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Biomedical subjects

Thomas Hartung

Publications and source records attributed to Thomas Hartung.

At least 109 records · Page 6Linked to original sources
PubMed2003

Characterization of the cytokine immune response in children who have experienced an episode of typical hemolytic-uremic syndrome.

The lipopolysaccharide (LPS) of enterohemorrhagic Escherichia coli (EHEC) and Shiga toxin together substantially contribute to the pathophysiology of typical hemolytic-uremic syndrome (HUS). Both factors have been shown to be immune stimulators and could play a key role in the individual innate immune response, characterized by proinflammatory and anti-inflammatory cytokines. By use of a whole blood stimulation model, we therefore compared the LPS- and superantigen-induced cytokine responses in children who had been having recovering from an acute episode of typical HUS for at least 6 months (group 1) with those in controls, who consisted of patients seen in the pediatric neurology outpatient department for routine examination (group 2). Samples were analyzed for cytokine protein levels and the levels of mRNA production. LPS stimulation revealed lower levels of interleukin 10 (IL-10) (P < 0.05) and increased levels of gamma interferon (P < 0.05) and increased ratios of pro- and anti-inflammatory cytokines (P < 0.05 for the IL-1beta/IL-10 ratio; P < 0.05 for the tumor necrosis factor alpha/IL-10 ratio) in group 1. In addition superantigen stimulation showed decreased IL-2 levels in group 1 (P < 0.01). Our results suggest an alteration of the cytokine response characterized by high proinflammatory cytokine levels and low anti-inflammatory cytokine levels as well as low levels of IL-2 production in children who have experienced an episode of typical HUS. We hypothesize that this altered immune response is not a residual effect of the infection but a preexisting characteristic of the patient. This could be one reason why individuals infected with EHEC are potentially predisposed to a systemic disease (HUS).

Case-Control Studies↗
PubMed2003

Borrelia burgdorferi-induced tolerance as a model of persistence via immunosuppression.

If left untreated, infection with Borrelia burgdorferi sensu lato may lead to chronic Lyme borreliosis. It is still unknown how this pathogen manages to persist in the host in the presence of competent immune cells. It was recently reported that Borrelia suppresses the host's immune response, thus perhaps preventing the elimination of the pathogen (I. Diterich, L. Härter, D. Hassler, A. Wendel, and T. Hartung, Infect. Immun. 69:687-694, 2001). Here, we further characterize Borrelia-induced immunomodulation in order to develop a model of this anergy. We observed that the different Borrelia preparations that we tested, i.e., live, heat-inactivated, and sonicated Borrelia, could desensitize human blood monocytes, as shown by attenuated cytokine release upon restimulation with any of the different preparations. Next, we investigated whether these Borrelia-specific stimuli render monocytes tolerant, i.e. hyporesponsive, towards another Toll-like receptor 2 (TLR2) agonist, such as lipoteichoic acid from gram-positive bacteria, or towards the TLR4 agonist lipopolysaccharide. Cross-tolerance towards all tested stimuli was induced. Furthermore, using primary bone marrow cells from TLR2-deficient mice and from mice with a nonfunctional TLR4 (strain C3H/HeJ), we demonstrated that the TLR2 was required for tolerance induction by Borrelia, and using neutralizing antibodies, we identified interleukin-10 as the key mediator involved. Although peripheral blood mononuclear cells tolerized by Borrelia exhibited reduced TLR2 and TLR4 mRNA levels, the expression of the respective proteins on monocytes was not decreased, ruling out the possibility that tolerance to Borrelia is attributed to a reduced TLR2 expression. In summary, we characterized tolerance induced by B. burgdorferi, describing a model of desensitization which might mirror the immunosuppression recently attributed to the persistence of Borrelia in immunocompetent hosts.

Animals↗
PubMed2003

ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies.

The European Centre for the Validation of Alternative Methods (ECVAM) has restructured its services by directly targeting the animal tests that need to be replaced. In view of the short time-lines for making available and implementing validated methods, ECVAM is offering to steer the process by bringing together the inputs of stakeholders and encouraging the early involvement of regulators. In essence, steering groups formed by ECVAM senior staff, and complemented with external experts, will carry out the project management and will coordinate the various inputs.

Administration, Topical↗
PubMed2003

Comparison of the reactivity of human and rabbit blood towards pyrogenic stimuli.

A comparison between humans and rabbits was performed based on stimulation of whole blood with well-known pyrogens from Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The reactivity was measured as release of IL-1 beta and IL-8 by ELISA. The reactivity of the two species towards LPS was found to be similar, whereas their reactivity towards LTA differed considerably. Differences between the levels of IL-1 beta and IL-8 release were observed in both species. This finding suggests that the In vitro Pyrogen Test (IPT) which uses human blood to detect contaminations, e.g. of injectable drugs, might predict the human reaction to the contamination better than the "gold standard" rabbit pyrogen test.

Animals↗
PubMed2002

Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.

