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A deep feedforward network has been used to predict the flight level with look ahead time up to six minutes for climbing flights. Representing features were developed from operational Mode S Enhanced Surveillance data. In parallel, using certified A340 and A319 Full Flight Simulators, complete climb profiles from Take Off to Top of Climb have been recorded. Certain parameters were modified, e.g. Take Off weight or head- and tailwind conditions. The features from the Full Flight Simulator were fed to the neural network which has been trained with the operational data. The predictions of the flight level in both cases are compared. Because the parameters in the simulator were known and controlled, it allows us to infer the settings of the operational traffic and to improve the features by adding wind information and recognizing the acceleration phase.

■ Improvement of Ground TP - Use of the correct speed profile, in particular climb CAS (below crossover altitude) or Mach (above crossover altitude) - Inclusion of the projected dynamic CAS/ Mach conversion altitude (cross over altitude) - Use of the correct climb or descent profile including vertical rates (could be computed from the EPP data as climb rates are not contained in the EPP reports) - Information on the aircraft FMS and guidance mode ■ Improvement of Air TP - ATC constraint information or handover conditions

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83(+) monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity.

Bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2) act synergistically to induce cell death in tumour cells of various origins with concomitant stimulation of the immune system. Here, we investigated the mechanisms involved in such actions and examined structural requirements of PtdIns-homologues to inhibit tumour cells in combination with bv-sPLA2. Renal cancer cells were treated with bv-sPLA2 alone or in combination with PtdIns-homologues. Inhibitory effects on [(3)H] thymidine incorporation and intracellular signal transduction pathways were tested. Reaction products generated by bv-sPLA2 interaction with PtdIns(3,4)P2 were identified by mass spectrometry. Among the tested PtdIns-homologues those with a phosphate esterified to position 3 of the inositol head group, were most efficient in cooperating with bv-sPLA2 to block tumour cell proliferation. Growth inhibition induced by the combined action of bv-sPLA2 with either PtdIns(3,4)bisphosphate or PtdIns(3,4,5)trisphosphate were synergistic and accompanied by potent cell lysis. In contrast, PtdIns, which lacked the phosphate group at position 3, failed to promote synergistic growth inhibition. The combined administration of PtdIns(3,4)P2 and bv-sPLA2 abrogated signal transduction mediated by extracellular signal regulated kinase 1 and 2 and prevented transduction of survival signals mediated by protein kinase B. Surface expression of the epidermal growth factor (EGF)-receptor was reduced after PtdIns(3,4)P2-bv-sPLA2 administration and associated with a blockade of EGF-induced signalling. In addition, mass spectroscopy revealed that bv-sPLA2 cleaves PtdIns(3,4)P2 to generate lyso-PtdIns(3,4)P2. In conclusion, we suggest that the cytotoxic activity mediated by PtdIns(3,4)P2 and bv-sPLA2 is due to cell death that results from disruption of membrane integrity, abrogation of signal transduction and the generation of cytotoxic lyso-PtdIns(3,4)P2.

We evaluated tumor cell growth modulation by bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate as well as potential cooperative effects. In addition, the immunomodulatory impact of tumor cell treatment was examined by monitoring changes in phenotype and function of monocyte-derived dendritic cells (moDCs) cocultured with pretreated tumor cells. Bv-sPLA2 or phosphatidylinositol-(3,4)-bisphosphate alone displayed moderate effects on the proliferation of A498 renal cell carcinoma cells, T-47D breast cancer cells, DU145 prostate cancer cells and BEAS-2B transformed lung cells. However, when bv-sPLA2 was coadministered with phosphatidylinositol-(3,4)-bisphosphate a potent inhibition of [3H] thymidine incorporation into all tested cell lines occurred. This inhibition was due to massive cell lysis that reduced the number of cells with proliferative capacity. Importantly, tumor cell lysates generated with bv-sPLA2 plus phosphatidylinositol-(3,4)-bisphosphate induced maturation of human moDCs demonstrated by enhanced expression of CD83 and improved stimulation in allogeneic mixed leukocyte reactions. Our data demonstrate that bv-sPLA2 and phosphatidylinositol-(3,4)-bisphosphate synergistically generate tumor lysates which enhance the maturation of immunostimulatory human monocyte-derived dendritic cells. Such tumor lysates which represent complex mixtures of tumor antigens and simultaneously display potent adjuvant properties meet all requirements of a tumor vaccine.

Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. DCs acquire immunity-initiating capacity only after a process of maturation usually induced by ligands that bind to members of the tumor necrosis factor (TNF) or toll-like receptor families. Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 ester bond of glycerophospholipids, regulates a variety of cellular functions including migration of endothelial cells and neurite outgrowth. In the present study we investigated the role of sPLA2 in DC biology. We report that human monocyte-derived DC cultures lack sPLA2 activity but respond to exogenous sPLA2. sPLA2 alone and in cooperation with TNF-α and interleukin 1 β (IL-1β) induced fatty acid release from DC membranes, which was accompanied by upregulation of surface markers and by an increase in the migratory and immunostimulatory capacity of the DCs. Our findings indicate that secreted enzymes such as sPLA2 can contribute to DC maturation and emphasize the role of lipid mediators in the regulation of immune responses. This observation may also have implications for DC-based vaccine development.

In this phase I/II study, we evaluated the feasibility, safety and efficacy of allogeneic dendritic cells (DCs) with or without cyclophosphamide in the treatment of patients with metastatic renal cell carcinoma (RCC). Immunomagnetic beads were used to isolate CD14(+) monocytes from healthy donor leukapheresis products, and CD83(+) antigen-pulsed monocyte-derived DCs (moDCs) loaded with tumor lysate and keyhole limpet hemocyanin (KLH) were generated. Twelve patients were treated with allogeneic moDCs alone, while ten patients also received cyclophosphamide on days 4 and 3 prior to vaccination. Of the 22 patients enrolled, 20 received full treatment consisting of at least three vaccinations at monthly intervals. Two mixed responses with substantial tumor regression were observed. In 3 patients, disease stabilization occurred, in 13 patients disease progressed and 4 patients were lost to follow-up. Overall, immune responses against KLH and tumor lysate were weak or absent; however, the strongest increases in antigen-independent and KLH-specific responses were observed in the 2 patients with mixed responses. In addition, 1 of them showed a substantial increase in oncofetal antigen (OFA)-specific IFN-gamma production. Importantly, the 2 mixed responders and 1 patient with stable disease belonged to the cyclophosphamide group. Median overall survival in the cyclophosphamide group was 23.2 and 20.3 months in the group that received allogeneic moDCs alone. Allogeneic immunotherapy with moDCs is feasible and well tolerated. However, the immunogenicity of allogeneic moDCs is clearly less pronounced than that of autologous moDC immunotherapy. Cyclophosphamide may have the capacity to augment DC-induced antitumor immunity.

