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The effect of 10 Hz transcranial alternating current stimulation (tACS) on corticomuscular coherence
(2013)
Synchronous oscillatory activity at alpha (8–12 Hz), beta (13–30 Hz), and gamma (30–90 Hz) frequencies is assumed to play a key role for motor control. Corticomuscular coherence (CMC) represents an established measure of the pyramidal system's integrity. Transcranial alternating current stimulation (tACS) offers the possibility to modulate ongoing oscillatory activity. Behaviorally, 20 Hz tACS in healthy subjects has been shown to result in movement slowing. However, the neurophysiological changes underlying these effects are not entirely understood yet. The present study aimed at ascertaining the effects of tACS at 10 and 20 Hz in healthy subjects on CMC and local power of the primary sensorimotor cortex. Neuromagnetic activity was recorded during isometric contraction before and at two time points (2–10 min and 30–38 min) after tACS of the left primary motor cortex (M1), using a 306 channel whole head magnetoencephalography (MEG) system. Additionally, electromyography (EMG) of the right extensor digitorum communis (EDC) muscle was measured. TACS was applied at 10 and 20 Hz, respectively, for 10 min at 1 mA. Sham stimulation served as control condition. The data suggest that 10 Hz tACS significantly reduced low gamma band CMC during isometric contraction. This implies that tACS does not necessarily cause effects at stimulation frequency. Rather, the findings suggest cross-frequency interplay between alpha and low gamma band activity modulating functional interaction between motor cortex and muscle.
The present study investigates the hemispheric contributions of neuronal reorganization following early single-sided hearing (unilateral deafness). The experiments were performed on ten cats from our colony of deaf white cats. Two were identified in early hearing screening as unilaterally congenitally deaf. The remaining eight were bilaterally congenitally deaf, unilaterally implanted at different ages with a cochlear implant. Implanted animals were chronically stimulated using a single-channel portable signal processor for two to five months. Microelectrode recordings were performed at the primary auditory cortex under stimulation at the hearing and deaf ear with bilateral cochlear implants. Local field potentials (LFPs) were compared at the cortex ipsilateral and contralateral to the hearing ear. The focus of the study was on the morphology and the onset latency of the LFPs. With respect to morphology of LFPs, pronounced hemisphere-specific effects were observed. Morphology of amplitude-normalized LFPs for stimulation of the deaf and the hearing ear was similar for responses recorded at the same hemisphere. However, when comparisons were performed between the hemispheres, the morphology was more dissimilar even though the same ear was stimulated. This demonstrates hemispheric specificity of some cortical adaptations irrespective of the ear stimulated. The results suggest a specific adaptation process at the hemisphere ipsilateral to the hearing ear, involving specific (down-regulated inhibitory) mechanisms not found in the contralateral hemisphere. Finally, onset latencies revealed that the sensitive period for the cortex ipsilateral to the hearing ear is shorter than that for the contralateral cortex. Unilateral hearing experience leads to a functionally-asymmetric brain with different neuronal reorganizations and different sensitive periods involved.
Human deep sleep is characterized by reduced sensory activity, responsiveness to stimuli, and conscious awareness. Given its ubiquity and reversible nature, it represents an attractive paradigm to study the neural changes which accompany the loss of consciousness in humans. In particular, the deepest stages of sleep can serve as an empirical test for the predictions of theoretical models relating the phenomenology of consciousness with underlying neural activity. A relatively recent shift of attention from the analysis of evoked responses toward spontaneous (or “resting state”) activity has taken place in the neuroimaging community, together with the development of tools suitable to study distributed functional interactions. In this review we focus on recent functional Magnetic Resonance Imaging (fMRI) studies of spontaneous activity during sleep and their relationship with theoretical models for human consciousness generation, considering the global workspace theory, the information integration theory, and the dynamical core hypothesis. We discuss the venues of research opened by these results, emphasizing the need to extend the analytic methodology in order to obtain a dynamical picture of how functional interactions change over time and how their evolution is modulated during different conscious states. Finally, we discuss the need to experimentally establish absent or reduced conscious content, even when studying the deepest sleep stages.
In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in in vitro experiments on mast cells of the human mast-cell line HMC-1 the effects of the physical stimuli; mechanical stress was applied by superfusion of the cells with hypotonic solution, heat was applied by incubation of the cells at 52°C, and red laser light of 657 nm was used for irradiation. We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca2+ rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.
Human adipose tissue derived stem cells promote liver regeneration in a rat model of toxic injury
(2013)
In the light of the persisting lack of donor organs and the risks of allotransplantations, the possibility of liver regeneration with autologous stem cells from adipose tissue (ADSC) is an intriguing alternative. Using a model of a toxic liver damage in Sprague Dawley rats, generated by repetitive intraperitoneal application of retrorsine and allyl alcohol, the ability of human ADSC to support the restoration of liver function was investigated. A two-thirds hepatectomy was performed, and human ADSC were injected into one remaining liver lobe in group 1 (n = 20). Injection of cell culture medium performed in group 2 (n = 20) served as control. Cyclosporine was applied to achieve immunotolerance. Blood samples were drawn weekly after surgery to determine liver-correlated blood values. Six and twelve weeks after surgery, animals were sacrificed and histological sections were analyzed. ADSC significantly raised postoperative albumin (P < 0.017), total protein (P < 0.031), glutamic oxaloacetic transaminase (P < 0.001), and lactate dehydrogenase (P < 0.04) levels compared to injection of cell culture medium alone. Transplanted cells could be found up to twelve weeks after surgery in histological sections. This study points towards ADSC being a promising alternative to hepatocyte or liver organ transplantation in patients with severe liver failure.
Retroviral vectors are powerful tools in clinical gene therapy as they integrate permanently into the target cell genome and thus guarantee long-term expression of transgenes. Therefore, they belong to the most frequently used application platforms in clinical gene therapy involving a broad range of different target cells and tissues. However, stable genomic integration of retroviral vectors can be oncogenic, as reported in several animal models and in clinical trials. In particular, γ-retroviral vectors, which derive from naturally mutagenic γ-retroviruses, integrate semirandomly into the host genome with regard to the target sequence, but have a preference for regions of active transcription and regulatory elements of transcriptionally active genes. The integration can result in overexpression of adjacent genes or disruption of ‘target’ gene expression. Moreover, γ-retroviral integration can cause modified transcripts and proteins through alternative or aberrant splicing or through premature termination of transcription.
Initially, the event of insertional mutagenesis and subsequent induction of leukemia by the genotoxicity of a γ-retroviral vector was described in a mouse model after genetic modification of hematopoietic stem cells (HSCs). Vector-related activation and overexpression of the oncogene ecotropic viral integration site-1 (Evi1) fostered clonal outgrowth and leukemogenesis. Additional genotoxic events of γ-retroviral vectors were observed in clinical HSC gene therapy trials for X-linked severe combined immune deficiency (SCID-X1), chronic granulomatous disease (X-CGD), and Wiskott-Aldrich Syndrome (WAS). But, genotoxicity induced by γ-retroviral vectors has never been described in clinical gene therapy trials involving adoptive transfer of genetically modified mature T lymphocytes. This fact is surprising, since T cells are long-lived and have a high capacity of self-renewal.
In a previous study, the susceptibility towards oncogenic transformation of mature T cells and HSCs after genetic modification was compared. It could be demonstrated that T-cell receptor (TCR)-polyclonal mature T cells are far less prone to transformation after γ-retroviral transfer of (proto-)oncogenes in vivo than HSCs. Additional experiments revealed that TCR-oligoclonal (OT-I and P14) mature T cells are transformable in the same setting and give rise to mature T-cell lymphomas (MTCLs).
In the present thesis, the susceptibility of mature T cells towards insertional mutagenesis was investigated. Within the first part of the thesis, retroviral integration sites (RISs) from 33 murine MTCLs were retrieved and subsequently analyzed in terms of integration pattern, detection of common integration sites (CIS) and gene ontology (GO). As these bioinformatic results demonstrated that insertional mutagenesis most likely contributed to mature T-cell lymphomagenesis, the susceptibility of mature T cells was directly assessed in a mouse model. Therefore, murine TCR-oligoclonal OT-I T cells were transduced with an enhanced green fluorescent protein (EGFP) encoding γ-retroviral vector and gene-modified T cells were transplanted into RAG1-/- mice. After 16 months, including one round of serial transplantation, a case of MTCL emerged. Tumor cells were characterized by CD3, CD8, TCR and ICOS expression. Integration site analysis via ligation-mediated polymerase chain reaction (LM-PCR) revealed a proviral insertion in the Janus kinase 1 (Jak1) gene. Subsequent overexpression of Jak1 could be demonstrated on transcriptional and protein level. Furthermore, T-cell lymphoma cells were characterized by an activated Jak/STAT-pathway as signal transducer and activator of transcription 3 (STAT3) was highly phosphorylated. The overexpression of Jak1 was causally implicated in tumor growth promotion as specific pharmacological inhibition of Jak1 using Ruxolitinib significantly prolonged survival of mice transplanted with these Jak1-activated tumor cells. A concluding systematic metaanalysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis.
This was the first reported case of an insertional mutagenesis event in mature T cells in vivo. Thus, the results obtained in this thesis underline the importance of long-term monitoring of genetically modified T cells in vivo and the evaluation of vector toxicology and safety in T-cell based gene therapies. In particular, the transduction of T cells with a recombinant TCR or CAR (chimeric antigen receptor) bears a risk enhancement, as normal T-cell homeostasis is perturbed besides the general risk of insertional mutagenesis.
The objective of this systematic review was to assess tooth wear against ceramic crowns in posterior region in vitro and in vivo. An electronic PubMed search was conducted to identify studies on tooth wear against ceramic crowns in posterior region. The selected studies were analyzed in regard to type of crowns, natural antagonist, measuring protocol and outcome. From a yield of 1 000 titles, 43 articles were selected for full-text analysis; finally, no in vitro and only five in vivo studies met the inclusion criteria. As there is heterogeneity in design, used measuring method, ceramics and analysis-form, a meta-analysis was not possible. Results of these studies are very controversial which makes a scientifically valid comparison impossible. This review indicated that some all-ceramic crowns are as wear friendly as metal-ceramic crowns. Up to now, it has been impossible to associate tooth wear with any specific causal agent. The role of ceramic surface treatment that might be responsible for the changing in rate of tooth wear seems undetermined as yet through clinical trials. The literature reveals that studies on this topic are subject to a substantial amount of bias. Therefore, additional clinical studies, properly designed to diminish bias, are warranted.
Choriocarcinoma is the most malignant tumor of gestational trophoblastic disease arising from any gestation. It has a tendency toward relapse as well as metastasis. Here, a case of relapsed high-risk choriocarcinoma (FIGO stage IV, WHO score 12) in a 37-year-old female presenting with vaginal bleedings is described. Relapse developed at the site of the surgical scar from hysterectomy that had been performed 2 years earlier. Although the patient was treated with aggressive chemotherapy, she was in a bad general condition and died from infection and liver insufficiency.
Background: Self-management support is a key component of effective chronic care management, yet in practice appears to be the least implemented and most challenging. This study explores whether and how self-management support is integrated into chronic care approaches in 13 European countries. In addition, it investigates the level of and barriers to implementation of support strategies in health care practice.
Methods: We conducted a review among the 13 participating countries, based on a common data template informed by the Chronic Care Model. Key informants presented a sample of representative chronic care approaches and related self-management support strategies. The cross-country review was complemented by a Dutch case study of health professionals’ views on the implementation of self-management support in practice.
Results: Self-management support for chronically ill patients remains relatively underdeveloped in Europe. Similarities between countries exist mostly in involved providers (nurses) and settings (primary care). Differences prevail in mode and format of support, and materials used. Support activities focus primarily on patients’ medical and behavioral management, and less on emotional management. According to Dutch providers, self-management support is not (yet) an integral part of daily practice; implementation is hampered by barriers related to, among others, funding, IT and medical culture.
Conclusions: Although collaborative care for chronic conditions is becoming more important in European health systems, adequate self-management support for patients with chronic disease is far from accomplished in most countries. There is a need for better understanding of how we can encourage both patients and health care providers to engage in productive interactions in daily chronic care practice, which can improve health and social outcomes.
Objectives: This study explores whether and how self-management support (SMS), a key element of well-coordinated chronic care, is integrated into existing chronic care approaches in 13 European countries.
Methodology: An expert review was conducted using a data template informed by the Chronic Care Model. Key informants (researchers and policymakers) from the 13 countries presented a sample of exemplary chronic care approaches and related SMS strategies. This was complemented by interviews with 27 Dutch care professionals investigating SMS implementation in practice.
Results: SMS remains relatively underdeveloped in Europe. Country-specific strategies are similar in involved providers (nurses) and settings (primary care), yet differ considerably in mode, format and materials used. SMS focuses mainly on patients medical and behavioral management, and less on emotional management. According to Dutch providers, barriers in financing and medical culture (e.g. length of consultation, patient-doctor communication) hamper implementation of SMS as an integral part of chronic care.
Conclusion: While Europe might increasingly be talking the talk of patient participation in chronic care, it appears far from walking the walk. Care professionals experience difficulties in operationalizing SMS in their daily routines. Stronger integration with the health promotion field may help patients and professionals to engage in productive partnerships.
Objective: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink−/−) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency.
Methods: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation.
Results: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink−/− larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink−/− larvae. There was also marked microglial activation in pink−/− larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis.
Interpretation: Pink1−/− zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD. Ann Neurol 2013;74:837–847
Celiac disease (CD) is an immune-mediated enteropathy that is characterized by intraepithelial lymphocytosis, crypt hyperplasia, and villous atrophy. Prevalence is high and has been estimated to range between 0.5% and 1.5%. Capsule endoscopy (CE) has a sensitivity and specificity of approximately 90%. CD is an important differential diagnosis for diagnostic workup for anemia, malabsorption, or diarrhea, and must be recognized reliably by the investigator. Moreover, CE is the preferred method to screen for complications in CD, such as enteropathy-associated T-cell lymphoma, ulcerative jejunitis, and small bowel adenocarcinoma. This article is part of an expert video encyclopedia.
Die offensichtlichen Ähnlichkeiten zwischen Neurasthenie und Burnout dürfen den wissenschaftlichen Blick jedoch nicht dazu verleiten, vorschnell von einer substantiellen Identität auszugehen. Es ist keineswegs gleichgültig, unter welchem Namen ein Leiden amtiert. Vielmehr gehe ich davon aus, dass die Etablierung eines neuen Begriffs ein Ereignis ist, das genauer in den Blick genommen zu werden verdient, weil es auf eine veränderte Problemlage hinweist. Die Frage lautet also, wie genau sich das Verhältnis zwischen Neurasthenie und Burnout darstellt, was diese beiden Begriffe trennt und verbindet, welche semantischen Bedeutungsebenen sich in ihnen jeweils abgelagert haben und welche Rückschlüsse sich aus der Untersuchung dieser Bedeutungsschichten für das Verständnis unserer Gegenwart möglicherweise ziehen lassen. Der erste Schritt einer solchen Fragestellung muss immer darin bestehen, die Phänomene gegeneinander zu legen und sie auf ihre Gemeinsamkeiten und Unterschiede hin zu untersuchen, um auf dieser Grundlage eine schärfere Kontur ihrer Besonderheiten zu erlangen - was im Folgenden geschehen soll.
Zieht man die vergeblichen Versuche der Mediziner und Philosophen in Betracht, eine sowohl praktikable, theoretisch konsistente und ethisch vertretbare Definition von Krankheit zu erarbeiten, wird die Differenz von Krankheitsbegriff und -metapher insgesamt problematisch. Der aktuell gebräuchliche, systemtheoretische Krankheitsbegriff bezieht sich beispielsweise gar nicht mehr auf Lebewesen, sondern auf die Kopplung bio-psycho-sozialer Systeme. Die Bindestriche in diesem Begriff übernehmen gewissermaßen die Funktion der Metapher: den Sprung über Kategoriengrenzen. Wenn Metaphern in den Wissenschaften grundsätzlich keinen guten Ruf genießen, gilt dies insbesondere für Krankheitsmetaphern. Zuletzt ist ihre Bedeutung für die pseudo-medizinische Legitimation und Durchführung des Holocaust umfangreich erforscht worden. Mit dem Paradigmenwechsel von der Hygiene zur Bakteriologie wurden Krankheiten demnach nicht mehr auf äußere Umstände, sondern auf labortechnisch identifizierbare Kontagien zurückgeführt. Diese medizinische 'Visualisierung des Feindes', welche Robert Koch durch die Markierung von Gewebeschnitten mit Annelinfarbe entscheidend voranbrachte, bot zusammen mit der monokausalen und ontologisierenden Erklärung von Krankheiten große metaphorische Anschlussflächen für die (bio-)politischen Diskurse, die sich seit dem 19. Jahrhundert mehr und mehr mit der Sicherung und Steigerung des Lebens beschäftigten.
The prevalence of food allergies has increased in the westernized countries during the past decades. Clinical manifestations of food allergies involve the skin (e.g. atopic dermatitis), the respiratory tract (e.g. rhinitis, and asthma), the ocular area (e.g. conjunctivitis), the gastrointestinal tract (e.g. food-protein-induced enterocolitis syndrome, food-induced proctocolitis, and eosinophilic gastroenteropathies), and the cardiovascular system (e.g. anaphylaxis). A curative treatment of these diseases has not been established yet. Oral immunotherapy (OIT) has gained attention as a potential therapy for food allergies. Continuous feeding of allergenic diet applied in the model described here mirrors to a certain extent an OIT treatment. It might be therefore useful to investigate efficacy and safety of OIT pre-clinically.
Mouse models have been widely used to analyse novel treatment approaches. Unfortunately, most of them have focussed on IgE-mediated hyperreactivity. Only a limited number of mouse models presenting mixed IgE- and non-IgE-mediated gastrointestinal symptoms and inflammation upon allergen-challenge are available. To study the mechanisms underlying the induction of food-induced gastrointestinal inflammation and subsequent oral tolerance induction, a mouse model of food-induced gastrointestinal allergy was established. BALB/c mice were sensitised with Ovalbumin (OVA) plus ALUM and subsequently challenged by feeding a diet containing egg white (EW diet). During the first seven days on EW diet, OVA-sensitised mice (OVA/ALUM EW mice) developed gastrointestinal symptoms (e.g. weight loss, ruffed fur, soft stool and less mobility) and inflammation in the small intestines accompanied by a strong induction of OVA-specific IgE antibodies and mouse mast cell protease-1 (mMCP-1). Proliferation of CD4+ T cells from spleen of OVA/ALUM EW mice was reduced compared controls. The result indicated that feeding EW diet induced T cell tolerance systemically. In contrast, CD4+ T cells isolated from MLN of OVA/ALUM EW mice showed stronger proliferation upon OVA stimulation in vitro than mice OVA-sensitised but fed a conventional diet, indicating that tolerance was not induced by short-term EW diet. Histological analysis of the small intestinal tissue of OVA/ALUM EW mice revealed strong inflammation present in the duodenum, jejunum and ileum at this time point.
