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Thomas Hartung

Publications and source records attributed to Thomas Hartung.

At least 55 records · Page 3Linked to original sources
PubMed2005

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

Animals↗
PubMed2005

Substrate specific metabolism by polymorphic cytochrome P450 2D6 alleles.

A comparative metabolism study was performed for bufuralol, dextromethorphan, imipramine, mianserin, sparteine, tamoxifen, haloperidol and two drug candidates (Rec27/0110 and Rec15/2739) on V79 cells genetically engineered to express human cytochrome P450 (CYP) variants 2D6*1, 2D*2, 2D*9 and 2D*17. Unexpectedly, the CYP2D6*17 dependent metabolism profile of haloperidol and Rec27/0110 were found to differ from all other substrates tested. Some of these known standard substrates are frequently applied in marker reactions for CYP2D6 and with these standard substrates, CYP2D6*1 is known to be the most active form. In both cases of haloperidol and Rec27/0110 the variant form CYP2D6*17 had equal or higher activity compared to the CYP2D6*1 form. Results obtained with the V79 cells were confirmed using microsomal preparation of yeast cells expressing the variants CYP2D6*1 and CYP2D6*17 and CYP2D6 inhibitor quinidine. In conclusion, there is no general rule for a variant dependent metabolism profile by cytochrome P450 2D6 indicating that the activity profile of the CYP2D6 alleles may be substrate specific, thus may be reflected in pharmacokinetics consequences for individuals.

Alleles↗
PubMed2005

Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance.

Lipopolysaccharide (LPS) tolerance is a state of refractoriness towards a second stimulation by LPS after a preceding stimulation. LPS is recognized by Toll-like receptor-4 (TLR-4), which belongs to a group of pattern recognition receptors mediating activation of innate immunity by microbial components. To date, it is not known in detail to what extent other TLR-dependent stimuli also induce tolerance and whether preceding and challenging stimuli are interchangeable. We have examined tolerance induction in detail for lipoteichoic acid (LTA), LPS and CpG-DNA, which are recognized by TLR-2, -4 and -9, respectively. In RAW264.7 macrophages, all three stimuli induced tolerance towards a subsequent challenge with the same stimulus used for priming, as well as cross-tolerance towards subsequent challenge with other stimuli signalling via different TLRs. However, whereas LPS/LTA cross-tolerance was also functional in an in vivo model of galactosamine (GalN)-primed liver damage, pretreatment with CpG only protected against GalN/CpG challenge and failed to induce cross-tolerance for LPS and LTA. CpG-DNA pretreatment even enhanced tumour necrosis factor (TNF)-alpha production and liver damage upon subsequent challenge with LPS or LTA. Stimulation with CpG-DNA resulted in a peculiar sensitization for interferon (IFN)-gamma secretion. The data indicate that, in contrast to in vitro macrophage desensitization, the in vivo consequences of repeated TLR stimulation greatly differ amongst different TLR ligands.

Animals↗
PubMed2005

Inhibition of microglial inflammation by the MLK inhibitor CEP-1347.

CEP-1347 is a potent inhibitor of the mixed lineage kinases (MLKs), a distinct family of mitogen-activated protein kinase kinase kinases (MAPKKK). It blocks the activation of the c-Jun/JNK apoptotic pathway in neurons exposed to various stressors and attenuates neurodegeneration in animal models of Parkinson's disease (PD). Microglial activation may involve kinase pathways controlled by MLKs and might contribute to the pathology of neurodegenerative diseases. Therefore, the possibility that CEP-1347 modulates the microglial inflammatory response [tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1)] was explored. Indeed, the MLK inhibitor CEP-1347 reduced cytokine production in primary cultures of human and murine microglia, and in monocyte/macrophage-derived cell lines, stimulated with various endotoxins or the plaque forming peptide Abeta1-40. Moreover, CEP-1347 inhibited brain TNF production induced by intracerebroventricular injection of lipopolysaccharide in mice. As expected from a MLK inhibitor, CEP-1347 acted upstream of p38 and c-Jun activation in microglia by dampening the activity of both pathways. These data imply MLKs as important, yet unrecognized, modulators of microglial inflammation, and demonstrate a novel anti-inflammatory potential of CEP-1347.

