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Background The purpose of the trial was to evaluate the safety and performance of the new Protégé stent in the treatment of common and/or internal carotid artery stenoses. Methods The Protégé® GPS stent is a self-expanding Nitinol stent system. It is mounted on a 6 Fr 0.018” (6-9mm stent) or 7 Fr, 0.035” (10mm stent) over-the-wire-delivery system and includes a new stent release system which allows exact placement of the stent. Seventyseven patients were enrolled in the trial. Study patient assessments were conducted clinically and by duplex scan at baseline, peri-procedure, discharge, one and six months post procedure. Results Seventyseven lesions were treated. Thirtyone lesions were symptomatic, 46 lesions were asymptomatic. The procedure was technically successful in 76 (99%) lesions. The percentage of stenosis was reduced from 86 ± 7 % to 16 ± 8 %. One procedure failed because the embolic protection device could not be retrieved and the patient was sent to surgery. Within 30 days there were 4 (5.2%) Major Adverse Neurological Events (MANEs). Three of the MANEs were major strokes (3.9%), one a minor stroke. The fifth MANE occurred prior to the six month follow-up visit; this patient had a major stroke 75 days after the procedure and died 36 days later. One additional death occurred due to urosepsis. Conclusions The trial shows that the Protégé stent satisfies safety and performance criteria for the treatment of carotid artery stenosis. The complication rate was comparable to the incidence of these events in other recent carotid stent and endarterectomy studies.
Aims: The purpose of this study was to evaluate the feasibility and short-term efficacy of transcatheter paravalvular leak closure using different occlusion devices. Methods and Results: Twenty one patients underwent transcatheter closure of either aortic or mitral paravalvular leak from June 2002 to February 2006 using the Amplatzer PDA, ASD or VSD occluder. All patients had symptoms and signs of haemolysis and/or cardiac decompensation with dyspnoea. Implantation of a device was technically successful in twenty patients (95 %). Immediate residual leak was found in seventeen patients (85 %). Significant shunting persisted in nine cases during follow up (45 %). Permanent leaflet obstruction was observed in one patient. Severe complications during follow up led to early death in one patient and surgical intervention in three. A successful second catheter treatment was performed in another three patients. The event-free survival from reoperation, death and stroke at the end of the observation period was 80 %. Conclusion: Transcatheter closure of paravalvular leaks is a technically feasible, but demanding procedure. Residual leaks are common and may worsen pre-existing haemolysis. Due to the significant ongoing morbidity in this group of patients and the complexity of follow up individual patient results differ considerably. Nevertheless, it is possible to achieve some symptomatic relief, thus an interventional approach should be discussed as a potential treatment option for those patients with a limited defect and who are not deemed suitable for another operation.
The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The aim of my Ph.D. thesis was to approach the mechanisms as well as the characteristics of macrophage phenotype alterations induced by AC, and to elucidate a possible connection between tumor cell apoptosis and TAM generation. In the first part of my studies, I investigated the impact of AC on macrophage viability. I could show that macrophage survival against pro-apoptotic agents increased after the interaction with AC. Protection of macrophages against cell death required activation of phosphatidylinositol-3 kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ signaling, and correlated with Bcl-XL and Bcl-2 up-regulation as well as Ser136-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. AC released the bioactive lipid sphingosine-1-phosphate (S1P), dependent on sphingosine kinase (SphK) 2, as a survival messenger. These data indicated an active role of AC in preventing cell destruction in their neighborhood. My next aim was to elucidate the mechanism of S1P production by AC. During cell death, SphK 2 was cleaved at its N-terminus by caspase-1. Thereupon, the truncated but enzymatically active fragment of SphK 2 was released from cells. This release was coupled to phosphatidylserine exposure, a hallmark of apoptosis and a crucial signal for the phagocyte/apoptotic cell interaction. Thus, I observed a link between common signaling events during apoptosis and the extracellular production of S1P, which is known to affect immune cell attraction and polarization as well as angiogenesis in cancer. In the next part of my studies, I asked for a correlation between tumor cell apoptosis and TAM polarization. During co-culture of human macrophages with human breast cancer carcinoma cells (MCF-7), the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α; and interleukin (IL)-12-p70 production, but increased formation of IL-8 and IL-10. Alternative macrophage activation required tumor cell death, because a co-culture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. These phenotype alterations were also achieved with conditioned media from apoptotic tumor cells, which again argued for a soluble factor being involved. Knock-down of SphK2, but not SphK1, to attenuate S1P formation in MCF-7 cells, repressed the otherwise observed alternative macrophage polarization during co-culture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P was characterized by suppression of pro-inflammatory nuclear factor (NF)-κB DNA binding. These findings suggested that tumor cell apoptosis-derived S1P contributes to the macrophage polarization present in human tumors. To validate these in vitro data, I used an in vivo tumor model to clarify the relevance of SphK2 and S1P in tumor development. The growth of, as well as blood vessel infiltration into SphK2 knock-down MCF-7 (MCF-7-siSphK2) xenografts in nude mice was markedly decreased in comparison to control MCF-7 xenografts. In contrast, macrophage infiltration was similar or even more pronounced. These data provided a first hint for an in vivo role of SphK2-derived S1P in macrophage polarization associated with tumor promotion. In summary, these data indicate a new mechanism how AC themselves shape macrophage polarization, which results in the termination of inflammatory responses and macrophage survival. Furthermore, my studies present evidence that human tumors may utilize this mechanism to foster growth via increased angiogenesis.
Background: False aneurysms at the puncture site develop in up to 8 % after catheter procedures. They can be treated surgically or by ultrasound guided manual compression. A new method is to inject thrombin into the aneurysm under ultrasound guidance. We evaluated safety and efficacy of this approach in a multicenter registry. Methods: In 595 consecutive patients (age: 31-94 years, median 70) a pseudoaneurysm (593 femoral arteries, 2 brachial arteries) was diagnosed 0 to 250 days (median 3 days) after a catheter procedure. The diameter of the aneurysm ranged from 0.5 x 0.5 x 0.5 (L x W x D) to 8x11x16 cm (median 2 x 2 x1.6 cm). 20 U to 4000 U of thrombin solution (median 400 U) were injected percutaneously into the aneurysm under ultrasound guidance. Results: The procedure was technically successful in 587/595 (99%) patients. The aneurysms were thrombosed after the first injection in 531 patients (89 %). Thirty-eight (6%) patients needed a second injection and 8 (1%) patients a third injection because residual flow in the aneurysm was visible at follow-up. In 4 additional patients (0.7%) the thrombosis of the aneurysms was delayed and occurred only after 24 hours to 7 days. 6 (1%) patients surgery was performed after successful closure of the aneurysm to remove the resulting haematoma. The overall technical success rate was 99% (587/595) and clinical success was achieved in 572/595 (96%) patients. Eight (1%) other patients underwent surgery due to thrombin injection failure. Complications occurred in 9 patients (1.5%): Intravascular thrombus formation (n=3), deep venous thrombosis (n=3), pulmonary embolism due to deep venous thrombosis (n=1), transient paresthesia in the leg during injection (n=3). Conclusion: Ultrasound guided thrombin injection is a safe, painless, effective and rapid alternative to treat false aneurysms. Complications and recurrent pseudoaneurysms are very rare. It has become the treatment of choice in our institution.
All living organisms exhibit daily fluctuations in biochemical, physiological and behavioural parameters driven by endogenous oscillators, residing in the organism itself. In mammals, the core circadian oscillator is located in the paired suprachiasmatic nuclei (SCN) of the hypothalamus. Circadian rhythm generation in the SCN depends upon the expression of clock genes interacting in positive and negative transcriptional/translational feedback loops. The SCN governs the timing of peripheral circadian oscillators by neuronal pathways and by neuroendocrine mechanisms. An important neuroendocrine hand of the core circadian oscillator is melatonin, which is produced in and secreted from the pineal gland night by night. The adenohypophysis represents a peripheral circadian oscillator and the secretion of one of its hormones, prolactin, is known to be regulated by melatonin. The aim of the present study was to analyze a putative influence of melatonin on the activity state and diurnal variations of identified cell types in the hypophysis. Particular attention was paid to lactotroph, gonadotroph and pars intermedia cells. Experiments were performed with young male mice of different strains: melatonin-proficient C3H, melatonin-deficient C57BL, melatonin-proficient C3H with targeted deletions of the Mel1a receptor (MelaaBB), Mel1b receptor (MelAAbb) or both receptors (Melaabb). Cells producing prolactin (PRL), follicle stimulating hormone (FSH) were immunocytochemically identified and the presence of phosphorylated CREB protein (pCREB) and clock gene protein PER1 was demonstrated by double immunolabeling at different time points during the light/dark cycle in melatonin deficient, melatonin proficient and melatonin receptor knockout mice. Melatonin influence on Prl mRNA levels was investigated by means of in situ hybridization. At night the percentage of lactotroph cells showing a positive nuclear pCREB- and PER1-immunoreaction is significantly smaller in C57BL than in C3H mice. In both mouse strains, the percentage of pCREB –immunoreactive cells is minimal in the early morning and gradually increases to reach a maximum in the late night. PER1 levels show a parallel temporal variation in C3H, but in C57BL, they are drastically reduced in the early afternoon. The percentage of FSH-immunoreactive cells showing pCREB immunoreaction was significantly lower in the melatonin-deficient C57Bl mice than in the melatonin-proficient C3H mice during the second part of the day and during the night. In each strain, the percentage of FSH-immunoreactive cells was lowest at the early morning and gradually increases until the maximum at late night. In wild type (MelAABB) and MelAAbb mice the percentage of lactotroph cells with nuclear pCREB immunoreactions varied significantly over 24 h period, whereas in MelaaBB and Melaabb mice no significant differences were found between the five time points analyzed. The number of Prl mRNA expressing cells was significantly higher in MelaaBB and MelAAbb than in their wild type (MelAABB) littermates. pCREB levels in the pars intermedia did not show rhythmic variation in wild type or Melaabb animals, but wild type mice had higher pCREB levels than Melaabb. The observation that, during darkness, the percentage of lactotroph cells with nuclear pCREB immunoreaction is significantly higher in C3H than in C57BL mice suggests the existence of a distinct cell population that is under the control of melatonin-dependent intrapituitary signaling. Results with melatonin receptor knockout mice indicate that Mel1a and Mel1b melatonin receptors are involved in the control of the activity state of lactotroph cells, but to a differing degree. Analysis of cells expressing Prl mRNA showed that inhibitory action on the Prl expression is mostly mediated through the Mel1a receptor. The significant difference between pCREB immunoreaction in gonadotroph cells of C3H and C57BL mice might suggest that, like lactotrophes, FSH cells represent a heterogeneous population and only a subpopulation is under control of melatonin signaling. The present study is first to show that melatonin signaling also affects pCREB levels in pars intermedia of mice.
To determine the effects of inhaled IL-10 at different doses and different time points on the pulmonary and systemic inflammatory response during endotoxemia, 48 ventilated, anaesthetized rats (mean body weight ± standard deviation, 500 ± 33g) were randomly assigned to six groups (n = 8, each). Interleukin-10 was nebulised either prior to or following the intravenous injection of LPS (5mg/kg) at two doses (5.0 mycro-g or 0.5 mycro-g) in our groups. Eight rats received the same insult with no further treatment (LPS-only group). Another eight rats served as controls without endotoxemia but with aerosolized phosphate-buffered saline, the solvent of IL-10 (Sham group). Concentrations of TNF-alpha, IL-1beta, IL-6, and IFN-gamma were analyzed in plasma and bronchoalveolar lavage fluid (BALF). In addition, the nitrite release from ex-vivo cultured alveolar macrophages was determined. As compared to the LPS-only group, the concentrations of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IFN-gamma in plasma were significantly reduced in the group, which inhaled 5 mycro-g IL-10 before LPS injection (p< 0.0125). Spontaneous nitrite release from exvivo cultured alveolar macrophages was suppressed in this group (p< 0.0125). Inhalation of 0.5 mycro-g IL-10 before LPS injection and both dosages of IL-10 inhalation (5 mycro-g or 0.5 mycro-g) after LPS injection did not significantly influence either inflammatory cytokine concentrations in BALF, in plasma or the nitrite release from ex-vivo cultured alveolar macrophages. In this study, inhaled IL-10 only demonstrated anti-inflammatory effects when it was administered at 5 mycro-g prior to the induction of experimental endotoxemia. Interleukin-10 aerosol had no effect when it was given either following induction of endotoxemia or given at a lower dosage (which here was 0.5 mycro-g) either before or following injection of lipopolysaccharide.
Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.