Interestingly, the observed symptoms in OVA/ALUM EW mice resolved spontaneously after 7 days on EW diet, if the feeding was continued. In the next steps the CD4+ T cell-mediated immune response after 28 days continuous EW diet was assessed and revealed that tolerance was induced systemically as well as locally. This was shown by reduced proliferation and cytokine secretion of CD4+ T cells from MLN of OVA/ALUM EW mice after long-term EW diet. However, the inflammation in the jejunum was aggravated instead of resolved at this time point of allergenic diet. Our results suggest that application of OIT in food-allergic patients with gastrointestinal inflammation may need to be reconsidered, since continuous administration of allergenic food may aggravate inflammation in the local tissue. Interestingly, only the jejunum was affected by a worsened condition, whereas duodenum and ileum resolved inflammation. In accordance to the observed jejunal inflammation mMCP-1 levels in the sera were not changed. Allergen-specific IgE levels did not reach baseline level after long-term EW diet, although they were reduced compared to levels in mice after 7 days on EW diet. This result suggests that residual OVA-specific IgE antibodies would promote the jejunal inflammation by sustained activation of mast cells. Furthermore, our results suggest that IL-4 produced by activated Th2 cells could be an effector molecule to induce intestinal inflammation.
The second part of this thesis was aimed at verifying the hypothesis that IgE-mediated mast cell activation is a major effector mechanism in induction of chronic inflammation induced by long-term EW diet. For that mice deficient for FcεRI, a high affinity IgE receptor, were used. These mice were sensitised with OVA and fed EW diet as described for WT mice. Although FcεRI-deficient mice showed an intact Th2 immunity with IgE production, weight loss in the receptor-deficient mice was moderately induced by EW diet compared to WT mice, suggesting that this clinical symptom during the acute phase of allergic response is associated with IgE-mediated mechanisms. Surprisingly, the deficient mice presented comparable intestinal inflammation on day seven of EW diet as WT mice did. However, if EW diet was continued, recovery of intestinal inflammation was observed in FcεRI-deficient mice in contrast to WT mice. These results suggest that the induction of intestinal inflammation is not IgE-dependent. Nevertheless, this does not rule out a potential role of mast cells in the inflammation, because of their IgE-independent activation pathways. It also suggests the involvement of T cell-mediated mechanisms during induction of jejunal inflammation. Interestingly, the aggravated inflammation seen after long-term EW diet in WT mice seems to be IgE-dependent, considering that it was not observed in FcεRI-deficient mice. The elevated number of mast cells in the intestine of WT mice further led to a hypothesis that their continuous activation might be responsible for the chronification of allergic inflammation observed after long-term EW diet. In the context of OIT it further implies that IgE might be a poor prognostic factor for recovery of intestinal inflammation during and after an OIT treatment. In the third part of this thesis regulatory mechanisms employed by the immune system were analysed. Initial results from CD4+ T cells isolated from MLN from OVA/ALUM EW mice showed elevated IL-10 levels in their supernatants after short-term EW diet. IL-10-deficient mice were used to analyse the effect of this immunosuppressive cytokine in the mouse model presented here. However, IL-10-deficient mice tend to develop a strong Th1-dominated immune response. Nevertheless, an accelerated weight loss and slight inflammation of the jejunum was observed after short-term EW diet. Analysis of OVA-specific proliferation and cytokine production CD4+ T cells from Spleen and MLN of IL-10-deficient mice on EW diet suggested that systemic as well as local tolerance was induced after short-term and long-term EW diet feeding, respectively. The result suggests that IL-10 is dispensable for induction of T cell tolerance in our mouse model.
However, the presence of functionally active Tregs was observed during this study in WT mice fed short-term EW diet, suggesting that Tregs might have an important role in regulating the systemic or local immune response. T cell deletion as an alternative immune regulatory mechanism was also observed. Additionally, the efficacy of continuous EW diet (mirroring to a certain extent an OIT treatment) in induction of permanent tolerance was assessed. In OVA-sensitised WT mice continuous allergenic diet was stopped after resolution of clinical symptoms and reintroduced after a defined period on conventional diet. Evaluating the weight development showed that reintroduction of EW diet induced weight loss again, but not as pronounced as seen after short-term EW diet. Also the CD4+ T cell-mediated response was elevated again upon allergen stimulation in vitro. The results suggested that permanent tolerance was not induced in the chosen feeding regime.
The mouse model established and analysed here was used to investigate inflammatory and regulatory mechanisms underlying food-induced gastrointestinal allergy. It presents clinical symptoms and intestinal inflammation (Burggraf et al., 2011). This model is easy to be reproduced in different laboratories, and is useful for testing novel therapy approaches (Schülke et al., 2011; Bohnen et al., 2013). It further provides an opportunity to investigate basic mechanisms underlying OIT. This therapy approach is currently extensively investigated and our mouse model would help to understand the therapeutic mechanism of OIT.
Background: Lung ultrasound has become an emerging tool in acute and critical care medicine. Combined theoretical and hands-on training has been required to teach ultrasound diagnostics. Current computer technology allows for display, explanation, and animation of information in a remote-learning environment.
Objective: Development and assessment of an e-learning program for lung ultrasound.
Methods: An interactive online tutorial was created. A prospective learning success study was conducted with medical students using a multiple-choice test (Trial A). This e-learning program was used as preparation for a certified course followed by an evaluation of trained doctors (Trial B) by linear analogue scales. Pretests were compared with postcourse tests and sustainability tests as well as a posttest of a one-day custom classroom training.
Results: In Trial A, during the learning success study (n = 29), the increase of correct answers was 11.7 to 17/20 in the post-test and to 16.6/20 in the sustainability test (relative change 45.1%, P < 0.0001). E-learning almost equalled scores of classroom-based training regarding gain and retention of factual knowledge. In Trial B, nineteen participating doctors found a 79.5% increase of knowledge (median, 95% CI: 69%; 88%).
Conclusion: The basics of lung ultrasound can be taught in a highly effective manner using e-learning.
Neural oscillations at low- and high-frequency ranges are a fundamental feature of large-scale networks. Recent evidence has indicated that schizophrenia is associated with abnormal amplitude and synchrony of oscillatory activity, in particular, at high (beta/gamma) frequencies. These abnormalities are observed during task-related and spontaneous neuronal activity which may be important for understanding the pathophysiology of the syndrome. In this paper, we shall review the current evidence for impaired beta/gamma-band oscillations and their involvement in cognitive functions and certain symptoms of the disorder. In the first part, we will provide an update on neural oscillations during normal brain functions and discuss underlying mechanisms. This will be followed by a review of studies that have examined high-frequency oscillatory activity in schizophrenia and discuss evidence that relates abnormalities of oscillatory activity to disturbed excitatory/inhibitory (E/I) balance. Finally, we shall identify critical issues for future research in this area.
In unserem visuell geprägten Kulturkreis bestimmt das Gesicht die Identität eines Menschen - doch ab wann kann von einer Gesichtsauflösung gesprochen werden? Deutliche Abweichungen von der in der Gesellschaft bekannten und deshalb akzeptierten Norm lassen sich als Gesichtsdefekte, Gesichtsdeformitäten oder eben als Gesichtsauflösung betrachten. Ursächlich können beispielsweise Unfälle, Infektionen, Tumorerkrankungen oder angeborene Fehlbildungen sein. Der Alterungsprozess sollte hingegen nicht als Gesichtsauflösung interpretiert werden. Hier handelt es sich um eine Strukturanpassung des Gewebes. Aufgabe der Medizin bzw. in diesem Falle der gesichtschirurgischen Profession ist es, dem Vorgang der Gesichtsauflösung entgegen zu wirken. Sie bietet die Möglichkeit, durch rekonstruktive Verfahren das Gesicht in seiner Ursprünglichkeit zu erhalten bzw. wieder herzustellen. Besteht im Falle von Gesichtsdefekten keine Möglichkeit mehr, durch operative Techniken eine adäquate Wiederherstellung zu gewährleisten, so kann auf die Versorgung mit künstlichen Gesichtsteilen zurück gegriffen werden. Ziel ist es, ein dysmorphes (fehlgestaltetes) Gesicht in ein wieder erkennbares, also in dem soziokulturellen Umfeld des Betroffenen akzeptiertes Gesicht umzuwandeln. Angestrebt wird, das durch die unterschiedlichen Ursachen geschädigte Gesicht im Rahmen ästhetischer Aspekte wieder herzustellen. Dadurch soll dem Betroffenen die problemlose Integration in sein soziales Umfeld ermöglicht werden. Für den Betroffenen kann dadurch ein Teil der bekannten Normalität wieder hergestellt sowie ein akzeptables Dasein vermittelt werden.
Gesichtsauflösungen
(2013)
Zu Beginn des 18. Jahrhunderts wird aus einem wiedergeöffneten Stollen der Bergwerke von Falun in Schweden der nahezu unversehrte Leib eines verschütteten Mannes geborgen. Die verblüfften Bergleute blicken in ein frisches Gesicht, in "die noch unveränderten Gesichtszüge eines verunglückten Jünglings" – wie es in der Quelle, in Gotthilf Heinrich von Schuberts Ansichten von der Nachtseite der Naturwissenschaften (1808) heißt. Gleichsam eingelegt in Kupfervitriolwasser hat das Gesicht in 300 Ellen Tiefe überdauert, doch kann dessen Konservierung der Berührung mit Luft und Licht und mithin dem Auge der Umstehenden nicht standhalten. Gesicht und Körper lösen sich auf, zerfallen zu Staub, doch erst, nachdem ein altes Mütterchen "an Krücken und mit grauem Haar" den Konservierten erkennt und an ihre Brust drücken kann: Nicht die Mutter, nein sie ist die Braut des Jünglings von vor fünfzig Jahren, und ihr eingefallenes, "verwelktes" Gesicht kontrastiert der Wirkung des seinen. Die Geschichte vom konservierten Bergmanngesicht ist eine der bekanntesten Kombinationen von Literatur und anorganischer Chemie. Johann Peter Hebel und E.T.A. Hoffmann haben diese Geschichte der Flüssigkeiten als eine Art Antidoton gegen die Auflösungserscheinungen des Gesichts erzählt, die zumindest bei Hebel von einer Verwirrung der Chemikalien und ihrer Wirkungen geprägt ist. Denn die schnelle Auflösung, freilich nach vollzogener Identifikation durch die alte Braut, widerspricht dem chemischen Prozess, durch die jedoch deutlich werden soll, dass der unversehrte Kopf in Vitriol, seine Erkennbarkeit und Präsenz nur um den Preis der Sichtbarkeit zu haben ist: Allein als ein den Blicken und Interaktionen mit den Menschen entzogener Kopf bleibt er sich gleich, ist er intakt, unvergänglich. Damit wird gleichsam auf dem Haupt des aus der Zeitlichkeit herausgefallenen Bergmanns ein Spiel um natürliche und erworbene Konservierungsmodalitäten durch das Vitriol des Berges einerseits und das Gedächtnis der ergrauten Braut andererseits ausgetragen. Als Garanten gegen die Auflösung buhlen sie um die Haltbarkeit und physische Integrität des Menschen, genauer um das Bild von ihm. Durch eine "Verwirrung der Chemikalien", die im Übertrag von der Quelle zur Prosaerzählung erfolgte, wird der Konservierungsstoff Kupfervitriol oft als Eisenvitriol oder folgenreicher und entgegen der Nomenklatur gar als Schwefelsäure überliefert. In dieser starken Säure allerdings hätten sich nicht nur Gesicht und Körper des Bergmanns in maximal zwei Stunden, sondern auch das ganze Bergwerk zersetzt. Im konservierten Bergmann als der Geschichte einer aufgeschobenen Auflösung konvergieren einige Themenfelder der folgenden Beiträge: Neben dem Entzug und der Bergung eines unversehrten Antlitzes, dem eine Art facelifting zuteil wurde, sind es die Umstände seiner Erinnerung und die Bedingungen zu seiner Wiedererkennung, seine Fragilität und sein Zerfall. Themen, in die an dieser Stelle ein wenig eingeführt werden soll.
Bbackground: We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes’ (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).
Methods: The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan–Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis.
Results: With a mean follow-up of 25 months (range, 2.3–63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049).
Conclusion: The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.
Trotz zunehmender Verbesserungen in der Diagnostik und Therapie von Krebserkrankungen leiden onkologische Patienten häufig unter gravierenden tumorund therapiebedingten Symptomen und Nebenwirkungen wie Fatigue, Reduktion der Leistungsfähigkeit und Lebensqualität (Courneya, 2003a; Crevenna et al., 2002; Ferriset al., 2009). Zahlreiche Untersuchungen und Übersichtsarbeiten zeigen, dass körperliche Aktivität in den verschiedenen Phasen der Krebstherapie möglich ist und zu einer Reduktion der Nebenwirkungen sowie zu einer Verbesserung der Lebensqualität und Leistungsfähigkeit führen kann (Cramp & Byron-Daniel, 2012; Jones & Peppercorn, 2010; Mishra et al., 2012b; Schmitz et al., 2010). In aktuellen Leitlinien wird körperliche Aktivität deshalb als wichtige supportive Therapiemaßnahme während der Akuttherapie und im Rahmen der Nachsorge sowie Rehabilitation empfohlen. Analog der zunehmenden Individualisierung medizinischer Diagnostik- und Therapiestrategien in der Onkologie (z. B. vergleichbare oder sequentielle Therapieregime, targeted therapies, Patientenwunsch), gibt es inzwischen auch im Bereich der Sportmedizin Forderungen nach individuell angepassten, effektiven körperlichen Trainingsprogrammen (Jensen et al., 2011). Bei der Erarbeitung dieser Bewegungsangebote sollten Informationen zur Einschätzung der körperlichen Leistungsfähigkeit sowie zu den individuellen persönlichen und medizinischen Voraussetzungen der Betroffenen berücksichtigt werden. Entsprechend muss bei der Planung der körperlichen Aktivität auch die aktuelle Behandlungsphase im Rahmen der onkologischen Therapien einbezogen werden. Neben der zeitlichen Einteilung der Therapiephasen in Akut- oder Rehabilitationsphase gibt es die Möglichkeit, den Therapieprozess in Abhängigkeit der Heilungsaussicht einzuordnen. Dabei wird die Prognose einer Tumorerkrankung in Abhängigkeit des Tumorstadiums, des Lymphknotenbefalls und der möglichen Metastasierung in einen heilbaren (kurativen) und nicht heilbaren (palliativen) Therapieansatz eingestuft. Während ein Großteil der Studien die Wirkung bewegungstherapeutischer Interventionen bei Patienten mit kurativem Therapieansatz untersucht, gibt es bisher nur sehr wenig Untersuchungen bei unheilbar kranken Tumorpatienten (Albrecht & Taylor, 2012). Infolgedessen sind Aussagen zu prognosebezogenen Informationen über die individuelle Leistungsfähigkeit und zu unterschiedlichen physischen und psychischen Reaktionenaufgrund körperlicher Aktivität bei dieser Patientengruppe bisher nur bedingt möglich und erlauben folglich keine zielgruppenspezifischen Empfehlungen.
Angesichts dieses Forschungsdefizits ist das Kernziel der vorliegenden Arbeit, mögliche Unterschiede von Lebensqualität, Fatigue und aerober Kapazität (VO2peak) in Abhängigkeit der Heilungsaussicht (kurativ/palliativ) initial zu identifizieren und gleichzeitig die jeweiligen Veränderungen im Rahmen der Intervention über den Gesamtuntersuchungszeitraum zu überprüfen.
Initial konnten 300 onkologische Patienten (histologisch gesichertes Malignom) mit unterschiedlichen Krebsentitäten, in verschiedenen Behandlungsphasen, mit bekannter klinischer Heilungsprognose (kurativ/palliativ) und unter Berücksichtigung definierter Ein- und Ausschlusskriterien in die Untersuchung eingeschlossen werden. Mit dem Ziel einer individuellen Sportberatung und Trainingsplangestaltung absolvierten die Studienteilnehmer eine sportmedizinische Gesundheits- und Leistungsdiagnostik zur Ermittlung der Ausdauerleistungsfähigkeit und Bestimmung des Trainingsbereichs. Die Messungen erfolgten auf dem Fahrradergometer (0W; 25W Inkrement; 3 Minuten) und umfassten Herzfrequenz, Blutdruck, maximale Sauerstoffaufnahmefähigkeit (VO2peak), Laktatkonzentration und subjektives Belastungsempfinden (Borg Skala). Baseline- und identische Wiederholungs-untersuchungen nach 4-6 und nach 16-20 Wochen dienten gleichzeitig der Erfassung der subjektiven Parameter Lebensqualität und Fatigue (EORTC QLQ-C30) (Aaronson et al., 1993). Der Trainingsplan wurde unter Einbeziehung persönlicher Präferenzen, individueller Leistungsfähigkeit und Empfehlungen zur Gesundheitsprävention der WHO (als Orientierung für den Trainingsumfang von 150 min/Wo.) erstellt und dem Patienten in einem ca. 20minütigen Beratungsgespräch erläutert. Art, Umfang und Häufigkeit des mindestens mit moderater Intensität absolvierten Ausdauertrainings wurde durch die Patienten in einem Trainingstagebuch dokumentiert. Die Gruppenzuteilung erfolgte in Abhängigkeit der Heilungsprognose (kurativ/palliativ) unter Verwendung des TNM-Systems. Patienten mit der Prognose „heilbar“ wurden der kurativen Stichprobe zugeteilt, während Patienten mit histologisch gesichertem Nachweis von Metastasen (M1) als palliativ eingestuft wurden.
Referenzwerte waren für die VO2peak: alters- und geschlechtsentsprechende Normdaten (Median) des American College of Sports Medicine und für die Daten des EORTCQLQ-C30: das Manual „EORTC QLQ-C30 Reference Values“ einer EORTCArbeitsgruppe (Scott, 2008). Die Dateneingabe und die Aufbereitung der Rohdaten erfolgte mit Hilfe von Microsoft Excel. Für die statistische Auswertung wurden alle statistischen Analysen anschließend mithilfe der Statistikprogramme SPSS 19.0 (SPSS Inc., Chicago, IL, USA) und „BIAS für Windows“, Version 10, 2012, Universität Frankfurt) durchgeführt. Das Signifikanzniveau wurde a priori auf p<0,05 festgelegt.
Insgesamt 158 Patienten (99 kurativ, 59 palliativ; 54,9±11,1 Jahre, 108 ♀, 50 ♂) nahmen an allen drei Untersuchungen teil. Der parameterfreie Mann-Whitney-Test zeigte sowohl für Lebensqualität als auch Fatigue-Symptomatik keine signifikanten Unterschiede bei der Eingangsuntersuchung zwischen kurativen und palliativen Teilnehmern. Für die VO2peak ergab der parametrische T-Test ebenfalls keine Unterschiede bei den Initialwerten. Nach Abschluss der Intervention zeigten sich in beiden Patientengruppen sowohl bei der Lebensqualität als auch der Fatigue-Symptomatik signifikante Verbesserungen über den gesamten Untersuchungszeitraum. Anschließende post-hoc-Tests ergaben keine signifikanten Gruppenunterschiede bezüglich der Entwicklung während der verschiedenen Untersuchungszeiträume und der Differenz von Initial- und Abschlusswert. Die Varianzanalyse mit Messwiederholung (Anova) zeigte sowohl für Kurativ- als auch Palliativpatienten signifikante Veränderungen der VO2peak über die Zeit. Einen Haupteffekt im Bezug auf die Gruppe oder eine Interaktion von Zeit und Gruppe gab es dabei nicht. Folglich entwickelten sich beide Gruppen über den Untersuchungszeitraum vergleichbar.
Die vorliegenden Ergebnisse zeigen, dass die Heilungsprognose, kurativ oder palliativ, keinen unterschiedlichen Einfluss auf die Trainierbarkeit der Betroffenen zu haben scheint. Körperliches Training führte bei beiden Patientengruppen dieser Studie zu signifikanten Verbesserungen der Zielparameter. Ein Vergleich der vorliegenden Daten mit bisherigen Untersuchungsergebnissen ist aufgrund der aktuell geringen Anzahl an Studien mit Palliativpatienten und einer bisher nicht einheitlichen Palliativ-Definition schwierig.
Die sporttherapeutische Beratung, welche neben der Vermittlung von Trainingsumfang und –intensität insbesondere Trainingsziele und deren Wirksamkeit aufzeigen soll, kann Patienten und ihrem Umfeld helfen, den Stellenwert von körperlichem Training zuverstehen und bestenfalls die Compliance erhalten. Darüber hinaus kann die allgemeine Leitlinien-Empfehlung von 150 Minuten moderates Ausdauertraining pro Woche als grober Richtwert bestätigt werden. Unterschiedlich hohe Trainingsumfänge in Abhängigkeit initialer Leistungsfähigkeit weisen indessen darauf hin, dass individuelle Trainingsempfehlungen zu bevorzugen sind. Als Konsequenz aus diesen Ergebnissen ist zu empfehlen, dass sich zukünftig körperliche Aktivität als unverzichtbarer Bestandteil des supportiven Therapieangebotes für Krebspatienten mit fortgeschrittener Erkrankung, speziell bei palliativ eingestuften Patienten, etabliert.
Weitere Untersuchungen zu diesem Thema sollten insbesondere darauf abzielen, Dosis-Wirkungs-Zusammenhänge zu ermitteln und diese in symptom- und entitätsspezifische Empfehlungen zu integrieren.
Medizinstudium, Examina und die Berufstätigkeit sind stressig; belastbare Daten zum allgemeinen und spezifischen Stress während des Medizinstudiums liegen nur in geringem Ausmaß vor. Wir haben die Stressbelastung und Resilienz der Frankfurter Medizinstudenten in den Kohorten 1. vorklinisches Semester, 1. klinisches Semester und PJ-Eintritt erhoben (Trierer Inventar zum chronischen Stress TICS, altersnormierter Mittelwert = 50; Resilienz-Skala RS11, kein Optimum, hohe Werte weisen auf Resilienz hin); an der Studie nahmen jeweils mehr als 90% der entsprechenden Kohorte teil. Während zu Studienbeginn der Summenwert (altersnormierter T-Wert) bei 56% lag, fiel dieser im 1. klin. Semester auf 54%, und stieg zum PJ nur gering wieder an. Unter den Subskalen fiel auf, dass Überlastung, Überforderung und chronische Besorgnis parallel zum Gesamtscore abfielen, die Subskala Unzufriedenheit jedoch zunahm (1. vorklin. Semester 53%, 1. klin. Semester 55%, PJ 58%). Die höchsten Werte in der PJ-Gruppe fanden sich ebenfalls für die Subskalen soziale Überlastung, Mangel an sozialer Anerkennung und Soziale Spannungen. Niedrigere Stressskala-Werte zeigten sich nach dem Staatsexamen M1 in den Subskalen Überlastung, Erfolgsdruck, Überforderung, soziale Isolierung, chronische Besorgnis und dem Summenscore. Überraschenderweise fiel der Summenwert der Resilienz vom 1. vorklinischen und 1. klinischen Semester (80,7%) auf 76,7% vor dem PJ-Eintritt, dieser Abfall zeigte sich für alle 11 Einzelitems dieses Fragebogens in gleicher Weise. Während eine Abnahme der Belastungsabhängigen Skalen Überlastung und Überforderung nach dem Staatsexamen M1 erwartet worden war, überraschte die im Gruppenvergleich abnehmende Resilienz bei den Studenten vor dem Praktischen Jahr. Ebenso überraschend war die fast kontinuierliche Zunahme auf der Subskala Unzufriedenheit während des Studiums. Inwieweit diese Differenzen auf das Studium zurückzuführen sind oder auf eine überzufällige Häufung bei den Studienabbrechern, wird in einer prospektiven Fortführung dieser Studie untersucht.
Die derzeitige Regelung der Zulassung zum Medizinstudium berücksichtigt die Abiturnote und Wartezeiten; universitäre Parameter können diese Kriterien modifizieren. Hierzu zählen z.B. die Leistungskurswahl, wie es an der Goethe-Universität Frankfurt gehandhabt wird. Im Rahmen der Untersuchung zu Stress und Resilienz bei Medizinstudenten haben wir bei den Studenten des 1. vorklinischen Semesters soziodemographische Daten erhoben, die einen Kohortenvergleich erlauben. Die chronische Stressbelastung wurde mit dem Trierer Inventar zum chronischen Stress TICS erhoben (T-Wert von 50 entspricht dem altersnormierten Durchschnitt), die Resilienz mit der Skala RS11 (keine Normwerte, hohe Werte weisen auf Resilienz hin); an der Studie nahmen 90% der Studienanfänger teil. Neben dem Summenscore für Stress wurden die 9 Subskalen Überlastung, Überforderung, Unzufriedenheit, Erfolgsdruck, Soziale Überlastung, soziale Spannungen, Soziale Isolierung, Mangelnde Soziale Anerkennung und Chronische Besorgnis erhoben. Signifikant höhere Werte bei Studentinnen (n=234) als bei Studenten (n=111) fanden sich für die Skalen Überlastung, Überforderung und chronische Besorgnis, mit dem größten Unterschied bei chronischer Besorgnis (♀ T-Wert von 56 der Altersnorm, ♂ 51). Bei älteren Studienanfängern (n=89, Alter >21 Jahre) fand sich eine leichte Korrelation mit den Skalen Soziale Überlastung und Mangel an sozialer Anerkennung; auch im Gruppenvergleich haben ältere Studenten mit einem T-Wert von 55 einen signifikant höheren Wert als junge Studenten (T-Wert 50). Auch die Notwendigkeit, das Studium ganz (n=86) oder teilweise (n=58) selbst zu finanzieren, erhöht die Werte auf den Skalen Soziale Überlastung, Soziale Spannung, Mangel an sozialer Anerkennung sowie den Summenscore. Keinen Einfluss hatten Parameter wie „nichtdeutsche Hochschulzugangsberechtigung“, dagegen finden sich bei Studenten mit einer nicht-deutschen Muttersprache (n=61) und Sprachschwierigkeiten (n=12) häufiger überfordert, überlastet, sozial nicht anerkannt, chronisch besorgt und gestresst. Ein erhöhter Stress bei Medizin-Anfängern wird bei Frauen, älteren Studienanfängern sowie eigener Finanzierung gesehen. Überraschend war der geringe Einfluss von Parametern wie Deutsch als Fremdsprache, oder kulturelle Faktoren, die über einen oder beide Elternteile außerhalb Deutschlands oder der EU erfasst wurden.
ß1-integrins are essential for angiogenesis but the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. BRAG2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of BRAG2 in EC and angiogenesis and the underlying molecular mechanisms remains unclear. siRNA-mediated BRAG2-silencing reduced EC angiogenic sprouting and migration. BRAG2-siRNA-transfection differentially affected a5ß1- and aVß3-integrin function: specifically, BRAG2-silencing increased focal/fibrillar adhesions and EC adhesion on ß1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the aVß3-integrin-ligand, vitronectin. Consistent with these results, BRAG2-silencing enhanced surface expression of a5ß1-integrin, while reducing surface expression of aVß3-integrin. Mechanistically, BRAG2 mediated recycling of aVß3-integrins and endocytosis of ß1-integrins and specifically of the active/matrix bound a5ß1-integrin present in fibrillar/focal adhesions (FA), suggesting that BRAG2 contributes to the disassembly of FA via ß1-integrin-endocytosis. Arf5 and Arf6 are promoting downstream of BRAG2 angiogenic sprouting, ß1-integrin-endocytosis and the regulation of FA. In vivo silencing of the BRAG2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitral injection of plasmids containing BRAG2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveals that BRAG2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating ß1-integrin internalization and associates for the first time the process of ß1-integrin endocytosis with angiogenesis.
So far clinical human immunodeficiency virus (HIV) therapy is limited to non-curative treatments. However, as recently shown, alternative approaches such as HIV gene therapy have the potential to functionally cure the disease (e.g. the hematopoietic stem cell (HSC)-transplantation with a CCR5Δ32 homozygous transplant) (1). In contrast to the highly personalized medical treatment applied in the ‘Berlin case’, more broadly applicable approaches are currently under intensive investigation.
One example is the adeno-associated-virus (AAV)-mediated delivery of in vivo secreted antiviral entry inhibitors (iSAVE), the concept of which is based on the direct in vivo administration of a broadly applicable highly potent antiviral gene (here: a C46-derived entry inhibitory peptide interfering with HIV-1 membrane fusion). The AAV-based gene delivery is believed to overcome several limitations of gene therapeutic treatments based on ex vivo lentiviral trials in the past. It is (i) targeting differentiated HIV target cells (i.e. liver and differentiated lymphatic cells) reducing the risk of genotoxicity compared to stem cell-based trials, (ii) overcoming the limitation of a low number of genetically modifiable cells as in lentivirally based ex vivo transduction strategies (i.e. limited modifiable cell number due to culture conditions and lower vector titers) and (iii) using the safe AAV vector system, which has not been associated with major genotoxicity in men. (iv) Most importantly, the concept of secretable entry inhibitors does not require transduction of large amounts of cells due to the protective bystander effect. Thus, iSAVE might be a treatment principle for HIV infection that might be able to cure patients irrespective of their viral isolates or adherence.
Accordingly, the iSAVE concept could aim at two different sites in the patient for the production of antiviral transgenes, either the systemic production via suitable producer cells (e.g. hepatocytes) or the local production in the lymphatic system.
In a first approach, we are able to efficiently target hepatocytes using the natural AAV serotype 8 to express high plasma levels of secretable antiviral entry inhibitors in order to systemically suppress viral replication. In this setting we could show that iSAVE peptides are highly expressed in hepatocytes. However, plasma levels of iSAVE were insufficient when using a secretable peptide as sole antiviral transgene.
As a second treatment strategy, the iSAVE project aimed to deliver antiviral genes directly to the site of viral replication, the lymphatic system. Here, (i) a panel of naturally occurring AAV serotypes as well as (ii) AAV retargeting approaches were employed to design a highly efficient and selective AAV vector variant for gene delivery into the lymphatic system after intravenous vector administration.
In detail, (i) screening of the natural occurring serotypes revealed that the AAV serotype 1 (AAV-1) was best in targeting splenic tissue in two humanized mouse models, however at a very low level. After systemic AAV-1 vector administration neither transduction of human lymphocytes did occur nor was iSAVE expressed in the lymphatic system in a humanized mouse model.
(ii) In a second approach, we modified the well-characterized AAV-2 serotype in a tropism-defining region of its capsid gene by insertion of human peripheral blood lymphocytes (hPBL)-tropic peptide ligands. These in turn were selected by M13 in vivo phage display and by in vivo AAV peptide display. Selected variants were cloned and tested for hPBL transduction in vitro. Although the selected variants did not show increased expression efficacies compared to AAV-2 WT, it still might be possible that the selected variant are more specific for hPBLs as these conditions have not been tested.
As these selection processes required a humanized mouse model that comprises a functional lymphatic system, we established the previously described Trimera mouse model in our lab (2). We found that this mouse model could be further improved to allow engraftment of a lower number of gene-modified (gm) human T cells as in the classical Trimera model. These modified Trimera mice (mT3 mice) were conditioned by inclusion of cyclophosphamide (CTX) to the irradiation-conditioning scheme of the classical Trimera model.
Comparison of mT3 mice with established NSG and DKO mice in an adoptive gm T cell transplantation setting revealed that NSG mice were the most robust model providing high reproducibility in human T cell engraftment. MT3 mice allowed a substantial, yet more variable engraftment of gm T cells. Besides comparing engraftment kinetics, the graft quality (i.e. clonality and cytokine milieu) was analyzed. Again, NSG mice showed the most balanced homeostatic repopulation three weeks after transplantation, while mT3 mice were prone to Th1-type, oligloclonal repopulation, indicating an early onset of xenograft-versus-host disease. Finally, the lymphatic infiltration was analyzed. As expected, mT3 mice provided the most intact lymphatic structures, although the normal lymphatic morphology was not restored.
In conclusion, it was demonstrated in this work that AAV-mediated iSAVE gene therapy faces specific limitations depending on the respective targeting approach
In the systemic approach, iSAVE peptides have to be further optimized in terms of transgene design itself, as high-level accumulation in murine plasma was not feasible for the short iSAVE precursor. In the local, lymphatic targeting approach, AAV-mediated expression faces its limits in targeting specificity but foremost expression efficacy. Thus, the AAV vector itself needs further optimization for sufficient local iSAVE expression levels. Independently from the AAV-related approaches, a novel humanized mouse model was established in this work. Despite drawbacks regarding repopulation variability and set-up complexity, the novel mT3 mouse model comprised improved secondary lymphatic structures for adoptive T cell transfer, which might be an interesting platform for studies in lymphoma or leukemia therapy.
FIAS Scientific Report 2012
(2013)
Background: Characterisation of risk groups who may benefit from pneumococcal vaccination is essential for the generation of recommendations and policy.
Methods: We reviewed the literature to provide information on the incidence and risk of invasive pneumococcal disease (IPD) in at-risk children in Europe and North America. The PubMed database was searched using predefined search terms and inclusion/exclusion criteria for papers reporting European or North American data on the incidence or risk of IPD in children with underlying medical conditions.
Results: Eighteen references were identified, 11 from North America and 7 from Europe, with heterogeneous study methods, periods and populations. The highest incidence was seen in US children positive for human immunodeficiency virus infection, peaking at 4167 per 100,000 patient-years in 2000. Studies investigating changes in incidence over time reported decreases in the incidence of IPD between the late 1990s and early 2000s. The highest risk of IPD was observed in children with haematological cancers or immunosuppression. Overall, data on IPD in at-risk children were limited, lacking incidence data for a wide range of predisposing conditions. There was, however, a clear decrease in the incidence of IPD in at-risk children after the introduction of 7-valent pneumococcal conjugate vaccine into immunisation programmes, as previously demonstrated in the general population.
Conclusion: Despite the heterogeneity of the studies identified, the available data show a substantial incidence of IPD in at-risk children, particularly those who are immunocompromised. Further research is needed to determine the true risk of IPD in at-risk children, particularly in the post-PCV period, and to understand the benefits of vaccination and optimal vaccination schedules.
Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD.
Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.
Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (~ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.
Inhibition of the proteasome is considered as a promising strategy to sensitize cancer cells to apoptosis. Recently, we demonstrated that the proteasome inhibitor Bortezomib primes neuroblastoma cells to TRAIL-induced apoptosis. In the present study, we investigated whether Bortezomib increases chemosensitivity of neuroblastoma cells. Unexpectedly, we discover an antagonistic interaction of Bortezomib and microtubule-interfering drugs. Bortezomib significantly attenuates the loss of cell viability and induction of apoptosis on treatment with Taxol and different vinca alkaloids but not with other chemotherapeutics, that is, Doxorubicin and Cisplatinum. Importantly, Bortezomib inhibits G2/M transition by inhibiting proteasomal degradation of cell cycle regulatory proteins such as p21, thereby preventing cells to enter mitosis, the cell cycle phase in which they are most vulnerable to antitubulin chemotherapeutics. Consequently, Bortezomib counteracts Taxol-induced mitotic arrest and polyploidy, as shown by reduced expression of PLK1 and phosphorylated histone H3. In addition, Bortezomib antagonizes Taxol-mediated degradation of MCL-1 during mitotic arrest by preventing cells to enter mitosis and by inhibiting the proteasome. Downregulation of MCL-1 is critically required for Taxol-induced apoptosis, as overexpression of a phosphomutant MCL-1 variant, which is resistant to degradation, significantly diminishes Taxol-triggered apoptosis. Vice versa, attenuation of Bortezomib-mediated accumulation of MCL-1 by knockdown of MCL-1 significantly enhances Taxol/Bortezomib-induced apoptosis. Thus, Bortezomib rescues Taxol-induced apoptosis by inhibiting G2/M transition and mitigating MCL-1 degradation. The identification of this antagonistic interaction of Bortezomib and microtubule-targeted drugs has important implications for the design of Bortezomib-based combination therapies.
In der vorliegenden Arbeit wird untersucht, wie das Gehirn Bewusstsein erzeugt. Diese Frage wird als eines der größten Rätsel der heutigen Wissenschaft angesehen: Wie kann es sein, dass aus der Aktivität der Nervenzellen unsere subjektive Welt entsteht? Es ist offensichtlich nicht einfach, diese Frage wissenschaftlich zu untersuchen. Eine der vorgeschlagenen Strategien für die Untersuchung von Bewusstsein behauptet, dass man zunächst die neuronalen Korrelate des Bewusstseins finden sollte (Koch, 2004). Einer Definition zufolge sind die neuronalen Korrelate des Bewusstseins die kleinste Menge neuronaler Prozesse, die hinreichend für eine bestimmte bewusste Erfahrung sind (zum Beispiel für die bewusste Erfahrung des Blaubeergeschmacks). Manche behaupteten, die Entdeckung der neuronalen Korrelate des Bewusstseins würde es erlauben, dem Rätsel des Bewusstseins näher zu kommen (Crick & Koch, 1990). Nur wie soll man die neuronalen Korrelate des Bewusstseins finden? Eine relativ einfache Strategie dafür wurde schon vor mehr als 20 Jahren beschrieben. Es sollten einfach experimentelle Bedingungen erschaffen werden, in welchen ein Reiz manchmal bewusst wahrgenommen wird und manchmal nicht (Baars, 1989). Solche Analysen, die Bedingungen mit und ohne bewusste Wahrnehmung vergleichen, werden als „Kontrastierungsanalyse“ bezeichnet (da zwei Bedingungen miteinander kontrastiert werden). Es existieren viele verschiedene experimentelle Paradigmen, bei welchen man den Reiz unter denselben Bedingungen präsentieren kann, so dass er bei manchen Versuchsdurchgängen bewusst wahrgenommen wird, bei anderen nicht (Kim & Blake, 2005). Mit solchen experimentellen Paradigmen kann man angeblich die neuronalen Korrelate des Bewusstseins finden, wenn man a) bei jedem Durchgang die Versuchsperson fragt, ob oder was die Versuchsperson bei dem Durchgang wahrgenommen hat und b) gleichzeitig die neuronalen Prozesse misst (zum Beispiel mit EEG, MEG oder fMRT). Anschließend kann man die erhobenen neuronalen Daten unter den Bedingungen mit und ohne bewusste Wahrnehmung vergleichen.
Mittlerweile gibt es viele Studien, in denen solche experimentelle Paradigmen – und damit die Kontrastierungsanalyse – angewendet wurden. Insofern könnte man glauben, die neuronalen Korrelate des Bewusstseins seien schon gefunden worden. Allerdings ist dies nicht der Fall. Es existiert in der Literatur weiterhin Uneinigkeit darüber, ob die Korrelate des Bewusstseins früh oder spät in der Zeit liegen, und ob die Korrelate in sensorischen Arealen oder eher im hierarchisch höheren fronto-parietalen Kortex zu finden sind.
Nach unserer Meinung sind die experimentellen Paradigmen, die üblicherweise zum Auffinden der neuronalen Korrelate des Bewusstseins verwendet werden, nicht spezifisch genug, um diese eindeutig zu lokalisieren. Eher glauben wir, dass die klassische Kontrastierungsanalyse auch andere Prozesse als Ergebnisse hervorbringt und uns deshalb prinzipiell nicht zu den neuronalen Korrelaten des Bewusstseins führen kann.
Im Kapitel 2 wird erklärt, wieso die typischen experimentellen Paradigmen nicht die neuronalen Korrelate des Bewusstseins ausfindig machen können. Wir behaupten, dass der Vergleich neuronaler Daten aus experimentellen Bedingungen mit und ohne bewusste Wahrnehmung auch die neuronalen Prozesse widerspiegeln könnte, die bewussten Wahrnehmungen entweder vorausgehen oder folgen. Es ist beispielsweise bekannt, dass neuronale Prozesse vor Auftreten des Reizes darüber bestimmen können, ob der Reiz bewusst wahrgenommen wird oder nicht (Busch, Dubois, & VanRullen, 2009; Mathewson, Gratton, Fabiani, Beck, & Ro, 2009). Wenn man experimentelle Bedingungen mit und ohne bewusster Wahrnehmung miteinander vergleicht, werden auch solche Prozesse als Ergebnis auftauchen, obwohl diese zeitlich klar vor dem Reiz stattfinden und deshalb keine neuronalen Korrelate des Bewusstseins sein können. Es ist natürlich einfach zu entscheiden, dass diese Prozesse, die schon vor dem Reiz stattfinden, der bewussten Wahrnehmung vorausgehen müssen, aber es ist unmöglich zu sagen, ob ein neuronaler Prozess 100 oder 200 Millisekunden nach der Präsentation des Reizes immer noch ein Vorläuferprozess ist schon ein neuronales Korrelat des Bewusstseins darstellt. Deshalb ist die typische Kontrastierungsanalyse nicht spezifisch genug und wir wissen nicht, ob neuronale Prozesse, die durch die Kontrastierungsanalyse aufgedeckt werden, direkt die neuronalen Korrelate des Bewusstseins oder eher Prozesse vor der bewussten Wahrnehmung widerspiegeln.
Nicht nur die Vorläuferprozesse der bewussten Warnehmung stellen ein Problem dar. Auch Konsequenzen der bewussten Verarbeitung werden durch die Kontrastierungsanalyse gefunden. Beispielsweise wurden im medialen Temporallappen Neurone gefunden, die nur dann feuern, wenn ein Patient eine Person auf einem Bild bewusst erkennt, aber nicht feuern, wenn der Patient die Person auf dem Bild nicht bewusst wahrnimmt (Quiroga, Mukamel, Isham, Malach, & Fried, 2008). So könnte man vorerst meinen, dass das Feuern dieser Neurone das neuronale Korrelat des Bewusstseins sein könnte. Nach einer Läsion, sprich neuronalen Schädigung des medialen Temporallappens kann man die Welt jedoch weiterhin bewusst wahrnehmen (man hat jedoch Probleme mit dem Gedächtnis und Wiedererkennen). Insofern kann das Feuern dieser Neurone nicht das neuronale Korrelat des Bewusstseins sein und ist eher ein Beispiel für die Konsequenz der bewussten Verarbeitung. Wir behaupten, dass es noch viele andere solcher Vorläuferprozesse und Konsequenzen gibt, die notwendigerweise als Ergebnis bei der Kontrastierungsanalyse auftauchen, und also ist die typische Kontrastierungsanalyse extrem unspezifisch bezüglich der neuronalen Korrelate des Bewusstseins. In anderen Worten: Die typische Kontrastierungsanalyse, bei welcher man experimentelle Bedingungen mit und ohne bewusste Wahrnehmung miteinander vergleicht, wird uns nicht helfen die neuronalen Korrelate des Bewusstseins zu finden.
Wir glauben, dass neue experimentelle Paradigmen entwickelt werden sollten, um die neuronalen Korrelate des Bewusstseins ausfindig zu machen. Wahrscheinlich gibt es kein einfaches Experiment, mit dem man die Vorläuferprozesse und Konsequenzen vollständig vermeiden kann, um damit direkt die neuronalen Korrelate des Bewusstseins zu bestimmen. Eher braucht man viele verschiedene Experimente, die Schritt für Schritt unser Wissen über die neuronalen Korrelate des Bewusstseins erweitern.
In der vorliegenden Arbeit (in Kapiteln 3, 4 und 5) wird ein neues experimentelles Paradigma angewandt. Dieses Paradigma wird nicht alle oben erwähnten Probleme lösen, wird aber hoffentlich erlauben, einige Vorläuferprozesse der bewussten Wahrnehmung von den neuronalen Korrelaten des Bewusstseins auseinanderzuhalten. Der Vorteil unseres experimentellen Paradigmas besteht darin, dass die bewusste Wahrnehmung durch zwei verschiedene Vorläuferprozesse beeinflusst wird. Die Versuchspersonen müssen auf schnell präsentierten und mittels Rauschens undeutlich gemachten Bildern eine Person detektieren. Die experimentellen Bedingungen sind derart gestaltet, dass die Versuchspersonen nicht bei jedem Durchgang die Person auf dem Bild wahrnehmen können. Damit können wir den Wahrnehmungsprozess manipulieren. Bei einer Manipulation variieren wir den Anteil des Rauschens auf dem Bild und damit die sensorische Evidenz. Je weniger Rauschen, desto besser können die Versuchspersonen die Bilder wahrnehmen und desto öfter sehen sie auch bewusst die Person auf dem Bild. Bei der anderen experimentellen Manipulation der Wahrnehmung werden einige Bilder den Versuchspersonen vorher klar und ohne Rauschen gezeigt. Damit erschafft man Wissen über bestimmte Bilder, die später mit Rauschen präsentiert werden. Man kann zeigen, dass solch bestehendes Wissen tatsächlich die Wahrnehmung beeinflusst. Wenn die Versuchspersonen bestehendes Wissen über ein Bild haben, ist es wahrscheinlicher, dass sie die Person auf dem Bild bewusst wahrnehmen. Damit haben wir zwei verschiedene Vorläuferprozesse – sensorische Evidenz und bestehendes Wissen, die beide die bewusste Wahrnehmung beeinflussen. Beide Vorläuferprozesse erhöhen den Anteil der Durchgänge, in welchen die Versuchspersonen die Person auf dem Bild bewusst wahrnehmen.
Mit diesem experimentellen Paradigma möchten wir einige Aussagen über die neuronalen Korrelate des Bewusstseins testen. Wenn über einen neuronalen Prozess behauptet wird, dass er einem neuronalen Korrelat des Bewusstseins entspricht, müsste dieser Prozess von den beiden manipulierten Vorläuferprozessen in ähnlicher Weise beeinflusst werden, da bewusste Wahrnehmung durch beide manipulierten Vorläuferprozessen in ähnlicher Weise erleichtert wird. Wenn aber der Prozess, über den behauptet wird, er sei ein neuronales Korrelat des Bewusstseins, nicht durch beide Manipulationen geändert wird, kann dieser Prozess kein neuronales Korrelat des Bewusstseins sein, da er nicht beeinflusst wird, obwohl die bewusste Wahrnehmung geändert wurde.
Mit diesem experimentellen Paradigma und dieser Logik haben wir zwei unterschiedliche neuronale Prozesse getestet, von denen behauptet wird, dass sie den neuronalen Korrelaten des Bewusstseins entsprechen könnten. In Kapitel 3 wurde untersucht, ob lokale kategorienspezifische Gammabandaktivität die neuronalen Korrelate des Bewusstseins reflektieren könnte. In Kapitel 4 wurde mit diesem experimentellen Paradigma untersucht, ob die neuronale Synchronisierung dem neuronalen Korrelat des Bewusstseins entsprechen könnte.
Unsere Arbeit im Kapitel 3 baut auf der von Fisch und Kollegen (2009) auf. Fisch und Kollegen (2009) zogen aus ihrer experimentellen Arbeit den Schluss, dass lokale kategorienspezifische Gammabandaktivität die neuronalen Korrelate des Bewusstseins reflektieren könnte. Sie hatten Elektroden auf dem visuellen Kortex von Epilepsiepatienten implantiert und von diesen Elektroden die Gammabandaktivität abgeleitet. Im ersten Schritt suchten sie nach Elektroden, die kategorienspezifische Antworten zeigen. Bei den kategorienspezifischen Elektroden ist die Gammabandaktivität abhängig vom präsentierten Stimulusmaterial. Zum Beispiel kann man bei einer Elektrode auf dem Fusiform Face Area starke Gammabandaktivität nur dann messen, wenn ein Gesicht auf dem Bild zu sehen ist. Die Autoren benutzten solche kategorienspezifischen Elektroden, um nach den neuronalen Korrelaten des Bewusstseins zu suchen. Sie zeigten den Patienten Bilder von Gesichtern, Häusern und Objekten, die direkt nach der kurzen Präsentation maskiert wurden, so dass die Patienten nur bei manchen Durchgängen erkannten, was auf dem Bild war, bei anderen Durchgängen nicht. Dies entspricht der typischen Kontrastierungsanalyse. Die Ergebnisse haben klar gezeigt, dass bei diesen kategorienspezifischen Elektroden die Gammabandaktivität erhöht wurde, als die Patienten bewusst wahrnahmen, was auf dem Bild zu sehen war. Aus diesen Ergebnissen zogen die Autoren den Schluss, dass lokale kategorienspezifische Gammabandaktivität dem neuronalen Korrelat des Bewusstseins entspricht. Diese Aussage wollten wir mit unserem experimentellen Paradigma testen.
Um diese Behauptung zu untersuchen, erhoben wir sehr ähnliche Daten wie Fisch et al. (2009) und analysierten die Daten auf ähnliche Weise. Unsere experimentelle Frage war, ob die lokale kategorienspezifische Gammabandaktivität durch unsere beiden Manipulationen – sensorische Evidenz und bestehendes Wissen – in ähnlicher Weise erhöht wird. Dies sollte der Fall sein, wenn die lokale kategorienspezifische Gammabandaktivität dem neuronalen Korrelat des Bewusstseins entspricht, da sensorische Evidenz und bestehendes Wissen beide den Anteil der Durchgänge, in welchen die Versuchsperson die Person auf dem Bild bewusst wahrnimmt, erhöhen. Dieses Ergebnis wurde nicht gefunden. Stattdessen fanden wir, dass die lokale kategorien-spezifische Gammabandaktivität nur durch sensorische Evidenz erhöht wurde, bestehendes Wissen aber keinen Effekt auf diese Aktivierung hatte. Da bestehendes Wissen auch den Anteil der Durchgänge mit bewusster Wahrnehmung erhöht, die kategorienspezifische Gammabandaktivität aber nicht durch bestehendes Wissen erhöht wurde, kann man schlussfolgern, dass die kategorienspezifische Gammabandaktivität nicht die neuronalen Korrelate des Bewusstseins reflektieren kann.
Als nächstes (Kapitel 4) haben wir die Hypothese getestet, dass Synchronizität dem neuronalen Korrelat des Bewusstseins entspricht. Um diese Idee zu testen, maßen wir mittels Magnetoenzephalographie die magnetischen Felder des Gehirns, schätzten aus diesen Daten mittels Beamforming die neuronalen Aktivitätsquellen und quantifizierten die Synchronizität zwischen diesen Quellen. Wenn die interareale Synchronizität dem neuronalen Korrelat des Bewusstseins entspräche, sollte die Synchronizität für Bedingungen mit mehr sensorischer Evidenz und mit bestehendem Wissen erhöht sein. Dies wurde nicht beobachtet. Wir fanden, dass Synchronizität (gemittelt über die Quellen) nur bei den Bildern erhöht war, für die bestehendes Wissen vorlag. Ein ähnlicher Effekt für sensorische Evidenz wurde nicht gefunden. Insofern können wir sagen, dass unsere Befunde dagegen sprechen, dass neuronale Synchronizität den Mechanismus für Bewusstsein darstellt. Allerdings können wir das in diesem Fall auch nicht völlig ausschließen, denn Synchronizität könnte die Informationsverarbeitung auf einem kleineren Maßstab koordinieren als wir es mit dem MEG messen können (Singer, in press).
Im Kapitel 5 untersuchten wir, wie schnell bestehendes Wissen bewusste Verarbeitung beeinflussen kann. Um dies herauszufinden machten wir uns die intraindividuellen Unterschiede der perzeptuellen Leistung zu Nutze. Wir fanden, dass bestehendes Wissen bewusste Verarbeitung schon innerhalb der ersten 100 Millisekunden nach der Präsentation des Reizes beeinflusst. Wir beobachteten auch, dass ein größerer perzeptueller Effekt des bestehenden Wissens in geringerer neuronaler Aktivität in Durchgängen mit bestehendem Wissen hervorruft. Diese Ergebnisse sind im Einklang mit Theorien, die besagen, dass unsere Wahrnehmung bestehendes Wissen nutzt, um vorherzusagen, wie die visuelle Welt sich ändert und um die neuronalen Antworten zu verringern (Friston, 2010).
In der vorliegenden Arbeit wurde diskutiert, warum die typische Kontrastierungsanalyse uns nicht zu den neuronalen Korrelaten des Bewusstseins führen kann. Wir schlugen vor, dass neue experimentelle Paradigmen nötig sind, um näher an die neuronalen Korrelate des Bewusstseins heranzukommen. Es wurde ein neues Paradigma benutzt, um zwischen Vorläuferprozessen und neuronalen Korrelate des Bewusstseins zu unterscheiden. Mit diesem Paradigma wurden zwei sehr unterschiedliche Hypothesen getestet und gefunden, dass die kategorienspezifische Gammabandaktivität nicht die neuronalen Korrelate des Bewusstseins widerspiegeln kann. Wir hoffen, dass unsere Experimente eine Entwicklung von vielen weiteren und besseren experimentellen Paradigmen stimuliert, die zwischen den Vorläuferprozessen, den Konsequenzen und den eigentlichen Korrelaten des Bewusstseins unterscheiden können. Wenn man über die Kontrastierungsanalyse hinausgeht, kann man die gegenwärtigen Theorien des Bewusstseins testen und damit Schritt für Schritt näher an die neuronalen Grundlagen des Bewusstseins kommen.
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.
Opportunities to treat infection with hepatitis C virus (HCV) are evolving rapidly. From the introduction of interferon-α monotherapy in 1992 to the approval of telaprevir- and boceprevir-based triple therapies with pegylated interferon-α and ribavirin in 2011, the chances of curing patients infected with HCV genotype 1 have improved from <10% to approximately 70%. Significant further improvements are on the horizon, which may well cure virtually all hepatitis C patients with an all-oral, interferon-free regimen in the very near future. These exciting developments are reviewed in the present article.
Calcium-deficiency rickets (CDR) is a metabolic bone disease in children that is characterized by impaired mineralization and severe bone deformities. As CDR is often an endemic phenomenon that is almost exclusively restricted to tropical areas, environmental conditions are currently considered to be a possible predisposing factor for the CDR. Apart from a lack of macronutrients and micronutrients, an oversupply of potentially toxic elements (PTEs) in the soil-plant pathway of the CDR areas is thought to be involved in the aetiology of CDR. This study is the first to comprehensively analyze the impact of the environment on Ca deficiency and the resulting CDR.
To analyze the impact of the environment on CDR in developing countries, a rural region near Kaduna City, northern Nigeria, was chosen as a study area. From this area, cases of CDR have been reported since the early 2000s with a prevalence rate of 5%. Within this study area, 11 study sites, including areas with a high CDR prevalence (HR), a low CDR prevalence (LR) and no CDR prevalence (NR), were visited. In these HR, LR and NR study sites, the bedrock was investigated and the types of parent materials were identified. Local farmers were interviewed to determine the type and intensity of the land use. The soil types were determined along toposequences. The soil textures as well as the clay mineral fractions were determined. The pH values were measured, and the contents of organic carbon (OC) were determined. The potential cation-exchange capacity (CECpot) and the base saturation (BS) were analyzed. Furthermore, the total and plant-available macronutrient, micronutrient and PTE concentrations were measured in the soils. The drinking water was analyzed for pH values and the concentrations of Ca, Se and F were measured. The maize was analyzed for the Ca, Mg, K and P, Se and phytic acid (PA) contents.
The field and laboratory analyses on the bedrock showed that the HR, LR and NR study sites near Kaduna City, northern Nigeria, were underlain by Older Granites. A direct link between the distribution of the bedrock, the parent materials and the prevalence of CDR was not found. Interviews with the local farmers showed that the land use in the Kaduna study area is dominated by the cultivation of cash crops and food crops. Field analyzes on the soil types in the Kaduna study area showed that the distribution of the soil types is highly dependent on the topography and the distribution of the parent materials. In near vicinity to the inselbergs, Lixisols had developed on grus slope deposits. In the lower pediment and plain positions, Acrisols had developed on grus slope deposits and pisolite slope deposits. In the upper plains, Plinthosols had developed on pisolite slope deposits and in the river valleys, Fluvisols had developed on river deposits. Such soil types and soil type distributions are typical for granite-underlain areas in the northern guinea savanna of West Africa. Similarly, the physical soil conditions were representative for the soils of the northern guinea savanna: sandy topsoils, clayey subsoils and relatively high contents of kaolinite clay minerals in the clay fractions. With regard to the geochemical composition, no significant difference was found between the soils of the Kaduna study area and the soils of other granite-underlain areas in West Africa. Only the concentrations of P were considerably low in the soils of the Kaduna study area. However, P deficiency is a typical phenomenon in West African savanna soils and is not restricted to CDR areas. The micronutrient concentrations in the soils were low, but not critically low. Laboratory analyses on the amounts of PTEs showed that compared to worldwide background levels and international critical limits the PTE concentrations were very low in the soils of the Kaduna study area. In the drinking water, neither a significant lack of macronutrients and micronutrients, nor a noticeable oversupply of PTEs was found. The maize in the HR, LR and NR study sites contained normal contents of Mg, K and P, low contents of Ca and Se as well as slightly elevated concentrations of PA compared to West African food composition tables. Comparisons between the mineral contents of traditional and modern maize cultivars showed that the traditional maize cultivars contained significantly higher contents of Ca and noticeably lower concentrations of PA than the modern maize cultivars.
A direct link between the environmental conditions and the CDR in the Kaduna study area was considered unlikely, as neither a statistically significant lack of macronutrients and micronutrients, nor a statistically significant oversupply of PTEs was found in the environment of this area. Instead, the results indicated that the nutrition rather than the environmental conditions that impacts the prevalence of CDR.
Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.
The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that - after first-line in vitro assays - characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.
Objectives: To describe changes in costs of managing hospitalised patients with acute myeloid leukaemia (AML) after chemotherapy in Germany over 3 yr, with a special focus on prophylaxis and treatment patterns as well as resource use related to invasive fungal infections (IFI).
Methods: The study was conducted as a retrospective, single-centre chart review in patients with AML hospitalised for chemotherapy, neutropenia and infections after myelosuppressive chemotherapy from January 2004 to December 2006 in Germany. The following resource utilisation data were collected: inpatient stay, mechanical ventilation, parenteral feeding, diagnostics, systemic antifungal medication and cost-intensive concomitant medication. Direct medical costs were calculated from hospital provider perspective.
Results: A total of 471 episodes in 212 patients were included in the analysis. Occurrence of IFI decreased from 5.9% in 2004 to 1.9% in 2006. Mean (± standard deviation) hospital stay decreased from 28.7 ± 17.9 d in 2004 to 22.4 ± 11.8 d in 2006. From 2004 to 2006, the use of a single antifungal drug increased from 30.4% to 46.9%, whereas the use of multiple antifungal drugs decreased from 24.4% to 13.1%. The use of liposomal amphotericin B declined between 2004 and 2006 (21.4% vs. 3.8%) and caspofungin between 2005 and 2006 (19.3% vs. 8.1%). Total costs per episode declined from €19051 ± 19024 in 2004 to €13531 ± 9260 in 2006; major reductions were observed in the use of antimycotics and blood products as well as length of hospital stay.
Conclusion: Analysis of real-life data from one single centre in Germany demonstrated a change in antifungal management of patients with AML between 2004/2005 and 2006, accompanied by a decline in total costs.
Gene therapy on the move
(2013)
The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders.
Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNFα) on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNFα is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3–4 days post lesion (dpl), but not for the induction of synaptic scaling at 1–2 dpl. Furthermore, laser capture microdissection combined with quantitative PCR demonstrates an increase in TNFα-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3–4 dpl. Immunostainings for the glial fibrillary acidic protein and TNFα suggest that astrocytes are a source of TNFα in our experimental setting. We conclude that TNFα-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons.
Polo-like kinase 1, a pivotal regulator of mitosis and cytokinesis, is highly expressed in a broad spectrum of tumors and its expression correlates often with poor prognosis, suggesting its potential as a therapeutic target. p53, the guardian of the genome, is the most important tumor suppressor. In this review, we address the intertwined relationship of these two key molecules by fighting each other as eternal rivals in many signaling pathways. p53 represses the promoter of Polo-like kinase 1, whereas Polo-like kinase 1 inhibits p53 and its family members p63 and p73 in cancer cells lacking functional p53. Plk1 inhibitors target all rapidly dividing cells irrespective of tumor cells or non-transformed normal but proliferating cells. Upon treatment with Plk1 inhibitors, p53 in tumor cells is activated and induces strong apoptosis, whereas tumor cells with inactive p53 arrest in mitosis with DNA damage. Thus, inactive p53 is not associated with a susceptible cytotoxicity of Polo-like kinase 1 inhibition and could rather foster the induction of polyploidy/aneuploidy in surviving cells. In addition, compared to the mono-treatment, combination of Polo-like kinase 1 inhibition with anti-mitotic or DNA damaging agents boosts more severe mitotic defects, effectually triggers apoptosis and strongly inhibits proliferation of cancer cells with functional p53. In this regard, restoration of p53 in tumor cells with loss or mutation of p53 will reinforce the cytotoxicity of combined Polo-like kinase 1 therapy and provide a proficient strategy for combating relapse and metastasis of cancer.
Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.
Protein quality control systems (PQC), i.e. UPS and aggresome-autophagy pathway, have been suggested to be a promising target in cancer therapy. Simultaneous pharmacological inhibition of both pathways have shown increase efficacy in various tumors, such as ovarian and colon carcinoma. Here, we investigate the effect of concomitant inhibition of 26S proteasome by FDA-approved inhibitor Bortezomib, and HDAC6, as key mediator of the aggresome-autophagy system, by the highly specific inhibitor ST80 in rhabdomyosarcoma (RMS) cell lines. We demonstrated that simultaneous inhibition of 26S proteasome and selective aggresome-autophagy pathway significantly increases apoptosis in all tested RMS cell lines. Interestingly, we observed that a subpopulation of RMS cells was able to survive the co-treatment and, upon drug removal, to recover similarly to untreated cells. In this study, we identified co-chaperone BAG3 as the key mediator of this recovery: BAG3 is transcriptionally up-regulated specifically in the ST80/Bortezomib surviving cells and mediates clearance of cytotoxic protein aggregates by selective autophagy. Impairment of the autophagic pathway during the recovery phase, both by conditional knock-down of ATG7 or by inhibition of lysosomal degradation by BafylomicinA1, triggers accumulation of insoluble protein aggregates, loss of cell recovery and cell death similarly to stable short harpin RNA (shRNA) BAG3 knock-down. Our results are the first demonstration that BAG3 mediated selective autophagy is engaged to cope with proteotoxicity induced by simultaneous inhibition of constitutive PQC systems in cancer cell lines during cell recovery. Moreover, our data give new insights in the regulation of constitutive and on demand PQC mechanisms pointing to BAG3 as a promising target in RMS therapy.
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.
Glioblastoma is the most common and most aggressive type of brain tumor in adults. In contrast to epithelial cancers, glioblastomas do not metastasize. While the major treatment challenge in epithelial cancers is not the primary tumor but metastasis, glioblastoma patients die of the primary tumor.
However, there is a common theme which underlies the malignant properties of progressed epithelial cancers and glioblastoma: invasion from the primary tumor into the surrounding tissue. In the case of epithelial cancers this is the first and necessary step to metastasis, whereas invasion leads inevitably to tumor recurrence after resection in the case of glioblastoma, causing it to be incurable.
A cellular program which has been described in detail to promote the invasive phenotype in epithelial tumors, is the epithelial-mesenchymal-transition (EMT). Differentiated neural cells are not epithelial, thus, strictly speaking, EMT does not occur in glioblastoma. However, the traits acquired in the process of EMT, especially invasiveness and stemness, are highly relevant to glioblastoma. One of the key transcription factors known to induce EMT in epithelial cancers is ZEB1, which has been described only marginally in the central nervous system so far. Here, I investigate the expression and function of ZEB1 in glioblastoma and during human fetal neural development.
ZEB1 mRNA was significantly upregulated in all histological types of glioma, including glioblastoma, when compared to normal brain. There was no correlation between ZEB1 mRNA levels and tumor grade. Immunohistochemical staining of glioma samples demonstrated that ZEB1 was highly expressed in the great majority of tumor cells. In the developing human brain, intense staining for ZEB1 could be observed in the ventricular and subventricular zone, where stem- and progenitor cells reside. ZEB1 positive cells included cells stained with stem- and progenitor markers like PAX6, GFAP and Nestin. In contrast, ZEB1 was never found in early neuronal cells as identified by TUBB3 staining.
To gain insight into ZEB1 function I generated a human fetal neural stem cell line and a glioblastoma cell line with ZEB1 knockdown, which were compared with their respective control cell lines. First, I found that ZEB1 does not regulate the micro RNA 200 family in either cell line, which has been described as an essential ZEB1 target in epithelial cancers. Second, regulated target genes were identified with a genome wide microarray. The third approach was to directly identify genomic binding sites of ZEB1 by chromatin immunoprecipitation sequencing (ChIP-seq). All three approaches showed that the ZEB1 transcriptional program is surprisingly similar in the neural stem cell line and the glioblastoma cell line. In contrast, it bears only little resemblance to the program described in epithelial cancers.
The most interesting, previously unrecognized ZEB1 target gene identified in this study is integrin b1. It was regulated after ZEB1 knockdown detected by microarray analysis, and has a ZEB1 binding site in its promoter region detected by ChIP-seq. Finally, I addressed the question whether ZEB1 influences tumor growth and invasiveness in a glioblastoma model. After intracranial xenotransplantation in mice, ZEB1 knockdown glioblastoma cells formed significantly smaller and less invasive tumors than control glioblastoma cells.
This study demonstrates that ZEB1 is widely expressed in glioma and relevant for glioblastoma growth and invasion. In contrast to what is known about ZEB1 function in epithelial cancers, ZEB1 is not associated with glioma progression, but instead seems to be an early and necessary event in tumorigenesis. Also with regard to ZEB1 target genes, ZEB1 functions differently in glioblastoma than in epithelial cancers. The two most important ZEB1 targets in epithelial cancers are E-cadherin and the miR-200 family members. Both are not relevant to ZEB1 function in glioblastoma. Interestingly, while the ZEB1 transcriptional program is different from the one described in epithelial cancers, it is highly similar in glioblastoma cells and fetal neural stem cells. This suggests that an embryonic pathway restricted to stem- and progenitor cells during development is reactivated in glioblastoma.
Previously known ZEB1 target genes were tissue specific and therefore seemed unlikely to mediate ZEB1 function in the central nervous system. However, the newly identified ZEB1 target gene integrin b1 is well known to play pivotal roles in both glioblastoma tumorigenesis and invasion as well as in neural stem cells. Additionally, integrin b1 is widely expressed and seems a likely ZEB1 target in other organs than the brain.
Taken together, I demonstrate that ZEB1 is a new regulator of glioblastoma growth and invasion. The transcriptional program of ZEB1 differs from the one in epithelial cancers but is strikingly similar to the one in neural stem cells. The newly identified ZEB1 target gene integrin b1 is likely to mediate crucial ZEB1 functios. Thus, this study identifies ZEB1 as a yet unrecognized player in glioblastoma and neural development. Furthermore, it sets the stage for more research which will help to deepen our understanding of ZEB1 function in the central nervous system and beyond.
C-Typ Lektin-ähnliche Rezeptoren (CTLRs) auf Lymphozyten des Immunsystems modulieren deren Effektorfunktionen wie Zytotoxizität oder Zytokinsekretion. Die Gene dieser Immunrezeptoren befinden sich in einer definierten genomischen Region, dem Natürlichen Killer Genkomplex (NKC), welcher im Menschen auf Chromosom 12 und in der Maus auf Chromosom 6 lokalisiert ist. Namensgebend für diesen Gencluster ist die erste Beschreibung von CTLRs auf Natürlichen Killerzellen (NK-Zellen), den Effektorlymphozyten des angeborenen Immunsystems. Einige NKC-kodierte CTLR, insbesondere Vertreter der C-Typ Lektin Familie 2 (CLEC2)-Rezeptorfamilie, werden jedoch auch in nicht-lymphozytären Zellen (z.B. humanes KACL in Keratinozyten, Maus Clr-f in Darmepithelzellen) vorgefunden und in Zusammenhang mit einer gewebsspezifischen Immunüberwachung gebracht. Bemerkenswerterweise sind die Lymphozytenassoziierten Rezeptoren dieser CLEC2-Proteine ebenso CTLRs, welche zudem eng benachbart zu den CLEC2-Proteinen im NKC kodiert sind, sodass es sich um genetisch gekoppelte Rezeptor-Liganden-Paare mit immunologischer Funktion handelt.
Zu Beginn der vorliegenden Arbeit richtete sich das Interesse auf ein bislang uncharakterisiertes Mitglied der CLEC2-Proteinfamilie (CLEC2L), das jedoch außerhalb des NKC und in unmittelbarer Nachbarschaft zu einem weiteren und ebenso uncharakterisierten CTLR (KLRG2) kodiert ist. Im Unterschied zu anderen Mitgliedern der CLEC2-Familie ist CLEC2L (wie auch KLRG2) in Säugetieren hochkonserviert. Im Rahmen dieser Arbeit wurde der Frage nachgegangen, ob CLEC2L wie andere Mitglieder der CLEC2-Proteinfamilie eine gewebsspezifische Expression aufweist, mit einem genetisch gekoppelten CTLR, d. h. mit KLRG2, interagiert und funktionell in Verbindung mit dem Immunsystem gebracht werden kann. Ziel dieser Arbeit war es somit, eine detaillierte Expressions- und Funktionsstudie zu CLEC2L durchzuführen.
Mittels quantitativer Echtzeit-PCR und in situ Hybridisierung konnte CLEC2L-RNA im humanen und Maus-Gehirn nachgewiesen werden. Da die Mengen dort das Expressionsniveau in anderen Organen bei Weitem überstiegen, wurde das CLEC2L-kodierte Protein als BACL (engl. Brain-Associated C-type Lectin) neu benannt. Ektop exprimiertes BACL bildet ähnlich wie viele andere CLEC2-Mitglieder ein disulfid-verknüpftes Homodimer auf der Zellmembran von Säugetierzellen. Um die endogene Proteinexpression dieses gehirnassoziierten „Waisen"-Rezeptors zu charakterisieren, wurde die BACL-Ektodomäne rekombinant produziert und als Immunogen zur Herstellung BACLspezifischer Antikörper eingesetzt. Mit diesen Antikörpern und einer Kombination immunologischer Techniken wie Immunhistochemie, Immunfluoreszenz und Immunpräzipitation konnte die Präsenz von BACL auf humanen und Maus-Neuronen des Gehirns mit einer besonders ausgeprägten Expression in Purkinje-Zellen zum ersten Mal gezeigt werden. Neben dem Gehirn wurden andere Bereiche des Nervensystems, darunter Spinalganglien und Retina, auf die Expression des BACL-Proteins untersucht. Hierbei konnte mittels Immunfluoreszenz und hochauflösender konfokaler Mikroskopie gezeigt werden, dass BACL mit Neuronenmembranen assoziiert ist. Die durchflusszytometrische Analyse von in vitro kultivierten Neurosphären untermauerte die Expression von endogenem BACL als membranständiges Oberflächenprotein.
Diverse Ansätze zur Identifizierung von Interaktionspartnern von BACL erbrachten letztlich keine eindeutigen Ergebnisse. KLRG2 wurde ursprünglich aufgrund seiner benachbarten genomischen Lokalisation als möglicher Rezeptor von BACL favorisiert, jedoch konnten weder Reporterassays noch durchflusszytometriebasierte Bindungsanalysen eine Interaktion dieser beiden Proteine aufzeigen. Auch die aus den massenspektrometrischen Analysen von humanen und Maus BACL-Immunpräzipitaten erhaltenen Kandidatenproteine konnten letztendlich nicht als Interaktionspartner von BACL eindeutig verifiziert werden.
Eine mögliche immunologische Bedeutung von BACL in vivo wurde im Rahmen von Tumorimplantationsexperimenten mit BACL-exprimierenden Tumorzellen untersucht. Hierbei wurde das Tumorwachstum von BACL- mit Kontroll-Transfektanten in C57BL/6 Mäusen verglichen. Der beobachtete Effekt des verlangsamten Tumorwachstums nach BACL-Überexpression war jedoch nicht auf BACL-Erkennung durch Lymphozyten zurückzuführen, wie anhand von immundefizienten Rag1-k.o. und NOD-SCID-gammak.o. Mäusen gezeigt werden konnte.
Insgesamt liefert diese Arbeit die Erstbeschreibung des bislang uncharakterisierten CTLRs BACL. Das Protein teilt strukturelle Merkmale mit Mitgliedern der CLEC2-Familie, unterscheidet sich jedoch deutlich durch (i) seine hohe Konservierung in Säugetieren, (ii) seine Kodierung außerhalb des NKC und (iii) seine pan-neuronale Expression. Die Erkenntnis, dass BACL in Maus- und in humanen Neuronen exprimiert wird, wirft die Frage nach seiner funktionellen Relevanz auf. Als Membranprotein könnte es eine wichtige Rolle in der neuronalen Kommunikation und bei zellulären Kontakten spielen. Die Frage nach der Funktion von BACL wird in zukünftigen Forschungsarbeiten zu klären sein.
Despite advances in allogeneic stem cell transplantation, BCR-ABL-positive acute lymphoblastic leukaemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, miR-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and overexpression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labelling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b overexpression, Bcl2-specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL-positive human ALL cell lines were more sensitive to pharmacological BCL2 inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real-time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL.
Object: Minimally invasive spine (MIS) procedures are increasingly being recognized as equivalent to open procedures with regard to clinical and radiographic outcomes. These techniques are also believed to result in less pain and disability in the immediate postoperative period. There are, however, little data to assess whether these procedures in combination with minimally invasive transforaminal interbody fusion (MI-TLIF) and percutaneous pedicle screw insertion are effective in complex cases of stenotic degenerative spondylolisthesis with severe facet joint osteoarthritis (FJO).
Methods: This study retrospectively reviewed all patients who underwent lumbar instrumentation, fusion and decompression for degenerative spondylolisthesis with severe stenosis and facet joint osteoarthritis (FJO) between June 2010 and June 2011. Blood loss, operative time and intraoperative complications were assessed in all surgically treated patients who were treated with MIS decompression, MI-TLIF and percutaneous transpedicular instrumentation. Clinical outcome was measured using the Oswestry Disability Index (ODI) and Visual Analogue Scale (VAS) for back pain, leg pain, and activity level. Satisfaction was assessed with VAS for satisfaction. Radiological follow-up includes x-ray films, computed tomography and in some cases magnetic tomography scan.
Results: Twenty four cases with severe stenotic changes accompanied by severe FJO were treated with minimally invasive procedure. The minimum follow-up was 6 months with a mean of 8 months. The mean preoperative ODI score was 46.8, decreasing to a mean of 23 postoperatively. The mean VAS leg and back pain scores were 67.5 improving to means of 25.8. Twenty one out of 24 cases experienced a clinical benefit according to VAS for satisfaction and ODI. Complications included wound healing disturbance (4%), CSF fistula (4%) and contralateral radiculopathy due to articular bone spurs (8%). The accuracy of pedicle screws was high and only one revision surgery was performed.
Conclusion: MIS for severe stenotic spondylolisthesis leads to adequate and safe decompression of lumbar stenosis and results in a highly significant reduction of symptoms and disability. MIS-TLIF and percutaneous pedicle screw insertion constitute a promising treatment alternative for patients with severe stenosis and facet joint osteoarthritis
In this manuscript a comprehensive coverage of recent developments in the drug therapy of vasospasm while providing the background information that neuroscientists need to understand its rationale. The range of new agents available for treatment of cerebral vasospasm is expanding rapidly along with rapid advances in pharmacology and physiology that are uncovering the mechanisms of this disease. Although there are many publications for treatment of cerebral vasospasm, most are focusing on different aspects of vasospasm treatment and many have limited value due to insufficient quality. Moreover, the complexity of this, in many cases deleterious condition, is enormous and the information needed to understand drug effects is accordingly often not readily available in a single source. A number of pharmacological and medical therapies are currently in use or being investigated in an attempt to reverse cerebral vasospasm, but only a few have proven to be useful. Current research efforts promise the eventual production of new medical therapies. At last, recommendations for the use of different treatment stages based on currently available clinical data are provided.
Background: Classical dance comprises gender specific movement tasks. There is a lack of studies which investigate work related traumatic injuries in terms of gender specific differences in detail.
Objective: To define gender related differences of occupational accidents.
Methods: Basis for the evaluation were occupational injuries of professional dancers from three (n = 785; f: n = 358, m: n = 427) state theatres.
Results: The incidence rate (0.36 per year) was higher in males (m: 0.45, f: 0.29). There were gender specific differences as to the localizations of injuries, particularly the spine region (m: 17.3%, f: 9.8%, p = 0.05) and ankle joint (m: 23.7%, f: 35.5%, p = 0.003). Compared to male dancers, females sustained more injuries resulting from extrinsic factors. Significant differences could specifically be observed with dance floors (m: 8.8%, f: 15.1%, p = 0.02). There were also significant gender differences observed with movement vocabulary.
Conclusion: The clearly defined gender specific movement activities in classical dance are reflected in occupational accidents sustained. Organisational structures as well as work environment represent a burden likewise to male and female dancers. The presented differences support the development of gender specific injury prevention measures.
Background: Only few studies deal with the workload of physical therapists and the health consequences, although this occupational group is quite important for the health care system in many industrialized countries (e.g. ca. 136 000 people are currently employed as physical therapists in Germany). Therefore, the current state of knowledge of work-related diseases and disorders of physical therapists is insufficient. The aim of the "Physical Therapist Cohort" (PTC) study is to analyze the association between work-related exposures and diseases among physical therapists in Germany. This article describes the protocol of the baseline assessment of the PTC study.
Methods/Design: A cross-sectional study will be conducted as baseline assessment and will include a representative random sample of approximately 300 physical therapists employed in Germany (exposure group), and a population-based comparison group (n = 300). The comparison group will comprise a sample of working aged (18–65 years) inhabitants of a German city. Variables of interest will be assessed using a questionnaire manual including questions regarding musculoskeletal, dermal, and infectious diseases and disorders as well as psychosocial exposures, diseases and disorders. In addition to subjective measures, a clinical examination will be used to objectify the questionnaire-based results (n = 50).
