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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.
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.
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.
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.
Glioblastoma multiforme accounts for more than 80% of all malignant gliomas in adults and a minor fraction of new annual cases occurs in children. In the last decades, research shed light onto the molecular patterns underlying human malignancies which resulted in a better understanding of the disease and finally an improved long term survival for cancer patients. However, malignancies of the central nervous system and especially glioblastomas are still related to poor outcomes with median survivals of less than 6 months despite extensive surgery, chemotherapy and radiation. Hence, a better understanding of the molecular mechanism driving and sustaining cancerous mutations in glioblastomas is crucial for the development of targeted therapies. Apoptosis, a form of programmed cell death, is an important feature of eukaryotic cells and crucial for the maintenance of multicellular homeostasis. Because apoptosis is a highly complex and tightly regulated signaling pathway, resisting apoptotic stimuli and avoiding cell death is a hallmark of the cancerous transformation of cells. Hence, targeting molecular structures to reestablish apoptotic signaling in tumor cells is a promising approach for the treatment of malignancies. Smac mimetics are a group of small molecular protein inhibitors that structurally derive from an intracellular protein termed Smac and selectively block Inhibitor of apoptosis (IAP) proteins, which are often aberrantly expressed in cancer. Several studies confirmed the antitumoral effects of Smac mimetics in different human malignancies, including glioblastoma, and give rationales for the development of potent Smac mimetics and Smac mimetic-based combination protocols. This study investigates the antitumoral activity of the bivalent Smac mimetic BV6 in combination with Interferon α. Latter is a well characterized cytokine with an essential role in immunity, cell differentiation and apoptosis. This study further aims to address the molecular mechanisms underlying the antitumoral activity of the combination treatment by using well established molecular cell death assays, flow cytometry, western blot analysis, genetic approaches and selective pharmacological inhibition. Since different Smac mimetics and Smac mimetic-based combination therapies are currently under clinical evaluations, findings of this study may have broad implications for the application of Smac mimetics as clinical cancer therapeutics.
The genetic mutation of the coagulation factor VIII (fVIII) results in a defective or missing protein, leading to a malfunctioning blood coagulation. The resulting disease is called hemophilia A. Depending on the severity of the mutation, affected patients experience an increased risk of pathologic bleeding after minor trauma or even sudden bleeding events. Substitution therapies with extrinsic fVIII exist using plasmatic or recombinant fVIII products. Due to an insufficient immune tolerance towards substituted fVIII, about 30 % of patients develop allogenic neutralizing antibodies (inhibitors) against substituted fVIII products. The gold standard of treating inhibitors is the immune tolerance induction (ITI), where patients are given frequent, high doses of fVIII to induce an immune tolerance. ITI therapy fails in about 30 % of patients. Mechanisms of action of ITI are part of research, as insufficient knowledge about mechanisms and prognostic factors complicate treatment. For example, the development of anti-idiotypic antibodies, which occur naturally as a regulatory mechanism of the immune system, are being studied. Such anti-idiotypes have been detected in immunoglobuline preparations and in patients after successful ITI.
Inhibitors interfere with fVIII function in coagulation by binding functional epitopes within fVIII domains. Inhibitors against the A2 and C2 domain are predominantly found, however also the C1 domain has been shown to be highly immunogenic in some patients. The polyclonality of inhibitors aggravates the understanding and treatment of these. The present project therefore focusses on the selection of synthetic anti-idiotypic antibodies to target inhibitors in patients. The phage display method was applied to, for one, isolate anti-idiotypic single chain variable fragments (scFvs) specific against human polyclonal anti-fVIII antibodies and second against two C1 domain-specific inhibitory monoclonal antibodies (mAbs).
In the first project, anti-fVIII antibodies were purified from human plasma to serve as target molecules. A previous project showed that using full plasma as a target did not yield anti-idiotypic antibodies from phage display. For the purification, protein A chromatography and fVIII coupled Affi Gel® chromatography were applied. The isolated antibodies were next used as targets for the selection of anti-idiotypic scFvs. Analysis revealed that none of the selected phages solely bound the anti-fVIII antibody target. Consequently, the test protocol was modified, which resulted in a reduction of unspecific binders. Yet, no target-specific binders were isolated from phage pools. Reason for this may have been the high diversity of the polyclonal antibody target and the limited diversity of the phage libraries.
The aim of the second project, was the selection and characterization of scFvs, that target the paratopes of C1 domain-specific mAbs GMA8011 and LE2E9. From a therapeutic viewpoint, the preparation of an anti-idiotypic antibody pool, tailored to each patient’s inhibitor population, could help neutralize inhibitors in patients. Ultimately, one GMA8011-specific scFv-carrying phage clone (H2C1) and two specifics to LE2E9 (H3G7, H3F10) were isolated. In further experiments, only the GMA8011-specific scFv showed competitive behavior in presence of fVIII, pointing towards an anti-idiotypic binding to the inhibitor paratope. The LE2E9-specific scFvs did not prevent binding of the inhibitor to fVIII. Hence, no anti-idiotypic behavior could be determined. For further characterization, selected scFvs were genetically fused to Fc antibody fragments and recombinantly produced. In this antibody format, all three scFvs showed concentration dependent binding to the target and the isotype control. The binding specificity to the target, observed in phage context, could not be reproduced. Competition experiments with fVIII confirmed that none of the scFvs bound the paratope of their target inhibitor.
The selection of anti-idiotypic scFvs from phage display libraries proves to be effortful. Polyclonal anti-fVIII antibodies purified from hemophilic plasma appear to be unsuitable as a target for phage display, likely due to the high diversity of the target molecules. Furthermore, the preparation of an individualized anti-idiotypic pools for patients by selecting scFvs against single inhibitory mAbs proves to be difficult. The selection of scFvs against anti-C1 inhibitors GMA8011 and LE2E9 produced three promising scFv-carrying phages. However, analysis could not detect anti-idiotypic behavior. Further research with inhibitors, monoclonal and polyclonal, and anti-idiotypic antibodies should be performed to bring better insight into the highly complex paratope-epitope interaction.
