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In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research., Competing Interests: The authors have declared that no competing interests exist.

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.

Purpose: Treatment of cancer using standard chemotherapy still offers a poor prognosis combined with severe side effects. Novel antibody-based therapies have been shown to overcome low efficiency and lack of selectivity by targeting cancer-associated antigens, such as aminopeptidase CD13., Methods: We isolated a high-affinity CD13-specific single-chain fragment variable (scFv13) from a phage display library of V-genes from mice immunized with soluble antigen. An immunotoxin comprising the scFv13 and a truncated version of the exotoxin A of Pseudomonas aeruginosa (ETA', scFv13-ETA') and a bispecific scFv targeting CD13 and CD16 simultaneously (bsscFv[13xds16]) was generated and investigated for their therapeutic potential., Results: Both fusion proteins bound specifically to target cells with high affinity. Furthermore, scFv13-ETA' inhibited the proliferation of human cancer cell lines efficiently at low concentrations (IC 50 values of 408 pM-7 nM) and induced apoptosis (40-85% of target cells). The bsscFv triggered dose-dependent antibody-dependent cell-mediated cytotoxicity, resulting in the lysis of up to 23.9% A2058 cells, 18.0% MDA-MB-468 cells and 19.1% HL-60 cells., Conclusion: The provided data demonstrate potent therapeutic activity of the scFv13-ETA' and the bsscFv[13xds16]. The CD13-specific scFv is therefore suitable for the direct and specific delivery of both cytotoxic agents and effector cells to cancer-derived cells, making it ideal for further therapeutic evaluation.

Antibody-drug conjugates (ADCs) can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs) with 1:1 stoichiometry while preserving antigen binding. Two (human and mouse) scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR) were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF). Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to improve the development of personalized medicines., Competing Interests: Disclosure Wolfgang Richter is an employee of Tube Pharmaceuticals GmbH. The authors report no other conflicts of interest in this work.

Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

Among all the sleep-related respiratory disorders, sleep apnoea is clinically the most important one because of its high incidence and potential cardiovascular sequelae. Treatment depends upon the risk profile of the individual patient; in severe cases or in risk patients nasal continuous superpressure ventilation is employed, whereas in milder cases theophylline has been successfully used alongside general measures. 103 patients were followed up during treatment on an outpatient basis for 2 to maximum 12 months. Initially a significant reduction of the apnoea index by a 50% average was achieved. This therapeutical effect remained largely constant with a slight average deterioration at the end of the observation period, whereas the subjective feeling tone also improved in most patients. The treatment course was prematurely discontinued in 34 patients. Good to very good success was achieved in a total of 43 patients, whereas 26 patients were non-responders either initially or as the treatment proceeded. Even though theophylline treatment is now well-tried in mild forms of sleep apnoea, predictors for its use in certain groups of patients must be determined before treatment is initiated.

We conducted a prospective study to perform classification according to responder types and follow-up during theophyllin treatment (500 mg/d) in a group of (up to now) 65 patients suffering from sleep apnoea. Changes in the apnoea index (AI) and clinical symptoms were taken as response criteria. A significant AI reduction was seen during a follow-up period of 3 months. At the last control measurement after 6 months no significant change has been detected so far. 10 patients were classified as Type I responders (improvement in initial AI 60%, over the total period at least 25%). No patient had a responder type II profile. 13 patients were nonresponders (improvement in initial AI and over the whole period not more than 25%, no improvement in the pattern of clinical symptoms). 13 patients dropped out of the trial due to non-compliance. Side effects typical of theophyllin leading to discontinuation of the treatment course occurred in 5 patients. 22 patients have not yet completed the course at the time of writing. Standardised questioning of patients showed a definite improvement in feeling tone, especially with regard to the tendency to fall asleep during the day and refreshed feeling in the morning. Nocturnal complaints occurred in only a few isolated cases. Hence, administration of theophyllin retard preparations in evening doses of 500 mg/d remains a meaningful therapeutic approach to sleep apnoea.

A review of the literature shows that more than 50% of examined patients suffering from coronary heart disease were also suffering from sleep-related apnea. We were able to diagnose a pathological sleep apnea in 9 out of 25 patients (36%) suffering from an angiographically confirmed coronary 2-vessel and 3-vessel disorder. Patients with this combination--this is the hypothesis derived from our study--are at risk due to nocturnal apnea-induced myocardial ischaemia and rhythmic disorders. In 15 patients with sleep apnea and coronary heart disease or small vessel disease, nocturnal polysomnography was conducted, in parallel a 6-channel ECG was recorded. The apnea index (second night) was on the average 33 phases/h, the maximal duration of an apnea phases being 120 seconds. The minimal blood gas saturation recorded during sleep was between 46 and 89% (median 76.0%). In 4 of the 15 patients it was possible to confirm myocardial ischaemia (correlated via REM and also via NREM) with a maximum duration of 60 seconds, mainly during the phases of maximal apnea activity and blood gas desaturation. On comparing the ventricular arrhythmias waking/sleep, the Lown class did not change in 12 patients; there was deterioration in 2 patients and in one patient a qualitative improvement during the sleep phase. Patients suffering from sleep-related respiratory disorders and coronary heart disease are at cardiac risk, the more so since long-lasting apneas can lead to conditions of hypoxia at the heart in pre-existing changes in the coronary arteries, restricted coronary reserves and reduced tolerance to hypoxia. Such hypoxia can in turn induce enhanced electrical instability and a disturbance of the contractile function.