Discussion: The study, which includes extensive data collection, provides a unique opportunity to study the prospective association of work-related exposures and associated complaints of physical therapists. Baseline results will give first clues with regard to whether and how prevalent main exposures of physiotherapeutic work and typical work areas of physical therapists are associated with the development of work-related diseases. Thereby, this baseline assessment provides the basis for further investigations to examine causal relationships in accordance with a longitudinal design.
Nanoparticles (particles sized between 1 and 100 nanometers) are more and more used in all fields of science and medicine for their physicochemical properties. As gold has traditionally been considered as chemically inert and biocompatible, in particular, gold nanoparticles have been established as valuable tools in several areas of biomedical research. But in contrast to the multitude of studies that addressed the clinical use of gold nanoparticles, only little is known about potential toxicological effects such as induction of inflammatory immune responses, possible apoptotic cell death or developmental growth inhibition in embryos. Therefore the present study performed a systematic review of toxicological data, especially experimentally acquired data concerning in-vivo-toxicity, published in the PubMed. It can be stated that the data in this area of research is still largely limited. Especially, knowledge about size-, charge- and surface-chemistry dependent in-vivo-toxicity is needed to predict the hazard potential of auric nanoparticles (AuNPs) for humans.
T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.
The neurophysiological changes associated with Alzheimer's Disease (AD) and Mild Cognitive Impairment (MCI) include an increase in low frequency activity, as measured with electroencephalography or magnetoencephalography (MEG). A relevant property of spectral measures is the alpha peak, which corresponds to the dominant alpha rhythm. Here we studied the spatial distribution of MEG resting state alpha peak frequency and amplitude values in a sample of 27 MCI patients and 24 age-matched healthy controls. Power spectra were reconstructed in source space with linearly constrained minimum variance beamformer. Then, 88 Regions of Interest (ROIs) were defined and an alpha peak per ROI and subject was identified. Statistical analyses were performed at every ROI, accounting for age, sex and educational level. Peak frequency was significantly decreased (p < 0.05) in MCIs in many posterior ROIs. The average peak frequency over all ROIs was 9.68 ± 0.71 Hz for controls and 9.05 ± 0.90 Hz for MCIs and the average normalized amplitude was (2.57 ± 0.59)·10(-2) for controls and (2.70 ± 0.49)·10(-2) for MCIs. Age and gender were also found to play a role in the alpha peak, since its frequency was higher in females than in males in posterior ROIs and correlated negatively with age in frontal ROIs. Furthermore, we examined the dependence of peak parameters with hippocampal volume, which is a commonly used marker of early structural AD-related damage. Peak frequency was positively correlated with hippocampal volume in many posterior ROIs. Overall, these findings indicate a pathological alpha slowing in MCI.
Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.
In zahlreichen, in der Einleitung bereits näher beschriebenen Studien konnte anxiolytische Wirksamkeit des patentierten ätherischen Öls Silexan sowohl im Tiermodell, als auch am Menschen zur Therapie subsyndromaler und generalisierter Angsterkrankungen demonstriert werden.
Das Ziel der vorliegenden Dissertation war es, den zugrunde liegenden pharmakologischen Wirkmechanismus zu untersuchen. Nach eingehender Analyse der Targets klassischer Anxiolytika, richtete sich der Fokus auf spannungsabhängige Calciumkanäle, die die Bindungsstelle des Gabapentinoids Pregabalin enthalten. Die mögliche Modulation der Kanäle wurde an PC-Zellen, einem Modell neuronaler Vorläuferzellen, murinen Synaptosomen, aber auch in primären Neuronen der für die Genese der Angsterkrankungen relevanten Hirnareale Hippocampus und Cortex untersucht. Die Kanalexpression, sowie - distribution in den unterschiedlichen Geweben wurde charakterisiert und die Frage erörtert, ob Silexan spezifische Modulation eines bestimmten Kanalsubtyps vermittelt oder unspezifisch alle Kanäle dieser Gattung inhibiert. Um dies näher zu beleuchten wurden elektrophysiologische Messungen an transfizierten Zellen durchgeführt, die jeweils nur einen Subtyp der Familie spannungsabhängiger Calciumkanäle exprimieren. Zur weiteren Aufklärung des Wirkmechanismus wurde auch die mögliche Involvierung G-Protein gekoppelter Rezeptoren ermittelt.
Der zweite Teil der vorliegenden Untersuchung widmet sich der Klärung potentieller antidepressiver Wirkungen durch mögliche neurotrophe Effekte. Primäre hippocampale Neurone und PC12-Zellen wurden hierzu u.a. auf prä- und postsynaptische Marker, Ausdifferenzierungsmarker, sowie Neuritenwachstum hin analysiert.
Background: High-producer TGFβ1 genotypes are associated with severe lung disease in cystic fibrosis (CF), but studies combining IL-8, TNFα-, and TGFβ1(+genotype) levels and their impact on CF lung disease are scarce.
Aim: Assessing the relationship between TGFβ1, IL-8, and TNF-α and lung disease in CF in an exacerbation-free interval.
Methods: Twenty four patients delta F508 homozygous (median age 20.5 y, Shwachman score 75, FEV1(%) 83) and 8 controls (median age 27.5 y) were examined. TGFβ1 was assessed in serum and induced sputum (IS) by ELISA, for IL-8 and TNF-α by chemiluminescence in IS and whole blood. Genotyping was performed for TGFβ1 C−509T and T+869C utilizing RFLP.
Results: TGFβ1 in IS (CF/controls median 76.5/59.1 pg/mL, P < 0.074) was higher in CF. There was a negative correlation between TGFβ1 in serum and lung function (LF) (FEV1 (r = −0.488, P = 0.025), MEF 25 (r = −0.425, P = 0.055), and VC (r = −0.572, P = 0.007)). Genotypes had no impact on TGFβ1 in IS, serum, and LF. In IS TGFβ1 correlated with IL-8 (r = 0.593, P < 0.007) and TNF-α (r = 0.536, P < 0.018) in patients colonized by bacteria with flagellin.
Conclusion: TGFβ1 in serum not in IS correlates with LF. In patients colonized by bacteria with flagellin, TGFβ1 correlates with IL-8 and TNF-α in IS.
The Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO) here presents its updated recommendations for the treatment of documented fungal infections. Invasive fungal infections are a main cause of morbidity and mortality in cancer patients undergoing intensive chemotherapy regimens. In recent years, new antifungal agents have been licensed, and agents already approved have been studied in new indications. The choice of the most appropriate antifungal treatment depends on the fungal species suspected or identified, the patient’s risk factors (e.g., length and depth of neutropenia), and the expected side effects. This guideline reviews the clinical studies that served as a basis for the following recommendations. All recommendations including the levels of evidence are summarized in tables to give the reader rapid access to the information.
Background: The pro-inflammatory status of the elderly triggers most of the age-related diseases such as cancer and atherosclerosis. Atherosclerosis, the leading cause world wide of morbidity and death, is an inflammatory disease influenced by life-style and genetic host factors. Stimuli such as oxLDL or microbial ligands have been proposed to trigger inflammation leading to atherosclerosis. It has recently been shown that oxLDL activates immune cells via the Toll-like receptor (TLR) 4/6 complex. Several common single nucleotide polymorphisms (SNPs) of the TLR system have been associated with atherosclerosis. To investigate the role of TLR-6 we analyzed the association of the TLR-6 SNP Pro249Ser with atherogenesis.
Results: Genotyping of two independent groups with CAD, as well as of healthy controls revealed a significant association of the homozygous genotype with a reduced risk for atherosclerosis (odds ratio: 0.69, 95% CI 0.51-0.95, P = 0.02). In addition, we found a trend towards an association with the risk of restenosis after transluminal coronary angioplasty (odds ratio: 0.53, 95% CI 0.24-1.16, P = 0.12). In addition, first evidence is presented that the frequency of this protective genotype increases in a healthy population with age. Taken together, our results define a role for TLR-6 and its genetic variations in modulating the inflammatory response leading to atherosclerosis.
Conclusions: These results may lead to a better risk stratification, and potentially to an improved prophylactic treatment of high-risk populations. Furthermore, the protective effect of this polymorphism may lead to an increase of this genotype in the healthy elderly and may therefore be a novel genetic marker for the well-being during aging.
The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21) which appears in about 12% of all de novo acute myeloid leukemias (AMLs). Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2) within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21)-positive leukemia.
Purpose: Scientific evidence is limited regarding the long-term (>10 years) outcomes of large enough numbers of implants (>500) to allow for reliable comparison of subgroups. The purpose of this study was to analyze the outcomes of dental implants placed in an active University Clinic setting and followed for up to 20 years.
Materials and Methods: Data documenting the implant placement, prosthetic reconstruction, and annual follow-up of patients treated at Frankfurt University were extracted from a Structured Query Language database and patients’ written records and evaluated statistically.
Results: Between April of 1991 and May of 2011, 12,737 ANKYLOS® (DENTSPLY Implants Manufacturing GmbH, Mannheim, Germany) implants were placed in 4,206 patients for a variety of clinical indications. The Kaplan–Meier cumulative survival rate (CSR) was 93.3% after 204 months. Most of the failures (198/1.6%) occurred during the first year after implant placement and before prosthesis delivery. A significantly higher (p < .001) number of implants placed in the mandible and in hard quality bone failed than those placed in the maxilla or in weak and normal quality bone. Female patients had significantly higher CSRs (93.7% 204 months) than male patients (92.8% 204 months/p = .029). The implants showed low rates of peri-implant bone loss after 204 months (horizontal: 21 mm: 85.7%, vertical: 21 mm: 85.2%).
Conclusion: ANKYLOS dental implants followed for up to 20 years have high CSRs and low rates of peri-implant bone loss.
Objective: The aim of the study was to analyse the psychometric properties of the EQ-5D in patients with social phobia.
Methods: We used a sample of 445 patients with social phobia with five measurement points over a 30 month period. The discriminative ability of the EQ-5D was analysed by comparing the patients' responses with the general population and between different disease severity levels. For test-retest reliability we assessed the level of agreement in patients' responses over time, when there was no change in the Liebowitz Social Anxiety Scale (LSAS). Construct validity was analysed by identifying correlations of the EQ-5D with more specific instruments. For responsiveness we compared the means of EQ VAS/EQ-5D index anchored on improved (deteriorated) health status and computed effect sizes as well as a receiver operating characteristic (ROC) curve.
Results: Compared to the general population, patients with social phobia reported more problems in the dimensions "usual activities", "pain/discomfort", and "anxiety/depression" and less problems in "mobility" and "self-care". The EQ-5D was able to distinguish between different disease severity levels. The test-retest reliability was moderate (intraclass correlation coefficient > 0.6). Correlations between the EQ-5D and other instruments were mostly small except for correlations with Beck Depression Inventory. The EQ-5D index seemed to be more responsive than the EQ VAS, but with only medium effect sizes (0.5 < effect size < 0.8) in the British EQ-5D index and only significant in patients with improved health status. The ROC analysis revealed no significant results.
Conclusions: The EQ-5D was moderately reliable and responsive in patients with improved health status. Construct validity was limited.
Trial registration: Current controlled trials ISRCTN53517394.
Inhibitor of Apoptosis (IAP) proteins are a family of proteins with antiapoptotic functions that contribute to the evasion of apoptosis, a form of programed cell death. IAP proteins are expressed at high levels in a variety of human cancers including childhood acute leukemia. This elevated expression has been associated with unfavorable prognosis and poor outcome. Therefore, IAP proteins are currently exploited as therapeutic targets for cancer drug discovery. Consequently, small-molecule inhibitors or antisense oligonucleotides directed against IAP proteins have been developed over the last years. Indeed, IAP antagonists proved to exhibit in vitro and in vivo antitumor activities against childhood pediatric leukemia in several preclinical studies. Thus, targeting IAP proteins represents a promising molecular targeted strategy to overcome apoptosis resistance in childhood leukemia, which warrants further exploitation.
Scientists who are members of an editorial board have been accused of preferentially publishing their scientific work in the journal where they serve as editor. Reputation and academic standing do depend on an uninterrupted flow of published scientific work and the question does arise as to whether publication mainly occurs in the self-edited journal. This investigation was designed to determine whether editorial board members of five urological journals were more likely to publish their research reports in their own rather than in other journals. A retrospective analysis was conducted for all original reports published from 2001–2010 by 65 editorial board members nominated to the boards of five impact leading urologic journals in 2006. Publications before editorial board membership, 2001–2005, and publications within the period of time as an editorial board member, 2006–2010, were identified. The impact factors of the journals were also recorded over the time period 2001–2010 to see whether a change in impact factor correlated with publication locality. In the five journals as a whole, scientific work was not preferentially published in the journal in which the scientists served as editor. However, significant heterogeneity among the journals was evident. One journal showed a significant increase in the amount of published papers in the ‘own’ journal after assumption of editorship, three journals showed no change and one journal showed a highly significant decrease in publishing in the ‘own’ journal after assumption of editorship.
The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.
Introduction: We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT.
Patients and Methods: The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher.
Results: Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11).
Conclusion: Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.
Despite numerous clinical studies, which have investigated the therapeutic potential of repetitive transcranial magnetic stimulation (rTMS) in various brain diseases, our knowledge of the cellular and molecular mechanisms underlying rTMS-based therapies remains limited. Thus, a deeper understanding of rTMS-induced neural plasticity is required to optimize current treatment protocols. Studies in small animals or appropriate in vitro preparations (including models of brain diseases) provide highly useful experimental approaches in this context. State-of-the-art electrophysiological and live-cell imaging techniques that are well established in basic neuroscience can help answering some of the major questions in the field, such as (i) which neural structures are activated during TMS, (ii) how does rTMS induce Hebbian plasticity, and (iii) are other forms of plasticity (e.g., metaplasticity, structural plasticity) induced by rTMS? We argue that data gained from these studies will support the development of more effective and specific applications of rTMS in clinical practice.
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18:1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18:1, 9-and 13-HODE and HETEs.
Background: Published estimates on age-dependent frequency of diabetes in cystic fibrosis (CF) vary widely, and are based mostly on older data. However, CF treatment and prevention of comorbidities changed over recent years. In many studies, definition of cystic fibrosis-related diabetes (CFRD) is not in line with current guideline recommendations. Therefore, we evaluated age-dependent occurrence of glucose abnormalities and associated risk factors in CF patients who participated in a multicenter screening program using oral glucose tolerance tests (OGTT).
Methods: Between 2001 and 2010, 43 specialized CF centers from Germany and Austria serially performed 5,179 standardized OGTTs in 1,658 clinically stable, non-pregnant CF patients with no prior steroid medication or lung transplantation. Age-dependent occurrence of impaired fasting glucose (IFG), impaired glucose tolerance (IGT), IFG+IGT, one (DGT) or two consecutive (CFRD) diabetic OGTTs was analyzed, using Kaplan Meier curves. Cox proportional-hazards models were created to elucidate the influence of sex or underweight.
Results: At baseline/last OGTT, median age was 15.9 years/18.2 years and 30.6%/31.8% of patients were underweight. 25% of patients showed IFG at age 14.3 years; IGT at age 16.3 years; IFG+IGT combined at age 17.7 years. DGT was observed in 25% of patients at age 22.6 years; CFRD at age 34.5 years. Females had a 3.54 [95% CI 1.23–10.18] times higher risk for CFRD; risk for DGT was 2.21 [1.22–3.98] times higher. Underweight was a risk factor for IGT (HR [95% CI]: 1.38 [1.11–1.71]) and IFG+IGT (1.43 [1.11–1.83]), and in males also for DGT (1.49 [1.09–2.04]).
Conclusions/Significance: If confirmation of diabetes by a second test is required, as recommended in current guidelines, age at CFRD diagnosis was higher compared to most previous studies. However, known risk factors for glucose abnormalities in CF were confirmed. Confirmation of diabetic OGT by a repeat test is important for a consistent diagnosis of CFRD.
Background: Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.
Design and Methods: We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated.
Results: A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.
Conclusions: The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.
Sequential dosing in chemosensitization : targeting the PI3K/Akt/mTOR pathway in neuroblastoma
(2013)
Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ~30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3β and inhibition of GSK3β, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death – at least in part – by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network.
Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner.
Functional responses of natural killer (NK) cells including eradication of “harmful” cells and modulation of immune responses are regulated by a broad variety of activating and inhibitory NK receptors. Whereas the leukocyte receptor complex (LRC) encodes for NK receptors of the immunoglobulin superfamily, genes of C-type lectin-like NK receptors are clustered in the mammalian natural killer gene complex (NKC). Besides the thoroughly studied C-type lectin-like receptors NKG2D, CD94/NKG2x, and members of the murine Ly49 subfamily, the NKC also encodes for NK receptors of the less characterized NKRP1 subfamily. The prototypic mouse NKRP1 receptor is Nkrp1c (also known as NK1.1), while human members of the NKRP1 subfamily are NKRP1A, NKp80, and NKp65. The latter are not straight homologs of mouse NKRP1 receptors, but share distinct subfamily-specific traits classifying them as members of the NKRP1 subfamily. Ligands of the human NKPR1 receptors are likewise C-type lectin-like glycoproteins belonging to the CLEC2 subfamily (i.e., LLT1, AICL, and KACL), and are encoded in the NKC in tight genetic linkage to their respective receptors. Similarly, certain members of the mouse NKRP1 subfamily interact with genetically coupled CLEC2 glycoproteins, while the reasons for this intriguing tight genetic linkage remain unknown. Recent studies provided new and unique insights into the expression, interaction, and signaling of NKRP1 receptors and their ligands, thereby substantially advancing our understanding of their function and biology. Here, we review our current knowledge on NKRP1 receptors and their genetically linked CLEC2 ligands with an emphasis on the human receptor/ligand pairs NKRP1A-LLT1, NKp80-AICL, and NKp65-KACL.
The efficient coding hypothesis posits that sensory systems of animals strive to encode sensory signals efficiently by taking into account the redundancies in them. This principle has been very successful in explaining response properties of visual sensory neurons as adaptations to the statistics of natural images. Recently, we have begun to extend the efficient coding hypothesis to active perception through a form of intrinsically motivated learning: a sensory model learns an efficient code for the sensory signals while a reinforcement learner generates movements of the sense organs to improve the encoding of the signals. To this end, it receives an intrinsically generated reinforcement signal indicating how well the sensory model encodes the data. This approach has been tested in the context of binocular vison, leading to the autonomous development of disparity tuning and vergence control. Here we systematically investigate the robustness of the new approach in the context of a binocular vision system implemented on a robot. Robustness is an important aspect that reflects the ability of the system to deal with unmodeled disturbances or events, such as insults to the system that displace the stereo cameras. To demonstrate the robustness of our method and its ability to self-calibrate, we introduce various perturbations and test if and how the system recovers from them. We find that (1) the system can fully recover from a perturbation that can be compensated through the system's motor degrees of freedom, (2) performance degrades gracefully if the system cannot use its motor degrees of freedom to compensate for the perturbation, and (3) recovery from a perturbation is improved if both the sensory encoding and the behavior policy can adapt to the perturbation. Overall, this work demonstrates that our intrinsically motivated learning approach for efficient coding in active perception gives rise to a self-calibrating perceptual system of high robustness.
