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1.  Skin cancer in solid organ transplant recipients: are mTOR inhibitors a game changer? 
While immunosuppressive agents are necessary to prevent the rejection of transplanted organs, and are a great medical success story for protecting against early allograft loss, graft and patient survival over the long term are diminished by side effects from these same drugs. One striking long-term side effect is a high rate of skin cancer development. The skin cancers that develop in transplant recipients tend to be numerous, as well as particularly aggressive, and are therefore a major contributor to morbidity and mortality in transplant recipients. An apparent reason for the high incidence of skin cancer likely relates to suppression of immune surveillance mechanisms, but other more direct effects of certain immunosuppressive drugs are also bound to contribute to cancers of UV-exposed skin. However, over the past few years, evidence has emerged to suggest that one class of immunosuppressants, mammalian target of rapamycin (mTOR) inhibitors, could potentially inhibit skin tumour formation through a number of mechanisms that are still being studied intensively today. Therefore, in light of the high skin cancer incidence in transplant recipients, it follows that clinical trials have been conducted to determine if mTOR inhibitors can significantly reduce these post-transplant skin malignancies. Here, the problem of post-transplant skin cancer will be briefly reviewed, along with the possible mechanisms contributing to this problem, followed by an overview of the relevant clinical trial results using mTOR inhibitors.
PMCID: PMC4332735
mTOR; Organ transplantation; Post-transplant malignancy; Calcineurin inhibitors; Skin cancer
2.  The influence of mTOR inhibitors on immunity and the relationship to post-transplant malignancy 
Transplantation Research  2013;2(Suppl 1):S2.
The known role of mammalian target of rapamycin (mTOR) in the immune response has been rapidly evolving, from what was once thought to be a simple immunosuppressive antiproliferative effect on T cells to a very complex central role that serves to integrate multiple signals given to T cells, B cells and antigen-presenting cells. The complexity of this topic is demonstrated by recent data suggesting that mTOR inhibition can either inhibit or promote certain aspects of immune responses, depending on the nature of the antigenic stimulus, and the environmental conditions cueing the cellular immunological players. There is even evidence that, under mTOR inhibition, an immune response to one foreign entity (for example, an organ transplant) may be simultaneously completely different to that of another (for example, tumour or microorganism). To understand how this might be possible, it is necessary to investigate the central role that mTOR seems to have in shaping the immune response. This review is aimed at examining how mTOR controls the development and function of key immune cells, and puts this information primarily in the context of organ transplant rejection and post-transplant malignancy.
PMCID: PMC3834556  PMID: 24565200
mammalian target of rapamycin; post-transplant malignancy; T cells; B cells; dendritic cells; organ transplantation
3.  Heart Grafts Tolerized Through Third-Party Multipotent Adult Progenitor Cells Can Be Retransplanted to Secondary Hosts With No Immunosuppression 
This study found that stable allograft survival could be achieved following third-party multipotent adult progenitor cell (MAPC) infusion in a rat model of fully allogeneic, heterotopic heart transplantation. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy and has prompted the initiation of a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.
Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.
PMCID: PMC3726139  PMID: 23836805
Immunosuppression; Mesenchymal stem cells; Transplantation; T cells
4.  Protective Vaccination against Papillomavirus-Induced Skin Tumors under Immunocompetent and Immunosuppressive Conditions: A Preclinical Study Using a Natural Outbred Animal Model 
PLoS Pathogens  2014;10(2):e1003924.
Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients. Hence, the burden of these clinical manifestations demands for a prophylactic approach. To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV). This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal. Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections. VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment. Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors. Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread. Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination. These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.
Author Summary
Organ transplant recipients (OTR) frequently suffer from fulminant warts that are induced by cutaneous human papillomaviruses (HPV). Moreover, some skin HPV types may also be involved in the development of non-melanoma skin cancer. Mimicking the situation of immunosuppressed OTR who acquire cutaneous HPV infections already in childhood, we explored the efficacy of a vaccine in infected animals that additionally underwent immunosuppression. We demonstrate for the first time the success of a vaccine against a skin papillomavirus in a natural outbred animal system, which completely prevents both benign and malignant skin tumor formation even under immunosuppressed conditions. Hence, our study provides the basis for clinical development of a vaccine against cutaneous HPV infections, which may be particularly useful in transplant recipients.
PMCID: PMC3930562  PMID: 24586150
5.  Reviewer acknowledgement 2013 
Contributing reviewers
The Editors of Transplantation Research would like to thank all our reviewers who have contributed to the journal in Volume 2 (2013).