Prostaglandin D2 (PGD2) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood and the data obtained so far are rather controversial. Here we investigated the effects of central PGD2 delivery and of systemic prostaglandin D synthase (PGDS) or cyclooxygenase (COX) inhibition on core body temperature (TC) and on prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Both PGE2 and PGD2 were detectable in CSF samples from control rats (6.2 ± 1.1 and 17.3 ± 3.1 pg/ml, respectively). Lipopolysaccharide (LPS) injection (50 μg i.p.) induced fever during the 5-hour observation period. Five hours after LPS injection, the levels of PGE2 and PGD2 were increased in the CSF about 90-fold (541.0 ± 47.5 pg/ml) and 5-fold (95.4 ± 23.1 pg/ml), respectively. Administration of PGD2 (50 - 500 ng) into the cisterna magna (i.c.m) evoked a delayed fever response in a dose-dependent manner that was accompanied by increased levels of PGE2 in the CSF. RT-PCR analyses revealed that the increased levels of PGE2 after PGD2 administration were not caused by up-regulation of COX-2 or microsomal prostaglandin E synthase 1 (mPGES-1) in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD2 considerably sustained the pyrogenic effects of i.c.m. administered PGE2. Pretreatment with a novel PGDS inhibitor, EDJ300520 (10 – 40 mg/kg p.o.), 1 h prior to the LPS injection impaired the LPS-induced increase of both PGD2 and PGE2 in the CSF and inhibited the fever response. In contrast, administration of EDJ300520 3 h after LPS injection did not ameliorate the LPS-induced fever. Accordingly, the concentration of PGE2 in the CSF was not decreased after EDJ300520 treatment. However, the CSF levels of PGD2 were reduced after administration of a high dose of EDJ300520 (40 mg/kg). We also investigated the effects of antipyretic drugs on the CSF levels of PGE2 and PGD2 during LPS-induced fever. Four antipyretic drugs with different mechanisms of action were used, including ibuprofen (5 - 20 mg/kg), celecoxib (10 - 50 mg/kg), SC560 5 - 20 mg/kg), and paracetamol (50 - 150 mg/kg). Each drug was used in three different doses and was orally administered 3 h after the LPS injection. All drugs were capable to attenuate the LPS-induced fever. The decrease of TC paralleled the reduction of PGE2 levels in the CSF. Of note, there was a tendency to reduced PGD2 levels in the CSF after treatment with the antipyretic drugs. However, only SC560 and the high dose of celecoxib (50 mg/kg) reduced the PGD2 levels significantly. In summary, our experiments underscore the pivotal role of PGE2 as the principal downstream mediator of fever. Moreover, we demonstrate that PGD2 is also involved in the mechanisms underlying fever. Our data suggest that PGD2 exerts an indirect pyrogenic effect by modulating the availability of PGE2 in the CSF. Additional studies are needed to explore the exact mechanism by
Molecular mechanism of intracellular signal transduction by the angiotensin-converting enzyme
(2007)
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensin system (RAS) and is crucially involved in the homeostasis of fluid and electrolyte balance and thus in the regulation of blood pressure. The zinc metallopeptidase is involved in the generation of angiotensin II, a potent vasoconstrictor and in the degradation of bradykinin, a potent vasodilator. It is worth noting that ACE more readily hydrolyzes bradykinin than it does angiotensin I thus culminating in the net physiological effect of the production of a vasoconstrictor and the decrease in the availability of a vasodilator. ACE inhibitors have become one of the most successful therapeutic approaches as a first line of therapy in hypertension, and are also widely used in treating heart failure, myocardial infarction, stroke, coronary artery disease and impaired left ventricular function. However, one unexpected clinically relevant finding related to ACE inhibitors is their ability to delay the onset of type II diabetes that was revealed by various large clinical trials. However, the mechanisms underlying these beneficial effects of ACE inhibitor therapy are currently unclear and cannot be explained by the prevention of angiotensin II formation or the attenuated degradation of bradykinin. Thus the potential beneficial effects attributed to ACE inhibitors may occur independent of reductions in blood pressure paving way for new and/or unknown mechanism. Our group has recently redefined ACE as a signal transduction molecule which upon binding to ACE inhibitor turns on a signalling cascade leading to phosphorylation of Ser1270 by CK2, activation of JNK and changes in gene expression in endothelial cells. However the mechanism by which ACE inhibitor initiates the signalling cascade was not clear. It was hypothesized that ACE, which is anchored to the membrane with a single transmembrane domain should dimerize prior to initiating further downstream signalling events in endothelial cells. Therefore, we sought to explore whether or not ACE forms dimers in endothelial cells and whether ACE dimerization is essential for the initiation of ACE signalling in endothelial cells. Using native gel electrophoresis, we found that ACE forms dimers in endothelial cells and that there is an increase in the dimer formation upon treatment of endothelial cells with ACE inhibitors. ACE homodimerization was also demonstrated using the split-ubiquitin system and chemical cross-linking experiments. ACE dimers are also formed in endothelial cells overexpressing the non-phosphorylatable ACE, wherein ACE signalling was abolished indicating that dimerization process is not influenced by the phosphorylation of the serine residue residing in the cytoplasmic tail. Monosaccharides like glucose, galactose and mannitol did not have any influence on ACE-inhibitor induced dimerization. Making use of different monoclonal antibodies directed to the epitopes of N-domain which harbours carbohydrate recognizing domain, also did not affect dimerization. However, inactivation of the C-domain active site by introducing mutation of the key histidine residues in HEMGH consensus sequences, which complexes the zinc ions, abolished enzyme dimerization both in the basal state and in response to ramiprilat. Mutation of the C-domain also resulted in the loss of ACE inhibitor-induced ACE signalling, that is we failed to observe ramiprilat-induced increase in the phosphorylation of the Ser1270 and the subsequent JNK activation. ACE-inhibitor induced dimerization precedes the phosphorylation of Ser1270 and activation of JNK. Thus the ACE-inhibitor induced dimerization via the C-domain of ACE represents the initial step in the ACE signalling pathway which involves the activation of JNK/c-Jun pathway and leading to the changes in the gene expression in endothelial cells. Our group previously identified ACE itself as well as cyclooxygenase-2 (COX-2) as two “ACE signalling-regulated” genes. To screen for additional genes regulated in a similar manner we used DNA microarray technology, to assess ramiprilat-induced changes in the endothelial cell gene expression. 21 genes were identified to be differentially regulated of which, 7 were upregulated and 14 were downregulated by ramiprilat. However, when screened at the protein level, we found no significant differences between the untreated control cells and those treated with ramiprilat. As several other cells and tissues possess a fully functional RAS we screened plasma samples from healthy volunteers as well as from patients with coronary artery disease for the proteins identified in the microarray. We observed that the cellular retinal binding protein-1 (CRBP-1) was detectable at low levels in plasma from patients and that ramipril markedly increased serum levels of this protein. Endothelial cells overexpressing CRBP-1 demonstrated increased RXRE and PPRE activity when stimulated with 9-cis retinoic acid and rosiglitazone respectively suggesting that CRBP-1 might affect gene expression via heterodimerization of PPAR elements with RXR elements by virtue of its function as a transport protein of retinoic acid. Studies aimed at determining the consequences of elevated CRBP-1 expression on endothelial cell homeostasis are ongoing. Although the RAS has been described in many other tissues apart from endothelial cells, ACE signalling has not yet been addressed in tissues such as monocytes/macrophages, which have an increased ACE expression in an atherosclerotic setting. We observed that upon stimulation of cultured ACE expressing monocytes with ramiprilat, JNK is activated suggesting the occurrence of ACE signalling in human monocytes. It is worth noting that ACE inhibitors delay the onset of type II diabetes in spite of moderate decrease in blood pressure. To further elucidate the mechanism underlying this effect, we found that ACE inhibitors increase the PPARgamma levels in the nuclear extracts of ACE expressing monocytes which were also reproduced in human endothelial cells overexpressing human somatic ACE. However, ramiprilat did not have any direct effect on the activity of a luciferase-coupled promoter containing several copies of the PPRE in human endothelial cells. These results contrasted with the actions of the PPARgamma agonist suggesting that ramiprilat enhances PPARgamma levels through an indirect mechanism. We next hypothesized that ramiprilat might increase the levels of 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) which is a natural ligand for PPARgamma via COX enzymes in monocytes. We observed that ramiprilat was able to decrease the diminution of COX-2 levels upto 48 hours of treatment but the levels of 15dPGJ2 were too low to be detected by ELISA. However ramiprilat enhanced the plasma levels of adiponectin, a downstream target of PPARgamma, which is a anti-atherogenic and anti-inflammatory adipokine, in patients with coronary artery disease. Though adiponectin is a PPARgamma-regulated gene, the observed increase in adiponectin might be attributed to the increase in RXR rather than via PPARgamma. Taken together, the results of this investigation have revealed that ACE inhibitors initiate ACE signalling by eliciting the dimerization of the enzyme, more specifically via its C-domain active centers. The ACE signalling cascade when activated leads to the enhanced expression of ACE, COX-2 and CRBP-1 which in turn favours the heterodimerization of PPARgamma with RXR and thus results in the increased expression of “PPARgamma regulated” genes such as adiponectin. The latter results provide a molecular basis for the observation that ACE inhibitors can delay the onset of type 2 diabetes in as much as it was possible to link ramipril with CRBP-1, RXR activity and the expression of adiponectin, an adipokine associated with improved insulin sensitivity. Further work is however required to elucidate the consequences of ACE inhibitors in monocytes and adipocytes as well as in intact animals.
Transcranial magnetic stimulation (TMS) is a non-invasive technique which can be used to study different intracortical excitatory and inhibitory neuronal circuits in the intact human being. In the primary motor cortex, there are essentially three different TMS measures of inhibitory neuronal circuits as determined by paired-pulse TMS: short-interval intracortical inhibition (SICI), long-interval intracortical inhibition (LICI) and interhemispheric inhibition (IHI). It was hypothesized that SICI is a GABAA receptor mediated inhibition (Ilic et al., 2002) whereas LICI and IHI are mediated by GABAB receptors (Daskalakis et al., 2002; McDonnell et al., 2006). Additionally, it was shown that these inhibitory circuits interact negatively, possible due to presynaptic GABAB receptor mediated inhibition (Sanger et al., 2001; Daskalakis et al., 2002). Which neuronal populations exactly underlie SICI, LICI and IHI, is not completely clear and by which mechanism these inhibitory circuits interact has never been tested pharmacologically so far. Thus, the effects of a single oral dose of Diazepam (DZP), a specific positive allosteric modulator at the GABAA receptor, and of Baclofen (BAC), a specific GABAB receptor agonist, on SICI, LICI and IHI as well as their interactions were tested here in a randomized, placebo controlled, double-blinded crossover study. SICI significantly increased after intake of DZP whereas BAC did not change SICI. Conversely, LICI significantly increased after intake of BAC but did not change after intake of DZP. IHI showed only a trend towards a decrease after intake of DZP but no change after intake of BAC. The interactions IHI-SICI, LICI-IHI and LICI-SICI were all negative at baseline. SICI and IHI were partially suppressed in the presence of IHI and LICI, respectively, and SICI in the presence of LICI was almost completely blocked. BAC did not change any of these interactions, whereas DZP significantly increased SICI in the presence of LICI. This study is the first to examine by means of pharmacological testing the complex interactions between different inhibitory circuits in the human motor cortex. The effects of DZP and BAC on SICI and LICI confirmed the notion that SICI is a GABAA receptor mediated intracortical inhibition whereas LICI depends on GABAB receptor mediated neurotransmission. The pharmacology of IHI at short interstimulus intervals of < 20 ms (12 ms in this study) remains still inconclusive and warrants further investigation. Findings further suggest that SICI, LICI and IHI represent three different inhibitory neuronal circuits which can be tested non-invasively by means of paired-pulse TMS. Furthermore, the data support the idea that the negative interactions IHI-SICI, LICI-IHI and LICI-SICI are most likely due to presynaptic GABAB receptor mediated autoinhibition.
In 1911 Eugen Bleuler (Bleuler, 1911) postulated that schizophrenia was a disorder resulting from inability to properly integrate mental processes. Around the same time, Carl Wernicke (Wernicke, 1894) proposed that psychosis might result from disruption of white matter tracts. Both of these statements can be considered early cornerstones of modern connectivity hypotheses developed towards the end of the twentieth century by such researchers as Karl Friston (1998) and Nancy Andreansen (1998). In the current work, the hypothesis that schizophrenia, rather than being a disorder or either anatomical or functional connectivity, is a disorder where both of these processes interact and influence the clinical presentation of patients, is examined. This is achieved through a detailed examination of a sample of chronic schizophrenia patients using a combination of functional and anatomical Magnetic Resonance Imaging techniques. The relationship of these measures to clinical symptoms is also explored. In the first study, anatomical connectivity at the whole-brain level is examined using Diffusion Tensor Imaging. The results of the study contribute to the previous literature on auditory hallucinations in schizophrenia and provide the first direct correlation between increased anatomical connectivity and increased severity of psychotic symptoms. The second study provides a thorough examination of the interhemispheric connectivity. This is achieved through a detailed examination of the corpus callosum using a combination of diffusivity and volumetric values. This is the first study to date where several anatomical methods are used in one sample. The results illustrate the importance of using different techniques to accurately characterize anatomical abnormalities observed in schizophrenia. In addition, contrary to previous research reports, the results of the current study imply that only specific sub-sections of the corpus callosum are affected by anatomical abnormalities. The pattern of these changes may influence clinical presentation of patients. Finally, functional connectivity at the whole-brain level is examined during resting-state using Independent Component Analysis. Similarly to the results of the anatomical examinations, it provides further supporting evidence that the pattern of disturbances observed in the current sample of schizophrenia patients examined herein reflects a combination of hypo- and hyperconnectivity. Moreover, the study further validates resting-state functional Magnetic Resonance Imaging as a reliable tool for examining functional abnormalities in schizophrenia.
Visual information is processed hierarchically in the human visual system. Early during processing basic features are analysed separately while at later stages of processing, they are integrated into a unified percept. By investigating a basic visual feature and following its integration at different levels of processing one can identify specific patterns. In certain visual impairments, these patterns can function defectively and their detailed study can clarify the cause of the visual deficit. Here we investigate orientation as a basic feature and use a property of the visual system called adaptation. Adaptation occurs as a decrease in the level of neural activity during repetitive presentation of the same stimulus. Psychophysical studies have shown that adaptation transfers interocularly, meaning that if only one eye is adapted the other eye shows also adaptation effects. Our aim was to investigate interocular transfer by means of functional magnetic resonance imaging (fMRI). Even though adaptation was demonstrated in the fMRI environment, the interocular transfer was never investigated in such a setup. First, we developed a method to measure interocular transfer of adaptation to gratings with fMRI. We then went further to test it in various groups of subjects. In normally sighted humans interocular transfer was present both in early (striate) as well as later visual areas (extrastriate). In subjects with impaired stereovision (with or without normal visual acuity) interocular transfer was absent in the investigated regions. Detailed analysis of the recorded differences between subjects with and subjects without stereovision was performed. The results of this analysis are presented in detail in this book. These results suggest that the neuronal mechanisms involved in the interocular transfer of pattern adaptation share, at least in part, the neural circuitry underlying binocular functions and stereopsis. We conclude that fMRI adaptation can be used for the assessment of cortical binocularity in humans with normal and impaired stereopsis. Further investigations are needed to address more subtle aspects of the lack of interocular transfer. Towards this purpose, through a fourth experiment we propose further directions that might shed more light on the issue of stereovision and its clinical implications. We show that carefully tuned variations in our experimental procedure might reveal other aspects of binocularity in the human visual system. We believe that the method we developed, apart from the interesting results shown here, has a high potential to be further used for other research questions. Following the above summarized ideas, the thesis comprises of three parts (chapters). The first chapter provides the main theoretical backgrounds of the visual system and of the MRI imaging technique, chapter two describes the experimental procedures while the results and their detailed discussion are detailed in chapter three.
Background and objectives: Constrictive pericarditis (CP) is the result of a spectrum of primary cardiac and non-cardiac conditions. Little data exists on the cause-specific survival after pericardiectomy in the modern era. The impact of pericardial calcification (CA) on survival is unclear. We sought to determine the association of etiology of CP, CA and other clinical variables with long-term survival after pericardiectomy. Methods: We analyzed the records of 163 patients who underwent pericardiectomy for CP over a 24-year period at a single center. The diagnosis of CP was established by surgical report. Vital status was obtained by the Social Security Death Index. The Kaplan Meier method was used to estimate overall survival and survival by etiology group. Cox proportional hazards regression analysis was performed to assess the effect of various causes for CP on longterm survival while adjusting for age. Results: The etiology of CP was idiopathic in 75 patients (46%), prior cardiac surgery in 60 (37%), radiation treatment in 15 (9%) and miscellaneous in 13 patients (8%). Vital status was obtained in 160 patients (98%). Median follow-up was 6.9 years (range: 0.8 to 24.5 years). Perioperative mortality for all patients was 6%. Idiopathic CP had the best prognosis (7 year survival: 88%, 95% confidence interval [CI] 76% to 94%) followed by postsurgical (66%, 95% CI 52% to 78%) and postradiation CP (27%, 95% CI 9% to 58%). In bootstrap-validated proportional hazards analyses, predictors of poor survival were prior radiation, worse renal function, higher pulmonary artery pressure (PAP), abnormal left ventricular (LV) systolic function, lower serum sodium level, and older age. Pericardial calcification had no impact on survival. Conclusion: Long-term survival after pericardiectomy for CP is determined by the underlying etiology of constriction, LV systolic function, renal function, serum sodium, and PAP. Patients with postsurgical as well as postradiation CP have a survival inferior to patients with idiopathic CP. Perioperative Mortality is low. The relatively good survival after pericardiectomy in patients with idiopathic CP emphasizes the safety of pericardiectomy in this group.
The µ-opioid receptor is the primary target structure of most opioid analgesics and thus responsible for the predominant part of their wanted and unwanted effects. Carriers of the frequent genetic µ-opioid receptor variant N40D (allelic frequency 8.2 - 17 %), coded by the single nucleotide polymorphism A>G at position 118 of the µ-opioid receptor coding gene OPRM1 (OPRM1 118A>G SNP), suffer from a decreased opioid potency and from a higher need of opioid analgesics to reach adequate analgesia. The aim of the present work was to identify the mechanism by which the OPRM1 118A>G SNP decreases the opioid potency and to quantify its effects on the analgesic potency and therapeutic range of opioid analgesics.
To elucidate the consequences of the OPRM1 118A>G SNP for the effects of opioid analgesics, brain regions of healthy homozygous carriers of the OPRM1 118A>G SNP were identified by means of functional magnetic resonace imaging (fMRI), where the variant alters the response to opioid analgesics after painful stimulation. Afterwards, the µ-opioid receptor function was analyzed on a molecular level in post mortem samples of these brain regions. Finally, the consequences of the OPRM1 118A>G SNP for the analgesic and respiratory depressive effects of opioids were quantified in healthy carriers and non-carriers of OPRM1 118A>G SNP by means of experimental pain- and respiratory depression-models.
To identify pain processing brain regions, where the variant alters the response to opioid analgesics after painful stimulation, we investigated the effects of different alfentanil concentration levels (0, 25, 50 and 75 ng/ml) on pain-related brain activation achieved by short pulses (300 msec) of gaseous CO2 (66% v/v) delivered to the nasal mucosa using a 3.0 T magnetic head scanner in 16 non-carriers and nine homozygous carriers of the µ-opioid receptor gene variant OPRM1 118A>G. In brain regions associated with the processing of the sensory dimension of pain (pain intensity), such as the primary and secondary somatosensory cortices and the posterior insular cortex, the activation decreased linearly in relation to alfentanil concentrations, which was significantly less pronounced in OPRM1 118G carriers. In contrast, in brain regions known to process the affective dimension of pain (emotional dimension), such as the parahippocampal gyrus, amygdala and anterior insula, the pain-related activation disappeared already at the lowest alfentanil dose, without genotype differences.
Subsequently, we investigated the µ-opioid receptor-expression ([3H]-DAMGO saturation experiments, OPRM1 mRNA analysis by means of RT-PCR), the µ-opioid receptor affinity ([3H]-DAMGO saturation and competition experiments) and µ-opioid receptor signaling ([35S]- GTPγS binding experiments) in post mortem samples of the human SII-region, as a cortical projection region coding for pain intensity, and lateral thalamus, as an important region for nociceptive transmission. Samples of 22 non-carriers, 21 heterozygous and three homozygous carriers of OPRM1 118A>G SNP were included into the analysis. The receptor expression and receptor affinity of both brain regions did not differ between non-carriers and carriers of the variant N40D. In non-carriers, the µ-opioid receptors of the SII-region activated the receptor bound G-protein more efficiently than those of the thalamus (factor 1.55-2.27). This regional difference was missing in heterozygous (factor 0.78-1.66) and homozygous (factor 0.66-1.15) carriers of the N40D variant indicating a reduced receptor-G-protein-coupling in the SII-region.
