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Institute
kurz und kn@pp news : Nr. 28
(2013)
Network or graph theory has become a popular tool to represent and analyze large-scale interaction patterns in the brain. To derive a functional network representation from experimentally recorded neural time series one has to identify the structure of the interactions between these time series. In neuroscience, this is often done by pairwise bivariate analysis because a fully multivariate treatment is typically not possible due to limited data and excessive computational cost. Furthermore, a true multivariate analysis would consist of the analysis of the combined effects, including information theoretic synergies and redundancies, of all possible subsets of network components. Since the number of these subsets is the power set of the network components, this leads to a combinatorial explosion (i.e. a problem that is computationally intractable). In contrast, a pairwise bivariate analysis of interactions is typically feasible but introduces the possibility of false detection of spurious interactions between network components, especially due to cascade and common drive effects. These spurious connections in a network representation may introduce a bias to subsequently computed graph theoretical measures (e.g. clustering coefficient or centrality) as these measures depend on the reliability of the graph representation from which they are computed. Strictly speaking, graph theoretical measures are meaningful only if the underlying graph structure can be guaranteed to consist of one type of connections only, i.e. connections in the graph are guaranteed to be non-spurious. ...
When studying real world complex networks, one rarely has full access to all their components. As an example, the central nervous system of the human consists of 1011 neurons which are each connected to thousands of other neurons. Of these 100 billion neurons, at most a few hundred can be recorded in parallel. Thus observations are hampered by immense subsampling. While subsampling does not affect the observables of single neuron activity, it can heavily distort observables which characterize interactions between pairs or groups of neurons. Without a precise understanding how subsampling affects these observables, inference on neural network dynamics from subsampled neural data remains limited.
We systematically studied subsampling effects in three self-organized critical (SOC) models, since this class of models can reproduce the spatio-temporal activity of spontaneous activity observed in vivo. The models differed in their topology and in their precise interaction rules. The first model consisted of locally connected integrate- and fire units, thereby resembling cortical activity propagation mechanisms. The second model had the same interaction rules but random connectivity. The third model had local connectivity but different activity propagation rules. As a measure of network dynamics, we characterized the spatio-temporal waves of activity, called avalanches. Avalanches are characteristic for SOC models and neural tissue. Avalanche measures A (e.g. size, duration, shape) were calculated for the fully sampled and the subsampled models. To mimic subsampling in the models, we considered the activity of a subset of units only, discarding the activity of all the other units.
Under subsampling the avalanche measures A depended on three main factors: First, A depended on the interaction rules of the model and its topology, thus each model showed its own characteristic subsampling effects on A. Second, A depended on the number of sampled sites n. With small and intermediate n, the true A¬ could not be recovered in any of the models. Third, A depended on the distance d between sampled sites. With small d, A was overestimated, while with large d, A was underestimated.
Since under subsampling, the observables depended on the model's topology and interaction mechanisms, we propose that systematic subsampling can be exploited to compare models with neural data: When changing the number and the distance between electrodes in neural tissue and sampled units in a model analogously, the observables in a correct model should behave the same as in the neural tissue. Thereby, incorrect models can easily be discarded. Thus, systematic subsampling offers a promising and unique approach to model selection, even if brain activity was far from being fully sampled.
Two generic mechanisms for emergence of direction selectivity coexist in recurrent neural networks
(2013)
Poster presentation: Twenty Second Annual Computational Neuroscience Meeting: CNS*2013. Paris, France. 13-18 July 2013.
In the mammalian visual cortex, the time-averaged response of many neurons is maximal for stimuli moving in a particular direction. Such a direction selective response is not found in LGN, upstream of the visual processing pathway, suggesting that cortical networks play a strong role in the generation of direction selectivity. Here we investigate the mechanisms for the emergence of direction selectivity in the recurrent networks of nonlinear firing rate neurons in layer 4 of V1 receiving the input from LGN. In the model the LGN inputs are characterized by different receptive field positions, and their relative temporal phase shifts are reversed for the stimuli moving in the opposite direction. We propose that two distinct mechanisms result in the neuronal direction selective response in these recurrent networks. The first one is a result of nonlinear feed-forward summation of several time-shifted inputs. The second mechanism is based on the competition between neurons for firing in a winner-take-all regime. Both mechanisms rely on inhibitory interactions in the connectivity matrix of lateral connections, but the second one involves inhibitory loops. Typically, the first mechanism results in lower selectivity values than the second, but the time-course of acquiring direction selective response is faster for the first mechanism. Importantly, the two mechanisms have different input frequency tuning. The first mechanism, based on the nonlinear summation, result in a relatively narrow tuning curve around the preferred frequency of the stimulus in the case of the moving grating. In contrast, the direction selectivity arising from the second mechanism depends only weakly on the input frequency, i.e. has a broader tuning curve. These differences allow us to provide the recipe for identifying in experiment which of the two mechanisms is used by a given direction selective neuron. We then analyze how the statistics of the connections in the random recurrent networks affect the relative contributions from these two mechanisms and determine the distributions of the direction selectivity values. We identify the motifs in the connectivity matrix, which are required for each mechanism and show that the minimal conditions for both mechanisms are met in a very broad set of random recurrent networks with sufficiently strong inhibitory connections. Thus, we propose that these mechanisms coexist in generic recurrent networks with inhibition. Our results may account for the recent experimental observations that direction selectivity is present in dark-reared mice and ferrets [1,2]. It can also explain the emergence of direction selectivity in species lacking a spatially organized direction selectivity map.
To truly appreciate the myriad of events which relate synaptic function and vesicle dynamics, simulations should be done in a spatially realistic environment. This holds true in particular in order to explain as well the rather astonishing motor patterns which we observed within in vivo recordings which underlie peristaltic contractionsas well as the shape of the EPSPs at different forms of long-term stimulation, presented both here, at a well characterized synapse, the neuromuscular junction (NMJ) of the Drosophila larva (c.f. Figure 1). To this end, we have employed a reductionist approach and generated three dimensional models of single presynaptic boutons at the Drosophila larval NMJ. Vesicle dynamics are described by diffusion-like partial differential equations which are solved numerically on unstructured grids using the uG platform. In our model we varied parameters such as bouton-size, vesicle output probability (Po), stimulation frequency and number of synapses, to observe how altering these parameters effected bouton function. Hence we demonstrate that the morphologic and physiologic specialization maybe a convergent evolutionary adaptation to regulate the trade off between sustained, low output, and short term, high output, synaptic signals. There seems to be a biologically meaningful explanation for the co-existence of the two different bouton types as previously observed at the NMJ (characterized especially by the relation between size and Po), the assigning of two different tasks with respect to short- and long-time behaviour could allow for an optimized interplay of different synapse types. We can present astonishing similar results of experimental and simulation data which could be gained in particular without any data fitting, however based only on biophysical values which could be taken from different experimental results. As a side product, we demonstrate how advanced methods from numerical mathematics could help in future to resolve also other difficult experimental neurobiological issues.
Neuronal dynamics differs between wakefulness and sleep stages, so does the cognitive state. In contrast, a single attractor state, called self-organized critical (SOC), has been proposed to govern human brain dynamics for its optimal information coding and processing capabilities. Here we address two open questions: First, does the human brain always operate in this computationally optimal state, even during deep sleep? Second, previous evidence for SOC was based on activity within single brain areas, however, the interaction between brain areas may be organized differently. Here we asked whether the interaction between brain areas is SOC. ...
"A manager in the minds of doctors" : a comparison of new modes of control in European hospitals
(2013)
Background: Hospital governance increasingly combines management and professional self-governance. This article maps the new emergent modes of control in a comparative perspective and aims to better understand the relationship between medicine and management as hybrid and context-dependent. Theoretically, we critically review approaches into the managerialism-professionalism relationship; methodologically, we expand cross-country comparison towards the meso-level of organisations; and empirically, the focus is on processes and actors in a range of European hospitals.
Methods: The research is explorative and was carried out as part of the FP7 COST action IS0903 Medicine and Management, Working Group 2. Comprising seven European countries, the focus is on doctors and public hospitals. We use a comparative case study design that primarily draws on expert information and document analysis as well as other secondary sources.
Results: The findings reveal that managerial control is not simply an external force but increasingly integrated in medical professionalism. These processes of change are relevant in all countries but shaped by organisational settings, and therefore create different patterns of control: (1) ‘integrated’ control with high levels of coordination and coherent patterns for cost and quality controls; (2) ‘partly integrated’ control with diversity of coordination on hospital and department level and between cost and quality controls; and (3) ‘fragmented’ control with limited coordination and gaps between quality control more strongly dominated by medicine, and cost control by management.
Conclusions: Our comparison highlights how organisations matter and brings the crucial relevance of ‘coordination’ of medicine and management across the levels (hospital/department) and the substance (cost/quality-safety) of control into perspective. Consequently, coordination may serve as a taxonomy of emergent modes of control, thus bringing new directions for cost-efficient and quality-effective hospital governance into perspective.
BACKGROUND: In two clinical trials, low-grade fever was observed more frequently after coadministration than after separate administration of two recommended routine pediatric vaccines. Since fever is an important issue with vaccine tolerability, we performed this open-label study on the efficacy and safety of prophylactic use of paracetamol (acetaminophen, Benuron(R)) in children administered routine 7-valent pneumococcal conjugate vaccine (PCV-7) coadministered with hexavalent vaccine (diphtheria-tetanus-acellular pertussis-hepatitis B, polio, Haemophilus influenzae type b vaccine [DTPa-HBV-IPV/Hib]) in Germany.
METHODS: Healthy infants (N = 301) who received a 3-dose infant series of PCV-7 and DTPa-HBV-IPV/Hib plus a toddler dose were randomly assigned 1:1 to prophylactic paracetamol (125 mg or 250 mg suppositories, based on body weight) at vaccination, and at 6--8 hour intervals thereafter, or a control group that received no paracetamol. Rectal temperature and local and other systemic reactions were measured for 4 days post vaccination; adverse events were collected throughout the study.
RESULTS: In the intent-to-treat population, paracetamol reduced the incidence of fever >=38[degree sign]C, but this reduction was only significant for the infant series, with computed efficacy of 43.0% (95% confidence interval [CI]: 17.4, 61.2), and not significant after the toddler dose (efficacy 15.9%; 95% CI: -19.9, 41.3); results were similar in the per protocol (PP) population. Fever >39[degree sign]C was rare during the infant series, such that there were too few cases for assessment. After the toddler dose, paracetamol effectively reduced fever >39[degree sign]C, reaching statistical significance in the PP population only (efficacy 79%; 95% CI: 3.9, 97.7). Paracetamol also reduced reactogenicity, but there were few significant differences between groups after any dose. No vaccine-related serious adverse events were reported.
CONCLUSIONS: Paracetamol effectively prevented fever and other reactions, mainly during the infant series. However, as events were generally mild and of no concern in either group our data support current recommendations to administer paracetamol to treat symptoms only and not for routine prophylaxis.Trial registration: NCT00294294.
Radiographic and safety details of vertebral body stenting : results from a multicenter chart review
(2013)
Background: Up to one third of BKP treated cases shows no appreciable height restoration due to loss of both restored height and kyphotic realignment after balloon deflation. This shortcoming has called for an improved method that maintains the height and realignment reached by the fully inflated balloon until stabilization of the vertebral body by PMMA-based cementation. Restoration of the physiological vertebral body height for pain relief and for preventing further fractures of adjacent and distant vertebral bodies must be the main aim for such a method. A new vertebral body stenting system (VBS) stabilizes the vertebral body after balloon deflation until cementation. The radiographic and safety results of the first 100 cases where VBS was applied are presented.
Methods: During the planning phase of an ongoing international multicenter RCT, radiographic, procedural and followup details were retrospectively transcribed from charts and xrays for developing and testing the case report forms. Radiographs were centrally assessed at the institution of the first/senior author.
Results: 100 patients (62 with osteoporosis) with a total of 103 fractured vertebral bodies were treated with the VBS system. 49 were females with a mean age of 73.2 years; males were 66.7 years old. The mean preoperative anterior-middle-posterior heights were 20.3-17.6-28.0 mm, respectively. The mean local kyphotic angle was 13.1°. The mean preoperative Beck Index (anterior edge height/posterior edge height) was 0.73, the mean alternative Beck Index (middle height/posterior edge height) was 0.63. The mean postoperative heights were restored to 24.5-24.6-30.4 mm, respectively. The mean local kyphotic angle was reduced to 8.9°. The mean postoperative Beck Index was 0.81, the mean alternative one was 0.82. The overall extrusion rate was 29.1%, the symptomatic one was 1%. In the osteoporosis subgroup there were 23.8% extrusions. Within the three months followup interval there were 9% of adjacent and 4% of remote new fractures, all in the osteoporotic group.
Conclusions: VBS showed its strengths especially in realignment of crush and biconcave fractures. Given that fracture mobility is present, the realignment potential is sound and increases with the severity of preoperative vertebral body deformation.
Background: Exercise seems to minimize prostate cancer specific mortality risk and treatment related side effects like fatigue and incontinence. However the influence of physical activity on the immunological level remains uncertain. Even prostate cancer patients undergoing palliative treatment often have a relatively long life span compared to other cancer entities. To optimize exercise programs and their outcomes it is essential to investigate the underlying mechanisms. Further, it is important to discriminate between different exercise protocols and therapy regimes.
Methods/Design: The ProImmun study is a prospective multicenter patient preference randomized controlled trial investigating the influence of a 24 week endurance exercise program in 80–100 prostate cancer patients by comparing patients undergoing Antiandrogen therapy combined with exercise (AE), Antiandrogen therapy without exercise (A), Chemotherapy with exercise(CE) or Chemotherapy without exercise (C). The primary outcome of the study is a change in prostate cancer relevant cytokines and hormones (IL-6, MIF, IGF-1, Testosterone). Secondary endpoints are immune cell ratios, oxidative stress and antioxidative capacity levels, VO2 peak, fatigue and quality of life. Patients of the intervention group exercise five times per week, while two sessions are supervised. During the supervised sessions patients (AE and CE) exercise for 33 minutes on a bicycle ergometer at 70-75% of their VO2 peak. To assess long term effects and sustainability of the intervention two follow-up assessments are arranged 12 and 18 month after the intervention.
Discussion: The ProImmun study is the first trial which primarily investigates immunological effects of a six month endurance exercise program in prostate cancer patients during palliative care. Separating patients treated with Antiandrogen therapy from those who are additionally treated with Chemotherapy might allow a more specific view on the influence of endurance training interventions and the impact of different therapy protocols on the immune function.
Trial registration: German Clinical Trials Register: DRKS00004739
Background: One of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells.
Methods: High quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50–100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated.
Results: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells.
Conclusion: Molecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay.
In der vorliegenden Arbeit konnte gezeigt werden, dass bestimmte neuronale microRNAs im Rückenmark und in den Spinalganglien konstitutiv exprimiert und nach peripherer Entzündung mit Formalin oder Zymosan differenziell reguliert werden. Bei der SNI-induzierten Neuropathie konnte indessen keine signifikante Regulation der untersuchten microRNAs nachgewiesen werden. Aufgrund der Lokalisation in den Neuronen der Schmerz-verarbeitenden Laminae I und II des Dorsalhorns des Rückenmarks und angesichts der Regulation in entzündlich stimulierten Neuronen und Mikroglia wurde der Fokus der Arbeit auf die Untersuchung von microRNA-124a gelegt. Anhand von Expressionsanalysen konnte gezeigt werden, dass eine periphere entzündliche Stimulation mit Formalin oder Zymosan microRNA-124a im Rückenmark inhibiert, die Expression pro-inflammatorischer und pro-nozizeptiver Gene hiernach ermöglicht und ein vermehrtes Schmerzverhalten bewirkt. Die funktionelle Relevanz von microRNA-124a wurde in vivo mittels intravenöser Applikation von microRNA-124a-Modulatoren bei einem Modell für entzündliche Schmerzen, dem Formalin-Modell untersucht. Dabei führte die Hemmung von microRNA-124a zu einem verstärkten Schmerzverhalten, welches mit einer Hochregulation verschiedener Entzündungsmarker einherging. Die Überexpression von microRNA-124a dagegen antagonisierte die Hochregulation entzündlicher Mediatoren und führte zu einer Schmerzhemmung. Darüber hinaus konnte in der vorliegenden Arbeit der antinozizeptive Effekt von microRNA-124a mit der Regulation der Epigenetik-regulierenden Targets MeCP2, HDAC5 und MYST2 assoziiert werden und u.a. über die Hemmung des neuromodulierenden, pro-inflammatorischen Peptids BDNF verifiziert werden. Die spezielle Darreichung von microRNA-124a könnte demzufolge einen vielversprechenden Ansatz zur Therapie chronisch-entzündlicher Schmerzen liefern. Zukünftig werden weitere Studien notwendig sein um die eindeutige Funktion, die individuelle Wirkung sowie die therapeutische Relevanz von microRNA-124a zu analysieren. Darüber hinaus müssten Dosis-Wirkungs-Beziehungen und Nebenwirkungsprofile für microRNA-124a erstellt werden, um potenzielle Risiken, Chancen und Vorteile der microRNA-Modulation hinsichtlich einer humanen Schmerztherapie bewerten zu können.
This study aimed at determining the recommended dose of the mammalian target of rapamycin inhibitor everolimus in combination with mitomycin C (MMC) in patients with previously treated metastatic esophagogastric cancer. In this phase I trial, patients received escalated doses of oral everolimus (5, 7.5, and 10 mg/day) in combination with intravenous MMC 5 mg/m2 every 3 weeks. Endpoints were the dose-limiting toxicity (DLT), safety, and response rates. Tumor tissues were tested for HER2-status and mutations in the PTEN, PIK3CA, AKT1, CTNNB1, and E-cadherin type 1 genes. Sixteen patients (12 male, four female) with gastric/gastroesophageal junction cancer were included. All patients were previously treated with a platinum-based chemotherapy. Treatment cohorts were: 5 mg/day, three patients; 7.5 mg/day, three patients; and 10 mg/day, 10 patients. No DLTs occurred during dose escalation. Most frequent grade 3 toxicities were leukopenia (18.8%) and neutropenia (18.8%). All other grade 3 toxicities were below 10%. No grade 4 toxicities occurred. Three (18.8%) patients experienced partial responses and four patients had stable disease (SD). Antitumor activity according to Response Evaluation Criteria In Solid Tumors (RECIST)-criteria was highest in the 10 mg/day cohort. No associations between HER2-status or detected mutations and response were observed. The recommended dose of everolimus combined with MMC is 10 mg/day. Encouraging signs of antitumor activity were seen (http://www.ClinicalTrials.gov; Clinical trial registration number: NCT01042782).
Important brain functions need to be conserved throughout organisms of extremely varying sizes. Here we study the scaling properties of an essential component of computation in the brain: the single neuron. We compare morphology and signal propagation of a uniquely identifiable interneuron, the HS cell, in the blowfly (Calliphora) with its exact counterpart in the fruit fly (Drosophila) which is about four times smaller in each dimension. Anatomical features of the HS cell scale isometrically and minimise wiring costs but, by themselves, do not scale to preserve the electrotonic behaviour. However, the membrane properties are set to conserve dendritic as well as axonal delays and attenuation as well as dendritic integration of visual information. In conclusion, the electrotonic structure of a neuron, the HS cell in this case, is surprisingly stable over a wide range of morphological scales.
Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.
Background: Hepatitis B coinfection is common in HIV-positive individuals and as antiretroviral therapy has made death due to AIDS less common, hepatitis has become increasingly important. Several drugs are available to treat hepatitis B. The most potent and the one with the lowest risk of resistance appears to be tenofovir (TDF). However there are several questions that remain unanswered regarding the use of TDF, including the proportion of patients that achieves suppression of HBV viral load and over what time, whether suppression is durable and whether prior treatment with other HBV-active drugs such as lamivudine, compromises the efficacy of TDF due to possible selection of resistant HBV strains.
Methods: A systematic review and meta-analysis following PRISMA guidelines and using multilevel mixed effects logistic regression, stratified by prior and/or concomitant use of lamivudine and/or emtricitabine.
Results: Data was available from 23 studies including 550 HBV/HIV coinfected patients treated with TDF. Follow up was for up to seven years but to ensure sufficient power the data analyses were limited to three years. The overall proportion achieving suppression of HBV replication was 57.4%, 79.0% and 85.6% at one, two and three years, respectively. No effect of prior or concomitant 3TC/FTC was shown. Virological rebound on TDF treatment was rare.
Interpretation: TDF suppresses HBV to undetectable levels in the majority of HBV/HIV coinfected patients with the proportion fully suppressed continuing to increase during continuous treatment. Prior treatment with 3TC/FTC does not compromise efficacy of TDF treatment. The use of combination treatment with 3TC/FTC offers no significant benefit over TDF alone.
Background and Aims: Mutations reducing the function of Nav1.7 sodium channels entail diminished pain perception and olfactory acuity, suggesting a link between nociception and olfaction at ion channel level. We hypothesized that if such link exists, it should work in both directions and gain-of-function Nav1.7 mutations known to be associated with increased pain perception should also increase olfactory acuity.
Methods: SCN9A variants were assessed known to enhance pain perception and found more frequently in the average population. Specifically, carriers of SCN9A variants rs41268673C>A (P610T; n = 14) or rs6746030C>T (R1150W; n = 21) were compared with non-carriers (n = 40). Olfactory function was quantified by assessing odor threshold, odor discrimination and odor identification using an established olfactory test. Nociception was assessed by measuring pain thresholds to experimental nociceptive stimuli (punctate and blunt mechanical pressure, heat and electrical stimuli).
Results: The number of carried alleles of the non-mutated SCN9A haplotype rs41268673C/rs6746030C was significantly associated with the comparatively highest olfactory threshold (0 alleles: threshold at phenylethylethanol dilution step 12 of 16 (n = 1), 1 allele: 10.6±2.6 (n = 34), 2 alleles: 9.5±2.1 (n = 40)). The same SCN9A haplotype determined the pain threshold to blunt pressure stimuli (0 alleles: 21.1 N/m2, 1 allele: 29.8±10.4 N/m2, 2 alleles: 33.5±10.2 N/m2).
Conclusions: The findings established a working link between nociception and olfaction via Nav1.7 in the gain-of-function direction. Hence, together with the known reduced olfaction and pain in loss-of-function mutations, a bidirectional genetic functional association between nociception and olfaction exists at Nav1.7 level.
Background: The Sphingosine-1-phosphate (S1P) signaling pathway is known to influence pathophysiological processes within the brain and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P signaling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors.
Methods: We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence.
Results: FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in reactive astrogliosis and larger post-synaptic densities as well as changes in the expression of vascular endothelial growth factor α (VEGF α). Within the periinfarct cortex, S1P is significantly increased compared to healthy brain tissue.
Conclusion: Besides its known neuroprotective effects in the acute phase of experimental stroke, the initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors.
Background and Aims: In patients with advanced liver cirrhosis due to chronic hepatitis C virus (HCV) infection antiviral therapy with peginterferon and ribavirin is feasible in selected cases only due to potentially life-threatening side effects. However, predictive factors associated with hepatic decompensation during antiviral therapy are poorly defined.
Methods: In a retrospective cohort study, 68 patients with HCV-associated liver cirrhosis (mean MELD score 9.18±2.72) were treated with peginterferon and ribavirin. Clinical events indicating hepatic decompensation (onset of ascites, hepatic encephalopathy, upper gastrointestinal bleeding, hospitalization) as well as laboratory data were recorded at baseline and during a follow up period of 72 weeks after initiation of antiviral therapy. To monitor long term sequelae of end stage liver disease an extended follow up for HCC development, transplantation and death was applied (240weeks, ±SD 136weeks).
Results: Eighteen patients (26.5%) achieved a sustained virologic response. During the observational period a hepatic decompensation was observed in 36.8%. Patients with hepatic decompensation had higher MELD scores (10.84 vs. 8.23, p<0.001) and higher mean bilirubin levels (26.74 vs. 14.63 µmol/l, p<0.001), as well as lower serum albumin levels (38.2 vs. 41.1 g/l, p = 0.015), mean platelets (102.64 vs. 138.95/nl, p = 0.014) and mean leukocytes (4.02 vs. 5.68/nl, p = 0.002) at baseline as compared to those without decompensation. In the multivariate analysis the MELD score remained independently associated with hepatic decompensation (OR 1.56, 1.18–2.07; p = 0.002). When the patients were grouped according to their baseline MELD scores, hepatic decompensation occurred in 22%, 59%, and 83% of patients with MELD scores of 6–9, 10–13, and >14, respectively. Baseline MELD score was significantly associated with the risk for transplantation/death (p<0.001).
Conclusions: Our data suggest that the baseline MELD score predicts the risk of hepatic decompensation during antiviral therapy and thus contributes to decision making when antiviral therapy is discussed in HCV patients with advanced liver cirrhosis.
Corneal scarring remains a major cause of blindness world-wide, with limited treatment options, all of which have side-effects. Here, we tested the hypothesis that topical application of Rosiglitazone, a Thiazolidinedione and ligand of peroxisome proliferator activated receptor gamma (PPARγ), can effectively block scar formation in a cat model of corneal damage. Adult cats underwent bilateral epithelial debridement followed by excimer laser ablation of the central corneal stroma to a depth of ~160 µm as a means of experimentally inducing a reproducible wound. Eyes were then left untreated, or received 50 µl of either 10 µM Rosiglitazone in DMSO/Celluvisc, DMSO/Celluvisc vehicle or Celluvisc vehicle twice daily for 2 weeks. Cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and post-mortem immunohistochemistry for alpha smooth muscle actin (αSMA). Impacts of the wound and treatments on optical quality were assessed using wavefront sensing and optical coherence tomography at 2, 4, 8 and 12 weeks post-operatively. In parallel, cat corneal fibroblasts were cultured to assess the effects of Rosiglitazone on TGFβ-induced αSMA expression. Topical application of Rosiglitazone to cat eyes after injury decreased αSMA expression and haze, as well as the induction of lower-order and residual, higher-order wavefront aberrations compared to vehicle-treated eyes. Rosiglitazone also inhibited TGFβ-induced αSMA expression in cultured corneal fibroblasts. In conclusion, Rosiglitazone effectively controlled corneal fibrosis in vivo and in vitro, while restoring corneal thickness and optics. Its topical application may represent an effective, new avenue for the prevention of corneal scarring with distinct advantages for pathologically thin corneas.
Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.
Hintergrund und Fragestellung: Patientensicherheit ist in den letzten Jahren zum intensiv diskutierten Thema geworden. Zudem rückt als potenzielle Basis der Patientensicherheit die Patientensicherheitskultur von Einrichtungen des Gesundheitswesens in den Fokus, bislang wurde dahingehend vor allem der stationäre Bereich untersucht. Ziel dieser Arbeit ist es, einen Einblick in den aktuellen Stand der Patientensicherheitskultur in Hausarztpraxen zu geben, und diesbezügliche Einflussfaktoren und Zusammenhänge aufzuzeigen. Dabei wurden insbesondere zwei Fragestellungen untersucht: 1. Lässt sich ein Zusammenhang aufzeigen zwischen einzelnen Praxis- und Teammerkmalen einerseits und den Ergebnissen in den Bereichen Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement andererseits? 2. Lassen sich durch einzelne Praxis- und Teammerkmale bzw. Sicherheitsklimafaktoren die Ausprägungen einer Praxis in bestimmten Patientensicherheitsindikatoren und dem Fehlermanagement einer Praxis vorhersagen?
Material und Methoden: In 60 allgemeinärztlich tätigen Praxen aus Hessen wurde die Patientensicherheitskultur anhand von drei Methoden gemessen. Dies waren 1. der auf Selbstauskunft beruhende „Fragebogen zum Sicherheitsklima“, 2. durch Praxisbegehungen und Interviews erfasste Patientensicherheitsindikatoren, sowie 3. detailliert analysierte Fehlerberichte. Die statistische Auswertung umfasste u.a. Korrelationsanalysen (Mann-Whitney-U, Wilcoxon-W, Spearman-Rangkorrelation) sowie multivariate schrittweise Regressionsanalysen.
Ergebnisse: Die Beurteilung des Sicherheitsklimas fiel über alle Praxen hinweg homogen positiv aus (acht von neun Sicherheitsklimafaktoren mit Mittelwerten von mind. vier von fünf Punkten). Bei den 12 Patientensicherheitsindikatoren ergaben sich differenziertere Werte (niedrigster Mittelwert: Indikator „Marcumartherapie“ mit 0,43 von 1, höchster Mittelwert: Indikator „Allergiehinweis“ mit 0,75 von 1). Es gingen 24 Berichte kritischer Ereignisse ein, die zu 79% als „Kein Fehlermanagement“ oder „Unbefriedigendes Fehlermanagement“ beurteilt wurden. Die Korrelations- und Regressionsanalysen zeigten Zusammenhänge auf, z.B. erzielten größere Praxisteams niedrigere Werte beim Patientensicherheitsklima und höhere Werte bei den Patientensicherheitsindikatoren im Vergleich zum Durchschnitt.
Diskussion: Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement sind in einer Hausarztpraxis mit den verwendeten Instrumenten messbar. Jedes der drei Instrumente misst einen anderen, wichtigen Bereich der Sicherheitskultur, wodurch jeweils unterschiedliche Einstellungen und Prozesse beleuchtet und anschließend auch beurteilt und verbessert werden können. In den Analysen zur Beantwortung der beiden Fragestellungen konnten Zusammenhänge und Vorhersagevariablen herausgearbeitet werden, allerdings waren diese Zusammenhänge zum Teil entgegengesetzt. Daraus ergibt sich die Hypothese, dass Praxis- und Teammerkmale als Voraussetzungen zu unterschiedlichen Ausprägungen von Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement führen können. Insgesamt könnte die Qualität der hausärztlichen Arbeit und die Sicherheit der Versorgung durch eine regelmäßige Reflektion der Praxisabläufe anhand der drei Messmethoden gesteigert werden.
Body Integrity Identity Disorder (BIID) ist eine bisher kaum erforschte Störung, bei der die Betroffenen den Wunsch beziehungsweise das Verlangen nach einer Körperbehinderung verspüren. In den meisten Fällen, wie auch in dieser Studie, ist eine Oberschenkelamputation die gewünschte Modifikation. Durch die Amputation erhoffen die Betroffenen endlich sie selbst zu werden, da sie sich mit ihrem realen Körperbild nicht identifizieren können. Ihr vorgestelltes Körperbild ist das eines Amputierten. Die Störung manifestiert sich bereits im Kindesalter. Im Laufe der Zeit nimmt das Verlangen der Amputation zu, so dass es neben der vermehrten Beschäftigung sogar zu lebensgefährlichen Selbstverletzungen im Zuge einer Verwirklichung kommen kann.
Die vorliegende Studie beschäftigt sich erstmalig mittels funktioneller Magnetresonanztomographie mit der neuronalen Repräsentation der Störung BIID beim Anblick des eigenen realen und des gewünschten amputierten Körpers. Für die Studie wurden Fotos von den Probanden und einer fremden Person gemacht und mit einer Software so modifiziert, dass die Probanden in sechs verschiedenen Kategorien sowohl sich selbst real, sowie amputiert und mit Prothese als auch die fremde Person real, amputiert und mit Prothese gezeigt bekamen. Dasselbe Design wurde auch einer gesunden Kontrollgruppe vorgeführt. Aufgrund der Datenmenge wird in dieser Studie nur der reale und der amputierte Körper berücksichtigt.
Es zeigen sich deutliche Aktivierungsunterschiede zwischen der BIID Gruppe und der Kontrollgruppe beim Anblick des eigenen realen Körpers und beim Anblick des eigenen amputierten Körpers. Beim Anblick des eigenen realen Körpers zeigt die Kontrollgruppe gegenüber der BIID Gruppe einen stärkeren Selbstbezug zu ihrem Körper durch Aktivierungen des medialen frontalen Gyrus, des postzentralen Gyrus oder der Amygdala und einen positiver valenzierten Anblick, der sich im Gruppenvergleich durch eine Mehraktivierung im superioren temporalen Gyrus ausdrückt sowie durch die Ergebnisse der post-fMRT-Fragebögen unterstützt wird. Beim Anblick des eigenen amputierten Körpers zeigt sich durch ein fronto-parietales Netzwerk der stärkere Selbstbezug bei der BIID Gruppe. Die deutliche emotionale Involviertheit wird repräsentiert durch große Teile des limbischen Systems sowie durch präfrontale Bereiche. Hinzu kommen Aktivierungen, die eine deutliche Beteiligung des episodisch-autobiographischen und prozeduralen Gedächtnisses zeigen. So ist eine vollständig geplante Bewegungsabfolge der BIID Probanden beim Anblick ihres amputierten Körpers anhand der aktivierten Areale darstellbar, einschließlich der Feinregulationen in den Basalganglien, dem Nucleus ruber und dem Kleinhirn.
Die Ergebnisse zeigen neuronale Netzwerke der Körperrepräsentation, bei denen fehlende Aktivierungen der BIID Probanden beim Anblick des eigenen realen Körpers auf eine Fehlfunktion hindeuten könnten. Sie zeigen aber auch ein Netzwerk aus Erinnerungen und erlernten Prozessen, die mit Hilfe des mesolimbischen Dopaminsystems zur Aufrechterhaltung der Störung beitragen könnten. Zusammengefasst lässt sich sagen, dass das amputierte Wunschkörperbild neuronal stark und breitgefächert verankert ist und viel dominanter repräsentiert ist als das reale Körperbild. Durch die dargestellten Aktivierungen und Regelkreise leiten sich vor allem neue therapeutische Ansätze ab, die zu einer Linderung der Symptome von BIID beitragen könnten und vielleicht auch neue Anstöße in Hinblick auf eine Heilung der Störung liefern.
RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.
Erkrankungen des Herz-Kreislauf-Systems sind für die größte Zahl der Todesfälle in Deutschland verantwortlich. Das liegt nicht zuletzt daran, dass das menschliche Herz kaum Selbstheilungskräfte besitzt. Wissenschaftler suchen deshalb nach Möglichkeiten, die Regenerationsfähigkeit des Organs zu steigern. Dabei helfen ihnen der Blick ins Tierreich und modernste molekularbiologische Verfahren.
Wenn Zellen zu Medikamenten werden : neue Zelltherapien verbessern die Heilungschancen bei Leukämien
(2013)
Die Transplantation von Zellen aus dem Knochenmark oder von Stammzellen aus dem Blut gehört zu den bekanntesten Therapien bei Leukämie. Doch dabei treten Immunreaktionen als Nebenwirkung auf. Deshalb nehmen Forscher seit Kurzem auch die Transplantation bestimmter Immunzellen in den Blick. Im Labor gentechnisch aufgerüstet, werden sie zu äußerst effizienten "Krebs-Medikamenten".
Von Killerzellen und Kurzzeitweckern : ein Laborbesuch bei der "AG Koch" im Georg-Speyer-Haus
(2013)
Eins vorweg: Killerzellen sind in dieser Geschichte die Guten. In Laborräumen der Sicherheitsstufe eins werden Kittel und Handschuhe getragen, um das Material vor den Wissenschaftlern zu schützen. Kurzzeitwecker und Handcremetuben sind allgegenwärtig. Und wie das Experiment ausgegangen ist, wird noch nicht verraten. Doch jetzt der Reihe nach.
Rückschläge werfen eine neue Technologie um Jahrzehnte zurück – besonders, wenn Menschenleben zu beklagen sind. Bei der Gentherapie wird aber oft vergessen, dass sie nur bei Patienten angewendet wird, für die es keine konventionelle Therapie mehr gibt. Nach der Euphorie und den Rückschlägen der Anfangsjahre können Forscher nun die ersten Erfolge vorweisen.
Im Frankfurter LOEWE-Zentrum für Zell- und Gentherapie arbeiten mehr als 100 Wissenschaftler daran, die Verfahren der Zell- und Gentherapie in ihrer Wirksamkeit zu steigern und sicherer zu machen. Dabei konzentrieren sie sich auf Herz-, Infektions- und Krebserkrankungen sowie Erkrankungen des Immunsystems. Wissenschaftler, die die Grundlagen erforschen, kooperieren eng mit den Medizinern in der Klinik. So können neue Methoden frühzeitig in die Behandlungsstrategien einfließen.
Je besser Forscher es verstehen, defekte Gene zu reparieren oder beliebige Körperzellen zu reprogrammieren, desto gefahrloser wird die Gen- und Stammzell-Therapie für Patienten, die an heute noch unheilbaren Krankheiten leiden. Gleichzeitig zeichnet sich damit die Möglichkeit ab, in ferner Zukunft vielleicht das Genom kommender Generationen zu verändern oder Menschen zu klonieren. Der Internist Prof. Hubert Serve und die Politikwissenschaftlerin Dr. Anja Karnein wagen im Gespräch mit den beiden Redakteurinnen des Wissenschaftsmagazins »Forschung Frankfurt« Dr. Anne Hardy und Ulrike Jaspers einen Ausblick jenseits aller aktuellen Debatten. Sie diskutieren aber auch über die Themen, die Patienten wie Wissenschaftler zurzeit unmittelbar berühren.
Gen- und Stammzelltherapie stehen für das, wovon die Medizin schon immer geträumt hat: geschädigtes Gewebe durch gesundes ersetzen und die Wirkung defekter Gene durch intakte Kopien korrigieren. Wie ist der Stand der weltweiten Forschung? Welche Hindernisse sind zu überwinden, damit mehr Patienten von der regenerativen Medizin profi tieren werden?
Krebserkrankungen des lymphatischen Systems verlaufen sehr unterschiedlich, sind schwer zu diagnostizieren und haben oft schlechte Prognosen. Eine Forschergruppe unter der Leitung der Goethe-Universität will nun die Entstehung »Reifer T-Zell-Lymphome« besser verstehen und damit die Heilungschancen erhöhen. Die kürzlich von der Deutschen Forschungsgemeinschaft (DFG) bewilligte Forschergruppe wird in den kommenden drei Jahren mit zwei Millionen Euro gefördert.
Gentests an Embryonen sind in sehr begrenzten Ausnahmefällen jetzt auch in Deutschland erlaubt. In den nächsten Monaten werden an wenigen ausgewählten Kliniken Zentren zur Präimplantationsdiagnostik (PID) eingerichtet. Die Klinik für Frauenheilkunde und Geburtshilfe am Frankfurter Universitätsklinikum rechnet sich gute Chancen aus, dass ihre Bewerbung erfolgreich verläuft, so ihr Direktor Prof. Sven Becker.
Rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) are among the most common pediatric sarcomas (Arndt et al., 2012). Despite sarcomas representing a highly heterogeneous group of tumors, ES and alveolar RMS (ARMS) typically share one common genetic characteristic, namely a specific chromosomal translocation (Helman and Meltzer, 2003; Lessnick and Ladanyi, 2012). These translocations generate fusion proteins, which are composed of two transcription factors (TF). Typically, one TF is a developmentally regulated factor that is essential for proper specification of a given lineage and provides the DNA-binding domain, while the partner TF contributes a transactivation domain that drives aberrant expression of target genes. Based on these common genetic characteristics, the first ESF-EMBO research conference entitled “Molecular Biology and Innovative Therapies in Sarcomas of Childhood and Adolescence” with special focus on RMS and ES was held at the Polonia Castle in Pultusk, Poland. The conference gathered 70 participants from more than 15 countries and several continents representing most research groups that are active in this field.
