Holger Rumpold

CD4+CD25+ Regulatory T Cells Inhibit Experimental Anti-Glomerular Basement Membrane Glomerulonephritis in Mice

CD4+CD25+ regulatory T cells (Treg) are of critical importance for the maintenance of tolerance. The kidney is frequently involved in autoimmune diseases, such as lupus erythematosus or glomerulonephritis (GN). Therefore, the therapeutic efficacy of Treg in a T cell-dependent murine model of experimental anti-glomerular basement membrane (anti-GBM) GN was tested. Transfer of 1 x 10(6) CD4+CD25+ T cells (day -1) into mice that were previously immunized with rabbit IgG (day -3) and subsequently received an injection of anti-GBM rabbit serum (day 0) significantly attenuated the development of proteinuria when compared with animals that received an injection of 1 x 10(6) CD4+CD25- T cells (control group). Treg injection induced a dramatic decrease of glomerular damage as well as a marked decrease of CD4+ T cell, CD8+ T cell, and macrophage infiltration. Of note, deposition of immune complexes was not prevented by Treg, showing that Treg rather inhibited cell-mediated organ damage than priming of the humoral immune response. Accordingly, a significant reduction of IFN-gamma, TNF-alpha, and TGF-beta1 mRNA in kidneys from animals that received Treg injection was observed. Tracking of enhanced green fluorescence protein-transgenic Treg revealed a predominant migration to secondary lymphoid organs with a significant increase of regulatory T cells (CD4+CD25+CD69-CD45RB(low)) in the lymph nodes. In contrast, enhanced green fluorescence protein-and FoxP3-positive cells by reverse transcription-PCR and CD4+CD25+CD69-CD45RB(low) T cells by flow cytometry in the kidney of nephritic animals were not detected. This report provides first evidence that Treg are potent suppressors of anti-GBM GN. Treg therefore might be of therapeutic value for the treatment of severe GN in humans.

Regulatory T-cells in the graft and the risk of acute graft-versus-host disease after allogeneic stem cell transplantation

Background. FOXP3+ regulatory T-cells (Treg) are important regulators of allo-reactivity and may therefore represent an important predictor for the risk of graft versus-host disease (GVHD) after allogeneic stem cell transplantation. Methods. To determine the clinical significance of Treg-content in stem cell grafts, we analyzed 58 human leukocyte antigen (HLA)-identical sibling donors (34 patients received myeloablative and 24 patients reduced intense conditioning regimens) and correlated the Treg frequency with clinical outcome after stem cell transplantation (SCT). Results. A mean value of 9.1×106 CD4+FOXP3+ Treg per kg body weight (bw) of the recipient was transplanted (ranging from 0.7 to 33.7×106 Treg/kg bw). Graft content of Treg correlated with mononuclear cells and CD3+ T-cells. Patients receiving low numbers of Treg (Treglow) after myeloablative conditioning for SCT had a significantly increased cumulative incidence of 76% for acute GVHD when compared with 23% for individuals receiving high numbers of Treg (Treghigh). This observation, however, was not made in patients after reduced intense conditioning-SCT. Notably, relapse rate was not significantly different between Treglow and Treghigh patients in either patient group and overall survival was even increased in Treghigh patients after myeloablative SCT. Finally, low Treg graft levels represent an independent prognostic factor in multivariate analysis for the appearance of acute GHVD. Conclusion. Donor-derived Treg might be of particular significance for the development of acute GVHD after myeloablative SCT using HLA-identical sibling donors.

Endothelial progenitor cells: a source for therapeutic vasculogenesis?

Angiogenesis has been defined as sprouting of blood vessels from pre‐existing vascular structures. Risau and coworkers defined the term vasculogenesis while studying the formation of new blood vessels in embryoid bodies. This process is characterized by the recruitment of endothelial progenitor cells (EPC) to sites of new vessel formation with subsequent differentiation of EPC into mature endothelial cells, extensively proliferating in situ. Data from recent years provided evidence that EPC also exist in the adult and contribute to new vessel formation, a process called postnatal vasculogenesis. The existence of EPC has been convincingly shown in both, animals and humans. They represent a perfect cellular progenitor cell population for the ex vivo generation of EC, which in turn serve as cellular source for therapeutic vasculogenesis or tumor targeting. This review provides an overview on this hot topic of cellular‐based therapeutic concepts and the therapeutic potential of ex vivo generated EPC.

Telomere length of in vivo expanded CD4 + CD25 + regulatory T-cells is preserved in cancer patients

Purpose: CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. Experimental Design: Treg and CD4+CD25− T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. Results: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. Conclusions: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.