Finally, the consequences of the alteration of µ-opioid receptor function in carriers and noncarriers of the genetic variant was investigated using pain- and respiratory depression-models. Therefore, 10 healthy non-carriers, four heterozygous and six homozygous carriers of the µ- opioid receptor variant N40D received an infusion of four different concentrations of alfentanil (0, 33.33, 66.66 and 100 ng/ml). At each concentration level, analgesia was assessed by means of electrically (5 Hz sinus 0 to 20 mA) and chemically (200 ms gaseous CO2 pulses applied to the nasal mucosa) induced pain, and respiratory depression was quantified by means of hypercapnic challenge according to Read and recording of the breathing frequency. The results showed that depending on the used pain model, both heterozygous and homozygous carriers of the variant N40D needed 2 – 4 times higher alfentanil concentrations to achieve the same analgesia as non-carriers. This increase seems to be at least for homozygous carriers unproblematic, because to reach a comparable respiratory depression as non-carriers, they needed 10-12 times higher alfentanil concentrations.
The results of this work demonstrate that the µ-opioid receptor variant N40D causes a regionally limited reduction of the signal transduction efficiency of µ-opioid receptors in brain regions involved in pain processing. Thus, the painful activation of sensory brain regions coding for pain intensity is not sufficiently suppressed by opioid analgesics in carriers of the variant N40D. Due to the insufficient suppression in hetero- and homozygous carriers of the variant N40D, the concentration of opioids has to be increased by a factor 2 - 4, in order to achieve the same analgesia as in non-carriers. At the same time, the respiratory depressive effects are decreased to a greater extent in homozygous carriers of the N40D variant as they need a 10 - 12 times higher opioid concentration to suffer from the same degree of respiratory depression as non-carriers. Due to the increased therapeutic range of opioid analgesics, an increase of the opioid dose seems to be harmless, at least for homozygous carriers of the N40D variant.
The physiology of our most complex organ, the brain, is still not comprehensively understood. The brain basically serves the processing, storing and binding of external and internal information, and thereby generates amazing phenomena like the understanding of oneself as an individual entitiy. How exactly information is encoded and represented, how individual neurons or networks of neurons actually interact, is a gigantic puzzle, whose pieces were collected since many decades. Subject of scientific discussions are the basic spatiotemporal structures of neuronal representations. Suggestions and observations reach hereby from simple rate coding of individual neurons to synchronous activity of larger ensembles. To approach answers to these questions, our working group has used a combination of different recording techniques that allowed for the comparison of neuronal interactions on different spatial scales. We focused on prefrontal neuronal interactions during visual short-term memory. Herefore two rhesus monkeys had been trained to perform a visual short-term memory task. We measured and recorded their neuronal activity by means of a microelectrode matrix that could be inserted into the cortex via a closable chamber, which had been previously implanted above prefrontal cortex. The acquired signal was separated into two components: a high-frequency component, that represents the spiking output activity of few neurons in the vicinity of each electrode tip (multi-unit activity), and a low-frequency component, that results from dendritic input activity of larger neuronal assemblies (local field potential). From one of the experimental animals we also recorded mass signals of even larger neuronal populations by means of small silverball electrodes, that had been implated into the skull above prefrontal cortex (skull EEG) in the context of a pilot project. In the first subproject, we analyzed the selectivity of output signals with respect to the memorized stimulus and task performance. We compared selectivities of local recording sites (multi-unit activity) with the selectivities of patterns created by the combined activity of all recording sites, thus representing the activity of large and distributed ensembles. Local neuronal activity correlated with the course of the visual short-term memory task, but was not highly discriminative with respect to different visual stimuli. We could show that the population activity was significantly more specific. Concerning task performance, we obtained the same result, albeit less pronounced. Further analyses revealed that the patterns of distributed ensemble activity were only partly based on realtime coordination of neuronal activity, and in addition, did not remain stable across the time course of the short-term memory task. In the second subproject, we focused on the oscillatory behavior of the local field potential. After a time-frequency analysis, we studied different frequency bands concerning stimulus selectivity and task performance of the monkey. We hereby found significant modulations of oscillations in the beta- and gamma-frequency range, that correlated with different periods of the task. Especially for oscillations in beta- and low-gamma-range, we observed phase-locking of oscillations between different recording sites, which could play an important role as internal clock to coordinate spatially separate activity. Local high-gamma oscillations themselves seemed to be important for the maintenance of information. These results could be partly confirmed by mass signals of EEG. In sum, our results support the hypothesis that information is represented in the brain by means of concerted activity of spatially distributed neuronal ensembles. This activity again appears to be coordinated by oscillatory activity in beta- and low-gamma-frequency ranges. A deeper understanding of central nervous information processing could contribute to better treatment of diseases like Parkinson’s, Alzheimer’s as well as epilepsy, and neuropsychiatric disorders like schizophrenia.
Macrophages show a remarkable functional plasticity, which enables them to change their phenotype in response to environmental signals. They are key players during infection by initiating inflammation through the release of proinflammatory mediators. Furthermore, macrophages contribute to the resolution of inflammation by phagocytosis of apoptotic granulocytes. Phagocytosis of apoptotic cells (AC) induces an anti-inflammatory phenotype in macrophages and protects them against apoptosis. However, mechanistic details provoking these phenotype alterations are incompletely understood. Therefore, the aim of my Ph.D. thesis was to investigate the molecular basis of anti-inflammatory macrophage polarization. In the first part of my studies, I investigated the expression of heme oxygenase (HO)-1 in macrophages following treatment with supernatants from AC. HO-1 catalyzes the first and rate-limiting step of heme degradation and potentially bears anti-inflammatory as well as anti-apoptotic potential. I was able to show biphasic upregulation of HO-1 by AC supernatants. The first phase of HO-1 induction at 6 h required activation of p38 MAPK and was accomplished by the bioactive lipid sphingosine-1-phosphate (S1P) engaging S1P receptor 1 (S1P1). However, the second wave of HO-1 induction at 24 h was attributed to autocrine signaling of vascular endothelial growth factor (VEGF) A, whose expression was facilitated by S1P. The release of VEGFA from macrophages was STAT1-dependent, whereas VEGFA itself acted on the macrophage HO-1 promoter via STAT1/STAT3 heterodimer binding. Knockdown of HO-1 revealed its relevance in promoting enhanced expression of the anti-apoptotic proteins B cell leukemia/lymphoma-2 (Bcl-2) and B cell leukaemia/lymphoma-x long (Bcl-XL), as well as the anti-inflammatory adenosine receptor A2A. MHC II and indoleamine 2,3-dioxygenase expression were also affected by ACsupernanatants, but were not HO-1 dependent. Unexpectedly, S1P1 was also upregulated following treatment with AC supernatants. Thus, I considered whether S1P1 induction could specifically be mediated by alternative macrophage activating factors. The expression of S1P1 was enhanced in the presence of the alternative activation stimuli IL-4 as well as IL-10, whereas it was unchanged following incubations with LPS, interferon-g or S1P. My next aim was to investigate the expression of the different S1P receptor isoforms in macrophages following treatment with supernatants form AC. While the expressions of S1P1 as well as S1P3 were induced by exposure to supernatants from AC, S1P2 expression was unaffected. As S1P1/3 and S1P2 are conflictively involved in the regulation of cell migration, I asked for a correlation between increased S1P receptor expression and enhanced migration rate. Indeed, macrophages showed enhanced motility following treatment with supernatants form AC, which was inhibited in S1P1 knockout macrophages. In summary, my findings indicate that HO-1, which is induced by AC-derived S1P, is critically involved in macrophage polarization towards an alternatively activated macrophage phenotype. S1P1 seems to represent a central checkpoint during macrophage activation. On the one hand, S1P1 is induced by supernatants form AC and promotes migration of macrophages. On the other hand, it mediates the induction of HO-1, which is accompanied by antiinflammatory as well as anti-apoptotic signaling. Furthermore, my studies provide evidence that upregulation of HO-1 and S1P1 in macrophages may contribute to the resolution of inflammation by establishing an anti-inflammatory macrophage phenotype and provoking macrophage migration along the vascular S1P gradient out of an inflammatory environment into the lymph.
It has been shown that stem and progenitor cells are therapeutically effective after i.v application. Yet, many aspects regarding intracellular signaling pathways which are involved in the homing and local action of these cells still have to be elucidated. In this work, it was aimed to investigate the role of the small GTPase Rap1 in adhesion activation in Hematopoietic Stem and Progenitor Cells (HSC/HPC) and in Mesenchymal Stem Cells (MSC). The potential role of Rap1 was assessed in, mice which were homozygote negative for the expression of the Rap1a gene. Peripheral blood lymphocyte counts as well as numbers of HPCs in the blood were decreased in Rap1a-/- mice compared to wild-type controls. Additionally the adhesion capability of HPCs from Rap1a-/- to the endothelial ligand, Vascular Cell Adhesion Molecule – 1 under shear stress was decreased. The hematopoietic repopulation potential of Rap1a-/- HPC was however not decreased in a competitive bone marrow transplantation model, indicating that deficiency of Rap1a in HSC/HPC does not negatively affect their ability to interact with the bone marrow microenvironment. In contrast, the isolation of MSC was not possible from Rap1a-/- bone marrow, indicating an altered situation in the bone marrow niche through changed stromal cell behaviour. Instead, Rap1a+/- MSC could be isolated and showed an adhesion deficit under shear stress. In contrast, no differences were noted in their differentiation potential. In a mouse homing model, the overall ability of the Rap1a+/- MSC to home to different tissues was found preserved. Finally, in a murine subcutaneous carcinoma model, cells with an HPC phenotype were observed to be present in the tumor microenvironment, and it was shown that they home directly to tumors. Since HPC isolated from bone marrow were able to differentiate into cells with a pro-angiogenic phenotype in vitro, HPC may be of relevance for neovascularization, as tumor-infiltrating progenitor cells. The results of the study should contribute to the understanding of the regulation of progenitor cell homing behaviour in situations simulating cell therapy approaches in preclinical situations.
Dendritic cells are the sentinels between the innate and the adaptive immunity. They are professionals that capture invading pathogens, recognize specific microbial structures and induce naïve T lymphocytes to polarize into a specific T cell subset. To initiate the T cell polarization DCs secrete cytokines which are induced upon Toll-like receptor activation by microbial structures. The recognition of these structures and the discrimination between non-self and self structures by TLRs is fine tuned, but under defined circumstances deregulation of immune responses appears. Consequently, this can result in immune disorders such as autoimmunity, chronic inflammatory diseases or cancer. In this thesis the investigations are focused on the regulation of the IL-12 family members IL-12p70 and IL-23 in DCs. The objective was to investigate three different endogenous and exogenous factors that regulate IL-12p70 or IL-23. In the first part Selenium, an essential trace element and important factor in several metabolic pathways including the cellular redox status and reactive oxygen species (ROS) dependent signaling was applied as supplement in immature Langerhans cell culture. Because Selenium also plays a role in the immune system the TLR-induced IL-23 production of the DCs upon Selenium treatment was analyzed. In the immature Langerhans cell line XS-52 the strongest inducer of IL-23 was TLR4 ligand LPS. Furthermore increased levels of TLR4-induced IL-23 in cells treated with Selenium were detected in a concentration dependent manner. Whereas the IL-23 subunit p40 was upregulated upon Selenium treatment the second subunit p19 was completely unaffected. This effect was detected on mRNA and protein level. In addition, as expected, IFN-gamma inhibited the TLR4-induced IL-23 secretion of both, Selenium treated and untreated cells. In the second part of this thesis p47phox, an organizing protein of the NADPH oxidase was analyzed regarding its potential to regulate IL-12p70 and/or IL-23 secreted by different DC subtypes. Since it was demonstrated that p47phox deficiency is associated with enhanced autoimmunity and chronic inflammation we wanted to prove whether it has a function in addition to that within the NADPH oxidase. We found some hints that p47phox may be interact with proteins of the TLR signaling pathway and thus we hypothesized that p47phox may have a function for the regulation of TLR-mediated cytokine production in DCs. In several experiments with DCs from the spleen of different p47phox deficient mice we detected an increased production of TLR9-induced IL-12p70 compared to wild type cells. In contrast TLR4 stimulation with LPS displayed no significant differences between p47phox deficient and wild type cells. In spleen cells IL-23 was not detected. Confirming the results of this new negative feedback by p47phox on IL-12p70 rats, with a single nucleotide polymorphism in the p47phox gene, were investigated. Interestingly this polymorphism is located in the phosphorylation site of IRAK4, an important kinase in the TLR pathway. In rats with a methionine residue at this position in the p47phox protein enhanced IL-12p70 level were found, compared to the rats with threonine, which can be phosphorylated by IRAK4. All analyzed mice and rats have defects in the NADPH oxidase function due to a non functional p47phox protein which results in a defective ROS production. To determine whether the observed negative feedback mechanism is connected to the lack of ROS production experiments with gp91phox deficient mice, which also have a defective NADPH oxidase function, were performed. In several experiments the enhanced IL-12p70 production in cells from p47phox deficient mice could be confirmed, but no differences between gp91phox deficient and wild type mice have been observed. In further studies was found that the inhibition of the NADPH oxidase function did not alter the negative feedback on TLR9-induced IL-12p70 secretion by p47phox. Interestingly upon treatment with the inhibitor a feedback mechanism in wild type cells also after TLR4 stimulation was observed. Hence, blocking a ROS-dependent TLR4 pathway by the inhibitor uncovered the LPS induced ROS-independent pathway of the TLR4 signaling. These findings strongly approve a NADPH oxidase/ROS-independent function of p47phox in DCs. Because splenic DCs do not secrete IL-23, in vitro differentiated DCs from the bone marrow were investigated regarding the negative feedback mechanism. In DCs from p47phox deficient mice, differentiated with GM-CSF, the upregulation of IL-12p70 was confirmed, whereas Flt3-L cultured DCs did not display the negative feedback. In contrast to IL-12p70 no difference for the IL-23 production between wild type and p47phox deficient cells has been detected. Thus, we concluded that IL-23 production is not regulated by p47phox. IL-12p70 is the major cytokine in the Th1 polarization whereas IL-23 is important for the maintenance and survival of Th17 cells. To prove whether the regulation of IL-12p70 influences the T cell response immunization experiments closely resembling the classical DTH-like protocols were performed. Groups of p47phox deficient and wild type mice received either PBS, OVA alone or mixed with TLR9 ligand CpG2216 in IFA s.c. to activate and polarize naïve T cells towards Th1 or Th17 cells. After ten days isolated lymph node cells were incubated in an ELISA spot assay with or without OVA and the frequency of IFN-gamma and IL-17 producing T cells was quantified. In vitro recall of OVA immunization of wild type and p47phox deficient mice resulted in an increased IFN-gamma and IL-17 frequency in the p47phox deficient cells. The combination with CpG2216 as adjuvant and inducer of the 3rd signal enhanced the frequency of IFN-gamma and IL-17 producing T cells in wild type mice significantly. However, in p47phox deficient cells the IFN-gamma and IL-17 response, being already detectable without in vitro OVA re-stimulation, was strongly augmented upon OVA restimulation. These findings confirmed our in vitro data for IL-12p70. Hence, the data supports our hypothesis that the p47phox dependent regulation of IL-12p70 and the consequences for the T cell response is an important mechanism to prevent uncontrolled immune responses. In the last part of this thesis the immunomodulatory property of vitamin D3 on the IL-12p70 production of DCs was examined. Since it was shown that VD3 influences the differentiation and maturation of monocytes and DCs, splenic DCs from C57BL/6 and BALB/c mice were investigated regarding their IL-12p70 production after VD3 treatment. Spleen cells, stimulated with LPS or CpG2216, exhibited a decreased IL-12p70 production when treated with VD3 before stimulation phase. In contrast treatment with VD3 only during TLR stimulation had no influence on the IL-12p70 production. Since it was demonstrated that VD3 stimulates the expression of p47phox mRNA cells from p47phox deficient mice were also treated with VD3. In initial experiments only a slight inhibition of IL-12p70 has been detected in p47phox deficient cells compared to the wild type. In summary the thesis displays three different possibilities to influence the TLR-induced cytokine secretion of DCs, although with different intensities and specificities.
