Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific ...mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K
(K-allele), which is expressed in >40% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progression-free survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K
was reduced slightly, but ligand-mediated EGFR activation was intact. We found a lack of glycan sialyation on EGFR-K
that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K
expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform.
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Deep brain stimulation (DBS) is a therapy of movement disorders including Parkinson's disease (PD). Commercially available electrodes for animal models of Parkinson's disease vary in geometry and ...material. We characterized such electrodes and found a drift in their properties within minutes and up to about 60
h after immersion in cell culture medium, both with and without a stimulation signal. Electrode properties could largely be restored by proteolytic treatment for platinum/iridium electrodes but not for stainless steel ones. Short-term drift and irreversible aging could be followed by impedance measurements. Aging was accompanied by metal corrosion and erosion of the plastic insulation. For both materials, the degradation rates depended on the current density at the electrode surfaces. Fourier analysis of the DBS pulse (60
μs, repetition rate 130
Hz) revealed harmonic frequencies spanning a band of more than three decades, with significant harmonics up to the MHz range. The band is located in a window imposed by electrode processes and capacitive cell membrane bridging at the low and high frequency ends, respectively. Even though electrode processes are reduced at higher frequencies they only vanish above 1
MHz and cannot be avoided. Therefore, the use of inert electrode materials is of special importance. The neurotoxicity of iron makes avoiding stainless steel electrodes imperative. Future developments need to avoid the use of corrosive materials and current density hot spots at the electrode surface, and to reduce low frequency components in the DBS pulses in order to diminish electrode processes.
Deep brain stimulation, the electric stimulation of basal ganglia nuclei, is a treatment for movement disorders such as Parkinson's disease. The underlying mechanisms are studied in animals, e.g. ...rodents. Designs and materials of commercially available microelectrodes, as well as experimentally applied driving signals vary tremendously. We used finite integration modeling to compare the electric field and current density distributions induced by various electrodes. Current density or field strength “hot spots”, which are located particularly at sites of high curvature and material interfaces coincided with corrosion and erosion at poles and insulation, respectively, as shown by scanning electron microscopy of stainless steel electrodes. Cell constants, i.e. geometry factors relating the electrode impedance to the specific medium conductivity, were calculated to determine the electrode voltage for a given stimulation current. Nevertheless, for electrodes of the same cell constant but of different geometry, current and field distributions may be very dissimilar. We found geometry-dependent limiting values of the stimulation current, above which electric tissue damage may occur. These values limit the reach of the stimulation signal for a given electrode geometry. Also, electrode geometries determine the shape of the stimulated tissue volume. This study provides tools for choosing the most appropriate geometry for targeting different-sized brain areas.
Die Basis für den therapeutischen Einsatz elektrischer Felder bei der Behandlung von Knochenbrüchen liegt in der Existenz von belastungsabhängigen elektrischen Potentialen im Knochen. Die zellulären ...Wirkungsmechanismen sind jedoch unverstanden. Um zu untersuchen, ob und wie elektrische Felder Wachstum und Differenzierung von Knochenzellen beeinflussen, wurden humane Osteosarcomazellen (HOS TE85) und aus Minischweinen isolierte primäre Osteoblasten mit elektrischen Feldern im Frequenzbereich von 0.1 Hz bis 100 kHz befeldet und verschiedene zelluläre Parameter zeitabhängig gemessen. Das elektrische Feld wurde mittels platinierten Platinelektroden appliziert, um ausreichend hohe Feldstärken bei gleichzeitig guter Kontrolle über die im Gewebe erzeugte Wellenform zu gewährleisten. Beim Einsatz von Elektroden müssen jedoch elektrochemische Reaktionen an der Elektrodenoberfläche berücksichtigt werden. Daher wurden verschiedene Elektroden für die in vivo- und in vitro-Stimulation mit elektrischen und numerischen Methoden untersucht und verglichen. Das führte einerseits zur Erarbeitung verschiedener Kriterien zur Auswahl geeigneter Elektrodenmaterialien und -geometrien. Andererseits konnten detaillierte Angaben zur effektiven Feldstärke und Feldverlauf in der Zellebene sowie zu elektrochemisch bedingten Nebenwirkungen gemacht werden. Es konnte gezeigt werden, dass elektrische Felder das Signalsystem und den Stoffwechsel von Knochenzellen im gesamten untersuchten Frequenzbereich beeinflussen. Die Richtung des Nettoeffekts war jedoch im oberen und im unteren Frequenzbereich unterschiedlich. Im Frequenzbereich über 1 kHz fanden wir immer eine Erhöhung des Wachstums und der Konzentration der "second messenger" und eine Herabsetzung der Konzentration der reaktiven Sauerstoffverbindungen (ROS). Bei Frequenzen unter 1 kHz wurde dagegen eine Erhöhung der Aktivität der alkalischen Phosphatase und des oxidativen Stress beobachtet. Signifikante Effekte traten jedoch nur bei Feldstärken oberhalb 100 V/m auf. Hinweise auf bevorzugte Frequenzen oder Wellenformen, die für "Fenstereffekte" sprechen würden, konnten nicht gefunden werden. Da alle zellulären Parameter an einem einheitlichen System gemessen wurden, ließen sich die Ergebnisse in ein Modell der feldinduzierten Signaltransduktion integrieren. Prinzipiell muss man von einem gemeinsamen Effekt mehrerer Signalwege ausgehen. Dabei kann die Erhöhung der intrazellulären Kalziumkonzentration als ein genereller Mechanismus der Feldwirkung angesehen werden. Dafür sind sowohl der Influx als auch die Freisetzung von Kalzium aus intrazellulären Speichern verantwortlich. Es konnte weiterhin gezeigt werden, dass auch cGMP und Prostaglandin E2, jedoch nicht cAMP an der Vermittlung der Feldwirkung beteiligt ist. Außerdem wurde die Translokation der mitogen-aktivierten Proteinkinase (MAPK) beeinflusst. Trotzdem sind die beobachteten Effekte relativ schwach im Vergleich zu publizierten klinischen Erfolgen. Es muss daher in vivo Verstärkungsmechanismen geben, die durch Untersuchungen an Zellkulturen nicht erfasst werden können.
