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T Hartung

Publications and source records attributed to T Hartung.

At least 19 recordsLinked to original sources
PubMed2006

In vitro tests to evaluate immunotoxicity: a preliminary study.

The implementation of Registration, Evaluation and Authorisation of new and existing Chemicals (REACH) will increase the number of laboratory animals used, if alternative methods will not be available. In the meantime, REACH promotes the use of in vitro tests and, therefore, a set of appropriated alternative testing methods and assessment strategies are needed. The immune system can be a target for many chemicals including environmental contaminants and drugs with potential adverse effects on human health. The aim of this study was to evaluate the predictivity of a set of in vitro assays to detect immunosuppression. The tests have been performed on human, rat and murine cells. Different endpoints have been assessed: cytotoxicity, cytokine release, myelotoxicity and mitogen responsiveness. For each of these endpoints IC50s values have been calculated. Six chemical substances, representative of the full range of in vivo responses and for which good human and/or animal data are available either from databases or literature, have been selected: two chemicals classified as not immunotoxic (Urethane and Furosemide), and four (tributyltin chloride (TBTC), Verapamil, Cyclosporin A, Benzo(a)pyrene) with different effect on immune system. All the tests confirmed the strong immunotoxic effect of TBTC as well as they confirmed the negative controls. For one chemical (Verapamil) the IC50 is similar through the different tests. The IC50s obtained with the other chemicals depend on the endpoints and on the animal species. The clonogenic test (CFU-GM) and the mitogen responsiveness showed similar IC50s between human and rodent cells except for Cyclosporin A and TBTC. All different tests classified the compounds analyzed in the same way.

Animals↗
PubMed2006

Bruton's tyrosine kinase together with PI 3-kinase are part of Toll-like receptor 2 multiprotein complex and mediate LTA induced Toll-like receptor 2 responses in macrophages.

Lipoteichoic acid (LTA) of Gram-positive bacteria initiates innate immune responses via Toll-like receptor-2 (TLR2), resulting in the activation of intracellular signaling and production of inflammatory cytokines in macrophages. Although Bruton's tyrosine kinase (Btk) is biologically important molecule implicated in immune regulation and recently in TLR signaling its importance for LTA-TLR2 mediated responses has not been evaluated. In this study, we detected Btk in the LTA signaling complex with TLR2 and PI 3-kinase (PI3K). The constitutive interaction of these proteins was mediated via PI3K Src homology (SH3) -domain. Both Btk and PI3K were activated by LTA stimulation and the LTA induced cytokine expression was differentially modulated by these kinases. LTA induced the activation of nuclear factor kappaB (NFkappaB), however, only Btk inhibition affected the LTA induced Ser536 phosphorylation and DNA-binding of NFkappaB. In conclusion, our results demonstrate that Btk and PI3K occupy important roles in TLR2-induced activation of macrophages, resulting in selective regulation of cytokines.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2006

A Staphylococcus aureus lipoteichoic acid (LTA) derived structural variant with two diacylglycerol residues.

Based on 1,2-O-isopropylidene-sn-glycerol five chiral building blocks containing differently modified glycerol residues were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl beta-gentiobioside carrying a phosphite residue at 6b-O position. Ligation of these five building blocks led to the desired glycerol phosphate backbone to which d-alanyl residues were attached, thus generating after O-deprotection the target molecule 2, a bisamphiphilic structural variant of Staphylococcus aureus LTA. This compound displayed higher potency in terms of cytokine release by human blood leukocytes than the monoamphiphilic variant LTA.

Carbohydrate Conformation↗
PubMed2006

Toward an evidence-based toxicology.

The increasing demands on toxicology of large-scale risk assessment programmes for chemicals and emerging or expanding areas of chemical use suggest it is timely to review the toxicological toolbox. Like in clinical medicine, where an evidence-based medicine (EBM) is critically reviewing traditional approaches, toxicology has the opportunity to reshape and enlarge its methodology and approaches on the basis of compounded scientific knowledge. Such revision would have to be based on structured reviews of current practice, ie, assessment of test performance characteristics, mechanistic understanding, extended quality assurance, formal validation and the use of integrated testing strategies. This form of revision could optimize the balance between safety, costs and animal welfare, explicitly stating and, where possible, quantifying uncertainties. After a self-critical reassessment of current practices and evaluation of the thus generated information, such an evidence-based toxicology (EBT) promises to make better use of resources and to increase the quality of results, facilitating their interpretation. It shall open up hazard and also risk assessments to new technologies, flexibly accommodating current and future mechanistic understanding. An EBT will be better prepared to answer the continuously growing safety demands of modern societies.

