8 research outputs found
Trauma induces apoptosis in human thoracolumbar intervertebral discs
BACKGROUND: Vertebral fractures resulting from high energy trauma often comprise the risk of posttraumatic degenerative changes in the affected intervertebral discs (IVD). Particularly in conservatively treated patients, or in cases after implant removal of an exclusively posterior stabilization, consecutive disc degeneration and the associated functional losing of the spinal segment clearly represent detrimental treatment results. In this regard, apoptosis of IVD cells has been suggested to be involved in the critical changes of the extracellular matrix. METHODS: To investigate whether fractures of the vertebrae induce apoptosis in the affected IVD, disc tissue from patients (n = 17) undergoing open reduction and internal fixation of thoracolumbar spine fractures were analysed in regards to caspase activity, apoptosis-receptor expression levels and gene expression of apoptosis-regulating proteins such as Bax and Bcl-2. Healthy IVD tissue (n = 3) obtained from patients undergoing surgical resection of adjacent vertebrae were used as control samples. RESULTS: In contrast to healthy control IVD tissues, samples from traumatic thoracolumbar IVD showed positive TUNEL staining and a significant increase of caspase-3/7 activity. Interestingly, analyses of the initiator caspase-8 and -9 revealed significantly increased activation levels compared to control values, suggesting the coexistent activation of both the extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) apoptosis pathway. Accordingly, expression levels of the Fas receptor (FasR) mRNA were significantly increased. Although the TNF receptor I (TNFR I) was only slightly upregulated, corresponding TNFα from trauma IVD presented significantly increased mRNA expression values. Furthermore, traumatic IVD cells demonstrated significantly reduced expression of the mitochondria-bound anti-apoptotic Bcl-2, thereby maintaining baseline transcriptional levels of the pro-apoptotic Bax protein when compared to control IVD cells. CONCLUSION: Our data suggest that thoracolumbar fractures induce early caspase-dependent apoptosis in IVD cells of the affected intervertebral disc, in part, by downregulation of the anti-apoptotic protein Bcl-2 (intrinsic apoptosis pathway), as well as signalling via the death receptor complex (TNFR I and FasR)
Development of a Ribozyme an DNAzyme mediated therapeutical strategy against allergy and asthma
GesamtdissertationDie komplexe Pathogenese allergischer Erkrankungen und des Asthma bronchiale
macht die Entwicklung neuer TherapieansÀtze notwendig. Von grundlegender
pathophysiologischer Bedeutung fĂŒr die Entstehung allergischer Erkrankungen
sind die Zytokine IL-4 und IL-13. In der IL-4- und IL-13-vermittelten
Signaltransduktion nimmt der Transkriptionsfaktor STAT6 eine zentrale Stellung
ein. Untersuchungen an stat6-knockout-MĂ€usen zeigen, daĂ stat6 fĂŒr die
AusprÀgung des allergischen PhÀnotypes wesentlich ist. Mit der Anwendung von
Ribozymen und DNAzymen wird eine therapeutische Strategie auf NukleinsÀure-
Basis zur spezifischen Spaltung der mRNA des Transkriptionsfaktors stat6 der
Maus verfolgt. Die in der vorliegenden Arbeit verwendeten mstat6-spezifischen
Ribozyme besitzen eine GUC-Konsensussequenz und 6 nt lange Bindungsarme.
Potentielle Ribozym-Spaltstellen werden auf der Grundlage von
SekundÀrstrukturberechnungen der mstat6-RNA identifiziert und liegen in
einzelstrĂ€ngigen Loop-Strukturen. FĂŒr eine stabile Expression werden die fĂŒr
die Ribozyme kodierenden Oligonuleotide in die Expressionskassette pGvaL
kloniert. Insgesamt finden drei mstat6-spezifische Ribozyme Verwendung, deren
AktivitÀt unter zellfreien Bedingungen getestet wird. Alle drei untersuchten
Ribozyme sind in-vitro nicht aktiv. Unter Verwendung eines Multiplex-
AktivitÀts-Assays wird auf der RNA des stat6 der Maus ein Screening nach
zugĂ€nglichen DNAzym-Spaltstellen vorgenommen. Die dafĂŒr eingesetzten DNAzyme
haben eine RU-Konsensussequenz (R = A, G) und besitzen Bindungsarme mit einer
LĂ€nge von jeweils 9 nt. FĂŒr die Lokalisation der Bindungsstellen ist die
thermodynamische StabilitĂ€t âG0 der DNAzym-Substrat-Heteroduplex entscheidend.