Obesity is considered as a type of chronic inflammation. It enhances the risk of developing cardiovascular disease, diabetes, and some cancers. The key players in the induction of inflammation in adipose tissue are macrophages. However the mechanism of macrophage activation in obese fat tissue is still not fully understood. Elevated level of saturated fatty acids in adipose tissue promotes inflammation and insulin resistance. Exposure of macrophages to saturated fatty acids stimulates pro-inflammatory c-Jun N-terminal kinase (JNK), nuclear factor kappa B (NF-kB) signaling, and production of pro-inflammatory cytokines, such as IL-6, IL-8, IL-1β, and TNFα. Palmitate is a major saturated free fatty acid released by adipocytes. It activates inflammatory pathways through Toll-like receptors (TLR) 2 and 4, provokes endoplasmic reticulum (ER) stress and increases levels of diacylglycerols (DAGs) and ceramides. Saturated fatty acids also affect cellular oxidative metabolism. Thus, mitochondrial fatty acid oxidation reduces ER-stress and expression of inflammatory cytokines in palmitate-treated macrophages. On the other hand mitochondrial reactive oxygen species (ROS) promote palmitate-mediated pro-inflammatory cytokine production. Recently, mitochondrial functions were linked to their morphology. Mitochondrial fission has been reported in β-cells and myocytes in response to high levels of glucose and free fatty acids, and was associated with disruption of mitochondrial functions, increased ROS level, and cell death. The aim of this study was to investigate the role of mitochondrial fragmentation in palmitate-induced inflammation in human macrophages. In our settings fatty acids, independently of their saturation, affected mitochondrial morphology. Mixtures of long chain saturated and unsaturated fatty acids as well as triglyceride-rich lipoprotein lipolysis products promoted mitochondrial fission. Mitochondrial fragmentation in palmitate-treated macrophages revealed a time- and concentration-dependent character, and was reversible upon palmitate removal. This observation, together with unaltered levels of mitochondrial protein and DNA content, and intact mitochondrial respiration, suggested that mitochondria were not damaged and were functionally active. Mechanistically, palmitate-induced mitochondrial fragmentation was not regulated by ER stress or loss of mitochondrial membrane potential. However, inhibition of palmitate incorporation into mitochondrial membrane phospholipids decreased mitochondrial fragmentation. Other approach to prevent mitochondrial fission was the inhibition of dynamin-related protein 1 (DRP1) activity, which drives mitochondrial fission by forming ring- like structures around mitochondria and constricting mitochondrial membranes. Palmitate altered mitochondrial membrane lipid composition and promoted DRP1-oligomerization. The inhibition of palmitate-induced mitochondrial fragmentation enhanced mitochondrial ROS production, c-Jun phosphorylation, and upregulated expression of pro-inflammatory cytokines. Taken together, these results suggest that mitochondrial fragmentation is a protective mechanism attenuating palmitate-induced inflammatory responses. Future experiments will be required to investigate the role of mitochondrial fragmentation in obesity-associated diseases in vivo.
Cancer is one of the leading causes of death across all countries and its diagnosis still yields fear for the affected patient. Although treatment of cancer has made marvelous progress compared to the agents available thirty years ago, a cure for cancer, however, is still a distant prospect. Modern therapy still is a burden for many patients due to heavy side effects. With the development of agents targeting specific molecular targets on cancer cells, a new field of cancer therapy was opened and a small success story in the history of cancer began.
Aurora kinases represent a relatively new target in cancer therapy. The kinase is a essential part of mitosis and cell cycle progression and its overexpression has been shown to be related to many kinds of malignancies. Allosteric inhibition of a kinase is an increasing pre-clinical approach not yet established in the treatment of patients. In this thesis, we combine allostery with another innovative approach that is drug repurposing. If repurposed, a drug can be permitted to fast track drug admission to clinical trials.
I set up a screening of 1280 FDA approved drugs to identify small molecule compounds that affect the binding of Aurora kinase A and its main physiologic binding partner, TPX2. Further, I characterized the positive hits in vitro for their capabilities to displace TPX2 from Aurora A, to inhibit Aurora kinase activity, to thermally stabilize the protein and performed assays to determine their dissociation constant. Last but not least, I tested the compounds in cells for their effect on the cell viability and cell cycle via flow cytometry. Comparing the hit-compounds with controls I found that ATP-competitive AurA inhibitor MLN 8237 strongly displaces the interaction of Aurora A with TPX2.
Summarized, we identified eight hit compounds allosterically affecting Aurora A, but no compound proved to be active in all assays. Just one compound, PS 731, identified in another screening performed by our group and further characterized in this thesis remains interesting, especially when put in context with recent publications released in the time between the start of experiments for this thesis and its finalization.
How much we trust our own decisions, knowledge or perceptions influences our behavior in many everyday situations. Normally the confidence we have in our decisions is rather accurate, but under certain circumstances the subjective evaluation of a decision and its objective quality can differ heavily. Subjectively over- or underestimating the quality of decisions can lead to disadvantageous behavior. Little is known about how this feeling of confidence about a decision is generated. Is it computed automatically with the decision or does it arise in a different process?
This thesis is based on a publication that contributed to the investigation of this question by comparing the influence of two different forms of spatial attention on decision confidence. Visual spatial attention is a cognitive mechanism that serves to select parts of the visual field, leading to more accurate decisions about the attended items. It can be either voluntarily controlled (endogenous) or reflexively driven by external events (exogenous). In an orientation-matching task participants performed better in both attentional conditions than in a control condition without directed attention. Additionally, we found that only endogenous, but not exogenous attention led the subjects to overestimate the quality of their performance. The possible implications of this “relative overconfidence” were discussed with respect to the theoretical framework of spatial attention and decision confidence. The present findings support the idea that decision confidence is generated in a distinct metacognitive process. Possible ideas for further neurophysiological research are proposed. The thesis concludes with an attempt to integrate the discussion into a broader context of medical research on certain neuropsychiatric symptoms and conditions.
After entorhinal deafferentiation of the hippocampal dentate gyrus a reinnervation of the denervated neurons by axon collaterals can be observed. This process takes place in a matter of weeks. However, the overall functional effect on the hippocampal network is still unclear.
