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Biomedical subjects

Thomas Hartung

Publications and source records attributed to Thomas Hartung.

At least 19 recordsLinked to original sources
PubMed2007

In vitro pyrogen test--A new test method for solid medical devices.

Medical devices manufactured for implantation into humans must be free of any contamination with viable bacteria. However, remnants of dead bacteria and bacterial components alone may induce an inflammatory immune response. Pyrogen tests for such inflammatory contaminations are generally performed either by determining the content of lipopolysaccharide in rinsing solutions of batch samples by limulus amoebocyte lysate assay, by injecting the rinsing solutions into rabbits or by implanting batch samples into rabbits and measuring change of body temperature. In this study, we show that the in vitro pyrogen test (IPT), which measures the release of the inflammatory cytokine IL-1beta in fresh or cryopreserved human whole blood, can be used to assess the pyrogenic contamination of implantable medical devices. This test was used to check neurosurgical implants, namely aneurysm clips, as a proof of principle. Owing to the direct contact of the test material with the blood cells, this test does not require rinsing procedures, which have variable efficacy. The use of human blood ensures the detection of all substances that are pyrogenic for humans and reflects their relative potency. The safety of the products as delivered could be confirmed. The effects of sterilization and depyrogenization procedures on intentional pyrogenic contaminations of samples could be followed. This new application of the already internationally validated method promises to replace further rabbit pyrogen tests. It generates extremely sensitive results with an extended range of detectable pyrogenic contaminants.

Equipment and Supplies↗
PubMed2006

An in vitro pyrogen safety test for immune-stimulating components on surfaces.

Due to the discovery of novel surgical techniques and new materials medical devices are increasingly used. Contact of these biomaterials with human tissue or blood commonly leads to inflammation of varying degrees, sometimes resulting in severe health problems. Possible causes are limited biocompatibility or pyrogenic contamination of the material. We adapted the recently validated in vitro pyrogen test (IPT), based on human whole blood cytokine release, to test the safety of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1beta is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram-positive (lipoteichoic acid, LTA), peptidoglycan (cell wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10 pg/ml for LPS, 10 ng/ml for zymosan and 1 microg/ml for LTA and peptidoglycan from different strains. Furthermore, we have shown that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.

Adjuvants, Immunologic↗
PubMed2006

Synthesis of Staphylococcus aureus lipoteichoic acid derivatives for determining the minimal structural requirements for cytokine induction.

For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b-O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1-4) could be readily obtained and provided for biological studies.

Cytokines↗
PubMed2006

International validation of pyrogen tests based on cryopreserved human primary blood cells.

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.

Animal Testing Alternatives↗
PubMed2006

Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae.

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.

Animals↗
PubMed2006

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Acute-Phase Proteins↗
PubMed2006

Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting.

Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogen-associated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.

CD36 Antigens↗
PubMed2006

Highly purified lipoteichoic acid induced pro-inflammatory signalling in primary culture of rat microglia through Toll-like receptor 2: selective potentiation of nitric oxide production by muramyl dipeptide.

In contrast to the role of lipopolysaccharide from Gram-negative bacteria, the role of Gram-positive bacterial components in inducing inflammation in the CNS remains controversial. We studied the potency of highly purified lipoteichoic acid and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to lipoteichoic acid triggered a significant time- and dose-dependent production of pro-inflammatory cytokines (tumour-necrosis factor-alpha, interleukin-1beta, interleukin-6) and nitric oxide. Muramyl dipeptide strongly and selectively potentiated lipoteichoic acid-induced inducible nitric oxide synthase expression and nitric oxide production. However, it did not have any significant influence on the production of pro-inflammatory cytokines. As bacterial components are recognised by the innate immunity through Toll-like receptors (TLRs) we showed that lipoteichoic acid was recognised in microglia by the TLR2 and lipopolysaccharide by the TLR4, as cells isolated from mice lacking TLR2 or TLR4 did not produce pro-inflammatory cytokines and nitric oxide upon lipoteichoic acid or lipopolysaccharide stimulation, respectively. Lipoteichoic acid-induced glia activation was mediated by p38 and ERK1/2 MAP kinases, as pretreatment with inhibitor of p38 or ERK1/2 decreased lipoteichoic acid-induced cytokine release, iNOS mRNA expression and nitric oxide production. The observed pro-inflammatory response induced by lipoteichoic acid-activated microglia could play a major role in the inflammatory response of CNS induced by Gram-positive bacteria.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2006