Animals↗
PubMed2005

Highly purified lipoteichoic acid from gram-positive bacteria induces in vitro blood-brain barrier disruption through glia activation: role of pro-inflammatory cytokines and nitric oxide.

The co-culture of bovine brain capillary endothelial cells and rat primary glial cells was established as an in vitro blood-brain barrier model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid and muramyl dipeptide induced injury of blood-brain barrier structure and function. We found that highly purified lipoteichoic acid disrupted blood-brain barrier integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance and high permeability to fluorescein isothiocyanate-inulin observed in the presence of lipoteichoic acid-activated glial cells were potentiated by muramyl dipeptide and could be reversed only when glial cells were activated by lipoteichoic acid at 10 microg/ml but not with a higher lipoteichoic acid concentration (30 microg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction proteins occludin and claudin after lipoteichoic acid treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by lipoteichoic acid. Lipoteichoic acid-activated glial cells produced nitric oxide and pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) that contributed to lipoteichoic acid-induced blood-brain barrier disruption, since the direct treatment of the endothelial monolayer with tumor necrosis factor-alpha or interleukin-1beta increased blood-brain barrier permeability, whereas the pre-treatment of lipoteichoic acid-activated glial cells with antibodies against these two cytokines blocked lipoteichoic acid effects. Additionally, nitric oxide was also involved in blood-brain barrier damage, since the nitric oxide donor itself (diethylenetriamine-nitric oxide adduct) increased blood-brain barrier permeability and inducible nitric oxide synthase inhibitor (1400W) partially reversed lipoteichoic acid-induced trans-endothelial electrical resistance decrease.

Actins↗
PubMed2005

The effects of solvents on embryonic stem cell differentiation.

Dimethyl sulfoxide (DMSO) and ethanol are common organic solvents used for dissolving lipophilic substances for in vitro testing. However, DMSO is known to induce differentiation in embryonic stem (ES) and embryonic teratocarcinoma (EC) cells. In order to clarify if solvents like DMSO and ethanol have an influence on in vitro developmental toxicity test systems, the presented study has evaluated their effects on differentiation by using different test systems. ES and EC cells were transfected with a construct containing the mTert promoter combined with the green fluorescent protein gene (GFP). A down-regulation of mTert, a marker for undifferentiated cells, results in a lower expression of GFP, which could be measured by flow cytometry. Taking the specific characteristics of ES and EC cells into account this effect could be a hint for the interaction of DMSO with embryonic development. Additionally, the effects of the solvents ethanol and DMSO on Oct-4 expression, another marker for undifferentiated cells, were measured in wild-type ES cells. Both selected molecular markers demonstrated an induction of differentiation after exposure to DMSO; in wild-type ES cells at a concentration of 0.125% and in transgenic EC cells at a concentration of 0.25% DMSO. All other differences from controls, including those which attained a level of statistical significance, were minor or not dosage related in degree, or were not consistent over time and are, therefore, considered to be of little toxicological importance. In addition, a cytotoxicity test demonstrated that the solvents affected the employed molecular markers in non-cytotoxic concentrations. The ES cells were the most sensitive towards the cytotoxic effects of the solvent DMSO while the EC cells were more sensitive when treated with the solvent ethanol.

Animals↗
PubMed2004

Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern.

The development of in vitro methods for regulatory embryotoxicity testing is challenging since the understanding of chemical effects on the mammalian development is still poor. The aim of the project is to identify marker genes during in vitro cell differentiation of murine embryonic stem cells, in order to predict chemical effects on cell differentiation of specific target tissues. The present study is focusing on the expression pattern by using semi-quantitative reverse transcriptase (RT)-PCR of key genes involved in cardiomyocytes development; i.e. Oct-4, Brachyury, Nkx2.5 and alpha myosin heavy chain (alpha-MHC). Two reference chemicals with well-known in vivo data have been analysed by using this approach: retinoic acid and lithium chloride. Retinoic acid has been selected as a teratogen affecting several target tissues, whereas lithium chloride has been described to affect the development of the cardiovascular system. We demonstrate that retinoic acid already affects in the early stage of germ layer formation, which was demonstrated by a change of Oct-4 and Brachyury gene expression. As we expected, the expression of cardiac specific genes (Nkx2.5, alpha-MHC) has been also modified. In contrary, the Oct-4 and Brachyury expression was not changed by lithium treatment. In this case, we observed a modification in the normal gene expression pattern, for alpha-MHC and Nkx2.5, demonstrating that lithium chloride affects the later stage of heart development. These data suggest that the inclusion of selective target organ genes in an established embryotoxicity test allows to predict effects of chemicals and drugs to the heart development.

