Biomedical subjects

Stefan Bauer

Publications and source records attributed to Stefan Bauer.

At least 37 records · Page 2Linked to original sources

PubMed2005

Intraoperative transit time flow measurement: off-pump versus on-pump coronary artery bypass.

BACKGROUND: Off-pump coronary artery bypass grafting (OPCAB) has attracted increasing attention. Performing the anastomosis off-pump is technically more demanding. The objective of the study is to assess the quality of anastomosis in OPCAB in comparison with conventional on-pump coronary artery bypass grafting using the transit time flow measurement. METHODS: Four hundred forty-five patients operated on using OPCAB technique were included in the study. For each patient in this group a similar patient from the on-pump coronary artery bypass grafting population was selected according to the number of grafts, bypass material, and target coronary arteries. The mean flow and the pulsatile index were measured in every bypass graft in both groups. RESULTS: The average pulsatile index in OPCAB was 2.09 +/- 1.03 (mean flow, 39 +/- 22.63 mL/min), whereas with on-pump coronary artery bypass grafting it was 1.9 +/- 0.98 (mean flow, 44.19 +/- 23.58 mL/min); p = 0.005. Subgroup analysis showed significantly lower mean flows and higher pulsatile index with OPCAB in grafts to the obtuse marginal, diagonal, and right coronary artery, but not to the left anterior descending territory. CONCLUSIONS: The quality of the anastomosis performed using the OPCAB technique might be jeopardized by less accessibility as in the case of lateral and posterior wall coronary arteries. Techniques to optimize the accessibility of the coronary artery like combining sling support with cup stabilizers, together with systematic training, should be strongly considered in OPCAB. Whenever there is good accessibility of the coronary artery as in the case of the left anterior descending, the anastomosis performed under OPCAB has a quality as good as that performed using the conventional technique.

Aged↗

PubMed2005

Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes.

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-kappaB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1-10 expression profile. First, reverse transcription-polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1-10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-kappaB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-kappaB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.

Biological Transport↗

PubMed2004

Toward a systems approach to understanding plant cell walls.

One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.

Cell Division↗

PubMed2004

Murine TLR2 expression analysis and systemic antagonism by usage of specific monoclonal antibodies.

Cellular recognition of immuno-stimulatory microbial products alarming the host immune system upon infection, as well as endogenous molecular patterns representing perturbation of regular homeostasis such as through necrosis of host cells is mediated by innate pattern recognition receptors to which toll-like receptors (TLRs) belong. A variety of agonists has been attributed to TLR2. We raised monoclonal antibodies (mAbs) toward the murine TLR2 extracellular domain (mT2ECD) in order to analyze murine TLR2 expression. Murine macrophages were stained TLR2-specifically with distinct mAbs as shown by flow cytometry, immuno precipitation, and immuno-cytochemical analysis. TLR2-specific murine macrophage activation was inhibited through pre-incubation with a mAb mT2.4 while another mTLR2-specific mAb mT2.7 did not affect cell activation through TLR2. Plasmon resonance based analysis showed inhibition of lipopeptide binding to mT2ECD if complex formation with mT2.4 preceded binding analysis. Systemic induction of IL-6, IL-12p40, and GROalpha/KC release to the serum upon lipopeptide challenge of mice was inhibited by systemic administration of mT2.4. Furthermore, 120 mg/kg of mT2.4 protected mice from lethal shock-like syndrome in an experimental low-dose model of septic shock. This result validates blockage of cell surface TLR2 for inhibition of immune cell over-activation upon microbial challenge.

Animals↗

PubMed2004

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways.

Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-alpha, and whether the production of IFN-alpha in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-alpha in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-alpha was produced by other cell types. Moreover, IFN-alpha production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-alpha in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-alpha production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-alpha in response to HSV-1 via both TLR9-independent and -dependent pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity.

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which 'trim' polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-kappaB (nuclear factor kappaB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IkappaBalpha (inhibitor of kappaB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKgamma (IkappaB kinase gamma)/NEMO (NF-kappaB essential modifier) signal adaptors relies on their modification with 'nonclassical' forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-kappaB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.

