Jörg Hennenlotter

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.

Effective Treatment of Neurogenic Detrusor Dysfunction by Combined High-Dosed Antimuscarinics without Increased Side-Effects

OBJECTIVES Patients with neurogenic bladder dysfunction demonstrate an insufficient treatment outcome under dosage-escalated monotherapy. With the objectives of continence and normalised bladder pressure, safe and tolerable non-invasive treatment alternatives were evaluated by using combined antimuscarinics. METHODS Twenty-seven patients who were previously registered in a doubled antimuscarinics study were enrolled in this study. The patients demonstrated urodynamic-proven neurogenic bladder dysfunction with incontinence, reduced bladder capacity, and increased intravesical pressure, resulting from spinal cord injury (n=21); spinal cord dysplasia (myelomeningocele; n=3); multiple sclerosis (n=2), and viral encephalomyelitis (n=1). On the basis of the initial study treatment, they were allocated into three groups and treated with two antimuscarinics. Before enrollment, at 4 wk, and at 6 mo, patients underwent urodynamics and recorded bladder diaries, including side-effects. RESULTS In all three groups, significant changes were noted at the 4-wk follow-up. Incontinence events decreased from an average of 7 to 1 event per day. The average median bladder capacity (180-393 ml) and reflex volume (125-335 ml) increased; detrusor compliance also improved (average, 15-33 ml/cm H2O). Seven patients reported side-effects; two discontinued the successful treatment. Two other patients did not reach satisfactory amelioration of the detrusor dysfunction. CONCLUSION With combined high-dosage antimuscarinic medications, 85% of the patients who previously demonstrated unsatisfactory outcome with dosage-escalated monotherapy were treated successfully. The appearance of side-effects was comparable to that of normal-dosed antimuscarinics. Further studies are required to investigate the long-term pharmacological and physiological background of our findings.

Distorted Relation between mRNA Copy Number and Corresponding Major Histocompatibility Complex Ligand Density on the Cell Surface

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to T-cells and is thus crucial for triggering specific immune responses against viral infections or cancer. Up to now, there has been no evidence for a correlation between levels of mRNA (the “transcriptome”) and the density of MHC-peptide complexes (the “MHC ligandome”) on cells. Because such dependences are of intrinsic importance for the detailed understanding of translation efficiency and protein turnover and thus for systems biology in general and for tumor immunotherapy in practical application, we quantitatively analyzed the levels of mRNA and corresponding MHC ligand densities in samples of renal cell carcinomas and their autologous normal kidney tissues. Relative quantification was carried out by gene chip analysis and by stable isotope peptide labeling, respectively. In comparing more than 270 pairs of gene expression and corresponding peptide presentation ratios, we demonstrate that there is no clear correlation (r = 0.32) between mRNA levels and corresponding MHC peptide levels in renal cell carcinoma. A significant number of peptides presented predominantly on tumor or normal tissue showed no or only minor changes in mRNA expression levels. In several cases, peptides could even be identified despite the virtual absence of the respective mRNA. Thus we conclude that a majority of epitopes from tumor-associated antigens will not be found in approaches based mainly on mRNA expression studies as mRNA expression reflects a distorted picture of the situation on the cell surface as visible for T-cells.

Novel Multi-Peptide Vaccination In Hla-A2+ Hormone Sensitive Patients With Biochemical Relapse of Prostate Cancer

A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment.

Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR–presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. Results: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNγ. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. Conclusions: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.

Annexin A3 in Urine: A Highly Specific Noninvasive Marker for Prostate Cancer Early Detection