BACKGROUND In advanced stages of prostate cancer, the phosphatidylinositol-3′ kinase (PI3K)/Akt signaling cascade, one of the major survival pathways in the cell, is frequently constitutively activated due to mutation or loss of the tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Using cell culture models representing different tumor stages, we explored the effect of inhibition of this survival pathway on the induction of apoptosis. METHODS Inhibition of the survival kinase Akt and induction of apoptosis was analyzed in androgen-insensitive DU145 and PC-3 cells, in androgen-responsive LNCaP, and in androgen-independent long-term androgen-ablated LNCaP-abl cells representing therapy-resistant prostate cancer cells. Activated Akt was determined by immunoblotting using a phospho-Akt specific antibody. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry and measurement of cleavage of the caspases substrate poly-ADP-ribose polymerase (PARP). RESULTS IGF-1, EGF, and heregulin but not PDGF or activators of protein kinase A induced phosphorylation of Akt in DU145 cells and activation was completely blocked by the PI3K inhibitor LY294002. In the hormone-responsive prostate cancer cell line LNCaP that has a constitutively switched-on Akt kinase, LY294002 caused a dose- and time-dependent Akt inhibition, which was absent in long-term androgen-ablated LNCaP sublines. In agreement with the resistance to inhibition of the PI3K/Akt pathway, long-term androgen-ablated LNCaP sublines remained relatively resistant to induction of cell death by LY294002 or the cytotoxic drug etoposide. Inhibition of the PI3K/Akt pathway restored the sensitivity of long-term androgen-ablated cells to induction of apoptosis by a cytotoxic drug almost completely. CONCLUSION These results suggest that long-term androgen ablation therapy for prostate cancer reinforces the PI3K/Akt pathway and impedes its inhibition thus contributing to increased resistance of tumor cells to induction of apoptosis. With regard to treatment of therapy-refractory prostate cancer, these findings suggest effectiveness of a combination of cytotoxic treatment and inhibition of the PI3K-Akt survival pathway in tumor cells after failure of androgen-ablation therapy. © 2003 Wiley-Liss, Inc.

The androgen receptor (AR) is the key regulatory element of androgen signaling in the cell. It mediates action of androgens and is therefore essential for growth, function and differentiation of the human male urogenital tract. Genetic alterations in the AR gene may cause impaired development resulting in androgen insensitivity syndromes (AIS) or in neurodegenerative diseases like Kennedy syndrome. Besides the crucial role in the process of virilization during embryogenesis and puberty, the AR also plays an important role in the adult man as the intracellular mediator of androgen action. Androgen withdrawal and/or AR blockade is the main choice of treatment of nonorgan-confined prostate cancer. Unfortunately, this treatment is only palliative and a majority of these tumors recur and progress to an androgen-independent and therapy-resistant stage. Recent findings gave new insight into the molecular structure and function of the AR and improved our understanding about prostate cancer progression, consequently resulting in the development of novel treatments. It has become evident that the AR is a nuclear transcription factor that can be activated ligand-dependently by androgens as well as ligand-independently by other hormones and various growth factors, respectively. Moreover, it was shown that the interaction of the AR with other proteins of the intracellular signal transduction cascade may promote prostate tumor growth. This review will summarize the most important findings about the AR and the androgen signaling pathway to improve the understanding of prostate diseases and novel treatment strategies that may be useful in the clinic.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen-independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to approximately 2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer.

BACKGROUND Androgen receptor (AR) transcriptional activity is modulated by cofactor proteins. They act as costimulators, corepressors, or bridging proteins, and a disbalanced expression may contribute to the altered activity of the AR in advanced prostate cancer. We investigated the expression of a series of steroid receptor cofactors in prostate cancer cell lines, including several LNCaP sublines, and in prostate stromal cells. METHODS Expression of cofactors was analyzed by means of RT-PCR in PC-3, Du-145, LNCaP, three sublines of LNCaP established after long-term androgen deprivation, and two strains of primary prostate stroma cells. Expression in LNCaP and LNCaP-abl cells (which represented an advanced tumor cell) was analyzed employing semiquantitative RT-PCR. RESULTS Ten of the 12 cofactors tested were expressed in all cells analyzed (AIB1, ARA54, ARA70, CBP, cyclin D1, Her2/neu/erbB2, BAG-1/M/L, SRC-1, SMRT, and TIF2). Only ARA55 and FHL2 mRNAs were not detected in all cells. ARA55 mRNA was absent in LNCaP cells, LNCaP sublines, and DU-145 cells; FHL2 was not expressed in LNCaP cells and its derivatives. The expression pattern was identical in LNCaP cells, and the long-term androgen ablated LNCaP sublines. Moreover, comparison of expression levels in LNCaP and LNCaP-abl cells revealed a slight reduction in LNCaP-abl cells but no gross differences. CONCLUSIONS Prostatic cells express a great number of steroid receptor cofactors. AR activity thus seems to be modulated in a very complex way in prostate cells. Prostate 45:124–131, 2000. © 2000 Wiley-Liss, Inc.

