Jens Bedke

Physical and Biological Characterization of Superparamagnetic Iron Oxide- and Ultrasmall Superparamagnetic Iron Oxide-labeled Cells: A Comparison

Rationale:Superparamagnetic iron-oxide particles are used frequently for cellular magnetic resonance imaging and in vivo cell tracking. The purpose of this study was to compare the labeling characteristics and efficiency as well as toxicity of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) for 3 cell lines. Methods:Using human fibroblasts, immortalized rat progenitor cells and HEP-G2-hepatoma cells, dose- and time-dependence of SPIO and USPIO uptake were evaluated. The amount of intracellular (U)SPIO was monitored over 2 weeks after incubation by T2-magnetic resonance relaxometry, ICP-mass-spectrometry, and histology. Transmission-electronmicroscopy was used to specify the intracellular localization of the endocytosed iron particles. Cell death-rate and proliferation-index were assessed as indicators of cell-toxicity. Result:For all cell lines, SPIO showed better uptake than USPIO, which was highest in HEP-G2 cells (110 ± 2 pg Fe/cell). Cellular iron concentrations in progenitor cells and fibroblasts were 13 ± 1pg Fe/cell and 7.2 ± 0.3pg Fe/cell, respectively. For all cell lines T2-relaxation times in cell pellets were below detection threshold (<3 milliseconds) after 5 hours of incubation with SPIO (3.0 μmol Fe/mL growth medium) and continued to be near the detection for the next 6 days. For both particle types and all cell lines cellular iron oxide contents decreased after recultivation and surprisingly were found lower than in unlabeled control cells after 15 days. Viability and proliferation of (U)SPIO-labeled and unlabeled cells were not significantly different. Conclusions:The hematopoetic progenitor, mesenchymal fibroblast and epithelial HEP-G2 cell lines accumulated SPIO more efficiently than USPIO indicating SPIO to be better suited for cell labeling. However, the results indicate that there may be an induction of forced cellular iron elimination after incubation with (U)SPIO.

A novel CXCL8 protein-based antagonist in acute experimental renal allograft damage.

Acute renal allograft damage is caused by early leukocyte infiltration which is mediated in part by chemokines presented by glycosaminoglycan (GAG) structures on endothelial surfaces. CXCL8 can recruit neutrophils and induce the firm arrest of monocytes on activated endothelial cells. A human CXCL8-based antagonist (dnCXCL8) designed to generate a dominant-negative mutant protein with enhanced binding to GAG structures and reduced CXCR1/2 receptor binding ability was tested in models of early allograft injury. The agent displayed enhanced binding to GAG structures in vitro and could antagonize CXCL8-induced firm adhesion of monocytes as well as neutrophils to activated microvascular endothelium in physiologic flow assays. In a rat model of acute renal damage, dnCXCL8 treatment limited proximal tubular damage and reduced granulocyte infiltration. In a Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat acute renal allograft model, dnCXCL8 was found to reduce monocyte and CD8+ T-cell infiltration into glomeruli and to limit tubular interstitial inflammation and tubulitis in vivo. Early treatment of allografts with agents like dnCXCL8 may help reduce acute allograft damage and preserve renal morphology and thereby help limit chronic dysfunction.

Role of xanthine oxidoreductase in experimental acute renal-allograft rejection.

Background. Increased oxygen radical production may not only contribute to posttransplant ischemia-reperfusion injury but also to acute rejection of renal allografts. Xanthine oxidoreductase (XOR) may constitute a relevant reactive oxygen species (ROS) source. The study was conducted (1) to determine ROS production as well as oxidant and antioxidant enzyme activities in renal grafts and (2) to modulate acute rejection by tungsten administration, a specific inhibitor of XOR. Methods. Syngraft (Lewis to Lewis, Fisher344 to Fisher344) and allograft (Fisher344 to Lewis) kidney transplantations were performed with or without tungsten administration. Analysis was performed at day 1, 3, or 9 posttransplantation. Results. Generation of ROS was enhanced, being 10-fold higher in renal allografts versus control kidneys at day 9 (P <0.01); this was associated with histologic signs of acute rejection. Oxygen radicals were generated to a significant degree by enhanced XOR activity, which increased more than 10-fold in renal allografts at day 9 posttransplantation; XOR protein in glomeruli and tubulointerstitium was also elevated in allo-grafts. In addition, NADPH oxidase activity increased significantly in allografts. The activity of antioxidant enzymes tended to decrease. Tungsten treatment resulted in a pronounced reduction of XOR activity and ROS production, without any effect on NADPH-oxidase activity; mononuclear cell infiltration and rejection signs were significantly ameliorated at day 9 post-transplantation by selective inhibition of XOR. Conclusions. A major part of ROS generation in acute rejection was contributed by XOR. ROS are not only associated with but also contribute to acute allograft rejection because inhibition of XOR alleviated rejection phenomena.

