Monocytes were isolated from peripheral blood mononuclear cells, transferred into chemotaxis chambers and subjected to a defined oxygen gradient with or without the addition of CCL26. Cell migration was recorded and secretome analyses were performed. Results Cell migration recordings revealed directed migration of monocytes towards the source of hypoxia. Analysis of the monocyte secretome demonstrated a reduced secretion of 70% (19/27) of the analyzed cytokines under hypoxic conditions. The most down-regulated factors were CCL26 (− 99%), CCL1 (− 95%), CX3CL1 (− 95%), CCL17 (− 85%) and XCL1 (− 83%). Administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26. Conclusions Hypoxia induces directed migration of human monocytes in-vitro. Autocrine/paracrine released CCL26 is involved in the hypoxia-mediated monocyte migration and may represent a target molecule for the modulation of monocyte migration in-vivo.

Objectives The sequence of initial tissue ischaemia and consecutive blood flow restoration leads to ischaemia/reperfusion (I/R) injury, which is typically characterized by a specific inflammatory response. Migrating monocytes seem to mediate the immune response in ischaemic tissues and influence detrimental as well as regenerative effects during I/R injury. Materials and Methods To clarify the role of classical monocytes in I/R injury, isolated human monocytes were subjected to I/R in vitro (3 hours ischaemia followed by 24 hours of reperfusion). Cellular resilience, monocyte differentiation, cytokine secretion, as well as influence on endothelial tube formation, migration and cell recovery were investigated. Results We show that I/R supported an enhanced resilience of monocytes and induced intracellular phosphorylation of the prosurvival molecules Erk1/2 and Akt. FACS analysis showed no major alteration in monocyte subtype differentiation and surface marker expression under I/R. Further, our experiments revealed that I/R changes the cytokine secretion pattern, release of angiogenesis associated proteins and MMP-9 activity in supernatants of monocytes exposed to I/R. Supernatants from monocytes subjected to I/R attenuated endothelial tube formation as indicator for angiogenesis as well as endothelial cell migration and recovery. Conclusion In summary, monocytes showed no significant change in cellular integrity and monocyte subtype after I/R. Functionally, monocytes might have a rather detrimental influence during the initial phase of I/R, suppressing endothelial cell migration and neoangiogenesis.

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N = 14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p < 0.001) while C-MYC and KLF4 were downregulated (p < 0.001 and p < 0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1β, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

Backround Employing growth factor-induced partial reprogramming in vitro, peripheral human blood monocytes can acquire a state of plasticity along with expression of various markers of pluripotency. These so-called programmable cells of monocytic origin (PCMO) hold great promise in regenerative therapies. The aim of this translational study was to explore and exploit the functional properties of PCMO for allogeneic cell transplantation therapy in critical limb ischemia (CLI). Methods Using our previously described differentiation protocol, murine and human monocytes were differentiated into PCMO. We examined paracrine secretion of pro-angiogenic and tissue recovery-associated proteins under hypoxia and induction of angiogenesis by PCMO in vitro. Allogeneic cell transplantation of PCMO was performed in a hind limb ischemia mouse model in comparison to cell transplantation of native monocytes and a placebo group. Moreover, we analyzed retrospectively four healing attempts with PCMO in patients with peripheral artery disease (PAD; Rutherford classification, stage 5 and 6). Statistical analysis was performed by using one-way ANOVA, Tukey’s test or the Student’s t test, p < 0.05. Results Cell culture experiments revealed good resilience of PCMO under hypoxia, enhanced paracrine release of pro-angiogenic and tissue recovery-associated proteins and induction of angiogenesis in vitro by PCMO. Animal experiments demonstrated significantly enhanced SO 2 saturation, blood flow, neoangiogenesis and tissue recovery after treatment with PCMO compared to treatment with native monocytes and placebo. Finally, first therapeutic application of PCMO in humans demonstrated increased vascular collaterals and improved wound healing in patients with chronic CLI without exaggerated immune response, malignant processes or extended infection after 12 months. In all patients minor and/or major amputations of the lower extremity could be avoided. Conclusions In summary, PCMO improve angiogenesis and tissue recovery in chronic ischemic muscle and first clinical results promise to provide an effective and safe treatment of CLI.

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+-induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs relies on multiple non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-β, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct-pathway Tregs.