Beta-lactam antibiotic-induced release of lipoteichoic acid from Staphylococcus aureus leads to activation of neutrophil granulocytes.

BACKGROUND: Polymorphonuclear neutrophil granulocytes (PMN) are phagocytes of the first line of antimicrobial defense. Previously we demonstrated that lipoteichoic acid (LTA) from Staphylococcus aureus (S. aureus) directly activates neutrophil granulocytes. Others have reported that exposure of S. aureus to beta-lactam antibiotics leads to LTA release. In the present study we addressed the question whether exposure of S. aureus to beta-lactam antibiotics or antibiotics of other groups results in the generation of PMN-stimulating activity and whether this activity can be attributed to LTA. METHODS: S. aureus were exposed to flucloxacillin, a beta-lactam antibiotic or to the protein synthesis-inhibitors erythromycin and gentamicin, or to ciprofloxacin, a gyrase inhibitor. Supernatants of the antibiotic-treated bacteria were assayed for their LTA content and for their effect on PMN functions. RESULTS: We observed that exposure of S. aureus to flucloxacillin and, to a lesser degree to ciprofloxacin, but not to erythromycin or gentamicin led to LTA release. Co-incubation of neutrophil granulocytes with LTA-containing supernatants led to PMN activation as assed by morphological changes, release of IL-8, delay of spontaneous apoptosis and enhanced phagocytic activity. Depletion of LTA from the supernatants markedly reduced their PMN-activating capacity. CONCLUSION: The findings suggest that, via the activation of PMN, antibiotic-induced LTA release from S. aureus leads to enhanced antimicrobial activity of the innate immune defense mechanisms.

Apoptosis↗
PubMed2006

In vitro pyrogen test for toxic or immunomodulatory drugs.

Pyrogenic contaminations of some classes of injectable drugs, e.g. toxic or immunomodulatory as well as false-positive drugs, represent a major risk which cannot yet be excluded due to the limitations of current tests. Here we describe a modification of the In vitro Pyrogen Test termed AWIPT (Adsorb, Wash, In vitro Pyrogen Test), which addresses this problem by introducing a pre-incubation step in which pyrogenic contaminations in the test sample are adsorbed to albumin-coated beads. After rinsing, the beads are incubated with human whole blood and the release of the endogenous pyrogen interleukin-1beta is measured as a marker of pyrogenic activity. Intentional contaminations with lipopolysaccharide were retrieved from the chemotherapeutic agents paclitaxel, cisplatin and liposomal daunorubicin, the antibiotic gentamicin, the antifungal agent liposomal amphotericin B, and the corticosteroid prednisolone at lower dilutions than in the standard in vitro pyrogen test. This represents a promising new approach for the detection of pyrogenic contamination in drugs or in drugs containing interfering additives and should lead to improved safety levels.

Adsorption↗
PubMed2006

Endogenous cortisol determines the circadian rhythm of lipopolysaccharide-- but not lipoteichoic acid--inducible cytokine release.

