Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

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.

Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

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-EVMreg) 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-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. RESULTS: Mreg and L-EVMreg 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-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg 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-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg 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-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study.

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.

Inhibition of regulated cell death by cell-penetrating peptides.

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.

A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease.

With an overall prevalence of 10-20%, gallstone disease (cholelithiasis) represents one of the most frequent and economically relevant health problems of industrialized countries. We performed an association scan of >500,000 SNPs in 280 individuals with gallstones and 360 controls. A follow-up study of the 235 most significant SNPs in 1,105 affected individuals and 873 controls replicated the disease association of SNP A-1791411 in ABCG8 (allelic P value P(CCA) = 4.1 x 10(-9)), which was subsequently attributed to coding variant rs11887534 (D19H). Additional replication was achieved in 728 German (P = 2.8 x 10(-7)) and 167 Chilean subjects (P = 0.02). The overall odds ratio for D19H carriership was 2.2 (95% confidence interval: 1.8-2.6, P = 1.4 x 10(-14)) in the full German sample. Association was stronger in subjects with cholesterol gallstones (odds ratio = 3.3), suggesting that His19 might be associated with a more efficient transport of cholesterol into the bile.

Clinical management of patients receiving cell-based immunoregulatory therapy.

Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.

IFN-γ-induced iNOS expression in mouse regulatory macrophages prolongs allograft survival in fully immunocompetent recipients.

Mouse monocytes exposed to macrophage colony-stimulating factor (M-CSF) and interferon-γ (IFN-γ) were driven to a novel suppressor phenotype. These regulatory macrophages (M regs) expressed markers distinguishing them from M0-, M1-, and M2-polarized macrophages and monocyte-derived dendritic cells (DCs). M regs completely suppressed polyclonal T cell proliferation through an inducible nitric oxide synthase (iNOS)-dependent mechanism. Additionally, M regs eliminated cocultured T cells in an allospecific fashion. In a heterotopic heart transplant model, a single intravenous administration of 5 × 10(6) donor-strain M regs before transplantation significantly prolonged allograft survival in fully immunocompetent recipients using both the stringent C3H-to-BALB/c (32.6 ± 4.5 versus 8.7 ± 0.2 days) and B6-to-BALB/c (31.1 ± 12 versus 9.7 ± 0.4 days) strain combinations. Nos2-deficient M regs did not prolong allograft survival, proving that M reg function in vivo is iNOS-dependent and mediated by living cells. M regs were detectable for at least 2 weeks postinfusion in allogeneic recipients. In their origin, development, phenotypic relationship with other in vitro-derived macrophages and functions, there are solid grounds to assert a near-equivalence of mouse and human M regs. It is concluded that mouse M regs represent a novel, phenotypically distinct subset of suppressor macrophages. Clinical applications of M reg therapy as an adjunct immunosuppressive therapy are currently being investigated within The ONE Study.

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Large extracellular vesicles derived from human regulatory macrophages (L-EVMreg) attenuate CD3/CD28-induced T-cell activation in vitro.

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-EVMreg) 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-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg 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-EVMreg. 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-EVMreg 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 × 106 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-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. KEY MESSAGES: Mreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µm L-EVMreg exhibit phosphatidylserine positivity L-EVMreg suppress CD4+ and CD8+ T-cells L-EVMreg hold clinical potential in T-cell-related diseases.

EGF and HB-EGF enhance the proliferation of programmable cells of monocytic origin (PCMO) through activation of MEK/ERK signaling and improve differentiation of PCMO-derived hepatocyte-like cells.

BACKGROUND: Hepatocyte-like cells (NeoHepatocytes) generated from a peripheral blood monocyte-derived stem cell-like cell (the PCMO) are a promising alternative for primary hepatocytes in cell transplantation studies to cure liver diseases. However, to be therapeutically effective NeoHepatocytes are needed in large quantities. It was the aim of the present study to investigate i) whether the proportion of actively proliferating NeoHepatocytes can be enhanced by supplementing the PCMO differentiation medium (containing M-CSF, IL-3, and human serum) with either EGF or HB-EGF and ii) which signaling pathway underlies the promitotic effect. RESULTS: EGF and HB-EGF enhanced cell proliferation of PCMOs as demonstrated by increased expression of cycle control genes (ABL, ANAPC2, CDC2, CDK4, CDK6), phosphorylation of the retinoblastoma protein, and increased PCMO cell numbers after stimulation with EGF or HB-EGF. EGF also raised the number of monocytes expressing the proliferation marker Ki67. PCMOs expressed the EGF receptors EGFR (ERBB1) and ERBB3, and expression of both increased during PCMO generation. Phosphoimmunoblotting of PCMOs indicated that both EGF and HB-EGF activated MEK-1/2 and ERK1/2 in a concentration-dependent fashion with the effect of EGF being more prominent. EGF treatment further decreased expression of p47phox and increased that of Nanog indicating enhanced dedifferentiation and pluripotency, respectively. Treatment with both EGF and HB-EGF resulted in NeoHepatocytes with improved functional parameters. CONCLUSIONS: The results suggested that the addition of EGF or HB-EGF to PCMO differentiation medium superactivates MEK/ERK signaling which then increases both PCMO proliferation, number, and functional differentiation of PCMO-derived NeoHepatocytes.