PURPOSE In prostate cancer cases the early diagnosis of tumors carrying a high risk of progression is of the utmost importance. There is an urgent clinical need to avoid unnecessary biopsies and subsequent overtreatment. We validated annexin A3 as a diagnostic marker for prostatic disease in typical clinical populations and relevant segments, such as patients with a negative digital rectal examination and low prostate specific antigen. MATERIALS AND METHODS We performed a blinded clinical study (ClinicalTrials.gov Identifier NCT00400894) from September 2005 to January 2007 in 591 patients who were continuously recruited from 4 European urological clinics. Urine was obtained directly after digital rectal examination and the annexin A3 concentration in urine was quantified by Western blot. Statistical analysis included combinations of annexin A3 with total, percent free, complexed and percent complexed prostate specific antigen. RESULTS Combined readouts of prostate specific antigen and urinary annexin A3 were superior to all others with an area under the ROC curve of 0.82 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.83 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.81 in all patients. The best performing prostate specific antigen derivative was percent free prostate specific antigen with an area under the ROC curve of 0.68 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.72 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.73 in all patients. Annexin A3 has an inverse relationship to cancer and, therefore, its specificity was much better than that of prostate specific antigen. CONCLUSIONS Annexin A3 quantification in urine provides a novel noninvasive biomarker with high specificity. Annexin A3 is complementary to prostate specific antigen or to any other cancer marker. It has a huge potential to avoid unnecessary biopsies with a particular strength in the clinically relevant large group of patients who have a negative digital rectal examination and prostate specific antigen in the lower range of values (2 to 10 ng/ml).

Simultaneous Infiltration of Polyfunctional Effector and Suppressor T Cells into Renal Cell Carcinomas

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4(+) Th1, Th2, and Th17 and CD8(+) Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patients peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4(+) cells of the Th1 lineage and in effector memory CD8(+) cells. Additionally, several populations of CD4(+) and CD8(+) regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses.

Differentiation of urinary calculi with dual energy CT: effect of spectral shaping by high energy tin filtration.

Objectives:In dual energy (DE) computed tomography (CT), spectral shaping by additional filtration of the high energy spectrum can theoretically improve dual energy contrast. The aim of this in vitro study was to examine the influence of an additional tin filter for the differentiation of human urinary calculi by dual energy CT. Materials and Methods:A total of 36 pure human urinary calculi (uric acid, cystine, calciumoxalate monohydrate, calciumoxalate dihydrate, carbonatapatite, brushite, average diameter 10.5 mm) were placed in a phantom and imaged with 2 dual source CT scanners. One scanner was equipped with an additional tin (Sn) filter. Different combinations of tube voltages (140/80 kV, 140/100 kV, Sn140/100 kV, Sn140/80 kV, with Sn140 referring to 140 kV with the tin filter) were applied. Tube currents were adapted to yield comparable dose indices. Low- and high energy images were reconstructed. The calculi were segmented semiautomatically in the datasets and DE ratios (attenuation@low_kV/attenuation@high_kV) and were calculated for each calculus. DE contrasts (DE-ratio_material1/DE-ratio_material2) were computed for uric acid, cystine and calcified calculi and compared between the combinations of tube voltages. Results:Using exclusively DE ratios, all uric acid, cystine and calcified calculi (as a group) could be differentiated in all protocols; the calcified calculi could not be differentiated among each other in any examination protocol. The highest DE ratios and DE contrasts were measured for the Sn140/80 protocol (53%–62% higher DE contrast than in the 140/80 kV protocol without additional filtration). The DE ratios and DE contrasts of the 80/140 kV and 100/Sn140 kV protocols were comparable. Conclusion:Uric acid, cystine and calcified calculi could be reliably differentiated by any of the protocols. A dose-neutral gain of DE contrast was found in the Sn-filter protocols, which might improve the differentiation of smaller calculi (Sn140/80 kV) and improve image quality and calculi differentiation in larger patients (Sn140/100 kV). However, even with the improved spectral separation of the Sn-filter protocols, the DE ratios of calcified calculi are not sufficiently distinct to allow a differentiation within this group.

Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer

BackgroundFibronectin 1 (FN1) is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC) and to determine a prognostic relevance for optimal clinical management.Methods212 renal tissue samples (109 RCC, 86 corresponding tissues from adjacent normal renal tissue and 17 oncocytomas) were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was performed using quantitative real time PCR, three endogenous controls, renal proximal tubular epithelial cells (RPTEC) as biological control and the ΔΔCt method for calculation of relative quantities.ResultsMean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues (p < 0.001; ANOVA). Furthermore, tissue specific mean FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC (p = 9×10-5; Wilcoxon rank sum test). Patients with advanced disease had higher FN1 expression when compared to organ-confined disease (p < 0.001; Wilcoxon rank sum test). Applying subgroup analysis we found a significantly higher FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group (p = 0.02 vs. p = 0.2; Wilcoxon rank sum test). There was an increased expression in RCC compared to oncocytoma (p = 0.016; ANOVA).ConclusionsTo our knowledge, this is the first study to show that FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC.