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A recent report showed PINK1 transcript levels to be up- or down-regulated by the gain or loss of Ataxin-2 function, respectively, in human blood, in a human neural cell line and in mouse tissues. These observations may have profound implications for the regulation of cell growth and may be medically exploited for the treatment of cancer and neural atrophy...
Alzheimer’s disease (AD) is the most common form of dementia in the elderly; important risk factors are old age and inheritance of the apolipoprotein E4 (APOE4) allele. Changes in amyloid precursor protein (APP) binding, trafficking, and sorting may be important AD causative factors. Secretase-mediated APP cleavage produces neurotoxic amyloid-beta (Aβ) peptides, which form lethal deposits in the brain. In vivo and in vitro studies have implicated sortilin-related receptor (SORL1) as an important factor in APP trafficking and processing. Recent in vitro evidence has associated the APOE4 allele and alterations in the SORL1 pathway with AD development and progression. Here, we analyzed SORL1 expression in neural stem cells (NSCs) from AD patients carrying null, one, or two copies of the APOE4 allele. We show reduced SORL1 expression only in NSCs of a patient carrying two copies of APOE4 allele with increased Aβ/SORL1 localization along the degenerated neurites. Interestingly, SORL1 binding to APP was largely compromised; this could be almost completely reversed by γ-secretase (but not β-secretase) inhibitor treatment. These findings may yield new insights into the complex interplay of SORL1 and AD pathology and point to NSCs as a valuable tool to address unsolved AD-related questions in vitro.
Background: Research has implicated that changes in zinc (Zn) metabolism may be associated with the biological underpinnings of eating disorders, in particular anorexia nervosa. However, to date research on the role of Zn in patients with bulimia nervosa (BN) is scarce.
Objective: We aimed to explore serum Zn concentrations in young patients with BN, with a focus on the stage of the disorder, comparing acutely ill and recovered patients with BN with healthy controls.
Methods: Serum Zn concentrations were obtained from healthy controls and from acutely ill and remitted young patients with BN. Mean duration of remission was 4.0±3.5 years.
Results: Remitted patients showed elevated serum Zn concentrations when compared to controls (Cohen’s d=2.022), but concentrations were still in the normal range. Acutely ill patients also had higher serum Zn levels when compared to controls (all values still being within the reference range, Cohen’s d=0.882). There was no difference between acutely ill and remitted patients with BN in serum Zn concentrations. Of note, remitted patients had a significantly higher body weight when compared to the other two groups. Overall, there were no significant differences in dietary preferences with regard to Zn containing foods between the groups.
Conclusion: The present study provides preliminary evidence that the underlying factors for changes in Zn serum concentrations in young patients with BN do not vary with regard to the stage of illness (acute versus remitted BN). Further prospective research is needed in order to disentangle the possible interplay between serum Zn status and bulimic eating behaviors.
Hypoxia-induced miR-210 displays a pro-survival, cytoprotective and pro-angiogenic role in several in vitro systems. In vivo, we previously found that miR-210 inhibition increases ischemic damage. Here we describe the generation of a versatile transgenic mouse model allowing the evaluation of miR-210 therapeutic potential in ischemic cardiovascular diseases. We generated a Tet-On miR-210 transgenic mouse strain (TG-210) by targeted transgenesis in the ROSA26 locus. To functionally validate miR-210 transgenic mice, hindlimb ischemia was induced by femoral artery dissection. Blood perfusion was evaluated by power Doppler while tissue damage and inflammation were assessed by histological evaluation. We found that miR-210 levels were rapidly increased in TG-210 mice upon doxycycline administration. miR-210 overexpression was maintained over time and remained within physiological levels in multiple tissues. When hindlimb ischemia was induced, miR-210 overexpression protected from both muscular and vascular ischemic damage, decreased inflammatory cells density and allowed to maintain a better calf perfusion. In conclusion, we generated and functionally validated a miR-210 transgenic mouse model. Albeit validated in the context of a specific cardiovascular ischemic disease, miR-210 transgenic mice may also represent a useful model to assess the function of miR-210 in other physio-pathological conditions.
Patient-reported outcomes (PROs) such as quality of life and work productivity are important for measuring patient's experience. We assessed PROs during and after treatment of hepatitis C virus (HCV) patients.Data were obtained from a phase 3 open label study of sofosbuvir and ribavirin (SOF + RBV) with and without interferon (IFN). Patients completed 4 PRO assessment instruments (SF-36, Functional Assessment of Chronic Illness Therapy-Fatigue, Chronic Liver Disease Questionnaire- HCV, Work Productivity and Activity-Specific Health Problem) before, during, and after treatment.A total of 533 patients with chronic HCV were enrolled; 28.9% treatment-naïve, 23.1% cirrhotic, 219 received IFN + SOF + RBV and 314 received IFN-free SOF + RBV. At baseline, there were no differences in PROs between the IFN-free and IFN-containing treatment arms (all P > 0.05). During treatment, patients receiving IFN + SOF + RBV had a substantial impairment in their PROs (up to -24.4% by treatment week 12, up to -8.3% at week 4 post-treatment). The PRO decrements seen in the SOF + RBV arm were smaller in magnitude (up to -7.1% by treatment week 12), and all returned to baseline or improved by post-treatment week 4. By 12 weeks after treatment cessation, patients who achieved sustained viral response-12 showed some improvement of PRO scores regardless of the regimen (up to +7.1%, P < 0.0001) or previous treatment experience. In multivariate analysis, the use of IFN was independently associated with lower PROs.IFN-based regimens have a profoundly negative impact to PROs. By contrast, the impact of RBV on these PROs is relatively modest. Achieving HCV cure is associated with improvement of most of the PRO scores.
The former and current multiple sclerosis (MS) classifications are essential for describing different phenotypes and disease dynamics. To establish personalized treatment regimes, further clinical and paraclinical parameters have to be considered such as imaging, cerebrospinal fluid (CSF) findings, past disease-modifying therapies (DMTs), and disease activity under these therapies. In clinical practice, this information is often difficult to overview. Especially, patients with a long course of disease offer an extensive medical history so that comprehending all of the necessary information can be very time consuming.
Introduction: The 2017 update to the Global Initiative for Obstructive Lung Disease (GOLD) strategy document includes recommendations for treatment intensification or step-down in chronic obstructive pulmonary disease (COPD), although recognises that limited supporting information is available.
DACCORD is an ongoing observational, non-interventional study, recruiting patients following COPD maintenance treatment change or initiation, a subset of whom were receiving a long-acting β2-agonist (LABA) plus a long-acting muscarinic antagonist (LAMA) fixed-dose combination (FDC) on entry. Since there were no requirements in terms of prior medication (and no washout before commencing LABA/LAMA FDC), this provides an opportunity to generate "real world" data to test the GOLD 2017 recommendations.
