[Show abstract][Hide abstract] ABSTRACT: Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS(+)) and STRO-1-negative (MACS(-)) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS(+) and MACS(-) cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS(+) and MACS(-) cell fractions showed plastic adherence. MACS(+) cells, in contrast to MACS(-) cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS(+) cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS(-) cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS(+) cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS(-) cells demonstrated slight osteogenic potential. Unstimulated MACS(+) cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS(-) cells demonstrated higher expression of osteonectin (P<0.05; Mann-Whitney). The present study is the first to compare gingival MACS(+) and MACS(-) cell populations demonstrating that MACS(+) cells, in contrast to MACS(-) cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS(+) technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS(+) cells are a unique renewable source of multipotent stem/progenitor cells.International Journal of Oral Science advance online publication, 26 September 2014; doi:10.1038/ijos.2014.41.
International Journal of Oral Science 09/2014; · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new cell-based medicinal product containing human regulatory macrophages, known as Mreg_UKR, has been developed and conforms to expectations of a therapeutic drug. Here, Mreg_UKR was subjected to pharmacokinetic, safety pharmacology, and toxicological testing, which identified no adverse reactions. These results would normally be interpreted as evidence of the probable clinical safety of Mreg_UKR; however, we contend that, owing to their uncertain biological relevance, our data do not fully support this conclusion. This leads us to question whether there is adequate scientific justification for preclinical safety testing of similar novel cell-based medicinal products using animal models. In earlier work, two patients were treated with regulatory macrophages prior to kidney transplantation. In our opinion, the absence of acute or chronic adverse effects in these cases is the most convincing available evidence of the likely safety
of Mreg_UKR in future recipients. On this basis, we consider that safety information from previous clinical investigations of related cell products should carry greater weight than preclinical data when evaluating the safety profile of novel cell-based medicinal products. By extension, we argue that omitting extensive preclinical safety studies before conducting small-scale exploratory clinical investigations of novel cell-based medicinal products data may be justifiable in some instances.
[Show abstract][Hide abstract] ABSTRACT: Objective: The vision of potential autologous cell therapy for the cure of diabetes encourages ongoing research. According to a previously published protocol for the generation of insulin-producing cells from human monocytes, we analyzed whether the addition of growth factors could increase insulin production. This protocol was then transferred to a non-human primate model by using either blood- or spleen-derived monocytes. Methods: Human monocytes were treated to dedifferentiate into programmable cells of monocytic origin (PCMO). In addition to the published protocol, PCMOs were then treated with either activin A, betacellulin, exendin 3 or 4. Cells were characterized by protein expression of insulin, Pdx-1, C-peptide and Glut-2. After identifying the optimal protocol, monocytes from baboon blood were isolated and the procedure was repeated. Spleen monocytes following splenectomy of a live baboon were differentiated and analyzed in the same manner and calculated in number and volume. Results: Insulin content of human cells was highest when cells were treated with activin A and their insulin content was 13 000 µU/1 million cells. Insulin-producing cells form primate monocytes could successfully be generated despite using human growth factors and serum. Expression of insulin, Pdx-1, C-peptide and Glut-2 was comparable to that of human neo-islets. Total insulin content of activin A-treated baboon monocytes was 16 000 µU/1 million cells. Conclusion: We were able to show that insulin-producing cells can be generated from baboon monocytes with human growth factors. The amount generated from one spleen could be enough to cure a baboon from experimentally induced diabetes in an autologous cell transplant setting.