Animals↗
PubMed2005

Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis.

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.

Animals↗
PubMed2005

Critical evaluation of urine-based PCR assay for diagnosis of Lyme borreliosis.

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at--20 degrees C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.

Adult↗
PubMed2005

The detection of differentiation-inducing chemicals by using green fluorescent protein expression in genetically engineered teratocarcinoma cells.

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.

Animal Testing Alternatives↗
PubMed2005

Optimisation of pyrogen testing in parenterals according to different pharmacopoeias by probabilistic modelling.

The rabbit test to detect pyrogenic contamination in parenterals is crucial to ensure patient safety. The pharmacopoeial tests in Europe, the US and Japan are based on the fever reaction of rabbits, but differ in their experimental design and in their algorithms to assess contamination. Employing an international reference endotoxin, fever can be induced in rabbits. Data from 171 rabbits built the base for probabilistic modelling of the fever reaction and for the comparison of the pharmacopoeial tests. The rabbit fever reaction could be modelled as a function of the amount of injected endotoxin (per kg body weight) by linear regression. Combining the pharmacopoeial algorithms of the rabbit pyrogen test with the developed model allowed analysis of differences regarding test results and animal consumption. This showed that the assessment of pyrogenic contamination strongly depends on the respective pyrogen test stipulated by regulations. Additionally, the approach was used to develop a new experimental design. Two specific versions of this design resulted in a reduction of the number of animals used by about 30% while the safety of the test was maintained. A need for harmonisation is evident, allowing optimisation of the experimental design, which promotes animal welfare.

Algorithms↗
PubMed2005

Structure/function relationships of lipoteichoic acids.

The role of lipoteichoic acids (LTAs) from Gram-positive bacteria as immunostimulatory molecules was controversial for many years, as inadequate preparation methods as well as heterogeneous and endotoxin-contaminated commercial preparations led to conflicting results. An improved purification methodology for LTA now yields potent bioactive and chemically defined material, which is currently being characterized in various models. A synthetic analogue of Staphylococcus aureus LTA has proven the structure/function relationship. The key role of D-alanine esters for the immune response of LTA was confirmed by synthetic derivatives. The glycolipid anchor of LTA plays a central role analogous to the lipid A of LPS. Methodological aspects and criteria for quality assessment of LTA preparations are discussed.

Lipopolysaccharides↗
PubMed2005

Developmental toxicity testing from animal towards embryonic stem cells.

Developmental toxicology is the study of undesirable effects on the development of the organism, which may result from exposure before conception, from the period of prenatal development, or postnatally during the time of sexual maturation. The principal manifestations of developmental toxicity include: embryolethality, malformations, growth retardation, and functional impairment. In 2001, the European Commission published the future chemicals policy entitled "White Paper: Strategy for a Future Chemicals Policy". The new regulation requires a toxicological evaluation with strong emphasis on reproductive toxicity, by using in vitro methods, especially for those chemicals marketed at more than 1 ton per year. For this reason, the establishment of in vitro models capable of detecting major undesirable manifestations in the fetus, are urgently required. The aim of the present review is to explore the capacity of existing in vitro systems, based on embryonic stem (ES) cells, to identify embryotoxicity with a focus on specific effects such as teratogenicity and growth retardation. In addition, we discuss the possibility to adapt the mouse ES cells based tests to human ES cells, avoiding inter-species variations in developmental toxicity studies and address related ethical issues. Considering the different manifestations of developmental toxicity, only a battery of in vitro tests will provide the necessary information for regulatory developmental toxicity assessment.

Animal Testing Alternatives↗
PubMed2004

Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes.

