Online monitoring of metabolism and morphology of peptide-treated neuroblastoma cancer cells and keratinocytes.

Antimicrobial peptides are promising anti-cancer agents with a unique mode of action. We established the usage of a chip-based sensor to monitor the dynamic interplay between cells on the chip and peptides and compared it with endpoint tests. Human neuroblastoma cancer cells and spontaneously immortalized non-cancer keratinocytes were perfused with representative peptides (NK-2, NK11, and melittin). The sensor system enabled continuous recording of cell layer impedance (adhesion/confluence), oxygen consumption (respiration) and extracellular acidification (glycolysis) and provided insights in cell damage, stress response and recovery. Cells responded differentially to peptide treatment. During perfusion, peptides accumulated on the cell surface until they reached a critical concentration. Preceding to cell death, melittin triggered glycolysis, suggesting stress response. NK-2 induced no change in energy metabolism, but led to an increase in impedance, i.e. a temporarily altered morphology, which appeared to be an excellent parameter to detect subtle structural changes of cell layers.

In vitro system for the prediction of hepatotoxic effects in primary hepatocytes.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

The neuronal 5-HT3 receptor network after 20 years of research--evolving concepts in management of pain and inflammation.

The 5-HT3 receptor is a pentameric ligand-gated cation channel which is found in the central and peripheral nervous system and on extraneuronal locations like lymphocytes, monocytes and fetal tissue. Five monomer subtypes, the 5-HT(3A-E) subunits, have been identified which show differences in the amino-terminal and the transmembrane region. The functional relevance of different receptor compositions is not yet clarified. 5-HT3 receptors are located predominantly in CNS regions that are involved in the integration of the vomiting reflex, pain processing, the reward system and anxiety control. The preferential localization on nerve endings is consistent with a physiological role of 5-HT3 receptors in the control of neurotransmitter release such as dopamine, cholecystokinin, glutamate, acetylcholine, GABA, substance P, or serotonin itself. 5-HT3-receptor agonists cause unpleasant effects like nausea and anxiety, and no clinical use has been considered. In contrast, the introduction of 5-HT3-receptor antagonists for chemotherapy-induced vomiting was extremely successful. After development of other gastrointestinal indications like postoperative vomiting and diarrhea-predominant irritable bowel syndrome recent research focuses on rheumatological indications such as fibromyalgia, rheumatoid arthritis and tendinopathies. Positive effects have also been observed for pain syndromes such as chronic neuropathic pain and migraine. These effects seem to be related to substance P-mediated inflammation and hyperalgesia. Furthermore, antiinflammatory and immunomodulatory properties have been observed for 5-HT3-receptor antagonists which might explain promising findings in systemic sclerosis and other immunological conditions. For all of these innovative indications the optimal dosing schedule is a crucial issue, since a bell-shaped dose-response curve has been observed repeatedly for 5-HT3-receptor antagonists, particularly in CNS effects.

Online monitoring of cell metabolism for studying pharmacodynamic effects.

To characterize modes of action of substances and their cytotoxic effects Bionas GmbH has developed a new screening system to allow the continuous recording of how an active substance can act (Bionas 2500 analyzing system). In the pharmaceutical industry it is important to acquire as much information as possible about the metabolic effects of an active substance. Most classical pre-clinical studies are very expensive and time-consuming. Often they are so-called end-point tests which require many individual tests before approximate statements can be made about how an effect takes its course. With the Bionas 2500 analyzing system metabolically relevant data including oxygen consumption, acidification rate and the adhesion (cell impedance) of cells can be measured in parallel, online and label-free. Using e.g. ion-sensitive field effect-transistors (ISFET) and electrode structures it is possible to observe metabolic parameters non-invasively and continuously over longer periods of time. The system has already been established for several cell models, cell lines as well as primary cells. It also offers the advantage that regenerative effects can be observed during the same test run.