Purpose: To assess the levels of inflammatory and angiogenic cytokines in undiluted vitreous from treatment-naïve patients with macular edema secondary to nonischemic branch retinal vein occlusion (BRVO), with flow cytometric bead array (CBA) and to correlate the results with subjective and multiple spectral-domain optical coherence tomography (SD-OCT) parameters.
Methods: A total of 43 eyes from 43 patients (mean age 69.7 years, 23 male) were divided into groups of new, "fresh" (n = 28; mean duration after onset 4.1 months) and older BRVO (n = 15; 11.6 months). Because of macular edema, these patients underwent an intravitreal therapy combining a single-site 23 g core vitrectomy with bevacizumab and dexamethasone. Undiluted vitreous was then analyzed for interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor isoform A (VEGF-A) levels with CBA and correlated with visual acuity (VA), clinical parameters of BRVO (type and perfusion status), and morphologic parameters, such as central macular thickness, central retinal thickness, thickness of the neurosensory retina, thickness of the serous retinal detachment, and the disruption of the ellipsoid line (photoreceptor inner and outer segments) and the external limiting membrane, as measured with SD-OCT. Twenty-eight undiluted vitreous samples from patients with idiopathic, nonuveitis vitreous floaters served as the controls.
Results: The mean IL-6 was 23.2 pg/mL (standard deviation, ±48.8), MCP-1 was 602.6 (±490.3), and VEGF-A was 161.8 (±314.3), and this was higher than in the control group, which had a mean IL-6 of 6.2 ± 3.4 pg/mL (P = 0.17), MCP-1 of 253.2 ± 73.5 (P < 0.0000001), and VEGF-A of 7.0 ± 4.9 (P < 0.003). In all BRVO samples, IL-6 correlated positively with MCP-1 and VEGF-A (correlation coefficient r = 0.79 and r = 0.46, respectively). VEGF-A was the only cytokine to correlate significantly with SD-OCT parameters (thickness of the neurosensory retina r = 0.31; disruption of the ellipsoid line r = 0.33). In the older BRVO group, there was a positive correlation between cytokines (IL-6 with MCP-1, r = 0.77; Il-6 with VEGF-A, r = 0.68; MCP-1 and VEGF-A, r = 0.68), whereas only IL-6 correlated with MCP-1 in the fresh group (r = 0.8).
Conclusion: The inflammatory markers and VEGF-A were elevated in the vitreous fluid of patients with BRVO, and these correlated with one another. VEGF-A was more often correlated with the morphologic changes assessed by SD-OCT, whereas the inflammatory markers had no significant influence on SD-OCT changes.
Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.
The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.
Author Summary: The live-attenuated yellow fever vaccine has been administered to more than 600 million people worldwide and is considered to be one of the most successful viral vaccines ever produced. Following injection, the apathogenic vaccine virus replicates in the vaccinee and induces antibodies that mediate virus neutralization and subsequent protection from disease. In principle, many different antibodies are induced by viral antigens, but it is becoming increasingly clear that only a subset of them is capable of inactivating the virus, and some antibody populations appear to dominate the immune response. However, to date there has been very little information on individual-specific variations of immunodominance and how such variations can affect the functionality of antibody responses. In our study, we addressed these issues and analyzed the fine specificities of antibodies induced by YF vaccination as well as the contribution of different antibody subsets to virus neutralization in 51 vaccinees. We demonstrate an extensive degree of individual variation with respect to immunodominance of antibody populations and their contribution to virus neutralization. Such variations can have an impact on vaccine-mediated protection, and thus insight into this phenomenon can provide leads for novel strategies in modern vaccine design.
Men and women differ substantially regarding height, weight, and body fat. Interestingly, previous work detecting genetic effects for waist-to-hip ratio, to assess body fat distribution, has found that many of these showed sex-differences. However, systematic searches for sex-differences in genetic effects have not yet been conducted. Therefore, we undertook a genome-wide search for sexually dimorphic genetic effects for anthropometric traits including 133,723 individuals in a large meta-analysis and followed promising variants in further 137,052 individuals, including a total of 94 studies. We identified seven loci with significant sex-difference including four previously established (near GRB14/COBLL1, LYPLAL1/SLC30A10, VEGFA, ADAMTS9) and three novel anthropometric trait loci (near MAP3K1, HSD17B4, PPARG), all of which were significant in women, but not in men. Of interest is that sex-difference was only observed for waist phenotypes, but not for height or body-mass-index. We found no evidence for sex-differences with opposite effect direction for men and women. The PPARG locus is of specific interest due to its link to diabetes genetics and therapy. Our findings demonstrate the importance of investigating sex differences, which may lead to a better understanding of disease mechanisms with a potential relevance to treatment options.
Natural Killer Gene Complex (NKC)–encoded C-type lectin-like receptors (CTLRs) are expressed on various immune cells including T cells, NK cells and myeloid cells and thereby contribute to the orchestration of cellular immune responses. Some NKC-encoded CTLRs are grouped into the C-type lectin family 2 (CLEC2 family) and interact with genetically linked CTLRs of the NKRP1 family. While many CLEC2 family members are expressed by hematopoietic cells (e.g. CD69 (CLEC2C)), others such as the keratinocyte-associated KACL (CLEC2A) are specifically expressed by other tissues. Here we provide the first characterization of the orphan gene CLEC2L. In contrast to other CLEC2 family members, CLEC2L is conserved among mammals and located outside of the NKC. We show that CLEC2L-encoded CTLRs are expressed as non-glycosylated, disulfide-linked homodimers at the cell surface. CLEC2L expression is fairly tissue-restricted with a predominant expression in the brain. Thus CLEC2L-encoded CTLRs were designated BACL (brain-associated C-type lectin). Combining in situ hybridization and immunohistochemistry, we show that BACL is expressed by neurons in the CNS, with a pronounced expression by Purkinje cells. Notably, the CLEC2L locus is adjacent to another orphan CTLR gene (KLRG2), but reporter cell assays did neither indicate interaction of BACL with the KLRG2 ectodomain nor with human NK cell lines or lymphocytes. Along these lines, growth of BACL-expressing tumor cell lines in immunocompetent mice did not provide evidence for an immune-related function of BACL. Altogether, the CLEC2L gene encodes a homodimeric cell surface CTLR that stands out among CLEC2 family members by its conservation in mammals, its biochemical properties and the predominant expression in the brain. Future studies will have to reveal insights into the functional relevance of BACL in the context of its neuronal expression.
Background: Remote ischemic preconditioning (RIPC) has been shown to enhance the tolerance of remote organs to cope with a subsequent ischemic event. We hypothesized that RIPC reduces postoperative neurocognitive dysfunction (POCD) in patients undergoing complex cardiac surgery.
Methods: We conducted a prospective, randomized, double-blind, controlled trial including 180 adult patients undergoing elective cardiac surgery with cardiopulmonary bypass. Patients were randomized either to RIPC or to control group. Primary endpoint was postoperative neurocognitive dysfunction 5–7 days after surgery assessed by a comprehensive test battery. Cognitive change was assumed if the preoperative to postoperative difference in 2 or more tasks assessing different cognitive domains exceeded more than one SD (1 SD criterion) or if the combined Z score was 1.96 or greater (Z score criterion).
Results: According to 1 SD criterion, 52% of control and 46% of RIPC patients had cognitive deterioration 5–7 days after surgery (p = 0.753). The summarized Z score showed a trend to more cognitive decline in the control group (2.16±5.30) compared to the RIPC group (1.14±4.02; p = 0.228). Three months after surgery, incidence and severity of neurocognitive dysfunction did not differ between control and RIPC. RIPC tended to decrease postoperative troponin T release at both 12 hours [0.60 (0.19–1.94) µg/L vs. 0.48 (0.07–1.84) µg/L] and 24 hours after surgery [0.36 (0.14–1.89) µg/L vs. 0.26 (0.07–0.90) µg/L].
Conclusions: We failed to demonstrate efficacy of a RIPC protocol with respect to incidence and severity of POCD and secondary outcome variables in patients undergoing a wide range of cardiac surgery. Therefore, definitive large-scale multicenter trials are needed.
Trial Registration: ClinicalTrials.gov NCT00877305
Diffusion tensor imaging (DTI) based assessment of white matter fiber tract integrity can support the diagnosis of Alzheimer’s disease (AD). The use of DTI as a biomarker, however, depends on its applicability in a multicenter setting accounting for effects of different MRI scanners. We applied multivariate machine learning (ML) to a large multicenter sample from the recently created framework of the European DTI study on Dementia (EDSD). We hypothesized that ML approaches may amend effects of multicenter acquisition. We included a sample of 137 patients with clinically probable AD (MMSE 20.6±5.3) and 143 healthy elderly controls, scanned in nine different scanners. For diagnostic classification we used the DTI indices fractional anisotropy (FA) and mean diffusivity (MD) and, for comparison, gray matter and white matter density maps from anatomical MRI. Data were classified using a Support Vector Machine (SVM) and a Naïve Bayes (NB) classifier. We used two cross-validation approaches, (i) test and training samples randomly drawn from the entire data set (pooled cross-validation) and (ii) data from each scanner as test set, and the data from the remaining scanners as training set (scanner-specific cross-validation). In the pooled cross-validation, SVM achieved an accuracy of 80% for FA and 83% for MD. Accuracies for NB were significantly lower, ranging between 68% and 75%. Removing variance components arising from scanners using principal component analysis did not significantly change the classification results for both classifiers. For the scanner-specific cross-validation, the classification accuracy was reduced for both SVM and NB. After mean correction, classification accuracy reached a level comparable to the results obtained from the pooled cross-validation. Our findings support the notion that machine learning classification allows robust classification of DTI data sets arising from multiple scanners, even if a new data set comes from a scanner that was not part of the training sample.
Background: Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC).
Methodology/Principal Findings: Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis.
Conclusions/Significance: Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.
Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver.
Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.
Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.
CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.
Objectives Tumour recurrence of glioblastoma multiforme (GBM) after initial treatment with surgical resection, radiotherapy and chemotherapy is an inevitable phenomenon. This retrospective cohort study compared the efficacy of interstitial high dose rate brachytherapy (HDR-BRT), re-resection and sole dose dense temozolomide chemotherapy (ddTMZ) in the treatment of recurrent glioblastoma after initial surgery and radiochemotherapy.
Design Retropective cohort study.
Setting Primary level of care with two participating centres. The geographical location was central Germany.
Participants From January 2005 to December 2010, a total of 111 patients developed recurrent GBM after initial surgery and radiotherapy with concomitant temozolomide. The inclusion criteria were as follows: (1) histology-proven diagnosis of primary GBM (WHO grade 4), (2) primary treatment with resection and radiochemotherapy, and (3) tumour recurrence/progression.
Interventions This study compared retrospectively the efficacy of interstitial HDR-BRT, re-resection and ddTMZ alone in the treatment of recurrent glioblastoma.
Primary and secondary outcome measures Median survival, progression free survival and complication rate.
Results Median survival after salvage therapy of the recurrence was 37, 30 and 26 weeks, respectively. The HDR-BRT group did significantly better than both the reoperation (p<0.05) and the ddTMZ groups (p<0.05). Moderate to severe complications in the HDR-BRT, reoperation and sole chemotherapy groups occurred in 5/50 (10%), 4/36 (11%) and 9/25 (36%) cases, respectively.
Conclusions CT-guided interstitial HDR-BRT attained higher survival benefits in the management of recurrent glioblastoma after initial surgery and radiotherapy with concurrent temozolomide in comparison with the other treatment modalities. The low risk of complications of the HDR-BRT and the fact that it can be delivered percutaneously in local anaesthesia render it a promissing treatment option for selected patients which should be further evaluated.
Purpose: To correlate inflammatory and proangiogenic key cytokines from undiluted vitreous of treatment-naïve central retinal vein occlusion (CRVO) patients with SD-OCT parameters.
Methods: Thirty-five patients (age 71.1 years, 24 phakic, 30 nonischemic) underwent intravitreal combination therapy, including a single-site 23-gauge core vitrectomy. Twenty-eight samples from patients with idiopathic, non-uveitis floaterectomy served as controls. Interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF-A) levels were correlated with the visual acuity (logMar), category of CRVO (ischemic or nonischemic) and morphologic parameters, such as central macular thickness-CMT, thickness of neurosensory retina-TNeuro, extent of serous retinal detachment-SRT and disintegrity of the IS/OS and others.
Results: The mean IL-6 was 64.7pg/ml (SD ± 115.8), MCP-1 1015.7 ( ± 970.1), and VEGF-A 278.4 ( ± 512.8), which was significantly higher than the control IL-6 6.2 ± 3.4pg/ml (P=0.06), MCP-1 253.2 ± 73.5 (P<0.0000001) and VEGF-A 7.0 ± 4.9 (P<0.0006). All cytokines correlated highly with one another (correlation coefficient r=0.82 for IL-6 and MCP-1; r=0.68 for Il-6 and VEGF-A; r=0.64 for MCP-1 and VEGF-A). IL-6 correlated significantly with CMT, TRT, SRT, dIS/OS, and dELM. MCP-1 correlated significantly with SRT, dIS/OS, and dELM. VEGF-A correlated not with changes in SD-OCT, while it had a trend to be higher in the ischemic versus the nonischemic CRVO group (P=0.09).