PMCID: PMC3913949  PMID: 24499571
6.  Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study 
Immune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.
Six leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.
Optimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.
Local performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.
PMCID: PMC3827923  PMID: 24160259
Immune monitoring; Organ transplantation; Cell therapy; Tolerance; Kidney; Flow cytometry
7.  IL-13 signaling via IL-13Rα2 triggers TGF-β1-dependent allograft fibrosis 
Allograft fibrosis still remains a critical problem in transplantation, including heart transplantation. The IL-13/TGF-β1 interaction has previously been identified as a key pathway orchestrating fibrosis in different inflammatory immune disorders. Here we investigate if this pathway is also responsible for allograft fibrosis and if interference with the IL-13/TGF-β1 interaction prevents allograft fibrosis.
FVB or control DBA/1 donor hearts were transplanted heterotopically into DBA/1 recipient mice and hearts were explanted at day 60 and 100 post-transplantation. Cardiac tissue was examined by Masson’s trichrome staining and immunohistochemistry for CD4, CD8, CD11b, IL-13, Fas ligand, matrix metalloproteinase (MMP)-1, MMP-13, β2-microglobulin, and Gremlin-1. Graft-infiltrating cells were isolated and analyzed by flow cytometry. IL-13 and TGF-β1 levels were determined by enzyme-linked immunosorbent assay (ELISA) and the amount of collagen was quantified using a Sircol assay; IL-13Rα2 expression was detected by Western blotting. In some experiments IL-13/ TGF-β1 signaling was blocked with specific IL-13Rα2 siRNA. Additionally, a PCR array of RNA isolated from the allografts was performed to analyze expression of multiple genes involved in fibrosis.
Both groups survived long-term (>100 days). The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100. Furthermore, elevated IL-13 levels (P = 0.0003) and numbers of infiltrating IL-13+ cells (P = 0.0037), together with an expression of IL-13Rα2, were detected only within allografts. The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094). The allograft fibrosis found in these experiments was accompanied by upregulation of multiple profibrotic genes, which was confirmed by immunohistochemical stainings of allograft tissue. Blockage of the IL-13/TGF-β1 interaction by IL-13Rα2 siRNA led to lower numbers of CD11bhighGr1intermediateTGF-β1+, CD4+, CD8+, and CD11b+ cells, and prevented collagen deposition (P = 0.0018) within these allografts.
IL-13 signaling via IL-13Rα2 induces TGF-β1 and causes allograft fibrosis in a murine model of chronic transplant rejection. Blockage of this IL-13/TGF-β1 interaction by IL-13Rα2 siRNA prevents cardiac allograft fibrosis. Thus, IL-13Rα2 may be exploitable as a future target to reduce allograft fibrosis in organ transplantation.
PMCID: PMC4016099  PMID: 24143891
IL-13; IL-13Rα2; TGF-β1; Allograft fibrosis; Heart transplantation
8.  Extended pancreas donor program – the EXPAND study rationale and study protocol 
Simultaneous pancreas kidney transplantation (SPK), pancreas transplantation alone (PTA) or pancreas transplantation after kidney (PAK) are the only curative treatment options for patients with type 1 (juvenile) diabetes mellitus with or without impaired renal function. Unfortunately, transplant waiting lists for this indication are increasing because the current organ acceptability criteria are restrictive; morbidity and mortality significantly increase with time on the waitlist. Currently, only pancreas organs from donors younger than 50 years of age and with a body mass index (BMI) less than 30 are allocated for transplantation in the Eurotransplant (ET) area. To address this issue we designed a study to increase the available donor pool for these patients.
This study is a prospective, multicenter (20 German centers), single blinded, non-randomized, two armed trial comparing outcome after SPK, PTA or PAK between organs with the currently allowed donor criteria versus selected organs from donors with extended criteria. Extended donor criteria are defined as organs procured from donors with a BMI of 30 to 34 or a donor age between 50 and 60 years. Immunosuppression is generally standardized using induction therapy with Myfortic, tacrolimus and low dose steroids. In principle, all patients on the waitlist for primary SPK, PTA or PAK are eligible for the clinical trial when they consent to possibly receiving an extended donor criteria organ. Patients receiving an organ meeting the current standard criteria for pancreas allocation (control arm) are compared to those receiving extended criteria organ (study arm); patients are blinded for a follow-up period of one year. The combined primary endpoint is survival of the pancreas allograft and pancreas allograft function after three months, as an early relevant outcome parameter for pancreas transplantation.