In an effort to investigate this effect of axonal sprouting on the neuronal network of the dentate gyrus we compared the electrophysiological response of the dentate gyrus after electric stimulation in wild-type mice (WT mice) with a normal post-lesion sprouting, with genetically modified mice with an overexpression of the growth-protein CAP23 (cytoskeleton-associated protein 23). CAP23 overexpressing mice (CAP23tg mice) are known to have an enhanced axonal growth and sprouting after lesion.
The mice (both the WT as well as the CAP23tg mice) were deeply anesthetized and a lesion of the perforant path was induced stereotactically with a wire knife. After that the mice were permitted to survive for 4-6 weeks for partial reinnervation of the dentate gyrus before they were again operated and evoked potentials were measured (extracellular recordings of evoked potentials in the dentate gyrus). Non-lesioned litter-mate mice were taken as reference. The sprouting and the correct position of the electrodes was confirmed histologically.
For electrophysiological investigation we assessed laminar profiles and calculated a current-source density (CSD). In lesioned CAP23tg mice compared to lesioned WT mice this CSD-analysis revealed a significant enhancement of the current sink in the area of deafferentiation (outer molecular layer) and a significant excitation in the granule-cell layer.
Our results show that axonal sprouting seems to enhance the excitability of granule-cells. Thus, even if an enhanced axonal sprouting might accelerate the reinnervation of denervated dendrites after lesion, but it also leads to posttraumatic hyperexcitability of the neuronal network. In a therapeutic approach of fascilitating axonal sprouting this hyperexcitability has to be taken into consideration.
Throughout the entire life, new neurons of the granule cell type (GCs) are continu-ously generated in the mammalian hippocampal dentate gyrus (DG). As a part of the limbic system, the hippocampus is concerned with spatial and declarative memory for-mation. Increasing evidence exists, that adult born granule cells (ABGCs) play an im-portant role in this process. An especially critical period, when these ABGCs are 4-6 weeks old, has come into the focus of research. It is during this specific time-span that the ABGCs express enhanced excitability and synaptic plasticity as well as a lowered threshold for the induction of long term potentiation (LTP), a mechanism associated to learning and memory formation.
This study investigates the time course and dynamics of synaptic integration in ABGCs and mature GCs together with which differences exist between them at various cell ages. Furthermore, spine plasticity following high frequency stimulation (HFS) is analysed focusing on a critical phase of enhanced excitability in 4-5 week old ABGCs.
In this thesis, two approaches at studying the synaptic integration and structural plas-ticity of ABGCs in rats were investigated. This work was performed on fixed brain ma-terial that was provided by two laboratories that performed the in vivo labelling, stimu-lation procedures and brain fixation. In the first project, 6, 12 and 35 weeks old XdU-labelled ABGCs were studied. Adult rats were exposed to an enriched environment and received unilateral intrahippocampal delta burst stimulation (DBS) and LTP induction. The ABGCs and a control population of mature GCs were immunohistologically ana-lysed for Egr1 (early growth response 1) expression. Egr1 is an immediate early gene (IEG), expressed after LTP induction and marks neuronal excitation.
It was found, that unilateral stimulation of the perforant path of the hippocampus re-sults in an increase of Egr1 expression in ABGCs of both hemispheres. It could be shown that the enhanced expression intensity of Egr1 in ABGCs is not a usual state of young GCs but a reaction to DBS. ABGCs from unstimulated control animals and mature GCs expressed lower levels of Egr1. Interestingly, the stimulation induced a similar degree of Egr1 expression intensity in all ABGC age groups. Furthermore, it was found that young ABGC from the infrapyramidal dentate gyrus (DG) express a higher excita-bility than those from the suprapyramidal DG.
In the second project, fixed brain sections were analysed. They stemmed from rat brains containing 28 and 35 day old ABGC that had been transfected with intrahippo-campal RV-GFP (retroviral-green fluorescent protein) injections and had received uni-lateral high frequency stimulation of the medial perforant path in vivo. Nuclear Egr1 expression intensity was analysed in a cell specific manner. Dendritic spine size was measured in the inner-, middle- and outer molecular layer (IML, MML, OML). It was found that in ABGC, stimulation induced Egr1 expression increase is lower than in ma-ture GC. Following HFS, a significant homosynaptic spine enlargement was observed in the MML indicating homosynaptic LTP, while heterosynaptic spine shrinkage was found in the adjacent IML and OML. The latter corresponds to heterosynaptic long term depression (LTD). Homosynaptic plasticity describes an input-specific potentiation of synapses that received direct activation. The weakening of synapses not stimulated dur-ing homosynaptic potentiation is oppositely coined heterosynaptic plasticity1.
A positive correlation between an increase in nuclear Egr1 expression intensity and spine enlargement due to homosynaptic plasticity induced by HFS could be shown. Concomitant heterosynaptic plasticity, as indicated by spine shrinkage was observed. Spine shrinkage in the IML and OML showed a negative correlation to a decrease in Egr1 intensity.
Taken together, the results provide detailed information on the gradual integration of ABGC with ongoing maturation. Cell specific proof for homo- and heterosynaptic plas-ticity following HFS was found in the critical period of synaptic integration of ABGCs.
The present study aimed to assess the tissue response to the SYMBIOS® resorbable collagen membrane SR, which is derived from bovine Achilles tendon, and compare it to the physiological wound healing of a sham operation as a control.
An ex vivo analysis was performed using injectable platelet-rich fibrin (i-PRF), that is gained by the centrifugation of human venous blood and contains fibrin, leukocytes and platelets, to elucidate the membrane permeability and interactions with human cells and plasma proteins. In the in vivo study, a subcutaneous implantation model was established in Wistar rats to evaluate the cellular reactions for up to 30 days after membrane implantation. Histochemical, immunohistochemical and histomorphometric analyses were performed to assess the cellular inflammatory response, vascularization pattern and cell infiltration capacity.
In the ex vivo study, i-PRF components including fibrin, leukocytes and platelets penetrated the membrane after just 15 minutes. Within the observation period, the cellular reaction in the early phase, which included the first 3 days, produced only mononuclear cells. From 10 to 30 days , the formation of multinucleated giant cells (MNGCs) was induced by the collagen membrane. CD-68 positive cells (macrophages) occurred in a high number on day 3, and the number decreased over time up to day 30. Along with the reduction in the number of CD-68 positive cells, the number of MNGCs increased significantly. The presence of MNGCs was accompanied by significantly increased vascularization within the central region of the membrane, and only mononuclear cells (MNCs) did not produce vascularization. In contrast, the accumulated MNGCs were located on the membrane surface. The control group reflected the physiological process of wound healing, as MNGCs did not form over the 30 day period, and a significantly lower level of vascularization was observed compared with the test group.
