A comparison of medical education in Germany and the United States: from applying to medical school to the beginnings of residency.

Both Germany and the United States of America have a long tradition of science and medical excellence reaching back as far as the nineteenth century. The same tribute must be paid to the medical educational system in both countries. Despite significant initial similarities and cross-inspiration, the paths from enrolling in a medical university to graduating as a medical doctor in Germany and the US seem to have become much different. To fill a void in literature, the authors' objective therefore is to delineate both structures of medical education in an up-to-date review and examine their current differences and similarities. Recent medical publications, legal guidelines of governmental or official organizations, articles in media, as well as the authors' personal experiences are used as sources of this report. Tuition loans of over $200,000 are not uncommon for students in the US after graduating from medical schools, which are often private institutions. In Germany, however, the vast majority of medical universities are tax-funded and, for this reason, free of tuition. Significant differences and surprisingly multiple similarities exist between these two systems, despite one depending on government and the other on private organizations. Germany currently employs an integrated medical curriculum that typically begins right after high school and consists of a 2-year long pre-clinical segment teaching basic sciences and a 4-year clinical segment leading medical students to the practical aspects of medicine. On the other hand, the US education is a two-stage process. After successful completion of a Bachelor's degree in college, an American student goes through a 4-year medical program encompassing 2 years of basic science and 2 years of clinical training. In this review, we will address some of these similarities and major differences.

Clinical Development and Commercialization of Advanced Therapy Medicinal Products in the European Union: How Are the Product Pipeline and Regulatory Framework Evolving?

The research and development of advanced therapy medicinal products (ATMPs) has been active in Europe and worldwide during recent years. Yet, the number of licensed products remains low. The main expected legal change in the near future in the European Union (EU) concerns the regulation on clinical trials (536/2014), which will come into force in 2018. With this new framework, a more harmonized and swift process for approval of clinical trials is anticipated, which is expected to support the entry of new innovations into the EU market. A survey on ATMPs in clinical trials during 2010-2015 in the EU was conducted in order to study the trends of ATMP development since the earlier survey published in 2012. According to the results, the number of clinical trials using ATMPs is slowly increasing in the EU. Yet, the focus is still in early development, and the projects are mainly carried out by small and medium-sized enterprises, academia, and hospitals. Oncology is the main area of clinical development. Yet, the balance between cell-based products and gene therapy medicinal products in this area may be changing in the future due to the new T-cell technologies. Many limitations and challenges are identified for ATMP development, requiring proportionate regulatory requirements. On the other hand, for such a novel field, the developers should be active in considering possible constraints and actively engage with authorities to look for solutions. This article provides up to-date information on forthcoming regulatory improvements and discusses the main challenges hampering the commercialization of ATMPs in the EU.

Optimizing adenoviral transduction of endothelial cells under flow conditions.

PURPOSE: To target adenoviral vectors to cells of the vasculature and shielding vectors from inactivation by the immune system. METHODS: Complexes of reporter gene expressing adenoviral vectors with positively charged magnetic nanoparticles were formed by electrostatic interaction in presence or absence of additional negatively charged poly(ethylene glycol)-based polymer. Transduction of HUVEC was analyzed in vitro under flow. Protection from inactivation by the immune system was analyzed by pre-incubation of AdV and complexes with neutralizing antibodies and subsequent reporter protein analysis of infected cells. RESULTS: Physical association of AdV with MNP and polymers was demonstrated by radioactive labelling of components and co-sedimentation in a magnetic field. Ad-MNP+/-polymer resulted in efficient transduction of HUVEC, depending on MOI and flow rate in presence of magnetic field, whereas no transduction was observed without complex formation with MNP or in absence of magnetic field. Association with MNP did result in protection from neutralizing antibodies, with slightly increased protection provided by the polymer. CONCLUSIONS: Complex formation of AdV with MNP is a viable means for targeting of vectors to areas of magnetic field gradient. Additional coating with polymer might proof useful in protection from inactivation by the immune system.

YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

Tetracycline-regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects.

OBJECTIVE: Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete. METHODS: Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system. RESULTS: Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions. CONCLUSION: The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.

Tissue inhibitor of metalloproteinases-1-induced scattered liver metastasis is mediated by host-derived urokinase-type plasminogen activator.

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP-1-induced modulation of a pro-metastatic microenvironment in the liver. Indeed, in livers of uPA-ablated mice elevated TIMP-1 levels did not trigger HGF signalling and did not promote metastasis of a murine T-lymphoma cell line. In contrast, lack of tumour cell-derived uPA induced by gene silencing did not interfere with this pro-metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP-1 levels. This newly identified co-operation between TIMP-1 and host uPA suggests that therapies, simultaneously interfering with pro- and anti-proteolytic pathways may be beneficial for patients with metastatic disease.