The EXPAND Study has been initiated to investigate the hypothesis that locally allocated extended criteria organs can be transplanted with similar results compared to the currently allowed standard ET organ allocation. If our study shows a favorable comparison to standard organ allocation criteria, the morbidity and mortality for patients waiting for transplantation could be reduced in the future.
Trial registration
Trial registered at: NCT01384006
PMCID: PMC3716891  PMID: 23816330
Pancreas transplantation; Organ allocation; Extended donor criteria; Rejection
9.  Cytokines Mediating the Induction of Chronic Colitis and Colitis-Associated Fibrosis 
Mucosal immunology  2008;1(0 1):S24-S27.
To investigate the immunopathogenesis of inflammation-associated fibrosis we analyzed the chronic colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intra-rectal trinitrobenzene sulfonic acid (TNBS). We showed first in this model that an initial Th1 response involving IL-12p70 and IFN-γ subsides after three weeks to be supplanted by an IL-23/IL-25 response beginning after 4–5 weeks. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a Th2 response, particularly IL-13, which reaches a plateau at 8–9 weeks. We then show that IL-13 production results in the induction of an IL-13 receptor formerly thought to function only as a decoy receptor, IL-13Rα2, and this receptor is critical to the production of TGF-β1 and the onset of fibrosis. Thus, if IL-13 signaling through this receptor is blocked by administration of soluble IL-13Rα2-Fc, or by administration of IL-13Rα2–specific siRNA, TGF-β1 is not produced and fibrosis does not occur. These studies show that in chronic TNBS colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell-surface-expressed IL-13 receptor to induce TGF-β1.
PMCID: PMC3673699  PMID: 19079223
10.  Treatment-emergent adverse events after infusion of adherent stem cells: the MiSOT-I score for solid organ transplantation 
Trials  2012;13:211.
Cellular therapy after organ transplantation is emerging as an intriguing strategy to achieve dose reduction of classical immunosuppressive pharmacotherapy. Here, we introduce a new scoring system to assess treatment-emergent adverse events (TEAEs) of adherent stem cell therapies in the clinical setting of allogeneic liver transplantation (for example, the MiSOT-I trial Eudract CT: 2009-017795-25).
The score consists of three independent modalities (set of parameters) that focus on clinically relevant events early after intravenous or intraportal stem cell infusion: pulmonary toxicity, intraportal-infusional toxicity and systemic toxicity. For each modality, values between 0 (no TEAE) and 3 (severe TEAE) were defined. The score was validated retrospectively on a cohort of n=187 recipients of liver allografts not receiving investigational cell therapy between July 2004 and December 2010. These patients represent a control population for further trials. Score values were calculated for days 1, 4, and 10 after liver transplantation.
Grade 3 events were most commonly related to the pulmonary system (3.5% of study cohort on day 4). Almost no systemic-related TEAEs were observed during the study period. The relative frequency of grade 3 events never exceeded 5% over all modalities and time points. A subgroup analysis for grade 3 patients provided no descriptors associated with severe TEAEs.
The MiSOT-I score provides an assessment tool to score specific adverse events that may occur after adherent stem cell therapy in the clinical setting of organ transplantation and is thus a helpful tool to conduct a safety study.
PMCID: PMC3543274  PMID: 23151227
Adherent adult stem cells; Mesenchymal stem cells; Multipotent adult progenitor cells; Solid organ transplantation; Immunotherapy; Scoring adverse events; Phase I trial
11.  STAT5b as Molecular Target in Pancreatic Cancer—Inhibition of Tumor Growth, Angiogenesis, and Metastases12 
Neoplasia (New York, N.Y.)  2012;14(10):915-925.
The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.
PMCID: PMC3479837  PMID: 23097626
12.  Friend or foe 
Oncoimmunology  2012;1(7):1159-1160.
For many years, β-defensins were best known for their antimicrobial activity. However, β-defensins also exert immunomodulatory functions, such as the chemotactic recruitment of immune cells via chemokine receptors. We demonstrated that mouse β-defensin 14 recruits CCR6+ B cells into fibrosarcomas, resulting in enhanced angiogenesis and tumor development.
