[Medical habilitation in German-speaking countries : Quantitative assessment of content and elaboration of habilitation guidelines]. / Medizinische Habilitation im deutschsprachigen Raum : Quantitative Untersuchung zu Inhalt und Ausgestaltung der Habilitationsrichtlinien.

BACKGROUND: Habilitation defines the qualification to conduct self-contained university teaching and is the key for access to a professorship at German, Austrian and Swiss universities. Despite all changes implemented in the European higher education systems during the Bologna process, it is the highest qualification level issued through the process of an university examination and remains the core concept of scientific careers in these countries. In the field of medicine, this applies not only to scientific staff at the universities but also to those medical doctors aiming at a clinical career track. OBJECTIVES: To provide a quantitative analysis of the scientific, didactic, and procedural criteria for medical habilitation in German-speaking countries. MATERIALS AND METHODS: Based on the guidelines of all 43 medical academic institutions, the criteria which candidates are required to fulfil prior to habilitation as well as formal requirements related to the habilitation procedure itself have been acquired and quantitatively analyzed. RESULTS: Having evaluated all habilitation guidelines by means of 87 items, the quantitative analysis revealed significant differences in terms of number, kind and scale of criteria stated therein. Most habilitation guidelines scarcely define the capabilities applicants have to prove: concerning the scientific qualifications on types of publications for instance, no item was mentioned in more than half of all habilitation guidelines. CONCLUSION: Based on this data analysis, the authors discuss the related literature and describe five main distinguishing areas of habilitation guidelines in terms of the set of the formal and procedural framework as well as the prequalification and postqualification criteria imposed on habilitation candidates. There are therefore substantial differences in the organization of the habilitation for medicine.

Transfection of dendritic cells (DCs) with the CIITA gene: increase in immunostimulatory activity of DCs.

Dendritic cells (DCs) are the major antigen-presenting cells. They are able to present tumor antigens to immunologic effector cells. MHC class II molecules on DC surfaces play an important role in priming effector cells against tumor cells and their antigens. The transactivator CIITA (MHC class II transactivator) is a non-DNA-binding transactivator, which regulates the expression of MHC class II, HLA-DM, and invariant chain and behaves as a master controller of constitutive and inducible MHC class II gene activation. Here, we transfected DCs with the CIITA gene using a novel transfection technique. The vector system consisted of a plasmid bound to an adenovirus via poly-L-lysine, which is covalently bound to a UV-irradiated adenovirus. After transfection, expression of MHC class II on DCs increased from 27% to 75% on day 2 after transfection. Transfected DCs were co-cultured with immunologic effector cells. Cytotoxicity of effector cells against tumor cells increased after co-culture with transfected DCs to 63% compared to 15% with effector cells co-cultured with irrelevantly transfected DCs (P=.037). This effect was dependent on the timing and period of co-culture. In conclusion, transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs. We can further conclude that DCs could be efficiently transfected with the CIITA gene. Transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs and may have a major impact on immunotherapeutic protocols for patients with cancer.

Therapeutic vaccination against metastatic carcinoma by expression-modulated and immunomodified autologous tumor cells: a first clinical phase I/II trial.

Therapeutic vaccination of tumor patients with cytokine gene-transfected tumor cells leads to tumor regression in animal models but has so far not resulted in significant clinical benefit. We and others demonstrated that tumor cells transfected to mediate overexpression of a cytokine gene activate immunologic effector cells for an improved proliferation rate and significantly higher antitumoral cytotoxic activity. Here, we performed a pilot study of therapeutic vaccination in patients with metastatic disease. Autologous tumor cells were simultaneously transfected with novel minimalistic, immunogenically defined, gene expression constructs (MIDGE) for overexpression of the two cytokines interleukin 7 (IL-7) and GM-CSF and newly designed double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM) as a Th1-promoting and NK cell-stimulating adjuvant. Transfection was performed ex vivo by ballistomagnetic gene transfer. Patients received four subcutaneous injections of at least 1 x 10(6) of their expression-modulated and immunomodified autologous tumor cells. Ten patients have been enrolled in the study protocol. In all patients no adverse effects could be detected. IL-7 and interferon gamma levels were elevated in the serum of the patients after treatment. Interestingly, cytotoxicity of patient-derived PBLs increased significantly during treatment. All 10 patients had progressive disease when entering our protocol. One complete, one partial, and one mixed response with progression of abdominal metastases and regression of lung metastases were observed. Two patients showed a stable disease after treatment and five patients remained in progressive disease. Our observations confirm the capability of autologous expression-modified and immunomodulated tumor cell vaccines to stimulate a strong immune response in patients with metastatic cancer even in the presence of a large tumor burden.

Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both populations.