Methods: To reduce heterogeneity, the current analyses include patients receiving indacaterol/glycopyrronium at baseline, and who, prior to the study, were receiving no COPD maintenance medication ("none"), LABA or LAMA monotherapy ("mono"), LABA plus inhaled corticosteroid (ICS; "LABA/ICS"), or triple therapy ("triple"). At the baseline visit, data collected included: demographic and disease characteristics; COPD Assessment Test (CAT); and exacerbations in the 6 months prior to entry. At 3, 6, 9 and 12 months data on exacerbations were collected, with CAT recorded at 3 and 12 months.
Results: A total of 2724 patients were included in the baseline analyses: 795, 954, 598 and 377 in the "none", "mono", "LABA/ICS" and "triple" subgroups, respectively. There were no clinically relevant differences in baseline demographics between the four groups. In terms of disease characteristics, the "triple" group had the highest proportion of patients with a disease duration of more than 1 year since diagnosis and with severe/very severe airflow limitation, but a similar percentage of non-exacerbators compared to the "none" group.
Over the 1-year follow-up, the majority of patients in all four subgroups did not exacerbate (exacerbation rates 0.16, 0.19, 0.21, and 0.26 in the "none", "mono", "LABA/ICS" and "triple" groups, respectively). At 12 months, 61.4%, 65.0%, 71.0% and 52.4% of patients had a clinically relevant improvement in CAT score.
Conclusions: Overall, the results support the GOLD recommendations in suggesting that a switch from a mono-bronchodilator or LABA plus ICS to LABA/LAMA FDC is a valid treatment option for patients with COPD. The results also validate the use of a LABA/LAMA FDC as initial maintenance treatment for COPD, and provide first "real world" evidence to support the newly added "step down" recommendation (from triple to LABA/LAMA FDC).
The disruption of coupling between brain areas has been suggested as the mechanism underlying loss of consciousness in anesthesia. This hypothesis has been tested previously by measuring the information transfer between brain areas, and by taking reduced information transfer as a proxy for decoupling. Yet, information transfer is a function of the amount of information available in the information source—such that transfer decreases even for unchanged coupling when less source information is available. Therefore, we reconsidered past interpretations of reduced information transfer as a sign of decoupling, and asked whether impaired local information processing leads to a loss of information transfer. An important prediction of this alternative hypothesis is that changes in locally available information (signal entropy) should be at least as pronounced as changes in information transfer. We tested this prediction by recording local field potentials in two ferrets after administration of isoflurane in concentrations of 0.0%, 0.5%, and 1.0%. We found strong decreases in the source entropy under isoflurane in area V1 and the prefrontal cortex (PFC)—as predicted by our alternative hypothesis. The decrease in source entropy was stronger in PFC compared to V1. Information transfer between V1 and PFC was reduced bidirectionally, but with a stronger decrease from PFC to V1. This links the stronger decrease in information transfer to the stronger decrease in source entropy—suggesting reduced source entropy reduces information transfer. This conclusion fits the observation that the synaptic targets of isoflurane are located in local cortical circuits rather than on the synapses formed by interareal axonal projections. Thus, changes in information transfer under isoflurane seem to be a consequence of changes in local processing more than of decoupling between brain areas. We suggest that source entropy changes must be considered whenever interpreting changes in information transfer as decoupling.
The view that tumors consist of a homogenous mass of clonal derived cells has dramatically changed in recent years. Tumors harbor an enormous heterogeneity of cells with distinct capabilities and functions. The heterogeneity originates from a differentiation hierarchy of tumor cells, similar to normal tissue organization of stem-cell driven organs, but also from clonal succession of subpopulations by randomly acquired genetic mutations and epigenetic changes. Both scenarios are certainly not mutually exclusive, and also stem and progenitor cells underlie mutational selection. Intratumoral heterogeneity is a major challenge for cancer treatment and disease monitoring. Functional studies revealed that not all tumor cells have the same ability to initiate tumor growth upon transplantation in receptive animal models. The tumorinitiating cells (TICs) were called cancer stem cells due to their similarities to normal tissue stem cells in their molecular and functional properties. They can renew themselves long-term and give rise to tumor cells lacking cancer stem cell properties. However, it is worth stressing here that TICs do not necessarily originate from stem cells, but may have regained stem cell properties. TICs caught major attention since they may provide important steps in the progression of malignant diseases, such as epithelial-to-mesenchymal transition, dissemination, long-term persistence, therapy resistance, and relapse of the disease. The prospective identification of TICs using distinct surface markers would allow their molecular and functional characterization, the design of detection methods for diagnosis and prognosis, and the development of targeted therapies against these detrimental cells. While functional evidence for the existence of TICs were provided for many tumor entities, their marker profile still remains largely undefined and controversial. ...
After initial formation, the heart tube grows by addition of second heart field progenitor cells to its poles. The transcription factor Isl1 is expressed in the entire second heart field in mouse, and Isl1-deficient mouse embryos show defects in arterial and venous pole development. The expression of Isl1 is conserved in zebrafish cardiac progenitors; however, Isl1 is required for cardiomyocyte differentiation only at the venous pole. Here we show that Isl1 homologues are expressed in specific patterns in the developing zebrafish heart and play distinct roles during cardiac morphogenesis. In zebrafish, isl2a mutants show defects in cardiac looping, whereas isl2b is required for arterial pole development. Moreover, Isl2b controls the expression of key cardiac transcription factors including mef2ca, mef2cb, hand2 and tbx20. The specific roles of individual Islet family members in the development of distinct regions of the zebrafish heart renders this system particularly well-suited for dissecting Islet-dependent gene regulatory networks controlling the behavior and function of second heart field progenitors in distinct steps of cardiac development.
Objective: This study examines the interrelations of parenting practices, emotional climate, and household chaos in families with children with and without ADHD. In particular, indirect pathways from children’s ADHD symptomatology to inadequate parenting and negative emotional climate via household chaos were investigated. Method: Parenting, emotional climate, and household chaos were assessed using questionnaires and a speech sample of parents of 31 children with and 53 without ADHD, aged 7 to 13 years. Results: Group differences were found for certain parenting dimensions, the parent–child relationship, critical comments, and household chaos. While we found significant indirect effects between children’s ADHD and certain parenting dimensions through household chaos, no effects were found for any aspect of emotional climate. Conclusion: Children’s ADHD symptoms translate into inadequate parenting through household chaos, which underlines the need for interventions to improve household organization skills in parents of children with ADHD.
Information processing performed by any system can be conceptually decomposed into the transfer, storage and modification of information—an idea dating all the way back to the work of Alan Turing. However, formal information theoretic definitions until very recently were only available for information transfer and storage, not for modification. This has changed with the extension of Shannon information theory via the decomposition of the mutual information between inputs to and the output of a process into unique, shared and synergistic contributions from the inputs, called a partial information decomposition (PID). The synergistic contribution in particular has been identified as the basis for a definition of information modification. We here review the requirements for a functional definition of information modification in neuroscience, and apply a recently proposed measure of information modification to investigate the developmental trajectory of information modification in a culture of neurons vitro, using partial information decomposition. We found that modification rose with maturation, but ultimately collapsed when redundant information among neurons took over. This indicates that this particular developing neural system initially developed intricate processing capabilities, but ultimately displayed information processing that was highly similar across neurons, possibly due to a lack of external inputs. We close by pointing out the enormous promise PID and the analysis of information modification hold for the understanding of neural systems
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.