PMCID: PMC3494629  PMID: 23170263
angiogenesis; antimicrobial peptide; chemokine receptor 6; defensin; lymphotoxin β-receptor; tumor growth
13.  Alternative approaches to myeloid suppressor cell therapy in transplantation: comparing regulatory macrophages to tolerogenic DCs and MDSCs 
Several types of myeloid suppressor cell are currently being developed as cell-based immunosuppressive agents. Despite detailed knowledge about the molecular and cellular functions of these cell types, expert opinions differ on how to best implement such therapies in solid organ transplantation. Efforts in our laboratory to develop a cell-based medicinal product for promoting tolerance in renal transplant patients have focused on a type of suppressor macrophage, which we call the regulatory macrophage (M reg). Our favoured clinical strategy is to administer donor-derived M regs to recipients one week prior to transplantation. In contrast, many groups working with tolerogenic dendritic cells (DCs) advocate post-transplant administration of recipient-derived cells. A third alternative, using myeloid-derived suppressor cells, presumably demands that cells are given around the time of transplantation, so that they can infiltrate the graft to create a suppressive environment. On present evidence, it is not possible to say which cell type and treatment strategy might be clinically superior. This review seeks to position our basic scientific and early-stage clinical studies of human regulatory macrophages within the broader context of myeloid suppressor cell therapy in transplantation.
PMCID: PMC3561050  PMID: 23369628
Regulatory macrophage; M reg; Cell-based medicinal product; The ONE Study
15.  Deletion of Foxp3+ regulatory T cells in genetically targeted mice supports development of intestinal inflammation 
BMC Gastroenterology  2012;12:97.
Mice lacking Foxp3+ regulatory T (Treg) cells develop severe tissue inflammation in lung, skin, and liver with premature death, whereas the intestine remains uninflamed. This study aims to demonstrate the importance of Foxp3+ Treg for the activation of T cells and the development of intestinal inflammation.
Foxp3-GFP-DTR (human diphtheria toxin receptor) C57BL/6 mice allow elimination of Foxp3+ Treg by treatment with Dx (diphtheria toxin). The influence of Foxp3+ Treg on intestinal inflammation was tested using the CD4+ T-cell transfer colitis model in Rag−/− C57BL/6 mice and the acute DSS-colitis model.
Continuous depletion of Foxp3+ Treg in Foxp3-GFP-DTR mice led to dramatic weight loss and death of mice by day 28. After 10 days of depletion of Foxp3+ Treg, isolated CD4+ T-cells were activated and produced extensive amounts of IFN-γ, IL-13, and IL-17A. Transfer of total CD4+ T-cells isolated from Foxp3-GFP-DTR mice did not result in any changes of intestinal homeostasis in Rag−/− C57BL/6 mice. However, administration of DTx between days 14 and 18 after T-cell reconstitution, lead to elimination of Foxp3+ Treg and to immediate weight loss due to intestinal inflammation. This pro-inflammatory effect of Foxp3+ Treg depletion consecutively increased inflammatory cytokine production. Further, the depletion of Foxp3+ Treg from Foxp3-GFP-DTR mice increased the severity of acute dSS-colitis accompanied by 80% lethality of Treg-depleted mice. CD4+ effector T-cells from Foxp3+ Treg-depleted mice produced significantly more pro-inflammatory cytokines.
Intermittent depletion of Foxp3+ Treg aggravates intestinal inflammatory responses demonstrating the importance of Foxp3+ Treg for the balance at the mucosal surface of the intestine.
PMCID: PMC3449180  PMID: 22849659
16.  Open new possibilities in Transplantation Research 
PMCID: PMC3552568  PMID: 23369410
17.  Safety and feasibility of third-party multipotent adult progenitor cells for immunomodulation therapy after liver transplantation--a phase I study (MISOT-I) 
Liver transplantation is the definitive treatment for many end-stage liver diseases. However, the life-long immunosuppression needed to prevent graft rejection causes clinically significant side effects. Cellular immunomodulatory therapies may allow the dose of immunosuppressive drugs to be reduced. In the current protocol, we propose to complement immunosuppressive pharmacotherapy with third-party multipotent adult progenitor cells (MAPCs), a culture-selected population of adult adherent stem cells derived from bone marrow that has been shown to display potent immunomodulatory and regenerative properties. In animal models, MAPCs reduce the need for pharmacological immunosuppression after experimental solid organ transplantation and regenerate damaged organs.