Animals↗

PubMed2004

Targeted bioactivity of membrane-anchored TNF by an antibody-derived TNF fusion protein.

We describe the generation and characterization of a fusion protein consisting of a humanized anti-fibroblast-activating protein (anti-FAP) Ab and human TNF replacing the IgG1 CH2/CH3 Fc domain. The construct was generated by recombinant DNA technology and preserved its IgG1-derived dimeric structure with the TNF molecule linked as a dimer. Expression in CHO cells was optimized in serum-free medium under GMP conditions to achieve production levels up to 15 mg/liter. Recognition of the FAP Ag by the construct was as good as that by the parental anti-FAP Ab. TNF signaling was induce able via both TNF receptor types. When acting in solution, the Ab-linked TNF dimer exhibited a 10- to 20-fold lower activity compared with recombinant trimeric TNF. However, after binding to FAP-expressing cells, immobilized anti-FAP-TNF dimer was equivalent to membrane-anchored TNF with regard to bioactivity. Amplification of TNF-related pathways by mimicking the membrane-integrated TNF signaling was detectable in various systems, such as apoptosis induction or tissue factor production. The difference in TNF receptor type 1 and 2 signaling by the anti-FAP-TNF construct correlated well with its Ag-bound or -soluble status. Translating the approach into a xenograft animal model (BALB/c nu/nu mice), we demonstrated low toxicity with measurable antitumor efficacy for the TNF fusion protein after i.v. application. Immunohistochemical analysis of tumor sections showed restricted TNF-mediated macrophage recruitment to the targeted tissue in a time- and dose-dependent manner. These data warrant transfer of the anti-FAP-TNF immunocytokine into clinical trials for the treatment of FAP-positive tumors.

Animals↗

PubMed2004

Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice.

How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.

Animals↗

PubMed2004

Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner.

Toll-like receptors (TLR) recognize bacterial and viral components, but direct interaction of receptor and ligand is unclear. Here, we demonstrate that TLR9 binds directly and sequence-specifically to single-stranded unmethylated CpG-DNA containing a phosphodiester backbone. TLR9-CpG-DNA interaction occurs at the acidic pH (6.5-5.0) found in endosomes and lysosomes. By sequence comparison we identified a potential CpG-DNA binding domain homologous to that described for methyl-CpG-DNA binding proteins. Amino acid substitutions in this region abrogated CpG-DNA binding and led to loss of NF-kappaB activation. Furthermore, chloroquine and quinacrine, therapeutic agents for autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, directly blocked TLR9-CpG-DNA interaction but not TLR2-Pam3Cys binding. Our results demonstrate direct binding of TLR9 to CpG-DNA and suggest that the therapeutic activity of chloroquine and quinacrine in autoimmune diseases may be due to its activity as a TLR9 antagonist and inhibitor of endosomal acidification.

Amino Acid Sequence↗

PubMed2004

Targeting split vaccines to the endosome improves vaccination.

Compared to 'live' vaccines, the immunogenicity of 'split' vaccines based on recombinant antigen (Ag) is poor, presumably because exogeneous recombinant Ag fails to efficiently access the major histocompatibility complex (MHC) class I processing pathway needed for 'cross-presentation'. Here we discuss recent evidence that targeting ligands of the Toll-like receptor 9 together with proteinaceous Ag to the endosome of dendritic cells conveys immunogenicity to Ag similar in magnitude to 'live' vaccines that produce Ag. Enforced endocytosis of Ag together with the adjuvant effect of Toll-like receptor 9 ligands might be key for the efficient cross-presentation of exogeneous Ag as well as for effective cross-priming of MHC class I restricted CD8 T effector cells.

Adjuvants, Immunologic↗

PubMed2004

Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes.

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Animals↗

PubMed2003

Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation of DNA-conjugated antigens but essential for cross-priming of CD8 T cells.

Covalent linkage of immunostimulatory CpG DNA to OVA results in CpG DNA-aided cross-presentation of OVA by dendritic cells (DCs). In vivo, cross-presentation is conditional for cross-priming of OVA-specific CD8 T cells. In this study, we investigated the involvement of the CpG DNA receptor Toll-like receptor (TLR)9 in CpG DNA-aided cross-presentation and cross-priming. Although CpG DNA-aided cross-presentation is not altered in TLR9-deficient cells, TLR9 is required for maturation of APC allowing cross-priming, as resulting in CTL function. These findings imply that TLR9 does not trigger endocytosis of CpG-OVA conjugates, but activates DCs downstream of endocytosis.

