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Biomedical subjects

Klaus Heeg

Publications and source records attributed to Klaus Heeg.

At least 19 recordsLinked to original sources
PubMed2006

Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation.

Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

Animals↗
PubMed2006

Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling.

Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-gamma plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-gamma signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-gamma-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-gamma receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-gamma on parasite replication. The inhibitory effects of T. gondii on IFN-gamma were diminished in macrophages from SOCS-1-/- mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite's immune evasion strategies.

Animals↗
PubMed2006

Differences in innate immune responses upon stimulation with gram-positive and gram-negative bacteria.

BACKGROUND AND OBJECTIVES: Host recognition pathways for gram-negative and gram-positive bacteria comprise pattern recognition receptors among which Toll-like receptors (TLRs) play a pivotal role. TLRs share common signaling pathways yet exhibit specificity as well. Periodontal disease is initiated and maintained in the first line by gram-negative but also gram-positive bacterial infection of the gingival sulcus. To date only limited information is available on whether gram-positive and gram-negative bacteria induce different host responses (strength or quality). MATERIALS AND METHODS: To elucidate these differential effects we focused on proinflammatory cytokine releases by assessing ex vivo stimulation of whole blood with heat-killed gram-negative and gram-positive bacteria and thereof derived microbial products associated with distinct TLRs. Tumor necrosis factor-alpha and interleukin-8 release were measured in the supernatants by enzyme-linked immunosorbent assay. In addition, innate immune responses of peritoneal macrophages from mice lacking TLR2 and TLR4 were tested. RESULTS: We observed that gram-negative and gram-positive species induced distinct patterns of cytokine production. Gram-negative species produced higher amounts of tumor necrosis factor-alpha while gram-positive species released higher amounts of the chemokine interleukin-8. Data from TLR knockout mice and TLR-transfected HEK cells revealed a somehow specific role of TLR4 and TLR2 for the recognition of gram-negative and gram-positive bacteria, respectively, an observation that goes along with the dominant recognition of the respective pathogen associated molecular patterns lipopolysaccharide and lipoteichoic acid. CONCLUSIONS: The results show that gram-negative and gram-positive bacterial species induce different patterns of immunoregulatory activity, which might be the result of activation of different TLRs.

Animals↗
PubMed2006

Activation of toll-like receptor 9 by DNA from different bacterial species.

Toll-like receptor 9 (TLR-9) recognizes unmethylated CpG dinucleotides which are abundant in prokaryotic DNA and yet are rare in eukaryotic DNA. Little is known about the significance of TLR-9 in terms of recognition of different bacterial DNA species. In this study HEK293 cells stably transfected with human TLR-9 were used to analyze the immunostimulatory properties of 15 bacterial DNA preparations. In addition, bacterial genome data were analyzed for the frequency of unmethylated cytosine-guanosine ([CG]) dinucleotides. We observed that DNA samples of different bacteria showed considerable differences in their potential to stimulate TLR-9. This correlated with the frequency of [CG] dinucleotides. Based upon data from our experiments the estimate of immunostimulatory bacterial DNA concentrations translated to as high as 10(9) bacteria/ml. Application of the transfection reagent DOTAP resulted in a more efficient delivery of DNA into the cell, and this went along with increased TLR-9 activation. The data indicate that bacterial DNA preparations from different species differ in their capacity to activate TLR-9, which is dependent on the individual [CG] content. Moreover, increased intracellular delivery results in a marked enhancement of immunostimulation.

Animals↗
PubMed2005

Possible risk for re-colonization with methicillin-resistant Staphylococcus aureus (MRSA) by faecal transmission.

An increase in the frequency of methicillin-resistant Staphylococcus aureus (MRSA) as an important causative agent of nosocomial infections is observed worldwide. Unfortunately, in spite of optimal hygienic conditions (barrier isolation, screening, decontamination), patients often remain MRSA positive or are detected as "re-colonized" at readmission. The objective of our study was to clarify if this is due to an undetected colonization of the gastrointestinal tract, which could possibly lead to re-colonization after primary successful decontamination. Therefore, all MRSA strains were collected from 290 in-patients of a university hospital over a period of 2 years. A surprisingly high number (24.1% of all) was isolated from stool samples. Even 13.1% of the total collection could be first observed in this material before detecting MRSA in other materials of these patients. To evaluate the epidemiology of these isolates, pulsed-field gel electrophoresis (PFGE) was used. On the basis of PFGE restriction types one main clone and 11 singular clones could be identified. Additionally, for six individual patients MRSA isolates from stool specimens were indistinguishable from other isolates from different locations. We show here that colonization of the gastrointestinal tract with MRSA apparently could play an important role in spreading MRSA via faecal contamination. Hence, we suggest that stool colonization with MRSA could be the source of a so far unrecognized transmission of MRSA within individual patients and within the population. Therefore, our findings imply a modification in the hygienic strategies for handling decontamination and therapy of MRSA patients.