We recently purified lipoteichoic acid (LTA) from Staphylococcus aureus to more than 99% purity by a novel preparation method and deduced its structure with the first nuclear magnetic resonance (NMR) of a complete LTA. In contrast to Gram-negative lipopolysaccharides, this LTA requires the toll-like receptor (TLR)-2 and not TLR-4 for cytokine induction in monocytes and macrophages. To elucidate the structure-function relationships for LTA from S. aureus, the lipid anchor was prepared by either acidic hydrolysis of native LTA or chemical synthesis (gentiobiosyl-sn-dimyristoylglycerol). Next, a complete LTA molecule with six glycerophosphate units carrying four alanine plus one N-acetyl-glucosamine substituent was synthesized, which displayed the same potency to activate monocytes as native LTA. However, 100-1,000 times higher concentrations of the lipid anchor were required for cytokine induction. It is worthy to note that replacing D-alanine with L-alanine blunted the effect indicating stereoselective recognition. The structure identification of this synthesized and biologically active LTA was proven by NMR and matrix-assisted laser desorption-ionization mass spectrometry. We concluded that the lipid anchor, with its fatty acids, represents an integral part of the immunostimulatory activity of LTA, but requires additional structural components on the polyglycerophosphate backbone.

Animals↗
PubMed2002

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase.

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.

Enzyme Activation↗
PubMed2002

Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid.

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.

Animals↗
PubMed2002

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.

Animals↗
PubMed2002

Cytokine induction by purified lipoteichoic acids from various bacterial species--role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release.

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.

Acute-Phase Proteins↗
PubMed2002

Granulocyte colony-stimulating factor attenuates LPS-stimulated IL-1beta release via suppressed processing of proIL-1beta, whereas TNF-alpha release is inhibited on the level of proTNF-alpha formation.

In the presence of granulocyte colony-stimulating factor (G-CSF), the release of IL-1beta and TNF-alpha by LPS-stimulated human whole blood was suppressed. Via measurement of cytokine mRNA, inactive precursor and mature protein, we investigated whether this inhibition occurs at the transcriptional, translational or post-translational level of cytokine production. G-CSF inhibited IL-1beta release, but the formation of proIL-1beta was not attenuated, indicating that G-CSF interferes with the proteolytic processing of proIL-1beta. Since the release of IL-1beta in LPS-stimulated whole blood was blocked by the caspase-1 inhibitor YVAD-cmk, processing of proIL-1beta appears to depend on caspase-1 activity. The conclusion that G-CSF inhibits caspase-1 activity was supported bythe finding that the release of IL-18 was also inhibited by G-CSF, similar to IL-1beta release. Intracellular caspase-1 activity in monocytes was measured by flow cytometry with the cell-permeablecaspase substrate Asp(2)-rhodamine. In the presence of G-CSF the cleavage of this substrate was inhibited by more than 50%. G-CSF had no effect on LPS-induced doubling of caspase-1 mRNA, indicating that G-CSF affects caspase-1 activation and not its formation. For TNF-alpha another mechanism of G-CSF action was identified: TNF-alpha as well as proTNF-alpha formation were inhibited by G-CSF, butG-CSF had no influence on LPS-induced TNF-alpha mRNA level. We therefore suggest that G-CSF causes translational silencing of LPS-induced TNF-alpha mRNA.

Granulocyte Colony-Stimulating Factor↗
PubMed2002

Coronary microembolization: the role of TNF-alpha in contractile dysfunction.

Coronary microembolization is a frequent complication of atherosclerotic plaque rupture in acute coronary syndromes and during coronary interventions. Experimental coronary microembolization results in progressive contractile dysfunction associated with a local inflammation. We studied the causal role of tumor necrosis factor-alpha (TNF-alpha) in the progressive contractile dysfunction resulting from coronary microembolization. Anesthetized dogs were subjected to either coronary microembolization with infusion of 3.000 microspheres (42 microm diameter) per ml coronary inflow into the left circumflex coronary artery (n=9), or to intracoronary infusion of recombinant human TNF-alpha without microembolization (n=4), or to treatment with anti-murine TNF-alpha sheep antibodies prior to microembolization (n=4). Posterior systolic wall thickening (PWT; sonomicrometry) decreased from 21.1+/-5.3% (s.d.) at baseline to 5.5+/-2.2% (P<0.05) at 8 h after microembolization. Infarct size (1.8+/-1.9%; TTC and histology) and the amount of apoptosis (<0.1%; TUNEL and DNA-laddering) were small. TNF-alpha at the protein level (WEHI cytolytic assay) was increased and localized to leukocytes (immunostaining), which were increased in number (quantitative histology). In situ hybridization for TNF-alpha mRNA identified viable cardiomyocytes surrounding the microinfarcts as the major source of TNF-alpha. Supporting the role of TNF-alpha, infusion of TNF-alpha without microembolization decreased PWT from 27.3+/-6.9% at baseline to 10.1+/-4.9% after 8 h (P<0.05); in contrast, in the presence of TNF-alpha antibodies, microembolization no longer reduced PWT (19.3+/-7.0% at baseline v 16.9+/-5.0% at 8 h). In conclusion, TNF-alpha is the mediator responsible for the profound contractile dysfunction following coronary microembolization.