To investigate the circadian rhythm of inducible cytokine release and a potential pacemaker role of endogenous cortisol, cortisol levels as well as cytokine release from ex vivo LPS-stimulated blood were assessed at 4-h intervals over 24 h in 11 volunteers. We found a significant diurnal variation for IFN-gamma and IL-8, and a tendency for TNF, all inversely correlated to the serum cortisol levels, but no evidence for such a rhythm for IL-1beta and IL-6. In vitro IC(50) values for cytokine inhibition by hydrocortisone (HC) corresponded to the observed rank order for circadian rhythmicity. mRNA analyses revealed that this was due to a reduction of gene transcription. These effects of HC were significantly reversed by the glucocorticoid receptor antagonist RU486. Supplementation of HC in vivo to maintain morning cortisol levels throughout the day blunted the circadian rhythm of ex vivo LPS-induced cytokines. Surprisingly, no significant diurnal variation for any investigated cytokine was found in the same volunteer group upon stimulation with lipoteichoic acid (LTA), the gram-positive counterpart to LPS. Furthermore, 10-50-fold higher HC concentrations as compared to LPS were required to block LTA-induced cytokine release. LTA, in contrast to LPS, failed to activate Jun kinase, a reported target for HC action.

Abortifacient Agents, Steroidal↗
PubMed2006

Thiazolidinedione bioactivation: a comparison of the bioactivation potentials of troglitazone, rosiglitazone, and pioglitazone using stable isotope-labeled analogues and liquid chromatography tandem mass spectrometry.

Troglitazone, a thiazolidinedione (TZD) type insulin sensitizer for the treatment of diabetes, was withdrawn from the U.S. market after several fatal cases of hepatotoxicity. Although the mechanism(s) of these idiosyncratic adverse reactions are not completely understood, circumstantial evidence suggests at least a partial contribution of reactive metabolite formation. Despite isolated case reports of hepatotoxicity, the other TZD derivatives pioglitazone and rosiglitazone are comparatively safe. Herein, we report on the bioactivation potential of these drugs and their TZD ring isotope-labeled 2-(15)N-3,4,5-(13)C(3) analogues in rat and human liver microsomes supplemented with glutathione (GSH). Screening for GSH adducts as surrogate markers for reactive intermediate formation was performed by liquid chromatography tandem mass spectrometry. Chemical characterization of the GSH conjugates was conducted by acquisition of their respective product ion spectra and the comparison between unlabeled and stable isotope-labeled TZD derivatives. The data suggest that all drugs undergo bioactivation processes via a common metabolic activation on the TZD ring, yielding disulfide type GSH conjugates as evidenced by the loss of labeled positions in the TZD moiety. Additional bioactivation processes leading to GSH adducts not involving TZD ring scission were evident for troglitazone. In human liver microsomes at low substrate concentrations, only troglitazone yielded a predominant GSH adduct not involving TZD ring scission. This property may contribute, together with other factors such as the relatively high dose administered as well as its potential to induce hepatic cholestasis and oxidative stress, to the hepatotoxicity of this drug.

Animals↗
PubMed2006

Lipoteichoic acid (LTA) from Staphylococcus aureus stimulates human neutrophil cytokine release by a CD14-dependent, Toll-like-receptor-independent mechanism: Autocrine role of tumor necrosis factor-[alpha] in mediating LTA-induced interleukin-8 generation.

OBJECTIVE: In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils. SUBJECTS: Isolated human neutrophils from healthy volunteers. INTERVENTIONS: Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies. MEASUREMENTS: Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction. CONCLUSIONS: The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Analysis of Variance↗
PubMed2006

Cell activation by ligands of the toll-like receptor and interleukin-1 receptor family depends on the function of the large-conductance potassium channel MaxiK in human macrophages.

Performing patch-clamp experiments on human macrophages, we show that the K(+) channel MaxiK is activated by lipopolysaccharide, peptidoglycan, and interleukin-1. Cytokine production initiated by several Toll-like receptor (TLR) ligands and by interleukin-1 is inhibited by MaxiK blockade. This provides evidence for functional association of the MaxiK channel and TLR signaling complexes.

Cell Line↗
PubMed2006

D-alanyl ester depletion of teichoic acids in Lactobacillus plantarum results in a major modification of lipoteichoic acid composition and cell wall perforations at the septum mediated by the Acm2 autolysin.

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.

Alanine↗
PubMed2006

Identification of Borrelia burgdorferi ribosomal protein L25 by the phage surface display method and evaluation of the protein's value for serodiagnosis.

The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.

Adult↗