Conclusions: The inflammatory cytokines were more often correlated with morphologic changes assessed by SD-OCT, whereas VEGF-A did not correlate with CRVO-associated changes in SD-OCT. VEGF inhibition alone may not be sufficient in decreasing the inflammatory response in CRVO therapy.
The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to β-tricalcium phosphate (β-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/β-TCP composites showed a trend of increased stiffness compared with the controls (β-TCP without protein). N-SHh/β-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/β-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage.
We present the case of an aphasic 77-year-old stroke patient with left distal M1 occlusion who received rt-PA for thrombolysis while on oral anticoagulant treatment with dabigatran (150 mg b.i.d.). Coagulation parameters were normal (thrombin time 20 s, aPTT 20 s, INR 1.08) and the patient improved from an NIHSS of 15 to 5 within 24 h with sonographic evidence of M1 recanalization. She did not develop intracranial bleeding complications but showed unusually large diffuse skin ecchymoses. In our report, we give an overview of all reported cases of thrombolysis under dabigatran anticoagulation and discuss the questions of medication adherence under novel oral anticoagulants (NOA) and the safety of NOA in terms of secondary intracerebral hemorrhage after stroke.
Background: The incidence of pulmonary failure in trauma patients is considered to be influenced by several factors such as liver injury. We intended to assess the association of various potential predictors of pulmonary failure following thoracic trauma and liver injury.
Methods: Records of 12,585 trauma patients documented in the TraumaRegister DGU® of the German Trauma Society were analyzed regarding the potential impact of concomitant liver injury on the incidence of pulmonary failure using uni- and multivariate analyses. Pulmonary failure was defined as pulmonary failure of ≥ 3 SOFA-score points for at least two days. Patients were subdivided according to their injury pattern into four groups: group 1: AIS thorax < 3; AIS liver < 3; group 2: AIS thorax ≥ 3; AIS liver < 3; group 3: AIS thorax < 3; AIS liver ≥ 3 and group 4: AIS thorax ≥ 3; AIS liver ≥ 3.
Results: Overall, 2643 (21%) developed pulmonary failure, 12% (n= 642) in group 1, 26% (n= 697) in group 2, 16% (n= 30) in group 3, and 36% (n= 188) in group 4. Factors independently associated with pulmonary failure included relevant lung injury, pre-existing medical conditions (PMC), sex, transfusion of more than 10 units of packed red blood cells (PRBC), Glasgow Coma Scale (GCS) ≤ 8, and the ISS. However, liver injury was not associated with an increased risk of pulmonary failure following severe trauma in our setting.
Conclusions: Specific factors, but not liver injury, were associated with an increased risk of pulmonary failure following trauma. Trauma surgeons should be aware of these factors for optimized intensive care treatment.
In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.
Introduction: Vasospastic brain infarction is a devastating complication of aneurysmal subarachnoid hemorrhage (SAH). Using a probe for invasive monitoring of brain tissue oxygenation or blood flow is highly focal and may miss the site of cerebral vasospasm (CVS). Probe placement is based on the assumption that the spasm will occur either at the dependent vessel territory of the parent artery of the ruptured aneurysm or at the artery exposed to the focal thick blood clot. We investigated the likelihood of a focal monitoring sensor being placed in vasospasm or infarction territory on a hypothetical basis.
Methods: From our database we retrospectively selected consecutive SAH patients with angiographically proven (day 7–14) severe CVS (narrowing of vessel lumen >50%). Depending on the aneurysm location we applied a standard protocol of probe placement to detect the most probable site of severe CVS or infarction. We analyzed whether the placement was congruent with existing CVS/infarction.
Results: We analyzed 100 patients after SAH caused by aneurysms located in the following locations: MCA (n = 14), ICA (n = 30), A1CA (n = 4), AcoA or A2CA (n = 33), and VBA (n = 19). Sensor location corresponded with CVS territory in 93% of MCA, 87% of ICA, 76% of AcoA or A2CA, but only 50% of A1CA and 42% of VBA aneurysms. The focal probe was located inside the infarction territory in 95% of ICA, 89% of MCA, 78% of ACoA or A2CA, 50% of A1CA and 23% of VBA aneurysms.
Conclusion: The probability that a single focal probe will be situated in the territory of severe CVS and infarction varies. It seems to be reasonably accurate for MCA and ICA aneurysms, but not for ACA or VBA aneurysms.
Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.
Im Rahmen des Vortrags wird zunächst ein Überblick über die aktuell angewandten Linsensysteme in der (refraktiven) Kataraktchirurgie sowie im Rahmen des RLA gesprochen. Hierzu zählen die monofokalen sphärischen Standardlinsen, aber auch asphärische, torische und multifokale Implantate.
Speziell wird auf die Ergebnisse einer Studie zu einem neuen multifokalen-torischen Implantat eingegangen. Der Schwerpunkt des Vortrages liegt dann weiterhin auf den trifokalen Korrektionsmöglichkeiten. Zum einen wird die sog. binokulare Trifokalität, bei der zwei multifokale Intraokularlinsen unterschiedlicher Addition implantiert werden, besprochen. Durch die Anpassung jeweils eines Auges an den Intermediär- bzw. Nahbereich soll so bei verringerten optischen Phänomenen ein deutliches Sehen in drei Hauptdistanzen ermöglicht werden. Weiterhin befasst sich der Vortrag aber auch mit den neuen echten trifokalen Optiksystemen, welche ebenfalls deutliches Sehen in verschiedenen Entfernungen gewährleisten können.
Im dritten Teil des Vortrages werden aktuelle Langzeitergebnisse aus einer FDA Studie zur Evaluation einer kammerwinkelgestützten phaken Intraokularlinse, mit speziellem Augenmerk auf den cornealen Endothelzellverlust, sowie eine neuartige sulcusgestütze phake Intraokularlinse mit zentralem Loch zur Glaukomvermeidung vorgestellt.
Years of endemic infections with highly pathogenic avian influenza (HPAI) A subtype H5N1 virus in poultry and high numbers of infections in humans provide ample opportunity in Egypt for H5N1-HPAIV to develop pandemic potential. In an effort to better understand the viral determinants that facilitate human infections of the Egyptian H5N1-HPAIVvirus, we developed a new phylogenetic algorithm based on a new distance measure derived from the informational spectrum method (ISM). This new approach, which describes functional aspects of the evolution of the hemagglutinin subunit 1 (HA1), revealed a growing group G2 of H5N1-HPAIV in Egypt after 2009 that acquired new informational spectrum (IS) properties suggestive of an increased human tropism and pandemic potential. While in 2006 all viruses in Egypt belonged to the G1 group, by 2011 these viruses were virtually replaced by G2 viruses. All of the G2 viruses displayed four characteristic mutations (D43N, S120(D,N), (S,L)129Δ and I151T), three of which were previously reported to increase binding to the human receptor. Already in 2006–2008 G2 viruses were significantly (p<0.02) more often found in humans than expected from their overall prevalence and this further increased in 2009–2011 (p<0.007). Our approach also identified viruses that acquired additional mutations that we predict to further enhance their human tropism. The extensive evolution of Egyptian H5N1-HPAIV towards a preferential human tropism underlines an urgent need to closely monitor these viruses with respect to molecular determinants of virulence.
Introduction: Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial.
Methods: HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in-situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro.
Results: Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, p<0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, p<0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (p=0.004), but not in HER2-positive/ESR1-negative tumors.
Conclusions: Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group.
Background: Critical organ shortage results in the utilization of extended donor criteria (EDC) liver grafts. These marginal liver grafts are prone to increased ischemia reperfusion injury (IRI) which may contribute to deteriorated graft function and survival. Experimental data have shown that the calcineurin inhibitor tacrolimus exerts protective effects on hepatic IRI when applied intravenously or directly as a hepatic rinse. Therefore, the aim of the present study is to examine the effects of an ex vivo tacrolimus perfusion on IRI in transplantation of EDC liver grafts.
Methods/Design: The TOP-Study (tacrolimus organ perfusion) is a randomized multicenter trial comparing the ex vivo tacrolimus perfusion of marginal liver grafts with placebo. We hypothesize that a tacrolimus rinse reduces IRI, potentially improving organ survival following transplantation of EDC livers. The study includes livers with two or more EDC, according to Eurotransplant International Foundation’s definition of EDC livers. Prior to implantation, livers randomized to the treatment group are rinsed with tacrolimus at a concentration of 20 ng/ml in 1000 ml Custodiol solution and in the placebo group with Custodiol alone. The primary endpoint is the maximum serum alanine transamninase (ALT) level within the first 48 hours after surgery; however, the study design also includes a 1-year observation period following transplantation. The TOP-Study is an investigator-initiated trial sponsored by the University of Munich Hospital. Seven other German transplant centers are participating (Berlin, Frankfurt, Heidelberg, Mainz, Münster, Regensburg, Tübingen) and aim to include a total of 86 patients.
Discussion: Tacrolimus organ perfusion represents a promising strategy to reduce hepatic IRI following the transplantation of marginal liver grafts. This treatment may help to improve the function of EDC grafts and therefore safely expand the donor pool in light of critical organ shortage.
Trial register: EudraCT number: 2010-021333-31, ClinicalTrials.gov identifier: NCT01564095
Background: Castleman’s disease is a rare lymphoproliferative disorder. It typically presents as mediastinal masses and causes a wide range of clinical symptoms. Histologically, Castleman’s disease is classified as either a hyalinic vascular or plasma cell variant. The prognosis mainly depends on the histological type and broadly varies. We herein report our sonographic findings in a patient with Castleman’s disease, including gray-scale ultrasonography, color Doppler ultrasonography, and sonoelastography ultrasonography, which have not been previously reported in the literature. These findings allowed for a preoperative diagnosis and avoidance of overly aggressive therapy.
Case presentation: A 28-year-old European female patient with unicentric Castleman’s disease of hyalinic vascular type (HV) restricted to the axilla was referred to us because of a 4-month history of a painless, solitary mass located in the left axilla. The patient’s medical history was unremarkable.
Conclusion: Castleman’s disease is a pathologic entity of unknown etiology and pathogenesis. In this case report of unicentric HV-type CD, we demonstrate that typical sonographic findings can lead to a preoperative diagnosis of Castleman’s disease. Core needle biopsy usually allows for a final diagnosis and helps to avoid unnecessary operations and overtreatment.
Background: The introduction of fast-track treatment procedures following cardiac surgery has significantly shortened hospitalisation times in intensive care units (ICU). Readmission to intensive care units is generally considered a negative quality criterion. The aim of this retrospective study is to statistically analyse risk factors and predictors for re-admission to the ICU after a fast-track patient management program.
Methods: 229 operated patients (67 ± 11 years, 75% male, BMI 27 ± 3, 6/2010-5/2011) with use of extracorporeal circulation (70 ± 31 min aortic crossclamping, CABG 62%) were selected for a preoperative fast-track procedure (transfer on the day of surgery to an intermediate care (IMC) unit, stable circulatory conditions, extubated). A uni- and multivariate analysis were performed to identify independent predictors for re-admission to the ICU.
Results: Over the 11-month study period, 36% of all preoperatively declared fast-track patients could not be transferred to an IMC unit on the day of surgery (n = 77) or had to be readmitted to the ICU after the first postoperative day (n = 4). Readmission or ICU stay signifies a dramatic worsening of the patient outcome (mortality 0/10%, mean hospital stay 10.3 ± 2.5/16.5 ± 16.3, mean transfusion rate 1.4 ± 1,7/5.3 ± 9.1). Predicators for failure of the fast-track procedure are a preoperative ASA class > 3, NYHA class > III and an operation time >267 min ± 74. The significant risk factors for a major postoperative event (= low cardiac output and/or mortality and/or renal failure and/or re-thoracotomy and/or septic shock and/or wound healing disturbances and/or stroke) are a poor EF (OR 2.7 CI 95% 0.98-7.6) and the described ICU readmission (OR 0.14 CI95% 0.05-0.36).
Conclusion: Re-admission to the ICU or failure to transfer patients to the IMC is associated with a high loss of patient outcome. The ASA > 3, NYHA class > 3 and operation time >267 minutes are independent predictors of fast track protocol failure.
Over the last century, humans from industrialized societies have witnessed a radical increase in some dental diseases. A severe problem concerns the loss of dental materials (enamel and dentine) at the buccal cervical region of the tooth. This “modern-day” pathology, called non-carious cervical lesions (NCCLs), is ubiquitous and worldwide spread, but is very sporadic in modern humans from pre-industrialized societies. Scholars believe that several factors are involved, but the real dynamics behind this pathology are far from being understood. Here we use an engineering approach, finite element analysis (FEA), to suggest that the lack of dental wear, characteristic of industrialized societies, might be a major factor leading to NCCLs. Occlusal loads were applied to high resolution finite element models of lower second premolars (P2) to demonstrate that slightly worn P2s envisage high tensile stresses in the buccal cervical region, but when worn down artificially in the laboratory the pattern of stress distribution changes and the tensile stresses decrease, matching the results obtained in naturally worn P2s. In the modern industrialized world, individuals at advanced ages show very moderate dental wear when compared to past societies, and teeth are exposed to high tensile stresses at the buccal cervical region for decades longer. This is the most likely mechanism explaining enamel loss in the cervical region, and may favor the activity of other disruptive processes such as biocorrosion. Because of the lack of dental abrasion, our masticatory apparatus faces new challenges that can only be understood in an evolutionary perspective.
