Biomedical subjects

Klaus Heeg

Publications and source records attributed to Klaus Heeg.

29 records · Page 2Linked to original sources

PubMed2003

DNA from periodontopathogenic bacteria is immunostimulatory for mouse and human immune cells.

Although bacterial DNA (bDNA) containing unmethylated CpG motifs stimulates innate immune cells through Toll-like receptor 9 (TLR-9), its precise role in the pathophysiology of diseases is still equivocal. Here we examined the immunostimulatory effects of DNA extracted from periodontopathogenic bacteria. A major role in the etiology of periodontal diseases has been attributed to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros. We therefore isolated DNA from these bacteria and stimulated murine macrophages and human gingival fibroblasts (HGF) in vitro. Furthermore, HEK 293 cells transfected with human TLR-9 were also stimulated with these DNA preparations. We observed that DNA from these pathogens stimulates macrophages and gingival fibroblasts to produce tumor necrosis factor alpha and interleukin-6 in a dose-dependent manner. Methylation of the CpG motifs abolished the observed effects. Activation of HEK 293 cells expressing TLR-9 which were responsive to bDNA but not to lipopolysaccharide confirmed that immunostimulation was achieved by bDNA. In addition, the examined bDNA differed in the ability to stimulate murine macrophages, HGF, and TLR-9-transfected cells. DNA from A. actinomycetemcomitans elicited a potent cytokine response, while DNA from P. gingivalis and P. micros showed lower immunostimulatory activity. Taken together, the results strongly suggest that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros possesses immunostimulatory properties in regard to cytokine secretion by macrophages and fibroblasts. These stimulatory effects are due to unmethylated CpG motifs within bDNA and differ between distinct periodontopathogenic bacteria strains. Hence, immunostimulation by DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros could contribute to the pathogenesis of periodontal diseases.

Aggregatibacter actinomycetemcomitans↗

PubMed2003

Synergistic and antagonistic interactions between LPS and superantigens.

Superantigens trigger polyclonal activation of T lymphocytes with cytokine release that eventually may lead to lethal cytokine syndrome (toxic shock). In contrast, bacterial components that are recognized by Toll-like receptors (e.g. LPS or CpG DNA) primarily target macrophages and dendritic cells. We have analyzed whether superantigens and TLR ligands interact with each other. We found that superantigens synergize with LPS in an IFN-gamma-dependent pathway. More important, we found compelling evidence that superantigens prime the innate immune cell system to a subsequent challenge with endotoxin. This sensitization was critically dependent on T-cell derived IFN-gamma. When we analyzed the underlying molecular mechanisms, we additionally found that TLR stimulation enhanced IFN-gamma-mediated cellular responses. Moreover, TLR ligands induced proteins of the SOCS family thus shutting off IFN-gamma-mediated cellular activation. Since IFN-gamma is synthesized by T cells after superantigen triggering, these results show that superantigen and TLR pathways are interconnected and regulate each other. They further show that the outcome of this interaction may include activation as well as down-regulation of the respective response pattern.

Animals↗

PubMed2003

Suppressors of cytokine signaling proteins in innate and adaptive immune responses.

Suppressors of cytokine signaling (SOCS) proteins have been identified as important mediators of negative regulatory circuits within cytokine receptor signaling. They are induced upon stimulation by an increasing set of cytokines as well as further immunological stimuli and are capable to inhibit Janus kinases and signal transducer and activator of transcription signaling. Inhibition is mediated by interfering directly with signal transduction at the receptor as well as targeting of associated molecules for proteosomal degradation. Targeted gene deletion approaches have revealed the importance of SOCS mediated termination of cytokine signaling during normal cellular activation. In addition to their function as classical feedback inhibitors SOCS proteins display a broad panel of inhibitory activity thereby mediating cross-talk modulation between different stimuli. The consequences for regulation of innate and adaptive immune responses are thus obvious. Finally, there are emerging data showing involvement of SOCS proteins in various immune diseases. Modulating SOCS activity could be a promising new approach for molecular therapeutic strategies.

Animals↗

PubMed2002

Immunostimulatory CpG-DNA activates murine microglia.