This finding showed dynamic changes in the cellular reaction, which indicated a relationship between macrophage fusion and MNGC formation, and vascularization of the collagen membrane is circumstantial evidence of a reaction to a foreign body. However, the collagen membrane was able to maintain its structure and integrity over time, showing no signs of premature breakdown and disintegration due to the specific porosity of its membrane structure.
Therefore, we questioned whether the biomaterial-induced formation of MNGCs should be accepted as a biomaterial-induced cellular reaction that is able to restore vascularization or as an adverse reaction. Therefore, extensive preclinical and clinical studies are needed to investigate the type of MNGCs that form in response to the membrane material studied here.
Cancer microenvironment is now recognized as a critical regulator of all stages of cancer development. Beside the tumor vasculature and tumor-infiltrating immune cells, other stromal cells such as cancer-associated fibroblasts (CAFs) regulate tumor growth. Fibroblasts are ubiquitous cells in connective tissue, where they shape the extracellular matrix (ECM). Fibroblasts are usually quiescent but get activated when tissue homeostasis is disturbed. Then, activated fibroblasts rebuild the ECM and communicate with local cells to participate in wound repair. These repair properties can go awry when being unchecked, which can lead to fibrosis and subsequently cancer development. CAFs can promote cancer development by fostering tumor cell growth, polarizing immune cells to an immunosuppressive phenotype, and crosslinking collagen to enable tumor cell invasion. Molecular mechanisms of CAF activation, thus, need to be understood to target these cells in tumors. Prostanoid prostaglandin E2 (PGE2) is viewed as a pro-tumor lipid mediator as suggested by studies pharmacologically or genetically targeting the enzymes producing PGE2, such as microsomal PGE synthase-1 (mPGES-1) in tumor models. Similar to CAFs, PGE2 drives tumor cell growth and tumor-associated immune suppression. Therefore, I hypothesized that PGE2 may play a role in CAF activation.
This hypothesis was tested in two mouse models of breast cancer (orthotopic grafting model, and polyoma middle T oncogene transgenic model), besides using isolated mammary gland (MG) fibroblasts in vitro. As expected, given the pro-tumor function of PGE2, knocking out mPGES-1 reduced the growth of oncogene-driven and transplanted mammary tumors. Surprisingly, CAF density was markedly increased when mPGES-1 was depleted. Importantly, despite reduced primary tumor growth, I observed enhanced lung metastasis upon mPGES-1depletion. Using MG-derived fibroblasts in vitro furthermore revealed that treatment with PGE2 reduced a TGFβtriggered CAF-like activation state. Importantly, bioinformatics analysis of a human breast cancer patient dataset revealed a negative correlation of a PGE2 production signature with fibroblast marker genes. In a next step I investigated if the increased CAF infiltrate was connected to the reduced tumor growth upon depletion of PGE2. To unravel this, I first asked through which E prostanoid (EP) receptor PGE2 signals in fibroblasts. MG fibroblasts mainly expressed EP3, and EP3 KO fibroblasts showed a hyper-proliferative and activated phenotype, indicating EP3 as the main PGE2 receptor in MG fibroblasts. Co-injecting of EP3 KO MG fibroblasts and tumor cells in WT mice suppressed tumor growth, whereas co-injection of WT fibroblasts with tumor cell in mPGES-1 KO mice increased tumor growth. These data indicate that PGE2 restricts CAF levels through EP3, which supports tumor growth. Whole transcriptome mRNAsequencing of WT and mPGES-1 KO FACS-sorted CAFs combined with immunohistochemical data suggested a role of p38 mitogen-activated protein kinase (MAPK) in the modulation of fibroblast activation by PGE2.
In summary, I showed in two breast cancer models that mPGES-1 depletion delays breast cancer progression, which is probably driven by the EP3-PGE2 signaling axis in host stroma. PGE2 appears to be a potent anti-fibroblast activation agent in tumors via EP3 and downstream p38 MAPK signaling. This study therefore hits the dogmatic perception of the general pro-tumor nature of PGE2; showing that PGE2 might be a double-edged mediator that can promote tumor growth at the primary site by restricting CAF expansion, which may in turn hinder infiltration of tumor cells to a secondary site.
Acute and chronic inflammation play a pivotal role in various diseases, such as rheumatoid arthritis, atherosclerosis, bacterial as well as viral infections and therefore are an everyday-challenge in clinical practice. In this context, biologically active products of the cyclooxygenases and the prostanoid synthases, e.g. prostaglandins, critically contribute to various aspects of the inflammatory response in almost every tissue of the body. Emerging evidence over the past decades has demonstrated that these mediators are not only responsible for a pro-inflammatory response, but also show anti-inflammatory and pro-resolving properties. The relevance of biologically active lipids in this context is strengthened by the clinical efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs), e.g. Aspirin®, which block the biosynthesis of the mediators via the cyclooxygenase (COX) enzymes. Notably, microsomal prostaglandin E synthase-1 (mPGES-1)-derived prostaglandin E2 (PGE2) is a well-studied, functionally versatile PG, which promotes its effects via specific G protein-coupled receptors (GPCRs). Activation of these receptors elicits an internal signal transduction cascade, including activation of the adenylyl cyclase (AC). Active AC contributes to an elevated intracellular cyclic adenosine monophosphate (cAMP) level, which in turn activates the transcription factor cAMP response element-binding protein (CREB) via phosphorylation.