Animals↗
PubMed2004

The use of embryonic stem cells for regulatory developmental toxicity testing in vitro--the current status of test development.

The future chemical policy of the European Union as well as the 7th amendment of the cosmetic directive is calling for the development of alternative tests to animal experimentation for toxicological safety testing. In the field of embryotoxicity one of the most promising in vitro models are based on embryonic stem cells. The embryonic stem cell test has already been validated in comparison to in vivo results in an international blind collaboration study. The presented review is discussing the use, limitations and further needs for the test in order to be fully suitable for regulatory acceptance. In this context, it is summarized which requirements for an in vitro embryotoxicity test have to be fulfilled for regulatory toxicity testing. In addition, an overview about the current status of test development of other embryonic stem cell tests is presented. Several workshops have reviewed the progress of in vitro tests for developmental toxicity testing. A general consensus of these workshops has been that one single test will not be sufficient to cover all manifestations of developmental toxicity. The establishment of a test battery for developmental toxicity is required. This will be even more challenging since the principles of a composing test strategy and its validation have not been defined yet. Finally, the unique possibility of combining the human embryonic stem cell technology and the microarray techniques might lead to a deeper understanding of the toxicological mechanisms of human developmental toxicants.

Abnormalities, Drug-Induced↗
PubMed2003

[Infestation of ticks with Borrelia, and Lyme borreliosis in Germany].

Currently, borreliosis is the most common tick-borne infectious disease in Europe and the USA. In Europe, three species of Borrelia are known to be pathogenic for humans, each of which apparently give rise to different clinical symptoms. Diagnosis is based in particular on the clinical presentation and history of exposure, and, as a rule, is confirmed by serological methods. It must, however, be remembered that the humoral immune response may appear relatively late, so at an early stage, patients are still serologically negative. Depending on the stage involved, oral or peroral antibiotic treatment is applied--however, eradication of the pathogen is unsuccessful in some cases. In future, adjuvant immunotherapy might close the therapeutic gap.

Anti-Bacterial Agents↗
PubMed2002

Molecular aspects of anti-inflammatory action of G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine which stimulates the production of neutrophils in the bone marrow and modulates cellular functions of mature neutrophils. Besides neutrophils and their precursors, monocytes are direct target cells of G-CSF action. G-CSF influences monocyte functions in an anti-inflammatory way: The stimulation of monocytes with G-CSF results in an attenuation of LPS-induced release of IL-1beta, TNF-alpha, IL-12 and IL-18. G-CSF exerts its biological functions on neutrophils and monocytes via membrane-bound receptors. Seven different human G-CSF receptor isoforms have been described which are generated by alternative splicing. The physiologic roles of these isoforms and the expression pattern on various cell types are still unknown. The signal transduction pathway of G-CSF receptors involves the activation of JAK tyrosine kinases and STAT transcription factors and the ras/mitogen-activated protein kinase pathway.

Animals↗
PubMed2002

Comparison and validation of novel pyrogen tests based on the human fever reaction.

The absence of pyrogens (fever-inducing contamination) in injectable drugs is a crucial safety control because pyrogens can pose a life-threatening risk to the patient. The field of applications for pyrogen-testing is expanding and becoming more diverse due to innovative high-tech products such as medical devices, cellular therapies and species-specific agents (e.g. recombinant proteins). The overall aim of the consortium is to develop, evaluate and validate a method based on the human fever reaction to replace the rabbit pyrogen test and the Limulus assay. The network brings together the six most prominent test systems developed recently in Europe for trans-national comparison and subsequent validation of the most promising models. This pre-competitive development will initiate further applications and exploitations for new fields of pyrogen testing such as cellular therapies, medical devices and pyrogen pollution control in the work place. A method for introduction into the European Pharmacopoeia will be developed as a replacement for the rabbit pyrogen test for end-product control.