CD34+/CD133- circulating endothelial precursor cells (CEP) : Characterization, senescence and in vivo application

Circulating endothelial precursor cells (CEP) are interesting candidates for the treatment of ischemic diseases and for tumor targeting/imaging. We isolated a homogeneous population of CEP from CD34(+)/CD133(-) cells of peripheral blood that can be expanded easily on collagen-type-I coated plastic. CEP displayed a phenotype of mature endothelial cells (vWF, CD31, CD34, VEGF-R2, CD105, CD146) similar to that of cord-blood CEP and umbilical vein endothelial cells. They bound UEA-1 lectin, incorporated acetylated LDL and formed tube-like structures with capillary lumens in vitro. Weibel-Palade bodies were observed by electron microscopy. After 40-60 cell population doublings, CEP cultures underwent a terminal growth arrest, had shorter telomeres, up-regulated cell cycle inhibitory proteins, such as p21(CIP1) and stained positive for senescence-associated-beta galactosidase. During the whole expansion period CEP retained their endothelial phenotype and a normal karyotype. CEP had the capacity to home to ischemic tissue in vivo after systemic injection in nude rats. The convenient expandability, the homogenous phenotype, the functional cellular senescence program, the regular karyotype and the homing capacity to ischemic myocardium suggest autologous CEP cultures as a safe and promising tool for cell-based therapeutic approaches in targeting ischemic tissue and tumors.

Intratumoral interferon regulatory factor (IRF)-1 but not IRF-2 is of relevance in predicting patient outcome in ovarian cancer

IRF‐1 and IRF‐2 expression was determined by real‐time PCR in 138 ovarian cancer samples and 30 healthy ovarian biopsies and was correlated with the expression of other relevant immunologic parameters and common clinicopathologic variables. Regulation of IRF‐1 and IRF‐2 was evaluated by cytokine treatment of various ovarian cancer cell lines, human peritoneal mesothelial cells and ovarian surface epithelium. IRF‐1 but not IRF‐2 was constitutively over‐expressed in 5 of 7 ovarian cancer cell lines. Both IRFs were inducible with IFN‐γ and to a lesser extent with IL‐1 or TNF‐α, but not with IL‐6. Epidermal growth factor (EGF) treatment down‐regulated both IRFs. In ovarian cancer samples only IRF‐1, but not IRF‐2 mRNA, was up‐regulated when compared with healthy ovarian tissue. IRF‐1 but not IRF‐2 expression was significantly associated with interferon (IFN)‐γ and forkhead box P3 (FoxP3). In univariate survival analysis, strong expression of IRF‐1 and IRF‐2 predicted improved disease‐free survival (DFS) and overall survival (OS). In Cox regression analyses, IRF‐1 retained independent prognostic significance for DFS and OS and IFN‐γ for OS. In contrast to other solid tumors, IRF‐2 expression cannot be regarded as a classic oncoprotein associated with poor prognosis in ovarian cancer. Of the immunologic parameters investigated, intratumoral IRF‐1 expression is the most powerful independent predictor of a favorable clinical outcome.

siRNA-Mediated Knock-Down of P-Glycoprotein Expression Reveals Distinct Cellular Disposition of Anticancer Tyrosine Kinases Inhibitors

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5), indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.

Iron metabolism and iron supplementation in cancer patients

SummaryIron deficiency and iron deficiency-associated anemia are common complications in cancer patients. Most iron deficient cancer patients present with functional iron deficiency (FID), a status with adequate storage iron, but insufficient iron supply for erythroblasts and other iron dependent tissues. FID is the consequence of the cancer-associated cytokine release, while in absolute iron deficiency iron stores are depleted resulting in similar but often more severe symptoms of insufficient iron supply. Here we present a short review on the epidemiology, pathophysiology, diagnosis, clinical symptoms, and treatment of iron deficiency in cancer patients. Special emphasis is given to intravenous iron supplementation and on the benefits and limitations of different formulations. Based on these considerations and recommendations from current international guidelines we developed recommendations for clinical practice and classified the level of evidence and grade of recommendation according to the principles of evidence-based medicine.

Drug Transporter-Mediated Protection of Cancer Stem Cells From Ionophore Antibiotics

Ionophore antibiotics were reported to selectively kill cancer stem cells and to overcome multidrug resistance, but mechanistic studies of the significance of drug transporters for treatment with these compounds are lacking. We applied chemosensitivity testing of well‐characterized human cancer cell lines to elaborate on whether drug transporters are involved in protection from the cytotoxic effects of the ionophore antibiotics salinomycin and nigericin. Our experiments demonstrated that ionophore antibiotics were ineffective against both stem‐like ovarian cancer side population cells (expressing either ABCB1 or ABCG2) and K562/Dox‐H1 cells, which constitute a genetically defined model system for ABCB1 expression. Considering that cancer stem cells often express high levels of drug transporters, we deduced from our results that ionophore antibiotics are less suited to cancer stem cell‐targeted treatment than previously thought.

DyeCycle violet used for side population detection is a substrate of P‐glycoprotein

Cancer stem cells (CSC) are increasingly recognized as key target cells for cancer therapy because they are both tumorigenic and chemoresistant and, therefore, can give rise to recurrent disease. Side population (SP) sorting using an ultraviolet (UV) laser is an established method to isolate CSC based on ABC drug transporter‐dependent (e.g., ABCG2) Hoechst 33342 efflux. We here show that Vybrant® DyeCycle™ Violet (DCV), a DNA‐binding fluorophor allowing SP sorting using a non‐UV laser (such as violet laser), is transported via P‐glycoprotein (PgP). Because PgP might be particularly abundant in multidrug‐resistant cancer cells rather than bona fide CSC, investigators using DCV should be aware that this strategy might also detect PgP‐expressing non‐CSC.