While the role of PGE2 in the inflammatory context has been well-documented in previous literature, relatively little is known about CREB-dependent transcriptional changes in inflammation. Therefore, the aim of this study was to investigate the effect of mPGES-1-derived PGE2 on CREB-mediated transcriptional changes specifically in murine wild-type (WT) and mPGES-1 knock-out (KO) macrophages in an inflammatory context. To address this issue, bone marrow-derived macrophages (BMDMs) were treated with either the bacterial cell wall component lipopolysaccharide (LPS) in combination with interferon-γ (IFN-γ) or the yeast extract zymosan. To analyze effects on CREB activation we determined protein expression profiles of relevant PGE2-synthesizing enzymes, i.e. COX-2 and mPGES-1, as well as activity of the downstream transcription factor CREB. The activity of mPGES-1 was simultaneously determined by the analysis of the prostanoid kinetics. Under these experimental conditions we showed that COX-2 is strongly induced, and we also observed elevated activated CREB levels in WT as well as in mPGES-1 KO macrophages. Further, both LPS+IFN-γ and zymosan increased expression of mPGES-1 in WT but not in mPGES-1-deficient macrophages. These findings go in hand with largely similar alterations in the PGD2, TXB2, PGF2α profiles in WT and mPGES-1 KO macrophages upon stimulation. Of note, an elevated PGE2 production was also observed in mPGES-1-deficient macrophages at later stages upon inflammatory conditions. Subsequently, potential CREB-regulated targets were identified in macrophages upon inflammatory stimuli after 16 h by chromatin immunoprecipitation (ChIP) followed by Next-Generation-Sequencing (NGS). Surprisingly, despite equal levels of pCREB the characterization of CREB binding sites revealed different targetome profiles between WT and mPGES-1 KO macrophages. Specifically, the fatty acid metabolic processes-associated targets appeared to be selectively lost in mPGES-1-deficient vs. WT macrophages. We further validated one of those targets, i.e. the endoplasmic reticulum lipid raft-associated protein 1 (Erlin1), at the mRNA expression level, which indeed was differentially transcribed in response to different PGE2 synthesizing conditions.
Mechanistically, CREB is a well-characterized phosphorylation-dependent transcription factor in cell survival, proliferation, differentiation, and immune responses. Yet, our understanding of the functions of CREB in inflammation, specifically with respect to its activation by PGE2, is insufficient. Due to its biological relevance in inflammation it clearly requires additional studies to shed light on the details of CREB activation in macrophages to provide possibilities of therapeutic interventions.
Durch Implementierung eines effizienten Früherkennungsprogramms ist die Inzidenz des Zervixkarzinoms in Industrienationen seit 2005 auf konstant niedrigem Niveau. Ungeachtet dessen ist das Zervixkarzinom mit deutlich höheren Inzidenzraten und weniger als 50% Gesamtüberleben in den nicht industrialisierten Staaten die vierthäufigste Tumorentität der Frau weltweit.
Zur Behandlung des lokal fortgeschrittenen Zervixkarzinoms (FIGO Stadium IIb bis IVa bzw. Ib2/IIa2 mit mehreren histologischen Risikofaktoren) besteht nach aktueller Leitlinie (Stand 2014) und internationalem Konsens Indikation zur platinhaltigen Radiochemotherapie (RCT), subsequent gefolgt von einer (High Dose-Rate) Brachytherapie (HDR-BT). Unter diesen Umständen beträgt die lokale Kontrolle für Patientinnen mit lokal fortgeschrittenem Tumor zwischen 74% und 85%.
Dennoch stagnieren Gesamtüberleben und das spezifische Überleben bezogen auf verschiedene klinische Endpunkte, sodass die Entwicklung neuer Behandlungsstrategien und Therapieoptionen, insbesondere zur Behandlung rezidivierter und metastasierter Erkrankungsstadien, angezeigt ist. Darüber hinaus spielen im Gegensatz zu anderen Tumorentitäten molekulare Marker sowohl als prädiktive als auch als therapeutische Targets bei der Behandlung des Zervixkarzinoms eine bislang untergeordnete Rolle, während molekular-zielgerichtete Therapien in der modernen Krebstherapie einen immer größeren Stellenwert einnehmen.
Ziel der hier vorliegenden Arbeit ist es, neue Biomarker für das Zervixkarzinom und dessen Ansprechen auf simultane Radiochemotherapie und anschließende Brachytherapie zu identifizieren.
Zu diesem Zweck untersuchten wir in einem Patientenkollektiv von 74 Patientinnen mit histologisch gesichertem Zervixkarzinom (FIGO Ib - IVb) prätherapeutisch gewonnenes Biopsiegewebe. Mittels immunhistochemischer Methoden wurde die Expression von Polo-like Kinase 3 (PLK3) und phosphoT273 Caspase-8 erfasst und quantifiziert. Die Ergebnisse wurden anschließend mit klinischen bzw. histo-pathologischen Charakteristika, einschließlich der p16INK4a Expression und den klinischen Endpunkten lokales progressionsfreies- und Fernmetastasen-freies
Überleben bzw. dem tumorspezifischem und dem Gesamtüberleben nach kurativ intendierter Therapie korreliert.
Hierbei konnte zunächst eine signifikante Korrelation zwischen der PLK3 und pT273 Caspase-8 Expression beobachtet werden (p = 0.009). Darüber hinaus war PLK3 signifikant mit dem N-Status (p = 0.046), dem M-Status (0.026) und dem FIGO-Stadium (p = 0.001) assoziiert, wohingegen die pT273 Caspase8-Expression signifikant mit der Tumorgröße (T-Stadium) korreliert war. Bezogen auf univariate Überlebenszeitanalysen war eine erhöhte PLK3-Expression signifikant mit einer geringeren Rate an Fernmetastasen (DMFS p = 0.009) sowie einem signifikant verlängertem tumorspezifischen und Gesamtüberleben assoziiert (CSS p = 0.001, OS p = 0.003). Vergleichbare Ergebnisse konnten auch für die pT273-Caspase 8 Expression mit einer verringerten Metastasierungsrate (p=0.021) und verbessertem tumorspezifischem (p<0,001) sowie Gesamtüberleben (p=<0.001) gezeigt werden. In den multivariaten Analysen verblieb die pT273-Caspase 8-Expression mit einem signifikant verbesserten Gesamtüberleben (p=0.001).
Zusammenfassend belegen diese Daten erstmals eine signifikante Korrelation zwischen einer erhöhten prä-therapeutischen PLK3 und pT273 Caspase 8- Expression und einem zu favorisierenden klinischen Verlauf nach mit Radiochemotherapie behandeltem Zervixkarzinom.