According to economic theory, the technology policy of a country should be guided by the respect of both, the cumulative character of new technologies and the specific national innovation system. In this context, the present article compares French and German technology policies. The industrial laser technology is chosen as a comparative case study in order to highlight the different approaches in the two countries. Having identified the technology policies in the two countries, their respective successes are compared to what would be expected from theory. The relatively greater success of the industrial laser technology in Germany is in accordance with the theoretical conclusions as it goes hand in hand with a more « dynamic » technological system which is more adapted to the characteristics of this new technology. Furthermore, it is confirmed that the two countries dispose of different national innovation systems and it seems that the German success in this case is like-wise based on a better fit of the laser technology to a diffusion-oriented innovation system., Selon la théorie économique, la politique technologique d'un État doit être guidée par le respect à la fois du caractère cumulatif de technologies nouvelles et de son propre système national d'innovation. Dans cette optique, cet article compare les politiques technologiques françaises et allemandes. En particulier, la technologie du laser industriel est choisie comme étude de cas comparative pour mettre en évidence les approches différentes des deux pays. Une fois les politiques technologiques des deux pays identifiées, leurs réussites respectives sont comparées aux conclusions délivrées par la théorie. Ainsi, la réussite relativement plus importante de la technologie du laser industriel en Allemagne se révèle conforme aux conclusions théoriques car elle va de pair avec un système technologique plus « dynamique » tenant mieux compte des caractéristiques de cette technologie nouvelle. De plus, il est confirmé que les deux pays disposent de systèmes nationaux d'innovation différents et il semble que la réussite allemande dans ce cas est également due à une correspondance plus grande de la technologie du laser à un système d'innovation orienté vers la diffusion technologique., Wolter Achim, Putz Thomas, Schmied Helwig. Le développement inégal de la technologie des lasers industriels en France et en Allemagne - Quelques enseignements pour la politique technologique. In: Revue d'économie industrielle, vol. 76, 2e trimestre 1996. pp. 91-113.