The therapeutic use of electric fields in the treatment bone fracture healing is based on the existence of different load dependent electric potentials in bone. However, the cellular mechanisms of field action are still not understood. We investigated the effect of electric fields on proliferation and differentiation of human osteosarcoma cells (HOS TE85) and primary osteoblasts isolated from mini pigs. The cells were exposed to electric fields (0.1 Hz - 100 kHz) and various cellular parameters were measured. The field was applied using platinized platinum electrodes ensuring sufficiently strong electric fields and a well controlled wave form in the tissue. Nevertheless, in this case electrochemical reactions which occur at the electrode surface have to be taken into account. That’s why various electrodes for in vivo and in vitro stimulation are characterized by electrical and numerical methods leading not only to criteria for choosing electrode materials and geometries, but also providing detailed knowledge about the electric field induced in the tissue and electrochemically induced side effects. We found that cellular signaling as well as proliferation and differentiation of bone cells are influenced by electric fields in the whole investigated frequency range. Proliferation and the second messenger concentrations are increased at frequencies above 1 kHz, whereas below 1 kHz the alkaline phosphatase activity is increased. Significant effects were found only for electric fields above 100 V/m, but we could not find any hints on preferred frequencies or wave forms ruling out "window effects". Because all cellular parameters were measured within one experimental system, the results can be summarized in a signal transduction model for the action of electric fields. Generally, a common action of multiple signaling pathways has to be assumed. The increase of the intracellular calcium concentration can be considered as a general mechanism of field action. This increase is caused by calcium release from intracellular calcium stores as well as influx of extracellular calcium. Additionally, the concentration of cGMP and prostaglandin E2, but not that of cAMP is increased by the electric field. Furthermore, the translocation of the mitogen activated protein kinase (MAPK) is influenced. Nevertheless, the field effects found are weak compared to those published in clinical studies. We suppose that there are amplifying mechanisms in vivo which could not be seen during in vitro investigations.
The effect of extracellular calcium on human osteoblast-like cells (HOS) has been demonstrated. An experimental setup was used for applying defined rates of change in the extracellular calcium ...concentration. The intracellular calcium concentration was monitored using the fluorescence dye fura-2. HOS cells showed qualitatively different responses of the intracellular calcium concentration to changes of the extracellular calcium concentration depending on its changing rate. A small rate caused only a small and slow increase of the intracellular calcium concentration, whereas faster changes are able to cause a rapid transient increase followed by a sustained elevation of the internal calcium level. Surprisingly, both an increasing as well as a decreasing external calcium concentration is able to cause cellular responses. These signals could be reduced by the IP3-inhibitor neomycin. We propose that the G-protein dependent signalling pathway of HOS cells can not only sense the extracellular calcium concentration but also its time derivative.
In the present paper, the induction of calcium signals in neuroblastoma cells, cells of T-cell leukemia, and osteogenic sarcoma cells were investigated in relation to the UVA irradiation used in ...fluorescence microscopy. Methods were developed to measure both the mean UVA irradiance and the intensity profile in the UVA-illuminated area of the microscope. This allowed us to calculate the applied UVA radiant exposure of the cells during each experiment. This investigation was undertaken because of the conflicting results in the literature on the effects of electromagnetic fields on the signals of the calcium-sensitive fluorescence probe FURA-2 in lymphocytes. Taking into account that each group used a different system with different optics and lamps, these conflicting results are now at least partially understandable. Our measurements indicate that in a typical experiment with FURA-2 the cells were irradiated with up to $776\ {\rm kJ}\ {\rm m}^{-2}$ during 25 min of exposure to UVA light. This causes changes in intracellular free Ca2+ concentrations ( Ca2+ i). Designating cells in which the Ca2+ i was distinctly increased during the experiment as "responding", we found Hill-type dependences on the irradiance. Jurkat cells showed a 50% response even at $10\ {\rm kJ}\ {\rm m}^{-2}$ and osteosarcoma cells at about $60\ {\rm kJ}\ {\rm m}^{-2}$, whereas neuroblastoma cells even at the maximum possible dose responded only minimally. In the case of neuroblastoma cells, we found a dependence of this effect on the CO2 partial pressure during the preincubation. An electrical treatment with an a.c. field (5 kHz sinusoidal, amplitude modulation 16 Hz 100%, $800\ {\rm V}\ {\rm m}^{-1}$, 5 min) had a significant effect on intracellular calcium in neuroblastoma cells only in the case of cells that were not pretreated with CO2 with high fluences of UVA irradiation. In conclusion, these results indicate that the possibility of UVA artifacts must be considered in all experiments using fluorescence microscopy. Furthermore, our results lead to the hypothesis that oxidative stress could be the link between UVA and electric-field effects.
