Biomedical subjects

Thomas Hartung

Publications and source records attributed to Thomas Hartung.

At least 127 records · Page 7Linked to original sources

PubMed2002

Inhibition of cyclooxygenase-2 by natriuretic peptides.

The atrial natriuretic peptide (ANP) has been suggested to possess immunomodulatory potential because of its property to alter macrophage functions via its guanylate-cylcase- coupled A-receptor (NPR-A), such as inhibiting the expression of inducible nitric oxide synthase or TNF-alpha. The aim of this study was to investigate whether ANP influences COX-2. COX-2 expression in murine macrophages and in mice was induced by lipopolysaccharide. Release of PGE(2) and thromboxane B(2) was significantly reduced in the presence of ANP. C-type natriuretic peptide (CNP) also significantly reduced PGE(2)-accumulation in macrophages. Northern and Western blots showed that ANP attenuates COX-2 mRNA and protein. Reduction of neither COX-2 nor of PGE(2) production was significantly abrogated by an NPR-A antagonist, suggesting a pathway independent of cGMP. Furthermore, dibutyryl-cGMP did not affect PGE(2)-accumulation. cANF, the specific ligand for the natriuretic peptide (NP) clearance-receptor (NPR-C), significantly inhibited PGE(2)-production. Because some biological activities of ANP have been reported to be mediated via an NPR-C-mediated inhibition of adenylate-cyclase, we determined cAMP levels. ANP, CNP, and cANF significantly attenuated intracellular cAMP. In summary, ANP was shown to attenuate PGE(2)-production of lipopolysaccharide-activated macrophages predominantly via the NP clearance-receptor. ANP reduces COX-2-protein and -mRNA levels. The inhibition seems to be mediated via NPR-C and related to an attenuation of cAMP production.

Animals↗

PubMed2002

Induction and regulation of endogenous granulocyte colony-stimulating factor formation.

Granulocyte colony-stimulating factor (G-CSF) is one of the most prominent endogenous proteins in broad clinical use. While its biological and clinical effects are relatively well studied, little is known about its endogenous formation in health and disease. However, such knowledge is crucial to decide in which situations G-CSF should be applied efficiently in the clinic, ie. when endogenous production does not suffice. The dramatic changes induced by G-CSF in the differential blood cell count are directly immunomodulatory, strengthening the innate defence by multiplying neutrophilic granulocytes. A multitude of further immunomodulatory effects contribute to the regulation of the concerted host defence. In this review, following a short introduction into the biology of G-CSF, the available data on endogenous formation in a number of animal models and human diseases is compiled. The cellular sources and inducers of G-CSF formation are reviewed and the regulation of G-CSF expression on both the transcriptional and translational level are discussed. The emerging understanding of the role and regulation of endogenous G-CSF formation opens up possibilities to define therapeutic windows as well as targets for diagnostics or drug development. Lastly, the modulation of G-CSF formation by various pharmacological agents alerts to putative side effects of these drug treatments.

Adjuvants, Immunologic↗

PubMed2002

Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14.

BACKGROUND: The pro-inflammatory potential of lipoteichoic acid (LTA) from Staphylococcus aureus is controversial. The present study was undertaken to examine the ability of highly purified and characterized S. aureus LTA to stimulate the production of pro-inflammatory cytokines in human leukocytes at both mRNA and protein level, and to study the involvement of Toll-like receptors (TLRs) and CD14 in this response. MATERIAL/METHODS: Purified LTA was administered to whole human blood ex-vivo (or primary adherent monocytes) and the cytokine response assessed in plasma by EIA. Cytokine mRNA was measured by RT-PCR on leukocyte subsets isolated following stimulation. To study the involvement of specific receptors for LTA signaling, CHO cells transfected with CD14 and/or TLR2, TLR4 were used, as well as antibodies directed against these receptors. RESULTS: Addition of highly purified LTA to human whole blood or primary adherent human monocytes elicited a time and concentration dependent release of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6 and IL-8. Messenger RNA encoding TNF-alpha, IL-1 beta and IL-6 seemed to be accumulated in monocytes and T cells, but not in granulocytes and B cells. Expression of TLR2, but not TLR4, in chinese hamster ovary cells conferred responsiveness to LTA. However, antibodies directed towards TLR2 (clone TL2.1) or TLR4 (clone HTA125) failed to inhibit TNF-a release induced by LTA in both the whole blood model and in adherent monocytes. In contrast, blockade of the CD14 receptor with MAb18D11 strongly attenuated the LTA induced release of TNF-alpha in both models. CONCLUSIONS: We propose that (i) LTA from S. aureus triggers the release of cytokines during staphylococcal infections, and (ii) both monocytes and T cells contribute to cytokine production induced by LTA. TLR2 may mediate cellular activation by LTA, but the functional significance of this receptor during staphylococcal infections remains elusive.