Animals↗
PubMed2002

Interaction of lipoteichoic acid and CpG-DNA during activation of innate immune cells.

The innate immune system recognizes pathogen-associated molecular patterns (PAMP) to cope with evolving infections. Toll-like receptors (TLRs) play a pivotal role in recognition of PAMPs. In the course of infection not a single but rather a full panel of different microbial components interacts with distinct TLRs simultaneously. Only limited information is available on effects of combinations of TLR agonists. Here, we have analyzed the effects of lipoteichoic acid (LTA), CpG-DNA and combinations thereof on innate immune cells in vitro. Although proinflammatory cytokines like TNF-alpha were induced by these agonists in quite similar amounts, CpG DNA was superior in its potency to induce IL-12p40 reflecting important differences in the biological valence of LTA and CpG-DNA. When given in combination, LTA and CpG-DNA were additive in induction of TNF-alpha, IL-6 and nitric oxide in RAW 264 macrophages, peritoneal macrophages and dendritic cells. Additive effects were also observed in regard to TNF-alpha mRNA. In contrast, LTA suppressed IL12p40 secretion induced by CpG-DNA in RAW cells and peritoneal macrophages but not in dendritic cells. Intracellular signal cascades (NFkappaB and p38 MAP kinase) showed additive effects after simultaneous triggering. mRNA expression ofTLRs showed only minor regulation after CpG or LTA application and thus does not account for the additive/suppressive effects observed. These results indicate that the consequences of interaction of innate immune cells with microbial pattern depend on the responding cell type and might be differential for certain effector mechanisms. Thus, the pathogen-characteristic panel of TLR ligands will induce pathogen-specific innate responses decisive for the inflammatory reactions.

Animals↗
PubMed2002

Structural decomposition and heterogeneity of commercial lipoteichoic Acid preparations.

Fractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% for Streptococcus pyogenes, 16% for Bacillus subtilis, and 75% for Staphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine and N-acetylglucosamine substituents. All preparations contained-to varying degrees-non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA from Bacillus subtilis and S. pyogenes but not LTA from S. aureus induced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.

Bacillus subtilis↗
PubMed2002

Toll-like receptor 4 (TLR4)-deficient murine macrophage cell line as an in vitro assay system to show TLR4-independent signaling of Bacteroides fragilis lipopolysaccharide.

Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4 independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine were found when cells were stimulated with E. coli LPS. Bacteroides fragilis-derived LPS, however, can effectively stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate immune response.

Animals↗
PubMed2002

Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR.

A LightCycler-based PCR protocol was developed which targets the ospA gene for the identification and quantification of the different Borrelia burgdorferi sensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three different B. burgdorferi sensu lato genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of different Borrelia species in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence of B. burgdorferi sensu lato infection in Ixodes ricinus ticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution of B. burgdorferi species by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas was B. afzelii (53%), followed by B. garinii (18%) and B. burgdorferi sensu stricto (11%); 0.8% of the infecting Borrelia could not be identified.

Animals↗
PubMed2002

Comparison of eleven commercial tests for Chlamydia pneumoniae-specific immunoglobulin G in asymptomatic healthy individuals.

The seroprevalence of anti-Chlamydia pneumoniae-specific immunoglobulin G (IgG) antibodies is high in the adult population. Experience is required to perform a microimmunofluorescence test (MIF), the current "gold standard" for serological diagnosis, and the assay still lacks standardization. Partially automated enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), which are more standardized and for which the reading of results is less subjective, have been developed. The different commercially available serological tests differ in their sensitivities and specificities, depending primarily on the antigen used. Therefore, we evaluated 11 different tests (10 were species specific, 1 was genus specific) for IgG antibodies using serum samples of 80 apparently healthy volunteers. The interpretation of the results was based on the results of the gold standard, MIF: a sample was judged positive if it was positive by at least three of the four different MIFs. Based on this internal standard, we found that 71% of the samples were positive, while 8% were false positive by some tests. The correlations between the results of the different MIFs ranged from 83 to 99%, and the correlations between the results of the MIFs and the different ELISAs and EIAs ranged from 78 to 98%. Comparison of the IgG titers measured by MIF showed good agreement (r = 0.76 to 0.91). This analysis revealed that some ELISAs and EIAs fail to detect low IgG titers. The specificities of the species-specific tests varied from 95 to 100%, and the sensitivities varied from 58 to 100%. These results indicate that serological assays for the detection of anti-C. pneumoniae-specific IgG vary greatly in their sensitivities and specificities. MIF must still be considered the best method for the detection of IgG in apparently healthy subjects, but the sensitivities and specificities of new ELISAs approximate those of MIFs.

Adult↗
PubMed2002

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

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests, based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.

Animal Testing Alternatives↗