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In recent years, a number of functional and structural neuroimaging studies have investigated the neural bases of aggressive and violent behaviour in children and adolescents. Most functional neuroimaging studies have persued the hypothesis that pathological aggression is a consequence of deficits in the neural circuits involved in emotion processing. There is converging evidence for abnormal neural responses to emotional stimuli in youths with a propensity towards aggressive behaviour. In addition, recent neuroimaging work has suggested that aggressive behaviour is also associated with abnormalities in neural processes that subserve both the inhibitory control of behaviour and the flexible adaptation of behaviour in accord with reinforcement information. Structural neuroimaging studies in children and adolescents with conduct problems are still scarce, but point to deficits in brain structures involved in the processing of social information and in the regulation of social and goal-directed behaviour. The indisputable progress that this research field has made in recent years notwithstanding, the overall picture is still rather patchy and there are inconsistencies between studies that await clarification. Despite this, we attempt to provide an integrated view on the neural abnormalities that may contribute to various forms of juvenile aggression and violence, and discuss research strategies that may help to provide a more profound understanding of these important issues in the future. Keywords: aggression, violence, conduct disorder, fMRI, brain imaging, psychiatry
Short-term memory requires the coordination of sub-processes like encoding, retention, retrieval and comparison of stored material to subsequent input. Neuronal oscillations have an inherent time structure, can effectively coordinate synaptic integration of large neuron populations and could therefore organize and integrate distributed sub-processes in time and space. We observed field potential oscillations (14–95 Hz) in ventral prefrontal cortex of monkeys performing a visual memory task. Stimulus-selective and performance-dependent oscillations occurred simultaneously at 65–95 Hz and 14–50 Hz, the latter being phase-locked throughout memory maintenance. We propose that prefrontal oscillatory activity may be instrumental for the dynamical integration of local and global neuronal processes underlying short-term memory.
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.
Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT1-, H2O2-, and PYK2-dependent manner. Low concentrations (1–100 µmol/liter) of H2O2 stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 µmol/liter H2O2 induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H2O2-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS–/– mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H2O2. In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2y/– mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2y/– mice. Collectively, endothelial PYK2 activation by Ang II and H2O2 causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin–angiotensin system and elevated redox stress.
The peroxisome proliferator activated receptor gamma (PPARgamma) plays an eminent role during alternative activation of macrophages and resolution of inflammation. As an antiinflammatory signaling molecule, it seems likely that it is tightly regulated dependent on the state of the immune response. There is growing evidence that PPARgamma expression is reduced during inflammation, whereas molecular mechanisms are illdefined. Even though, its role in immunosuppression is getting more definite. Apoptotic cells (AC) provoke an active repression of pro-inflammatory responses inter alia by the inhibition of pro-inflammatory cytokine expression or attenuated generation of reactive oxygen species (ROS). The reduced formation of ROS was attributed to PPARgamma activation, while mechanisms behind the reduced cytokine expression remained unclear. Therefore, my Ph.D. thesis addressed the role of PPARgamma during inhibited cytokine synthesis in response to AC and the regulation of PPARgamma expression during an inflammatory response, which was initiated by lipopolysaccharide (LPS) exposure. In the first part of the thesis, I investigated the role of PPARgamma in coordinating the attenuation of pro-inflammatory cytokine expression in response to AC. Exposing murine RAW264.7 macrophages to AC prior to LPS-stimulation, reduced NFKB transactivation and lowered target gene expression of e.g. TNFalpha and IL-6 compared to controls. In macrophages over-expressing a dominant negative (d/n) mutant of PPARgamma, NFKB transactivation in response to LPS was restored, while using macrophages from myeloid lineage-specific conditional PPARgamma knock-out mice proved that PPARgamma transmitted the anti-inflammatory response