Differential roles of Smad2 and Smad3 in the regulation of TGF-ß1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1.

BACKGROUND: Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-ß)/Smad signalling pathway, eventually resulting in loss of TGF-ß-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-ß are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-ß type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-ß1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-ß1-mediated growth inhibition and cell migration (chemokinesis). RESULTS: SiRNA-mediated silencing of Smad3 in the TGF-ß responsive PDAC cell line PANC-1 reduced TGF-ß1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-ß1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-ß1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-ß signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-ß growth inhibition. CONCLUSIONS: In malignant cells with a functional TGF-ß signalling pathway Rac1 antagonizes the TGF-ß1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-ß signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.

Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning.

Hematopoietic stem cells have been successfully employed for tolerance induction in a variety of rodent and large animal studies. However, clinical transplantation of fully allogeneic bone marrow or blood-borne stem cells is still associated with major obstacles, such as graft-versus-host disease or cytoreductive conditioning-related toxicity. Here we show that when rat embryonic stem cell-like cells of WKY origin are injected intraportally into fully MHC-mismatched DA rats, they engraft permanently (>150 days) without supplementary host conditioning. This deviation of a potentially alloreactive immune response sets the basis for long-term graft acceptance of second-set transplanted WKY cardiac allografts. Graft survival was strictly correlated with a state of mixed chimerism, which required functional thymic host competence. Our results provide a rationale for using preimplantation-stage stem cells as vehicles in gene therapy and for the induction of long-term graft acceptance.

Tolerance in clinical transplantation: progress, challenge or just a dream?

INTRODUCTION: The achievement of clinical operational tolerance (COT) is still considered a major goal in the academic field of solid organ transplantation. Even COT is feasible and safe in selected cases after liver transplantation, in the clinical arena of solid organ transplantation, tolerance remains, for the most part, a concept rather than a reality. CHALLENGES: Although modern immunosuppression regimens have effectively handled acute rejection, nearly all organs except the liver commonly suffer chronic immunologic damage that impairs organ function, threatening patient and allograft survival. Strong arguments in favour of conducting clinical tolerance trials are the high number of grafts still lost due to chronic rejection, the burden of serious adverse effects from immunosuppressants which causes considerable cardiovascular morbidity and mortality, respectively, and the fact that sporadic tolerance can be observed in rare cases where non-adherence to immunosuppressive regimens is linked with a state of long-lasting organ tolerance. Whereas molecule-based regimens have been largely ineffective, cell-based tolerance protocols have delivered some encouraging results to achieve COT. DISCUSSION: In combination with donor bone marrow-derived stem cells, some encouraging results in COT development were reported lately for renal transplantation as well. However, less toxic conditioning protocols and more experience by use of cell products with regulatory properties in combination with synergizing immunosuppressive drugs is required to launch future tolerance trials for a broader spectrum of potential transplant candidates. New methods in immunomonitoring including biomarkers, microarray-based genetic tolerance signatures and functional assays may pave the way to achieve COT in upcoming clinical trials.

A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma.