Journal of Clinical Research in Pediatric Endocrinology 06/2014; 6(2):93-9.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor (TGF)-β1 promotes progression of pancreatic ductal adenocarcinoma (PDAC) by enhancing epithelial-mesenchymal transition, cell migration/invasion, and metastasis, in part by cooperating with the small GTPase Rac1. Prompted by the observation of higher expression of Rac1b, an alternatively spliced Rac1 isoform, in pancreatic ductal epithelial cells and in patients with chronic pancreatitis vs. PDAC, as well as in long-time vs. short-time survivors among PDAC patients, we asked whether Rac1b might negatively affect TGF-β1 prometastatic function. Interestingly, the non-malignant pancreatic ductal epithelial cell line H6c7 exhibited a higher ratio of active Rac1b to total Rac1b than the TGF-β1-responsive PDAC cell lines Panc-1 and Colo357. Notably, siRNA-mediated silencing of Rac1b increased TGF-β1/Smad-dependent migratory activities in H6c7, Colo357, and Panc-1 cells, while ectopic overexpression of Rac1b in Panc-1 cells attenuated TGF-β1-induced cell motility. Depletion of Rac1b in Panc-1 cells enhanced TGF-β1/Smad-dependent expression of promoter-reporter genes and of the endogenous Slug gene. Moreover, Rac1b depletion resulted in a higher and more sustained C-terminal phosphorylation of Smad3 and Smad2, suggesting that Rac1b is involved in Smad2/3 dephosphorylation/inactivation. Since pharmacologic or siRNA-mediated inhibition of Smad3 but not Smad2 was able to alleviate the Rac1b siRNA effect on TGF-β1-induced cell migration, our results suggests that Rac1b inhibits TGF-β1-induced cell motility in pancreatic ductal epithelial cells by blocking the function of Smad3. Moreover, Rac1b may act as an endogenous inhibitor of Rac1 in TGF-β1-mediated migration and possibly metastasis. Hence, it could be exploited for diagnostic/prognostic purposes or even therapeutically in late-stage PDAC as an antimetastatic agent.
[Show abstract][Hide abstract] ABSTRACT: A prospective identification of the estimated 20-50% of pediatric LTX recipients developing operational tolerance would be of great clinical advantage. So far markers of immune tolerance - T-cell subpopulations or gene expression profiles - have been investigated only retrospectively in successfully weaned patients. Fifty children aged 8-265 months (median 89) were investigated 1-180 months (median 44) after LTX under ongoing immunosuppression. T-cell subpopulations were measured during regular post-transplant visits using FACS (Vδ1- vs. Vδ2-γδ-T cells and Tregs). A Vδ1/Vδ2-γδ-T-cell ratio ≥1.42 previously reported in operational tolerance was found in 12 of 50 (24%) patients. In analogy, a Treg count ≥44 per μL was found in 35 of 50 (70%) patients and a Treg proportion ≥2.23% of CD3(+) -T cells in 39 of 50 (78%) patients. Only 9 of 50 patients (18%) fulfilled both criteria. The parameters Vδ1/Vδ2-γδ-T-cell ratio and Tregs were not significantly correlated to each other or with donor type or immunosuppression. Vδ1/Vδ2-γδ-T-cell ratio was more stable in serial examinations compared with Treg analyses. The observed proportion of 18% pediatric LTX patients with potential operational tolerance is in accordance with previous reports. However, clinical experience shows that rejections may happen even after long-time weaning of immunosuppression. This suggests that operational tolerance is a dynamic process, with uncertain prediction by Vδ1/Vδ2-γδ-T-cell ratio and/or Tregs under immunosuppression.
[Show abstract][Hide abstract] ABSTRACT: During the last decade it was realized that stem cell-based therapies hold an enormous therapeutic potential, improving the life of patients with conditions ranging from neurodegenerative and traumatic diseases to regenerative medicine requiring replacement of complex structures such as bones and teeth. Based on their ability to regenerate and/or repair damaged tissue and eventually restore organ function, multiple types of stem/progenitor cells have been discovered. In the field of periodontal regeneration and tooth engineering, several types of adult multipotent mesenchymal stem cells from various sources are currently being investigated. These include the bone marrow stromal stem cells (BMSSCs), adipose-derived stromal cells (ADSCs), dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from human exfoliated deciduous teeth (SHEDs), stem cells from the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone proper-derived stem cells, and gingival stem cells. The potential of these different MSCs as precursors for regenerative purposes in the dental field is discussed in this chapter.