Role of Orphan G-protein-coupled receptor GPRC5B in smooth muscle contractility and differentiation
(2019)
G protein coupled receptors (GPCRs) are the largest family of cell-surface receptors encoded in the human genome. They mediate the cellular responses to a wide variety of stimuli, ranging from light, odorants, and metabolic cues to hormones, neurotransmitters, and local mediators. Upon ligand binding, the GPCR undergoes conformational changes resulting in the activation of heterotrimeric G-proteins belonging to the families Gs, Gi/o, Gq/11, G12/13, which in turn mediate the downstream signaling. While most of the 360 non-olfactory GPCRs are well studied, approximately 120 GPCRs are still considered "orphan", meaning that their mechanism of activation and biological function is unknown. GPCRs have been functionally described in the regulation of almost all organ systems, and their dysregulation has been implicated in the pathogenesis of a multitude of diseases. In the vascular system, the contractile tone of vessels is crucially regulated by GPCRs. Substances that act through G12/13- and Gq/11-coupled GPCRs are associated with facilitation of contraction, while Gs-coupled GPCRs are usually associated with the induction of relaxation. Furthermore, while Gq/11 pathway activation promotes proliferation and dedifferentiation of vascular smooth muscle cells (VSMC), G12/13 and Gs signaling pathways promote expression of contractile proteins and differentiation.
The functional properties of VSMC depend on the anatomical location, and a recent single-cell expression analysis showed that VSMC from different vascular beds have different patterns of GPCR expression. Interestingly, smooth muscle cells (SMCs) from resistance arteries not only express various GPCRs for known modulators of vascular tone, but also a number of orphan GPCRs. These results suggest a potential role of orphan GPCRs in the modulation of blood pressure. Orphan GPCR GPRC5B was one of the GPCRs enriched in resistance arteries, and this receptor was also upregulated in dedifferentiated aortal SMC. The function of GPRC5B in these types of SMC is currently unknown. In vitro studies suggested that GPRC5B negatively regulates obesity, inflammation, insulin secretion and fibrotic activity, but there are no data available with respect to its function in regulation of vascular tone or other SMC functions.
Our study aimed at the identification of the specific functions of GPRC5B in SMC. To do so, we generated a SMC-specific GPRC5B-deficient mouse line by crossing Gprc5bfl/fl mice with smooth muscle-specific, tamoxifen-inducible Myh11-CreERT2 mice. We found that SMC-specific deletion of GPRC5B did neither affect myogenic tone in pressure myography, nor the response to the contractile agonists in wire myography. In contrast, vessel relaxation in response to prostacyclin analogues cicaprost and iloprost, which act on the prostacyclin receptor IP, were increased. These results suggested a selective improvement of IP receptor signaling. The IP receptor is coupled to Gs protein, it promotes vasorelaxation and acts as a restraint on platelet activation. Using overexpression of IP and GPRC5B in HEK cells, we found that GPRC5B physically interacts with the IP receptor and controls IP trafficking and membrane localization. Furthermore, we found that membrane IP receptor expression was increased in GPRC5B-deficient human aortic SMC and in resistance vessels of SMC-specific GPRC5B. To investigate the importance of increased IP-mediated signaling in SMC in vivo, we measured blood pressure in two mouse models of hypertension. We found that SMC-deletion of Gprc5b resulted in a significant reduction of blood pressure compared with control mice, which suggested that Gprc5b negatively regulated relaxation in hypertensive disease by decreasing IP mediated relaxation. In line with this notion we found that application of the IP antagonist Cay10441 largely abrogated the beneficial effect of GPRC5B inactivation in this hypertension model. Another important function of the IP receptor is the regulation of SMC differentiation, which led us to investigate the differentiation state of GPRC5B-deficient SMC. We found that deletion of GPRC5B enhanced expression of contractile genes and reduced expression of proliferative markers. This improved differentiation was, at least partially, due to increased IP signaling in SMC. Moreover, in a mouse model of atherosclerosis SMC-specific deletion of Gprc5b reduced plaque area and contributed to a more stable fibrous cap by promoting differentiation.
In conclusion, deletion of GPRC5B in SMC significantly improved contractility and differentiation by increasing IP receptor membrane availability and signaling.
Langzeitbeobachtung der Therapie von Hämophilie A-Patienten mit einem humanen Faktor VIII-Konzentrat
(2019)
This doctoral thesis entitled “Long-term surveillance of the therapy of haemophilia A patients with a human plasma-derived factor VIII concentrate” was performed to assess the influence of the chronic long-term therapy with a human plasma-derived factor VIII concentrate in daily clinical practice on the health of haemophilia A patients.
Haemophilia A is a chronic disease, caused by a congenital deficiency of coagulation factor VIII, which requires life-long haemostatic treatment. The severity of bleedings, as the main clinical feature of haemophilia A, is generally correlated with the residual activity of coagulation factor VIII.
Until recently, factor VIII preparations, used to replace the deficient factor VIII, were the only treatment option for haemophilia A. Development of inhibitory antibodies against factor VIII is the most serious complication associated with the use of factor VIII products, rendering the administered factor VIII ineffective.
To date, all novel treatments still rely on some factor VIII replacement therapy. At least in the near future and probably for longer, (concomitant) therapy with factor VIII concentrates will continue to be necessary for treatment of haemophilia A, emphasising the continuous need for efficacy and safety data in terms of pharmacovigilance on factor VIII replacement therapy.
Medicines to treat haemophilia A, are authorised for use, when evidence of its efficacy and safety is limited to data of a small number of investigated patients during short-term observation periods of about six months, and thus have not been systematically assessed in all patient groups until marketing authorisation. Long-term efficacy and safety data from post-marketing surveillance are important to prove that a chronic treatment is efficacious and safe in the real-life setting by monitoring “real-life” patients of all age groups, rather than a carefully selected patient population. Medical and scientific analyses of such long-term data are crucial to detect, understand, and potentially prevent the harm resulting from (new) adverse drug reactions, including those, which only rarely occur and therefore are difficult to detect.