Following a several-day incubation in medium containing IL-3 and M-CSF to generate a more plastic intermediate "reprogrammed multipotent cells of monocytic origin (RMCMO)," peripheral blood mononuclear cells (PBMCs) can be efficiently converted to hepatocyte-like cells (neohepatocytes) and insulin-producing cells. However, continuous efforts are devoted to enhancing the proliferative capacity of these multipotent cells while maintaining or further increasing their redifferentiation potential. In the present work, PBMCs were transfected with one pluripotency gene (SOX2) and the resulting RMCMO compared to standard RMCMO with respect to cell viability, proliferative activity, and redifferentiation potential. Ectopic SOX2 expression increased the number of viable RMCMO, activated cell cycle genes, and enhanced proliferation as shown by quantitative RT-PCR and Ki67 immunofluorescent staining, respectively. Redifferentiation of RMCMO derived from SOX2-transfected PBMCs to neohepatocytes was more complete in comparison to control cells as revealed by higher urea and glucose secretion, increased activity of cytochrome P450 isoforms, and a phase II enzyme, while the same was true for insulin-producing cells as assessed by the expression of INS, PDX1, and GLUT2 and glucose-stimulated insulin secretion. Our results indicate that SOX2 transfection increases both multipotency and proliferation of RMCMO, eventually allowing production of neohepatocytes and insulin-producing cells of higher quality and quantity for transplantation purposes.