Ongoing liver inflammation in patients with chronic hepatitis C and sustained virological response
(2017)
Background: Novel direct-acting antiviral DAA combination therapies tremendously improved sustained virologic response (SVR) rates in patients with chronic HCV infection. SVR is typically accompanied by normalization of liver enzymes, however, hepatic inflammation, i.e. persistently elevated aminotransferase levels may persist despite HCV eradication. Aim: To investigate prevalence and risk factors for ongoing hepatic inflammation after SVR in two large patient cohorts.
Methods: This post-hoc analysis was based on prospectively collected demographic and clinical data from 834 patients with SVR after HCV treatment with either PegIFN- or DAA-based treatment regimens from the PRAMA trial (n = 341) or patients treated at our outpatient clinic (n = 493).
Results: We observed an unexpected high prevalence of post-SVR inflammation, including patients who received novel IFN-free DAA-based therapies. Up to 10% of patients had ongoing elevation of aminotransferase levels and another 25% showed aminotransferase activity above the so-called healthy range. Several baseline factors were independently associated with post-SVR aminotransferase elevation. Among those, particularly male gender, advanced liver disease and markers for liver steatosis were strongly predictive for persistent ALT elevation. The use of IFN-based antiviral treatment was independently correlated with post-SVR inflammation, further supporting the overall benefit of IFN-free combination regimens.
Conclusion: This is the first comprehensive study on a large patient cohort investigating the prevalence and risk factors for ongoing liver inflammation after eradication of HCV. Our data show a high proportion of patients with ongoing hepatic inflammation despite HCV eradication with potential implications for the management of approximately one third of all patients upon SVR.
Purpose: To evaluate the effect of reduced z-axis scan coverage on diagnostic performance and radiation dose of neck CT in patients with suspected cervical abscess.
Methods: Fifty-one patients with suspected cervical abscess were included and underwent contrast-enhanced neck CT on a 2nd or 3rd generation dual-source CT system. Image acquisition ranged from the aortic arch to the upper roof of the frontal sinuses (CTstd). Subsequently, series with reduced z-axis coverage (CTred) were reconstructed starting at the aortic arch up to the orbital floor. CTstd and CTred were independently assessed by two radiologists for the presence/absence of cervical abscesses and for incidental and alternative findings. In addition, diagnostic accuracy for the depiction of the cervical abscesses was calculated for both readers. Furthermore, DLP (dose-length-product), effective dose (ED) and organ doses were calculated and compared for CTred and CTstd, using a commercially available dose management platform.
Results: A total of 41 abscesses and 3 incidental/alternative findings were identified in CTstd. All abscesses and incidental/alternative findings could also be detected on CTred resulting in a sensitivity and specificity of 1.0 for both readers. DLP, ED and organ doses of the brain, the eye lenses, the red bone marrow and the salivary glands of CTred were significantly lower than for CTstd (p<0.001).
Conclusions: Reducing z-axis coverage of neck CT allows for a significant reduction of effective dose and organ doses at similar diagnostic performance as compared to CTstd.
Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient’s own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF) was compared to standard platelet-rich plasma (PRP) with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g) for 3 min) were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), collagen1 (COL1) and fibronectin (FN). The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA) levels of PDGF, TGF-β, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.
Clustering of cardiovascular risk factors and carotid intima-media thickness : the USE-IMT study
(2017)
Background: The relation of a single risk factor with atherosclerosis is established. Clinically we know of risk factor clustering within individuals. Yet, studies into the magnitude of the relation of risk factor clusters with atherosclerosis are limited. Here, we assessed that relation.
Methods: Individual participant data from 14 cohorts, involving 59,025 individuals were used in this cross-sectional analysis. We made 15 clusters of four risk factors (current smoking, overweight, elevated blood pressure, elevated total cholesterol). Multilevel age and sex adjusted linear regression models were applied to estimate mean differences in common carotid intima-media thickness (CIMT) between clusters using those without any of the four risk factors as reference group.
Results: Compared to the reference, those with 1, 2, 3 or 4 risk factors had a significantly higher common CIMT: mean difference of 0.026 mm, 0.052 mm, 0.074 mm and 0.114 mm, respectively. These findings were the same in men and in women, and across ethnic groups. Within each risk factor cluster (1, 2, 3 risk factors), groups with elevated blood pressure had the largest CIMT and those with elevated cholesterol the lowest CIMT, a pattern similar for men and women.
Conclusion: Clusters of risk factors relate to increased common CIMT in a graded manner, similar in men, women and across race-ethnic groups. Some clusters seemed more atherogenic than others. Our findings support the notion that cardiovascular prevention should focus on sets of risk factors rather than individual levels alone, but may prioritize within clusters.
Eosinophilic cholangitis is a potentially underdiagnosed etiology in indeterminate biliary stricture
(2017)
AIM: To investigate presence and extent of eosinophilic cholangitis (EC) as well as IgG4-related disease in patients with indeterminate biliary stricture (IBS).
METHODS: All patients with diagnosis of sclerosing cholangitis (SC) and histopathological samples such as biopsies or surgical specimens at University Hospital Frankfurt from 2005-2015 were included. Histopathological diagnoses as well as further clinical course were reviewed. Tissue samples of patients without definite diagnosis after complete diagnostic work-up were reviewed regarding presence of eosinophilic infiltration and IgG4 positive plasma cells. Eosinophilic infiltration was as well assessed in a control group of liver transplant donors and patients with primary sclerosing cholangitis.
RESULTS: one hundred and thirty-five patients with SC were included. In 10/135 (13.5%) patients, no potential cause of IBS could be identified after complete diagnostic work-up and further clinical course. After histopathological review, a post-hoc diagnosis of EC was established in three patients resulting in a prevalence of 2.2% (3/135) of all patients with SC as well as 30% (3/10) of patients, where no cause of IBS was identified. 2/3 patients with post-hoc diagnosis of EC underwent surgical resection with suspicion for malignancy. Diagnosis of IgG4-related cholangitis was observed in 7/135 patients (5.1%), whereas 3 cases were discovered in post-hoc analysis. 6/7 cases with IgG4-related cholangitis (85.7%) presented with eosinophilic infiltration in addition to IgG4 positive plasma cells. There was no patient with eosinophilic infiltration in the control group of liver transplant donors (n = 27) and patients with primary sclerosing cholangitis (n = 14).
CONCLUSION: EC is an underdiagnosed benign etiology of SC and IBS, which has to be considered in differential diagnosis of IBS.