The project Labour Market in Touch: new non-routine skills via mobile game-based learning is implemented from November 2010 until October 2012, involves people from 8 project partners in 7 European countries (SE, LT, IT, UK, FR, AT, BG), enables nearly 1600 person-days resources, involves more than 600 employees in more than 100 SME all over Europe is supported by the EC in the Lifelong Learning Programme . This 2 years project aims to spread an innovative approach enabling new generations of workers from business services SMEs to develop their crucial non-routine skills via mobile Learning. On the bases of the EU recommendations described in the document “New skills for new jobs”, the partners will design an ad hoc mobile training environment teaching crucial non routine skills in a labour market, flexible and technological oriented way. The partners will design an ad hoc innovative m-learning kit for working adults about crucial “non-routine” skills, exploiting the new technologies of smartphones for creating open and mobile didactical environments. Designing the Game-Models In the course of the project the top 10 European skills for Competitiveness in the future business service SMEs have been identified. An ebook illustrating the top 10 crucial transversal skills for non-routine tasks according to the European employers and managers was issued in August 2011. Such skills will be translated into learning outcomes – defined in terms of knowledge, skills and competence needed by employees for performing non-routine tasks. Based on the skills and learning outcomes 30 situational learning cases for competitiveness will be elaborated and a report describing the most crucial non routine tasks for competitiveness are developed. Advantages of pervasive games The basic idea is to use the potential of the mobile phone communication channel and combine it with the possibilities of digital learning games in order to create a new and improved learning environment. The concept of pervasive gaming lends itself to this purpose, in that the real world fuses with the virtual world (Walther, 2005). In this context, one can speak of pervasive game learning, an expansion of mobile game-based learning. Pervasive games take place in the real world, and the player communicates with his fellow players and the remote control system via wireless technologies. Various communication channels are available for this, among them the mobile phone. Additional core technologies needed for pervasive gaming are portable displays, which render the digital content tangible in the real world independent of location, and sensor technologies, such as cameras, through which the status of the player can be ascertained (Benford et al., 2005). These technological prerequisites are fulfilled by modern mobile phones, which therefore offer an ideal medium for the execution of pervasive games. Design decisions The basic idea is to use the potential of the mobile phone communication channel and combine it with the possibilities of case-study-based learning and digital learning games in order to create a new and improved learning environment. The use of the potentials of pervasive games lends itself to this purpose. Unfortunately, although the use of pervasive games has numerous advantages, it is also often associated with high development and implementation costs and consequently a limited mass appeal. Technologically complex equipment is often needed, which makes practicable and seamless application in a learning context nearly impossible. Based on this problem, the objective of this section is to develop a pervasive game that does not require costly equipment, such as head-mounted displays. Value is placed above all on a high degree of mass appeal and economic feasibility in the learning context, because it is only under these basic conditions that comprehensive practical use is realistic. The Game-Models Game Model Quiz Learning goals are decided by the game author: Content goals: eg Facts (after Prensky, 2001) Activity goals: eg Questions (after Prensky, 2001) Process goals: eg Remember (after Anderson and Krathwohl, 2001) Key objectives of game-play: gain points for fast and correct answers Game Model Simulation Learning goals are decided by the game author: Content goals: eg Facts (after Prensky, 2001) Activity goals: eg Simulation (after Prensky, 2001) Process goals: eg Decision making (after Anderson and Krathwohl, 2001) Key objectives of game-play: gain points for good, fast, independent decision-making learn from the quality of decisions reflect on success of one's decision-making strategy. The player is confronted with a scenario where there are 4 critical cases to handle. Game Model Story telling Non-routine skill: decision making The user reads the story and has to take two decisions, in two different moments of the story, by clicking on the answer-button. The feedback is the result of the combination of the two choices: selecting first the A answer and then the A or B answers the user goes to the best feedback; instead, selecting the C answer he goes to the second best feedback; selecting first the B answer, the user can reach all the three feedbacks: the best choosing the A answer at the second decision moment, the second best choosing the B answer and the third best choosing the C answer. selecting first the C answer, and then the A answer the user can reach the second best feedback; instead, choosing the B or C answer he reaches the third best feedback. Game Model Labytinth Non-routine skill: strategic thinking In the first screen is described a company typical troubled situation. To solve the problem is necessary to talk to different divisions or to other members of the company In the game screen is shown a map which represent the plant of the company with the work positions of the characters. Aim of the game is to find the path to solve the problem by selecting in the correct order the divisions or the characters to talk to. Game Model Planning Non-routine skill: team work At the beginning of the game we have the description of the scenario. The user has to help the main character to choose the activities concerned in improving team work among the ones listed. Only four of the six listed activities are actually necessary.

Feasibility of a new air traffic management concept is examined which might increase the efficiency of en-route operations by up to 100%. The concept follows the idea of a sector-less airspace organization. Aircraft entering the airspace (e.g. the German airspace or the entire European Airspace) will be assigned to one controller who is in charge of controlling the aircraft from entry point (or TMA exit point) to exit point of the airspace (or TMA entry point). First simple real time simulations revealed very promising results. The concept seems to enable an easy transition into a user preferred trajectory (direct-to) operations whilst at the same time increasing ATCO's efficiency and airspace capacity.

This paper reports on an experiment conducted with airline pilots on a full flight simulator. The objective was a) to confirm the feas ibility of airborne spacing with speed and lateral managed modes and b) to assess the compatibility of airborne spacing and continuous descent approach (CDA). The overall feedback was globally positive and consistent with previous experiments. All pilots found airborne spa cing feasible from cruise until 2000ft, although more sensitive during the approach phase than in initial descent. The speed and lateral managed mode were well accepted. The pilots agreed that performing a CDA while maintaining spacing was feasible. The spacing was w ell maintained within the tolerance of 5s in initial descent (95% containment within ±2.1s). However, a cross-track error in approach led to larger deviation (95% containment within ±5s) and extreme values outside the tolerance. The cost induced was in the order of 115kt additional speed changes for the complete descent phase. In the terminal area, the r oute structure and altitudes were already optimised to allow a continuous descent from FL100.