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-DNA) triggers innate immune cells through the pattern recognition receptor Toll-like receptor 9 (TLR-9). CpG-DNA possesses potent immunostimulatory effects on macrophages, dendritic cells, and B lymphocytes. Therefore, CpG-DNA contributes to inflammation during the course of bacterial infections. In contrast to other TLR-dependent microbial patterns, CpG-DNA is a strong inductor of IL-12. Thus, it acts as a Th1-polarizing agent that can be utilized as potent vaccine adjuvant. To assess the role of CpG-DNA in immune reactions in the CNS, we analyzed the effects of CpG-DNA on microglial cells in vitro and in vivo. Primary microglial cells as well as microglial cell lines express TLR-9 mRNA. Consequently, CpG-DNA activated microglial cells in vitro and induced TNF-alpha, IL-12p40, IL-12p70, and NO. Furthermore, MHC class II, B7-1, B7-2, and CD40 molecules were up-regulated. In addition, phagocytic activity of microglia was enhanced. After intracerebroventricular injection of CpG-DNA, microglial cells were activated and produced TNF-alpha and IL-12p40 transcripts, as shown by in situ hybridization. These results indicate that microglia is sensitive to CpG-DNA. Thus, bacterial DNA containing CpG motifs could not only play an important role during infections of the CNS, but also might trigger and sustain Th1-dominated immunopathogenic reactions.

Adjuvants, Immunologic↗

PubMed2002

Phosphodiester CpG oligonucleotides as adjuvants: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo.

Bacterial DNA and oligonucleotides (ODN) containing CpG-motifs strongly activate cells of the immune system. Accordingly CpG-DNA is a powerful adjuvant in vaccination protocols for B-cell as well as for cytotoxic T-cell responses. A decisive propensity of CpG-DNA is its capacity to induce preferentially T helper type 1 (Th1)-dominated immune responses. To exert its function CpG-DNA has to be taken up by responsive cells, e.g. antigen-presenting cells (APC). The rate of uptake is influenced by the DNA's backbone modification and critically determines activity of CpG-DNA. CpG ODN with a phosphothioate backbone (PTO) are currently used for most in vivo and in vitro studies, since PTO modification protects ODN from the attack of nucleases. However, after administration of PTO-modified CpG-ODN long-lasting effects including lymphadenopathy as well as sustained local interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) production have been reported. To circumvent these restrictions we investigated the effects of DNA sequence as well as DNA backbone modification on cellular uptake and resulting immunostimulation. We show here that uptake of phosphodiester (PO)-CpG-ODN can be strongly enhanced by poly guanosine runs added at the 3' end of the ODN. In addition these ODN showed an improved immunostimulatory activity in vivo and in vitro. This included protection of mice against lethal Th2-dependent leishmaniasis as well as priming of antigen specific Th1 responses. More importantly, guanosine-rich PO-CpG-ODN neither induced lymphadenopathy nor prolonged cytokine production after local administration. Since these improved PO ODN are efficient in vitro and in vivo and lack long lasting undesired effects they could be used preferably as adjuvants in vaccination protocols.

Adjuvants, Immunologic↗

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

Effective postexposure treatment of retrovirus-induced disease with immunostimulatory DNA containing CpG motifs.

Therapeutic strategies for the treatment of acute retroviral infections have relied mainly on antiviral drugs. In this study we used the Friend virus model system to demonstrate that enhancement of the immune system can also have dramatic therapeutic effects. Since resistance to Friend virus-induced leukemia in mice is associated with T helper cell type 1 (Th1) immune responses, we enhanced these responses in susceptible mice by treatment with synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN). Treatments begun at 4 days postinfection increased recovery from 6% in the control group to 74% in the CpG-treated group. CpG-mediated recovery was associated with a significant reduction of viral loads in the blood and spleens of treated mice compared to those of control animals. The treatment promoted Th1-type cytokine production by splenocytes of Friend virus-infected mice and augmented Friend virus-specific cytotoxic T-cell responses, but no influence on the virus-specific neutralizing antibody response was observed. Friend virus-specific CD8(+) T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice prevented their recovery. Our results demonstrate that CpG-ODN therapy can significantly enhance virus-specific cellular immune responses and prevent retrovirus-induced disease. These findings may have implications for antiviral therapy in general.

Adjuvants, Immunologic↗

PubMed2002

Immunopharmacology of CpG DNA.

Recognition of danger of infection by innate immune cells is a prerequisite to combat infections and to activate T and B cells. Pathogen-associated molecular patterns (PAMP) play a fundamental role in this process. PAMPs are sensed by at least ten different Toll-like receptors (TLR). Within the realm of PAMPs, CpG DNA that is recognized by TLR-9 has an outstanding propensity to induce a milieu that favors activation of T lymphocytes and biases Th1-dominated immune responses. Therefore CpG DNA has become a promising immuno-therapeutical candidate to assist and to direct immune responses such as in vaccination or modulation of allergic responses. As opposed to other PAMPs, CpG DNA can be synthesized with defined purity and base composition. Moreover, chemical substitutions can confer new qualities to synthetic CpG DNA.