Patients enrolled in this phase I, single-arm, single-center safety and feasibility study (n = 3-24) will receive 2 doses of third-party MAPCs after liver transplantation, on days 1 and 3, in addition to a calcineurin-inhibitor-free "bottom-up" immunosuppressive regimen with basiliximab, mycophenolic acid, and steroids. The study objective is to evaluate the safety and clinical feasibility of MAPC administration in this patient cohort. The primary endpoint of the study is safety, assessed by standardized dose-limiting toxicity events. One secondary endpoint is the time until first biopsy-proven acute rejection, in order to collect first evidence of efficacy. Dose escalation (150, 300, 450, and 600 million MAPCs) will be done according to a 3 + 3 classical escalation design (4 groups of 3-6 patients each).
If MAPCs are safe for patients undergoing liver transplantation in this study, a phase II/III trial will be conducted to assess their clinical efficacy.
PMCID: PMC3166276  PMID: 21798013
18.  DX5+NKT cells display phenotypical and functional differences between spleen and liver as well as NK1.1-Balb/c and NK1.1+ C57Bl/6 mice 
BMC Immunology  2011;12:26.
Natural killer T cells represent a linkage between innate and adaptive immunity. They are a heterogeneous population of specialized T lymphocytes composed of different subsets. DX5+NKT cells are characterized by expression of the NK cell marker DX5 in the context of CD3. However, little is known about the phenotype and functional capacity of this unique cell population. Therefore, we investigated the expression of several T cell and NK cell markers, as well as functional parameters in spleen and liver subsets of DX5+NKT cells in NK1.1- Balb/c mice and compared our findings to NK1.1+ C57Bl/6 mice.
In the spleen 34% of DX5+NKT cells expressed CD62L and they up-regulated the functional receptors CD154 as well as CD178 upon activation. In contrast, only a few liver DX5+NKT cells expressed CD62L, and they did not up-regulate CD154 upon activation. A further difference between spleen and liver subsets was observed in cytokine production. Spleen DX5+NKT cells produced more Th1 cytokines including IL-2, IFN-γ and TNF-α, while liver DX5+NKT cells secreted more Th2 cytokines (e.g. IL-4) and even the Th17 cytokine, IL-17a. Furthermore, we found inter-strain differences. In NK1.1+ C57Bl/6 mice DX5+NKT cells represented a distinct T cell population expressing less CD4 and more CD8. Accordingly, these cells showed a CD178 and Th2-type functional capacity upon activation.
These results show that DX5+NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity.
PMCID: PMC3097004  PMID: 21529347
19.  Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80+CD11bhighGr1low macrophages  
The Journal of Clinical Investigation  2011;121(5):1692-1708.
Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13–producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11bhighGr1low M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate–induced colitis, F4/80+CD11bhighGr1intermediate macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88–deficient (Myd88–/–) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88–/– mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11bhighGr1intermediate macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.
PMCID: PMC3083803  PMID: 21519141
20.  Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition 
BMC Cancer  2010;10:668.
Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer.
Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression.
The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer.
In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.
PMCID: PMC3003660  PMID: 21129190
21.  Study Protocol: A Pilot Study to Determine the Safety and Efficacy of Induction-Therapy, De Novo MPA and Delayed mTOR-Inhibition in Liver Transplant Recipients with Impaired Renal Function. PATRON-Study 
BMC Nephrology  2010;11:24.
Patients undergoing liver transplantation with preexisting renal dysfunction are prone to further renal impairment with the early postoperative use of Calcineurin-inhibitors. However, there is only little scientific evidence for the safety and efficacy of de novo CNI free "bottom-up" regimens in patients with impaired renal function undergoing liver transplantation. This is a single-center study pilot-study (PATRON07) investigating safety and efficacy of CNI-free, "bottom-up" immunosuppressive (IS) strategy in patients undergoing liver transplantation (LT) with renal impairment prior to LT.
Patients older than 18 years with renal impairment at the time of liver transplantation eGFR < 50 ml/min and/or serum creatinine levels > 1.5 mg/dL will be included. Patients in will receive a CNI-free combination therapy (basiliximab, MMF, steroids and delayed Sirolimus). Primary endpoint is the incidence of steroid resistant acute rejection within the first 30 days after LT. The study is designed as prospective two-step trial requiring a maximum of 29 patients. In the first step, 9 patients will be included. If 8 or more patients show no signs of biopsy proven steroid resistant rejection, additional 20 patients will be included. If in the second step a total of 27 or more patients reach the primary endpoint the regimen is regarded to be safe and efficient.