Dendrites form predominantly binary trees that are exquisitely embedded in the networks of the brain. While neuronal computation is known to depend on the morphology of dendrites, their underlying topological blueprint remains unknown. Here, we used a centripetal branch ordering scheme originally developed to describe river networks—the Horton-Strahler order (SO)–to examine hierarchical relationships of branching statistics in reconstructed and model dendritic trees. We report on a number of universal topological relationships with SO that are true for all binary trees and distinguish those from SO-sorted metric measures that appear to be cell type-specific. The latter are therefore potential new candidates for categorising dendritic tree structures. Interestingly, we find a faithful correlation of branch diameters with centripetal branch orders, indicating a possible functional importance of SO for dendritic morphology and growth. Also, simulated local voltage responses to synaptic inputs are strongly correlated with SO. In summary, our study identifies important SO-dependent measures in dendritic morphology that are relevant for neural function while at the same time it describes other relationships that are universal for all dendrites.
Tight regulation of inflammation is very important to guarantee a balanced immune response without developing chronic inflammation. One of the major mediators of the resolution of inflammation is the transcription factor: the nuclear factor erythroid 2-like 2 (Nrf2). Stabilized following oxidative stress, Nrf2 induces the expression of antioxidants as well as cytoprotective genes, which provoke an anti-inflammatory expression profile, and is crucial for the initiation of healing. In view of this fundamental modulatory role, it is clear that both hyper- or hypoactivation of Nrf2 contribute to the onset of chronic diseases. Understanding the tight regulation of Nrf2 expression/activation and its interaction with signaling pathways, known to affect inflammatory processes, will facilitate development of therapeutic approaches to prevent Nrf2 dysregulation and ameliorate chronic inflammatory diseases. We discuss in this review the principle mechanisms of Nrf2 regulation with a focus on inflammation and autophagy, extending the role of dysregulated Nrf2 to chronic diseases and tumor development.
Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1−/−-MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1−/−-MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1−/−-MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1−/−-MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.
The efficacy of antiviral treatment for chronic hepatitis C virus (HCV) infection is determined by measuring HCV RNA at specific time points throughout therapy using highly sensitive and accurate HCV RNA assays. This study compared the performances of two recently developed real-time PCR HCV RNA assays, cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and cobas HCV for use on the cobas 4800 system (cobas 4800 HCV), with those of two established assays, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2 (CAP/CTM v2) and the Cobas TaqMan HCV test, version 2 for use with the High Pure system (HPS/CTM v2). The limits of detection (LODs) and linearity at lower concentrations (5 to 1000 IU/ml) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1 to 4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1 to GT 4. Results from cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/ml difference between expected and observed results) with LODs of 8.2 IU/ml and 11.7 IU/ml, respectively, for GT 1. The new assays showed excellent agreement with results from CAP/CTM v2 and HPS/CTM v2 in samples with quantifiable viral loads. The concordances using the 6 million IU/ml cutoff were high among all four assays (90 to 94%). In conclusion, the cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear and correlate well with the established Roche assays used in clinical practice.
The Gini index is a measure of the inequality of a distribution that can be derived from Lorenz curves. While commonly used in, e.g., economic research, it suffers from ambiguity via lack of Lorenz dominance preservation. Here, investigation of large sets of empirical distributions of incomes of the World’s countries over several years indicated firstly, that the Gini indices are centered on a value of 33.33% corresponding to the Gini index of the uniform distribution and secondly, that the Lorenz curves of these distributions are consistent with Lorenz curves of log-normal distributions. This can be employed to provide a Lorenz dominance preserving equivalent of the Gini index. Therefore, a modified measure based on log-normal approximation and standardization of Lorenz curves is proposed. The so-called UGini index provides a meaningful and intuitive standardization on the uniform distribution as this characterizes societies that provide equal chances. The novel UGini index preserves Lorenz dominance. Analysis of the probability density distributions of the UGini index of the World’s counties income data indicated multimodality in two independent data sets. Applying Bayesian statistics provided a data-based classification of the World’s countries’ income distributions. The UGini index can be re-transferred into the classical index to preserve comparability with previous research.
Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD, the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deficiency of SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming, reflecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by treatment with the selective serotonin reuptake inhibitor fluoxetine. In addition to the previously suggested involvement of cortico-striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specific result of SPRED2 deficiency and not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identifies SPRED2 as a promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical circuitry involved in the pathogenesis of OCD.
Tumor-associated macrophages, angiogenesis, and tumor cell migration in oral squamous cell carcinoma
(2017)
Objective: To investigate the relationship between tumor-associated macrophages (TAMs), neovascularization, and tumor cell migration in oral squamous cell carcinoma (OSCC) of an African subpopulation.
Materials and Methods: Twenty OSCC paraffin blocks underwent immunohistochemistry to TAM1 (CCR7), TAM2 (CD206), Twist, E-cadherin, N-cadherin, and CD34. The relative percentage of CCR7 + and CD206 + cells to overall immune cell population was calculated for three high power fields and an average was taken. TAM-related microvessel density (MVD) was determined as the mean of the three recorded values. Cases that had no CD34 + vessels adjacent to the TAMs region were regarded as having an MVD score of 0.
Results: Ten cases (50%) expressed greater CCR7 activity than CD206, seven cases (35%) expressed approximately equal activity of CCR7 and CD206, while three cases (15%) expressed greater activity of CD206 than CCR7. Twist expression was strong in some cases with strong N-cadherin and weak E-cadherin, but the expression of Twist was not consistently high in all cases that expressed strong N-cadherin and weak E-cadherin.
Conclusions: TAMs distribution suggested antitumor activity and the potential for tumor metastasis was only partly due to Twist-mediated epithelial–mesenchymal transition.
Background: Treatment of patients presenting with possible acute myocardial infarction (AMI) is based on timely diagnosis and proper risk stratification aided by biomarkers. We aimed at evaluating the predictive value of GDF-15 in patients presenting with symptoms suggestive of AMI.
Methods: Consecutive patients presenting with suspected AMI were enrolled in three study centers. Cardiovascular events were assessed during a follow-up period of 6 months with a combined endpoint of death or MI.
Results: From the 1818 enrolled patients (m/f = 1208/610), 413 (22.7%) had an acute MI and 63 patients reached the combined endpoint. Patients with MI and patients with adverse outcome had higher GDF-15 levels compared with non-MI patients (967.1pg/mL vs. 692.2 pg/L, p<0.001) and with event-free patients (1660 pg/mL vs. 756.6 pg/L, p<0.001). GDF-15 levels were lower in patients with SYNTAX score ≤ 22 (797.3 pg/mL vs. 947.2 pg/L, p = 0.036). Increased GDF-15 levels on admission were associated with a hazard ratio of 2.1 for death or MI (95%CI: 1.67–2.65, p<0.001) in a model adjusted for age and sex and of 1.57 (1.13–2.19, p = 0.008) adjusted for the GRACE score variables. GDF-15 showed a relevant reclassification with regards to the GRACE score with an overall net reclassification index (NRI) of 12.5% and an integrated discrimination improvement (IDI) of 14.56% (p = 0.006).
Conclusion: GDF-15 is an independent predictor of future cardiovascular events in patients presenting with suspected MI. GDF-15 levels correlate with the severity of CAD and can identify and risk-stratify patients who need coronary revascularization.
