[Show abstract][Hide abstract] ABSTRACT: Background:
We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC).
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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[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.
Full-text · Article · Sep 2014 · International Journal of Oral Science
[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.
Full-text · Article · Jun 2014 · Journal of Clinical Research in Pediatric Endocrinology
[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.
No preview · Article · Jun 2013 · Pediatric Transplantation
[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.
[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.
Full-text · Article · Aug 2012 · Molecular Therapy
[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.
Preview · Article · Aug 2012 · Cell Communication and Signaling
[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.
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.
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.
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.
Full-text · Article · Jul 2012 · Journal of Plastic Reconstructive & Aesthetic Surgery