If a CNI-free-"bottom-up" IS strategy is safe and effective, this may be an innovative concept in contrast to classic top-down strategies that could improve the patient short and long-time renal function as well as overall complications and survival after LT. The results of PATRON07 may be the basis for a large multicenter RCT investigating the new "bottom-up" immunosuppressive strategy in patients with poor renal function prior to LT. NCT00604357
PMCID: PMC2945344  PMID: 20840760
22.  A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma 
BMC Cancer  2010;10:190.
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.
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.
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 NCT00355862
(EudraCT Number: 2005-005362-36)
PMCID: PMC2889889  PMID: 20459775
23.  Restoration of Tumor Immune Surveillance via Targeting of IL-13Receptor-α2 
Cancer research  2008;68(9):3467-3475.
In previous studies we described a “counter-immunosurveillance” mechanism initiated by tumor-activated, IL-13-producing NKT cells that signal Gr-1+ cells to produce TGF-β1, a cytokine that suppresses the activity of tumor-inhibiting cytolytic CD8+ T cells. Here we show that in two tumor models (the CT-26 metastatic colon cancer and the 15-12RM fibrosarcoma regressor models) this counter-surveillance mechanism requires the expression of a novel IL-13 receptor, IL-13Rα2, on Gr-1intermediate cells, since down-regulation of IL-13Rα2 expression or the AP-1 signal generated by the receptor via in vivo administration of specific siRNA or decoy oligonucleotides leads to loss of TGF-β1 production. Furthermore, acting on prior studies showing that IL-13Rα2 expression is induced (in part) by TNF-α, we show that receptor expression and TGF-β1 production is inhibited by administration of a TNF-α neutralizing substance, TNF-αR-Fc (etanercept). Taking advantage of this latter fact, we then demonstrate in the CT-26 model that counter-immunosurveillance could be inhibited, anti-CT-26-specific CD8+ cytolytic activity restored, and CT-26 metastatic tumor nodules greatly decreased by administration of TNF-αR-Fc. Corroborative data was obtained using the 15-12RM fibrosarcoma model. These studies point to the prevention of metastatic cancer with an available agent with already known clinically acceptable adverse effects and toxicity.
PMCID: PMC2746996  PMID: 18451175
24.  ENMD-1198, a novel tubulin-binding agent reduces HIF-1alpha and STAT3 activity in human hepatocellular carcinoma(HCC) cells, and inhibits growth and vascularization in vivo 
BMC Cancer  2008;8:206.
Hepatocellular carcinoma (HCC) represents a highly vascularized tumor entity and the process of angiogenesis is essential for the growth of HCC. Importantly, the pro-angiogenic transcription factors HIF-1α and STAT3 have been implicated in HCC progression, thus representing interesting targets for molecular targeted therapy. We hypothesized that therapeutic inhibition of HIF-1α could be achieved by using a novel tubulin-binding agent (ENMD-1198). ENMD-1198 is an analog of 2-methoxyestradiol (2ME2) with antiproliferative and antiangiogenic activity.
The human HCC cell lines HUH-7 and HepG2 were used for experiments. Effects of ENMD-1198 on constitutive and inducible (hypoxia, growth factors) activation of signaling cascades, including HIF-1α and STAT3, were investigated by Western blotting. Changes in VEGF expression were determined by real-time PCR. Effects of ENMD-1198 on cancer cell migration and invasion were evaluated in in vitro-assays. The growth-inhibitory effects of ENMD-1198 (200 mg/kg/day) were determined in a subcutaneous tumor model (HUH-7).
ENMD-1198 inhibited the phosphorylation of MAPK/Erk, PI-3K/Akt and FAK. Moreover, activation of HIF-1α and STAT3 was dramatically reduced by ENMD-1198, which resulted in lower VEGF mRNA expression (P < 0.05). In addition, tumor cell migratory and invasive properties were significantly inhibited (P < 0.05, for both). In vivo, treatment with ENMD-1198 led to a significant reduction in tumor growth, tumor vascularization, and numbers of proliferating tumor cells (P < 0.05 for all).
The novel microtubule destabilizing agent ENMD-1198 is suitable for inhibiting HIF-1α and STAT3 in human HCC cells and leads to reduced tumor growth and vascularization in vivo. Hence, inhibition of HIF-1α and STAT3 could prove valuable for therapy of hepatocellular carcinoma.
PMCID: PMC2496914  PMID: 18651980

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