Actins↗

PubMed2002

Potential for immune reconstitution through G-CSF treatment of HIV patients.

New treatment strategies for HIV/AIDS are very successful in reducing viral load. However, reconstitution of the immune system takes about one year and may be insufficient or remain incomplete. During this time the patient remains prone to opportunistic infections as a result of the complex immune dysfunction caused by the virus. Recombinant granulocyte colony-stimulating factor (G-CSF) has diverse immunomodulatory properties which may be beneficial in aiding immune reconstitution.

AIDS-Related Opportunistic Infections↗

PubMed2001

[A new application for the human whole blood test: development of an assay to assess the health risk of air-borne microbial contaminations].

The pathogenic properties of environmental microorganisms as well as pyrogens as fragments of those bacteria (especially endotoxins) for humans is increasingly recognised. Various clinical syndromes are described after contact with airborne microbial contaminants via the respiratory tract: Sick-building-syndrome, humidifier lung (a form of hypersensitive pneumonitis), "Monday sickness" etc. Air-conditioning and ventilation systems intensify this problem as well as storage of compost within the household which represents a considerable source of airborne pollutants. In 1995 a new method for the detection of pyrogenic (fever-inducing) hazardous substances was described by Hartung and Wendel. This whole blood assay utilises the natural reaction of the immune system in order to detect a broad spectrum of pyrogens very sensitively in the relevant species. Injectable drugs are the main area of application in which this innovative test has already proven effective and is currently validated for inclusion into European Pharmacopoeia. In co-operation with the FU Berlin we could demonstrate in ventilation systems in animal stables that the whole blood pyrogen test can also detect airborne environmental microorganisms very sensitively. The filtration technique for collection of these germs is an established method for air-conditioning and ventilation systems. In co-operation with the FU Berlin (Institut für Tier-und Umwelthygiene) and the filter producer Sartorius this method is currently developed for the detection of airborne contaminations.

Air Microbiology↗

PubMed2001

[Effect of the preservative thiomersal on the release of interleukin-1 beta from human peripheral blood cells].

Several vaccines have been tested in the human whole blood pyrogen test as an alternative to the rabbit pyrogen test. As reported previously by our group, the alternative test system is basically applicable to vaccines. The widely used conservative thiomersal is influencing the test system. Surprisingly, the induction of fever inducing cytokines by endotoxin contaminations and other pyrogens can be suppressed by thiomersal.

Animal Testing Alternatives↗

PubMed2001

[Good cell culture practice (GCCP)--an initiative for standardization and quality control of in vitro studies. The establishment of an ECVAM Task Force on GCCP].

Cultured human and animal cells are increasingly used as the basis for simplified, direct test systems that have the potential to be more controllable and more reproducible than in vivo test systems. However, if a biological test system is simplified to fundamental levels then it is paramount that the essential components of such a reduced systems are closely defined and reproducible. Thus, minimal requirements for quality standards in cell and tissue culture have to be defined. It is the aim of this GCCP initiative to establish principles for standardisation, rationalisation, and international harmonisation of cell and tissue culture laboratory practices. Therefore, in analogy to Good Laboratory Practice (GLP), a Good Cell Culture Practice (GCCP) was initiated at the 3rd World Congress on Alternatives and Animal Use in the Life Sciences, Bologna, 29. August-2. September 1999. This "Bologna Statement on Good Cell Culture Practice" was presented, discussed, and refined in a Workshop, and a final version was approved at the closing ceremony of the Congress by the scientific audience. Based on the Bologna Statement, an ECVAM Task Force on GCCP was initiated, that is chaired by Thomas Hartung and Sandra Ceocke, in which experts in the field should elaborate minimal requirements for quality standards in cell culture. It is the intention of the GCCP Guidelines to encourage consensus among all concerned with the use of in vitro systems, in order to establish and maintain best laboratory practices, to promote effective quality control systems, to facilitate education and training, to support journal editors, and to help any authorities who need to interpret and apply conclusions based on in vitro data.

Animal Testing Alternatives↗

PubMed2001

Three Rs potential in the development and quality control of pharmaceuticals.