Adjuvants, Immunologic↗

PubMed2002

CpG DNA in the prevention and treatment of infections.

Microbial infection is sensed by Toll-like receptors (TLRs) on innate immune cells. Among the ten so far defined TLRs, TLR9 and its ligand are peculiar. TLR9 recognises bacterial DNA characterised by the abundance of unmethylated CpG dinucleotides, which distinguish bacterial DNA (CpG DNA) from mammalian DNA. Moreover, TLR9 shows a restricted cellular and subcellular pattern of expression. In contrast to other TLR agonists, CpG DNA is superior in activation of dendritic dells and induction of costimulatory cytokines such as interleukin (IL)-12 and IL-18. This qualifies CpG DNA as a Th1-promoting adjuvant. During infection, recognition of CpG DNA of intracellular pathogens skews and fine-tunes the ongoing immune response and induces long-lasting Th1 milieus. Thus, CpG DNA might play an important role in driving the immune system to a Th1 profile, preventing undesired Th2 milieus that might favour induction of allergic responses. Since CpG DNA can be synthesised with high purity and sequence fidelity, synthetic CpG DNA will become an important agent for Th1 instruction and be an effective adjuvant during vaccination.

Adjuvants, Immunologic↗

PubMed2002

Signal integration following Toll-like receptor triggering.

Innate immune cells sense infectious danger through recognition of pathogen-associated molecular patterns (PAMP). Toll-like receptors (TLRs) play a pivotal role in the recognition of PAMP. Although various TLRs with distinct ligand specificities are expressed almost completely and contemporaneously by innate immune cells, and although central key signal transduction pathways of TLRs are shared, the innate response pattern is not uniform. Innate immune cells organize their response dependent on spatial and temporal requirements and mount a pathogen-specific and microbial-adopted reaction. Thus, after triggering of TLRs, complex signaling interactions take place that control and shape the reactivity patterns of innate immune cells. Here we discuss the central signaling pathways of TLRs and their interconnection with other signaling pathways, with emphasis on Janus kinases/signal transducers and activators of transcription JAK/STAT)-dependent intracellular signals. Cis and trans acting signal convergence mechanisms and mutual influence of TLR and cytokine signaling pathways determine the response profile. Hence, in innate immune cells, integration and processing of microbial-dependent and immunologic signals is achieved within individual cells to ascertain an appropriate immune response. Knowledge of these complex mechanisms will help to clarify the critical events during initiation of infection, which in turn may lead to new strategies to control and overcome infectious disease.

Animals↗

PubMedDate not supplied

Antineoplastic effect of immunostimulatory DNA (CpG-ODN) in a murine C57-BL6/MB-49 transitional cell carcinoma model.

BACKGROUND: Intravesical BCG installation is the standard of care in the prophylaxis of recurrent intermediate and high-risk transitional cell carcinoma (TCC), but its mode of action has not yet been elucidated. However, a Th-1 biased immune response is postulated Cell culture and animal models demonstrated the efficacy of synthetic CpG-oligodeoxynucleotides (ODN) as inducer and adjuvant for a strong Th1-response. The purpose of our study was to evaluate the antineoplastic effect of locally administered CpG ODN in a subcutaneous murine bladder cancer model. MATERIALS AND METHODS: A subcutaneous murine TCC model was established in female C57/BL6 mice using the corresponding syngeneic MB49 TCC cell line. Three groups of 5 animals received a cell suspension, standardized for 1x10(6) cells/50 microl, injected s.c. into the right and left flank. Group I received 10 nmol of CpG-ODN only into the right cell depot. Group II received 10 nmol of GpC ODN. Group III served as untreated control and received only PBS. The animals were examined at various time points after injection until sacrifice on day 14. Tumor or scar tissue were excised, weighed and examined histopathologically (HE-stain). RESULTS: Tumor sizes and weights showed no side differences. The average tumor weight on day 14 was 171 mg (SD +/- 8.9), 110 mg (SD +/- 19.2) and 18 mg (SD +/- 6.1), respectively, in groups III, II and I (p<0.05). Histopathology revealed solid vital epithelial tumors in group III and reduced vital tumor mass with central necrosis and moderate mononuclear infiltration in group II. Group I showed almost complete tumor necrosis and a considerable mononuclear inflammatory response. CONCLUSION: Immunostimulatory DNA has promising antineoplastic activity in a murine subcutaneous TCC-model. The histological findings suggest an immunologically-mediated mode of action. Further investigations are necessary to elucidate the immunological response.

Adjuvants, Immunologic↗