BACKGROUND: The potential anti-cancer effects of mammalian target of rapamycin (mTOR) inhibitors are being intensively studied. To date, however, few randomised clinical trials (RCT) have been performed to demonstrate anti-neoplastic effects in the pure oncology setting, and at present, no oncology endpoint-directed RCT has been reported in the high-malignancy risk population of immunosuppressed transplant recipients. Interestingly, since mTOR inhibitors have both immunosuppressive and anti-cancer effects, they have the potential to simultaneously protect against immunologic graft loss and tumour development. Therefore, we designed a prospective RCT to determine if the mTOR inhibitor sirolimus can improve hepatocellular carcinoma (HCC)-free patient survival in liver transplant (LT) recipients with a pre-transplant diagnosis of HCC. METHODS/DESIGN: The study is an open-labelled, randomised, RCT comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing LT for HCC. Patients with a histologically confirmed HCC diagnosis are randomised into 2 groups within 4-6 weeks after LT; one arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol and the second arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol for the first 4-6 weeks, at which time sirolimus is initiated. A 21/2 -year recruitment phase is planned with a 5-year follow-up, testing HCC-free survival as the primary endpoint. Our hypothesis is that sirolimus use in the second arm of the study will improve HCC-free survival. The study is a non-commercial investigator-initiated trial (IIT) sponsored by the University Hospital Regensburg and is endorsed by the European Liver and Intestine Transplant Association; 13 countries within Europe, Canada and Australia are participating. DISCUSSION: If our hypothesis is correct that mTOR inhibition can reduce HCC tumour growth while simultaneously providing immunosuppression to protect the liver allograft from rejection, patients should experience less post-transplant problems with HCC recurrence, and therefore could expect a longer and better quality of life. A positive outcome will likely change the standard of posttransplant immunosuppressive care for LT patients with HCC. TRIAL REGISTER: Trial registered at http://www.clinicaltrials.gov: NCT00355862(EudraCT Number: 2005-005362-36).

Customized cell-based treatment options to combat autoimmunity and restore beta-cell function in type 1 diabetes mellitus: current protocols and future perspectives.

Type 1 diabetes mellitus (T1D) is considered a classical autoimmune disease which commonly starts during childhood but may appear later in adulthood in a proportion of 30-40% of affected individuals. Its development is based on a combination of a genetic predisposition and autoimmune processes that result in gradual destruction of the beta-cells of the pancreas and cause absolute insulin deficiency. Evidence for an autoimmune origin of T1D results from measurable islet beta-cell autoantibody directed against various autoantigens such as proinsulin or insulin itself, glutamic acid decarboxylase 65, the islet tyrosine phosphatase IA-2, and the islet-specific glucose-6-phosphatase catalytic subunit-related protein. In addition, T-cell lines with specificity for insulin or glutamic acid decarboxylase have been identified within peripheral blood lymphocytes. Importantly, in most instances the pathogenesis of T1D comprises a slowly progressive destruction of beta-cell tissue in the pancreas preceded by several years of a prediabetic phase where autoimmunity has already developed but with no clinically apparent insulin dependency. Unless immunological tolerance to pancreatic autoantigens is re-established, diabetes treated by islet cell transplantation or stimulation/regeneration of endogenous beta-cells would remain a chronic disease secondary to immune suppression related morbidity. Hence, if islet cell tolerance could be re-induced, a major clinical hurdle to curing diabetes by islet cell neogenesis may be overcome. Targeted immunotherapies are currently explored in a variety of clinical studies and hold great promise for causative treatment to readjust the underlying immunologic imbalance with the goal to cure the disease. This chapter will outline possible treatment options to stop or reverse the beta-cell-specific autoimmune and inflammatory process within pancreatic islets. Special emphasis is given to stem cells of embryonic, mesenchymal, and haematopoietic origin, which, besides their use for regenerative purposes, possess potent immunomodulatory functions and thus have the potential to suppress the autoimmune response. At the end of this chapter we will introduce a novel type of in vitro modified monocytes with immunosuppressive and anti-inflammatory properties. These tolerogenic monocytes provide a feasible option to be used as autologous cellular transplants to halt autoimmunity and to protect still viable beta-cells within Langerhans islets.

The programmable cell of monocytic origin (PCMO): a potential adult stem/progenitor cell source for the generation of islet cells.

Adult stem or programmable cells hold great promise in diseases in which damaged or non-functional cells need to be replaced, such as in type 1 diabetes. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into pancreatic beta-cell-like cells capable of synthesizing insulin. The two-step phenotypic conversion commences with growth factor-induced partial reprogramming during which the cells acquire a state of plasticity along with expression of various markers of pluripotency. These cells, termed "programmable cells of monocytic origin" (PCMOs), can then be induced with appropriate differentiation media to become insulin-producing cells (NeoIslet cells). Expression profiling of transcription factors known to determine endocrine and beta-cell development in vivo indicated that NeoIslet cells resemble cells with an immature beta-cell phenotype. Current efforts focus on establishing culture conditions that (i) increase the plasticity and proliferation potential of PCMOs by enhancing the reprogramming process and (ii) improve insulin production by mimicking in vivo lineage specification and normal pancreatic endocrine development. Combining these two strategies has great potential in generating large amounts of blood-derived cells suitable for both autologous and allogeneic therapy of type 1 diabetes.