The peroxisome proliferator activated receptor gamma (PPARgamma) plays an eminent role during alternative activation of macrophages and resolution of inflammation. As an antiinflammatory signaling molecule, it seems likely that it is tightly regulated dependent on the state of the immune response. There is growing evidence that PPARgamma expression is reduced during inflammation, whereas molecular mechanisms are illdefined. Even though, its role in immunosuppression is getting more definite. Apoptotic cells (AC) provoke an active repression of pro-inflammatory responses inter alia by the inhibition of pro-inflammatory cytokine expression or attenuated generation of reactive oxygen species (ROS). The reduced formation of ROS was attributed to PPARgamma activation, while mechanisms behind the reduced cytokine expression remained unclear. Therefore, my Ph.D. thesis addressed the role of PPARgamma during inhibited cytokine synthesis in response to AC and the regulation of PPARgamma expression during an inflammatory response, which was initiated by lipopolysaccharide (LPS) exposure. In the first part of the thesis, I investigated the role of PPARgamma in coordinating the attenuation of pro-inflammatory cytokine expression in response to AC. Exposing murine RAW264.7 macrophages to AC prior to LPS-stimulation, reduced NFKB transactivation and lowered target gene expression of e.g. TNFalpha and IL-6 compared to controls. In macrophages over-expressing a dominant negative (d/n) mutant of PPARgamma, NFKB transactivation in response to LPS was restored, while using macrophages from myeloid lineage-specific conditional PPARgamma knock-out mice proved that PPARgamma transmitted the anti-inflammatory response delivered by AC. Domain analysis revealed that amino acids 32-250 are essential for inhibition of NFKB. Mutation of a SUMOylation (SUMO: small-ubiquitin related modifier) site in this region (K77R) and interfering SUMOylation by silencing the SUMO E3 ligase PIAS1 (protein inhibitor of activated Stat1) eliminated AC-provoked NFKB inhibition and concomitant TNFalpha expression. Chromatin-immunoprecipitation assays demonstrated that AC prevented the LPS-induced removal of nuclear receptor co-repressor (NCoR) from the KB response element within the TNFalpha promoter. I concluded that AC induce PPARgamma SUMOylation to attenuate the removal of NCoR, thereby blocking transactivation of NFKB. This contributes to an anti-inflammatory phenotype shift in macrophages in response to AC, by lowering pro-inflammatory cytokine production. The second part addressed molecular mechanisms responsible for reduced PPARgamma expression upon LPS exposure. PPARgamma gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer’s disease is associated with impaired PPARgamma expression. Initiation of an inflammatory response by exposing primary human macrophages to LPS revealed a rapid decline of PPARgamma1 expression. PPARgamma1 mRNA decrease was prevented by inhibition of NFKB and also after pre-treatment with the PPARgamma agonist rosiglitazone, suggesting a NFKB-dependent pathway, because activated PPARgamma is known to inhibit NFKB transactivation. Since promoter activities were not affected by LPS, I focused on mRNA stability and noticed a decreased PPARgamma1 mRNA half-life. RNA stability is often regulated via 3’ untranslated regions (UTRs). Therefore, I analyzed the impact of the PPARgamma-3’UTR by luciferase assays. LPS significantly reduced luciferase activity of pGL3-PPARgamma-3’UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion of a potential miR-27a/b binding site within the 3’UTR completely restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, whereas the mature miR-27 mimicked the effect of LPS. MiR-27b was at least partially induced by NFKB, thus correlating with NFKB-dependent PPARgamma1 mRNA decrease. Since deletion of the miR-27 site also containing an AU-rich element (ARE) completely abrogated LPS-induced reduction but inhibition of miR-27b only partially restored PPARgamma1 mRNA expression, I suggested an additional implication of an ARE-binding protein. I provide evidence that LPS induces miR-27b, which in turn destabilizes PPARgamma1 mRNA. Understanding the molecular mechanism of PPARgamma mRNA destabilization, might help to rationalize inflammatory diseases associated with impaired PPARgamma expression. Even though, further experiments are needed to clarify the potential involvement of ARE-binding proteins.
Sepsis is caused by infection and often followed by an overwhelming inflammatory response. This can lead to shock, organ failure and even death. Each year approximately 60,000 people die in Germany due to sepsis. There is good evidence that sepsis is associated with failure of the hypothalamic-pituitary-adrenal-axis. In patients with sepsis, glucocorticoids (e.g. corticosterone, cortisol) released from adrenal glands play an essential role in preventing an excessive pro-inflammatory response. Adrenal insufficiency occurs in a large number of patients with septic shock and is associated with an increased mortality. In the innate immune system, Toll-like receptors (TLRs) play a crucial role in its onset by recognizing pathogenassociated molecules. It is well known that there are interactions between the immune and endocrine stress systems; glucocorticoids and TLRs regulate each other in a bi-directional way. Therefore, a coordinated response of the adrenal and immune system is of vital importance for survival during severe inflammation. This experimental study focuses on the role of TLR-2, TLR-4 and TLR-9 during adrenal stress. The results show that in mice, the absence of TLR-2 and TLR-4, but not TLR-9 leads to altered adrenal morphology, relating to size and cellular structure. However, this alteration does not appear to compromise the phenotype of TLR knock-out mice. Mice deficient of TLR-2, 4 and 9 are not able to respond adequately to inflammatory stress induced by their potential ligands lipopolysaccharide (LPS), lipoteichoic acid (LTA) or cytidine phosphate guanosine-oligodeoxynucleotides (CpG-ODN). This impaired adrenal stress response appears to be associated with a decrease in systemic and intra-adrenal cytokine expressions. Taken together, these results suggest that TLR-2, 4 and 9 are key players in the immuno-endocrine response during inflammation and SIRS. In conclusion, TLRs play a crucial role in the immune-adrenal crosstalk. This close functional relationship needs to be considered in the treatment of inflammatory diseases where an intact adrenal stress response is required. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis
To date it is not clear at which stage of differentiation mature T cell leukaemia/lymphoma is initiated. Previous studies in our group showed that mature T cells are relatively resistant to transformation. We wanted to further investigate the transformation potential of NPM-ALK, p21SNFT and the viral oncoprotein Tax on mature T cells. First, we analyzed the effects on T cell growth in vitro after transducing human T cell lines with gammaretroviral vectors encoding these genes. No growth or proliferation promoting effect of all three genes was observed. In the second part of the project, we transduced murine, mature T cells and/or haematopoietic stem cells (HPCs/HSCs) and transplanted these cells into Rag-1 deficient recipients. All mice transplanted with NPM-ALK transduced monoclonal mature T cells (OT-1) developed leukaemia/lymphoma. In contrast, only few NPM-ALK transduced polyclonal T cell and HPC/HSC transplanted mice developed leukaemia/lymphoma. From the p21SNFT group, only two mice transplanted with transduced OT-1 T cells developed leukaemia/lymphoma, which showed high eGFP and interestingly CD19 expression. No malignancies were observed in Tax transplanted animals so far. Furthermore, the recipients do not show any eGFP marking in the periphery. In conclusion, our results show that compared to polyclonal T cells, monoclonal T cells are transformable after gammaretroviral transfer of NPM-ALK and p21SNFT.
Clinical application of transcranial Doppler for detection of cerebral emboli during cardiac surgery
(2010)
Objective: Neurologic injury is one of the most damaging complications for cardiac surgery. How to decrease neurologic impairment by improving perioperative monitoring remains a challenge for both cardiac surgeons and anesthetists. For this reason, transcranial doppler (TCD) has been widely used in cerebral monitoring during cardiac surgery. In this study, two experiments of clinical application of TCD for detection of cerebral emboli during cardiac surgery were to be done. One was “Solid and gaseous cerebral emboli during valvular surgery are significantly reduced with axillary artery cannulation”. The other was “Do intraoperative cerebral embolic signals differ between valvular surgery (VS) and CABG”. Methods: In experiment one, 20 valve and combined procedures with aortic cannulation (AoC group) were compared to 18 procedures with axillary cannulation (AxC group) in a prospective non-randomized study. In experiment two, 18 VS patients and 18 CABG patients were matched by extracorporeal circulation (ECC) time retrospectively. Intraoperative monitoring of both middle cerebral arteries was performed with TCD discriminating between solid and gaseous embolic signals (ES). Results: In experiment one, the AxC group had less solid ES than the AoC group (38±22 vs 55±25, P<0.05), but no significant difference was found in gaseous (501±271 vs 538±333, P>0.05) and total (539 ± 279 vs 593 ± 350, P>0.05) ES. The AxC group had less solid ES during arterial cannulation (2.1±1.5 vs 6.6±3.6, P<0.05) and during aortic cross-clamp time (4.4 ±3.1 vs 10.2 ± 5.1, P<0.05) than the AoC group. During ECC, gaseous ES was not significantly different between groups (398±210 vs 448±291, P>0.05). However, AxC showed less gaseous ES (85±68 vs 187±148, P<0.05) and less gaseous ES per minute (1.8±1.5 vs 4.5±3.2, P<0.05) during weaning off extracorporeal circulation than the AoC group. No significant difference in gaseous ES (313±163 vs 261±189, P>0.05) and gaseous ES per minute (3.1±2.2 vs 2.8±2.2, P>0.05) was found between groups from bypass start to aortic declamping. No neurologic complications occurred. In experiment two, no significant difference was found in solid (38±20 vs 40±26, P>0.05) or gaseous (457±263 vs 412±157, P>0.05) ES between the VS and CABG group during the whole recording time. During ECC, solid ES (20±10 vs 24±19, P>0.05) and gaseous ES (368±230 vs 317±157, P>0.05) were comparable between groups. Specifically, during weaning off ECC, the VS group had more gaseous ES/min (5.6±3.6 vs 3.1±1.2, P<0.05) than the CABG group. But this difference in gaseous ES/min was not significant during the period from bypass start to aortic declamping (2.5±1.8 vs 3.0±1.8, P>0.05). Conclusion: Cerebral embolization does occur during cardiac surgery. Through these two experiments, we demonstrated the feasibility and importance of clinical application of transcranial doppler for detection of cerebral emboli during cardiac surgery. Due to the diversity in clinical application of TCD, it is impossible to compare the number of ES between different research centers. More unified standards should be drawn in order to make wider clinical application possible. Up till now, no robust evidence shows the correlation between intraoperative ES and postoperative neurological impairment. The research on intraoperative ES and postoperative neurological impairment should rely on a complete concept.
Despite sensible guidelines for the use of opioid analgesics, respiratory depression remains a significant risk with a possibility of fatal outcomes. Clinicians need to find a balance of analgesia with manageable respiratory effects. The ampakine CX717 (Cortex Pharmaceuticals, Irvine, CA, USA), an allosteric enhancer of glutamate-stimulated AMPA receptor activation, has been shown to counteract opioid-induced respiratory depression in rats while preserving opioid-induced analgesia. Adopting a translational approach, we orally administered 1500 mg of CX717 to 16 male healthy volunteers in a placebo controlled double-blind study. Starting 100 min after CX717 or placebo intake, alfentanil was administered by computerized intravenous infusion targeting a plateau of effective alfentanil plasma concentrations of 100 ng/ml. One hour after start of opioid infusion, its effects were antagonized by intravenous injection of 1.6 mg of the classical opioid antidote naloxone. Respiration was quantified prior to drug administration (baseline), during alfentanil infusion and after naloxone administration by (i) counting the spontaneous respiratory frequency at rest and (ii) by employing hypercapnic challenge with CO2 rebreathing that assessed the expiratory volume at a carbon dioxide concentration in the breathable air of 55% (VE55). Pain was quantified at the same time points, immediately after assessment of respiratory parameters, by (i) measuring the tolerance to electrical stimuli (5 Hz sine increased by 0.2 mA/s from 0 to 20 mA and applied via two gold electrodes placed on the medial and lateral side of the mid-phalanx of the right middle finger) and (ii) by measuring the tolerance to heat (increased by 0.3°C/s from 32 to 52.5°C applied to a 3 x 3 cm2 skin area of the left volar forearm, after sensitization with 0.15 g capsaicin cream 0.1%). CX717 was tolerated by all subjects without side effects that would have required medical intervention. We observed that CX717 was approximately as effective as naloxone in reversing the opioid induced reduction of the respiratory frequency. Despite the presence of high plasma alfentanil concentrations, the respiratory frequency decreased only by 8.9 ± 22.4% when CX717 was pre-administered, which was comparable to the 7.0 ± 19.3% decrease observed after administration of naloxone. In contrast, after placebo pre-administration the respiratory rate decreased by 30.0 ± 21.3% (p=0.0054 for CX717 versus placebo). In agreement with this, periods of a very low respiratory frequency of <= 4 min-1 under alfentanil alone were shortened by ampakine pre-dosing by 52.9% (p=0.0182 for CX717 versus placebo). Furthermore, VE55 was decreased during alfentanil infusion by 55.9 ± 16.7% under placebo preadministration but only by 46.0 ± 18.1% under CX717 pre-administration (p=0.017 for CX717 versus placebo). Most importantly, in contrast to naloxone, CX717 had no effect on opioid induced analgesia. Alfentanil increased the pain tolerance to electrical stimuli by 68.7 ± 59.5% with placebo pre-administration. With CX717 pre-administration, the increase of the electrical pain tolerance was similar (54.6 ± 56.7%, p=0.1 for CX717 versus placebo). Similarly, alfentanil increased the heat pain tolerance threshold by 24.6 ± 10.0% with placebo pre-administration. Ampakine co-administration had also no effect on the increase of the heat pain tolerance of the capsaicin-sensitized skin (23.1 ± 8.3%, p=0.46 for CX717 versus placebo). The results of this study allow us to draw the conclusion, that opioid induced ventilatory depression can be selectively antagonized in humans by co-administering an ampakine. This is the first successful translation of a selective antagonism of opioidinduced respiratory depression from animal research into application in humans. Ampakines, namely CX717, thus are the first selective antidote for opioid-induced respiratory depression without loss of analgesia, available for the use in humans.
Summary: Information and communication is critical to the successful management of infectious diseases because an effective communication strategy prevents the surge of anxious patients who have not been genuinely exposed to the pathogen ('low risk patients') affecting medical infrastructures (1) and the future transmission of the infectious agent (2). Surge of low risk patients: The arrival of large numbers of low risk patients at hospitals following an infectious diseases emergency would be problematic for three main reasons. First, it would complicate the situation at hospitals receiving exposed patients, delaying the treatment of the acutely ill, creating difficulties of crowd control and tying up medical resources. Second, for the low risk patients themselves, attending hospital following an infectious disease emergency might increase their risk of exposure to the agent in question. Third, the needs of low risk patients may be poorly attended to at hospitals which are already overstretched dealing with medical casualties. Future transmission: Obtaining early information about symptoms and isolating infected patients is the most effective strategy to interrupt the chain of infection in the public in the absence of specific prophylaxis or treatment. Particularly at the beginning of an outbreak, these nonpharmaceutical interventions play an important role in enabling the early detection of signs or symptoms and in encouraging passengers to adopt appropriate preventive behaviour in order to limit the spread of the disease. This thesis includes two papers dealing with this problem: The first part is a systemic literature review of information needs following an infectious disease emergency (Anthrax, SARS, Pneumonic Plague). The key question was: what are the information needs of the public during an infectious disease emergency? The second part is an empirical investigation of information needs and communication strategies at the airport during the early stage of the Influenza Pandemic. The key question here was: what communication strategies help to meet the information needs and to enable the public to behave appropriately and responsibly? Conclusions: Evidence from the anthrax attacks in the United States suggested that a surge of low risk patients is by no means inevitable. Data from the SARS outbreak illustrated that if hospitals are seen as sources of contagion, many patients with non-bioterrorism related health care needs may delay seeking help. Finally, the events surrounding the Pneumonic Plague outbreak of 1994 in Surat, India, highlighted the need for the public to be kept adequately informed about an incident to avoid creating rumours. Clear, consistent and credible information is key to the successful management of infectious disease outbreaks. The results of the empirical investigation suggested that the desire for information is a reflection of current anxiety and does not mirror the objective scientific assessment of exposure. The airport study showed that perceived information needs were directly related to anxiety – the least anxious did not require any further information, the most anxious reported significant information needs concerning medical treatment, public health management and the assessment of the ongoing situation – irrespective of their actual exposure. A communication strategy only focussing on the 'real' exposed individuals neglects the information needs of those worrying about having contracted the virus and seeking medical attendance. Effective communication strategies should enable the general public to detect early signs or symptoms and provide them with behaviour advice to prevent the further transmission of the infectious agent. These include the provision of clear information about the incident, the symptoms and what to do to prevent the further transmission, detailed and regularly updated information in various media formats (telephone, internet, etc.) and rapid triage at hospital entrances to guide patients to the appropriate medical infrastructures. Relevance: These research findings could contribute to a shift in the organisational and communicative approach responding to infectious diseases outbreaks and could be considered relevant for future risk communication and policy decision making.