Blood levels of Glial Fibrillary Acidic Protein (GFAP) in patients with neurological diseases
(2013)
Background and Purpose: The brain-specific astroglial protein GFAP is a blood biomarker candidate indicative of intracerebral hemorrhage in patients with symptoms suspicious of acute stroke. Comparably little, however, is known about GFAP release in other neurological disorders. In order to identify potential “specificity gaps” of a future GFAP test used to diagnose intracerebral hemorrhage, we measured GFAP in the blood of a large and rather unselected collective of patients with neurological diseases.
Methods: Within a one-year period, we randomly selected in-patients of our university hospital for study inclusion. Patients with ischemic stroke, transient ischemic attack and intracerebral hemorrhage were excluded. Primary endpoint was the ICD-10 coded diagnosis reached at discharge. During hospital stay, blood was collected, and GFAP plasma levels were determined using an advanced prototype immunoassay at Roche Diagnostics.
Results: A total of 331 patients were included, covering a broad spectrum of neurological diseases. GFAP levels were low in the vast majority of patients, with 98.5% of cases lying below the cut-off that was previously defined for the differentiation of intracerebral hemorrhage and ischemic stroke. No diagnosis or group of diagnoses was identified that showed consistently increased GFAP values. No association with age and sex was found.
Conclusion: Most acute and chronic neurological diseases, including typical stroke mimics, are not associated with detectable GFAP levels in the bloodstream. Our findings underline the hypothesis that rapid astroglial destruction as in acute intracerebral hemorrhage is mandatory for GFAP increase. A future GFAP blood test applied to identify patients with intracerebral hemorrhage is likely to have a high specificity.
Background: In a previous analysis (Int J Radiat Oncol Biol Phys 70:828-835,2010), we assessed whether an adjuvant supplementation with selenium (Se) improves Se status and reduces the radiation-induced side-effects of patients treated by adjuvant radiotherapy (RT) for cervical and uterine cancer. Now, a potential relation between the planning target volume (PTV) of the RT and the Se effect concerning radiation induced diarrhoea was evaluated in detail.
Methods: Whole blood Se concentrations had been measured in patients with cervical (n=11) and uterine cancer (n=70) after surgical treatment, during, and at the end of RT. Patients with initial Se concentrations of less than 84 μg/l were categorized as Se-deficient and randomized before RT to receive Se (as sodium selenite) per os on the days of RT, or to receive no supplement during RT. Diarrhoea was graded according to the Common Toxicity Criteria system (CTC, Version 2a). The evaluation of the PTV of the RT was ascertained with the help of a specialised computer-assisted treatment planning software used for radiation planning procedure.
Results: A total of 81 patients had been randomized for the initial supplementation study, 39 of which received Se [selenium group, SeG] and 42 serving as controls [control group, CG]. Mean Se levels did not differ between SeG and CG upon study initiation, but were significantly higher in the SeG compared to the CG at the end of RT. The actuarial incidence of at least CTC 2 radiation induced diarrhoea in the SeG was 20.5% compared to 44.5% in the CG (p=0.04). The median PTV in both groups was 1302 ml (916–4608). With a PTV of <= 1302 ml (n=41) the actuarial incidence of at least CTC 2 diarrhoea in the SeG was 22.3% (4 of 18 patients) compared to 34.8% (8 of 23 patients) in the CG (p=0.50). In patients with a PTV of > 1302 ml (n=40) the actuarial incidence of at least CTC 2 diarrhoea in the SeG was 19.1% (4 of 21 patients) versus 52.6% (10 of 19 patients) in the CG (p=0.046).
Conclusions: Se supplementation during RT was effective to improve blood Se status in Se-deficient cervical and uterine cancer patients, and reduces episodes and severity of RT-induced diarrhoea. This effect was most pronounced and significant in patients with large PTV (> 1302 ml).
Current theories of the pathophysiology of schizophrenia have focused on abnormal temporal coordination of neural activity. Oscillations in the gamma-band range (>25 Hz) are of particular interest as they establish synchronization with great precision in local cortical networks. However, the contribution of high gamma (>60 Hz) oscillations toward the pathophysiology is less established. To address this issue, we recorded magnetoencephalographic (MEG) data from 16 medicated patients with chronic schizophrenia and 16 controls during the perception of Mooney faces. MEG data were analysed in the 25–150 Hz frequency range. Patients showed elevated reaction times and reduced detection rates during the perception of upright Mooney faces while responses to inverted stimuli were intact. Impaired processing of Mooney faces in schizophrenia patients was accompanied by a pronounced reduction in spectral power between 60–120 Hz (effect size: d = 1.26) which was correlated with disorganized symptoms (r = −0.72). Our findings demonstrate that deficits in high gamma-band oscillations as measured by MEG are a sensitive marker for aberrant cortical functioning in schizophrenia, suggesting an important aspect of the pathophysiology of the disorder.
As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.
This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.
Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.
Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.
Neuronal activity differs between wakefulness and sleep states. In contrast, an attractor state, called self-organized critical (SOC), was proposed to govern brain dynamics because it allows for optimal information coding. But is the human brain SOC for each vigilance state despite the variations in neuronal dynamics? We characterized neuronal avalanches – spatiotemporal waves of enhanced activity - from dense intracranial depth recordings in humans. We showed that avalanche distributions closely follow a power law – the hallmark feature of SOC - for each vigilance state. However, avalanches clearly differ with vigilance states: slow wave sleep (SWS) shows large avalanches, wakefulness intermediate, and rapid eye movement (REM) sleep small ones. Our SOC model, together with the data, suggested first that the differences are mediated by global but tiny changes in synaptic strength, and second, that the changes with vigilance states reflect small deviations from criticality to the subcritical regime, implying that the human brain does not operate at criticality proper but close to SOC. Independent of criticality, the analysis confirms that SWS shows increased correlations between cortical areas, and reveals that REM sleep shows more fragmented cortical dynamics.
Vascular endothelial growth factors (VEGFs), initially thought to act specifically on the vascular system, exert trophic effects on neural cells during development and adulthood. Therefore, the VEGF system serves as a promising therapeutic target for brain pathologies, but its simultaneous action on vascular cells paves the way for harmful side effects. To circumvent these deleterious effects, many studies have aimed to clarify whether VEGFs directly affect neural cells or if the effects are mediated secondarily via other cell types, like vascular cells. A great number of reports have shown the expression and function of VEGF receptors (VEGFRs), mainly VEGFR-1 and -2, in neural cells, where VEGFR-2 has been described as the major mediator of VEGF-A signals. This review aims to summarize and compare the divergent roles of VEGFR-1 and -2 during CNS development and homeostasis.
"PULS." – a blog-based online-magazine for students of medicine of the Goethe University Frankfurt
(2013)
In the context of nationwide protests 2009 also students of the faculty of medicine/dentistry at Goethe-University in Frankfurt demanded more transparency and communication. To satisfy these demands, a web 2.0-tool offered an innovative solution: A blog-based online-magazine for students and other faculty-members. The online-magazine "PULS." is realized with the share-ware blog-software (wordpress version 3.1.3) and is conceived and written by an online-journalist. "PULS." is available from https://newsmagazin.puls.med.uni-frankfurt.de/wp/. The articles are generated from own investigations and from ideas of different groups of the faculty– deanship, students and lecturers. A user-analysis is conducted with the open-source software Piwik and considers the data security. Additionally, every year an anonymous online-user-survey (Survey Monkey) is conducted. "PULS." is continuously online since 14.02.2010 and has published 806 articles (state: 27.11.2012) and has about 2400 readers monthly. The content focuses on the needs of Frankfurt medical students. The close cooperation with different groups of the faculty - deanship, students and lecturers - furthermore guarantees themes relevant to the academic faculty. "PULS." flanks complex projects and decisions with background-information and communicates them understandable. The user-evaluation shows a growing number of readers and a high acceptance for the online-magazine, its themes and its style. The web 2.0-tool "Blog" and the web-specific language comply with media habits of the main target group, the students of the faculty medicine/dentistry. Thus, "PULS." has proven as a suitable and strategic instrument. It pushes towards a higher transparency, more communication and a stronger identification of the students with their faculty.
"PULS." - Ein Blog als Online-Magazin für Medizinstudierende der Goethe-Universität Frankfurt
(2013)
Im Herbst 2009 forderten Studierende im Rahmen landesweiter Proteste auch am Fachbereich Medizin/Zahnmedizin der Goethe-Universität Frankfurt mehr Transparenz und Kommunikation zu Angelegenheiten ihres Studiums. Einen innovativen Lösungsansatz, um diesen Forderungen nachzukommen, bietet eines der Web 2.0 Werkzeuge: ein auf einer Blog-Software basierendes Online-Magazin für Studierende und andere Mitglieder des Fachbereichs.
Das öffentlich zugängliche Online-Magazin "PULS." (https://newsmagazin.puls.med.uni-frankfurt.de/wp/) wird mit einer freien Blog-Software (wordpress Version 3.1.3.) realisiert und von einer Online-Redakteurin konzipiert und geschrieben. Die Beiträge entstehen nach eigenen Recherchen sowie aus Anregungen und Gesprächen mit verschiedenen Personengruppen des Fachbereichs. Die datenschutzkonforme Auswertung der Zugriffe erfolgt über eine open-source Webanalyse-Software (Piwik). Zusätzlich werden jährlich mit dem Online-Umfrage-Tool Survey Monkey die Nutzer anonym befragt.
"PULS." ist seit dem 14.02.2010 ununterbrochen online und hat seitdem 806 Beiträge (Stand: 27.11.2012) publiziert und wird von ca. 2400 Besuchern monatlich gelesen. Das Themenspektrum ist zentriert auf die Anliegen der Frankfurter Medizin- und Zahnmedizinstudierenden. Die enge Zusammenarbeit mit verschiedenen Gruppierungen des Fachbereichs – Dekanat, Studierende und Lehrende – garantiert darüber hinaus ein fachbereichs-relevantes Themenspektrum. Das Online-Magazin begleitet komplexe Projekte und Entscheidungen mit Hintergrundinformationen und kommuniziert sie verständlich. Eine jährliche Nutzer-Evaluierung zeigt eine wachsende Leserzahl und eine sehr hohe Zustimmung für das Online-Magazin, seine Inhalte und seinen Stil. Das Web 2.0-Medium "Blog" und seine web-typische Sprache entsprechen dem Medienverhalten der Zielgruppe, d.h. den Studierenden des Fachbereichs Medizin.
"PULS." hat sich als ein geeignetes und strategisches Instrument erwiesen, um größere Transparenz, mehr Kommunikation und letztendlich eine stärkere Identifikation der Studierenden mit ihrem Fachbereich voranzutreiben.
Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set.
Purpose: Metabolic changes upon antiangiogenic therapy of recurrent glioblastomas (rGBMs) may provide new biomarkers for treatment efficacy. Since in vitro models showed that phospholipid membrane metabolism provides specific information on tumor growth we employed in-vivo MR-spectroscopic imaging (MRSI) of human rGBMs before and under bevacizumab (BVZ) to measure concentrations of phosphocholine (PCho), phosphoethanolamine (PEth), glycerophosphocholine (GPC), and glyceroethanolamine (GPE).
Methods: 1H and 31P MRSI was prospectively performed in 32 patients with rGBMs before and under BVZ therapy at 8 weeks intervals until tumor progression. Patients were dichotomized into subjects with long overall survival (OS) (>median OS) and short OS (<median OS) survival time from BVZ-onset. Metabolite concentrations from tumor tissue and their ratios were compared to contralateral normal-appearing tissue (control).
Results: Before BVZ, 1H-detectable choline signals (total GPC and PCho) in rGBMs were elevated but significance failed after dichotomizing. For metabolite ratios obtained by 31P MRSI, the short-OS group showed higher PCho/GPC (p = 0.004) in rGBMs compared to control tissue before BVZ while PEth/GPE was elevated in rGBMs of both groups (long-OS p = 0.04; short-OS p = 0.003). Under BVZ, PCho/GPC and PEth/GPE in the tumor initially decreased (p = 0.04) but only PCho/GPC re-increased upon tumor progression (p = 0.02). Intriguingly, in normal-appearing tissue an initial PEth/GPE decrease (p = 0.047) was followed by an increase at the time of tumor progression (p = 0.031).
Conclusion: An elevated PCho/GPC ratio in the short-OS group suggests that it is a negative predictive marker for BVZ efficacy. These gliomas may represent a malignant phenotype even growing under anti-VEGF treatment. Elevated PEth/GPE may represent an in-vivo biomarker more sensitive to GBM infiltration than MRI.
In complex networks such as gene networks, traffic systems or brain circuits it is important to understand how long it takes for the different parts of the network to effectively influence one another. In the brain, for example, axonal delays between brain areas can amount to several tens of milliseconds, adding an intrinsic component to any timing-based processing of information. Inferring neural interaction delays is thus needed to interpret the information transfer revealed by any analysis of directed interactions across brain structures. However, a robust estimation of interaction delays from neural activity faces several challenges if modeling assumptions on interaction mechanisms are wrong or cannot be made. Here, we propose a robust estimator for neuronal interaction delays rooted in an information-theoretic framework, which allows a model-free exploration of interactions. In particular, we extend transfer entropy to account for delayed source-target interactions, while crucially retaining the conditioning on the embedded target state at the immediately previous time step. We prove that this particular extension is indeed guaranteed to identify interaction delays between two coupled systems and is the only relevant option in keeping with Wiener’s principle of causality. We demonstrate the performance of our approach in detecting interaction delays on finite data by numerical simulations of stochastic and deterministic processes, as well as on local field potential recordings. We also show the ability of the extended transfer entropy to detect the presence of multiple delays, as well as feedback loops. While evaluated on neuroscience data, we expect the estimator to be useful in other fields dealing with network dynamics.
