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Biomedical subjects

R Ehret

Publications and source records attributed to R Ehret.

At least 19 recordsLinked to original sources
PubMed2001

Multiparametric microsensor chips for screening applications.

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.

Biocompatible Materials↗
PubMed2001

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET.

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

Biosensing Techniques↗
PubMed2001

Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

Acetaldehyde↗
PubMed2000

Treatment of cells with detergent activates caspases and induces apoptotic cell death.

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Non-invasive measurement of cell membrane associated proton gradients by ion-sensitive field effect transistor arrays for microphysiological and bioelectronical applications.

The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

Biosensing Techniques↗
PubMed1998

Monitoring of cellular signalling and metabolism with modular sensor-technique: the PhysioControl-Microsystem (PCM).

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

Biosensing Techniques↗
PubMed1998

On-line control of cellular adhesion with impedance measurements using interdigitated electrode structures.

Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells from close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

Biosensing Techniques↗
PubMed1998

Isolated extracellular matrix-based three-dimensional in vitro models to study orthotopically cancer cell infiltration and invasion.

An initial event in colon cancer progression is the migration of epithelial cells through the basement membrane (BM) and the invasion of the colon submucosa, where tumour cells enter blood and lymph vessels to spread throughout the body. To interrupt this process would mean the prevention of metastasis. In order to investigate tumour cell invasion orthotopically in the human system, we established novel in vitro models which mimic normal human colon tissue (colon reproductions, CoRes) and primary colon carcinomas (artificial tumours, ArTs). These models are based on the isolated extracellular matrix (iECM) of the respective human tissues. Two isolation methods were established, the Digestion Method and the Lysis Method neither of which destroyed the characteristic architecture of the ECM found in the original tissues. BM components, i.e. laminin, fibronectin and collagen IV, were detectable in the iECM isolated with the Lysis Method but not those isolated with the Digestion Method. Scanning electron microscopic analysis of the normal colon iECM demonstrated that even if the BM was missing, the luminal surface consisted of densely packed ECM filaments which do not allow cell infiltration without degradation of the iECM. Furthermore, we demonstrated that iECM can be separately supplemented with different cell types, i.e. colorectal carcinoma cells, normal fibroblasts and immune cells at any desired concentration, combination and localisation. Therefore, these models could be used to determine the role of the BM and of the tumour cell/normal cell crosstalk in the infiltration process of human colorectal carcinoma cells.

Basement Membrane↗
PubMed1998

Microsensor-aided measurements of cellular signalling and metabolism on tumor cells. The cell monitoring system (cms(R)).

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Biosensing Techniques↗
PubMed1997

Human immunodeficiency virus glycoprotein-specific CD4+ cytotoxic T lymphocytes are involved in two types of cytotoxicity: antigen-specific and cell-cell fusion-related cell lysis.

Human immunodeficiency virus (HIV) glycoprotein-specific CD4+ cytotoxic T lymphocytes (CTLs) lyse target cells in an MHC-restricted calcium-dependent fashion similar to the mechanism used by CD8+ CTLs. However, contact of unprimed peripheral blood CD4+ T cells with HIV glycoprotein-expressing cells has been shown to cause, in addition to cell-cell fusion, rapid cytolysis that may resemble antigen-specific cytotoxicity in the chromium release assay. In this study, the ability of glycoprotein-specific CD4+ CTLs to undergo similar fusion-related cytolysis was examined. The data obtained demonstrate that in addition to antigen-specific calcium-dependent cytotoxicity, envelope-specific CD4+ CTLs are involved in fusion-related, calcium-independent cytolysis.

Amino Acid Sequence↗
PubMed1997

Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures.

A new method for on-line and real-time monitoring of concentration, growth and physiological state of cells in culture is described. This biosensor is based on impedance measurements of adherently growing cells on interdigitated electrode structures (IDES). The measurements can be performed for several days as there is no detectable electrical influence on the cells. The versatility of this new sensor is shown with some exemplary experiments. Cell density, growth and long-term behaviour of cells on the electrodes clearly change the impedance of the IDES. Both, the global influence of serum components (deprivation of foetal bovine serum) and the toxic effects of heavy metal ions (cadmium) result in changes of the sensor signal and can be visualized.

Animals↗
PubMed1997

Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1beta-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1beta-induced fever.

The vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.

Animals↗
PubMed1996

[Microsensory systems in cell biology basic research and medical diagnostics].

The development of an integrated sensor system, called the physiocontrol microsystem, is presented. It is suited for microscopy, and works with both adherent cell types and cultures growing in suspension, as well as with tissue biopsies. The central part, a miniaturized culture chamber equipped with differently constructed microsensors, allows continuous observation of important physiological parameters even in the course of long-lasting experiments. Besides a description of the physical components, the study provides a summary of selected applications of the physiocontrol microsystem in basic cellular research and biomedical diagnostics.

Animals↗