Biomedical subjects

Stefan Zimmermann

Publications and source records attributed to Stefan Zimmermann.

18 recordsLinked to original sources

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↗

PubMed2005

Risk factors for bacterial catheter colonization in regional anaesthesia.

BACKGROUND: Although several potential risk factors have been discussed, risk factors associated with bacterial colonization or even infection of catheters used for regional anaesthesia are not very well investigated. METHODS: In this prospective observational trial, 198 catheters at several anatomical sites where placed using a standardized technique. The site of insertion was then monitored daily for signs of infection (secretion at the insertion site, redness, swelling, or local pain). The catheters were removed when clinically indicated (no or moderate postoperative pain) or when signs of potential infection occurred. After sterile removal they were prospectively analyzed for colonization, defined as > 15 colony forming units. RESULTS: 33 (16.7%) of all catheters were colonized, and 18 (9.1%) of these with additional signs of local inflammation. Two of these patients required antibiotic treatment due to superficial infections. Stepwise logistic regression analysis was used to identify factors associated with catheter colonization. Out of 26 potential factors, three came out as statistically significant. Catheter placement in the groin (odds-ratio and 95%-confidence interval: 3.4; 1.5-7.8), and repeated changing of the catheter dressing (odds-ratio: 2.1; 1.4-3.3 per removal) increased the risk for colonization, whereas systemic antibiotics administered postoperatively decreased it (odds ratio: 0.41; 0.12-1.0). CONCLUSION: Colonization of peripheral and epidural nerve catheter can only in part be predicted at the time of catheter insertion since two out of three relevant variables that significantly influence the risk can only be recorded postoperatively. Catheter localisation in the groin, removal of the dressing and omission of postoperative antibiotics were associated with, but were not necessarily causal for bacterial colonization. These factors might help to identify patients who are at increased risk for catheter colonization.

Journal Article↗

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

Anthecotulide: purification, analytical data, absence from chamomile preparations, stability and reactivity, and anti-infective testing.

The sesquiterpene lactone anthecotulide (1) was extracted from Anthemis cotula in 0.7% w/w yield. A pure standard was prepared by preparative TLC and used for quantitative determination of 1 with a TLC scanner. The constitution of 1 was corroborated and the double-bond configuration determined. It was stable at room temparature in air and reacted very slowly with glutathione. It was not detectable in 34 chamomile (Matricaria recutita) preparations and did not show antibacterial activity against a range of clinically important strains at a concentration of <10 microg/mL.

Anthemis↗

PubMed2005

On the regenerative capacity of human skeletal muscle.

The proliferative capacity of organotypic muscle stem cells, the satellite cells, from nine healthy human donors aged between 2 and 78 years was investigated. There was a loss in proliferative capacity with age, but the oldest donors (76, 78 years) would still be able to replace their musculature several times. Depending on frequency of desmin-positive (i.e., myogenic) cells during prolonged expansion, myoblast cultures could be designated as stable or unstable. There was a weak correlation between mean telomere lengths (estimated by flow-FISH) and remaining mean population doublings until senescence. A bimodal distribution of mean telomere lengths was apparent in both stable and unstable myoblast cultures and could be assigned to populations of differently sized cells. Furthermore, due to the presence of nonmyogenic cells with longer telomeres, unstable cultures tended to show an increasing rather than decreasing mean telomeric length on expansion. Bimodal distributions in myoblast cultures could be due to hitherto undefined myoblast populations.

Adolescent↗

PubMed2005

Telomere dynamics in hematopoietic stem cells.

The hematopoietic system has an outstanding regenerative capacity which depends on a relatively small population of hematopoietic stem cells (HSC). In contrast to normal human cells, blood-forming stem cells, like most of their counterparts from other adult tissues, exhibit telomerase activity to a certain level. Nevertheless, this telomerase activity does not prevent telomere shortening in HSC, suggesting a restriction of their proliferative capacity. Here, we review recent studies on telomere dynamics in HSC of humans and mice. Furthermore, we discuss the impact of telomere manipulation in HSC for possible clinical applications and speculate on functions of telomerase beyond telomere lengthening.

Animals↗

PubMed2005

The level of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line.