Transferring immunoregulatory cells from a tolerant donor to nontolerant recipient as a means of establishing tolerance is a well-known technique in experimental immunology, but its clinical application is only now receiving serious attention.1 Several immunoregulatory cell-based products are currently being investigated as adjunct immunosuppressive agents in early-phase clinical trials in solid organ transplantation. One particularly promising candidate cell type is the regulatory macrophage (Mreg) cell. The Mreg cell represents a unique state of macrophage polarisation, which is distinguished from other activation states by a robust phenotype and potent T cell suppressor function.2 Human Mreg cells suppress mitogen-stimulated T cell proliferation in vitro through IFN-γ–induced indoleamine 2,3-dioxygenase activity, as well as contact-dependent deletion of activated T cells. In addition, Mreg cells drive the development of activated induced regulatory T cells that, in turn, suppress the proliferation and activity of effector T cells. Human Mreg cells derive from CD14+ peripheral blood monocytes when cultured in the presence of M-colony–stimulating factor and high concentrations of heat-inactivated human serum for more than 4 days before stimulation with IFN-γ. A proprietory Good Manufacturing Practice–compliant process for manufacturing a therapeutic product, known as Mreg_UKR, containing human Mreg cells has been established at a commercial pharmaceutical manufacturing facility in Germany.3 Mreg_UKR is currently being investigated in a phase I/II trial as a means of promoting immune regulation in kidney transplant recipients to facilitate safe minimization of maintenance immunosuppression (clinicaltrials.gov: NCT02085629). Partway into this study, manufacturing failure occurred in 3 runs from different clinical trial participants. These failures, which were characterised by low cell yield and above specification expression of CD80, could neither be attributed to procedural errors or “drift,” nor to donor-related factors. Therefore, a root cause analysis was undertaken, including a full characterisation of the current and previous lots of human serum. Serum for Mreg_UKR manufacture is sourced from a commercial provider in Germany, which supplies recalcified plasma-derived sera pooled from at least 20 male donors of blood group AB. Male-only serum is preferred to female serum because it is theoretically less likely to contain anti-HLA antibodies, which may be elicited by pregnancy. Before use in Mreg_UKR culture, human serum is heat-inactivated to destroy complement and then frozen to prevent degradation. Through the process development phase and clinical Mreg_UKR production runs, 3 separate charges of serum (designated I, II, and III) were used. No prominent differences were identified in the biochemical composition of the 3 serum lots (data not shown). Under research laboratory conditions, all 3 sera were capable of supporting the development of Mreg cells from monocytes isolated from 4 randomly selected, healthy donors of known HLA type; in particular, cell yield and CD80−/low expression did not vary significantly between Mreg cells grown in the 3 sera. To test whether presence of donor-specific anti-HLA antibodies could explain idiosyncratic process failures, the 3 heat-inactivated sera were screened using single-antigen bead arrays. Surprisingly, all 3 serum lots contained HLA class I– and class II–reactive antibodies at above-threshold levels (Suppl. 1, SDC,https://links.lww.com/TP/B393). Considering these antibodies were diluted by pooling of sera, it seems that 1 or more of the plasma donors were truly sensitised (Table 1). Testing a second, independent sample of AB serum charge III confirmed the results of the first single-antigen bead array screening (Suppl. 2, SDC,https://links.lww.com/TP/B393). To test the hypothesis that bead arrays had returned false-positive results owing to process-related artefacts, samples of AB serum charge III were screened before and after preparatory steps (ie, thawing, heat-inactivation and refreezing) made by the contract manufacturer; however, no effect of was processing identified (Suppl. 2, SDC,https://links.lww.com/TP/B393).TABLE 1: Summary of Mreg_UKR manufacturing runs under research laboratory and clean room conditionsHypothetically, anti-HLA antibodies could affect Mreg cell development by opsonising the cells, leading to activation of other Fc receptor–bearing monocytes, or by inappropriately activating monocytes by retrograde signal transduction through MHC molecules themselves.4 However, in 5 separate production runs under research or clean room conditions, Mreg cells were found to develop normally in the presence of monocyte donor-specific antibodies, in so far as Mreg cell yield and phenotype were within specification. In another instance, a product failed yield and CD80 specifications despite the donor not expressing any identified target antigens. Although anti-HLA antibodies present in AB serum were not detrimental to Mreg cell growth and development, we were concerned that Mreg cells generated in the presence of donor-specific antibodies may be opsonized at the time of harvest. The effects of antibody binding to leucocytes in circulation are well known: opsonized cells are susceptible to complement-mediated lysis, killing by NK cells or destruction by phagocytes; hence, the presence of anti-HLA antibody during cell production must be considered as a possible cause of treatment failure.5 To test whether anti-HLA antibodies present in AB serum were capable of fixing complement, an accredited diagnostic laboratory performed mixed and B cell crossmatches using cells from a DR4-positive donor and serum charge III (Suppl. 2, SDC,https://links.lww.com/TP/B393). Despite the presence of DR4-specific antibody detected by single-antigen bead array, no complement-dependent cytotoxicity was observed. A low concentration of anti-HLA antibody in our AB serum is the most likely explanation for this discrepancy, although we have not formally excluded false-positive signals from the bead array or false-negative results from crossmatching assays owing to some unknown factor in our AB serum. By presenting these observations, we hope to draw others' attention to the unexpected presence of anti-HLA antibodies in certain commercially available human AB sera. These antibodies did not disturb Mreg cell development in culture and did not give positive crossmatch results; however, we cannot exclude that Mreg cell-bound antibodies have an adverse effect on Mreg cell survival or function after administration to patients. Our findings argue for substitution of human AB serum for platelet lysates or serum-free medium in clinical production of cell therapies wherever feasible. Where no alternative to serum exists, which is the case for Mreg_UKR production, we recommend screening new charges of serum for anti-HLA antibodies by bead array and crossmatching against a panel of cell donors who express relevant antigens. A risk assessment by the Sponsor of the ONEmreg12 trial concluded that, because there is no evidence of a detrimental effect of culturing Mreg cells using crossmatch-negative serum, irrespective of the presence of anti-HLA antibodies detected by bead array, it is justified to continue using such sera for Mreg_UKR manufacture.