Advances in biochemical engineering/biotechnology 09/2012; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.Molecular Therapy (2012); doi:10.1038/mt.2012.168.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Cell Communication and Signaling 08/2012; 10(1):23. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Composite tissue allotransplantation (CTA) is a newly emerging field of transplantation. Immunological research in CTA has been intensified due to the recent clinical success of hand and face transplantation. Establishing immunological tolerance by adoptive transfer of ex vivo cultured tolerance-inducing cell types is of growing interest. Transplant acceptance-inducing cells (TAICs) are a type of deactivated immunoregulatory macrophages. METHODS: A total of 36 allogeneic hind limb transplantations in the rat were performed in six groups. Group A (Lewis (LW) → Brown-Norway (BN)) received Lewis-donor-derived TAICs locally (i.m.). Group B (LW → BN) received Lewis-donor-derived TAICs systemically (i.v.) and group C (Sprague Dawley (Sp-D) → BN) served as a control group receiving Lewis-donor-derived TAICs systemically (i.v.). Groups D (LW → BN), E (LW → BN), and F (BN → BN) also served as control groups with group D receiving no immunosuppression, group E receiving FK506 and prednisolone and group F receiving no immunosuppression with isograft transplantations (BN → BN). The timing of rejection was assessed by clinical observation and histological findings. RESULTS: Rejection of the allogeneic hind limb occurred on average 7.7 days after transplantation in group A and 7.4 days in group B. Rejection was significantly delayed (Log-rank test, p < 0.01) compared to groups C and D, where rejection of the allogeneic hind limb occurred on average 5.8 days and 5.6 days after transplantation. No rejection was seen in groups E and F. CONCLUSION: For the first time, TAICs have been applied in a CTA model and demonstrated a significant immunosuppressive effect. Even though the immunomodulatory effect is relatively modest, the results of this study justify subsequent research on TAIC therapy to improve experimental and clinical outcome after CTA.
Journal of Plastic Reconstructive & Aesthetic Surgery 07/2012; · 1.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adult stem or programmable cells hold great promise in diseases in which damaged or non-functional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells whose phenotypes resemble specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion the monocytes downregulate monocyte/macrophage differentiation markers being indicative of partial dedifferentiation and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency. These cells were termed “programmable cells of monocytic origin” (PCMOs). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMOs in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies
Stem Cells and Cancer Stem Cells. 04/2012; 6(4):367-375.
[Show abstract][Hide abstract] ABSTRACT: In the search for an ideal minimally-invasive multipotent postnatal stem cells' source, the aim of the present study was to isolate and characterize multipotent postnatal stem/progenitor cells from the human alveolar bone proper tissue of the oral cavity. Cells were isolated from human alveolar bone parts, immunomagnetically sorted using STRO-1 antibodies and characterized flow cytometrically for the expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1 surface markers. Colony-formation and multilineage differentiation potential were tested. Mineralized tissue marker expression was examined using real time polymerase chain reaction (PCR). The cells were plastic-adherent and showed colony-formation. Cells expressed the surface markers CD73, CD90, CD105, STRO-1 and CD146/MUC18, while lacking the expression of the hematopoietic markers CD14, CD34 and CD45. Cells could be differentiated into osteoblastic, adipocytic and chondroblastic lineages. Unstimulated cells expressed alkaline phosphatase (ALP), type I, III and V collagens, osteonectin and osteocalcin in a very distinctive pattern. This study presents a practical and minimally-invasive scheme for the isolation of multipotent postnatal stem/progenitor cells from the human alveolar bone tissue of the oral cavity.
