Biomedical subjects

Albrecht Wendel

Publications and source records attributed to Albrecht Wendel.

26 records · Page 2Linked to original sources

PubMed2001

Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.

Apoptosis↗

PubMed1999

New Applications of the Human Whole Blood Pyrogen Assay (PyroCheck).

The absence of pyrogens in injectable drugs is an indispensable safety control because contaminants causing fever pose a life-threatening risk to the patient resulting in the worst case in death by shock. When fever- inducing agents, i.e.pyrogens, come into contact with the immunocompetent cells in blood, these cells release mediators which transmit the fever signal to the thermoregulatory centre of the brain. The Phamocopoeia lists currently two test systems for pyrogenicity: 1. The in vivo rabbit pyrogen test which measures the fever reaction following injection of the sample to the animals. 2. The in vitro Limulus Amebocyte Lysate assay (LAL) which measures the coagulation in a lysate prepared from the blood of the horseshoe crab specifically initiated by endotoxins, i.e. cell wall components from Gram-negative bacteria. The new test presented here (PyroCheck) exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after a fixed incubation time. The technology presently listed in the Pharmacopoeia is limited to parenteralia (rabbit test: biologicals and pharmaceuticals, LAL: predominantly pharmaceuticals). In the EU Medical Devices Directive from 1995 the rabbit pyrogen test for medical products is in some cases requested. (in some cases LAL of an eluate from the device). However, pyrogen-testing needs to cover also innovative high-tech products such as medical devices (implants, medical plastic materials, dialysis machines), cellular therapies and species-specific agents (e.g. recombinant proteins). Here we report that the human blood test PyroCheck is suitable for testing filters in air quality control as well as for assessing medical devices and biocompatibility (dialysate fluid), i.e. that it can be extended to a wide spectrum of applications.

Pyrogens↗

PubMed1998

[Evaluation and further development of a pyrogenicity assay based on human whole blood]

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever inducing contaminations) they release mediators transmitting the fever reaction within the organism. A new pyrogen test exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. In case of pyrogen contamination, the formation of interleukin-1 is induced, which is determined by ELISA. According to the various pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. In contrast to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the blood assay is not restricted to endotoxins of Gram-negative bacteria and is not to the same extent disturbed by endotoxin-binding blood proteins. Here, interim results of the ongoing optimisation and prevalidation are demonstrated. Preliminary data of the evaluation for biological and pharmaceutical drugs are presented.

Journal Article↗

PubMed1998

[Is replacement of animal experimentation in pharmacology a goal or a social constraint, utopia or reality?]

Animal experimentation in pharmacology is evaluated with respect to in vitro alternatives. Overall, the judgement comes clearly in favour of the alternative methods, needless to say in a field where most animal experimentation occurs. The authors conclude that the discipline considerably profits from switching to in vitro models, even though the complete replacement of animal experimentation is unrealistic.

Journal Article↗

PubMed1998

[Development and evaluation of a pyrogen test based on human whole blood]

When cells of the immune system, especially blood monocytes and macrophages, come into contact with pyrogenic (fever-inducing) contaminations, they secrete messenger molecules which initiate an hyperthermic reaction in the organism. Of this group of endogenous pyrogens, most is known about interleukin-1 (IL-1). A new pyrogen test makes use of this reaction as a system for detection: The substances which are to be screened are incubated with a small volume of blood from a healthy donor. Any pyrogens present induce the production of IL-1 which can be detected by ELISA. This test has a higher sensitivity and is more economical than the conventional pyrogen test in rabbits and furthermore reflects the reaction of the relevant species. In contrast to the customary alternative method, the Limulus amoebocyte lysate test (LAL), this test is not restricted to endotoxins from Gram-negative bacteria and is also not hindered by substances which bind endotoxins, such as blood proteins, to the same extent. Consequently, more than 50 non-endotoxin pyrogens have already been traced by this test. The whole blood test is even superior to the LAL in regard to the detection of endotoxins: in a comparison of about 60 endotoxins, there was a correlation of the potency of the individual endotoxins between the whole blood test and the pyrogen test in rabbits, but neither test correlated with the LAL test. In some cases, endotoxins with equal effects in the LAL test differed in potency in the human blood model by a factor of 10 000. A method has been developed by which cryopreserved blood can be put to use in the test. In this way, blood donations from a donor can be pre-tested so that uniform material may be employed in the test. This test opens up entirely new perspectives on pyrogen testing for Gram-positive or fungal pyrogens as well as in medicinal products. In addition, it could fill the dangerous security gap which might result from the limitations of testing medications and blood products with LAL only. The project was supported by ZEBET, D-Berlin, BMBF, D-Bonn, and set, D-Mainz.