delivered by AC. Domain analysis revealed that amino acids 32-250 are essential for inhibition of NFKB. Mutation of a SUMOylation (SUMO: small-ubiquitin related modifier) site in this region (K77R) and interfering SUMOylation by silencing the SUMO E3 ligase PIAS1 (protein inhibitor of activated Stat1) eliminated AC-provoked NFKB inhibition and concomitant TNFalpha expression. Chromatin-immunoprecipitation assays demonstrated that AC prevented the LPS-induced removal of nuclear receptor co-repressor (NCoR) from the KB response element within the TNFalpha promoter. I concluded that AC induce PPARgamma SUMOylation to attenuate the removal of NCoR, thereby blocking transactivation of NFKB. This contributes to an anti-inflammatory phenotype shift in macrophages in response to AC, by lowering pro-inflammatory cytokine production. The second part addressed molecular mechanisms responsible for reduced PPARgamma expression upon LPS exposure. PPARgamma gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer’s disease is associated with impaired PPARgamma expression. Initiation of an inflammatory response by exposing primary human macrophages to LPS revealed a rapid decline of PPARgamma1 expression. PPARgamma1 mRNA decrease was prevented by inhibition of NFKB and also after pre-treatment with the PPARgamma agonist rosiglitazone, suggesting a NFKB-dependent pathway, because activated PPARgamma is known to inhibit NFKB transactivation. Since promoter activities were not affected by LPS, I focused on mRNA stability and noticed a decreased PPARgamma1 mRNA half-life. RNA stability is often regulated via 3’ untranslated regions (UTRs). Therefore, I analyzed the impact of the PPARgamma-3’UTR by luciferase assays. LPS significantly reduced luciferase activity of pGL3-PPARgamma-3’UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion of a potential miR-27a/b binding site within the 3’UTR completely restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, whereas the mature miR-27 mimicked the effect of LPS. MiR-27b was at least partially induced by NFKB, thus correlating with NFKB-dependent PPARgamma1 mRNA decrease. Since deletion of the miR-27 site also containing an AU-rich element (ARE) completely abrogated LPS-induced reduction but inhibition of miR-27b only partially restored PPARgamma1 mRNA expression, I suggested an additional implication of an ARE-binding protein. I provide evidence that LPS induces miR-27b, which in turn destabilizes PPARgamma1 mRNA. Understanding the molecular mechanism of PPARgamma mRNA destabilization, might help to rationalize inflammatory diseases associated with impaired PPARgamma expression. Even though, further experiments are needed to clarify the potential involvement of ARE-binding proteins.
It has been shown that stem and progenitor cells are therapeutically effective after i.v application. Yet, many aspects regarding intracellular signaling pathways which are involved in the homing and local action of these cells still have to be elucidated. In this work, it was aimed to investigate the role of the small GTPase Rap1 in adhesion activation in Hematopoietic Stem and Progenitor Cells (HSC/HPC) and in Mesenchymal Stem Cells (MSC). The potential role of Rap1 was assessed in, mice which were homozygote negative for the expression of the Rap1a gene. Peripheral blood lymphocyte counts as well as numbers of HPCs in the blood were decreased in Rap1a-/- mice compared to wild-type controls. Additionally the adhesion capability of HPCs from Rap1a-/- to the endothelial ligand, Vascular Cell Adhesion Molecule – 1 under shear stress was decreased. The hematopoietic repopulation potential of Rap1a-/- HPC was however not decreased in a competitive bone marrow transplantation model, indicating that deficiency of Rap1a in HSC/HPC does not negatively affect their ability to interact with the bone marrow microenvironment. In contrast, the isolation of MSC was not possible from Rap1a-/- bone marrow, indicating an altered situation in the bone marrow niche through changed stromal cell behaviour. Instead, Rap1a+/- MSC could be isolated and showed an adhesion deficit under shear stress. In contrast, no differences were noted in their differentiation potential. In a mouse homing model, the overall ability of the Rap1a+/- MSC to home to different tissues was found preserved. Finally, in a murine subcutaneous carcinoma model, cells with an HPC phenotype were observed to be present in the tumor microenvironment, and it was shown that they home directly to tumors. Since HPC isolated from bone marrow were able to differentiate into cells with a pro-angiogenic phenotype in vitro, HPC may be of relevance for neovascularization, as tumor-infiltrating progenitor cells. The results of the study should contribute to the understanding of the regulation of progenitor cell homing behaviour in situations simulating cell therapy approaches in preclinical situations.
