Biomedical subjects

Sven Klaschik

Publications and source records attributed to Sven Klaschik.

6 recordsLinked to original sources

PubMed2006

CpG-mediated changes in gene expression in murine spleen cells identified by microarray analysis.

Unmethylated CpG motifs interact with Toll-like receptor 9 (TLR9), triggering an innate immune response characterized by the production of cytokines, chemokines and immunoglobulins. Microarray analysis of cDNA from murine spleen cells stimulated with CpG oligodeoxynucleotides (ODN) identified reproducible changes in gene expression over time. Eight genes are significantly up-regulated 2h post CpG ODN stimulation, most of which contribute to the induction of innate or adaptive immune responses. Network analysis indicates that TNF and NFKB1 are key regulators of gene expression at this early time point. At 4h, IL1B in addition to TNF and NFKB1 play dominant roles in the up-regulation of immune gene expression, whereas by 8h this function is mediated by TNF, IFNG, and MYC. Genes responsible for down-regulating CpG-induced responses were also identified, dampening what would otherwise be a continuous positive feedback loop. This work provides novel insights into the regulatory process embedded in the gene expression profile induced by CpG ODN, identifies novel genes associated with CpG-induced immune stimulation, and clarifies the breadth of the immune response elicited via TLR9.

Adjuvants, Immunologic↗

PubMed2005

Cytokine promoter polymorphisms in severe sepsis.

The need to develop individualized risk profiles and drug therapy regimens motivates interest in genetic studies of critically ill patients. Gene promoter variants may predict interindividual variability in response to inflammatory stimuli, such as infection and trauma. Genomic variations also may affect gene expression profiles, as well as the structure and production of proteins. The genes involved in inflammation are numerous, as are genomic variations within most of those genes. Cytokine genes involved in inflammatory cascades are important candidate genes that may determine the extent of a person's response to injury. Understanding the genetic determination of the inflammatory process includes the possibility of developing valuable diagnostic tools and new therapeutic approaches in severe sepsis. To date, specific patterns of markers of genomic variation reliably indicating at-risk populations do not exist. Evaluation of possible genomic markers for risk stratification of patients with sepsis and persons at high risk of developing organ failure has begun at a level of well-powered genetic epidemiological research. Cytokine promoter variants may contribute substantially to studies of genetic predisposition of sepsis because they operate in a gene region of high regulatory activity.

Animals↗

PubMed2005

Intracellular detection of macrophage migration inhibitory factor in peripheral blood leukocytes.

The oxidoreductase MIF is currently discussed as a new promising target of immunomodulatory therapy in patients with severe sepsis. An increasing body of evidence attributes an important role especially to intracellular MIF for regulation of endotoxin responsiveness as well as regulation of nuclear transcription factors. Up to now, measurement of MIF relied on ELISA techniques, lacking the ability to directly measure intracellular MIF and distinguish between different leukocyte subpopulations. Therefore, we developed a sensitive and robust flow cytometry-based method to reliably detect intracellular levels of MIF. This method can readily be applied in cultured cells as well as in subsets of human leukocytes in whole blood. Intracellular MIF content of whole-blood leukocyte subsets is detected simultaneously, and is individually determined for T-lymphocytes, B-lymphocytes, macrophages, and granulocytes, respectively. When tested in an ex vivo whole-blood stimulation system using PMA/Ionomycin the intracellular MIF content doubled in CD3+ T-lymphocytes and increased threefold in CD14+ macrophages. Baseline intracellular MIF levels of different leukocyte subpopulations were quantified in 22 healthy blood donors. Baseline intracellular MIF levels in T-lymphocytes are twice as high compared to those in B-lymphocytes and macrophages.

Enzyme-Linked Immunosorbent Assay↗

PubMed2005

Therapeutic potential of oligonucleotides expressing immunosuppressive TTAGGG motifs.

Synthetic oligodeoxynucleotides (ODNs) expressing immunosuppressive TTAGGG motifs downregulate the production of proinflammatory and Th1 cytokines. The ability of these "suppressive ODNs" to slow or prevent the development of diseases characterized by over-exuberant immune stimulation was examined. Suppressive ODNs significantly reduced disease severity in murine models of arthritis, lupus, and LPS-induced toxic shock. These beneficial effects were accompanied by a significant reduction in serum autoantibody and cytokine levels. Underlying these protective effects was the ability of suppressive ODNs to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade central to the initiation and/or perpetuation of these disease states. These findings suggest that suppressive ODNs might find use in the treatment of acute and chronic diseases characterized by excessive immune stimulation.

Amino Acid Motifs↗

PubMed2002

Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria.

We developed a consensus real-time PCR protocol that enables us to detect spiked bacterial 16S DNA from specimens such as water, urine, plasma, and sputum. The technique allows an exact Gram stain classification of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes. All tested bacteria were identified correctly, and none gave a false-positive signal with the opposite Gram probe.

Bacterial Infections↗

PubMed2002

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.

Artifacts↗