The intention of a pharmaceutical company is to develop new, efficient products quick and with a minimum of costs. Compared to in vitro methods, animal experiments in general consume much more time and resources (costs as well as time to the market) than in vitro methods. Therefore, the use of whole animal models depends primarily on the judgement of their efficacy in the screening process, but the willingness to incorporate in vitro methods in general is high and is furthered by new developments such as high-throughput screening. Nevertheless, in vitro tests might be politically promoted by increasing their costs (quality controls, requested housing conditions) and duration (time to start of an experiment, sequential performance). Which models are favoured by industry to include them in a screening process: They have to be based on our most recent understanding of the respective disease, well characterised to allow interpretation of results and require only limited development time. All these aspects argue in favour of collaboration between industry and academia, where our understanding of pathophysiology is generated and mechanism based models are developed and characterised. However, technology transfer towards industry represents a bottle-neck for industrial use of these new in vitro models. New platforms to promote this transfer should be developed in order to bring together developer and user of novel in vitro systems and promote demonstration projects. Financing of such collaborations is not the key problem (the development of a single drug makes up to 500 million $) but the dilemma of publication of results: The development advantage compared to competitors depends on the exclusive use of novel models. The protection of intellectual property rights and the public interest in spreading alternatives to animal experiments must be balanced, e.g. by delayed but indispensable publication or advantages for companies employing alternatives in the regulatory approval process for a new drug. Quality control of therapeutic drugs (except hormones and blood products) represents a minor field of animal consumption with the exception of pyrogenicity testing. Despite considerable progress due to the introduction of the Limulus assay which represents the most successful in vitro alternative in use so far. However, some limitations of this in vitro test might be overcome in the near future by the currently validated human whole blood assay. During the last few years considerable progress has been made in the replacement (and deletion) of animal tests required for the potency and safety testing of hormones. This has been made possible by biotechnical production methods, by better-defined products, and because physico-chemical methods can be used for the potency testing of these products. In general, the better defined a drug is, the easier chemical, physical or in vitro techniques can be used for batch control. Control authorities should therefore urge the use of highly standardised components.

Animal Testing Alternatives↗

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

[First Results in the prevaluation of the human whole blood assay for pyrogens in biological pharmaceuticals]

When establishing the whole blood assay for pyrogens in our laboratory we did several investigations on the standard pyrogen to be used. Because of the hydrophobicity of the lipopolysaccharid (LPS) it is difficult to get a homogeneous solution. Therefore we used ultrasonic and biphenyl hydroxyl amine chloride for a better dispergation. Surprisingly the reactivity of the LPS in the whole blood assay for pyrogens dropped the more the LPS was dispergated. That's right inverse to the situation in the LAL. There are first experiences in the whole blood assay for pyrogens with biological pharmaceuticals. This includes immunoglobulins, vaccines and coagulation factors. Except of the vaccines and albumin the rabbit assay is the only way to test these pharmaceuticals for pyrogens. The albumins show a little amplification of the reactivity of LPS in the whole blood assay for pyrogens. The testing of albumin in the whole blood assay for pyrogens showed highly reproproducible results and a detection limit under 50 pg/ml LPS. The immunoglobulins show an amplifying effect as well. Immunoglobulins spiked with LPS were detected as pyrogenic in all cases. Some vaccines show a slight pyrogenity in the whole blood assay which gets visible when diluting the sample. Undiluted vaccines may cover a LPS contamination in the whole blood assay for pyrogens.

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↗

PubMed1995

[The sophisticated process of validation]

Current concepts on the efficient performance of validation studies are summarized. From the experience of recent attempts to validate alternative methods to animal experiments, the difficult procedure of validation should be refined. The applications of toxicological tests (safety testing) and pharmacological methods (identification of putative drugs) require different validation stategies. Suggestions as to an improved design of test development, evaluation, prevalidation, definitive validation as well as final assessment of the result are presented.

Journal Article↗

PubMed1993

[Development of a cell culture model for septic shock multiorgan failure]

Septic shock remains a clinical problem difficult to manage. Several animal models have been established which closely reflect symptoms and course of septic shock. Regarding therapeutic approaches, the possibilities pharmacological intervention are unsatisfactory and call for further investigation. By now, the induction of multiorgan failure in experimental animals has been considered as a successful approach. These experiments impose considerable suffering to animals. The complexity of the molecular events taking place in vivo further restricts the possibility to define rationales for therapeutic intervention. These two aspects prompted the development of a cell-culture model which shares basic mechanisms with the whole-animal experiment. In this cell model using the coculture of parenchymal and non-parenchymal rat-liver cells, endotoxins, i.e. compounds from bacterial cell walls, induce an acute hepatocyte damage. Data obtained with the cell-culture model system are summarized in order to validate the cell-model in comparison to animal pathology, i.e. septic liver failure. Major mechanistic analogies offer the opportunity to develop a useful alternative to animal experiments in the field of septic shock.

Journal Article↗