This paper reports on an experiment conducted with airline pilots on a full flight simulator. The objective was to assess the effect of different positions in chain of aircraft under airborne spacing. Pilots reported several benefits: improved situation awareness, reduction of communications, and better accuracy of achieved spacing. They reported however an increase of workload, requiring for most of them an automatic mode. Despite the basic assistance provided, pilots could maintain the spacing well within tolerances (5 seconds), with on average one action per minute, but with unnecessary speed changes. The relation between required performance and cost induced will be further investigated.

Objective: While previous reports clearly demonstrated antiproliferative effects of IL-4 on renal cell carcinoma (RCC) in vitro, the administration of IL-4 to patients with metastatic RCC in clinical trials could not recapitulate the promising preclinical results. In the present study we wanted to examine the context of IL-4 action and to establish conditions of enhanced IL-4 efficacy. Methods: Primary and permanent human RCC cells were cultured in either serum-supplemented or chemically defined, serum-free culture medium in the presence or absence of cytokines. Cell proliferation was assessed as [3H]-thymidine incorporation. Cell apoptosis was measured using the fluorescent DNA intercalator 7-aminoactinomycin D and flow cytometry. In addition, culture media conditioned by RCC were subjected to cytokine antibody array and cytokine multiplex analysis. Results: Our results indicate that the previously reported antiproliferative effects of IL-4 are serum-dependent. Under serum-free conditions, IL-4 failed to exhibit growth-inhibitory effects or was even growth-stimulatory. In a chemically defined, serum-free medium (AIM-V), however, IL-4 inhibited the TNF-α induced proliferation of RCC. IL-4 and TNF-α synergistically induced apoptosis of RCC as well as a complex cytokine response by RCC, which included the synergistic upregulation of RANTES and MCP-1. Conclusions: IL-4 alone has little effect on the spontaneous proliferation of RCC but can prevent the enhancement of proliferation induced by growth promoters like FBS and TNF-α. The concomitant growth inhibitory, apoptosis-inducing, and cytokine-enhancing effects of IL-4 in combination with TNF-α on RCC support the view that Th2 cytokines may be required for productive immune responses against RCC.

CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7-53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.

We describe the generation of monocyte-derived dendritic cells (MoDC) from leukapheresis products using the CliniMACS system from Miltenyi Biotec. In a clinical setting, the method turned out to be feasible for the generation of clinical-grade MoDC from patients with metastatic renal-cell carcinoma. MoDC generated with this system exhibited a fully mature phenotype as well as high migratory and T-cell stimulatory capacity.

We describe the generation of monocyte-derived dendritic cells (MoDC) from leukapheresis products using the CliniMACS system from Miltenyi Biotec. In a clinical setting, the method turned out to be feasible for the generation of clinical-grade MoDC from patients with metastatic renal-cell carcinoma. MoDC generated with this system exhibited a fully mature phenotype as well as high migratory and T-cell stimulatory capacity.

Objective Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. Prostaglandin E2 (PGE2), a member of the eicosanoid family of arachidonic acid derivatives, is widely used to enhance the TNF-α-driven maturation of human monocyte-derived DCs (moDCs) both in basic research and in clinical settings. However, PGE2 is known to rapidly undergo nonenzymatic dehydration to produce PGA2, a member of the cyclopentenone PGs, which have been implicated in anti-inflammatory processes. Methods In a side-by-side analysis we therefore compared the influence of PGE2 and PGA2 on the TNF-α-induced maturation of human moDCs. Phenotypic changes, migratory responses towards MIP-3β, and T-cell responses induced by the differentially matured moDCs were assessed. Results We found that PGA2 is nearly as potent as PGE2 in costimulating the TNF-α-induced phenotypic maturation of human moDCs. Both PGE2 and PGA2 further enhanced the migratory and T-cell-stimulatory capacity of TNF-α-treated moDCs. Maturation of moDCs with either PGE2 or PGA2 resulted in enhanced IFN-γ, TNF-α, and IL-5 production and repressed IL-10 production in allogeneic mixed leukocyte cultures. PGE2 was always more potent than PGA2. Conclusions Our data suggest that some of the effects attributed to PGE2 may in fact be mediated by its degradation product PGA2. This work also demonstrates that cyclopentenone PGs may have pro-inflammatory properties and that both PGE2 and PGA2 can contribute to the development of Th1-type immune responses.