Atherosclerosis is accompanied by infiltration of macrophages to the intima of blood vessels. There they engulf oxLDL (oxidized low-density lipoproteins) and differentiate to foam cells. These cells are known as major promoters of atherosclerosis progression. In initial experiments I could demonstrate that foam cell formation caused a severe loss in the ability to produce IFNA (interferon A) in response to stimulation with the bacterial cell wall component LPS (lipopolysaccharide). Since IFNA is discussed to have anti-atherosclerotic potential and has the capability to induce immune tolerance, its inhibition in foam cells might promote the atherosclerotic process. For this reason the aim of my PhD project was to clarify the underlying molecular mechanisms that attenuate LPS-induced IFNA expression in foam cells. LPS activates TLR4 (Toll-like receptor 4) in macrophages. Downstream this receptor two distinct signaling pathways are activated, namely a MyD88 (myeloid differentiation primary response gene 88)-dependent and a TRIF (TIR-domain-containing adapter-inducing IFNA)-dependent one. Foam cell formation targeted the TRIF-dependent TLR4 signaling pathway, as seen by loss of IRF3 activation and IFNA expression inhibition, whereas MyD88-initiated NFBB (nuclear factor 'B-light-chain-enhancer' of activated B-cells) activation and subsequent TNF@ (tumor necrosis factor @) expression remained unaltered. The TRIF signaling cascade results in transactivation of the transcription factor IRF3 (interferon regulatory factor 3), the main activator of IFNA expression. This event demands IRF3 phosphorylation by TBK1 (TANK-binding kinase 1), whereas TBK1 needs to be recruited to TRAF3 (TNF receptor associated factor 3) by the scaffold protein TANK (TRAF family member-associated NFBB activator) for its activation. This work allowed to propose the following scheme: OxLDL utilizes SR-A1 (scavenger receptor A1) to activate IRAK4 (interleukin-1 receptor-associated kinase 4), IRAK1 and Pellino3. Active IRAK1 and Pellino3 associate with TRAF3 and Pellino3 promotes mono-ubiquitination of the adaptor molecule TANK. Mono-ubiquitination of TANK interrupts TBK1 recruitment to TRAF3 and thereby abrogates phosphorylation and transactivation of IRF3 as well as subsequent expression of IFNA. In this study I provide evidence for a negative regulatory role of Pellino3 for TRIF-dependent TLR4 signaling. This expands the current knowledge of the interplay between pathways downstream scavenger and Toll-like receptors. Due to the multifaceted roles of TLR4 signaling in pathology, the new TRIF-signaling inhibitor Pellino3 might be of importance as therapeutical target for disease intervention.
One of the earliest and most striking observations made about HIV is the extensive genetic variation that the virus has within individual hosts, particularly in the hypervariable regions of the env gene which is divided into 5 variable regions (V1-V5) and 5 more constant (C1-C5) regions. HIV evolves at any time over the course of an individual’s infection and infected individuals harbours a population of genetically related but non-identical viruses that are under constant change and ready to adapt to changes in their environment. These genetically heterogeneous populations of closely related genomes are called quasispecies [65]. Tuberculosis or tubercle forming disease is an acute and/or chronic bacterial infection that primarily attacks the lungs, but which may also affect the kidneys, bones, lymph nodes, and brain. The disease is caused by Mycobacterium tuberculosis (MTB), a slow growing rod-shaped, acid fast bacterium. It is transmitted from person to person through inhalation of bacteria-carrying air droplets. Worldwide, one person out of three is infected with Mycobacterium tuberculosis – two billion people in total. TB currently holds the seventh place in the global ranking of causes of death [73]. In 2008, there were an estimated 9.4 (range, 8.9–9.9 million) million incident cases (equivalent to 139 cases per 100 000 population) of TB globally [75]. A complex biological interplay occurs between M. tuberculosis and HIV in coinfected host that results in the worsening of both pathologies. HIV promotes progression of M. tuberculosis either by endogenous reactivation or exogenous reinfection [77, 78] and, the course of HIV-1 infection is accelerated subsequent to the development of TB [80]. Active TB is associated with an increase in intra-patient HIV-1 diversity both systemically and at the infected lung sites [64,122]. The sustainability or reversal of the HIV-1 quasispecies heterogeneity after TB treatment is not known. Tetanus toxoid vaccinated HIV-1 infected patients developed a transient increase in HIV-1 heterogeneity which was reversed after few weeks [121]. Emergence of a heterogeneous HIV-1 population within a patient may be one of the mechanisms to escape strong immune or drug pressure [65,128]. The existence of better fitting and/or immune escape HIV-variants can lead to an increase in HIV-1 replication [129,130]. It might be that TB favourably selected HIV-1 variants which are sources for consistent HIV-1 replication. Understanding the mechanisms underlying the impacts of TB on HIV-1 is essential for the development of effective measures to reduce TB related morbidity and mortality in HIV-1 infected individuals. In the present study we studied whether the increase in HIV-1 quasispecies diversity during active TB is reversed or preserved throughout the course of antituberculous chemotherapy. For this purpose Two time point HIV-1 quasispecies were evaluated by comparing HIV-1 infected patients with active tuberculosis (HIV-1/TB) and HIV-1 infected patients without tuberculosis (HIV-1/non TB). Plasma samples were obtained from the Frankfurt HIV cohort and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty five clones were sequenced from each patient. Various phylogenetic analyses were performed including tree inferences, intra-patient viral diversity and divergence, selective pressure, co-receptor usage prediction and two time point identity of quasispecies comparison using Mantel’s test. We found out from this study that: 1) Active TB sustains HIV-1 quasispecies diversity for longer period 2. Active TB increases the rate of HIV-1 divergence 3) TB might slow down evolution of X4 variants And we concluded that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV- 1 may be predominant for R5 viruses. The use of CCR5-coreceptor inhibitors for HIV-1/TB patients as therapeutic approach needs further investigation.
The pathophysiology of schizophrenia is still poorly understood. Investigating the neurophysiological correlates of cognitive dysfunction with functional neuroimaging techniques such as electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) is widely considered to be a possible solution for this problem. Working memory impairment is one of the most prominent cognitive impairments found in schizophrenia. Working memory can be divided into a number of component processes, encoding, maintenance and retrieval. They appear to be differentially affected in schizophrenia, but little is known about the neurophysiological disturbances which contribute to deficits in these component processes. The aim of this dissertation was to elucidate the neurophysiological underpinnings of the component processes of working memory and their disturbance in schizophrenia. In the first study the the neurophysiological substrates of visual working memory capacity limitations were investigated during encoding, maintenance and retrieval in 12 healthy subjects using event-related fMRI. Subjects had to encode up to four abstract visual shapes and maintain them in working memory for 12 seconds. Afterwards a test stimulus was presented, which matched one of the previously shown shapes in fifty percent of the trials. A bilateral inverted U-shape pattern of BOLD activity with increasing memory load in areas closely linked with selective attention, i.e. the frontal eye fields and areas around the intraparietal sulcus, was observed already during encoding. The increase of the number of stored items from memory load three to memory load four in these regions was negatively correlated with the increase of BOLD activity from memory load three to memory load four. These results point to a crucial role of attentional processes for the limited capacity of working memory. In the second study, the contribution of early perceptual processing deficits during encoding and retrieval to working memory dysfunction was investigated in 17 patients with schizophrenia and 17 healthy control subjects using EEG and event-related fMRI. A slightly modified version of the working memory task used in the fist study was employed. Participants only had to encode and maintain up to three items. In patients the amplitude of the P1 event-related potential was significantly reduced already during encoding in all memory load conditions. Similarly, BOLD activity in early visual areas known to generate the P1 was significantly reduced in patients. In controls, a stronger P1 amplitude increase with increasing memory load predicted better performance. These findings indicate that in addition to later memory related processing stages early visual processing is disturbed in schizophrenia and contributes to working memory dysfunction by impairing the encoding of information. In the third study, which was based on the same data set as the second study, cortical activity and functional connectivity in 17 patients with schizophrenia and 17 to healthy control subjects during the working memory encoding, maintenance and retrieval was investigated using event-related fMRI. Patients had reduced working memory capacity. During encoding activation in the left ventrolateral prefrontal cortex and extrastriate visual cortex was reduced in patients but positively correlated with working memory capacity in controls. During early maintenance patients switched from hyper- to hypoactivation with increasing memory load in a fronto-parietal network which included left dorsolateral prefrontal cortex. During retrieval right ventrolateral prefrontal hyperactivation was correlated with encoding-related hypoactivation of left ventrolateral prefrontal cortex in patients. Cortical dysfunction in patients during encoding and retrieval was accompanied by abnormal functional connectivity between fronto-parietal and visual areas. These findings indicate a primary encoding deficit in patients caused by a dysfunction of prefrontal and visual areas. The findings of these studies suggest that isolating the component processes of working memory leads to more specific markers of cortical dysfunction in schizophrenia, which had been obscured in previous studies. This approach may help to identify more reliable biomarkers and endophenotypes of schizophrenia.
Apoptotic cell (AC)-derived factors alter the physiology of macrophages (M Phi s) towards a regulatory phenotype that is characterized by enhanced production of anti-inflammatory mediators, an attenuated pro-inflammatory cytokine profile and reduced nitric oxide (NO) formation. Impaired NO production in response to ACs or AC-conditioned medium (CM) is facilitated by arginase II (ARG II) expression, which competes with inducible NO synthase for L-arginine. In this study, I investigated the signaling pathway that allowed CM to upregulate ARG II in M Phi s. A sphingolipid, further identified as sphingosine-1-phosphate (S1P), was required but authentic S1P alone only produced small effects. S1P acted synergistically with a so far unidentified factor to elicit high ARG II expression. S1P signaled through S1P receptor 2 (S1P2), since the S1P2-antagonist JTE013 and siRNA knock-down of S1P2 prevented ARG II upregulation. Further, inhibition and knock-down of extracellular signal-regulated kinase 5 (ERK5) attenuated CM-mediated ARG II protein induction. Exploring ERK5-dependent transcriptional regulation, promoter deletion and luciferase reporter analysis of the murine ARG II promoter (mpARG II) suggested the involvement of cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB). This was confirmed by EMSA analysis and decoyoligonucleotides scavenging CREB, thereby preventing it from activating target genes and thus, blocking ARG II expression. I concluded that AC-derived S1P binds to S1P2 and acts synergistically with other factors to activate ERK5 and concomitantly CREB. This signaling cascade shapes an anti-inflammatory M Phi phenotype by ARG II induction. Further investigations of ERK5-dependent CREB activation suggested an indirect mechanism implying that ERK5 inhibited phosphodiesterase 4 (PDE4) and thus, prevented hydrolysis of cAMP. Since S1P-dependent ERK5 activation presumably inhibited PDE4, subsequent cAMP accumulation led to enhanced PKA activity and CREB-mediated transcription. The unidentified factor(s) besides S1P probably provoked the required elevation of cAMP production in M Phi s. Indeed, pharmacological inhibition of cAMP-producing adenylyl cyclase with SQ22536 as well as cAMP-dependent protein kinase A (PKA) with KT5720 suggested cAMP to be involved in CM-mediated ARG II up-regulation. Furthermore, forskolin-dependent activation of adenyly cyclase and simultaneous rolipram-mediated inhibition of PDE4 mimicked CM-induced ARG II expression. Considering these findings, I propose that one or several unidentified factors in CM provoke cAMP production in M Phi s. In parallel, AC-derived S1P activates ERK5, which inhibits PDE4-dependent cAMP hydrolysis, further raising intracellular cAMP levels. Thus, unrestricted continuous cAMP signaling via PKA/CREB, results in a time-dependent and sustained ARG II induction.
The role of gamma oscillatory activity in magnetoencephalogram for auditory memory processing
(2010)
Recent studies have suggested an important role of cortical gamma oscillatory activity (30-100 Hz) as a correlate of encoding, maintaining and retrieving auditory, visual or tactile information in and from memory. It was shown that these cortical stimulus representations were modulated by attention processes. Gamma-band activity (GBA) occurred as an induced response peaking at approximately 200-300 ms after stimulus presentation. Induced cortical responses appear as non-phase-locked activity and are assumed to reflect active cortical processing rather than passive perception. Induced GBA peaking 200-300 ms after stimulus presentation has been assumed to reflect differences between experimental conditions containing various stimuli. By contrast, the relationship between specific oscillatory signals and the representation of individual stimuli has remained unclear. The present study aimed at the identification of such stimulus-specific gamma-band components. We used magnetoencephalography (MEG) to assess gamma activity during an auditory spatial delayed matching-to-sample task. 28 healthy adults were assigned to one of two groups R and L who were presented with only right- or left-lateralized sounds, respectively. Two sample stimuli S1 with lateralization angles of either 15° or 45° deviation from the midsagittal plane were used in each group. Participants had to memorize the lateralization angle of S1 and compare it to a second lateralized sound S2 presented after an 800-ms delay phase. S2 either had the same or a different lateralization angle as S1. After the presentation of S2, subjects had to indicate whether S1 and S2 matched or not. Statistical probability mapping was applied to the signals at sensor level to identify spectral amplitude differences between 15° and 45° stimuli. We found distinct gamma-band components reflecting each sample stimulus with center frequencies ranging between 59 and 72 Hz in different sensors over parieto-occipital cortex contralateral to the side of stimulation. These oscillations showed maximal spectral amplitudes during the middle 200-300 ms of the delay phase and decreased again towards its end. Additionally, we investigated correlations between the activation strength of the gamma-band components and memory task performance. The magnitude of differentiation between oscillatory components representing 'preferred' and 'nonpreferred' stimuli during the final 100 ms of the delay phase correlated positively with task performance. These findings suggest that the observed gamma-band components reflect the activity of neuronal networks tuned to specific auditory spatial stimulus features. The activation of these networks seems to contribute to the maintenance of task-relevant information in short-term memory.
Purpose of the Study: The purpose of the current study was to evaluate the role of radiofrequency (RF) and microwave (MW) ablation in the treatment of pulmonary neoplasms. Materials and Methods: From March 2004 to January 2009, 164 patients (92 males, 72 females; mean age 59.7 years, SD: 10.2) underwent computed tomography (CT)-guided percutaneous RFA of pulmonary malignancies. RFA was performed on 248 lung lesions (20 primary lesions and 228 metastatic lesions) in 248 sessions (one lesion per session). Tumors were pathologically proven and were classified as primary lung neoplasms in 20 patients (non-small cell lung cancer) and as metastatic lung neoplasms in 144 patients. RFA was performed using: a) CelonProSurge bipolar internally cooled applicator b) RITA®StarburstTMXL. From December 2007 to October 2009, 80 patients (30 males, 50 females; mean age 59.7 years, range: 48-68, SD: 6.4) underwent computed tomography (CT) guided percutaneous MW ablation of pulmonary metastases from variable histopathological primaries. MW was performed on 130 lung lesions in 130 sessions (one lesion per session) using Valleylab TM system. Results: The overall success rate of RFA was 67.7% (168/248 lesions), with overall failure rate either due to tumor residue or recurrence on follow up in 32.3% (80/248) with mean time to tumor progress was 5.6 months SD: 2.99 (Range:1-18 months). Complete successful ablation was achieved in patients treated by MWA in 73.1% (95/130 lesions), with failure rate either due to tumor residue or recurrence on follow up in 26.9% (35/130) with mean time to tumor progress 6 months SD: 2.83 (Range:1-12months). Correlation of the histopathological type of the lesion and the end result of ablation therapy revealed insignificant correlation in both RFA and MWA (p > 0.1). The preablation tumor size was one of the most significant factors that determined the end result of ablation. In RFA successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 2.5 cm (110/140) in comparison to lesions of more than 2.5 cm in maximal axial diameter (58/108) (Fisher’s exact test: p < 0.0001). While in MW ablated lesions successful tumor ablation was significant statistically for lesions with maximal axial diameter up to 3 cm (90/110) in comparison to lesions of more than 3 cm in maximal axial diameter (5/20) (Fisher’s exact test: p < 0.001). The location of the lesion was another important factor that determined the end result of ablation. In both RFA and MWA successful ablation was significantly more correlated to peripheral lesions (RFA: 120/160, 80% / MWA: 80/100, 80%) than centrally located lesions (RFA: 48/88, 50%; MWA: 15/30, 50%) (Fisher’s Exact Test: p > 0.001). For successfully RFA ablated cases mean preablation tumor volumes 1.9 cc SD: 0.9 (range: 0.3 - 4.25 cc) while for failed cases the mean tumor volume was 3.7 SD: 2.4 (range: 0.8 – 6.8cc). For successfully MW ablated cases the mean preablation tumor volume: 2.4 cc SD: 2.2 (range: 0.25-8.2 cc) while for failed cases the mean tumor volume was 3.5 SD: 2.6 (range: 0.3 – 7.1 cc). In RFA the survival rates at 12, 24 and 36 months were 90%, 78% and 68% respectively while in MWA treated patients the survival rate within 12 months follow up period was 96% while at 20 month the survival rate was 77%. Complications associated with the ablation therapy were: a) procedure related mortality: 0.4% (1/248) in RFA due to massive pulmonary hemorrhage versus 0% (0/130) in MWA, b) pneumothorax: 11.3% (28/240) in RFA versus 8.5% (11/130) in MWA, c) pulmonary Hemorrhage: 17.7% (44 of 248 sessions) of which one patient had massive uncontrolled bleeding and immediate death versus 6.2% (8/130) in MWA, d) pleural effusion: 3.2 % (8 of 248 sessions) in RFA versus 3.8 % (6/130) in MWA, e) hemoptysis: 4% (10/248) in RFA versus 4.6% (6/130) in MWA ranging from mild tinged sputum to frank bleeding, f) infection: 0.4% (1/248) in RFA, versus 0% in MWA, and g) post ablation pain: 10% (25/248) in RFA versus 9.2% (12/130) in MWA. Pain was generally adequately controlled by analgesics. Conclusion: Radiofrequency and microwave ablation are effective minimally invasive tools and may be safely applied for management of lung malignancy. The success of ablation therapy is significantly correlated to the preablation tumor size, volume and tumor location.