Sepsis is caused by infection and often followed by an overwhelming inflammatory response. This can lead to shock, organ failure and even death. Each year approximately 60,000 people die in Germany due to sepsis. There is good evidence that sepsis is associated with failure of the hypothalamic-pituitary-adrenal-axis. In patients with sepsis, glucocorticoids (e.g. corticosterone, cortisol) released from adrenal glands play an essential role in preventing an excessive pro-inflammatory response. Adrenal insufficiency occurs in a large number of patients with septic shock and is associated with an increased mortality. In the innate immune system, Toll-like receptors (TLRs) play a crucial role in its onset by recognizing pathogenassociated molecules. It is well known that there are interactions between the immune and endocrine stress systems; glucocorticoids and TLRs regulate each other in a bi-directional way. Therefore, a coordinated response of the adrenal and immune system is of vital importance for survival during severe inflammation. This experimental study focuses on the role of TLR-2, TLR-4 and TLR-9 during adrenal stress. The results show that in mice, the absence of TLR-2 and TLR-4, but not TLR-9 leads to altered adrenal morphology, relating to size and cellular structure. However, this alteration does not appear to compromise the phenotype of TLR knock-out mice. Mice deficient of TLR-2, 4 and 9 are not able to respond adequately to inflammatory stress induced by their potential ligands lipopolysaccharide (LPS), lipoteichoic acid (LTA) or cytidine phosphate guanosine-oligodeoxynucleotides (CpG-ODN). This impaired adrenal stress response appears to be associated with a decrease in systemic and intra-adrenal cytokine expressions. Taken together, these results suggest that TLR-2, 4 and 9 are key players in the immuno-endocrine response during inflammation and SIRS. In conclusion, TLRs play a crucial role in the immune-adrenal crosstalk. This close functional relationship needs to be considered in the treatment of inflammatory diseases where an intact adrenal stress response is required. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis
The nervous system probably cannot display macroscopic quantum (i.e. classically impossible) behaviours such as quantum entanglement, superposition or tunnelling (Koch and Hepp, Nature 440:611, 2006). However, in contrast to this quantum "mysticism" there is an alternative way in which quantum events might influence the brain activity. The nervous system is a nonlinear system with many feedback loops at every level of its structural hierarchy. A conventional wisdom is that in macroscopic objects the quantum fluctuations are self-averaging and thus not important. Nevertheless this intuition might be misleading in the case of nonlinear complex systems. Because of a high sensitivity to initial conditions, in chaotic systems the microscopic fluctuations may be amplified upward and thereby affect the system’s output. In this way stochastic quantum dynamics might sometimes alter the outcome of neuronal computations, not by generating classically impossible solutions, but by influencing the selection of many possible solutions (Satinover, Quantum Brain, Wiley & Sons, 2001). I am going to discuss recent theoretical proposals and experimental findings in quantum mechanics, complexity theory and computational neuroscience suggesting that biological evolution is able to take advantage of quantum-computational speed-up. I predict that the future research on quantum complex systems will provide us with novel interesting insights that might be relevant also for neurobiology and neurophilosophy.
A survey on worries of pregnant women - testing the German version of the Cambridge Worry Scale
(2009)
Background: Pregnancy is a transition period in a woman's life characterized by increased worries and anxiety. The Cambridge Worry Scale (CWS) was developed to assess the content and extent of maternal worries in pregnancy. It has been increasingly used in studies over recent years. However, a German version has not yet been developed and validated. The aim of this study was (1) to assess the extent and content of worries in pregnancy on a sample of women in Germany using a translated and adapted version of the Cambridge Worry Scale, and (2) to evaluate the psychometric properties of the German version. Methods: We conducted a cross-sectional study and enrolled 344 pregnant women in the federal state of Baden-Wurttemberg, Germany. Women filled out structured questionnaires that contained the CWS, the Spielberger-State-Trait-Anxiety Inventory (STAI), as well as questions on their obstetric history. Antenatal records were also analyzed. Results: The CWS was well understood and easy to fill in. The major worries referred to the process of giving birth (CWS mean value 2.26) and the possibility that something might be wrong with the baby (1.99), followed by coping with the new baby (1.57), going to hospital (1.29) and the possibility of going into labour too early (1.28). The internal consistency of the scale (0.80) was satisfactory, and we found a four-factor structure, similar to previous studies. Tests of convergent validity showed that the German CWS represents a different construct compared with state and trait anxiety but has the desired overlap. Conclusions: The German CWS has satisfactory psychometric properties. It represents a valuable tool for use in scientific studies and is likely to be useful also to clinicians.