Journal of cranio-maxillo-facial surgery: official publication of the European Association for Cranio-Maxillo-Facial Surgery 03/2012; · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, a first direct estimate of the single base-pair substitution rate in the human germline was derived from genome-wide DNA sequence data. This result has shed new light upon the question of whether cutting-edge molecular genetic analysis could, in a paternity dispute, potentiate discrimination between two alleged fathers who are monozygotic (MZ) twins. Such paternity cases are not infrequent and usually receive a high level of public attention. We performed a 'thought experiment', the outcome of which strongly suggests that, by a combination of currently available laboratory techniques, paternity testing is indeed feasible in the context of MZ twins. Taking into consideration what is known about the biology of the human male germline, we would predict that >80% of the offspring of one twin brother would carry at least one germline mutation that would be detectable in the sperm of their father, but not in that of the other twin.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Immunology 09/2011; 187(5):2072-8. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both transforming growth factor (TGF)-ß and the non-receptor tyrosine kinase Src play major roles during tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis, but little is known about the signaling crosstalk between them. To interfere with Src function in vitro and in vivo many studies have employed the pharmacologic Src inhibitors PP2 and PP1. Both agents have recently been shown to be powerful inhibitors of TGF-ß receptor type I/ALK5 and type II. As this situation prohibited any definite conclusions with respect to the relative contribution of TGF-ß vs. Src signaling, we decided to reappraise a potential role of Src in TGF-ß1-mediated cellular responses using RNA and dominant-negative (dn) interference to block Src expression and function, respectively. In TGF-ß-responsive pancreatic ductal adenocarcinoma (PDAC) cells, we show that Src is activated by TGF-ß1 and that its specific inhibition strongly attenuated basal proliferation and enhanced TGF-ß1-mediated growth arrest. However, Src inhibition was unable to impair TGF-ß1-controlled EMT as evidenced by cell morphology and regulation of the epithelial marker E-cadherin. Despite its dispensibility for TGF-ß-induced EMT, specific inhibition of Src dramatically reduced basal and TGF-ß1-induced cell migration in Panc-1 cells as measured with a novel real-time migration assay (xCELLigence DP system). Biochemically, dnSrc inhibition failed to block TGF-ß1/ALK5-induced activation of Smad2 and Smad3, but partially inhibited transcriptional activation of TGF-ß/Smad-responsive reporter genes, and effectively blocked basal and TGF-ß1-induced activation of p38 MAPK. Together, the data provide evidence for a role of Src in the regulation of basal proliferation as well as in basal and TGF-ß1-mediated cell motility but not EMT in TGF-ß-responsive pancreatic (tumor) cells.
International Journal of Oncology 03/2011; 38(3):797-805. · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Langenbeck s Archives of Surgery 03/2011; 396(4):475-87. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-β (TβRI, TβRII) play major roles during tumorigenesis by regulating cell growth, migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo, however, they may exert non-specific effects on other kinases. In this study, we have evaluated PP2 and PP1 for their ability to inhibit TGFβ1-mediated responses in the TGF-β-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-b1-induced growth arrest and p21(WAF1) induction, while basal growth was enhanced by PP2 and PP1, and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-β1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin, and upregulation of the EMT-associated transcription factor Slug. Likewise, PP2 and PP1 but not SU6656 effectively blocked TGF-β1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-β/Smad-responsive reporters of a kinase-active TβRI mutant ectopically expressed in Panc1 cells. Interestingly, PP2 and PP1 strongly inhibited recombinant TβRI in an in vitro kinase assay, with PP1 being more potent and PP2 being nearly as potent as the established TβRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TβRII kinase. Together, these data provide evidence that PP2 and PP1 are powerful inhibitors of TβR function that can block TGF-β/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics of late stage metastatic disease.
Current cancer drug targets 03/2011; 11(4):524-35. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulatory macrophages (M regs) are a novel type of suppressor macrophage which may be a particularly suitable cell for inducing tolerance of solid organ transplants. In this article, we provide a detailed description of the generation of human M regs from peripheral blood monocytes and methods for the assessment of their phenotype. The uniqueness of the human M reg is best appreciated when the M reg is compared to macrophages in other states of activation; therefore, protocols are provided for generating five comparator macrophage types which have been used as cell type-specificity controls in our work.
[Show abstract][Hide abstract] ABSTRACT: Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β 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-β 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-β1-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-β1-mediated growth inhibition and cell migration (chemokinesis).
SiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-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-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-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-β 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-β growth inhibition.
In malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 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-β 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.
Molecular Cancer 01/2011; 10:67. · 5.40 Impact Factor