Tissue engineering of the anterior cruciate ligament-sodium dodecyl sulfate-acellularized and revitalized tendons are inferior to native tendons.

The acellularization of tendons using detergents (sodium dodecyl sulfate, Triton-X, tri-nitro-butyl-phosphate) is a new source of scaffolds for tissue engineering in anterior cruciate ligament (ACL) repair. In vitro testing demonstrated that acellular tendon scaffolds are biocompatible and show good biomechanical properties, but in vivo confirmation of these results is not yet available. Therefore, the aim of this study was to see in vivo if an acellular allogenic construct colonized with autologous fibroblasts improves the quality of ACL reconstruction. ACL replacement was performed in 31 New Zealand White rabbits using a standardized model. Fifteen animals received autologous semitendinosus tendon, whereas 16 animals were treated with a tissue-engineered construct. This construct was made by acellularization of allogenic semitendinosus tendons using sodium dodecyl sulfate and subsequent in vitro colonization with autologous fibroblasts. Eight weeks postoperatively, macroscopic, biomechanical (ultimate load to failure, elongation, stiffness; n = 8/9), and histological (n = 5) examinations were performed. Biomechanical testing showed decreasing strength of the constructs at 8 weeks after implantation compared with the direct postsurgical strength. However, tissue-engineered constructs (F = 19.7 +/- 20.3 N) were significantly weaker than autologous tendons (F = 61.2 +/- 31.2 N). Histologically, the autologous tendons showed signs of partial necrosis and tissue remodeling. The tissue-engineered constructs exhibited an inflammatory reaction and showed both repopulated and acellular regions. In conclusion, in vivo results were much more unfavorable than in vitro results had suggested. Further studies have to be performed to test if modifications of the acellularization process yield better results in vivo.

Non-viral VEGF(165) gene therapy--magnetofection of acoustically active magnetic lipospheres ('magnetobubbles') increases tissue survival in an oversized skin flap model.

Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF(165)-cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane-filled magnetic lipospheres ('magnetobubbles') from Tween60-coated magnetic nanoparticles, Metafectene, soybean-oil and cDNA and studied the effect in an oversized random-pattern-flap model in the rats (n= 46). VEGF-cDNA-magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre-operative. Flap survival and necrosis were measured 7 days post-operatively. Flap perfusion, VEGF-protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF-cDNA-magnetobubbles presented an increased flap survival of 50% and increased flap perfusion (P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated (P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile.

Therapeutic vaccination with an interleukin-2-interferon-gamma-secreting allogeneic tumor vaccine in patients with progressive castration-resistant prostate cancer: a phase I/II trial.

Immunotherapy with whole cell cancer vaccines has been tested in various tumor types. This study investigated the safety profile and antitumor activity of an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant human interleukin-2 and human interferon-gamma. Thirty HLA-A*0201-matched patients with progressive, castration-resistant prostate cancer received four intradermal injections on days 1, 15, 29, and 92, and then every 90 days, as long as no tumor progression occurred. Three patients received a dose level of 7.5 million cells, and 27 patients received 15 million cells per injection. The primary study criteria were safety and the difference in prostate-specific antigen doubling time (PSA-DT), determined in the pretreatment phase (before the start of vaccination) and in the trial treatment phase (during vaccination). No dose-limiting or autoimmune toxicity was seen. During vaccination there was a significant prolongation of the PSA-DT compared with the prevaccination period (prolongation from 63 to 114 days; p < 0.01; intention to treat). In addition, results showed a period of PSA stabilization of at least 12 weeks, together with stable bone scans in 12 of 30 patients, and 3 patients sustained a >50% decrease in PSA versus baseline. The median overall survival time from first vaccination was 32 months (mean value, 34 months). Immune monitoring revealed T cell stimulation in the majority of patients. This vaccine strategy was found to be safe and well tolerated and was accompanied by prolongation of PSA-DT. The results of this trial warrant clinical development of this vaccine.

Combined reporter gene PET and iron oxide MRI for monitoring survival and localization of transplanted cells in the rat heart.

UNLABELLED: There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation. METHODS: Human endothelial progenitor cells (HEPCs), derived from CD34+ mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by (124)I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs. RESULTS: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron- and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as (124)I accumulation. The (124)I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-5'-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation. CONCLUSION: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.

Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis.