2.1. Background & purpose The recent introduction of new technical innovations such as CT perfusion (CTP) and dual energy CT (DECT) increases the diagnostic abilities of CT for imaging of the head and neck (H&N). The aim of this work was to evaluate the role of CTP and DECT in head and neck imaging. The first part tests whether CTP can differentiate between malignant H&N tumors and surrounding muscle, and discusses the impact of arterial input selection and tumor region of interest (ROI) on CTP of H&N cancer. The second part of the study evaluates radiation dose and image quality of DECT of the H&N. Finally the use of DE derived weighted averaging to improve lesion delineation and image quality is discussed. 2.2. Patients and methods CT perfusion Retrospective analysis of CTP was done for a total number of 55 cases of H&N tumors. Perfusion parameters were calculated for 33 cases of squamous cell carcinoma (SCC) and compared to those of muscles. CTP parameters of 50 cases of H&N tumors calculated using different arterial input functions were compared. CTP was calculated for 28 SCC cases using the single dynamic CT section that shows maximal tumor dimension compared to using average values obtained from all tumor-containing dynamic CT sections. Dual energy CT of head and neck This prospective part of the study was further divided into 2 parts. In the first part 32 consecutive patients underwent DECT of the H&N and were compared to a standard single energy CT (SE) control group. Radiation doses were compared. Weighted-average images from raw data of the 2 DE tubes (weighting factor 0.3 from 80 kVp and 0.7 from 140 KVp) were compared to SE images. Image noise was compared at 5 anatomic levels. Two blinded readers compared subjective overall image quality on a 5-point grading scale. In the second part 35 proved SCC cases underwent DECT of the neck. Pure 140 kVp and 80 kVp image datasets as well as weighted-average images from raw data of the 2 DE tubes at weighting factors 0.3, 0.6, 0.8 (30%, 60% and 80% from 80 kVp raw data respectively) were reconstructed. Objective image noise, contrast to noise ratio (CNR) and subjective image quality were compared between the 5 image datasets. Results CT perfusion Tumor perfusion parameters were significantly higher than those of muscle (p <0.05). Significant high correlation with no significant differences between the means (p >0.05) were observed between perfusion parameters obtained using internal carotid artery (ICA) versus external carotid artery (ECA) and ipsilateral versus contralateral ICA. High correlation was observed between perfusion parameters calculated using one section with maximal tumor dimension and the average of multiple sections. Differences between the means were non significant, p values>0.05. The 95% limits of agreement between repeated measurements using average of multiple sections were slightly narrower for blood volume and permeability than those of repeated measurements using one section. Dual energy CT of head and neck CTDIvol was 12% lower with DE than SECT (p<0.0001). There were no significant differences in objective noise between DECT and SECT at any of the anatomic levels (p >0.05). There were no significant differences between DE- and SECT in attenuation measurements, all p values >0.05. No significant differences in subjective image quality scores were observed between DE- and SECT at any of the 5 anatomic levels (p >0.05). At weighting factor 0.6 the lesion CNR was significantly higher than at weighting factor 0.3 and at pure 140 kVp image dataset (p< 0.0001); while non significantly lower than at weighting factor 0.8 and pure 80 kVp (p=1.00). The 0.6 weighting factor was rated the best at subjective image quality and lesion delineation. 2.4. Conclusion In conclusion; this study demonstrated the ability of CTP to differentiate SCC from surrounding muscle tissue. The choice of arterial input selection has no significant impact on quantitative CTP of H&N tumors. CTP of SCC calculated from one section with maximal tumor dimensions and the average values from multiple sections are not significantly different. The second part of the study showed that DE scanning can be routinely used for H&N imaging; preserving high diagnostic image quality even when the radiation dose was lowered by 12%. Average weighting of DE raw data, with a weighting factor 0.6, results in significant improvement in both tumor delineation and image quality.
Aim: To study the changes in leiomyoma volume following uterine artery embolization (UAE) and to correlate these changes with the initial leiomyoma volume and location within the uterus and to evaluate the impact of preprocedural prediction of the best tube angle obliquity for visualization of the uterine artery origin using 3D-reconstructed contrast-enhanced MR angiography (CE-MRA) on the radiation dose, fluoroscopy time and contrast medium volume used during UAE. Materials and Methods: The study was performed in two parts. The first part was retrospectively done on 28 patients (age range: 37-57 years, mean: 48 years, SD: 4.81) in whom UAE was performed. All leiomyomas in all patients were evaluated. In total, 84 leiomyomas were evaluated. MRI studies were performed before, 3 months and 1 year after UAE. The volumes and location of each leiomyoma in each patient were evaluated in consensus by two radiologists. The second part included 40 consecutive patients (age range: 37-56 years, mean: 46 years, SD: 4.49) and was done in a controlled prospective/retrospective manner. In 20 sample patients (prospective part) pre-procedural prediction of the best tube angle obliquity was predicted using 3D-reconstructed CE-MRA and provided to the interventionalist. 3D-reconstruction was done using Inspace application. The radiation dose, fluoroscopy time and contrast medium volume for those patients were compared with the data of the last 20 procedures (control) performed by the same interventionalist (retrospective part). Results: For the first part the mean pre-embolization volume was 51.6 cm3 range:0.72-371.1cm3, SD=79.3). At 3-month follow-up 83 (98.8%) leiomyomas showed a mean volume reduction of 52.62% (range: 12.79–96.67%, SD=21.85) and 1 leiomyoma (1.2%) increased in volume. At 1-year follow-up 5 (6%) leiomyomas were not detectable, 72 (85.7%) showed a further mean of 20.5% (range: 2.52–58.72%, SD=11.92) volume reduction compared to the 3-month follow-up volume and 7 (8.3%) leiomyomas increased in volume. A statistically significant (p=0.026 at 3-month, p=0.0046 at 1-year) difference in percentage of volume change was observed based on leiomyoma location; submucous leiomyomas showed the largest volume reduction. The initial leiomyoma volume showed a weak negative correlation (Spearman's correlation-coefficient =-0.35 at 3m and -0.36 at 1y) with the leiomyoma volume change. For the second part the tube angle prediction resulted in a significant reduction of the radiation dose utilized (p<0.001), fluoroscopy time (p=0.002) and contrast medium volume (p<0.001) for the sample patients when compared with the control patients. The overall radiation dose was reduced from a mean of 11044 μGym2 to a mean of 4172.5 μGym2, fluoroscopy time was reduced from a mean of 15.45 minutes to 8.81 minutes and contrast medium volume was reduced from a mean of 135 ml to 75 ml. Conclusion: UAE results in significant leiomyoma volume reduction at 3-month and 1- year follow-up. The leiomyoma location plays an important role in volume changes while the initial leiomyoma volume plays a minor role. Pre-procedural prediction of the best tube angle obliquity for visualization of the origin of the uterine artery using 3D-reconstructed CE-MRA results in a significant reduction of the radiation dose, fluoroscopy time and contrast medium volume used during UAE.
Purpose: The aim of this retrospective study is to evaluate the long term implant survival at 5 years, periimplantary conditions and prosthetic maintenance requirements for implant supported mandibular removable dentures retained on only 2 Ankylos® implants placed interforaminally in the mandible and using only conical double crown attachments. Materials and methods: Using the database at the Faculty of Dentistry, University of Frankfurt a selection process was performed to choose patients receiving only 2 Ankylos® implants placed interforaminally in the mandible and using only conical double crown attachments. Implant survival, periimplant condition (periodontal bleeding, plaque index and probing depth), bone loss (from panoramic radiographs) and mobility (using Periotest®) were monitored annually following implant loading. In addition a detailed prosthetic maintenance list was created for each patient based on their yearly checkups and emergency appointments. 37 patients with edentulous mandibles (34 with complete dentures in the upper jaw and 3 with tissue-tooth borne coverdentures) received 2 interforaminal Ankylos® implants (67 in the canine region, 7 in 2nd incisor region). Results: Mean Periotest® values at 5 years (-1.97 ±2.24) were lower than at loading (-1.47 ±2.33). A drop was seen in the Periotest® readings after the first year of loading. The decrease in mean Periotest® values between PTV5 and PTV 1 were not statistically significant (Tukey-Kramer test: p>0.05)
14 patients (37.8%) displayed no resorption at all with an average of 0.801 mm mesially and 0.807mm distally after 5 years. The most increase in bone loss was seen after the first year of loading. There was a gradual increase in bone resorption after the first year of loading. The differences between both distal and mesial bone resorption level at five years and at one year after loading are not significant (Tukey-Kramer test: p<0.05) Plaque and bleeding index values were low at a mean of 0.97 ±0.86 and 0.59 ±0.77 respectively after 5 years of loading. The increase from the first year of loading till the 5th year of loading was significantly higher for plaque measurements but not for bleeding measurements (Tukey-Kramer Test: p<0.05 and p>0.05 respectively). Mean probing depth values were higher after 5 years (2.61 ±0.92 mm) in comparison to the values at loading (2.15 ±0.75 mm). The difference between average values at year 5 and year 1 was statistically significant (Tukey-Kramer test: p<0.05). The most occurring form of maintenance was minor adjustments such as pressure point (15 patient or 40,5%) and relining 11 patients or 29.7%). Teeth breaking off the denture were less common (4 patients or 10.8%). 5 decementations of primary crowns occurred in 4 patients (10.8%) within the 5 year observation time. Other major complications were 4 loose abutments in 3 patients (8.1%), 3 decementations of secondary copings in 3 patients (8.1%) and 1 case (2.7%) in which the prosthetic metal framework fractured. No fracture of abutments or primary crowns occurred during the investigation. Implant survival was 100% percent after 5 years ,1 implants did not fulfil Albrektsson’s success criteria and showed more than 0.2 mm of bone loss per year after the first year of loading with the first year giving a success rate of 98.8%. Conclusion: In conclusion this study has demonstrated that patients have a wider variety of options when it comes to choosing a reliable prosthesis in the lower jaw. Patients with financial limitations can be provided with a reliable prosthetic option using removable dentures retained by conical double crown attachments on 2 implants. The requirements for such a construction are a mechanically stable implant system and a mechanically stable framework. When these prerequirments are fulfilled, the patient can be satisfied with a prosthesis of superior quality to other attachment types and the dentist can rely on the fact that frequent maintenance which costs time and money can be eliminated or at least reduced. Through further innovation this type of construction can also reach patients who are lower down on the economic scale such as elderly patients and retirees.
Recent data indicate that reactive oxygen species (ROS) are produced in the nociceptive system during persistent pain and contribute to pain sensitization. Aim of this study was to investigate potential antinociceptive effects of ROS scavengers in different animal models of pain. Intrathecal injection of ROS scavengers 1-Oxyl-2,2,6,6-tetramethyl -4-hydroxypiperidine (TEMPOL) or Phenyl-N-tert-butylnitrone (PBN) significantly inhibited formalin-induced nociceptive behavior in mice, suggesting that ROS released in the spinal cord are involved in nociceptive processing. Formalin-induced nociceptive behavior was also inhibited by intraperitoneal injection of a combination of vitamin C and vitamin E, but not of vitamin C or vitamin E alone. Moreover, the combination of vitamin C and E dose-dependently attenuated mechanical allodynia in the spared nerve injury (SNI) model of neuropathic pain. The SNI-induced mechanical allodynia was also reduced after intrathecal injection of the combination of vitamin C and E, and western blot analyses revealed that vitamin C and E treatment can ameliorate the activation of p38 MAPK in the spinal cord and in DRGs. These data suggest that a combination of vitamin C and E can inhibit the nociceptive behavior in animal models of pain, and points to a role of the spinal cord as an important area of ROS production during nociceptive processing.
Working memory (WM) contributes to countless activities during everyday live: reading, holding a conversation, making tea and so on. The core processes of WM comprise the phases of encoding, maintenance and retrieval. Successful recognition of stored objects requires several subprocesses such as stimulus encoding and evaluation, memory search and the organisation of a decision and a response. Much research has focused on encoding and maintenance of information but little interest has been directed to the retrieval of information. This is why the present dissertation investigated the neuronal correlates of retrieval of previously stored information and its modulation by load and probe-item similarity.
Here memory load and probe-item similarity were manipulated in order to investigate the neuronal correlates of the recognition process using electroencephalography (EEG). We tested the hypothesis recognition is influenced differently by probe-item similarity and by memory load and that these factors are re Effected by distinct neuronal correlates. Furthermore we tested whether distinct neuronal responses could be related to a summed similarity model.
The analysis of high-density ERP recordings showed both a load effect (load 1>load 3) and a similarity effect In addition, there was an interaction between load and similarity. The load effect was present during the whole epoch and did not change over time, whereas the similarity effect showed two distinct components between 300-600ms. In contrast to the load effect the similarity effect changed its sign over time. For the rest component, match probes elicited the strongest ERP responses, whereas for the second component dissimilar probes yielded the strongest ERP responses. The timing of the similarity effect corresponded well with the early and late P3b complex. The P3b complex is associated with stimulus categorisation and evaluation (early subcomponent) and memory search and criterion testing (late subcomponent).
The results suggest that the difficulty of a task is not only determined by load but also enhanced by probe-item similarity. Since increasing the number of samples (i.e. memory load) can also increase the probe-item similarity (i.e. the probability that one of the samples is perceptually similar to the probe), an independent manipulation of both factors is indispensable to disentangle their particular impact on short-term recognition. Furthermore, I propose that the two distinct neural correlates of the P3b complex reeffects different stages of task processing connected with probe-item similarity. As suggested by summed similarity VI models, these components might reflect the subprocesses of similarity summation (early P3b) and criterion testing (late P3b).
NK cells are part of the innate immune system, and are important players in the body’s first defence line against virus-infected and malignantly transformed cells. While T cells recognize neoplastic cells in an MHC-restricted fashion, NK cells do not require prior sensitization and education about the target. In leukemia and lymphoma patients undergoing allogeneic hematopoietic stem cell transplantation not only T cells but also NK cells have been found to mediate potent graft-versus-tumor effects. Hence, autologous or donor-derived NK cells hold great promise for cancer immunotherapy. Since the generation of highly purified NK cell products for clinical applications is labor-intensive and time consuming, established human NK cell lines such as NK-92 are also being considered for clinical protocols. NK-92 cells display phenotypic and functional characteristics similar to activated primary NK cells. While NK-92 cells are highly cytotoxic towards malignant cells of hematologic origin, they do not affect healthy human tissues. NK-92 cells can be expanded under GMP-compliant conditions, and can therefore be provided in sufficient numbers with defined phenotypic characteristics for clinical applications. Safety of NK-92 cells for adoptive immunotherapy was already shown in two phase I/II clinical trials...