Telomerase is the ribonucleoprotein enzyme that maintains telomeres of eukaryotic chromosomes. Activation of telomerase is a common feature of the majority of human cancers, and inhibition of this enzyme has been proposed as a novel target for cancer therapeutics. Here, we investigated the effects of telomerase inhibition in the non-small cell lung cancer cell line NCI-H460, using a genetic approach by ectopic expression of dominant-negative (DN)-hTERT. Five clones were selected in which telomerase activity was completely abolished. As a result, telomere erosion was observed leading to proliferation arrest after a lag period of 20-28 population doublings. Although overall telomere length was similar between the different clones as measured by quantitative fluorescence in situ hybridization (Q-FISH), striking differences were found in telomere length of individual chromosomes. In particular, lack of individual telomeres and formation of end-to-end fusions were variable. Interestingly, this level of individual telomere dysfunction was positively correlated with the remaining life span of the different clones in vitro. In addition, the amount of telomere dysfunction induced by DN-hTERT was twice as high compared to the small molecule telomerase inhibitor BIBR1532, which induced growth arrest after >100 population doublings. Thus, pharmacological strategies that aim at inhibition of telomerase in cancer cells should take into account that not only overall telomere shortening, but rapid induction of a high level telomere dysfunction appears to be the crucial surrogate parameter for the development of future telomerase-based therapeutics.

Carcinoma, Non-Small-Cell Lung↗

PubMed2004

Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532.

Telomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a nonnucleosidic small molecule inhibitor, BIBR1532 (2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid), has been identified that is highly selective for inhibition of telomerase, resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in short-term culture assays. We observed a dose-dependent direct cytotoxicity in concentrations ranging from 30 to 80 microM. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed time-dependent individual telomere erosion, which was associated with loss of telomeric repeat binding factor 2 (TRF2) and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34(+) cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and must be dissected from telomerase-suppressed overall telomere shortening.

Acute Disease↗

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

Induction of callose in roots of Norway spruce seedlings after short-term exposure to aluminum.

Callose (1,3-beta-glucan) is a suggested physiological indicator of aluminum (Al) toxicity in crop plants. It is not known if callose serves a similar function in forest trees, because quantitative data on callose formation in tree roots are limited, particularly under controlled conditions. To evaluate callose as a physiological indicator of Al toxicity in tree roots, we quantified callose formation in roots of Norway spruce (Picea abies (L.) Karst.) seedlings. Seedlings were grown in simulated soil solutions in the presence or absence (control) of Al under controlled conditions. In seedlings grown in solutions containing 280 microM Al, callose concentrations in roots were twice as high as control values after 6 h of Al treatment and 5 times higher than control values after 1 day. Thereafter, root callose concentrations gradually decreased and were only twice as high as control values after 7 days. The presence of various Al concentrations in the simulated soil solutions indicated that callose was induced by a relatively low Al concentration (84 microM). We conclude that callose in tree roots is an indicator of Al toxicity.

Aluminum↗

PubMed2004

Effects of telomerase modulation in human hematopoietic progenitor cells.

Loss of telomeric repeats has been causally linked to replicative senescence and aging in human cells. In contrast to normal somatic cells, which are telomerase-negative, hematopoietic stem cells have low levels of telomerase, which can be transiently upregulated upon cytokine stimulation. To examine whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we overexpressed telomerase in CD34+ and AC133+ cord blood (CB) cells using retroviral vectors containing hTERT, the catalytic component of telomerase. Although the hTERT-transduced CB cells exhibited significantly elevated telomerase activity (approximately 10-fold), the mean telomere length was only increased up to 600 bp, which was in contrast to hTERT-transduced fibroblast cells gaining more than 2-kb telomeric repeats. Moreover, ectopic telomerase activity did not prevent overall telomere shortening, which was in the range of 1.3 kb in serum-free expansion culture. We also blocked endogenous telomerase activity by ectopic expression of dominant-negative hTERT. Whereas CB cells with absent telomerase activity showed reduced absolute numbers of colony-forming cells, we observed increased rates only for burst-forming units erythroid when the enzyme was overexpressed. These results suggest that telomere shortening in human hematopoietic progenitor cells cannot be compensated by increased levels of telomerase alone and is likely to be dependent on other factors, such as telomere binding proteins. Furthermore, telomerase function seems to be directly associated with the proliferative capacity of stem cells and may exert an additional role in lineage differentiation.

Antigens, CD34↗

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

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↗

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

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↗