Introduction Human regulatory macrophages (Mreg) have emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in living-donor kidney transplantation. A therapeutic cell product, known as Mreg_UKR, is under investigation in a Phase-I/II trial as a means of safely minimising maintenance immunosuppression. Mregs can be distinguished from macrophages in other polarisation states by their unique mode of derivation, a constellation of surface markers (Fig.1) and suppressor function; however, until now, no single marker was available to specifically and stably identify human Mregs. Materials and Methods By immunoprecipitation and MALDI-MS sequencing, dehydrogenase/reductase 9 (DHRS9), a little-studied retinol dehydrogenase of the SDR family of NAD(P)(H)-dependent oxidoreductases, was identified as the cognate antigen of a mouse monoclonal antibody raised against human Mreg lysates (Fig.2). DHRS9 expression within a panel of human monocyte-derived macrophages and dendritic cells (DC) was investigated by q-PCR, immunoblotting and flow cytometry. Results and Discussion DHRS9 expression discriminated human Mregs from a diverse panel of in vitro-derived macrophages (Fig.3A&B) and human monocyte-derived tolerogenic DC, including Tol-DC, Rapa-DC, DC-10 and PGE2-induced MDSC (Fig.3C&D).Treating Mregs with 100 ng/ml lipopolysaccharide for 24h did not extinguish DHRS9 expression. Expression of DHRS9 was acquired gradually during in vitro development of Mregs from CD14+ monocytes and was further enhanced by IFN-&ggr; stimulation. As a member of the SDR family of retinol dehydrogenases, DHRS9 may be responsible for conversion of retinol to retinal, which is further metabolised to retinoic acid by retinal dehydrogenases, including ALDH1A1 and ALDH1A2 (Fig.4A). Retinol is liberated from &bgr;-carotene through the action of beta-carotene monooxygenases, such as BCO2. Accordingly, Mregs express the enzymes necessary to convert retinol and &bgr;-carotene to retinoic acid (Fig.4B). The functional significance of DHRS9 expression in Mregs has not been firmly established; however, it is well-known that certain tissue-resident macrophage populations responsible for maintaining tissue homeostasis and preventing constitutive inflammation, such as those in the gut, suppress T cell reactions and induce Tregs through production of retinoic acid. A population of DHRS9+ human splenic macrophages was identified by immunohistochemistry. Although it cannot be inferred that these naturally-occurring DHRS9+ macrophages are a physiological equivalent of in vitro-derived Mregs, existence of these cells suggests that DHRS9 expression by cultured Mregs is not an artefact. Conclusion DHRS9 is a specific and stable marker of human Mregs that should be useful in future studies, especially searching for a natural counterpart of the in vitro-derived Mreg. Figure. No caption available. Figure. No caption available. Figure. No caption available. Figure. No caption available.

We have demonstrated previously that peripheral blood monocytes can be converted in vitro to a multipotent stem cell-like cell termed programmable cell of monocytic origin (PCMO) and subsequently into cells with chondrocyte-like phenotype. Here, we investigated whether PCMO could also be differentiated into osteoblast-like cells using growth factors with known osteoinductive potency. Following stimulation with BMP-2, BMP-7, IGF-1 or TGF-b1 for 7 and 14 days, PCMOs were analyzed for mRNA expression of collagen types I and V, alkaline phosphatase, osteocalcin, runt-related transcription factor2 (Runx2) and Osterix (Osx) by quantitative RT-PCR (qPCR) and the levels of collagen I in culture supernatants by ELISA. The expression of osteoblastic markers was evident, albeit at a different extent in cultures of PCMOs after treatment with the above-mentioned growth factors. Culture supernatants from PCMOs stimulated for 6e10 days with BMP-2, BMP-7, IGF-1 or TGF-b1 contained high levels of collagen type I, together with earlier data indicating synthesis and proper secretion. The findings suggest that PCMOs can transform into cells that are phenotypically similar to osteoblasts and identify these cells as osteochondroprogenitors. The possibility of differentiating PCMOs from peripheral blood in sizable quantities could be a novel way to obtain autologous bone-like substitutes without donor-site morbidity. (C) 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Development of the means to efficiently and continuously renew missing and non-functional proteins in diseased cells remains a major goal in modern molecular medicine. While gene therapy has the potential to achieve this, substantial obstacles must be overcome before clinical application can be considered. A promising alternative approach is the direct delivery of non-permeant active biomolecules, such as oligonucleotides, peptides and proteins, to the affected cells with the purpose of ameliorating an advanced disease process. In addition to receptor-mediated endocytosis, cell-penetrating peptides are widely used as vectors for rapid translocation of conjugated molecules across cell membranes into intracellular compartments and the delivery of these therapeutic molecules is generally referred to as novel prospective protein therapy. As a broad coverage of the enormous amount of published data in this field is unrewarding, this review will provide a brief, focused overview of the technology and a summary of recent studies of the most commonly used protein transduction domains and their potential as therapeutic agents for the treatment of cellular damage and the prevention of regulated cell death.