Therefore, data from two prospective surveillance studies investigating real-life therapies with the same human plasma derived factor VIII concentrate were combined and analysed retrospectively. It was hypothesised that the chronic long term therapy with a human plasma-derived factor VIII concentrate in daily clinical practice is effective, safe, and well tolerated with no unexpected adverse effect on the health of haemophilia A patients. It was the aim of this analysis to investigate the influence of the chronic long-term treatment with the factor VIII concentrate on the health of patients with severe as well as nonsevere haemophilia A including all age groups in a real-life setting. In addition, the influence of prophylactic factor VIII treatment or the switch to this regimen on the annual bleeding rate of all haemophilia A patients, and the long-term effects of this regimen on the patients’ annual bleeding rates were investigated.
Starting in 1998 until 2015, data of 1418 patient-years from 198 haemophilia A patients representing all age groups and haemophilia A severities were analysed. This study covered 18 years of documentation time with a mean observation period of more than seven years per patient. It is the longest study of a single factor VIII concentrate conducted so far, investigating the therapy of haemophilia A. The only observed side effects involved low incident factor VIII inhibitor formation in patients at risk (13 % of previously untreated patients, compared with usually about 30 %). Factor VIII inhibitor development was mainly transient, with low titers, and without clinical relevance. Any, even low frequent prophylaxis was found to be significantly better than on demand and had the greatest effect on the annual bleeding rate of patients, irrespective of their age or haemophilia A severity. Patients suffered during continuous prophylaxis from a very low bleeding rate (median 1.3 compared with 31.4 under on demand), down to no bleeding per year. Patients whose regimen changed to continuous prophylaxis benefitted most (median annual bleeding rate 1.1), irrespective of age or haemophilia A severity.
This analysis demonstrates that the chronic long-term therapy with the plasma-derived factor VIII concentrate in daily clinical practice is effective, safe, and well tolerated. Thus, data on efficacy and safety obtained during chronic long-term therapy with the human plasma-derived factor VIII concentrate reaffirm that there is no unexpected adverse effect on the health of haemophilia A patients.
These results support the therapeutic concept of a life-long prophylaxis of haemophilia A patients with a human plasma-derived factor VIII concentrate.
Characterization of SPRTN, the first mammalian metalloprotease that repairs DNA-protein-crosslinks
(2019)
DNA is constantly exposed to various endogenous and exogenous sources causing different kinds of DNA damage. To overcome this threat, cells have evolved various repair mechanisms. Impairments of these repair mechanisms result in diverse diseases. Ruijs-Aalfs syndrome is a monogenic disease characterized by accelerated ageing and carcinogenesis, typical features of impaired DNA repair and was shown to be caused by germline mutations of SPRTN, a newly identified and only partially understood protein. A role of SPRTN in DNA damage response was previously shown and an involvement in translesion synthesis (TLS) proposed. However, later discoveries revealed an essential function of SPRTN, being indispensible for embryonic development of vertebrates and cellular survival, whereby this function is independent of SPRTN’s proposed function in TLS. The essential function of SPRTN was proposed to be contained in its protease domain but remained unclear.
In this study we identify SPRTN as the first mammalian metalloprotease that repairs DNA-protein-crosslinks (DPCs). DPCs represent a specific type of DNA-lesions with bulky protein adducts covalently linked to DNA thereby being highly toxic as they potentially stall replication forks and lead to double strand breaks and genomic instability. DPC-repair remains only partially understood despite their frequent appearance and toxicity. With this study we discover and characterize a new mechanism of DPC-repair in mammalian cells - a proteolytic cleavage of the protein adduct by the metalloprotease SPRTN. Accordingly, a proteolytic activity of SPRTN is demonstrated and s SPRTN-recruitment to DNA upon DPC-induction displayed. Furthermore, SPRTN exhibits degradation of different proteins covalently bound to DNA in form of DPCs, but not of unbound fractions of the same protein substrates. Consequently, mutations of SPRTN’s proteolytic core as well as a mislocalization or depletion of SPRTN result in impaired DPC-repair. The importance of SPRTN-mediated DPC-removal is confirmed by a severely compromised response to DPC-inducing agents for cells with impaired SPRTN function. Additionally to the discovery of SPRTN’s essential function this study further provides an explanation of the molecular mechanism underlying Ruijs-Aalfs syndrome (RJALS), the segmental progeroid syndrome resulting from SPRTN mutation. The effects of the identified clinical mutations on the DPC-repair function of SPRTN are explained and a DPC-accumulation in cells carrying clinical SPRTN-mutation displayed. The obtained data provides sufficient evidence that an impaired DPC-repair is the pathophysiologic cause of RJALS-syndrome, confirming the importance of SPRTN’s newly identified function. In conclusion, SPRTN is the first identified mammalian metalloprotease with a DPC-repairing function and the impairment of SPRTN-mediated DPC-removal is the underlying mechanism of RJALS syndrome.
The carpal tunnel syndrome (CTS) is a chronic compression of the median nerve in the carpal tunnel, a condition in which the nerve is constricted especially under the flexor retinaculum (FR). The disease predominantly appears between 40 and 83 years of age. Women are significantly more often affected than men. The same applies to overweight people in comparison to normal weight people. Abnormal sensations at night, including paresthesias and dysesthesias, are classical CTS symptoms, predominately involving the middle fingers, later also the thumb. Diagnosis of CTS usually proceeds by motor nerve conduction study (mNCS) and determination of the distal motoric latency (DML). In conformity with electrophysiology, peripheral nerve ultrasonography has also attained an important diagnostic informative value. In principle, there is an open surgical procedure and an endoscopic carpal roof cleavage. The goal of therapy is the complete open division of the flexor retinaculum (FR) in order to relieve the median nerve from compression.
This work examines the morphological alterations of the median nerve at the site of the carpal tunnel after surgical decompression by means of high-resolution neurosonography in the scope of a prospective study. More than 100 patients were examined between October and December 2014 for planned decompressions surgery due to CTS. A total of 81 patients were prospectively included, 5 of which could not take part in the follow-up after six months and were excluded from this evaluation. A medical CTS case history, clinical examination findings, as well as a neurographic result were included. Patients with a relapse operation were not considered in this regard. Apart from a clinical examination and questioning of the patient three and six months after surgery, an electrophysiological examination and a high-resolution sonography of the median nerve were also carried out. Electroneurography and nerve sonography of the median nerve were applied to both hands. A prolonged distal motor latency of the median nerve amounting to 4ms, as well as a slowed nerve conduction velocity below the benchmark value of approx. 45m/s, were classified as pathological findings. In sonography, the largest cross-section area (CSA) of the median nerve was measured by applying transversal slicing to the distal transverse creases of the skin on the palmar surface of the wrist (rasceta) as well as 5cm proximal to the rasceta. The highest CSA values were determined visually. In cases of doubt several transversal slices were made until the highest CSA value could be identified.