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

Cell therapy approaches aiming at minimization of immunosuppression in solid organ transplantation.

PURPOSE OF REVIEW: Cell transplantation or administration of cellular products has escaped preclinical experimental status and will be integrated as a substantial component of future individualized treatment modalities for a broad scope of medical fields, including transplantation tolerance, cancer immunotherapies and auto-immune diseases. RECENT FINDINGS: Renal allograft tolerance has been successfully demonstrated using bone marrow transplantation with nonmyeloablative conditioning to induce transient hematopoietic chimerism, hereby exemplifying the immunomodulatory potential of living cells to reprogram an existing immune system to recognize and accept nonself major histocompatibility antigens expressed on the allogeneic donor organ. Strong efforts are currently undertaken to circumvent the necessity of hematopoietic chimerism induction by harnessing peripheral regulatory mechanisms. Potential candidate cell populations that bear immunomodulating and regulatory properties comprise regulatory T cells, dendritic cells and deactivated macrophages as well as stem cells of various origins, currently tested in different clinical transplantation tolerance trials. SUMMARY: Although transplantation tolerance is still on its way to be reliably accomplished in clinical settings, use of well specified and functionally characterized cellular therapeutics with regulatory properties is now entering the field of personalized medicine for transplanted patients to benefit from minimization protocols and less severe side affects related to conventional immunosuppressive drug treatment.

Investigation of the colorectal cancer susceptibility region on chromosome 8q24.21 in a large German case-control sample.

Human chromosome 8q24.21 has been implicated as a susceptibility region for colorectal cancer (CRC) as a result of genome-wide association and candidate gene studies. To assess the impact of molecular variants at 8q24.21 upon the CRC risk of German individuals and to refine the disease-associated region, a total of 2,713 patients with operated CRC (median age at diagnosis: 63 years) were compared with 2,718 sex-matched control individuals (median age at inclusion: 65 years). Information on microsatellite instability in tumors was available for 901 patients. Association analysis of SNPs rs10505477 and rs6983267 yielded allelic p-values of 1.42 x 10(-7) and 2.57 x 10(-7), respectively. For both polymorphisms, the odds ratio was estimated to be 1.50 (95% CI: 1.29-1.75) under a recessive disease model. The strongest candidate interval, outside of which significance dropped by more than 4 orders of magnitude, was delineated by SNPs rs10505477 and rs7014346 and comprised 17 kb. In a subgroup analysis, the disease association was found to be more pronounced in MSI-stable tumors (odds ratio: 1.71). Our study confirms the role of genetic variation at 8q24.21 as a risk factor for CRC and localizes the corresponding susceptibility gene to a 17 kb candidate region.

Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia.

Circulating progenitor cells home to sites of postnatal neovascularization and differentiate into endothelial cells but questions remain regarding the source of these cells. Indeed, a recent study suggests that nonbone marrow-derived cells may be even more important than bone marrow-derived cells in the setting of transplant arteriosclerosis. Thus, we aimed to thoroughly investigate the contribution of nonbone marrow-derived progenitor cells for neovascularization. We exclusively identified nonbone marrow-derived progenitor cells by combining a parabiosis model with reverse bone marrow transplantation followed by hindlimb ischemia. In this model, nonbone marrow-derived circulating progenitor cells attributed for 74+/-13% of the circulating progenitor cells that incorporated into the ischemic hindlimb. Increasing evidence suggests that organs such as small intestine and liver contain a considerable number of tissue resident progenitor cells and, thus, represent putative sources for nonbone marrow-derived progenitors. To track organ-derived progenitors, we transplanted sex-mismatched small intestine or liver, respectively, into rats followed by induction of hindlimb ischemia. These experiments show that organ-derived progenitor cells are contributing to postnatal vasculogenesis (intestine: 4.7+/-3.7%; liver: 6.3+/-2.2%). Based on the subsequent observation that liver-derived nonhematopoietic c-kit(+)CD45(-) progenitors are mobilized on induction of hindlimb ischemia, we prospectively isolated and intravenously infused these progenitors from murine livers. The isolated cells demonstrated a marked capacity for enhancing neovascularization and restoring blood flow to the ischemic hindlimb (no cells: 26.4+/-4.8% of normal blood flow; c-kit(+)CD45(-) cells: 67.0+/-8.0% of normal flow; P<0.01). In conclusion, we find that nonbone marrow-derived c-kit(+)CD45(-) progenitors contribute to postnatal neovascularization to an extent that is similar to that of bone marrow-derived progenitor cells. Intestine and liver represent a rich source for mobilized tissue-residing progenitor cells.