Reactive oxygen species are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases are important reactive oxygen species targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of protein-tyrosine phosphatase oxidation in signal transduction, the cell biological details of reactive oxygen species-catalyzed protein-tyrosine phosphatase inactivation have remained largely unclear, due to our inability to visualize protein-tyrosine phosphatase oxidation in cells. By combining proximity ligation assay with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor evokes transient oxidation on or close to RAB5+/ early endosome antigen 1− endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with platelet-derived growth factor receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that platelet-derived growth factor-dependent “redoxosomes,” contribute to proper signal transduction.
Background. Colorectal cancers (CRC) shed DNA into blood circulation. There is growing evidence that the analysis of circulating tumor DNA can be effectively used for monitoring of disease, to track tumor heterogeneity and to evaluate response to treatment. Case Presentation. Here, we describe two cases of patients with advanced CRC. The first case is about a patient with no available tissue for analysis of RAS mutation status. Liquid biopsy revealed RAS-wild-type and the therapy with anti-EGFR (epidermal growth factor receptor) monoclonal antibody cetuximab could be initiated. In the second case, the mutational profile of a patient with initial wild-type RAS-status was continually tracked during the course of treatment. An acquired KRAS exon 3 mutation was detected. The number of KRAS mutated fragments decreased continuously after the discontinuation of the therapy with EGFR-specific antibodies. Conclusion. Liquid biopsy provides a rapid genotype result, which accurately reproduces the current mutation status of tumor tissue. Furthermore, liquid biopsy enables close monitoring of the onset of secondary resistance to anti-EGFR therapy.
In der vorliegenden Arbeit sollte der Einfluss von langjährigem Lastkraftwagenfahren auf die posturale Kontrolle untersucht werden. Hierzu wurden die Auswirkungen verschiedener Einflussfaktoren, die mit langjährigem Lastkraftwagenfahren assoziiert sind, im Hinblick auf die posturale Kontrolle analysiert. In diesem Rahmen wurden auf einer Autobahnraststätte in Deutschland 180 (179m/1w) Lastkraftwagenfahrer im Alter von 21 bis 65 Jahren mit einer Berufserfahrung von 1 bis 45 Jahren untersucht. Mit Hilfe eines Fragebogens wurden Informationen über die Arbeitszeit und den Gesundheitszustand der Lastkraftwagenfahrer, wie beispielsweise bestehende Rückenschmerzen oder Sportverhalten, erhoben. Für die Messung der Parameter der posturalen Kontrolle in habitueller Standposition wurde die Druckmessplatte GP Multisens der Firma GeBioM (Münster, Deutschland) verwendet. Es wurde die prozentuale Druckverteilung im Vorfuß-Rückfußbereich für den linken und rechten Fuß gemessen. Des Weiteren konnte die Körperschwankung in frontaler (anterior/posterior) und sagittaler (medial/lateral) Ebene aufgezeichnet werden. Dadurch lassen sich unter anderem Rückschlüsse auf eine Verlagerung des Körperschwerpunktes ziehen. Die statistische Auswertung erfolgte mit Hilfe des Statistikprogrammes BiAS Version 11.0 (2015). Die erhobenen Daten wurden unter Verwendung des Kolmogoroff-Smirnoff-Lillifors-Tests auf Normalverteilung getestet. Das Signifikanzniveau lag bei α=5%. Daneben wurde ein zweiseitiges 95%-Konfidenzintervall sowie der Toleranzbereich der Daten berechnet. Da keine Normalverteilung der Daten vorlag, wurde der Kruskal Wallis Test mit anschließender Bonferoni-Holm Korrektur zum Vergleich der Daten eingesetzt. Die Korrelationen wurden durch einfache, lineare Korrelation nach Pearson oder Rang-Korrelation nach Spearman & Kendall berechnet. Die Studienteilnehmer wurden zusätzlich gemäß ihres BMI, ihrer bereits als Lastkraftwagenfahrer geleisteten Arbeitsjahre, nach bestehenden Rückenschmerzen und nach sportlicher Betätigung jeweils verschiedenen Gruppen zugeordnet. Die Gruppen wurden anschließend hinsichtlich der gemessenen Parameter miteinander verglichen. In Bezug auf ein zunehmendes Alter konnte eine signifikant zunehmende sagittale Schwankung (p ≤ 0,01) sowie eine erhöhte Belastung des rechten Fußes (p ≤ 0,01) gemessen werden. Im Vergleich zwischen normalgewichtigen und übergewichtigen Lastkraftwagenfahrern zeigten sich signifikante Unterschiede hinsichtlich der gemessenen Parameter der posturalen Kontrolle. Es konnte beobachtet werden, dass es mit zunehmenden BMI zu einer erhöhten Schwankung in frontaler (p ≤ 0,04) und sagittaler (p ≤ 0,001) Ebene kommt. Hinsichtlich der Fußdruckbelastung kam es zu keinen signifikanten Veränderungen zwischen den BMI-Gruppen. Lastkraftwagenfahrer mit mehr Arbeitsjahren zeigten erhöhte frontale und sagittale Körperschwankungen. Es lag eine signifikante, positive Korrelation zwischen der frontalen Schwankung und einer Zunahme der Arbeitsjahre (p ≤ 0,04) vor. Auch bei der sagittalen Schwankung bestand eine signifikante Korrelation (p ≤ 0,01). Lediglich bei der Belastung des rechten Vorfußes konnte eine signifikante Korrelation nachgewiesen werden (p ≤ 0,01), während bei den anderen Parametern der Fußbelastung keine signifikante Korrelation belegt werden konnte. Im Gruppenvergleich nach Arbeitsjahren zeigte sich, dass mit ansteigenden Arbeitsjahren als Lastkraftwagenfahrer zu einem Anstieg des BMI kommt. Im Vergleich von Lastkraftwagenfahrern mit und ohne Rückenschmerzen waren keine signifikanten Unterschiede bei den gemessenen Parametern der posturalen Kontrolle nachweisbar. Auch bei der Analyse des Einflusses von sportlicher Aktivität auf die posturale Kontrolle konnte kein Unterschied zwischen der Gruppe mit sportlicher Aktivität und ohne sportliche Aktivität gemessen werden.
Mit dieser Studie konnten die gesundheitsgefährdenden Einflüsse des Lastkraftwagenfahrens auf die posturale Kontrolle nachgewiesen werden. Vor allem das langjährige Sitzen und der erhöhte BMI haben einen Einfluss auf die Körperstabilität und stören die Kompensationsmechanismen der Aufrechterhaltung der posturalen Kontrolle. Die genauen Steuerungsmechanismen der Körperstabilität sind sehr komplex und wurden in dieser Studie nicht im Einzelnen analysiert. Um welche konkreten Strukturen es sich handelt, die durch das langjährige Lastkraftwagenfahren gestört werden, kann mit den Ergebnissen dieser Studie nicht erfasst werden.