Genetic or pharmacological ablation of toll-like receptor 2 (TLR2) protects against myocardial ischemia/reperfusion injury (MI/R). However, the endogenous ligand responsible for TLR2 activation has not yet been detected. The objective of this study was to identify HMGB1 as an activator of TLR2 signalling during MI/R. C57BL/6 wild-type (WT) or TLR2(-/-)-mice were injected with vehicle, HMGB1, or HMGB1 BoxA one hour before myocardial ischemia (30 min) and reperfusion (24 hrs). Infarct size, cardiac troponin T, leukocyte infiltration, HMGB1 release, TLR4-, TLR9-, and RAGE-expression were quantified. HMGB1 plasma levels were measured in patients undergoing coronary artery bypass graft (CABG) surgery. HMGB1 antagonist BoxA reduced cardiomyocyte necrosis during MI/R in WT mice, accompanied by reduced leukocyte infiltration. Injection of HMGB1 did, however, not increase infarct size in WT animals. In TLR2(-/-)-hearts, neither BoxA nor HMGB1 affected infarct size. No differences in RAGE and TLR9 expression could be detected, while TLR2(-/-)-mice display increased TLR4 and HMGB1 expression. Plasma levels of HMGB1 were increased MI/R in TLR2(-/-)-mice after CABG surgery in patients carrying a TLR2 polymorphism (Arg753Gln). We here provide evidence that absence of TLR2 signalling abrogates infarct-sparing effects of HMGB1 blockade.
Background and study objective: Focused lung ultrasound (LUS) examinations are important tools in critical care medicine. There is evidence that LUS can be used for the detection of acute thoracic lesions. However, no validated training method is available. The goal of this study was to develop and assess an objective structured clinical examination (OSCE) curriculum for focused thorax, trachea, and lung ultrasound in emergency and critical care medicine (THOLUUSE).
Methods: 39 trainees underwent a one-day training course in a prospective educational study, including lectures in sonoanatomy and -pathology of the thorax, case presentations, and hands-on training. Trainees' pre- and posttest performances were assessed by multiple choice questionnaires, visual perception tests by interpretation video clips, practical performance of LUS, and identification of specific ultrasound findings.
Results: Trainees postcourse scores of correct MCQ answers increased from 56 ± 4% to 82 ± 2% (mean± SD; P < 0.001); visual perception skills increased from 54 ± 5% to 78 ± 3% (P < 0.001); practical ultrasound skills improved, and correct LUS was performed in 94%. Subgroup analysis revealed that learning success was independent from the trainees' previous ultrasound experience.
Conclusions: THOLUUSE significantly improves theoretical and practical skills for the diagnosis of acute thoracic lesions. We propose to implement THOLUUSE in emergency medicine training.
Smoking cigarettes throughout pregnancy is one of the single most important avoidable causes of adverse pregnancy outcomes and it represents the first major environmental risk of the unborn. If compared with other risk factors in the perinatal period, exposure to tobacco smoke is considered to be amongst the most harmful and it is associated with high rates of long and short term morbidity and mortality for mother and child. A variety of adverse pregnancy outcomes are linked with cigarette consumption before and during pregnancy. Maternal prenatal cigarette smoke disturbs the equilibrium among the oxidant and antioxidant system, has negative impact on the genetic and cellular level of both mother and fetus and causes a large quantity of diseases in the unborn child. These smoking-induced damages for the unborn offspring manifest themselves at various times in life and for most only a very limited range of causal treatment exists. Education, support and assistance are of high importance to decrease maternal and fetal morbidity and mortality, as there are few other avoidable factors which influence a child's health that profoundly throughout its life. It is imperative that smoking control should be seen as a public health priority.
In the past century, scientists have realized that venoms are a source of a number of natural substances presenting a wide range of pharmacological properties and often displaying a high specificity for their targets. Thus, the field of toxinology came into being, which is defined as the study of toxic substances of biological origin. Toxins are found in a wide variety of animals, including fish, cone snails, scorpions, snakes, and even some mammals. To be classified as venom, these must contain substances, i.e. toxins, which disturb physiological processes and must be deliberately delivered to the target animal. Snakes have evolved one of the most sophisticated mechanisms for venom delivery. Envenomation by snakebite can induce and inhibit aggregation/agglutination of platelets as well as inhibit/activate hemostasis, but also disrupt other physiological functions via neurotoxins and angioneurin growth factors. Snake venoms contain a substantial amount of C-type lectin-related proteins (CLRPs) which are known to function, notably, as integrin inhibitors. CLRPs are heterodimers composed of homologous α and β subunits which can assemble either covalently or noncovalently to oligomers, resulting in αβ, (αβ)2 and (αβ)4 structures. Some of the main targets of CLRPs are membrane receptors, coagulation factors, and proteins essential to hemostasis. The platelet collagen receptors GPVI and α2β1 integrin as well as the von Willebrand factor receptor GPIb play important roles in platelet activation and aggregation and are considered main targets of antithrombotic drugs. In this thesis, the integrin α2β1 is particularly considered as it is the sole collagen-binding integrin on platelets. Reduced expression of this platelet receptor results in dysfunction of platelet responses. Equivalently, overexpression of α2β1 integrin results in an increased risk of thrombosis. As a result, selective inhibitors of the collagen-α2β1 interaction could give rise to effective antithrombotic drugs. Integrins are large receptors which mediate cell-cell contacts and the binding of cells to the extracellular matrix (ECM). Therefore, they play a role in physiological processes, e.g. hemostasis and immunity, as well as in pathological processes, e.g. tumor angiogenesis and atherosclerosis. 18 α and 8 β integrin subunits, with nine α subunits containing an additional A domain, associate non-covalently to form 24 heterodimers with distinct binding specificities. Integrin collagen receptors are a subclass of four receptors which all utilize the β1 subunit. The α2β1 integrin is a collagen-binding receptor expressed not only on platelets, but also on endothelial and epithelial cells. Consequently, this integrin is also essential for cell adhesion and migration playing a role in angiogenesis as well as tumor metastasis. To date, there are five known antagonists of α2β1 integrin: EMS16, rhodocetin, vixapatin, and most recently rhinocetin and flavocetin-A. The first four have been shown to be specific for the integrin α2A domain, the major collagen-binding domain. All these antagonists are CLRPs and present new leads for drug design. In the past few years, many insights into the structure and function of rhodocetin were obtained. Monoclonal antibodies proved to be advantageous in disclosing this information, making them not only useful as therapeutic agents, but also as tools for protein characterization. The venom of the Vipera palaestinae snake was recently shown to contain an α2β1 integrin inhibitor, which prevented the integrin from binding collagen. This inhibitor, called vixapatin, was the initial focus of this dissertation. Vixapatin’s interaction with the α2β1 integrin needed further characterization on a molecular and cellular level to assess its medical potential and monoclonal antibodies were to be used as a tool. Originally, vixapatin had been isolated by reversed-phase high-performance liquid chromatography. To avoid the stringency of this method, for this study, it was replaced with gentler chromatographic methods. First, the α2β1 integrin inhibitor was isolated from the crude snake venom with affinity chromatography using the α2A domain as bait, establishing a method to quickly screen venoms for α2β1-binding proteins which affect the collagenintegrin interaction. The applicability of this method to other snake venoms was shown by isolating an α2A domain-specific toxin from the venom of Trimeresurus flavoviridis. To allow further characterization of both these toxins, gel filtration and ion exchange chromatography were employed to purify the protein without the α2A domain. These classical protein purification methods resulted in similar separation patterns of both the V. palaestinae and T. flavoviridis venom proteins. Purified proteins exhibiting the potential of inhibiting integrinbinding to collagen were analyzed by two-dimensional gel electrophoresis. Both VP-i and flavocetin-A, the integrin inhibitors from V. palaestinae and T. flavoviridis, respectively, were shown to have more complex structures than was evident from the purification. Each consisted of four low-molecular-weight proteins which assembled into two bands (for VP-i) or one single band (for flavocetin-A) under non-reducing conditions. Mass spectrometry analyses revealed VP-i to belong to the family of CLRPs, just like vixapatin does. However, these two proteins differed in their primary sequences and only showed homology to one another. The toxin purified from T. flavoviridis revealed this toxin to be flavocetin-A, a heterodimeric CLRP which had so far only been shown to have GPIb-binding activity. At the time of flavocetin-A’s purification, flavocetin-B was co-purified; flavocetin-B consists of the same two α and β subunits, plus an additional γ subunit. As no sequence information is known to date for the γ subunit, it may be one of the additional proteins purified here, along with an additional δ subunit. Therefore, the toxin isolated here may actually consist of four different subunits forming a tetramer of two different heterodimers, generating an (αβ)2(γδ)2 structure. This proposed (αβ)2(γδ)2 flavocetin-A structure has binding sites for both α2β1 integrin and GPIb, with no sterical overlap, as shown by affinity chromatography using the α2A domain and the extracellular domain of the GPIb receptor. The potential of VP-i and flavocetin-A to inhibit integrin-binding to type I collagen was shown during purification: Both toxins efficiently bind to the integrin α2A domain; also, VP-i and vixapatin bind to the A domain with the same affinity. Surface plasmon resonance showed the interaction of flavocetin-A with the α2β1 integrin to be extremely strong and association to be very fast. Furthermore, both toxins were shown to inhibit binding of the wildtype integrin to collagen: VP-i and flavocetin-A acted antagonistically on cell adhesion and cell migration. Initially, the interaction between VP-i and α2β1 integrin was to be further characterized with the help of monoclonal antibodies. However, this proved problematic, the procedure requiring various optimizations. Although, after expert consultation, some monoclonal antibodies could be obtained, the cells were extremely sensitive and gave unsatisfactory results when tested as detection tools in Western blot and immunoassays. Concluding, two novel α2β1 integrin inhibitors were discovered: VP-i and flavocetin-A, which were purified using the same procedure and which have similar functions. Both are Ctype lectin-related proteins which effectively inhibit cell adhesion and migration. This underlines that nature has instrumentalized CLRPs to specifically inhibit α2β1 integrin. Further characterization of VP-i and flavocetin-A will be able to provide leads for future drug development.
Persistent neuropathic pain is a frequent consequence of peripheral nerve injuries, particularly in the elderly. Using the IntelliCage we studied if sciatic nerve injury obstructed learning and memory in young and aged mice, each in wild type and progranulin deficient mice, which develop premature signs of brain aging. Both young and aged mice developed long-term nerve injury-evoked hyperalgesia and allodynia. In both genotypes, aged mice with neuropathic pain showed high error rates in place avoidance acquisition tasks. However, once learnt, these aged mice with neuropathic pain showed a significantly stronger maintenance of the aversive memory. Nerve injury did not affect place preference behavior in neither genotype, neither in young nor aged mice. However, nerve injury in progranulin deficient mice impaired the learning of spatial sequences of awarded places, particularly in the aged mice. This task required a discrimination of clockwise and anti-clockwise sequences. The chaining failure occurred only in progranulin deficient mice after nerve injury, but not in sham operated or wildtype mice, suggesting that progranulin was particularly important for compensatory adaptations after nerve injury. In contrast, all aged mice with neuropathic pain, irrespective of the genotype, had a long maintenance of aversive memory suggesting a negative alliance and possibly mutual aggravation of chronic neuropathic pain and aversive memory at old age.
Gegenstand dieser Arbeit ist die Untersuchung der Genexpression der axonalen wachstumsassoziierten Proteine NAP-22, GAP-43 und CAP-23 in den dopaminergen Neuronen der Substantia nigra pars compacta (SNc), einer im Mesencephalon gelegenen Zellregion, relativ zu der Genexpression eines Referenzgens. Als Versuchstiere dienten sechs Monate alte, männliche CB6F1 Wildtypmäuse sowie sechs Monate alte, männliche CAP-23 transgene (CAP-23tg) Tiere, die das wachstumsassoziierte Protein CAP-23 überexprimieren. Die dopaminergen Zellen der SNc wurden zunächst morphologisch charakterisiert und ihre Ausdehnung im Mittelhirn durch alternierende Immunfärbungen der Tyrosinhydroxylase, einem Schlüsselenzym der Dopaminsynthese, sowie Toluidinblaufärbungen ermittelt. Anschließend wurden die Neurone durch die Methode der Lasermikrodissektion (LMD) im Zellverband isoliert. Hierfür war die Optimierung der Toluidinblaufärbung erforderlich, mit dem Ziel, sowohl eine gute Färbung der Neurone als auch eine hohe RNA-Nativität zu erzielen. Die RNA wurde isoliert und nach Integritätsprüfung in cDNA umgeschrieben. Daraufhin erfolgte die Analyse der Genexpression der beschriebenen Gene durch die quantitative Echtzeit-Polymerase-Kettenreaktion (qRT-PCR). Dabei war feststellbar, dass das Transgen Cap-23 in den transgenen Tieren stärker exprimiert wird als das Endogen Nap-22. Es zeigte sich jedoch kein signifikanter Unterschied der Expression von endogenem Nap-22 und endogenem Gap-43 in den CAP-23tg Tieren im Vergleich zu der Expression in den Wildtypmäusen. Das bedeutet, dass die Überexpression von Cap-23 in den transgenen Tieren die Expression der mRNA der beiden endogenen wachstumsassoziierten Proteine NAP-22 und GAP-43 nicht beeinflusst. Auf Grundlage der in dieser Arbeit vorgelegten Ergebnisse soll in Folgeexperimenten untersucht werden, inwieweit die Überexpression von CAP-23 die Reorganisationsfähigkeit dopaminerger Neuronen der SNc nach einer Schädigung beeinflusst, wie sie zum Beispiel beim Menschen im Verlauf des Morbus Parkinson beobachtet wird.
Abdominale Aortenaneurysmen sind in Industrienationen eine häufige Erkrankung der Personengruppe über 65 Jahre. Diese Dilatationen der abdominalen Aorta zeichnen sich durch eine lokale Inflammation aus, die mit der Infiltration von Immunzellen, dem Verlust von glatten vaskulären Muskelzellen und der Degeneration der extrazellulären Matrix einhergeht. Ursprünglich als Symptom einer Atherosklerose angesehen, sind die Ursachen dieser progressiv verlaufenden Erkrankung nach wie vor nicht vollständig verstanden; obwohl steigendes Alter, männliches Geschlecht, genetische Prädisposition, Rauchen und ein zuvor erlittener Myocardinfarkt als Risikofaktoren identifiziert werden konnten. Der lange Zeit asymptomatische Krankheitsverlauf, die Gefahr einer Ruptur mit häufig letalen Folgen und der Mangel einer effizienten pharmakologischen Therapie machen eine weitere Untersuchung dieser Erkrankung unabdingbar.
Diltiazem ist ein Inhibitor spannungssensitiver L Typ-Calciumkanäle, der seit über 25 Jahren zur Behandlung von arterieller Hypertonie, verschiedener Arrhythmien und Angina pectoris verwendet wird. Im Rahmen der vorliegenden Arbeit sollte untersucht werden, ob Diltiazem auch einen antianeurysmatischen Effekt besitzt. Eine vierwöchige subcutane Infusion des blutdrucksteigernden Hormons Angiotensin II führte nach vier Wochen zur Bildung abdominaler Aortenaneurysmen, sowie zu atherosklerotischen Gefäßveränderungen der thorakalen Aorta Apolipoprotein E (ApoE)-defizienter Mäuse. Eine parallele Therapie mit Diltiazem über das Trinkwasser konnte diese Entwicklung unabhängig vom arteriellen Blutdruck und damit unabhängig von der antihypertensiven Wirkung verhindern. Im Aortenbogen Diltiazem-behandelter Tiere konnte im Rahmen dieses in vivo-Modells nach sechs Tagen eine deutlich geringere lokale Expression proinflammatorischer Cytokine, wie Tumornekrosefaktor-a, Interleukin-1ß (IL1B) und Interleukin-6 (IL6), Chemokine, wie CCL2, und degenerativer Proteasen, wie der Matrix-Metalloprotease 9 (MMP9), festgestellt werden. Dies war die Folge einer reduzierten Anzahl von Macrophagen in der Gefäßwand. Zirkulierende proinflammatorische Cytokine, wie CCL12, konnten im Serum teilweise ebenfalls vermindert nachgewiesen werden.
Obwohl die antihypertensive Wirkung von Diltiazem in glatten vaskulären Muskelzellen vermittelt wird, war es nicht möglich, die Angiotensin II-induzierte Produktion von promigratorischem CCL2 und proinflammatorischem IL6 in isolierten Aortenringen ApoE-defizienter Mäuse oder in glatten vaskulären Muskelzellen der Ratte zu reduzieren. Diltiazem war zudem nicht in der Lage, die CCL2-induzierte Migration proinflammatorischer Ly6C+-Monocyten in vivo zu unterbinden. In isolierten peritonealen Macrophagen ApoE-defizienter Mäuse dagegen, konnte die IL6 induzierte Expression von IL1B- und CCL12-mRNA durch eine Inkubation mit Diltiazem verhindert werden. In der RAW264.7-Zelllinie, die morphologische und funktionelle Merkmale von Monocyten und Macrophagen aufweist, konnte die Dilitiazem-sensitive IL6-induzierte Expression von IL1B-mRNA in vitro ebenfalls nachgewiesen werden. Eine Stimulation mit IL6 war in diesen Zellen jedoch nicht ausreichend, um die Sekretion von IL1B-Protein auszulösen.
Thorakales Aortengewebe wies im Vergleich mit RAW264.7-Zellen eine veränderte Ausstattung spannungssensitiver Calciumkanäle auf. In letzteren fanden sich keine muskelzellspezifischen L-Typ-Calciumkanäle (CACNA1C), aber eine relevante Expression neuronaler P/Q-Typ-Calciumkanäle (CACNA1A). Mittels fluorimetrischer Bestimmung mit Fura-2AM konnte jedoch festgestellt werden, dass die intrazelluläre Calciumkonzentration Diltiazem-behandelter RAW264.7-Zellen unverändert war und der antiinflammatorische Effekt somit calciumunabhängig vermittelt wurde.
Diltiazem war nicht in der Lage, eine Lipopolysaccharid (LPS)-bedingte Inflammation in RAW264.7-Zellen zu unterbinden. Weder die LPS-induzierte Sekretion von IL1B Protein, noch die nucleäre Translokation des Transkriptionsfaktors NF-?B oder die Aktivierung des NF-?B-Promotors konnten durch eine Inkubation der Zellen mit Diltiazem verhindert werden. Diltiazem reduzierte jedoch, die IL6-induzierte Aktivierung des AP 1-Promotors unabhängig von der MAPK1-Phosphorylierung oder der Phosphorylierung und nucleären Translokation des Transkriptionsfaktors STAT3 zu unterbinden. Eine Unterdrückung von c-Jun N-terminale Kinase JNK- oder p38 Proteinkinase-vermittelten Signalwegen ist damit wahrscheinlich.
Das Pirinixinsäurederivat LP105 ist ein neuer Inhibitor der Arachidonat-5-Lipoxygenase (LOX5), der im Rahmen dieser Arbeit erstmals in vivo auf seine antianeurysmatischen Eigenschaften hin untersucht wurde. LOX5 katalysiert die Reaktion von Arachidonsäure zu Leukotrien A4 und kontrolliert damit einen wichtigen Schritt in der Synthese proinflammatorischer Leukotriene. LP105 war im Tiermodell nicht in der Lage die Angiotensin II-induzierte Bildung abdominaler Aortenaneurysmen in ApoE-defizienten Mäusen komplett zu unterbinden, führte aber über die Reduktion der vaskulären Inflammation zu einer deutlich verringerten Krankheitslast. LP105 selbst beeinflusste die mRNA-Expression verschiedener Enzyme des Arachidonsäuremetabolismus nicht, verstärkte jedoch durch die Blockade von LOX5 die Metabolisierung von Arachidonsäure über Arachidonat-15-Lipoxygenase und Cytochrom P450-Enzyme.