Dendritic cells (DCs) are major antigen-presenting cells. They are capable of capturing and processing tumor antigens, expressing lymphocyte costimulatory molecules, and secreting cytokines to initiate immune responses. Here, the authors tested the effect of cytokine-induced killer (CIK) cells, a population that includes CD3+CD56+ cells (natural killer T cells), with regard to their capacity to immunomodulate DCs. Cytokine-induced killer cells were cocultured with autologous DCs generated from peripheral blood mononuclear cells. Expression of markers typical for both populations was measured using flow cytometry, and secretion of interleukin (IL)-12 was determined using enzyme-linked immunosorbent assays. Cytotoxicity assays were performed to investigate the role of IL-12 and the importance of cell-cell interactions. Considering this, receptors for IL-12 and CD40 were blocked and cocultures were performed with cell culture inserts. Coculture of CIK cells led to a significant increase of DC-specific, costimulatory, and antigen-presenting molecules in DC cultures. In addition, coculture resulted in a dramatically increase of IL-12 secretion by DCs and to a significant increase in cytotoxic activity of CIK cells toward carcinoma cells. Blockage of IL-12 uptake decreased the cytolytic activity of CIK cells. Cytokine secretion was shown to be important for activation of CIK cells, and also cellular interactions between DCs and effector cells caused a higher cytolytic capacity. Interactions between DCs and CIK cells caused changes in the surface molecule expression of both populations, led to an increase of IL-12 secretion, and rendered an improved cytotoxic activity. The natural killer T cell subpopulation seems to be responsible for this effect. Therefore, coculture of DCs with CIK cells may have a major impact on immunotherapeutic protocols for patients with cancer.

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and primary lymphoma cells can be efficiently transfected using an adenoviral vector system. By adding cationic liposomes, the efficiency of adenoviral gene transfer to primary tumor cells could be further improved. This protocol may have an impact on the use of lymphoma cells in cancer gene therapy.

Increase in the immunostimulatory effect of dendritic cells by pulsing with serum derived from pancreatic and colorectal cancer patients.

Both we and others have observed a relative resistance of solid tumor cells to immunological effector cells in vitro, which may be one reason for the clinical phenomenon of resistance of patients with pancreatic carcinoma or other solid tumors to immunological therapeutic approaches. Dendritic cells (DC) are professional antigen-presenting cells which can process and present tumor-associated antigens such as CA 19-9. Here we tested DC pulsed with serum containing CA 19-9 for their capacity to stimulate immunological effector cells against pancreatic carcinoma cells. Coculture of immunological effector cells with DC led to a significant increase in cytotoxic activity as measured by a lactic dehydrogenase release assay. Most interestingly, cytotoxic activity against tumor cells was further increased using DC pulsed with patient-derived CA 19-9 containing serum. Similar results have been obtained using either autologous or allogeneic serum from patients with pancreas carcinoma. The effect of serum on the cytotoxicity of effector cells increased in a dose-dependent manner. Interestingly, heat inactivation led to a significant loss of immunostimulatory capacity of the serum. Cytotoxicity was partially inhibited by using an antibody directed against CA 19-9 on the surface of the target cells. Best results were obtained when adding CA 19-9 protein to CA 19-9 containing serum for pulsing of DC. In conclusion, DC pulsed with CA 19-9 containing serum increased the cytotoxic activity of immunological effector cells against pancreatic cancer cells. DC pulsed with CA 19-9 containing serum with or without additional exogenous CA 19-9 protein may have an impact on immunotherapeutic protocols for patients with CA 19-9 secreting tumors.

Increase of the immunostimulatory effect of dendritic cells by pulsing with CA 19-9 protein.

Previously, a relative resistance of solid tumor cells to immunologic effector cells was shown in vitro. This resistance could be one reason for the clinical phenomenon of resistance of patients with colon carcinoma or other solid tumors to immunologic therapeutic approaches. In this study, dendritic cells (DCs) pulsed with CA 19-9 protein were tested for their immunostimulatory capacity of immunologic effector cells against cells derived from colon and pancreatic carcinoma. Dendritic cell cultures coexpressed CMRF-44 and CD1a, markers typical of DCs, in 31.5% +/- 5.3% after 13 days of culture. Coculture of NK-like T lymphocytes with DCs led to a significant increase in cytotoxic activity, as measured using a lactate dehydrogenase release assay. Cytotoxic activity could be further increased using DCs pulsed with CA 19-9 protein. The effect of CA 19-9 on increasing the cytotoxic effect of NK-like T lymphocytes was dose dependent. Similarly, cocultivation of DCs with NK-like T cells derived from patients with metastatic pancreatic cancer and elevated CA 19-9 serum levels led to a significant increase in cytotoxic activity. In conclusion, DCs pulsed with CA 19-9 protein can increase the cytotoxic activity of immunologic effector cells against colon carcinoma and pancreatic cancer cells. Dendritic cells pulsed with CA 19-9 protein may have an important effect on immunotherapeutic protocols for patients with cancer.

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity.

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.