Mechanical and chemical stimulation have been shown to enhance in vitro chondrogenesis. The aim of this study was to analyze and compare combined physicobiochemical effects. Bovine articular chondrocytes were retrovirally transduced to express bone morphogenetic protein-2 (BMP-2) or left as naïve controls. Cells were seeded in three-dimensional polyurethane scaffolds and further cultured under static conditions or exposed to dynamic compression and shear in a joint-specific bioreactor. Four groups: control (A), load (B), BMP-2-infected (C), and BMP-2-infected plus load (D) were analyzed for DNA and glycosaminoglycan (GAG) content; collagen I, II, and X; aggrecan, (cartilage oligomeric protein (COMP), superficial zone protein, matrix metalloproteinase (MMP)-3; MMP-13 mRNA; histology; and immunohistochemistry at 7, 21, and 35 days post-seeding. Synergistic effects (D) were higher than the sum of the individual treatments (B and C) for GAG/DNA, collagen II, and COMP. Histology revealed a functional organization in D including an intense safranin O staining in C and D superior to that in A and B. Immunostaining for collagen II and aggrecan was detected in C and D and was strongest in D. The results show that both stimuli augment in vitro chondrogenesis better than in controls. Biochemical manipulation proved to be predominantly more effective than load, and synergistic effects were demonstrated.

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

Searching for the right timing of surgical delay: angiogenesis, vascular endothelial growth factor and perfusion changes in a skin-flap model.

BACKGROUND: The angiogenic potential of vascular endothelial growth factor (VEGF) and its oxygen pressure-dependent regulation suggest a strong connection between this growth factor and the 'delay phenomenon'. In this study we focused on the chronological changes in VEGF concentration and flap perfusion in order to optimise the duration of surgical delay. METHODS: The VEGF concentration in skin and underlying muscle was measured in oversized, random-pattern flaps on 38 male Sprague-Dawley rats after 3, 5 or 7 days of surgical delay. Additionally, flaps were raised 5 or 7 days past preconditioning. The effect on flap perfusion was measured using indocyanine green fluoroscopy and the size of surviving and necrotic areas of the flaps were analysed. Microvessel density was assessed using a monoclonal CD31 antibody, and vessel diameter and morphometry were appraised by means of corrosion casting. RESULTS: VEGF expression in the distal half of the flaps was significantly increased 3 days after preconditioning and perfusion was significantly enhanced after day 5. An interval of 5 days between preconditioning and flap transposition resulted in a significantly reduced average necrosis rate. Microvessel density was significantly increased and vessel diameters were enlarged (P<0.05). CONCLUSIONS: We illustrated the chronology of events from the ischaemic procedure to the rise in VEGF concentration and changes in flap perfusion, and demonstrated vasodilatation and the formation of new vessels. Most significantly, we were able to further specify the optimal length of surgical delay based on alterations on a molecular level as well as changes in vascularisation and perfusion.

Plasminogen activator inhibitor-2, but not cystatin C, inhibits the prometastatic activity of tissue inhibitor of metalloproteinases-1 in the liver.

Formation of multiple and scattered metastases in target organs, leading to disruption of organ functional integrity, is the death-determining step for most lethal cancers. In the clinic, elevated expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is often associated with increased aggressiveness of cancer. We demonstrated that elevated host expression of TIMP-1 leads to the promotion of scattered liver metastases in mice, associated with increased activity of cysteine proteases (CPs). This study aimed for reduction of TIMP-1-promoted experimental liver metastases of lacZ-tagged human fibrosarcoma cells by overexpression of cystatin C, a natural inhibitor of CPs, in the murine host. Although CP inhibition reduced TIMP-induced proteolytic activity, the TIMP-1-induced increase in total tumor cell burden in livers was not significantly reduced. However, overexpression of cystatin C in livers with elevated TIMP-1 led to the formation of large multicellular metastatic foci in 42% of the mice. This formation was associated with increased expression of plasminogen activators (PAs). Additional overexpression of plasminogen activator inhibitor-2 prevented the formation of macrometastatic foci as well as the TIMP-1-induced increase in total tumor cell burden. This demonstrates that PAs are crucial for the prometastatic activity of TIMP-1 and led to the assumption that patients with elevated TIMP-1 expression may benefit from an antiproteolytic treatment directed against PAs.

GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves.

BACKGROUND: Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats. METHODS: Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves). RESULTS: The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p=0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls. CONCLUSIONS: These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.

Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector.

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

BACKGROUND AND PURPOSE: Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1. METHODS AND MATERIAL: Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice. RESULTS: Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model. CONCLUSIONS: Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.