The role of peroxisome proliferator-activated receptor gamma during sepsis-induced lymphopenia
(2011)
Sepsis is one of the most common diseases on intensive care units all over the world and accounts there for the highest mortality rate. One of the hallmarks of sepsis is an accelerated T-cell apoptosis, resulting in a compromised immune state with the inability to eradicate pathogens. This promotes organ damage or even organ failure. A multiple organ dysfunction evolves, which often ends up in septic shock and death. Recently, it was shown that severe T-cell depletion correlates with sepsis mortality. When inhibiting T-cell apoptosis, an increased mouse survival was observed in experimental sepsis. ...
Identification of translationally deregulated proteins during inflammation-associated tumorigenesis
(2012)
The translation of mRNAs into proteins is an elaborate and highly regulated process. Translational regulation primarily takes place at the level of initiation. During initation the eukaryotic initiation factors (eIFs) form a complex that binds to the 5’end of the mRNA to scan for a start codon. Once recognized, the ribosome is recruited to the mRNA and protein synthesis starts. Initiation of translation can basically occur via two distinct mechanisms, i.e. cap-dependent and cap-independent that is mediated via internal ribosome entry sites (IRESs). The former is mediated by a 5’cap structure composed of a 7-methylguanylate which is added to every mRNA during transcription and recruits the initiation complex. IRES-dependent translation involves elements within the 5’untranslated region (UTR) of the mRNA that mostly bind IRES trans-acting factors (ITAFs) which associate either with the initiation complex or with the ribosome itself and consequently allow for internal initiation of translation.
During tumorigenesis the demand for proteins is increased due to rapid cell growth, which consequently requires enhanced translation. Many factors that regulate translation are overexpressed in tumors. Moreover, signaling pathways that trigger translation or further hyperactivated by the surrounding tumor microenvironment. This environment is largely generated by infiltration of immune cells such as macrophages that secrete cytokines and other mediators to promote tumorigenesis. As the effects of inflammatory conditions on the translation of specific targets are only poorly characterized, my study aimed at identifying translationally deregulated targets during inflammation-associated tumorigenesis.
For this purpose, I cocultured MCF7 breast tumor cells with conditioned medium of activated monocyte-derived U937 macrophages (CM). Polysome profiling and microarray analysis identified 42 targets to be regulated at the level of translation. The results were validated by quantitative PCR and one target - early growth response 2 (EGR2) - was chosen for in depth analysis of the mechanism leading to its enhanced translation.
In order to identify upstream signaling molecules causing enhanced EGR2 protein synthesis the cytokine profile of CM was analyzed and the impact of several cytokines on EGR2 translation was examined. Preincubation of CM with neutralizing antibodies revealed that lowering interleukin 6 (IL-6) had only little effect, whereas depletion of IL 1β significantly reduced EGR2 translation. This finding was corroborated by the fact that treatment with recombinant IL-1β enhanced EGR2 translation to virtually the same extend as CM. Further experiments revealed that this effect was mediated via the p38-MAPK signaling cascade.
Interestingly, I observed that the mTOR inhibitor rapamycin, which reduces cap-dependent translation, specifically stimulated EGR2 translation. This result argued for an IRES-dependent mechanism that might account for EGR2 translation. The use of bicistronic reporter assays verified this hypothesis. In line with the above mentioned results, CM, IL-1β and p38-MAPK induced EGR2-IRES activity.
Since IRESs commonly require ITAFs to mediate translation initiation, the binding of proteins to the 5’UTR was analyzed using mass spectrometry. Among others, several previously described ITAFs, such as polypyrimidine tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) were identified to directly bind to the EGR2-5’UTR. Furthermore, overexpression of hnRNP-A1 enhanced EGR2-IRES activity whereas a dominant negative form of hnRNP-A1 significantly decreased it, thus, showing its importance for EGR2 translation.
In summary, my data provide evidence that EGR2 expression can be controlled by IRES-dependent translational regulation, which is responsive to an inflammatory environment. The identified mechanism may not be exclusive for one target but might be representative for gene expression regulation mechanisms during tumorigenesis. This is of special interest for the treatment of cancer patients and development of more specific therapies to reduce tumor outcome.
1 Purpose of the Study:
The purpose of this retrospective study was to assess the volumetric changes of our institutional pediatric neuroblastoma in response to various therapeutic protocols.
2 Materials and Methods:
A retrospective study was conducted on children with neuroblastoma from different anatomical locations including suprarenal, paraspinal, pelvic, mediastinal and cervical neuroblastoma primaries. These children underwent tumor-stage based therapeutic protocols in Johann Wolfgang Goethe University Hospital, Frankfurt am Main, Germany, between January 1996 and July 2008. The study included 72 patients (44 males and 28 females). Patient demographics (age and gender), disease-related symptoms, laboratory results (tumor biomarkers including ferritin, neuron specific enolase, and urine catecholamine) and histopathological reports were collected from the electronic medical archiving system and subsequently analyzed.
Patients were classified into following groups according the anatomical origin of the primary neuroblastoma into:
1) Suprarenal neuroblastoma Group: This group included patients with neuroblastoma arising from the suprarenal gland. This group composed of 54 patients with male to female ratio (32:22).
2) Paravertebral neuroblastoma Group: This group composed of 6 male patients.
3) Mediastinal neuroblastoma Group: This group included patients with mediastinal neuroblastoma and composed of 3 patients (1 male and 2 females).
4) Pelvic neuroblastoma Group: This group included patients with pelvic neuroblastoma and composed of 6 patients (3 males and 3 females).
5) Cervical neuroblastoma Group: This group included patients with cervical neuroblastoma and composed of 2 male patients.
3 Results:
The mean volume of all suprarenal neuroblastoma group involved in the study before therapy was 176.62 cm3 (SD: 234.15) range: 239.4-968.9cm3. The mean initial volume of all suprarenal neuroblastoma group who underwent observation protocol was 86.0378 cm3 (SD: 114.44) range: 5.2-347.94cm3. Volumetric evaluation of suprarenal neuroblastoma following observation (Wait and See) protocol revealed continuous reduction of the tumor volumes in a statistically significant manner during the follow up periods up to 12 months with p value of less than 0.05. The volumetric changes afterwards were statistically insignificant.
The mean initial volume of all suprarenal neuroblastoma group who underwent primary surgery protocol was 42.4 cm3 (SD: 28.5) range: 7.5-90cm3. Complete surgical resection of the tumor was not feasible in all lesions due to local tumor extension and / or infiltration with the associated risk of injury of nearby organs or structures. However statistical analysis of the volumetric changes in the successive follow up periods did not reveal statistical significance.
Volumetric estimation of the tumor in the subsequent follow up periods revealed significant changes within the period first (3-9 month periods). The changes afterwards were statistically non significant. On the other hand, the mean initial volume of all suprarenal neuroblastoma group who underwent combined chemotherapy and Stem cell transplantation protocol only without surgical interference was 99.98cm3 (SD:46.2) range: 48.48-160.48 cm3. In this group the volumetric changes were variable and difference in volumes in follow up was statistically non significant during the follow up period.
The mean initial volume of all abdominal paravertebral neuroblastoma group was 249.197cm3 (SD: 249.63) range: 9.6-934cm3. The mean initial volume of all pelvic neuroblastoma group was 118.88cm3 (SD: 50.61) range: 73.4-173.4cm3. The mean initial volume of all mediastinal neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The mean initial volume of all cervical neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The volumetric measurements in the corresponding follow up periods according to the therapeutic protocol of abdominal paravertebral neuroblastoma, pelvic neuroblastoma, mediastinal and cervical neuroblastoma revealed significant change in the tumor volume within the early 3-6 months from the initial therapy while subsequently the tumor volumetric changes were statistically non significant.
4 Conclusion:
In conclusion, the role of MRI volumetry in the evaluation of tumor response is dependent on the risk adapted concept of neuroblastoma with the combination of different imaging modalities as well the therapeutic protocol. MRI Volumetry in addition to new protocols such as Whole-body imaging and 3D visualization techniques are gaining more importance and acceptance.
Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.
Aim: The aim of this study was to measure cortico-cortical connectivity in multiple sclerosis (MS) patients by TMS-evoked potential (TEP) latencies in EEG evoked by transcranial magnetic stimulation (TMS) of the hand area of the primary motor cortex of one hemisphere. TEPs were recorded on the stimulated- and at the homologue site in the non-stimulated contralateral hemisphere. Both interhemispheric directions were tested. Interhemispheric latencies of the two main reproducible TEPs, the positive component at 60 ms and the negative component at 100 ms (P60 and N100, respectively), were expected to be significantly prolonged in MS-patients compared to healthy volunteers.
Material and methods: The study compared interhemispheric propagation of P60 and N100 in groups of 12 patients with early-stage relapsing-remitting MS (RRMS) and 16 age- and gender-matched healthy controls. The study was approved by the Ethics Committee of the Medical Faculty of the Goethe-University of Frankfurt/Main and conformed to the latest revision of the Declaration of Helsinki of 2008. TEPs were recorded by means of EEG and their latencies were statistically evaluated in 10 channels around the stimulation site and in 10 corresponding electrodes in the non-stimulated contralateral hemisphere. Interhemispheric conduction time was calculated by the difference of TEP latency in non-stimulated vs. stimulated hemisphere.
Results: An ANOVA on interhemispheric conduction time showed a significant prolongation for the N100 from left to right hemisphere in MS compared to controls, while no group differences were found for the P60 and the N100 from right to left hemisphere.
Conclusion: The results provide first evidence that the N100 may constitute an interesting marker to measure interhemispheric conduction delays in early-stage RRMS. The specificity of the present finding and its relation to fiber tract pathology should be examined in further correlative analyses with diffusion tensor imaging and other structural MRI data.
Smoking tobacco throughout pregnancy is one of the single most important avoidable causes of adverse pregnancy outcomes. If compared with other risk factors in the perinatal period, exposure to tobacco smoke is considered to be amongst the most harmful. It is associated with high rates of long and short term morbidity and mortality for mother and child. Despite this importance until now a scientometric analysis about the development and the state of scientific knowledge about smoking and pregnancy has not been published. In order to close this gap this work was conceived. In this dissertation quantitative and qualitative data on this topic was analyzed using a variety of objective scientometric methods like the number of scientific contributions, the number of citations and the modified Hirsch-index (H-index). A collective volume of 10,043 entries covering a time period from 1900 to December 5, 2012 was obtained from the Web of Science (WoS) data base. Publishing activities of authors, institutions and countries, their cooperation, reception within the international scientific community and its reactions were interpreted and illustrated.
Myofacial Pain is the most common form of temporomandibular disorders (TMD), affecting principally women in reproductive age. The etiology of TMD is still controversial. Currently a multifactorial theory has received a great support among the scientific community. This theory draws attention to the interaction of psychological, neuromuscular and oral pathogenic factors. Objectives: to describe the possible etiological factors of the Myofacial Pain; and to evaluate the effectiveness of the current treatments for Myofacial Pain. Materials and methods: a narrative review of the etiological factors and epidemiological data of Myofacial Pain introduces this work. Thereafter the author presents five systematic reviews of RCTs which have been published during the last thirteen years (1999-2012) for the use of acupuncture, low level laser therapy, drugs, physiotherapeutical interventions, splint therapy, and psychosocial interventions in the treatment of Myofacial Pain. Moreover, the author reports a systematic review and meta-analysis of all the available literature of two modern approaches for the treatment of Myofacial Pain. A comparison between the “usual treatment” based on splint therapy and psychosocial interventions was conducted. Results: the author did not find sufficient evidence to support therapies based on one single intervention. However, the condition of the patients with myofacial pain could be treated more effectively with combined treatments. After comparing “usual treatment” with psychosocial interventions, the author observed a tendency of the latter to improve psychological outcomes, whereas the first one was slightly more effective to enhance clinical functional outcomes. In general, a high level of heterogeneity was observed among the included studies of the different systematic reviews. The quality of the studies is susceptible to be improved. Clinical implications: the author proposes core outcomes to be implemented within the research on myofacial pain in particular and temporomandibular disorders in general, in order to enable scientifical comparisons between different therapies.
Background: Alzheimer’s Disease (AD) is the most common form of dementia and one of the major diseases of old age, causing the impairment of cognitive functions. This disease does not only confront society with financial issues, but also puts severe stress on individuals suffering from AD and their relatives alike. One of the possible symptoms, commonly described in AD, is the impairment of learning as well as the recognition of face-name associations. Beginning at age 60, the chance to develop AD grows exponentially with increasing age, making age a major risk factor. Additionally, the e4 allele of the apolipoprotein E (APOE) polymorphism has been associated with the risk of developing AD when compared to the more common e3 allele. While strong evidence shows a stronger decline in cognitive function with rising age for e4 carriers, some studies demonstrated better cognitive function in e4 carriers at a young age.
This led to the postulation of the hypothesis of antagonistic pleiotropy of the APOE gene, wherein the e4 allele may benefit cognitive function in young carriers, yet leads to a faster decline at a later point in life, encouraging the development of cognitive dysfunction such as AD. Several functional magnetic resonance imaging (fMRI) studies, examining functional activation patterns, found APOE-related differences in key areas of episodic memory, such as the hippocampus, where e4 carriers show aberrant activation similar to AD patients. However, associative memory (encoding and retrieval of face name pairs) has not been well examined for APOE-related differences. Interaction effects of age and the APOE genotype, such as those postulated by the hypothesis of antagonistic pleiotropy, have not been addressed in face-name association tasks either.
Leading Question: Is it possible to detect interaction effects between age and APOE genotype on cognitive performance or neuronal activation patterns in healthy young and old participants during an fMRI face-name association task, supporting the hypothesis of antagonistic pleiotropy of the APOE genotype?
Methods: Participants were stratied by age, and APOE e4 carriers were randomly matched with homozygous e3 carriers. Neuropsychological examination (CVLT and CERAD) was administered. Participants underwent structural MRI analysis via voxelbased morphometry (VBM) as well as fMRI imaging during a face-name association task.
Results: Apart from strong age-related effects in cognitive function detected during neuropsychological testing, the behavioral data from the face-name association task as well as the structural MRI analysis did not show an association with the APOE genotype. Nevertheless, analysis of functional MRI data showed age- as well as APOE-dependent effects on activation patterns for the encoding and retrieval of face-name pairs, in absence of differences in cognitive performance. Further analysis showed eight clusters of significant age X APOE genotype interactions in areas previously associated with working and visual associative memory, including the fusiform gyri bilaterally. These interactions show different patterns, whereas a relative hypoactivation of young e4 carriers together with a hyperactivation of old e4 carriers is the most prominent.
Conclusions: With regard to the leading question, this study successfully found age X APOE interactions in a face-name pair retrieval task, although no interaction effects were present in the encoding task, structural analysis, or cognitive performance. The agemediated effect of the APOE e4 allele on functional activation patterns may be explained by the compensatory hypothesis, describing a relative hyperactivation of old e4 carriers as compensatory, and interpreting a relative hypoactivation of younger e4 participants as reduced effort to achieve the same cognitive performance as non carriers.
These findings present further evidence of an antagonistic pleiotropy of the APOE genotype, showing age-dependent effects of the e4 allele even in healthy carriers. Nevertheless, previously described differences in cognitive performance and brain structure, even in young participants, were not found. On the contrary, functional MRI analysis showed APOE-related differences in young and old participants, suggesting that this modality may be more sensitive in detecting APOE-mediated changes. Among the clusters, demonstrating an interaction effect, the fusiform gyri were most prominent, which might be due to its important role in visual associative memory. As previous studies indicate an early and strong involvement of this area due to AD pathology, this interaction effect of age and APOE genotype in healthy participants underlines the importance of this region in the development of AD, and should be the focus of further research. However, this research is also required to determine, how exactly the APOE genotype influences brain function in healthy humans, and to clarify its relationship to pathological processes facilitating the development of AD.
The small leucine-rich proteoglycan biglycan (Bgn) is a part of the extracellular matrix providing structure and enhancing fibril stability. In its soluble form, biglycan is able to bind and signal via the innate immune receptors Toll-like receptor (TLR) 2 and 4, thereby activating MAP-kinases and the NF-κB pathway. In macrophages soluble biglycan induces the secretion of several cytokines and chemokines, including TNF-α, CCL2, CXCL5 and CXCL13. A unique feature of biglycan is its ability to stimulate the secretion of mature IL-1β. By orchestrating TLR2 and 4 with the purinergic P2X4 and P2X7 receptor signalling biglycan triggers the activation of the NLRP3/ASC inflammasome, which in turn activates caspase-1 to cleave pro-IL-1β to mature IL-1β. Furthermore, in several inflammatory diseases an upregulated biglycan expression is found. Enhanced levels of biglycan could be measured in plasma and inflamed tissue. In mouse models of sepsis, lupus nephritis and renal ischemic reperfusion injury, biglycan-deficiency improved the disease outcome. Overexpression of soluble biglycan on the other hand increased immune cell infiltration into the kidney by inducing cytokine and chemokine expression in a TLR2/4-dependent manner. These studies emphasise its importance in inflammatory processes, especially in the kidney. Furthermore, the pro-inflammatory effects on macrophages and diseases established biglycan as a danger signalling molecule, yet its role as a soluble molecule in plasma was not further investigated.