Bacterial Typing Techniques↗
PubMed2005

Micromonas (Peptostreptococcus) micros: unusual case of prosthetic joint infection associated with dental procedures.

Micromonas (Peptostreptococcus) micros is frequently associated with periodontal disease as well as respiratory, gastrointestinal and female genitourinary tract infections, but only rarely has been reported as a pathogenic agent of prosthetic joint infections. Here we describe a case of a 63-year-old woman with prosthetic joint infection of total hip arthroplasty caused by the anaerobic species Micromonas micros, associated with tooth extraction. Samples obtained intraoperatively and from the oral cavity were positive for the presence of M. micros by culture and by real-time PCR. This case report indicates that infections of prosthetic joints can be associated with dental procedures and that sensitive molecular techniques are necessary for their routine diagnostic.

Aged↗
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

Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients.

BACKGROUND: Accurate laboratory tests for the detection and quantification of periodontopathogens in subgingival plaque samples of periodontal disease patients are becoming essential to study the pathogenesis of this polymicrobial condition. We used a real-time polymerase chain reaction (PCR) assay for the quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Dialister pneumosintes, Campylobacter rectus, and Micromonas micros as well as total eubacteria in subgingival plaque samples from individuals with periodontitis. METHODS: Eighty-three subgingival samples from periodontally diseased patients and 43 samples from periodontally healthy subjects were tested and the results of bacterial quantification were correlated to clinical parameters. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standards by real-time PCR. RESULTS: Results showed that patients as well as healthy subjects were positive for the presence of target periodontopathogens; however, median values were higher in samples from periodontitis subjects. In addition, a positive association was observed between colonization at high levels by P. gingivalis and M. micros and the presence of deep periodontal pockets. CONCLUSION: Real-time PCR provides a reliable high-throughput method for quantification of periodontopathogens and may be useful for understanding the complex etiology observed in periodontal diseases.

Case-Control Studies↗
PubMed2004

Poly-guanosine strings improve cellular uptake and stimulatory activity of phosphodiester CpG oligonucleotides in human leukocytes.

CpG-oligonucleotides (CpG-ODN) have been shown to exert strong immuno-stimulatory effects through activation of Toll-like receptor 9 (TLR-9). However, TLR-9 triggering takes place in endosomal compartments and thus CpG-ODN have to be taken up prior to signal transduction. We here report that 3'-poly-guanosine strings can improve cellular internalisation of phosphodiester but not of phosphorothioate CpG-ODN. Improved cellular uptake correlated with enhanced IL-6 secretion and proliferation of PBMC. Also, TLR-9 transfected HEK293 cells were activated more efficiently by poly-guanosine modified CpG-ODN. The results indicate that the synthesis of stimulatory CpG-ODN based on a phosphodiester backbone is feasible via such poly-guanosine substitutions. In addition we observed that phosphorothioate ODN were able to exert immunostimulatory effects independent of the presence of CpG motifs.

Adjuvants, Immunologic↗
PubMed2004

Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in innate immune cells.

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Toll-like receptors differentially induce nucleosome remodelling at the IL-12p40 promoter.

Toll-like receptors (TLRs) mediate recognition of microbial components. Despite activation of a shared set of signal transduction molecules, the biological effects of certain TLR agonists differ considerably. In macrophages and dendritic cells, stimulation by the prototypical stimuli CpG-DNA (TLR9), lipopolysaccharide (LPS; TLR4) and lipoteichoic acid (LTA; TLR2) resulted in striking differences in expression of IL-12. However, these stimuli induced similar amounts of the common proinflammatory cytokine TNFalpha. Surprisingly, an IL-12p40 promoter reporter construct was activated equally by CpG-DNA, LPS and LTA. Examinations of the chromatin structure of the endogenous IL-12p40 promoter revealed that nucleosome remodelling contributed to differential IL-12 induction. Upon stimulation, nucleosome architecture was changed to provide increased access to the IL-12p40 promoter. In dendritic cells, a differential induction of nucleosome remodelling at the IL-12p40 promoter was observed upon triggering with different TLR agonists. These results identify nucleosome remodelling as an additional restriction point in differential TLR signalling.

Animals↗
PubMed2004

CpG-DNA as immune response modifier.

Toll-like receptors (TLRs) have been recognized to play a fundamental role in the recognition of microbial pathogens and the activation of innate immunity. However, it has also turned out that triggering of TLRs with purified compounds or synthetic ligands represents a powerful means to modulate innate as well as adaptive immune responses. Among the substances currently under investigation as immune response modifiers CpG-DNA has gained particular interest. One reason for this is the feasibility to easily synthesize and modify immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODNs). This review deals with the structural needs of CpG-ODNs. CpG-ODNs are discussed as being composed of distinct building blocks (e.g. sequence strings, backbone modifications) giving rise to a concept of structural modules within CpG-ODNs.