Habitat destruction and deterioration are amongst the main drivers of biodiversity loss. Increasing demand for agricultural products, timber and charcoal has caused the rapid destruction of natural ...forests, especially in the tropics. The Taita Hills in southern Kenya are part of the Eastern Afromontane Biodiversity Hotspot and represent a highly diverse cloud forest ecosystem. However, the cloud forest suffers extremely from wood and timber exploitation and transformation into exotic tree plantations and agricultural fields. Existing conservation regulations and moratoriums aim to prevent further forest destruction. In this study, we analyzed land cover change and shifts in landscape configuration for a fraction of the Taita Hills, based on satellite imageries for the years 2003, 2011 and 2018. We found that the coverage of natural cloud forest further decreased between 2003 and 2018, despite the effort to conserve the remaining cloud forest patches and to reforest degraded areas by various conservation and management initiatives. In parallel, the proportion of exotic tree plantations and bushland strongly increased. Moreover, mean natural forest patch size decreased and the degree of interspersion with other land cover types increased notably. Logging bans for indigenous trees seem to have resulted in local opposition to the planting of indigenous trees and thereby hindered the recovering of the cloud forest. We suggest to enhance local awareness on the ecological value of the natural forest by community-based Conservation Forest Associations and to encourage the planting of indigenous tree species in farmer-owned woodlots. Besides, bottom-up management systems that allow for local participation in decision-making and benefit-sharing related to forest resources would be a way forward to achieve the sustainable use and conservation of the last remaining natural forest patches in the Taita Hills.
The growing interest in testosterone's effects on men's social behaviors, in particular aggressive, risk-taking, or status maintenance behaviors, is accompanied by a paucity of dose-dependent ...pharmacokinetic data. Examining the neurophysiological effects of transdermal testosterone typically includes a 4h delay before further brain-behavior measurements. Nevertheless, high heterogeneity regarding the timing of follow-up measurements and dosage remains. In a double-blind placebo-controlled design, we examined the short-term pharmacokinetic profile of 100-mg transdermal testosterone (Testotop
) to determine the optimal time for detecting testosterone-mediated effects. Across two studies, 35 healthy men received a single dose of testosterone and placebo in two separate sessions. In study one (n = 16), serum testosterone and cortisol were assessed serially every 30 min up to 2 h posttreatment. In study two (n = 19), we assessed serum testosterone and cortisol at baseline, 2 h, and 4.15 h (255 min) posttreatment. Relative to baseline and placebo, transdermal testosterone significantly increased total serum testosterone concentrations 90 min posttreatment, reaching maximum concentration between 2 h and 3 h posttreatment. Albeit elevated, serum testosterone levels gradually decreased between 2 h and 4 h following treatment. Transdermal testosterone did not suppress cortisol release. Instead, cortisol concentrations decreased according to cortisol's known circadian rhythm. Unlike previous findings showing significant testosterone concentration increases as soon as 60 min and as late as 3 h post 150-mg testosterone treatment, our 100-mg testosterone manipulation significantly increased testosterone concentrations 90 min following treatment. These pharmacokinetic data are important in facilitating the optimization of timing parameters for future testosterone challenge studies.
Land-use change remains the main threat to tropical forests and their dependent fauna and flora, and degradation of existing forest remnants will further accelerate species loss. Forest degradation ...may result directly from human forest use or through spatial effects of land-use change. Understanding the drivers of forest degradation and its effects on biodiversity is pivotal for formulating impactful forest management and monitoring protocols, but such knowledge is lacking for many biodiversity hotspots, such as the Taita Hills in southeast Kenya. Here we first quantify effects of social factors (human activity and presence) at plot and landscape level, forest management (gazetted vs. non-gazetted) and spatial factors (fragment size and distance to forest edge) on the vegetation structure of indigenous Taita forest fragments. Next, we quantify effects of degraded vegetation structure on arthropod abundance and diversity. We show that human presence and activity at both the plot and landscape level explain variation in vegetation structure. We particularly provide evidence that despite a national ban on cutting of indigenous trees, poaching of pole-sized trees for subsistence use may be simplifying vegetation structure, with the strongest effects in edge-dominated, small forest fragments. Furthermore, we found support for a positive effect of vegetation structure on arthropod abundance, although the effect of daily maximum temperature and yearly variation was more pronounced. Maintenance of multi-layered forest vegetation in addition to reforestation maybe a key to conservation of the endangered and endemic fauna of the Taita Hills.
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