Background: PINK1 deficiency causes the autosomal recessive PARK6 variant of Parkinson’s disease. PINK1 activates ubiquitin by phosphorylation and cooperates with the downstream ubiquitin ligase PARKIN, to exert quality control and control autophagic degradation of mitochondria and of misfolded proteins in all cell types.
Methods: Global transcriptome profiling of mouse brain and neuron cultures were assessed in protein-protein interaction diagrams and by pathway enrichment algorithms. Validation by quantitative reverse transcriptase polymerase chain reaction and immunoblots was performed, including human neuroblastoma cells and patient primary skin fibroblasts.
Results: In a first approach, we documented Pink1-deleted mice across the lifespan regarding brain mRNAs. The expression changes were always subtle, consistently affecting “intracellular membrane-bounded organelles”. Significant anomalies involved about 250 factors at age 6 weeks, 1300 at 6 months, and more than 3500 at age 18 months in the cerebellar tissue, including Srsf10, Ube3a, Mapk8, Creb3, and Nfkbia. Initially, mildly significant pathway enrichment for the spliceosome was apparent. Later, highly significant networks of ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing occurred. Finally, an enrichment of neuroinflammation factors appeared, together with profiles of bacterial invasion and MAPK signaling changes—while mitophagy had minor significance. Immunohistochemistry showed pronounced cellular response of Iba1-positive microglia and GFAP-positive astrocytes; brain lipidomics observed increases of ceramides as neuroinflammatory signs at old age.
In a second approach, we assessed PINK1 deficiency in the presence of a stressor. Marked dysregulations of microbial defense factors Ifit3 and Rsad2 were consistently observed upon five analyses: (1) Pink1 −/− primary neurons in the first weeks after brain dissociation, (2) aged Pink1 −/− midbrain with transgenic A53T-alpha-synuclein overexpression, (3) human neuroblastoma cells with PINK1-knockdown and murine Pink1 −/− embryonal fibroblasts undergoing acute starvation, (4) triggering mitophagy in these cells with trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and (5) subjecting them to pathogenic RNA-analogue poly(I:C). The stress regulation of MAVS, RSAD2, DDX58, IFIT3, IFIT1, and LRRK2 was PINK1 dependent. Dysregulation of some innate immunity genes was also found in skin fibroblast cells from PARK6 patients.
Conclusions: Thus, an individual biomarker with expression correlating to progression was not identified. Instead, more advanced disease stages involved additional pathways. Hence, our results identify PINK1 deficiency as an early modulator of innate immunity in neurons, which precedes late stages of neuroinflammation during alpha-synuclein spreading.
The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-α. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF-α resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-α. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF-α and IL-1β. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes.
The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, “3Y”) as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.
AKTIVA-MCI is a program for patients with mild cognitive impairment (MCI) that aims to enhance participation in cognitively stimulating leisure activities. Participation in cognitively stimulating activities seems to be a potential strategy for people with MCI delaying cognitive decline for a while. In total, 35 MCI patients were enrolled in the pilot study of whom 29 completed the whole program (16 female, 71.1±7.5 years; Mini Mental Status Examination score: 28±2.2). Daily activity protocols were used to measure the frequency of participation in cognitively stimulating activities during the program (12 sessions). Additional standardized psychometric tests and questionnaires were used to assess cognition, mood, and subjective memory decline. Analyses of the daily activity protocols showed that during the intervention participants increased the frequency of several cognitively stimulating leisure activities. Comparison of pre-post data indicates no changes in cognitive status, mood, and subjective memory decline. These findings indicate that the program is suitable for patients with MCI.
Drug induced liver injury (DILI) is a potentially serious adverse reaction in a few susceptible individuals under therapy by various drugs. Health care professionals facing DILI are confronted with a wealth of drug-unrelated liver diseases with high incidence and prevalence rates, which can confound the DILI diagnosis. Searching for alternative causes is a key element of RUCAM (Roussel Uclaf Causality Assessment Method) to assess rigorously causality in suspected DILI cases. Diagnostic biomarkers as blood tests would be a great help to clinicians, regulators, and pharmaceutical industry would be more comfortable if, in addition to RUCAM, causality of DILI can be confirmed. High specificity and sensitivity are required for any diagnostic biomarker. Although some risk factors are available to evaluate liver safety of drugs in patients, no valid diagnostic or prognostic biomarker exists currently for idiosyncratic DILI when a liver injury occurred. Identifying a biomarker in idiosyncratic DILI requires detailed knowledge of cellular and biochemical disturbances leading to apoptosis or cell necrosis and causing leakage of specific products in blood. As idiosyncratic DILI is typically a human disease and hardly reproducible in animals, pathogenetic events and resulting possible biomarkers remain largely undisclosed. Potential new diagnostic biomarkers should be evaluated in patients with DILI and RUCAM-based established causality. In conclusion, causality assessment in cases of suspected idiosyncratic DILI is still best achieved using RUCAM since specific biomarkers as diagnostic blood tests that could enhance RUCAM results are not yet available.
Background: To meet the requirements imposed by the time-dependency of acute stroke therapies, it is necessary 1) to initiate structural and cultural changes in the breadth of stroke-ready hospitals and 2) to find new ways to train the personnel treating patients with acute stroke. We aimed to implement and validate a composite intervention of a stroke team algorithm and simulation-based stroke team training as an effective quality initiative in our regional interdisciplinary neurovascular network consisting of 7 stroke units.
Methods: We recorded door-to-needle times of all consecutive stroke patients receiving thrombolysis at seven stroke units for 3 months before and after a 2 month intervention which included setting up a team-based stroke workflow at each stroke unit, a train-the-trainer seminar for stroke team simulation training and a stroke team simulation training session at each hospital as well as a recommendation to take up regular stroke team trainings.
Results: The intervention reduced the network-wide median door-to-needle time by 12 minutes from 43,0 (IQR 29,8–60,0, n = 122) to 31,0 (IQR 24,0–42,0, n = 112) minutes (p < 0.001) and substantially increased the share of patients receiving thrombolysis within 30 minutes of hospital arrival from 41.5% to 59.6% (p < 0.001). Stroke team training participants stated a significant increase in knowledge on the topic of acute stroke care and in the perception of patient safety. The overall course concept was regarded as highly useful by most participants from different professional backgrounds.
Conclusions: The composite intervention of a binding team-based algorithm and stroke team simulation training showed to be well-transferable in our regional stroke network. We provide suggestions and materials for similar campaigns in other stroke networks.
The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.
Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology
(2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
S1P provokes tumor lymphangiogenesis via macrophage-derived mediators such as IL-1β or lipocalin-2
(2017)
A pleiotropic signaling lipid, sphingosine-1-phosphate (S1P), has been implicated in various pathophysiological processes supporting tumor growth and metastasis. However, there are only a few descriptive studies suggesting a role of S1P in tumor lymphangiogenesis, which is critical for tumor growth and dissemination. Corroborating own data, the literature suggests that apoptotic tumor cell-derived S1P alters the phenotype of tumor-associated macrophages (TAMs) to gain protumor functions. However, mechanistically, the role of TAM-induced lymphangiogenesis has only been poorly described, mostly linked to the production of lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C) and VEGF-D, or transdifferentiation into lymphatic endothelial cells. Recent findings highlight a rather underappreciated role of S1P in tumor lymphangiogenesis, referring to the production of interleukin-1β (IL-1β) and lipocalin-2 (LCN2) by a tumor-promoting macrophage phenotype. In this review, we aim to provide to the readers with the current understanding of the molecular mechanism how apoptotic cell-derived S1P triggers TAMs to promote lymphangiogenesis.
Background: The Ebola virus has been responsible for numerous outbreaks since the 1970s, with the most recent outbreak taking place between 2014 and 2016 and causing an international public health emergency. Ebola virus disease (EVD) has a high mortality rate and no approved targeted treatment exists to date. A number of established drugs are being considered as potential therapeutic agents for the treatment of EVD.
Objective: We aimed to identify potential drug repositioning candidates and to assess the scientific evidence available on their efficacy.
Methods: We conducted a systematic literature search in MEDLINE, Embase, and other relevant trial registry platforms for studies published between January 1976 and January 2017. We included drug screening, preclinical studies, and clinical studies on repurposed drugs for the treatment of EVD. The risk of bias for animal studies and nonrandomized clinical studies was assessed. The quality of reporting for case series and case reports was evaluated. Finally, we selected drugs approved by established regulatory authorities, which have positive in vitro study outcomes and at least one additional animal or clinical trial.
Results: We identified 3301 publications, of which 37 studies fulfilled our inclusion criteria. Studies were highly heterogeneous in terms of study type, methodology, and intervention. The risk of bias was high for 13 out of 14 animal studies. We selected 11 drugs with potential anti-EVD therapeutic effects and summarized their evidence.
Conclusions: Several established drugs may have therapeutic effects on EVD, but the quality and quantity of current scientific evidence is lacking. This review highlights the need for well-designed and conducted preclinical and clinical research to establish the efficacy of potential repurposed drugs against EVD.
Background: Recognizing patients at risk for pulmonary complications (PC) is of high clinical relevance. Migration of polymorphonuclear leukocytes (PMN) to inflammatory sites plays an important role in PC, and is tightly regulated by specific chemokines including interleukin (IL)−8 and other mediators such as leukotriene (LT)B4. Previously, we have reported that LTB4 indicated early patients at risk for PC after trauma. Here, the relevance of LTB4 to indicating lung integrity in a newly established long-term porcine severe trauma model (polytrauma, PT) was explored.
Methods: mTwelve pigs (3 months old, 30 ± 5 kg) underwent PT including standardized femur fracture, lung contusion, liver laceration, hemorrhagic shock, subsequent resuscitation and surgical fracture fixation. Six animals served as controls (sham). After 72 h lung damage and inflammatory changes were assessed. LTB4 was determined in plasma before the experiment, immediately after trauma, and after 2, 4, 24 or 72 h. Bronchoalveolar lavage (BAL)-fluid was collected prior and after the experiment.
Results: Lung injury, local gene expression of IL-8, IL-1β, IL-10, IL-18 and PMN-infiltration into lungs increased significantly in PT compared with sham. Systemic LTB4 increased markedly in both groups 4 h after trauma. Compared with declined plasma LTB4 levels in sham, LTB4 increased further in PT after 72 h. Similar increase was observed in BAL-fluid after PT.
Conclusions: In a severe trauma model, sustained changes in terms of lung injury and inflammation are determined at day 3 post-trauma. Specifically, increased LTB4 in this porcine long-term model indicated a rapid inflammatory alteration both locally and systemically. The results support the concept of LTB4 as a biomarker for PC after severe trauma and lung contusion.
Purpose: To evaluate long-term outcome of three years and treatment patterns of patients suffering from severely drug-refractory epilepsy (SDRE).
Methods: This analysis was population-based and retrospective, with data collected from four million individuals insured by statutory German health insurance. ICD-10 codes for epilepsy (G40*) and intake of anticonvulsants were used to identify prevalent cases, which were then compared with a matched cohort drawn from the population at large. Insurance data were available from 2008 to 2013. Any patient who had been prescribed with at least four different antiepileptic drugs (AEDs) in an 18-month period was defined as an SDRE case.
Results: A total of 769 patients with SDRE were identified. Of these, 19% were children and adolescents; the overall mean age was 42.3 years, 45.4% were female and 54.6% male. An average of 2.7 AEDs per patient was prescribed during the first follow-up year. The AEDs most commonly prescribed were: levetiracetam (53.5%), lamotrigine (41.4%), valproate (41.3%), lacosamide (20.4%), and topiramate (17.8%). During 3-year follow-up, there was an annual rate of hospitalization in the range 42.7 to 55%, which was significantly higher than the 11.6–12.8% (p < 0.001) for the matched controls. Admissions to hospital because of epilepsy ranged between 1.7 and 1.9 per year, with an average duration for each epilepsy-caused hospitalization of 10–11.1 days. The number of comorbidities for SDRE patients was significantly increased compared with the matched controls: depression (28% against 10%), vascular disorders (22% against 5%), and injury rates were also higher (head 16% against 3%, trunk and limbs 16% against 8%). The 3-year mortality rate for SDRE patients was 14% against 2.1% in the matched cohort.
Conclusion: SDRE patients are treated with AED polytherapy for all of the 3-year follow-up period. They are hospitalized more frequently than the general population and show increased morbidity levels and a sevenfold increase in mortality rate over 3 years. Further examination is required of ways in which new approaches to treatment could lead to better outcomes in severely affected patients.
In der vorliegenden Studie wurden sieben verschiedene Biomarker (Survivin, Ki-S2, EGFR, Her2/neu, PTEN, p53, OSF-2) von Plattenepithelkarzinomen der Mundhöhle und des anterioren Oropharynx bezüglich ihres Expressionsverhaltens vor und nach Applikation einer intraarteriellen Induktionschemotherapie mit 150mg/m² Cisplatin und im Hinblick auf einen möglichen Zusammenhang des Expressionsverhaltens mit dem Chemotherapieresponse untersucht.
Mit Hilfe der TMA-Technologie wurde in Gewebeproben von 41 Patienten, welche am genannten Krebs erkrankt waren, die Expressionsraten immunhistochemisch bestimmt. Die Behandlung der Patienten erfolgte im Rahmen eines multimodalen Therapiekonzepts in der Klinik für Mund-, Kiefer- und Plastische Gesichtschirurgie am Klinikum der Johann Wolfgang Goethe-Universität in Frankfurt am Main. Alle Patienten hatten eine intraarterielle Induktionschemotherapie erhalten gefolgt von einer chirurgischen Intervention. Spätestens nach sechs Wochen erfolgte die adjuvante Bestrahlung beziehungsweise Radiochemotherapie (Maximalvariante). 56% der Patienten erhielten die Maximalvariante.