Adjuvants, Immunologic↗
PubMed2004

Quantitative detection of periodontopathogens by real-time PCR.

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗
PubMed2003

CpG oligonucleotides with modified termini and nicked dumbbell structure show enhanced immunostimulatory activity.

A series of 21 phosphodiester oligodeoxyribonucleotides containing the core sequence 5'-GACGTT-3' or related control sequences were prepared and tested for their immunostimulatory effect on murine macrophages. The range of structural modifications tested included substituents at 3'- or 5'-termini, N3-methylation of thymidine residues, and hexaethylene glycol linkers favoring nicked or cyclic dumbbell duplexes. Lipophilic and cationic substituents at the termini failed to increase the release of TNF-alpha and nitric oxide, but two new types of modification were found that enhance the stimulation of RAW264.7 macrophages. One is the substitution of the 5'-terminal hydroxyl group with an amino group, and the other is the introduction of linkers favoring nicked duplexes. Even for sequences without linkers, UV-melting analysis and two-dimensional NMR showed that the core sequence 5'-GACGTT-3' readily forms a duplex. The cyclic derivative of the most active nicked dumbbell sequence is inactive, however. Together these results suggest a recognition of both the 5'-terminus and the core of the CpG oligonucleotides by the putative receptor(s) and provide an entry into a class of modified oligonucleotides whose activity rivals that of phosphorothioates, but consists of synthetic compounds that are single stereoisomers.

Adjuvants, Immunologic↗
PubMed2003

TLR-induced negative regulatory circuits: role of suppressor of cytokine signaling (SOCS) proteins in innate immunity.

Recognition of pathogen-associated molecular pattern (PAMP) by PAMP receptors of innate immune cells leads to activation and initiation of innate and adaptive immune responses. While the initial steps have been elucidated recently, the mechanisms how innate immune responses are controlled and regulated are only poorly understood. Suppressor of cytokine signaling (SOCS) proteins seem to play an important role in this process. It has been recognized that SOCS proteins not only regulate JAK/STAT-dependent cytokine signaling pathways but are also induced by TLR stimuli and in addition might also regulate TLR signaling.

Animals↗
PubMed2003

Immunostimulatory DNA as adjuvant: efficacy of phosphodiester CpG oligonucleotides is enhanced by 3' sequence modifications.

The immunostimulatory activity of oligonucleotides containing CpG motifs is well established and represents the basis for an effective vaccine adjuvant. For use in vivo CpG ODN have to be protected from the attack of nucleases to ensure sustained effectiveness. This is usually accomplished by phosphothioate (PTO) modifications of the ODN's backbone. However, PTO modification may induce undesired effects. We, therefore, have attempted to enhance the immunostimulatory activity of CpG phosphodiester ODN by supplementation of the ODN's sequence. We report here, that addition of poly-guanosine runs to the 3'-end of a CpG-PO ODN conveys immunostimulatory activity to CpG-PO ODN in vivo and in vitro. Guanosine-rich PO ODN thus might be an alternative approach to develop effective and safe vaccine adjuvants.

Adjuvants, Immunologic↗
PubMed2003

Triggering of Toll-like receptors modulates IFN-gamma signaling: involvement of serine 727 STAT1 phosphorylation and suppressors of cytokine signaling.

Microbial stimuli activate cells of the innate immune system by triggering Toll-like receptors (TLR). Activation of macrophages and dendritic cells is further enhanced by secondary signals like IFN-gamma. Here we analyzed the interplay of IFN-gamma and TLR signaling in cells of the innate immune system. Using a STAT1-dependent reporter construct we show that IFN-gamma signaling can be enhanced as well as inhibited by simultaneous stimulation with either defined TLR agonists or whole-bacterial lysates. Short costimulation resulted in the amplification of IFN-gamma signaling and was attributable to the p38 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of signal transducer and activator of transcription (STAT)1 on serine 727. In contrast, prolonged co-incubation as well as pre-incubation with TLR agonists led to an inhibition of IFN-gamma signaling. TLR triggering induced expression of suppressor of cytokine signaling (SOCS)-1, SOCS-3 and cytokine-inducible SH2 domain-containing protein (CIS). Overexpression of SOCS-1 and, to a lesser extend, of SOCS-3 and CIS inhibited IFN-gamma signaling as measured by activation of STAT1. Moreover, pre-incubation with TLR-dependent stimuli impaired IFN-gamma-induced MHC class II regulation but enhanced CD40 and CD86 expression. Taken together, the results indicate a tight interplay between TLR and IFN-gamma signaling pathways which involve induction of SOCS proteins and serine phosphorylation of STAT1.

Antigens, CD↗