Journal Article↗

PubMed1998

[Development of a pyrogen test based on rabbit blood]

The testing of drugs for pyrogen contamination e.g. the exclusion of fever inducing impurities, was standardised in the Forties in the form of animal pyrogen assay. We recently proposed an alternative method using human whole blood from healthy donors. The substances to be tested are incubated in the presence of blood and the generation of endogenous pyrogens (Interleukin 1, IL-1) are measured reflecting the primary fever reaction in man. With this method it is possible to detect the reaction of the relevant species. However, pronounced differences were reported for the potency of several bacterial pyrogens in different species (e.g. up to factor 10.000 for an individual endotoxin in different mammals). As a part of the validation of the new method discrepancies between the whole blood system and the rabbit test might occur. In these cases it is not possible to distinguish between an in-vitro-artefact and species differences. Therefore, the goal of this project is the development of an in-vitro-test based on rabbit blood. For this purpose rabbit IL-1 was expressed. At present chickens and mice are immunized and possibly crossreacting anti-human-IL-1-antibodies are tested. In this way, an ELISA for the rabbit cytokin will be established. Using a temporarily commercially available rabbit TNF-ELISA, the feasibility of the test-system could already be shown: Pyrogens induced the generation of TNF in rabbit blood. In the human model, the endpoints IL-1 and TNF were very similar. The sensitivity of human blood and rabbit blood appeared to be very similar. However, individual pyrogens (only different endotoxins were tested up to now) showed considerable deviations. However, these results require the verification in the final test. The rabbit test in preparation will be implemented immediately into the ongoing evaluations by the Paul-Ehrlich-Institute (BMBF) and at the University of Heidelberg (European Pharmakopoea). Furthermore, such a test also represents an interesting alternative in the veterinarian medical field using the correct target species for pyrogenicity testing. This project war supported by the Foundation Research 3R, CH-Muensingen.

Journal Article↗

PubMed1996

[The formation of a mediator of inflammation]

Tumor necrosis factor-alpha (TNF-alpha) represents a central distal mediator of inflammation. It is a protein released upon infections, traumatic, lesions or autoimmune disorders from immunocompetent cells and thus maintains or enhances the inflammatory reaction. The determination of such mediators in experimentally challenged animals is a mean for testing the efficacy of putative drugs. Alternative attempts for the assessment of mediator release from cell cultures are limited by profound differences in the time course of mediator release in vitro. We checked the hypothesis that these kinetic differences are due to the lack of elimination of mediators formed and released in vitro. We used the release of TNF-alpha from liver macrophage cultures stimulated with the bacterial cell wall component endotoxin as a model. The discrepancy between the in vivo release of the cytokines during endotoxic shock in the rat and the in vitro release from Kupffer cells was confirmed. By using a continuous open perfusion system instead of a static culture, the simulation of an elimination resulted in a mediator release that closely resembled the kinetics seen in vivo. Perfusion cultures appear to be suitable for relevant in vitro screening models in drug development and testing.

Journal Article↗

PubMed1995

[Detection of Pyrogens using human whole blood]

The use of human whole blood as an in vitro model for detection of compounds with the potential to induce fever is described. As a readout, mediators (endogenous pyrogens) released from leukocytes such as tumor necrosis factor (TNF), interleukin I (IL-I), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) are quantitated. In fact, minute concentrations of pyrogens induced the release of mediators in amounts that were easily detectable. Endotoxins as well as other bacterial components induce this reaction. We used the antipyrogenic drug aspirin to show that in blood of volunteers the ex vivo stimulated PGE2 release was inhibited for several hours. This model seems superior to the rabbit pyrogen test or the Limulus assay for endotoxin. We propose to evaluate this system as an alternative in vitro method for pyrogen testing.

Journal Article↗