Die allgemeine pathologische Ansprechrate nach intraarterieller Induktion lag bei 39% (2%= pCR, 37%= pPR). 59% der Patienten zeigten kein Ansprechen auf die Induktion (pSD) und 2% zeigten eine Progression (pPD). Die Expressionsraten der einzelnen Biomarker wurden jeweils vor und nach intraarterieller Induktionschemotherapie bestimmt. Die statistische Analyse erfolgte univariat mittels Wilcoxon-Mann-Whitney-U-Test und Wilcoxon-matched-pairs-Test. Für Survivin, Ki-S2, OSF-2 und EGFR konnten signifikante Unterschiede nach Induktion verzeichnet werden, die Expressionsrate stieg für Survivin Kern (p= 0,0001), OSF-2 (OSF-2 Zytoplasma, p= 0,0067; OSF-2 Membran, p= 0,0001) und EGFR (p= 0,039) signifikant an. Für Ki-S2 nahm die Expressionsrate signifikant ab (p= 0,001).
In der Vergleichsgruppe Responder versus Non-Responder konnten sowohl in der diagnostischen Primärtumorbiopsie als auch im OP-Präparat des Primärtumors nach Induktion keine signifikanten Unterschiede zwischen Respondern und Non-Respondern in den Expressionsraten festgestellt werden. Für Survivin Zytoplasma lagen die Expressionsraten für die Responder-Patienten vor und nach Induktion im oberen Bereich. Für PTEN konnte bei 75% der Patienten ein Expressionsverlust verzeichnet werden. Damit konnte in der hier durchgeführten Studie kein Biomarker detektiert werden, welcher für eine signifikante Vorhersage zur Responserate herangezogen werden könnte.
Die Änderung der Expressionsraten von Biomarkern bei oralen und oropharyngealen Plattenepithelkarzinomen nach intraarterieller Induktionschemotherapie ist ein Befund, der an ähnliche Beobachtungen nach neoadjuvanter Chemotherapie z. B. beim Mammakarzinom anknüpft und darum weiter verfolgt werden sollte.
Background: In oldest-old patients (>80), few trials showed efficacy of treating hypertension and they included mostly the healthiest elderly. The resulting lack of knowledge has led to inconsistent guidelines, mainly based on systolic blood pressure (SBP), cardiovascular disease (CVD) but not on frailty despite the high prevalence in oldest-old. This may lead to variation how General Practitioners (GPs) treat hypertension. Our aim was to investigate treatment variation of GPs in oldest-olds across countries and to identify the role of frailty in that decision.
Methods: Using a survey, we compared treatment decisions in cases of oldest-old varying in SBP, CVD, and frailty. GPs were asked if they would start antihypertensive treatment in each case. In 2016, we invited GPs in Europe, Brazil, Israel, and New Zealand. We compared the percentage of cases that would be treated per countries. A logistic mixed-effects model was used to derive odds ratio (OR) for frailty with 95% confidence intervals (CI), adjusted for SBP, CVD, and GP characteristics (sex, location and prevalence of oldest-old per GP office, and years of experience). The mixed-effects model was used to account for the multiple assessments per GP.
Results: The 29 countries yielded 2543 participating GPs: 52% were female, 51% located in a city, 71% reported a high prevalence of oldest-old in their offices, 38% and had >20 years of experience. Across countries, considerable variation was found in the decision to start antihypertensive treatment in the oldest-old ranging from 34 to 88%. In 24/29 (83%) countries, frailty was associated with GPs’ decision not to start treatment even after adjustment for SBP, CVD, and GP characteristics (OR 0.53, 95%CI 0.48–0.59; ORs per country 0.11–1.78).
Conclusions: Across countries, we found considerable variation in starting antihypertensive medication in oldest-old. The frail oldest-old had an odds ratio of 0.53 of receiving antihypertensive treatment. Future hypertension trials should also include frail patients to acquire evidence on the efficacy of antihypertensive treatment in oldest-old patients with frailty, with the aim to get evidence-based data for clinical decision-making.
Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific gene functions.
Nutrition support is a necessary therapy for critically ill cardiac surgery patients. However, conclusive evidence for this population, consisting of well-conducted clinical trials is lacking. To clarify optimal strategies to improve outcomes, an international multidisciplinary group of 25 experts from different clinical specialties from Germany, Canada, Greece, USA and Russia discussed potential approaches to identify patients who may benefit from nutrition support, when best to initiate nutrition support, and the potential use of pharmaco-nutrition to modulate the inflammatory response to cardiopulmonary bypass. Despite conspicuous knowledge and evidence gaps, a rational nutritional support therapy is presented to benefit patients undergoing cardiac surgery.
Anticoagulation with warfarin and rivaroxaban ameliorates experimental autoimmune encephalomyelitis
(2017)
Background: In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE).
Methods: Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP139–151. Stable anticoagulation was maintained throughout the entire experiment. Mice without anticoagulation treated with the vehicle only were used as controls. The neurological deficit was recorded during the course of EAE, and histopathological analyses of inflammatory lesions were performed.
Results: In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed.
Conclusion: We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.
Treatment of refractory ascites with an automated low-flow ascites pump in patients with cirrhosis
(2017)
Background: Refractory ascites (RA) is a frequent complication of cirrhosis, requiring large volume paracentesis or placement of a transjugular intrahepatic portosystemic shunt (TIPSS). The automated low-flow ascites pump (alfapump, Sequana Medical AG, Zurich, Switzerland) is an innovative treatment option for patients with RA.
Aim: To assess safety and efficacy of this treatment in patients with a contraindication to TIPSS.
Methods: Fifty-six patients (43 males; mean age 62 years) from centres in Germany, Switzerland, UK and Spain were included and followed for up to 24 months. Complications, device deficiencies, paracentesis frequency and patient survival were recorded.
Results: At the time of this analysis, 3 patients completed the 24-month observation period, monitoring of 3 was ongoing, 9 underwent liver transplantation, 17 patients were withdrawn due to serious adverse events and 23 patients died. Most frequently observed technical complication was blocking of the peritoneal catheter. Twenty-three pump-related reinterventions (17 patients) and 12 pump exchanges (11 patients) were required during follow-up. The pump system was explanted in 48% of patients (in 17 patients due to serious adverse events, in 9 at the time of liver transplantation and in 1 due to recovery from RA). Median frequency of paracentesis dropped from 2.17 to 0.17 per month.
Conclusions: The alfapump can expand therapeutic options for cirrhotic patients with RA. Continuous drainage of ascites in a closed loop automated system led to significant reduction in paracentesis frequency. Technical and procedural improvements are required to reduce the rate of adverse events and reinterventions.
We are pleased to introduce the sixth Journal Club. This edition is focused on several relevant studies published in the last years in the field of Strength and Conditioning, chosen by our Editorial Board members and their colleagues. We hope to stimulate your curiosity in this field and to share with you the passion for sport and exercise seen from a scientific point of view. The Editorial Board members wish you an inspiring read.
Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56+ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56+ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.