Animal Use Alternatives↗
PubMed2001

Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria.

Infection with gram-positive bacteria is a major cause of pneumonia. Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung. Both proteins can bind to gram-positive bacteria. Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact. Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria. By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A. Unmodified PepG of Staphylococcus aureus was bound by SP-D. SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable. These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.

Animals↗
PubMed2001

Anti-TNF-neutralizing antibodies reduce pain-related behavior in two different mouse models of painful mononeuropathy.

We investigated the anti-hyperalgesic effect of neutralizing antibodies (AB) to tumor necrosis factor-alpha (TNF) in two murine models of neuropathy, the chronic constrictive sciatic nerve injury (CCI) which has a strong epineurial inflammatory component, and the partial sciatic nerve transection (PST), a 'pure' nerve injury model. In both models a single AB injection intra-operatively as well as on day 4 reduced thermal hyperalgesia significantly, whereas mechanical allodynia was only reduced with intraoperative but not with delayed treatment.

Animals↗
PubMed2001

Schwann cell apoptosis in experimental autoimmune neuritis of the Lewis rat and the functional role of tumor necrosis factor-alpha.

Schwann cell (SC) apoptosis may be a critical factor challenging nerve remyelination and regeneration in experimental autoimmune neuritis (EAN) in the Lewis rat. We therefore analyzed the fate of SC during high-dose antigen therapy of adoptive transfer-(AT-) EAN using rhP2 protein. P2 antigen therapy was associated with an increase of tumor necrosis factor (serum levels 1 h after intravenous (i.v.) injection and an augmentation of T-cell apoptosis. Antigen specific therapy had no clear effect on SC apoptosis. The effects on SC apoptosis were determined by morphological criteria or by in situ tailing (IST) followed by immunocytochemical analysis. Secondly, we neutralized TNF-alpha, released in abundance by antigen treatment but only in small concentrations during natural disease course. We found that the addition of a TNF-alpha neutralizing antiserum resulted in a significant decrease in the rate of SC apoptosis in vivo compared to animals treated with control antigen rhP0 or with rhP2 only.

Adoptive Transfer↗
PubMed2001

The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2.

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.

Animals↗
PubMed2001

Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators.

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent stimulation with LPS termed LPS desensitization or tolerance. To date, it is not known whether similar mechanisms of macrophage refractoriness are induced on contact with components of Gram-positive bacteria. In the present study, we demonstrate that pretreatment with highly purified lipoteichoic acid (LTA) results in suppression of cytokine release on restimulation with LTA in vitro and in vivo in both C3H/HeN and C3H/HeJ mice, but not in macrophages from Toll-like receptor (TLR)-2-deficient mice. Furthermore, desensitization in response to LPS or LTA exposure also inhibits responses to the other stimulus ("cross-tolerance"), suggesting that signaling pathways shared by TLR2 and TLR4 are impaired during tolerance. Finally, we show that LPS- or LTA-induced cross-tolerance is not transferred to hyporesponsive cells cocultured with LPS/LTA-responsive macrophages, showing that soluble mediators do not suffice for tolerance induction in neighboring cells.

Animals↗
PubMed2001

Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-kappaB translocation.

Recently Toll-like receptors (TLRs) have been found to be involved in cellular activation by microbial products, including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4, TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of recognition of lipoteichoic acids (LTAs) and related glycolipids has not been completely understood. In order to determine the role of TLRs in immune cell activation by these stimuli, experiments involving TLR-2-negative cell lines, TLR-expression plasmids, macrophages from TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as highly purified LTAs from Staphylococcus aureus and Bacillus subtilis exhibited TLR-2 dependence in nuclear factor kappaB activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4. Fractionation of the T. brennaborense glycolipids by hydrophobic interaction chromatography and subsequent cell stimulation experiments revealed two peaks of activity, one exhibiting TLR-2-, and a second TLR-4-dependence. Furthermore, we show involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids by dominant negative overexpression experiments. In summary, the results presented here indicate that TLR-2 is the main receptor for Treponema glycolipid and LTA-mediated inflammatory response.

Animals↗