Background: To compare the effect of aprotinin with the effect of lysine analogues (tranexamic acid and ε-aminocaproic acid) on early mortality in three subgroups of patients: low, intermediate and high risk of cardiac surgery.
Methods and Findings: We performed a meta-analysis of randomised controlled trials and observational with the following data sources: Medline, Cochrane Library, and reference lists of identified articles. The primary outcome measure was early (in-hospital/30-day) mortality. The secondary outcome measures were any transfusion of packed red blood cells within 24 hours after surgery, any re-operation for bleeding or massive bleeding, and acute renal dysfunction or failure within the selected cited publications, respectively.
Out of 328 search results, 31 studies (15 trials and 16 observational studies) included 33,501 patients. Early mortality was significantly increased after aprotinin vs. lysine analogues with a pooled risk ratio (95% CI) of 1.58 (1.13–2.21), p<0.001 in the low (n = 14,297) and in the intermediate risk subgroup (1.42 (1.09–1.84), p<0.001; n = 14,427), respectively. Contrarily, in the subgroup of high risk patients (n = 4,777), the risk for mortality did not differ significantly between aprotinin and lysine analogues (1.03 (0.67–1.58), p = 0.90).
Conclusion: Aprotinin may be associated with an increased risk of mortality in low and intermediate risk cardiac surgery, but presumably may has no effect on early mortality in a subgroup of high risk cardiac surgery compared to lysine analogues. Thus, decisions to re-license aprotinin in lower risk patients should critically be debated. In contrast, aprotinin might probably be beneficial in high risk cardiac surgery as it reduces risk of transfusion and bleeding complications.
The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.
Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a rare inherited childhood neurodegenerative disease that is caused by a mutation in the gene CLN3. The function of the protein produced by the gene has remained elusive, and therefore the disease mechanism of JNCL is as of yet unknown. The disease is fatal, and no cure is currently available. We believe that simvastatin shows promise as a possible treatment. Simvastatin is well tolerated in children, and as currently no other viable, less invasive treatment for JNCL exists, at least pilot-scale clinical trials for this new off-label use of simvastatin are warranted.
The protein CLN3 has been indicated to have several different subcellular localizations and functions, but conclusive evidence about its role in cellular metabolism is lacking. It is also unclear why the mutation causes the distinct phenotype of the JNCL disease. In order to bring lucidity to the issue, we set out to identify metabolic pathways related to the phenotype of JNCL by using Multi-Epitope Ligand Cartography (MELC) and the related field of toponomics. Toponomic methods are required to process the massive amount of data generated by the MELC runs in order to extract information from them.
Our disease model of choice was the CLN3Δex7/8 knock-in mouse. To separate cause from effect, we compared embryonal wild type and mutant mouse brains to their adult counterparts. The first analyses revealed progressively abnormal Combinatorial Molecular Patterns (CMPs, an unit of toponomic data) related to cholera toxin/ganglioside 1 (Ctx/GM1), which is a membrane microdomain marker.
Cholesterol is an essential part of microdomains, so we utilized filipin staining to see if there were actual changes in cholesterol concentration and localization between healthy and diseased animals. After the disturbance in cholesterol metabolism was verified, we investigated the metabolic pathway that synthesizes cholesterol, the mevalonate pathway. Simvastatin is a drug that specifically down-regulates the mevalonate pathway. Fish oil affects lipid homeostasis and has some effects similar to those of simvastatin, and both of these drugs have previously been studied for their effects on neurodegenerative diseases. After treatment of mice with these drugs, highperformance liquid chromatography (HPLC) measurements on the brain homogenate showed a decrease in levels of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), products of the mevalonate pathway, confirming the effect of these drugs on the brains of the animals. Analyses of motor function of the mice further supported the notion that simvastatin had a positive effect on the condition of the diseased animals.
CMP analyses from the simvastatin treated mice showed a rescue of the Ctx/GM1 CMPs, suggesting at least a partial restoration of membrane microdomain homeostasis. Filipin staining revealed reversion of the apparent cholesterol depletion in the adult mutant mouse hippocampus by simvastatin. Interestingly, an additional effect of the treatment was found: simvastatin also affected glutamate receptor homeostasis, especially as regarding to N-methyl-D-aspartate (NMDA) and alphaamino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors. This finding suggested that excitotoxicity could be a part of the disease process, and pointed towards glutamate receptors as possible therapy targets. This is in line with previous studies that have shown that attenuation of AMPA receptors and L voltage-dependent channels improve the phenotype of a JNCL mouse and cell model, respectively.
Simvastatin mediates many of its effects via downregulation of the mevalonate pathway products, such as isoprenoids and cholesterol. However, simvastatin also has multiple pleiotropic effects that include suppression of excitotoxicity and granting neuroprotection. It is apparent that simvastatin treatment has a positive effect on JNCL mice, but if its effects are mediated via cholesterol (and membrane microdomains), isoprenoids (and isoprenylated proteins) or via a fully cholesterol independent mechanism remains to be solved.
In this study we have shown that with the MELC method and toponomics it is possible to approach rare diseases with confounded disease mechanisms with a hypothesis-free approach, to identify possible drug targets, and to monitor the effects of the drugs on treated individuals. This should open up a new avenue in the research of the many diseases that so far have avoided all attempts at discerning their nature.
Introduction: Acute kidney injury (AKI) can evolve quickly and clinical measures of function often fail to detect AKI at a time when interventions are likely to provide benefit. Identifying early markers of kidney damage has been difficult due to the complex nature of human AKI, in which multiple etiologies exist. The objective of this study was to identify and validate novel biomarkers of AKI.
Methods: We performed two multicenter observational studies in critically ill patients at risk for AKI - discovery and validation. The top two markers from discovery were validated in a second study (Sapphire) and compared to a number of previously described biomarkers. In the discovery phase, we enrolled 522 adults in three distinct cohorts including patients with sepsis, shock, major surgery, and trauma and examined over 300 markers. In the Sapphire validation study, we enrolled 744 adult subjects with critical illness and without evidence of AKI at enrollment; the final analysis cohort was a heterogeneous sample of 728 critically ill patients. The primary endpoint was moderate to severe AKI (KDIGO stage 2 to 3) within 12 hours of sample collection.
Results: Moderate to severe AKI occurred in 14% of Sapphire subjects. The two top biomarkers from discovery were validated. Urine insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, a key mechanism implicated in AKI, together demonstrated an AUC of 0.80 (0.76 and 0.79 alone). Urine [TIMP-2].[IGFBP7] was significantly superior to all previously described markers of AKI (P <0.002), none of which achieved an AUC >0.72. Furthermore, [TIMP-2].[IGFBP7] significantly improved risk stratification when added to a nine-variable clinical model when analyzed using Cox proportional hazards model, generalized estimating equation, integrated discrimination improvement or net reclassification improvement. Finally, in sensitivity analyses [TIMP-2].[IGFBP7] remained significant and superior to all other markers regardless of changes in reference creatinine method.
Conclusions: Two novel markers for AKI have been identified and validated in independent multicenter cohorts. Both markers are superior to existing markers, provide additional information over clinical variables and add mechanistic insight into AKI. Trial registration: ClinicalTrials.gov number NCT01209169.
Background: The students' perception of working conditions in hospitals hasn't been subject of research in Germany so far. However the perception plays an important role talking about the sustainability of working conditions. The iCept Study wants to examine the perception of medical students compared to the perception of practicing physicians.
Methods: The perception will be investigated with a redesigned questionnaire based upon two established and validated questionnaires. The two samples built for this study (students and physician) will be chosen from members of the labor union Marburger Bund. The iCept-Study is designed as an anonymized online-survey.
Discussion: The iCept-Study is thought to be the basis of ongoing further investigations regarding the perception of working conditions in hospitals. The results shall serve the facilitation of improving working conditions.
Im Rahmen einer prospektiven Kohortenstudie wurde in der vorliegenden Arbeit die Epidemiologie und Ätiologie von Fieber während chemotherapiebedingter Neutropenie bei Patienten mit hämatologischen Neoplasien untersucht. Das diagnostische und therapeutische Vorgehen wurde beschrieben und die Übereinstimmung mit den Vorgaben einer hausinternen Leitlinie wurde evaluiert und der Verbrauch an antimikrobiellen Substanzen dokumentiert. Febrile Episoden konnten in 74% der Neutropenieepisoden dokumentiert werden; in 51% der Neutropenieepisoden kam es zu persistierendem Fieber ≥ 3 Tage. Eine dem Fieber zugrunde liegende Infektion wurde in 55% der Chemotherapiezyklen (Neutropeniedauer Median 16 Tage) mittels klinischer, apparativer und mikrobiologischer Diagnostik gefunden. Die häufigsten infektiösen Komplikationen waren Enteritiden mit Nachweis von C. difficile (11%), Enteritiden ohne Nachweis von C. difficile (29%), Pneumonien mit pilztypischen Infiltraten (12%) und Pneumonien mit unspezifischen Infiltraten (11%), sowie mikrobiologisch bestätigte Septikämien (18%). Im Vergleich zu Angaben in der Literatur wurde FUO in der vorliegenden Untersuchung häufiger diagnostiziert (11,8 Tage pro 1000 Patiententage vs. 8,2 Tage pro 1000 Patiententage). Die Gesamtmortalität des Kollektivs lag bei 7%. Die häufigste Todesursache war der septische Schock (8 von 5 Patienten; 63%), welcher sich auf dem Boden einer gramnegativen Septikämie mit multiresistenten Keimen entwickelt hatte (3 von 5 Septikämien; 60%). Die Mortalitätsrate war ähnlich hoch wie im Literaturvergleich bei Studien mit akuten myeloischen Leukämien. Prognoserelevante Faktoren, welche im Rahmen der vorliegenden Studie evaluiert wurden, sind durch schwer therapierbare Grunderkrankungen hervorgerufene prolongierte Neutropeniezeiten, sowie infektiöse Komplikationen, vor allem bei Nachweis multiresistenter Erreger.
Die Compliance bezüglich des diagnostischen und therapeutischen Vorgehens bei persistierendem Fieber betrug in der vorliegenden Arbeit zwischen 47% und 82%. Die Compliance mit den in der Leitlinie vorgeschlagenen antimikrobiellen prophylaktischen und initialen empirischen Therapien lag zwischen 83% und 85%. Der Gesamtverbrauch an antibiotischen und antimykotischen Substanzen war in der vorliegenden Studie höher als im Literaturvergleich (1647 DDD vs. 1140 DDD und 650 DDD vs. 430 DDD). Dabei fällt insbesondere der hohe Verbrauch an Reservemedikamenten gegen grampositive Bakterien auf. Allerdings fehlen aktuelle Vergleichsdaten vergleichbarer Patientenkollektive.
Bezüglich der Optimierung der diagnostischen Maßnahmen ist vor allem die Erhöhung der Sensitivität der Bronchoalveolären Lavage bei Pilzinfektionen, rechtzeitig und regelmäßig durchgeführte Surveillanceabstriche bei febriler Neutropenie, sowie die Diskussion der diagnostischen Ergebnisse und des weiteren therapeutischen Vorgehens mit infektiologischen Experten zu fordern. Patienten mit persistierendem Fieber - vor allem bei positivem Blutkulturnachweis - sollten, aufgrund des positiven prädiktiven Wertes erregerpositiver Blutkulturen für die Notwendigkeit einer intensivmedizinischen Betreuung und einer erhöhten Mortalität, Anlass sein, diagnostische und therapeutische Maßnahmen zu verbessern. Ziel ist es, durch verstärkten Einsatz diagnostischer Maßnahmen und Diskussion mikrobiologischer Befunde im Einzelfall, Reservesubstanzen seltener einzusetzen, gegebenenfalls Breitspektrum-Antibiotika oder ihre Kombinationen zu deeskalieren und somit die Kolonisation mit resistenten Erregern positiv zu beeinflussen, sowie das Ansprechen auf antimikrobielle Therapien zu optimieren.
Die Teilnahme an multizentrischen Surveillancestudien zur Inzidenzerfassung wichtiger, eindeutig definierter neutropener nosokomialer Infektionen wie radiologisch detektierter Pneumonien, mikrobiologisch bestätigter Septikämien und C. difficilepositiver Enteritiden, sowie zur Generierung von Antiinfektivaverbrauchszahlen, könnte helfen, die Beratung und Entscheidungsfindung am Krankenbett zu unterstützen. Auch könnten in diesem Rahmen Zahlen generiert werden, welche bisher zum Vergleich fehlten.
Die vorliegende Arbeit versteht sich im Rahmen der Erfassung von Ätiologie, Diagnostik und antimikrobieller Therapien bei febriler Neutropenie als Teil eines solchen Surveillanceprogrammes und hat somit eine Grundlagen für zukünftige Datenerhebungen geliefert.
kurz und kn@pp news : Nr. 27
(2013)
In the absence of apparent mutations, alteration of gene expression patterns represents the key mechanism by which normal cells evolve to cancer cells.
