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 Therapieansätze notwendig. Von grundlegender pathophysiologischer Bedeutung für 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-Mäusen zeigen, daß stat6 für die Ausprägung des allergischen Phänotypes wesentlich ist. Mit der Anwendung von Ribozymen und DNAzymen wird eine therapeutische Strategie auf Nukleinsäure- 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 Sekundärstrukturberechnungen der mstat6-RNA identifiziert und liegen in einzelsträngigen Loop-Strukturen. Für eine stabile Expression werden die für die Ribozyme kodierenden Oligonuleotide in die Expressionskassette pGvaL kloniert. Insgesamt finden drei mstat6-spezifische Ribozyme Verwendung, deren Aktivität unter zellfreien Bedingungen getestet wird. Alle drei untersuchten Ribozyme sind in-vitro nicht aktiv. Unter Verwendung eines Multiplex- Aktivitäts-Assays wird auf der RNA des stat6 der Maus ein Screening nach zugänglichen DNAzym-Spaltstellen vorgenommen. Die dafür eingesetzten DNAzyme haben eine RU-Konsensussequenz (R = A, G) und besitzen Bindungsarme mit einer Länge von jeweils 9 nt. Für die Lokalisation der Bindungsstellen ist die thermodynamische Stabilität ∆G0 der DNAzym-Substrat-Heteroduplex entscheidend. Die reziproke Korrelation von DNAzym-Aktivität und ∆G0 führt zur Auswahl von 41 DNAzym-Spaltstellen mit einem ∆G0 von jeweils ≤ \- 25 kcal/mol im Sequenzbereich von 150 - 600 bp sowie von 1,7 - 2 kb. Für die Auswahl des Sequenzbereiches 1,7 - 2 kb ist die Beschreibung von humanen stat6-Spleißvarianten von Bedeutung, da diese auf einer Deletion im Sequenzbereich von 1765 - 1863 bp beruhen. Von den 41 im Multiplex-Aktivitäts- Assay getesteten DNAzymen weisen acht DNAzyme eine Spaltungsaktivität auf. Die Testung aller acht DNAzyme in einem zellfreien System ergibt, daß sie über einen Zeitraum von 60 min aktiv sind. In weiterführenden Experimenten wird zu prüfen 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

Frühdiagnose der Chorea-Akanthozytose: orofaziale Dyskinesien, epileptische Anfälle und HyperCKämie

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 β-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