Although an increase of soluble biglycan in the circulation could be seen in several inflammatory diseases, the source is not fully unravelled. Previously it could be shown that macrophages and dendritic cells secrete soluble biglycan after stimulation with IL-6 and TGF-β1. However, since these cell are resident in organs and do not circulate in the blood stream their contribution to soluble biglycan levels in plasma is likely minor. Therefore, monocytes as precursor of both macrophages and dendritic cells were investigated as a possible source of circulating biglycan. Analysis of blood from septic patients revealed elevated soluble biglycan levels as well as an increased number of monocytes. Isolated monocytes from healthy volunteers incubated with the inflammatory cytokines IL-1β, IL-6 and TGF-β1 displayed increased biglycan mRNA expression and secretion of soluble biglycan into the supernatant, revealing monocytes as a producer of soluble biglycan in blood. Therefore this work was directed to further investigate the influence of soluble biglycan on circulating monocytes, with regard to sepsis.
Monocytes can be classified into three subtypes, while the classical monocytes express CD14 (CD14++CD16low), intermediate monocytes express both CD14 and CD16 (CD14++CD16+) and non-classical monocytes express mainly CD16 (CD14lowCD16++). The intermediate and non-classical monocytes make up about 10 % of all monocytes and are referred to as CD16-positive subtypes. The CD16-positive monocytes express higher levels of TNF-α and IL-1β upon stimulation and display different migration behaviour. In most inflammatory diseases an expansion of CD16-positive monocytes is observed, especially an increased number of intermediate monocytes frequently correlate with disease severity and mortality. Since septic patients had increased circulating biglycan levels and augmented CD16-positive monocytes, a possible correlation between these two parameters was investigated. Using FACS analysis of biglycan-stimulated monocytes from healthy donors revealed a significant shift from classical to intermediate and non-classical monocytes. This shift was mediated by increased expression of CD14 and CD16 on mRNA and protein levels upon biglycan treatment. Furthermore, biglycan induced the mRNA expression of the adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in CD14-positive monocytes. Four hours after biglycan stimulation an increased ICAM-1 protein expression on the cell surface of classical and intermediate monocytes was observed. Additionally, biglycan-treated CD14-positive monocytes rolled and attached to pre-stimulated endothelial cells to a greater extent compared to untreated monocytes. This demonstrates that biglycan not only triggers the expression of CD14 and CD16 but also induces a functional shift of monocytes. ...
Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Defects in flow-induced NO formation go along with endothelial dysfunction, initiation and progression of atherosclerosis as well as with arterial hypertension. Previous work has identified several mechanotransducing signaling processes involved in fluid shear stress-induced endothelial effects. But how fluid shear stress initiates the response is poorly understood. Here, I show in human and bovine endothelial cells that the G-protein Gq/G11 and the purinergic receptor P2Y2 mediate fluid shear stress-induced endothelial responses such as Ca2+ release, nitric oxide (NO) formation and the phosphorylation of platelet-endothelial-cell-adhesion-molecule-1 (PECAM-1), vascular endothelial growth factor-2 (VEGFR-2) and Akt kinase as well as activation of the endothelial NO synthase (eNOS). P2Y2 receptor is activated by adenosine triphosphate (ATP) which is released from endothelial cells under the influence of fluid shear stress. Arteries with P2Y2 or Gαq/Gα11 deficiency have impaired flow-induced dilatation. Mice with induced endothelium-specific deficiency of P2Y2 or Gαq/Gα11 develop hypertension which is accompanied by reduced eNOS activation. My data identify P2Y2 and Gq/G11 as a critical endothelial mechano-signaling pathway located upstream of mechanotransducing processes described so far. Moreover, I demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone and blood pressure.
The Hepatitis C virus (HCV) infects more than 170 million individuals worldwide and causes challenging HCV-related diseases. Unfortunately, there is no vaccine available. Therefore, a better understanding of the HCV life cycle is urgently needed to develop more effective and better tolerated therapies.
It has been reported that the secretory pathway plays an essential role for the release of HCV, and the SNARE complexes are a central factor controlling intracellular vesicular trafficking. Recently, our group observed that α-taxilin that binds to free syntaxin 4 prevents the SNARE complex formation and exerts an inhibitory effect on the release of HCV particles. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 is involved in the HCV life cycle.
An increased intracellular amount of syntaxin 4 was found in HCV-positive cells, while the level of syntaxin 4-specific transcripts was decreased as observed in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes (PHH). Since in HCV-positive cells a significant longer half-life of syntaxin 4 was found, the decreased expression is overcompensated, leading to the elevated amount of syntaxin 4. Overexpression of syntaxin 4 increases the amount of secreted infectious viral particles, while silencing of syntaxin 4 expression decreases the number of released viral particles, which indicates that HCV could use the SNARE-dependent secretory pathway for viral release. Confocal immunofluorescence microscopy and co-immunoprecipitation experiments revealed that syntaxin 4 interacts with HCV core and NS5A. To identify the binding domain, various mutants of syntaxin 4 were generated. Based on these mutants, it was found that the H3 domain of syntaxin 4 interacts with core. These data show that the t-SNARE protein syntaxin 4 is an essential cellular factor for HCV morphogenesis and secretion.
HCV induces autophagy, and in HCV-infected cells a major fraction of the de novo synthesized viral particles is not released but intracellularly degraded. Syntaxin 17 is an autophagosomal SNARE required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Therefore, we aim to investigate whether syntaxin 17 is a relevant factor for the HCV life cycle by regulating the fusion between autophagosomes and lysosomes. It was found that HCV-positive cells possess a decreased amount of syntaxin 17, and HCV reduces the intracellular level of syntaxin 17 by NS5A-mediated interruption of c-Raf signaling, which triggers the syntaxin 17 transcription, and by HCV-dependently induced autophagy. Overexpression of syntaxin 17 decreases the intracellular amount of viral particles and reduces the number of released infectious viral particles by favoring the formation of autolysosomes, in which HCV particles can be degraded. Vice versa, inhibition of syntaxin 17 expression by specific siRNAs results in an elevated amount of intracellular viral particles and increases the number of released viral particles by impaired autophagosome-lysosome fusion. Confocal immunofluorescence microscopy analyses show a fraction of core protein in autophagosomes as stained by lysotracker and the autophagy maker p62. These data identify syntaxin 17 as a novel factor controlling the release of HCV and reveal the autophagosome-autolysosome fusion as an essential step affecting the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular viral particles.
Taken together, these data identify the t-SNARE proteins syntaxin 4 and syntaxin 17 as essential cellular factors for HCV morphogenesis and secretion.
NOSTRIN belongs to the family of F-BAR proteins, which are multi-domain adaptor proteins that have emerged as important regulators of membrane remodeling and actin dynamics in a variety of vital cellular processes. They have been analyzed structurally and biochemically and overexpression studies have revealed their potential in inducing membrane curvature and tubulation. Several studies have begun to decipher the function of individual proteins, but the understanding of F-BAR protein functions in vivo is still quite limited. The F-BAR protein NOSTRIN is mainly expressed in endothelial cells and has originally been described as interaction partner of the endothelial nitric oxide synthase (eNOS), modulating eNOS subcellular localization. The phenotypic characterization of NOSTRIN knockout mice revealed decreased nitric oxide (NO) and cGMP levels, an increase in systolic blood pressure and an impairment of the acetylcholine-induced, NO-dependent relaxation of aortic rings from mice with global as well as endothelial cell-specific knockout of the NOSTRIN gene (ECKO) . These findings implied that NOSTRIN plays a role in regulating NO production in vivo, but the underlying molecular mechanisms were unclear. Therefore, this study was aimed at addressing the mechanism causing the inhibited vasodilation specifically upon stimulation with acetylcholine in NOSTRIN KO and ECKO mice, and at exploring additional roles of NOSTRIN in the signal transduction of endothelial cells.
The major acetylcholine receptor that mediates vessel relaxation upon stimulation with acetylcholine is the muscarinic acetylcholine receptor subtype M3 (M3R). In the present study NOSTRIN was identified as novel interaction partner of the M3R and important factor for the correct spatial distribution and functionality of the M3R. Moreover, it provides the first example of an F-BAR protein regulating a GPCR. Confocal immunofluorescence microscopy analysis of isolated aortae from NOSTRIN KO and WT mice indicated that NOSTRIN was necessary for the proper subcellular localization of the M3R and targeted it to the plasma membrane. A series of pulldown experiments revealed a direct interaction of NOSTRIN with the M3R. The binding required the SH3 domain of NOSTRIN and the third intracellular loop of the M3R, which has a recognized role in receptor regulation. The interaction of NOSTRIN with the M3R was confirmed by co-localization of NOSTRIN and the M3R upon overexpression in mammalian cells. Expression levels of the M3R as well as eNOS were not affected by the loss of NOSTRIN in accordance with the finding, that NOSTRIN impacts on the acetylcholine/eNOS signaling axis through regulation of the subcellular trafficking of its binding partners.
Furthermore, there were first indications for a role of NOSTRIN in facilitating the carbachol-induced calcium response in M3R-expressing cells, suggesting that NOSTRIN might influence M3R activation. in the absence of NOSTRIN, the function of the M3R in mammalian cells overexpressing the M3R was markedly impaired, resulting in abolition of the calcium response to the M3R agonist carbachol. In accordance, the activated eNOS fraction associated with the Golgi complex was markedly reduced in aorta explants from NOSTRIN knockout and ECKO mice. Moreover, NOSTRIN knockout inhibited the carbachol-induced, activating phosphorylation of eNOS in murine aortae as well as primary mouse lung endothelial cells confirming its role as important regulator of eNOS activity in vivo.
Background
Cochlear Implants (CIs) provide near normal speech intelligibility in quiet environments to individuals suffering from sensorineural hearing loss. Perception of speech in situations with competing background noise and especially music appraisal however are still insufficient. Hence, improving speech perception in ambient noise and music intelligibility is a core challenge in CI research. Quantitatively assessing music intelligibility is a demanding task due to its inherently subjective nature. However, previous approaches have related electrophysiological measurements to speech intelligibility, a corresponding relation to music intelligibility, can be assumed. Recent studies have investigated the relation between results obtained from hearing performance tests and Spread of Excitations (SoEs) measurements. SoE functions are acquired by measuring Electrically Evoked Compound Action Potentials (ECAPs) which represent the electrical response generated in the neural structures of the auditory nerve. The parameters designed to describe SoE functions are used to estimate the dispersal of the electric field in the cochlea. The quality of spatial separation of the electrical field generated by adjacent electrodes are assumed to correlate with hearing performance measures.
Aim of study
This study investigated the relation of parameters derived by ECAP measurements and perceptive skills which aim to access the level of speech and music intelligibility in CI users. In addition, the ratings assessed in a questionnaire on self-rated music intelligibility were correlated to a test battery consisting of measures for speech reception threshold (SRT) in noise (Oldenburger Satztest (OLSA)) and music intelligibility (Adaptive Melody-Pattern-Discrimination Test (AMPDT)). We hypothesised that results from this test battery correlated to subjective ratings and measures describing SoE functions.
Methods
The patient collective covered 17 well-experienced bilateral CI listeners (8 females, 9 males) between the age of 14 and 77 years with a minimum CI experience of two years. Music enjoyment and self-rated musicality was evaluated by means of a questionnaire. The AMPDT included two psychoacoustic tests: timbre difference discrimination threshold (TDDT) and background contour discrimination threshold (BCDT). The accentuation of harmonics in a foreground melody created a background melody. Accentuation was realised by sound level increment, frequency detuning and onset asynchrony. Subjects had to detect target intervals comprising both foreground and background melody by discriminating timbre differences in a Three-Interval Three-Alternative Forced-Choice (3I3AFC) procedure. In a One-Interval Two-Alternative Forced-Choice (1I2AFC) procedure, subjects had to classify the background melody’s contour. SoE was measured via a spatial forward-masking paradigm. A basal, medial and apical recording electrode was measured. Probe electrodes were one electrode position apical to the recording. The width of normalised SoE functions was calculated at their 25 % and 50 % level (excitation distance (DIST)). Furthermore, exponential functions were calculated for SoE profiles with more than three data points for each side. The OLSA assessed SRT in noise. The noisy environment was presented through an array of four loudspeakers (MSNF). The Fastl noise-condition allows to make use of gap listening representing the temporal characteristics of speech as a fluctuating noise. The OLnoise-condition is a continuous noise resulting in a maximum portion of masking.
Results
We found that background melody contour classiffication (BCDT) is more challenging to CI users than the detection of small perceptual timbre differences (TDDT). Background melody contour classification was possible with harmonic accentuation by sound level increment whereas accentuation by onset asynchrony was more demanding. CI users failed in background melody contour classification obtained by frequency detuning. SRTs assessed in the OLSA were significantly lower in the OLnoise than in the Fastl noise masking condition. A number of N = 90 SoE functions were acquired from ECAP measurements, in which N = 48 showed a clearly present ECAP response. The DIST at the 25 % and 50 % level was narrower for the basal than for the apical and medial electrode. SoE functions showed asymmetric profiles with larger amplitudes towards the basal end of the cochlea. Correlation analysis between the AMPDT, OLSA and DISTs showed no significant correlation. Correlation analysis between the AMPDT, OLSA and the questionnaire’s results could not prove that musical activities (music listening, singing or playing instruments) improve music intelligibility. However, CI supply has restored the importance of music, self-rated musicality and musical enjoyment in this study’s subjects.
Conclusions
The present study’s results imply that CI listeners are only able to detect distinct timbre alterations throughout the course of a musical piece whereas they cannot discriminate background melodies hidden in a pattern of complex harmonic sounds. Furthermore, SoE measurements do not seem to be an adequate tool to predict neither speech nor music intelligibility in CI listeners, contrary to our initial hypothesis. This finding is consistent with a number of studies who did not find a correlation between music or speech intelligibility and channel interactions assessed by SoE measurements. It can be concluded that albeit CI supply restores musical enjoyment in patients with sensorineural hearing loss, music perception is still poor and does not significantly improve by regular musical activities such as listening to music, singing or playing instruments.
Background: Minimally invasive coronary artery bypass grafting (MICS CABG) has been introduced to abstain from median sternotomy due to related comorbidities. The aim of this study is to report the long term results of three different MICS CABG strategies: Partial lower sternotomy (PLS), totally endoscopic coronary artery bypass grafting (TECAB) and anterolateral thoracotomy (ALT). Moreover we aimed to compare these surgical approaches in terms of quality of pain and pain intensity.
Methods: From 1997 to 2006, 126 patients underwent MICS CABG surgeries in our department through different surgical approaches: 43 PLS, 63 TECAB and 20 ALT. Preoperative characteristics were similar between groups. There were 90 males (71.4%) and 36 (28.6%) females with a mean age of 62±11 years (Range 36 to 90).
Results: There was no in-hospital mortality. Conversion to minithoracotomy was necessary in 2 (1.6%) patients and conversion to sternotomy was performed in 1 (0.8%) patient. Length of hospital stay was comparable in patients who underwent PLS or TECAB, but both groups had significantly shorter hospital stays than ALT patients (p<0.05). Two patients in group ALT developed temporary neurological complications postoperatively, which was significantly higher than that in groups TECAB (n=0) and PLS (n=0) (p<0.05). Mean follow-up was 12.2±2.1 (range 7.2 to 16.1) years with completed in 81.7 % of the patients. There were 17 late deaths. Freedom from graft problems was 87.5%, 86.5% and 94.7%; freedom from percutaneous coronary interventions (PCI) was 78.1%, 82.7% and 68.4% and freedom from Re-CABG was 100%, 96.1% and 94.7% in PLS, TECAB and ALT group, respectively. Pain intensity was similar between all three groups.
Conclusion: MICS CABG can be performed safely and effectively. Short and long-term outcomes of MICS CABG are comparable with those of the conventional CABG. There were no major differences regarding pain intensity between all three groups, although all three minimally invasive techniques have completely different surgical accesses.