The average age at which the disease was contracted amounted to 56.9 years. With one exception, all patients complained of nocturnal brachialgia before surgery (74, 96.2%). As far as neurological symptoms were concerned, 72 patients had paresthesias (93.6%) and 29 patients (37.7%) felt permanent numbness. A thenar atrophy of higher degree was diagnosed in two patients (2.6%). These complaints had improved in the patients surveyed in the scope of postoperative evaluations after three and six months.
Patients with motor deficits had a statistically significantly longer preoperative distal motor latency (10.5 ± 2.8ms vs. 6.5 ± 2.3ms). We observed an improvement of distal motor latency in 98% of the patients three months and six months after surgical decompression, displaying a statistically significant DML decrease from 6.6 ± 2.4ms to 4.8 ± 1.0ms and from 6.6 ± 2.4ms to 4.4 ± 1.0ms, respectively. There was a statistically significant correlation between the decrease of the nerve cross-section area and the decrease of distal motor latency.
At the time of the follow-up examination, three months after surgery, we were able to document a decrease in the CSA value in 80% of the patients. The mean CSA value decreased from 14.7 ± 4.4mm² to 12.4 ± 3.4 mm². Six months after surgical decompression the mean CSA value decreased from 14.3 ± 4.4mm² to 9.6 ± 2.3mm². Patients with a preoperative CSA value of ≥ 12mm² displayed a significantly greater relative reduction of their postoperative CSA value. Concerning all preoperative and postoperative parameters in patients who had undergone either open or endoscopic surgery, none revealed significant differences. Neither could an exploratory analysis (i.e. age, diabetic diseases) reveal any significant correlation between the parameters. Prior to surgery, a flattening of the median nerve or a loss of its fascicular structure (texture) had also been seen to exist in patients, apart from the nerve's larger cross-section area. Nerve sonography is an inexpensive and fast method. It is also extraordinarily reliable in the assessment of the CTS diagnosis and suits the necessary demands. We achieved a good efficiency with our sonographic examinations in the study presented here. New and improved developments show that high-resolution sonography will gain more and more significance in future CTS diagnostics.
The ubiquitin-related SUMO system represents a versatile post-translational modification pathway controlling a variety of cellular signalling networks. In mammalian cells, lysine residues of target proteins can be covalently modified with three SUMO isoforms (SUMO1, SUMO2 and SUMO3) resulting in conjugation of either single SUMO moieties or formation of poly-SUMO chains. Importantly, SUMO modification is a reversible process, where the deconjugation of SUMO from its substrates is mediated by SUMO proteases. In humans, the best-characterized subfamily is the SENP family of SUMO-specific isopeptidases comprised of SENP1-3 and SENP5-7. For undisturbed cellular signalling events, a proper balance of SUMO conjugation and deconjugation is crucial. SENPs fulfil the important function of counteracting SUMOylation. A key question is how the relatively low number of SENPs specifically controls the SUMOylation status of hundreds of cellular proteins.
The aim of this thesis was to uncover the regulation and substrate specificity of distinct SUMO isopeptidases in order to better understand their role in cellular signalling pathways.
In the first part of this work, we investigated the influence of hypoxia on SUMO signalling, in particular on the activity of SENPs. Importantly, we found that the catalytic activity of distinct SENPs (especially SENP1 and SENP3) is strongly but reversibly diminished under low oxygen. As a consequence, the SUMO modification of a specific subset of proteins is changed under hypoxia. We specifically identified proteins being hyperSUMOylated after 24 hours of hypoxia by SUMO1 immunoprecipitation followed by mass spectrometry. We further validated the transcriptional co-repressor BHLHE40 as hypoxic SUMO target and confirmed SENP1 as responsible isopeptidase for deconjugation of SUMOylated BHLHE40. We provide evidence that SUMO conjugation to BHLHE40 enhances its repressive functions on the expression of the metabolic master regulator PGC-1α. Therefore we propose a model where inactivation of SENP1 under hypoxia results in SUMOylated BHLHE40, possibly contributing to metabolic reprogramming under hypoxia.
To get insight into substrate selectivity of SENP family members, in particular SENP3 and SENP6, we choose a proteomic profiling strategy. For the identification of specific SUMO substrates controlled by SENP3, we applied a large-scale IP-MS approach in SENP3 KO and WT cells. The most strongly induced SUMO targets in the absence of SENP3 were key regulators of ribosome maturation. We identified factors involved in the remodelling of both 90S and 60S pre-ribosomes. SENP3 has already been described as being critically involved in maturation of the pre-60S subunit and 28S rRNA processing. Previously described SENP3-regulated master targets in this process are the ribosome maturation factors PELP1 and Las1L. Importantly, both were also identified as the most significantly regulated SENP3 targets in our unbiased proteomic approach. Importantly, however, enhanced SUMOylation was also detected on 90S-associated regulators, such as BMS1. Altogether, these data strengthen the functional link between SENP3 and ribosome biogenesis and point to a role of SENP3 beyond 60S maturation.
In addition to SENP3, we explored the substrate specificity of SENP6, which mainly acts on polymeric SUMO2/3 chains. Applying a proteomic profiling strategy, we were able to identify SENP6-controlled SUMO networks functioning in DNA damage response as well as chromatin organization. We demonstrated that SENP6 reverses polySUMOylation of several subunits of the cohesin complex, thereby regulating the SUMOylation status and chromatin association of this complex. Furthermore, we found a tight interaction of SENP6 with the hPSO4/PRP19 complex, involved in DNA damage response by activation of the ATR-CHK1 signalling cascade. In cells depleted of SENP6, we observe deficient recruitment of the co-activator ATRIP to chromatin which results in diminished CHK1 activation. We therefore illustrate a general role of SENP6 in the control of chromatin-associated protein networks involved in genome integrity and chromatin organization.