Adjuvants, Immunologic↗

PubMed2003

The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily.

Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll-like receptor (TLR) 7/MyD88-dependent signaling pathway. TLR7- and MyD88-deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7-deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R-848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG-DNA-driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen-associated molecular patterns in endosomal/lysosomal compartments.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Molecular markers in the evaluation of autologous chondrocyte implantation.

PURPOSE: With the help of molecular markers, it has become possible to quantify cartilage repair and degradation in joints. In this study, we attempt to determine whether or not molecular markers in synovial fluid can be helpful in defining the repair process following autologous chondrocyte implantation (ACI). TYPE OF STUDY: As part of a prospective clinical pilot study, 17 patients were evaluated before, as well as 6 weeks, 3, 6, and 12 months after the ACI. A synovial analysis was performed and molecular markers for bone and cartilage metabolism were determined. METHODS: A number of parameters, including pyridinium crosslink (PY), deoxypyridinolin (DPD), n-telopeptide (NTX) from type I collagen, MMP-1, MMP-3; TIMP-1, PICP, proteoglycan, and YKL-40 were analyzed. The levels were referenced to the total protein concentration of the synovial fluid. The synovial analyses were compared with clinical parameters (Larson score) and magnetic resonance imaging (MRI) examinations. RESULTS: The analysis of the data revealed differing trends for the various synovial markers over time. The most remarkable marker was found to be DPD, which increased continuously between surgery and week 12, only to disappear after the repair process had ceased 1 year after surgery. All molecular markers for cartilage degradation increased initially after surgery and dropped off below the original levels 3 to 6 months later. CONCLUSIONS: The evaluation revealed that the determination of marker levels can provide valuable information regarding the metabolism of bone and cartilage in a joint. They seem to provide a method for monitoring the repair process associated with the various treatment forms for chondral lesions.

Adipokines↗

PubMed2003

Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2.

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-gamma) secretion in the genital tract, and CpG ODN-treated IFN-gamma(-/-) mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.

Animals↗

PubMed2003

Risk in elderly patients after stentless versus stented aortic valve surgery.

Recent studies suggest that the hemodynamic advantage of stentless bioprostheses over the stented type improves long-term survival after aortic valve replacement, but the more complex and time-consuming implantation technique may increase the risks of operative death and postoperative complications. Between April 1996 and June 2001, 519 patients with a mean age of 76 +/- 5 years underwent aortic valve replacement using a stentless (Medtronic Freestyle, n = 277) or stented bioprosthesis (Medtronic Mosaic, n = 242). Multiple logistic regression analysis considering different patient populations revealed no increased risk of operative death, postoperative complications, or neurological impairment after implantation of a stentless bioprosthesis. Survival curves in respect of 367 patients who underwent aortic valve replacement up to September 2000 and were followed up for 3 years were not different (p = 0.98). As the patients were elderly, improved survival due to implantation of a stentless valve could not be demonstrated within this time span.

Aged↗

PubMed2002

Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments.

Recognition by innate immune cells of the pathogen associated molecular patterns (PAMP) lipopolysaccharide (LPS) from Gram-negative bacteria and bacterial CpG-DNA depends on Toll-like receptor4 (TLR4) and TLR9, respectively. To define differences in the response to these distinct PAMP we compared a key intracellular event, namely recruitment of myeloid differentiation marker 88 (MyD88) to the respective PAMP-initiated TLR signaling. Using MyD88-GFP fusion protein expressing macrophages we demonstrate that LPS and CpG-DNA trigger signaling from two different cellular locations: theformer at the cell membrane and the latter at the lysosomal compartment. While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG-DNA to activate TLR9. In support of these data TLR9 is not localized at the cell surface, but intracellularily. These data stress the need to characterize individual TLR at the very beginning of signal initiation in order to understand their diverse biological functions.

Adaptor Proteins, Signal Transducing↗