Survival Prediction of Clear Cell Renal Cell Carcinoma Based on Gene Expression Similarity to the Proximal Tubule of the Nephron

UNLABELLEDnThere is evidence that molecular features support subclassification of tumours, thereby improving prediction of patient outcome. Currently, two gene expression signatures (ccA/ccB and ClearCode34) have been established to classify clear cell renal cell carcinoma (ccRCC). Because RCC arises from nephron cell types, we aimed to explore its heterogeneity on a molecular level by comparing gene expression between tumour tissue and nephron regions. Based on genes that differ in expression between nephron regions, expression data of 479 ccRCCs and 212 papillary and 66 chromophobe RCCs from The Cancer Genome Atlas were correlated to those of nephron cell types. Cancer-specific survival (CSS) of ccRCC patients was significantly associated with gene expression similarity to the proximal tubules. Subsequently, a ccRCC risk score (S3-score) was established. Survival analyses indicated that the S3-score was significantly associated with CSS considering all cases of ccRCC, as well as metastatic and nonmetastatic ccRCC. Results could be validated in an independent cohort. The S3-score significantly improved the predictive ability of the ccA/ccB and ClearCode34 signatures, and the clinicopathologic-based stage, size, grade, and necrosis score (p [chi-square] = 1.56E-04). Intratumour heterogeneity of the S3-score was observed in 6 of 10 ccRCCs. In summary, the nephron-based S3-score enables prognostic risk stratification for ccRCC. Further studies are needed to evaluate its clinical utility.nnnPATIENT SUMMARYnWe developed a novel risk score for clear cell renal cell carcinoma to identify patients at risk of worse outcome that may improve patient care in the future.

Peroxisome Proliferator-Activated Receptor (PPAR)γ can inhibit chronic renal allograft damage

Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.

Suppression of chronic damage in renal allografts by Liver X receptor (LXR) activation relevant contribution of macrophage LXRα.

Liver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXRα (mLXRα-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow-derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXRα-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXRα-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1- and mannose receptor C type 1-positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXRα overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXRα in allograft rejection and prevention of fibrosis.

mRNA vaccine CV9103 and CV9104 for the treatment of prostate cancer

Among currently available vaccine strategies for cancer, nucleotide-based vaccination is an appealing treatment modality. Curevacs’ mRNA containing vaccines (RNActive®) combine the beneficial properties of sufficient antigen-expression, autologous immune-stimulation and a high flexibility with respect to production and application. CV9103 and CV9104 are novel RNActive®-derived anticancer vaccines for the treatment of patients with prostate cancer. After successful phase I/II studies with documentation of good tolerability and favorable immune-activation of CV9103, the vaccine CV9104 is currently undergoing clinical testing in specific clinical settings such as castration resistant prostate cancer and as a neoadjuvant agent in men with high risk prostate cancer prior to surgery. This review discusses the available preclinical and clinical data on the anticancer vaccination treatment with RNActive®-derived anticancer-vaccines CV9103 and CV9104.

Checkpoint modulation--A new way to direct the immune system against renal cell carcinoma.

The introduction of targeted therapies like the tyrosine kinase (TKI) and mammalian target of rapamycin (mTOR) inhibitors has improved patients´ survival in general. Nevertheless the prognosis remains limited. Therapies with a new mode of action are urgently warranted, especially those who would provoke long-term responders or long-lasting complete remissions as observed with unspecific immunotherapy with the cytokines interleukin-2 and interferon-α. In the recent years a deeper understanding of the underlying immunology of T cell activation led to the development of checkpoint inhibitors, which are mainly monocloncal antibodies and which enhances the presence of the co-stimulatory signals needed for T cell activation or priming. This review discusses the clinical data and ongoing studies available for the inhibition of the PD-1 (CD279) and CTLA-4 (CD152) axis in mRCC. In addition, potential future immunological targets are discussed. This approach of T-cell activation or re-activation by immunological checkpoint inhibition holds the inherent promise to directly affect the tumor cell and thereby to potentially cure a subset of patients with mRCC.

Comment on “Epigenetic activation of the drug transporter OCT2 sensitizes renal cell carcinoma to oxaliplatin”

Although the combination of decitabine and platinum drugs may be promising for therapy of renal cell carcinoma, the role of OCT2 needs further investigations. Although the combination of decitabine and platinum drugs may be promising for therapy of renal cell carcinoma, the role of OCT2 needs further investigations.

Viral macrophage inflammatory protein-II improves acute rejection in allogeneic rat kidney transplants.

PurposeDuring rejection, leukocytes are recruited from the peripheral circulation into the graft leading to the damage of endothelial cells, capillary perfusion failure and graft loss. Chemokines play a pivotal role in the recruitment of leukocytes to the endothelium. Viral macrophage inflammatory protein-II (vMIP-II), a human herpes virus-8 DNA-encoded protein, is a broad-spectrum chemokine antagonist. The aim of the study was to prove the beneficial activity of vMIP-II treatment on acute rat kidney allograft damage.MethodsHeterotopic rat kidney transplantation was performed in the Fischer 344 to Lewis transplantation model and animals were treated with vMIP-II (2xa0×xa015xa0µg or 100xa0µg/day) for 7xa0days. Rejection-induced damage was analyzed by histology, and microcirculatory changes within the graft were analyzed by in vivo microscopy.ResultsViral macrophage inflammatory protein-II significantly improved acute glomerular damage and tubulointerstitial inflammation and lowered the extent of vascular and tubulointerstitial damage of the treated allografts. Functional microcirculation of peritubular capillaries was significantly improved in vivo, and the firm adherence of leukocytes was significantly reduced by vMIP-II treatment.ConclusionsThe administration of the broad-spectrum antagonist vMIP-II improved acute renal allograft damage, mainly by a reduction in leukocyte recruitment with a subsequently improved renal cortical microcirculation in vivo.