Eine Infektion mit dem Hepatitis B Virus (HBV) kann bei 5-10 % der infizierten Erwachsenen und 70-90 % der infizierten Kinder chronisch verlaufen. Trotz einer verfügbaren Impfung gegen die Erkrankung sind heute nach Angaben der WHO weltweit etwa 350 Mio. Menschen chronisch HBV-infiziert [Lupberger and Hildt, 2007, Hollinger and Liang, 2001]. In 5-10 % der Fälle führt eine chronische Infektion zu einer Leberfibrose und Zirrhose, welche letztlich zur Ausbildung eines hepatozellulären Karzinoms (HCC) führen kann. HCCs sind die dritthäufigste karzinomassoziierte Todesursache weltweit [Blum, 2005]. Um Therapien gegen eine HBV-Infektion und das damit erhöhte Risiko einer HCC-Entstehung entwickeln zu können, müssen die einzelnen Schritte des HBV-Replikationszyklus verstanden sein. Wesentliche Schritte der frühen Infektionsphase, insbesondere der Rezeptor bzw. Rezeptorkomplex, welcher den Zelleintritt des Virus vermittelt sowie der Transport des Virusgenoms in den Zellkern, sind bisher noch unklar. Auch der Exportprozess und die Freisetzung der Viruspartikel ist bisher noch nicht im Detail verstanden. Es ist jedoch bekannt, dass die Viruspartikel unter Nutzung der zellulären ESCRT (endosomal sorting complex required for transport)-Maschinerie aus der Zelle freigesetzt werden [Lambert et al., 2007]. Auf der Suche nach Faktoren, die in diese Vorgänge involviert sind, konnte in dieser Arbeit das vesikeltransportassoziierte Protein α-Taxilin identifiziert werden. Der Einfluss von HBV auf die α-Taxilin-Bildung und seine mögliche Beteiligung am viralen Export wurden dabei näher charakterisiert. In HBV-positiven Zellen konnte in vivo und in vitro eine signifikante Steigerung der α-Taxilin-Expression nachgewiesen werden. Diese wird hierbei durch die HBV-Proteine HBx und LHBs über den Raf/Mek/Erk-Signalweg induziert [Glatzel, 2011]. Mithilfe von knockdown-Experimenten konnte beobachtet werden, dass α-Taxilin für den Export der Viruspartikel, nicht aber für den Export subviraler Partikel (SVPs) essentiell ist. Der Export der Virionen findet hierbei über das ESCRT-System statt. Den HBV-Strukturproteinen fehlen jedoch die für die Interaktion mit dem ESCRT-System essentiellen late-Domänen. Die Proteinstruktur von α-Taxilin dagegen weist diese late-Domänen auf. In dieser Arbeit konnte diese interaktionsvermittelnde Funktion von α-Taxilin zwischen dem Virus und dem ESCRT-System charakterisiert werden. Über eine Interaktion von α-Taxilin mit dem viralen LHBs-Protein auf der einen Seite und der tsg101-Komponente des ESCRT-I-Komplexes auf der anderen Seite agiert α-Taxilin als eine Art Linker zwischen dem ESCRT-System und HBV.
Darüber hinaus wurde Annexin A5 als zellulärer Interaktionspartner für α-Taxilin identifiziert [Röttger, 2011]. Es dirigiert α-Taxilin in einer Art shuttle-Funktion auf die Zellmembran suszeptibler Zellen und bindet es an deren Zelloberfläche. Diese Exposition von α-Taxilin nimmt während der Dedifferenzierung in Korrelation mit dem Suszeptibilitätsverlust primärer Hepatozyten ab. Eine Maskierung von α-Taxilin durch eine vorherige Inkubation der Zellen mit α-Taxilin-spezifischen Antikörpern konnte die Bindung und die Aufnahme der Viren inhibieren. Überexpressionsstudien bestätigten die essentielle Rezeptorfunktion von α-Taxilin. Die verstärkte Produktion von α-Taxilin führte zur Suszeptibilität der Zellen. Auch die Speziesspezifität der Bindung zwischen humanem α-Taxilin und HBV konnte in einem Co-Immunpräzipitationsexperiment mit den rezeptorbindenden Domänen von HBV, WHV und DHBV identifiziert werden.
In der vorliegenden Arbeit konnte somit zum ersten Mal eine Rezeptorfunktion von α-Taxilin bei der Aufnahme von HBV in die Wirtszelle nachgewiesen werden. Darüber hinaus schreiben die in dieser Arbeit gemachten Beobachtungen α-Taxilin eine essentielle Funktion für die Vermittlung des ESCRT-abhängigen Exports der Virionen aus der Zelle zu. Die hierbei gewonnen Erkenntnisse sind von hoher Relevanz für die weitere Erforschung der HBV-assoziierten Pathogenese und die Etablierung eines in vivo Infektions-Modells.
Die phylogenetisch hochkonservierte Jak/Stat‐Signaltransduktionskaskade repräsentiert eines der zentralen Säulen zellulärer Signalübertragung eukaryotischer Organismen. Ubiquitär im Organismus exprimiert und über eine Vielzahl von Zytokinen, Hormonen und Wachstumsfaktoren aktiviert, sind Stat‐Transkriptionsfaktoren maßgeblich an dem Erhalt der Physiologie und Homöostase von Organen und Geweben beteiligt. So sind die Mitglieder Stat5A und Stat5B (als homologe Proteine im Verbund als Stat5 bezeichnet) entscheidende Regulatoren des Immunsystems und der Hämatopoese, der Funktion und Entwicklung des Prostata‐ und Brustdrüsengewebes (Mammogenese) oder bestimmter Funktionen der Leber. Wie auch Stat3, konnten Stat5 Proteine in aberrant aktiver Form in verschiedensten Typen und Stadien humaner Tumore nachgewiesen werden, wo sie über die Expression ihrer Zielgene sowie über weitere nicht‐kanonische Funktionen im Zytoplasma und im Zellkern einer fortschreitend malignen Entartung entscheidend beitragen. Als Folge der Unterstützung essentieller Tumorgenese‐
Mechanismen, wie gesteigertes Zellwachstum, Apoptosehemmung, Migration und Metastasierung, Sauerstoff‐unabhängiger Energiestoffwechsel, Angiogenese oder Umgehung der Immunabwehr, entwickeln Tumore häufig eine Abhängigkeit gegenüber der gesteigerten Aktivität dieser Vertreter der Stat‐Proteinfamilie und reagieren mit einem Wachstumsstopp und Apoptoseinduktion auf ihre Inhibierung. Perspektivisch stellt die gezielte Interferenz mit aberranten, Tumortyp‐spezifischen Stat5‐Aktivitäten einen relevanten Ansatz in der personalisierten Therapie Stat5‐abhängiger Tumore, vorrangig leukämischen Ursprungs, dar. ...
The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment.
Introduction: We examined if a combination of proliferation markers and estrogen receptor (ER) activity could predict early versus late relapses in ER-positive breast cancer and inform the choice and length of adjuvant endocrine therapy.
Methods: Baseline affymetrix gene-expression profiles from ER-positive patients who received no systemic therapy (n = 559), adjuvant tamoxifen for 5 years (cohort-1: n = 683, cohort-2: n = 282) and from 58 patients treated with neoadjuvant letrozole for 3 months (gene-expression available at baseline, 14 and 90 days) were analyzed. A proliferation score based on the expression of mitotic kinases (MKS) and an ER-related score (ERS) adopted from Oncotype DX® were calculated. The same analysis was performed using the Genomic Grade Index as proliferation marker and the luminal gene score from the PAM50 classifier as measure of estrogen-related genes. Median values were used to define low and high marker groups and four combinations were created. Relapses were grouped into time cohorts of 0-2.5, 0-5, 5-10 years.
Results: In the overall 10 years period, the proportional hazards assumption was violated for several biomarker groups indicating time-dependent effects. In tamoxifen-treated patients Low-MKS/Low-ERS cancers had continuously increasing risk of relapse that was higher after 5 years than Low-MKS/High-ERS cancers [0 to 10 year, HR 3.36; p = 0.013]. High-MKS/High-ERS cancers had low risk of early relapse [0-2.5 years HR 0.13; p = 0.0006], but high risk of late relapse which was higher than in the High-MKS/Low-ERS group [after 5 years HR 3.86; p = 0.007]. The High-MKS/Low-ERS subset had most of the early relapses [0 to 2.5 years, HR 6.53; p < 0.0001] especially in node negative tumors and showed minimal response to neoadjuvant letrozole. These findings were qualitatively confirmed in a smaller independent cohort of tamoxifen-treated patients. Using different biomarkers provided similar results.
Conclusions: Early relapses are highest in highly proliferative/low-ERS cancers, in particular in node negative tumors. Relapses occurring after 5 years of adjuvant tamoxifen are highest among the highly-proliferative/high-ERS tumors although their risk of recurrence is modest in the first 5 years on tamoxifen. These tumors could be the best candidates for extended endocrine therapy.
Introduction: Langerhans Cell Histiocytosis (LCH) represents a rare benign disorder, previously designated as "Histiocytosis X", "Type II Histiocytosis" or "Langerhans Cell Granulomatosis". Clinical presentation includes osteolysis, ulcerations of skin and soft tissues but also involvement of the CNS is described.Because treatment concepts are not well defined the German Cooperative Group on Radiotherapy for Benign Diseases performed a retrospective analysis.
Methods and material: Eight closely cooperating centres collected patients' data of the past 45 years. As study endpoints disease free survival, recurrent disease, death and therapy related side effects were defined.
Results: A total of 80 patients with histologically proven LCH were irradiated within the past 45 years. According to the LCH classification of Greenberger et al. 37 patients had stage Ia, 21 patients stage Ib, 13 patients stage II and 9 patients stage IIIb and the median age was 29 years. The median Follow up was 54 months (range 9-134 months). A total of 39 patients had a surgical intervention and 23 patients a chemotherapy regimen.Radiation treatment was carried out with a median total dose of 15 Gy (range 3-50.4 Gy). The median single fraction was 2 Gy (range 1.8-3 Gy).Overall, 77% patients achieved a complete remission and 12.5% achieved a partial remission. The long-term control rate reached 80%. Within an actuarial overall 5-year survival of 90% no radiogenic side and late effects ≥EORTC/RTOG II° were observed.
Conclusion: In the present study a large collective of irradiated patients was analysed. Radiotherapy (RT) is a very effective and safe treatment option and even low RT doses show sufficient local control.
Background: A number of scientific papers on yellow fever have been published but no broad scientometric analysis on the published research of yellow fever has been reported. The aim of the article based study was to provide an in-depth evaluation of the yellow fever field using large-scale data analysis and employment of bibliometric indicators of production and quantity.
Methods: Data were retrieved from the Web of Science database (WoS) and analyzed as part of the NewQis platform. Then data were extracted from each file, transferred to databases and visualized as diagrams. Partially by means of density-equalizing mapping makes the findings clear and emphasizes the output of the analysis.
Results: In the study period from 1900 to 2012 a total of 5,053 yellow fever-associated items were published by 79 countries. The United States (USA) having the highest publication rate at 42% (n = 751) followed by far from Brazil (n = 203), France (n = 149) and the United Kingdom (n = 113). The most productive journals are the "Public Health Reports", the "American Journal of Tropical Medicine and Hygiene" and the "Journal of Virology". The gender analysis showed an overall steady increase of female authorship from 1950 to 2011. Brazil is the only country of the five most productive countries with a higher proportion of female scientists.
Conclusions: The present data shows an increase in research productivity over the entire study period, in particular an increase of female scientists. Brazil shows a majority of female authors, a fact that is confirmed by other studies.
Introduction: Postural control is a prerequisite to many everyday and sporting activities which requires the interaction of multiple sensorimotor processes. As long as we have no balance disorders, the maintenance of an erect standing position is taken for granted with automatic running control processes. It is well known that with increasing age or disease balance problems occur which often cause fall-related injuries. To assess balance performance, posturography is widely applied in which body sway is traditionally viewed as a manifestation of random fluctuations. Thus, the amount of sway is solely used as an index of postural stability, that is, less sway is an indication of better control. But, traditional measures of variability fail to account for the temporal organisation of postural sway. The concept of nonlinear dynamics suggests that variability in the motor output is not random but structured. It provides the stimulus to reveal the functionality of postural sway. This thesis evaluates nonlinear analysis tools in addition to classic linear methods in terms of age-related modifications of postural control and under different standing conditions in order to broaden the existing knowledge of postural control processes.
Methods: Static posturographic analyses were conducted which included the recording of centre of pressure (COP) time series by means of a force plate. Linear and nonlinear methods were used to quantify postural sway variability in order to evaluate both the amount and structure of sway. Classic time and frequency domain COP parameters were computed. In addition, wavelet transform (WT), multiscale entropy, detrended fluctuation analysis, and scaled windowed variance method were applied to COP signals in order to derive structural COP parameters. Two experiments were performed. 1) 16 young (26.1 ± 6.7 years), healthy subjects were asked to adopt a bipedal stance under single- and dual-task conditions. Three trials were conduced each with a different sampling duration: 30, 60, and 300 seconds [s]. 2) 26 young (28.15 ± 5.86 years) and 13 elderly (72 ± 7 years) subjects stood quietly for 60 s on five different surfaces which imposed different biomechanical constraints: level ground (LG), one foot on a step (ST), uphill (UH), downhill (DH), and slope (SL). Additional to COP recordings, limb load symmetry was assessed via foot pressure insoles.
Results: We found a higher sensitivity of structural COP parameters to modulations of postural control and partly an improved evaluation of sway dynamics in longer COP recordings. WT revealed a reweighing of frequency bands in response to altered standing conditions. Scaling exponents and entropy values of COP signals were task-dependent. Higher entropy values were found under the dual-task and condition ST. The time scales affected under the altered standing positions differed between groups and sway directions. Mainly larger posturograms were found in the elderly. Age effects were especially revealed in position ST and concerning medial-lateral COP signals. Load asymmetry was stronger in elderly subjects for LG, UH, and DH positions.
Discussion: Modifications of multiple time scales corresponds to an interplay of control subsystems to cope with the altered task demands. The affected time scales are age-dependent suggesting a change of control processes. Higher irregularity under the dual-task indicates a more complex motor output which is interpreted as less attentional investment into postural control. Larger complexity is evident for ST in contrast to LG position. ST obviously challenges lateral sway which is counteracted differently between groups. Load asymmetry suggests that especially elderly subjects adopt a step-initiation strategy.
Conclusion: A continued application of nonlinear methods is necessary to broaden the understanding of postural control mechanisms and to identify classifiers for balance dysfunctions. Structural COP parameters provide a more comprehensive indication of postural control system properties between groups and task demands. COP recordings of at least 60 s are recommended to adequately quantify COP signal structure. The analysis of postural strategies in everyday activities increases the ecological validity of postural control studies and can provide valuable information regarding the development of effective rehabilitation programs.
Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter of the inhibitory non-adrenergic non-cholinergic nervous system and influences the mammalian airway function in various ways. Hence known for bronchodilatory, immunomodulatory and mucus secretion modulating effects by interacting with the VIP receptors VPAC1 and VPAC2, it is discussed to be a promising target for pharmaceutical intervention in common diseases such as COPD and bronchial asthma. Here we examined the expression and transcriptional regulation of VPAC1 in the lungs of allergic mice using an ovalbumin (OVA) -induced model of allergic asthma. Mice were sensitized to OVA and challenged with an OVA aerosol. In parallel a control group was sham sensitized with saline. VPAC1 expression was examined using RT-PCR and real time-PCR studies were performed to quantify gene transcription. VPAC1 mRNA expression was detected in all samples of OVA-sensitized and challenged animals and control tissues. Further realtime analysis did not show significant differences at the transcriptional level.
Although the present studies did not indicate a major transcriptional regulation of VPAC1 in states of allergic airway inflammation, immunomodulatory effects of VPAC1 might still be present due to regulations at the translational level.
Purpose: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.
Experimental design: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.
Results: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.
Conclusions: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.
kurz und kn@pp news : Nr. 29
(2013)
Poster presentation: 28th Annual Scientific Meeting of the Society for Immunotherapy of Cancer (SITC)
Significant progress has been made over the last decade towards realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells, and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also continuously expanding cytotoxic cell lines such as NK-92 are being considered for adoptive cancer immunotherapy. High cytotoxicity of NK-92 has previously been shown against malignant cells of hematologic origin in preclinical studies, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. To enhance their therapeutic utility, we genetically modified NK-92 cells to express chimeric antigen receptors (CAR) specific for tumor-associated surface antigens. Such CAR were composed of a tumor-specific scFv antibody fragment fused via hinge and transmembrane domains to intracellular signaling moieties such as CD3 zeta chain, or composite fusion molecules also containing a costimulatory protein domain in addition to CD3 zeta. For development towards clinical applications, here a codon-optimized second generation CAR was constructed that consists of an ErbB2-specific scFv antibody domain fused via a linker to a composite CD28-CD3 zeta signaling domain. GMP-compliant protocols for vector production, lentiviral transduction and expansion of a genetically modified NK-92 single cell clone (NK-92/5.28.z) were established. Functional analysis of NK-92/5.28.z cells revealed high and stable CAR expression, selective cytotoxicity against ErbB2-expressing but otherwise NK-resistant tumor cells of different origins in vitro, as well as homing to ErbB2-expressing tumors in vivo. Furthermore, antigen specificity and selective cytotoxicity of these cells were retained in vivo, resulting in antitumoral activity against subcutaneous and intracranial glioblastoma xenografts in NSG mice. Ongoing work now focuses on the development of these cells for adoptive immunotherapy of ErbB2-positive glioblastoma.
BACKGROUND: Systemic thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the standard of acute stroke care. Its potential to increase the risk of secondary intracerebral hemorrhage, especially if administered late, has been ascribed to its proteolytic activity that has detrimental effects on blood-brain barrier (BBB) integrity after stroke. FTY720 has been shown to protect endothelial barriers in several disease models such as endotoxin-induced pulmonary edema and therefore is a promising candidate to counteract the deleterious effects of rt-PA. Besides that, every putative neuroprotectant that will be eventually forwarded into clinical trials should be tested in conjunction with rt-PA.
METHODS: We subjected C57Bl/6 mice to 3 h filament-induced tMCAO and postoperatively randomized them into four groups (n = 18/group) who received the following treatments directly prior to reperfusion: 1) vehicle-treatment, 2) FTY720 1 mg/kg i.p., 3) rt-PA 10 mg/kg i.v. or 4) FTY720 and rt-PA as a combination therapy. We measured functional neurological outcome, BBB disruption by quantification of EB extravasation and MMP-9 activity by gelatin zymography.
RESULTS: We observed a noticeable increase in mortality in the rt-PA/FTY720 cotreatment group (61%) as compared to the vehicle (33%), the FTY720 (39%) and the rt-PA group (44%). Overall, functional neurological outcome did not differ significantly between groups and FTY720 had no effect on rt-PA- and stroke-induced BBB disruption and MMP-9 activation.
CONCLUSIONS: Our data show that FTY720 does not improve functional outcome and BBB integrity in large hemispheric infarctions, neither alone nor in conjunction with rt-PA. These findings stand in contrast to a recently published study that showed beneficial effects of FTY720 in combination with thrombolysis in a thrombotic model of MCAO leading to circumscript cortical infarctions. They might therefore represent a caveat that the coadministration of these two drugs might lead to excess mortality in the setting of a severe stroke.
Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.