Gene expression is tightly regulated by posttranscriptional processes. Within this context, RNA-binding proteins (RBPs) represent fundamental factors, since they control mechanisms, such as mRNA-stabilization, -translation and -degradation. Human antigen R (HuR) was among the first RBPs that have been directly associated to carcinogenesis. HuR modulates the stability and translation of mRNAs which encode proteins facilitating various ‘hallmarks of cancer’, namely proliferation, evasion of growth suppression, angiogenesis, cell death resistance, invasion and metastasis. Furthermore, it is well established that tumor-promoting inflammation contributes to tumorigenesis. In this process, monocytes are attracted to the site of the tumor and educated towards a tumor-promoting macrophage phenotype. While HuR has been extensively studied in various tumor cell types, little is known about HuR in hepatocellular carcinoma (HCC). Thus, the aim of my work was to characterize the contribution of HuR to the development of cancer characteristics in HCC. I was particularly interested to investigate if HuR facilitates tumor-promoting inflammation, since a role for HuR has not been described in this context. To this end, I depleted HuR in HepG2 cells (HuR k/d) and used a co-culture model of HepG2 tumor spheroids and infiltrating monocytes to study the impact of HuR on the tumor microenvironment. I could show that depletion of HuR resulted in the reduction of cell numbers. Additionally, the expression of proliferation marker KI-67 and proto-oncogene c-Myc was reduced, supporting a proliferative role of HuR. Furthermore, exposure to cytotoxic staurosporine elevated apoptosis in HuR k/d cells compared to control cells. Concomitantly, the expression of the anti-apoptotic mediator B-cell lymphoma protein-2 (Bcl-2) was markedly reduced in the HuR k/d cells, pointing to an involvement of HuR in cell survival processes.
Accordingly, a pro-survival function of HuR was also observed in tumor spheroids, since HuR k/d spheroids exhibited a larger necrotic core region at earlier time points and showed elevated numbers of dead cells compared to control (Ctr.) spheroids. Interestingly, HuR k/d spheroids isplayed reduced numbers of infiltrated macrophages, suggesting that HuR contributes to a tumor-promoting, inflammatory microenvironment by recruiting monocytes/macrophages to the tumor site. Aiming at identifying HuR-regulated factors responsible for the recruitment of monocytes, I found reduced levels of the chemokine interleukin 8 (IL-8) in supernatants of HuR k/d spheroids, supporting a critical involvement of HuR in the chemoattraction of monocytes. Analyzing supernatants of co-cultures of macrophages and HuR k/d or Ctr. spheroids revealed additional differences in chemokine secretion patterns. Interestingly, protein levels of many chemokines were elevated in co-cultures of HuR k/d spheroids compared to control co-cultures. Albeit enhanced chemokine secretion was observed, less monocytes are recruited into HuR k/d spheroids, further underlining the necessity of HuR in cancer related monocyte/macrophage attraction and infiltration. Differences between chemokine profiles of mono- and co-cultured spheroids could be attributable to changes in spheroid-derived chemokines as a result of the crosstalk with the immune cells. Provided the chemokines originate from monocytes/macrophages, the different secretion patterns suggest that HuR contributes to the modulation of the functional phenotype of infiltrated macrophages, since the tumorenvironment is critically involved in the shaping of macrophage phenotypes. Regions of low-oxygen (hypoxia) represent another critical feature of tumors. Therefore, I next analyzed the impact of HuR on the hypoxic response. Loss of HuR attenuated hypoxia-inducible factor (HIF) 2α expression after exposure to hypoxia, while HIF-1α protein levels remained unaltered. Considering previous results of our group, showing that HIF-2α depletion (HIF-2α k/d) resulted in the enhanced expression of HIF-1α protein, I aimed to determine the involvement of HuR in the compensatory upregulation of HIF-1α protein in HIF-2α k/d cells. I could demonstrate that not only total HuR protein levels, but specifically cytoplasmic HuR was elevated in HIF-2α depleted cells pointing to enhanced HuR activity. Silencing HuR in HIF-2α deficient cells attenuated enhanced HIF-1α protein expression, thus confirming a direct role of HuR in the compensatory upregulation of HIF-1α. This as also reflected on HIF-1α target gene expression. I further investigated the mechanism underlying the compensatory HIF-1α expression in HIF-2α deficient cells. Analyzing HIF-1α mRNA expression, I excluded enhanced HIF1-α transcription and stability to account for elevated HIF-1α expression in HIF-2α k/d cells. HIF-1α promoter activity assays confirmed the mRNA data. Furthermore, HIF-1α protein half-life was not elevated in HIF-2α k/d cells compared to control cells, indicating that HIF-1α protein stability is not altered in HIF-2α k/d cells. Analysis of the association of HIF-1α with the translational machinery using polysomal fractionation finally revealed an increased istribution of HIF-1α mRNA in the heavier polysomal fractions in HIF-2α k/d cells compared to control cells. Since augmented ribosome occupancy is an indicator for more efficient translation, I propose enhanced HIF-1α translation as underlying principle of the compensatory increase in HIF-1α protein levels in HIF-2α k/d cells. In summary, my results demonstrate that HuR is critical for the development of cancer characteristics in HCC. Future work analyzing the impact of HuR on tumor-promoting inflammation, specifically macrophage attraction and activation could provide new trategies to inhibit macrophage-driven tumor progression. Furthermore, I provide evidence that HuR contributes to the hypoxic response by regulating the expression of HIF-1α and HIF-2α. Targeting single HIF-isoforms for tumor therapy should be carefully considered, because of their compensatory regulation when one α-subunit is depleted. Thus, therapeutic strategies targeting factors such as HuR that control both α-subunits and at the same time prevent compensation might be more promising.
Since most anticancer therapies including immunotherapy trigger programmed cell death in cancer cells, defective cell death programs can lead to treatment resistance and tumor immune escape. Therefore, evasion of programmed cell death may provide one possible explanation as to why cancer immunotherapy has so far only shown modest clinical benefits for children with cancer. A better understanding of the molecular mechanisms that regulate sensitivity and resistance to programmed cell death is expected to open new perspectives for the development of novel experimental treatment strategies to enhance the efficacy of cancer immunotherapy in the future.
Cataract surgery is one of the oldest and the most frequent outpatient clinic operations in medicine performed worldwide. The clouded human crystalline lens is replaced by an artificial intraocular lens implanted into the capsular bag. During the last six decades, cataract surgery has undergone rapid development from a traumatic, manual surgical procedure with implantation of a simple lens to a minimally invasive intervention increasingly assisted by high technology and a broad variety of implants customized for each patient’s individual requirements. This review discusses the major advances in this field and focuses on the main challenge remaining – the treatment of presbyopia. The demand for correction of presbyopia is increasing, reflecting the global growth of the ageing population. Pearls and pitfalls of currently applied methods to correct presbyopia and different approaches under investigation, both in lens implant technology and in surgical technology, are discussed.
Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding.
Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC’s collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter’s collagen-1α gene expression.
While necroptosis has for long been viewed as an accidental mode of cell death triggered by physical or chemical damage, it has become clear over the last years that necroptosis can also represent a programmed form of cell death in mammalian cells. Key discoveries in the field of cell death research, including the identification of critical components of the necroptotic machinery, led to a revised concept of cell death signaling programs. Several regulatory check and balances are in place in order to ensure that necroptosis is tightly controlled according to environmental cues and cellular needs. This network of regulatory mechanisms includes metabolic pathways, especially those linked to mitochondrial signaling events. A better understanding of these signal transduction mechanisms will likely contribute to open new avenues to exploit our knowledge on the regulation of necroptosis signaling for therapeutic application in the treatment of human diseases.
CSF and serum biomarkers focusing on cerebral vasospasm and ischemia after subarachnoid hemorrhage
(2013)
Delayed cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) remain severe complications after subarachnoid hemorrhage (SAH). Although focal changes in cerebral metabolism indicating ischemia are detectable by microdialysis, routinely used biomarkers are missing. We therefore sought to evaluate a panel of possible global markers in serum and cerebrospinal fluid (CSF) of patients after SAH.
CSF and serum of SAH patients were analyzed retrospectively. In CSF, levels of inhibitory, excitatory, and structural amino acids were detected by high-performance liquid chromatography (HPLC). In serum, neuron-specific enolase (NSE) and S100B level were measured and examined in conjunction with CVS and DCI. CVS was detected by arteriography, and ischemic lesions were assessed by computed tomography (CT) scans.
All CSF amino acids were altered after SAH. CSF glutamate, glutamine, glycine, and histidine were significantly correlated with arteriographic CVS. CSF glutamate and serum S100B were significantly correlated with ischemic events after SAH; however, NSE did not correlate neither with ischemia nor with vasospasm. Glutamate, glutamine, glycine, and histidine might be used in CSF as markers for CVS. Glutamate also indicates ischemia. Serum S100B, but not NSE, is a suitable marker for ischemia. These results need to be validated in larger prospective cohorts.
Background: This phase I/II-trial assessed the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of neoadjuvant radiochemotherapy (RCT) with docetaxel and oxaliplatin in patients with locally advanced adenocarcinoma of the oesophagogastric junction.
Methods: Patients received neoadjuvant radiotherapy (50.4 Gy) together with weekly docetaxel (20 mg/m2 at dose level (DL) 1 and 2, 25 mg/m2 at DL 3) and oxaliplatin (40 mg/m2 at DL 1, 50 mg/m2 at DL 2 and 3) over 5 weeks. The primary endpoint was the DLT and the MTD of the RCT regimen. Secondary endpoints included overall response rate (ORR) and progression-free survival (PFS).
Results: A total of 24 patients were included. Four patients were treated at DL 1, 13 patients at DL 2 and 7 patients at DL 3. The MTD of the RCT was considered DL 2 with docetaxel 20 mg/m2 and oxaliplatin 50 mg/m2. Objective response (CR/PR) was observed in 32% (7/22) of patients. Eighteen patients (75%) underwent surgery after RCT. The median PFS for all patients (n = 24) was 6.5 months. The median overall survival for all patients (n = 24) was 16.3 months. Patients treated at DL 2 had a median overall survival of 29.5 months.
Conclusion: Neoadjuvant RCT with docetaxel 20 mg/m2 and oxaliplatin 50 mg/m2 was effective and showed a good toxicity profile. Future studies should consider the addition of targeted therapies to current neoadjuvant therapy regimens to further improve the outcome of patients with advanced cancer of the oesophagogastric junction.
Trial Registration: NCT00374985
Background: Interferon and ribavirin therapy for chronic hepatitis C virus (HCV) infection yields sustained virological response (SVR) rates of 50–80%. Several factors such as non-1 genotype, beneficial IL28B genetic variants, low baseline IP-10, and the functionality of HCV-specific T cells predict SVR. With the pending introduction of new therapies for HCV entailing very rapid clearance of plasma HCV RNA, the importance of baseline biomarkers likely will increase in order to tailor therapy. CD26 (DPPIV) truncates the chemokine IP-10 into a shorter antagonistic form, and this truncation of IP-10 has been suggested to influence treatment outcome in patients with chronic HCV infection patients. In addition, previous reports have shown CD26 to be a co-stimulator for T cells. The aim of the present study was to assess the utility of CD26 as a biomarker for treatment outcome in chronic hepatitis C and to define its association with HCV-specific T cells.
Methods: Baseline plasma from 153 genotype 1 and 58 genotype 2/3 infected patients enrolled in an international multicenter phase III trial (DITTO-HCV) and 36 genotype 1 infected patients participating in a Swedish trial (TTG1) were evaluated regarding baseline soluble CD26 (sCD26) and the functionality of HCV-specific CD8+ T cells.
Results: Genotype 1 infected patients achieving SVR in the DITTO (P = 0.002) and the TTG1 (P = 0.02) studies had lower pretreatment sCD26 concentrations compared with non-SVR patients. Sixty-five percent of patients with sCD26 concentrations below 600 ng/mL achieved SVR compared with 39% of the patients with sCD26 exceeding 600 ng/mL (P = 0.01). Patients with sCD26 concentrations below 600 ng/mL had significantly higher frequencies of HCV-specific CD8+ T cells (P = 0.02).
Conclusions: Low baseline systemic concentrations of sCD26 predict favorable treatment outcome in chronic HCV infection and may be associated with higher blood counts of HCV-specific CD8+ T cells.
Apoptosis represents one of the most important forms of cell death in higher organisms and is typically dysregulated in human cancers, including pediatric tumors. This implies that ineffective engagement of cell death programs can contribute to tumor formation as well as tumor progression. In addition, the majority of cytotoxic therapeutic principles rely on the activation of cell death signaling pathways in cancer cells. Blockade of signaling networks that lead to cell death can therefore confer treatment resistance. A variety of genetic and epigenetic events as well as dysfunctional regulation of signaling networks have been identified as underlying causes of cell death resistance in childhood malignancies. Apoptosis pathways can be therapeutically exploited by enhancing proapoptotic signals or by neutralizing antiapoptotic programs. The challenge in the coming years will be to successfully transfer this knowledge into the development of innovative treatment approaches for children with cancer.