In advanced stages of prostate cancer, the phosphatidylinositol-3' kinase (PI3K)/Akt signaling cascade, one of the major survival pathways in the cell, is frequently constitutively activated due to mutation or loss of the tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Using cell culture models representing different tumor stages, we explored the effect of inhibition of this survival pathway on the induction of apoptosis.Inhibition of the survival kinase Akt and induction of apoptosis was analyzed in androgen-insensitive DU145 and PC-3 cells, in androgen-responsive LNCaP, and in androgen-independent long-term androgen-ablated LNCaP-abl cells representing therapy-resistant prostate cancer cells. Activated Akt was determined by immunoblotting using a phospho-Akt specific antibody. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry and measurement of cleavage of the caspases substrate poly-ADP-ribose polymerase (PARP).IGF-1, EGF, and heregulin but not PDGF or activators of protein kinase A induced phosphorylation of Akt in DU145 cells and activation was completely blocked by the PI3K inhibitor LY294002. In the hormone-responsive prostate cancer cell line LNCaP that has a constitutively switched-on Akt kinase, LY294002 caused a dose- and time-dependent Akt inhibition, which was absent in long-term androgen-ablated LNCaP sublines. In agreement with the resistance to inhibition of the PI3K/Akt pathway, long-term androgen-ablated LNCaP sublines remained relatively resistant to induction of cell death by LY294002 or the cytotoxic drug etoposide. Inhibition of the PI3K/Akt pathway restored the sensitivity of long-term androgen-ablated cells to induction of apoptosis by a cytotoxic drug almost completely.These results suggest that long-term androgen ablation therapy for prostate cancer reinforces the PI3K/Akt pathway and impedes its inhibition thus contributing to increased resistance of tumor cells to induction of apoptosis. With regard to treatment of therapy-refractory prostate cancer, these findings suggest effectiveness of a combination of cytotoxic treatment and inhibition of the PI3K-Akt survival pathway in tumor cells after failure of androgen-ablation therapy.

Androgens are pivotal regulators of prostate cell growth, differentiation and function, and their actions are believed to be involved in prostate cancer development. The androgen-signaling pathway in the prostate gland is therefore one of the possible sites of intervention in prostate cancer prevention efforts. The central element of androgen signaling in the cell is the androgen receptor (AR), a member of the superfamily of nuclear receptors. Binding of androgen to its ligand-binding domain transforms the receptor to an active transcription factor that regulates gene expression by interacting with specific regulatory elements in the promoters of genes. In addition to this genomic action, the AR also interacts with other signaling pathways through protein-protein interaction, for example with AP-1 or Ets transcription factors. It is not only the action of androgenic hormones, but also the interactions with growth factor and protein kinase A-signaling pathways that can induce activation of AR. Moreover, these ligand-independent activators act synergistically together with low concentrations of androgens. The effects of long-term androgen deprivation on androgen signaling have been investigated in the LNCaP cell culture system. Long-term culture in a steroid-free medium results in a subline showing a hyperreactive AR characterized by increased AR expression and enhanced AR transcriptional activity in an environment with low levels of androgen hormones. It is not yet clear if similar changes also occur in normal or premalignant prostate epithelial cells and are thus relevant for prevention trials which interfere with androgen hormone signaling.