Macrophages show a remarkable functional plasticity, which enables them to change their phenotype in response to environmental signals. They are key players during infection by initiating inflammation through the release of proinflammatory mediators. Furthermore, macrophages contribute to the resolution of inflammation by phagocytosis of apoptotic granulocytes. Phagocytosis of apoptotic cells (AC) induces an anti-inflammatory phenotype in macrophages and protects them against apoptosis. However, mechanistic details provoking these phenotype alterations are incompletely understood. Therefore, the aim of my Ph.D. thesis was to investigate the molecular basis of anti-inflammatory macrophage polarization. In the first part of my studies, I investigated the expression of heme oxygenase (HO)-1 in macrophages following treatment with supernatants from AC. HO-1 catalyzes the first and rate-limiting step of heme degradation and potentially bears anti-inflammatory as well as anti-apoptotic potential. I was able to show biphasic upregulation of HO-1 by AC supernatants. The first phase of HO-1 induction at 6 h required activation of p38 MAPK and was accomplished by the bioactive lipid sphingosine-1-phosphate (S1P) engaging S1P receptor 1 (S1P1). However, the second wave of HO-1 induction at 24 h was attributed to autocrine signaling of vascular endothelial growth factor (VEGF) A, whose expression was facilitated by S1P. The release of VEGFA from macrophages was STAT1-dependent, whereas VEGFA itself acted on the macrophage HO-1 promoter via STAT1/STAT3 heterodimer binding. Knockdown of HO-1 revealed its relevance in promoting enhanced expression of the anti-apoptotic proteins B cell leukemia/lymphoma-2 (Bcl-2) and B cell leukaemia/lymphoma-x long (Bcl-XL), as well as the anti-inflammatory adenosine receptor A2A. MHC II and indoleamine 2,3-dioxygenase expression were also affected by ACsupernanatants, but were not HO-1 dependent. Unexpectedly, S1P1 was also upregulated following treatment with AC supernatants. Thus, I considered whether S1P1 induction could specifically be mediated by alternative macrophage activating factors. The expression of S1P1 was enhanced in the presence of the alternative activation stimuli IL-4 as well as IL-10, whereas it was unchanged following incubations with LPS, interferon-g or S1P. My next aim was to investigate the expression of the different S1P receptor isoforms in macrophages following treatment with supernatants form AC. While the expressions of S1P1 as well as S1P3 were induced by exposure to supernatants from AC, S1P2 expression was unaffected. As S1P1/3 and S1P2 are conflictively involved in the regulation of cell migration, I asked for a correlation between increased S1P receptor expression and enhanced migration rate. Indeed, macrophages showed enhanced motility following treatment with supernatants form AC, which was inhibited in S1P1 knockout macrophages. In summary, my findings indicate that HO-1, which is induced by AC-derived S1P, is critically involved in macrophage polarization towards an alternatively activated macrophage phenotype. S1P1 seems to represent a central checkpoint during macrophage activation. On the one hand, S1P1 is induced by supernatants form AC and promotes migration of macrophages. On the other hand, it mediates the induction of HO-1, which is accompanied by antiinflammatory as well as anti-apoptotic signaling. Furthermore, my studies provide evidence that upregulation of HO-1 and S1P1 in macrophages may contribute to the resolution of inflammation by establishing an anti-inflammatory macrophage phenotype and provoking macrophage migration along the vascular S1P gradient out of an inflammatory environment into the lymph.