Die reziproke Korrelation von DNAzym-AktivitĂ€t und âG0 fĂŒhrt zur Auswahl von
41 DNAzym-Spaltstellen mit einem âG0 von jeweils †\- 25 kcal/mol im
Sequenzbereich von 150 - 600 bp sowie von 1,7 - 2 kb. FĂŒr die Auswahl des
Sequenzbereiches 1,7 - 2 kb ist die Beschreibung von humanen
stat6-SpleiĂvarianten von Bedeutung, da diese auf einer Deletion im
Sequenzbereich von 1765 - 1863 bp beruhen. Von den 41 im Multiplex-AktivitÀts-
Assay getesteten DNAzymen weisen acht DNAzyme eine SpaltungsaktivitÀt auf. Die
Testung aller acht DNAzyme in einem zellfreien System ergibt, daĂ sie ĂŒber
einen Zeitraum von 60 min aktiv sind. In weiterfĂŒhrenden Experimenten wird zu
prĂŒfen sein, ob die unter zellfreien Bedingungen aktiven DNAzyme auch in
Zellkulturen sowie allergischen Mausmodellen wirksam sind.The highly complex pathogenesis of allergic diseases such as asthma requires
the development of new and specific therapeutical approaches. Both
Interleukine-4 and Interleukine-13, respectively, are attributed to play an
essential role in the development of allergic asthma via activation of the
signal transducer and activator of transcription 6 (STAT6). Investigation of
stat6-deficient allergic mice demonstrated that stat6 is critical involved in
the allergic phenotype. By the application of ribozymes and DNAzymes a nucleic
acid based therapeutical strategy was choosen to investigate whether murine
stat6 mRNA could be specifically cleaved in-vitro. Murine stat6-specific
ribozymes adopted a GUC consensus sequence with binding arms of both 6 nt and
were developed on the basis of computational secondary structure calculation
of the murine stat6 transcript. For stable ribozyme expression a recently
described pGvaL expression cassette was used. However; all tested ribozymes
did not show any in-vitro activity. With the use of a multiplex activity assay
the murine stat6 transcript was screened for DNAzyme cleavage sites. The
converse correlation between the thermodynamic stability of the DNAzyme-
substrate heteroduplex and an improved DNAzyme activity revealed potential
cleavage sites on the murine stat6 transcript. Within 41 DNAzymes tested in
the multiplex assay eight DNAzymes showed distinct cleavage activities on the
transcript. Subsequently, the results were strongly confirmed in a cell-free
system. The used DNAzymes had RU consensus sequences with R = A, G and binding
arms of 9 nt. Additional in-vitro analyses provide evidence that these
stat6-specific DNAzymes were active over a time period of 60 minutes. Further
experiments have to test the murine stat6-specific DNAzymes in cell based
systems and appropriate allergic mice models
FrĂŒhdiagnose der Chorea-Akanthozytose: orofaziale Dyskinesien, epileptische AnfĂ€lle und HyperCKĂ€mie
Chorea-acanthocytosis is an uncommon neurodegenerative disorder. Early diagnosis is often challenging. The triad of orofacial dyskinesia, epileptic seizures, and hyperCKemia should alert neurologists of a neuroacanthocytosis syndrome. The diagnosis can be confirmed by detection of chorein deficiency or through molecular genetics (VPS13A mutation)
Biphasic onset of splenic apoptosis following hemorrhagic shock : critical implications for Bax, Bcl-2, and Mcl-1 proteins
INTRODUCTION: The innate immune response to trauma hemorrhage involves inflammatory mediators, thus promoting cellular dysfunction as well as cell death in diverse tissues. These effects ultimately bear the risk of post-traumatic complications such as organ dysfunction, multiple organ failure, or adult respiratory distress syndrome. In this study, a murine model of resuscitated hemorrhagic shock (HS) was used to determine the apoptosis in spleen as a marker of cellular injury and reduced immune functions. METHODS: Male C57BL-6 mice were subjected to sham operation or resuscitated HS. At t = 0 hours, t = 24 hours, and t = 72 hours, mice were euthanized and the spleens were removed and evaluated for apoptotic changes via DNA fragmentation, caspase activities, and activation of both extrinsic and intrinsic apoptotic pathways. Spleens from untreated mice were used as control samples. RESULTS: HS was associated with distinct lymphocytopenia as early as t = 0 hours after hemorrhage without regaining baseline levels within the consecutive 72 hours when compared with sham and control groups. A rapid activation of splenic apoptosis in HS mice was observed at t = 0 hours and t = 72 hours after hemorrhage and predominantly confirmed by increased DNA fragmentation, elevated caspase-3/7, caspase-8, and caspase-9 activities, and enhanced expression of intrinsic mitochondrial proteins. Accordingly, mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were inversely expressed within the 72-hour observation period, thereby supporting significant pro-apoptotic changes. Solely at t = 24 hours, expression of the anti-apoptotic Mcl-1 protein shows a significant increase when compared with sham-operated and control animals. Furthermore, expression of extrinsic death receptors were only slightly increased. CONCLUSION: Our data suggest that HS induces apoptotic changes in spleen through a biphasic caspase-dependent mechanism and imply a detrimental imbalance of pro- and anti-apoptotic mitochondrial proteins Bax, Bcl-2, and Mcl-1, thereby promoting post-traumatic immunosuppression
Protein expression of mitochondrial proteins after hemorrhagic shock (HS)
Western blot analysis of splenic Bax (left), Bcl-2 (middle), and Mcl-1 (right) expression compared to the housekeeping gene ÎČ-actin (lower part), detected in splenocytes of sham and controls (a) as well as in HS animals (b). Results are representative of at least three animals per group and controls. *< 0.05 as determined by analysis of variance (with Bonferroni/Dunn) test and Mann-Whitney test. Co, control.<p><b>Copyright information:</b></p><p>Taken from "Biphasic onset of splenic apoptosis following hemorrhagic shock: critical implications for Bax, Bcl-2, and Mcl-1 proteins"</p><p>http://ccforum.com/content/12/1/R8</p><p>Critical Care 2008;12(1):R8-R8.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2374615.</p><p></p