Lipopolysaccharides (LPS) are cell-surface components of Gram-negative bacteria and are microbe-/pathogen-associated molecular patterns in animal pathosystems. As for plants, the molecular mechanisms of signal transduction in response to LPS are not known. Here, we show that Arabidopsis thaliana reacts to LPS with a rapid burst of NO, a hallmark of innate immunity in animals. Fifteen LPS preparations (among them Burkholderia cepacia, Pseudomonas aeruginosa, and Erwinia carotovora) as well as lipoteichoic acid from Gram-positive Staphylococcus aureus were found to trigger NO production in suspension-cultured Arabidopsis cells as well as in leaves. NO was detected by confocal laser-scanning microscopy in conjunction with the fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, by electron paramagnetic resonance, and by a NO synthase (NOS) assay. The source of NO was addressed by using T-DNA insertion lines. Interestingly, LPS did not activate the pathogen-inducible varP NOS, but AtNOS1, a distinct NOS previously associated with hormonal signaling in plants. A prominent feature of LPS treatment was activation of defense genes, which proved to be mediated by NO. Northern analyses and transcription profiling by using DNA microarrays revealed induction of defense-associated genes both locally and systemically. Finally, AtNOS1 mutants showed dramatic susceptibility to the pathogen Pseudomonas syringae pv. tomato DC3000. In sum, perception of LPS and induction of NOS contribute toward the activation of plant defense responses.

Arabidopsis↗
PubMed2004

Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane.

The innate immune system utilises pattern recognition receptors in order to recognise microbial conserved molecular patterns. The family of Toll-like receptors (TLRs) has been shown to act as the main pattern recognition receptors for the innate immune system. Using biochemical as well as fluorescence imaging techniques, TLR2 and TLR4 were found to be recruited within microdomains upon stimulation by bacterial products. Furthermore their lateral diffusion in the cell membrane as determined by fluorescence recovery after photobleaching revealed that upon stimulation by bacterial products TLRs encounter barriers to their lateral movement, thus supporting the notion that specialised domains on the plasma membrane facilitate the innate recognition.

Animals↗
PubMed2004

Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi are lipid raft-dependent.

Lipoteichoic acid (LTA), a key cell wall component of Gram-positive bacteria, seems to function as an immune activator with characteristics very similar to lipopolysaccharide from Gram-negative bacteria. It has been shown that LTA binds CD14 and triggers activation via Toll-like receptor 2, but whether the activation occurs at the cell surface or internalization is required to trigger signaling has yet to be demonstrated. In this work we have investigated LTA binding and internalization and found that LTA and its receptor molecules accumulate in lipid rafts and are subsequently targeted rapidly to the Golgi apparatus. This internalization seems to be lipid raft-dependent because raft-disrupting drugs inhibited LTA/Toll-like receptor 2 colocalization in the Golgi. Similarly to lipopolysaccharide, LTA activation occurs at the cell surface, and the observed trafficking is independent of signaling.

Animals↗
PubMed2004

Activation of neutrophils and inhibition of the proinflammatory cytokine response by endogenous granulocyte colony-stimulating factor in murine pneumococcal pneumonia.

Granulocyte colony-stimulating factor (G-CSF) is considered to improve host defense during infection, via increased recruitment of and enhanced performance of neutrophils and subsequent inhibition of potentially harmful proinflammatory mediators. The present study sought to determine the role of endogenous G-CSF in host defense against pneumococcal pneumonia. Patients with unilateral community-acquired pneumonia demonstrated elevated concentrations of G-CSF in bronchoalveolar lavage fluid obtained from the infected, but not from the contralateral, site. Treatment of mice with pneumococcal pneumonia with an anti-G-CSF antibody reduced neutrophil counts in lung tissue and diminished CD11b expression on pulmonary neutrophils but increased the lung concentrations of tumor necrosis factor- alpha, interleukin-1 beta, and cytokine-induced neutrophil chemoattractant. Treatment with anti-G-CSF did not influence the outgrowth of pneumococci in lungs, the dissemination of the infection, or survival in murine pneumonia. During pneumococcal pneumonia, G-CSF is produced locally at the site of the infection, where it exerts both pro- and anti-inflammatory effects.

Adult↗
PubMed2004

Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells.

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2(-/-) mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2(-/-) macrophages. Although production of the proinflammatory cytokines TNF, IL-1alpha, and IL-1beta is normal, IL-10 release is severely impaired in the TLR2(-/-) mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2(-/-) mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.

Animals↗
PubMed2004

L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of Gram-positive bacteria, and activates the lectin pathway of complement.

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.

Adjuvants, Immunologic↗