Development of the means to efficiently and continuously renew missing and non-functional proteins in diseased cells remains a major goal in modern molecular medicine. While gene therapy has the potential to achieve this, substantial obstacles must be overcome before clinical application can be considered. A promising alternative approach is the direct delivery of non-permeant active biomolecules, such as oligonucleotides, peptides and proteins, to the affected cells with the purpose of ameliorating an advanced disease process. In addition to receptor-mediated endocytosis, cell-penetrating peptides are widely used as vectors for rapid translocation of conjugated molecules across cell membranes into intracellular compartments and the delivery of these therapeutic molecules is generally referred to as novel prospective protein therapy. As a broad coverage of the enormous amount of published data in this field is unrewarding, this review will provide a brief, focused overview of the technology and a summary of recent studies of the most commonly used protein transduction domains and their potential as therapeutic agents for the treatment of cellular damage and the prevention of regulated cell death.

In Brief Background We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC). Methods In a prospective-randomized open-label international trial, 525 LTx recipients with HCC initially receiving mammalian target of rapamycin inhibitor–free immunosuppression were randomized 4 to 6 weeks after transplantation into a group on mammalian target of rapamycin inhibitor–free immunosuppression (group A: 264 patients) or a group incorporating sirolimus (group B: 261). The primary endpoint was recurrence-free survival (RFS); intention-to-treat (ITT) analysis was conducted after 8 years. Overall survival (OS) was a secondary endpoint. Results Recurrence-free survival was 64.5% in group A and 70.2% in group B at study end, this difference was not significant (P = 0.28; hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.62; 1.15). In a planned analysis of RFS rates at yearly intervals, group B showed better outcomes 3 years after transplantation (HR, 0.7; 95% CI, 0.48-1.00). Similarly, OS (P = 0.21; HR, 0.81; 95% CI, 0.58-1.13) was not statistically better in group B at study end, but yearly analyses showed improvement out to 5 years (HR, 0.7; 95% CI, 0.49-1.00). Interestingly, subgroup (Milan Criteria-based) analyses revealed that low-risk, rather than high-risk, patients benefited most from sirolimus; furthermore, younger recipients (age ≤60) also benefited, as well sirolimus monotherapy patients. Serious adverse event numbers were alike in groups A (860) and B (874). Conclusions Sirolimus in LTx recipients with HCC does not improve long-term RFS beyond 5 years. However, a RFS and OS benefit is evident in the first 3 to 5 years, especially in low-risk patients. This trial provides the first high-level evidence base for selecting immunosuppression in LTx recipients with HCC. In a large prospective randomized open-label international trial, liver transplant recipients for HCC were randomized to receive non-mTOR-inhibitor-based-treatment or mTOR-inhibitor-based-treatment. Results indicate overall sirolimus does not improve long-term recurrence-free survival beyond 5 years while a benefit was seen in low-risk patients.

BACKGROUND:We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC). METHODS:In a prospective-randomized open-label international trial, 525 LTx recipients with HCC initially receiving mammalian target of rapamycin inhibitor-free immunosuppression were randomized 4 to 6 weeks after transplantation into a group on mammalian target of rapamycin inhibitor-free immunosuppression (group A: 264 patients) or a group incorporating sirolimus (group B: 261). The primary endpoint was recurrence-free survival (RFS); intention-to-treat (ITT) analysis was conducted after 8 years. Overall survival (OS) was a secondary endpoint. RESULTS:Recurrence-free survival was 64.5% in group A and 70.2% in group B at study end, this difference was not significant (P = 0.28; hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.62; 1.15). In a planned analysis of RFS rates at yearly intervals, group B showed better outcomes 3 years after transplantation (HR, 0.7; 95% CI, 0.48-1.00). Similarly, OS (P = 0.21; HR, 0.81; 95% CI, 0.58-1.13) was not statistically better in group B at study end, but yearly analyses showed improvement out to 5 years (HR, 0.7; 95% CI, 0.49-1.00). Interestingly, subgroup (Milan Criteria-based) analyses revealed that low-risk, rather than high-risk, patients benefited most from sirolimus; furthermore, younger recipients (age ≤60) also benefited, as well sirolimus monotherapy patients. Serious adverse event numbers were alike in groups A (860) and B (874). CONCLUSIONS:Sirolimus in LTx recipients with HCC does not improve long-term RFS beyond 5 years. However, a RFS and OS benefit is evident in the first 3 to 5 years, especially in low-risk patients. This trial provides the first high-level evidence base for selecting immunosuppression in LTx recipients with HCC.

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.